Sporothrix schenckii and RelatedSpecies of Ceratocystis u ...686 TRAVASSOS ANDLLOYD B O.. Go4s '-A 0 c. 4 w.~}.0.4.-a--J;A 's 'p, r v . eJ" FIG. 1. Invivomorphologyandin vitro morphologyofS

Vol. 44, No. 4MICROBIOLOGICAL REVIEWS, Dec. 1980, p. 683-7210146-0749/80/04-0683/39$02.00/0

Sporothrix schenckii and Related Species of Ceratocystis uLUIZ R )TRAVASSOS' AND KENNETH 6. LLOYD2*

Departamento de Microbiologiw Geral, Instituto de Microbiologia, Lniversidade Federal do Rio deJaneiro, Rio de Janeiro, RJ, 21491, Brasil1 and Memorial Sloan-Kettering Cancer Center, New York,

New York 100212

NTRODUCTCION ......................................................... 683ECOLOGY OF S. SCHENCKII AND CERATOCYST7S SPP. ........ ............. 684Methods of Isolation and Identification ............................. 684Environmental Factors and Biological Associations...... ... 685Epidemiology of Sporotrichosis ............. ..................... 687

MORPHOLOGICAL DIVERSITY AND ULTRASTRUCTURE ...... ............ 688S schenckii Cell Types In Vitro and In Vivo ................................. 688Dimorphism inS. schenckii ............................................. 689Ultrastructure and the Mycelium-to-Yeast Transition ....................... 690Cytochemistry of Cel Wall Structures ................... 691

BIOCHEMICAL ACIVITIES AND MAJOR CONSTITUENTS ........ ... 693Question of the Perfect Form of S. schenckii....... 696

CELLUILAR AND EXOCELLULAR POLYSACCHARIDES.697Methods for Isolation of Alkali-Soluble Polysaccharides... 697S. schenckii and Ceratocystis Rhamnomannans 698"3C Nuclear Magnetic Resonance Spectroscopy .............................. 700Acidic Rhamnomannan of Ceratocystis stenoceras....... 703Other Ceratocystis Polysaccharides ......................................... 705Galactose-Containing Polysaccharides 7..................................706S. schenckii Cell Wall Glucans ...................... ................ 707

IMMUNOCHEMISTRY OF S. SCHENCKII SURFACE ANTIGENS ....... 707Antigenic Determinants and Cross-Reactivities.... 707Determinants for Delayed-Type Hypersensitivity ........................... 709

NATURAL RESISTANCE AND IMMUNOLOGY IN SPOROTRICHOSIS. 710Experimental Infections in Animals.711Serological Tests for Diagnosis of Sporotrichosis ... ...... ....... 711

SUMMARY AND FUTUJRE PROSPECTS.713LITERATURE CITED.714

INTRODUCTION

Sporothrix schenckii Hektoen and Perkins(63), a dimnorphic fungus in the class Hyphomy-cetes, is the causative agent of human sporotri-chosis. Localized cutaneous- xrlUtsYus spo-rotrichosis is the most common form of infectionand is characterized by ulcerative lesions, asso-ciated regional lymphangitis, and lymphadenop-athy; hard, spherical nodules form along thelymphatic vessels. This disease can be diagnosedunequivocally by culturing the fungus and di-rectly identifying it by using specific fluorescentantibodies. Less easily recognizable forms ofspo-rotrichosis are the disseminated cutaneous-sub-cutaneous and the extracutaneous forms. Inthese, primary skin lesions are often absent, andsubcutaneous nodules may be present (scatteredover the entire body) or absent, as in some casesof sporotrichosis involving the lungs, joints,bones, and other organs (3, 60, 82, 100, 109, 151,176). Serological diagnosis is of special value insystemic disease.

The increasing incidence of extracutaneoussporotrichosis has been stressed (223). Dissemi-nated disease is usually associated with individ-uals with compromised immunological capaci-ties (13, 31, 100, 101, 212, 223). Healthy personsin endemic areas either are resistant to sporotri-chosis or may develop a localized cutaneousform. Self-limited sporotrichosis with sponta-neous cure has been reported (37, 64, 149). Spo-rotrichosis occurs on all continents. Humans anddomestic animals (1, 41, 95) can be affected.The name Sporotrichum for the agent of spo-

rotrichosis is inappropriate (26). S. schenckiidoes not resemble Sporotrichum aureum Link,the lectotype species of the genus Sporotrichum.The genus Sporothrix and the type species S.schenckii have been validated by Nicot andMariat (137). The variety S. schenckii var. lurieiAjello and Kaplan (2), which was isolated inSouth Africa, differs from the type species bythe unusual cell types which it forms in vivo.

Certain ascomycetes in the genus Ceratocystisare related to S. schenckii in several respects.

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Many species synthesize rhamnomannans (185,216), which are also the main surface antigens ofS. schenckii and are not found in most patho-genic fungi. Also, a few Ceratocystis species haveconidial forms of the Sporothrix type (119), andsome coexist with S. schenckii in the same hab-itats (115). Several Ceratocystis species are phy-topathogenic. Ceratocystis ulmi is the causativeagent of Dutch elm disease (22). Ceratocystisminor, which may be related phylogenetically toS. schenckii (126), is commonly found associatedwith various bark beetles in coniferous hosts(68). Ceratocystis stenoceras, whose conidialmorphology is indistinguishable from that of S.schenckii, is known to cause blue discolorationin conifers (59). Recently (216), the bipartitionof the genus Ceratocystis has been proposed.Those species with endogenous conidiogenesisor with Chalara states were considered Cera-tocystis sensu strictu, whereas those species withexogenous conidiogenesis or with Graphium-likestates were classified as Ophiostoma. Ascigerousspecies related to S. schenckii belong to thegenus Ophiostoma.Recent studies have contributed to a better

understanding of the surface antigens, cell wallstructure, systematic position, and ecology of S.schenckii. New models for experimental sporo-trichosis have appeared, and a better under-standing ofimmunological responses to S. schen-ckii is now possible. The influence of growthconditions and morphological differentiation oncell surface components has been analyzed ex-tensively. Because of the role of polysaccharidesin cell wall structure and antigenicity, these mol-ecules have been particularly emphasized. Thepresent work reviews and critically discussesthese results and stresses the unique character-istics of S. schenckii among pathogenic fungi. Italso reviews work on those Ceratocystis specieswhich are related taxonomically and biochemi-cally to S. schenckii.This review also summarizes the recent liter-

ature on S. schenckii, but it is designed primarilyfor medical mycologists who are willing to usevarious chemical, biochemical, and immuno-chemical methods to understand further thegeneral biology, taxonomy, virulence, and im-munological reactivity of pathogenic fungi. Italso draws attention to the value of '3C nuclearmagnetic resonance (NMR) spectroscopy as anaid in identifying polysaccharide structures,many of which are characteristic of a particularfungus or a morphological form of a certainfungus.

ECOLOGY OF S. SCHENCKII ANDCERATOCYSTIS SPP.

The primary habitats for S. schenckii and

Ceratocystis spp. are the soil and plants. Incontrast to Ceratocystis, S. schenckii is not phy-topathogenic. Preferential biological associa-tions or even strict host specificities among thesefungi have been recognized. For instance, C.ulmi is highly specific for elms. The ecology ofS. schenckii determines the epidemiology ofspo-rotrichosis. The conditions for the survival of S.schenckii in nature, such as temperature, hu-midity, and necessary nutrients, as well as theconditions favoring human infection, have beenstudied (106, 121).

Methods of Isolation and IdentificationS. schenckii is not easily detected by direct

observation of pathological specimens. If cellelements are visible after staining with periodicacid-Schiff reagent or other methods, they maystill be confused with cellular debris or otherfungi. Fluorescent rabbit anti-S. schenckii glob-ulins were shown to be useful in staining bothyeast and mycelial forms in cultures and clinicalspecimens (79, 83). The antibodies were ratherspecific since 47 strains of 21 heterologous spe-cies of fungi were not reactive (77). Fluorescentantibodies detected S. schenckii in smears oflesion exudates from 24 to 27 culturally positivepatients (78). S. schenckii cell types were cigar-shaped forms, but oval, elliptical, bacilliform,and globose cells were also observed. Because ofthe scarcity of cells present in such specimens,it was necessary to search several microscopicfields. Usually, the culturally negative cases werealso negative by the fluorescent antibody stain-ing method (77). S. schenckii and Cryptococcusneofornans were also localized in 4% formalde-hyde-fixed tissue sections (80) embedded in par-affim by an indirect immunoperoxidase stainingmethod (164). Because of the considerable stain-ing differential, identification of S. schenckiiyeast forms was possible.

Isolation of S. schenckii in culture can beachieved by using Sabouraud 2% dextrose agaror brain heart infusion agar supplemented withpenicillin, streptomycin, and cycloheximide (73)or chloramphenicol and cycloheximide (115,122). Mycelial cultures develop at 250C, whereasyeast cells are formed preferentially in brainheart infusion medium at 370C. In the mycelialphase this fungus grows rapidly as flat, moistcolonies which are at first dirty white andcoarsely tufted and then brown or black withwrinkled surfaces. Yeast colonies form at 370Cand are moist and cream colored. Other media(such as potato dextrose agar and Littman oxgallagar at room temperature or 37°C) can also beused to isolate S. schenckii (90).

Conversion to the yeast form is an importantstep in the identification of S. schenckii. This

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can be achieved by using special media, such asglucose cysteine blood agar at 37°C, by inocula-tion into animals, or by tissue culture. Whenmycelial fragments are inoculated into the testesof mice, the purulent material recovered after 2to 3 weeks contains yeast cells. Hempel andGoodman (65) used cultured guinea pig perito-neal macrophages and in 24 h obtained conver-sion of mycelial particles to yeast cells at 370Cin a CO2 incubator. In the identification ofstrains of S. schenckii and related Ceratocystisspp., several characteristics (such as conidialmorphology of the Sporothrix type, the abilityto grow in the presence of cycloheximide and at370C, and a requirement for thiamine) have beenconsidered relevant (119). The final identifica-tion of Ceratocystis is made by observation ofperithecium formation. In C. stenoceras, peri-thecia formed after the fungus was grown on amedium containing wood fragments or on asemisynthetic agar medium (119) for 10 days.Generally, these morphological structures arenot formed in Sabouraud medium. The Sporo-thrix conidial type is characterized by conidiaproduced sympodially at the apexes of the con-idiophores in rosette-like arrangements or in-serted directly onto the filaments. Conidia canbe detached easily, and thus slide cultures areusually required for observation of the typicalmorphology (Fig. 1).

Environmental Factors and BiologicalAssociations

S. schenckii is isolated most often from soil,live plants, or plant debris, wood, and straw, butit can also be found in insects, hair, water, air,and a variety of other sources (9, 16, 36, 163,181). Several domestic animals and rodents arecarriers of this fungus (95, 106, 127). Freitas etal. (43) described sporotrichosis in 12 dogs and8 cats, but these animals were not regarded asnatural reservoirs.

Isolates from nature have different character-istics. Strains isolated by Mackinnon (106) fromrotten palm tree trunks, dry grass from armnadilloand rodent holes or nests, and soil covered bymosses formned oval dark-pigmented radula-spores and numerous conidia. Multiple spiculeswere observed on the hyphae after conidial de-tachment. These strains grew at 370C and werepathogenic in mice. Strains isolated by Howardand Orr (67) from rat dung, wood, and soil didnot grow at 370C, formed spherical, dark-pig-mented conidia which could not be detachedeasily from hyphae, and were nonpathogenic.While tracing the source of contamination of apatient with sporotrichosis, Mariat (113) isolatedan S. schenckii strain from Aechmea (Bromeli-aceae), a decorative plant with thorny leaves, as

well as from the soil around the plant. This planthad been maintained in a compost made withhighly decayed manure or partially decayedbeech and oak leaves or both. Particles of Spo-rothrix (300 particles per g) were recognized inthe leaf litter but not in the manure. The strainisolated from the patient and the strain isolatedfrom the plant were compared. Whereas thestrain from the patient was in the yeast phase at370C and was pathogenic for golden hamstersand mice, the presumed wild-type strain isolatedfrom the soil around Aechmea grew faster at 30than at 370C, converted only partially to theyeast phase, and was nonpathogenic in animals.A strain of S. schenckii isolated from the air inParis (113) was also nonpathogenic in hamstersand mice.Correct identification of S. schenckii isolated

from natural environments is complicated bythe occurrence in the same habitats of morpho-logically related species of Ceratocystis and Gra-phium. Strains of low virulence which were iso-lated from the air and timbers at the Venterpostmine in Transvaal and were regarded as S.schenckii (36) were later identified as Graphium(24). Mackinnon (106) isolated strains similar topale strains of S. schenckii, but, unlike S. schen-ckii, these strains produced a downy whitishgrowth on two culture media. Some of thesestrains were identified as Ceratocystis sp.,whereas others had the morphology of Gra-phium. Cells similar to the cigar-shaped bodiesof S. schenckii have been observed in inoculatedmice, but the lesions which developed werenonevolutive and nonmetastatic. Mixed culturesof S. schenckii and Ceratocystis have also beenobtained (108). Although there may be nonpath-ogenic strains of S. schenckii, the criterion ofexperimental pathogenicity in mice and ham-sters and the characteristic evolution of the in-fection with metastases are of great help for finalidentifications of this fungus. In some instances,the virulence of strains isolated from nature issimilar to the virulence of strains isolated fromhumans (106). However, only 3 of 16 S. schenckiistrains isolated from contaminated trees and soilin Corse, France, were pathogenic in mice (115).Noteworthy was the high proportion of C. sten-oceras isolated in this study. S. schenckii strainswere isolated from Pinus laricio var. corsicana.C. stenoceras occurred predominantly in Euca-lyptus spp. To isolate these fungi, the plantfragments or leaves were ground to a thin pow-der, which was then suspended in distilled watercontaining 0.1% cycloheximide, 0.1% chloram-phenicol, and 0.02% Tween 80. After the super-natant of this suspension had settled for 15 min,it was inoculated onto Sabouraud agar contain-ing cycloheximide and chloramphenicol. Colo-

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FIG. 1. In vivo morphology and in vitro morphology of S. schenckii. (A) Abundance of spherical forms andof budding cells. (B) Giant cells. (C) Slide culture of a strain isolated from a human infection. (From Mariatet al. [118]; used with permission.)

nies of fungi showing the characteristic sympo-dulospores of S. schenckii were reisolated inSabouraud medium containing antibiotics, andthe formation of perithecia was then tested infavorable media (119). Since the only differencebetween strains with the same conidial mor-phology is the eventual formation of perithecia,it is conceivable that some S. schenckii strainswere indeed Ceratocystis and required specialconditions to form the fruiting bodies. The cri-terion of experimental pathogenicity in animalscould not be used in this case because most ofthe strains regarded as S. schenckii were non-pathogenic; however, one strain of C. stenocerascaused typical sporotrichosis in mice. The pos-sibility of mixed cultures of C. stenoceras and S.schenckii was not ruled out. It should be notedthat although conifers have been described ashabitats for C. stenoceras (59, 68, 74), strains ofthis ascomycete have been isolated from Euca-lyptus in Corsica, Mexico, and Guatemala (115,122) and from the tails of wild mammals (Cleth-rionomys glareolus, Apodemus sp., Sorex ara-neus, and Cricetus cricetus) in Alsace, France(115). These animals were not particularly as-sociated with a conifer-rich environment. In an-other study, C. stenoceras was not found inconifers in Manitoba, Canada (147).The isolation of S. schenckii from nature

seems to be more frequent in endemic zones ofsporotrichosis (39, 163), but isolations have alsobeen made in areas where the disease in humanshas not been reported (115). However, in onecase isolation from plants was not obtained inan endemic area (a small mountain village innorthern Peru) (49).The growth of S. schenckii in natural sub-

strates has also been studied. Well-seasoned tim-bers can support the growth of this fungus at arelative humidity of 95 to 100%; no growth wasobtained at 90% relative humidity. Growth wasfollowed by the appearance of a black discolor-ation in the wood (24), which was also observedin contaminated oat grains (16). S. schenckiialso caused rot in experimentally inoculated car-nations (15).The influence of climatic and other environ-

mental factors on the distribution of S. schenckiiin nature is not clear. Usually, the presence of S.schenckii is inferred from the number of casesof human sporotrichosis in an area. Since thisnumber is influenced to a large extent by theimmunological responses of different individualsin a population, by their activities or professions,and, particularly, by the amount of contactwhich they have with plants, soil, and othercontaminated materials, a correlation with theactual presence of S. schenckii in nature is dif-ficult to predict. As mentioned above, in zoneswhere human sporotrichosis is absent, S. schen-ckii can still be isolated frequently. On the otherhand, the geographic distribution of sporotri-chosis does not suggest preferential latitudes orspecial climatic conditions (150). However,Mackinnon (104, 105) tried to correlate the in-cidence of sporotrichosis with temperature andrelative humidity in a few cases. Sporotrichosisin Uruguay is more frequent in autumn andearly winter, seasons with frequent rainfalls, rel-ative humidities close to 100%, and temperatureranges of 16 to 20°C. Similar observations weremade by Silva (179) in Rio Grande do Sul, thesouthernmost state of Brazil, which bordersUruguay and has a similar climate. Findlay (39)

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observed that the fungus grew well on minepoles in South Africa at temperatures of 26 to27°C and relative humidities of 92 to 100%.Outside the mines, sporotrichosis was prevalentin a temperate highland plateau, which had arelative humidity of 65%. As pointed out byMackinnon (106), sporotrichosis is rare in zonesof little rainfall, such as central and northernChile, semiarid lands, high plateaus, and moun-tains. However, sporotrichosis is rare in Manaus(131), a city deep inside the Amazon region andthus in an environment of high relative humid-ity. Temperatures above 30°C probably repre-sent a limiting factor in tropical zones, as S.schenckii grows very poorly or not at all at 38 to38.5°C and converts to the yeast phase at tem-peratures of 28 to 35°C. Below 28°C only themycelial phase develops (111) with the forma-tion of conidia, which are presumably more ableto survive in nature than the yeast form. Thecases of sporotrichosis reported in Mexico (52,121) also had no apparent correlation with therainy season, as they were most frequent duringthe dry and cooler parts of the year. The suscep-tibility of S. schenckii yeast forms to tempera-tures above 370C is dramatized by the use oflocal heat in the treatment of cutaneous sporo-trichosis. Damp heat, provided by a water bathat 41°C or hot compresses, dry heat, such aswith the Japanese Kairo, and rubefacient drugshave been used successfully (44, 91, 107, 207). Atthe other extreme, prolonged low temperaturesdo not favor sporotrichosis, as this disease is rarein Canada (40).Besides C. stenoceras, which frequently is as-

sociated with S. schenckii in nature, other Cer-atocystis species produce sympodulosporogen-ous conidial states inistinguishable from those ofSporothrix (198). These include C. minor, Cer-atocystis multiannulata, Ceratocystis narcissi,Ceratocystis nigrocarpa, Ceratocystis perpar-vispora, and Ceratocystis pilifera, all of whichbelong to section 3 in the classification of Hunt(68). Based on deoxyribonucleic acid (DNA)properties, C. minor seems to be phylogeneti-cally closer to S. schenckii than the other Cer-atocystis species (126). C. minor has been iden-tified in several European countries, the UnitedStates, and Japan. It is commonly associatedwith conifers and is a phytopathogen (68).

Epidemiology of SporotrichosisWhen drinking wine amongst the rosesOr guzzling beer while throwing bricksOr playing games in bales of hayWhere lurks the tricky sporothrix,Beware, the price you pay for play,When you get struck by dread mycoses

-S. Vaisrub (213)

The incidence of sporotrichosis varies withthe region. In the United States, 350 cases wererecorded up to 1964 (169, 182), and in Japan 261cases were reported up to 1968 (7). This diseaseis practically nonexistent in Portugal, Germany(180, 220), and France (113). The highest inci-dence of sporotrichosis seems to be in southernBrazil and the central highlands of Mexico. Inindividual clinics in both of these countries, spo-rotrichosis accounted for 0.3 to 0.5% of all der-matoses (45, 168). Sporotrichosis is also commonin Venezuela, Colombia, and Uruguay. Out-breaks are usually related to working placeswhere contaminated materials are manipulatedand trauma to the skin is a likely occurrence.The occupations of 310 patients described in theliterature were classified by Mariat (113), whofound the highest incidence among farmers orallied workers (29.3%). Other groups with highproportions of cases were domestic workers andhousewives (19.7%), school children and stu-dents (13.5%), gardeners (8.4%), factory workers(5.4%), and several other individuals who did nothave contact with soil (teachers, policemen, etc.)(14.2%).The greatest epidemics which have been re-

ported involved gold mine workers in the Trans-vaal Mine, South Africa, who had been exposedto infected mine timbers. Up to 2,825 cases werereported between 1941 and 1944 (36, 64, 96).Treatment of the timbers with a fungicide ter-minated the epidemic.

Prairie hay is a common source of infectionfor epidemics in children. Other plants, such ascacti, corn, and particularly rose bushes, havebeen implicated (159). In New York (Long Is-land), infections were reported in workers onbulb farms where salt marsh hay (Spartinapaens) was used as a mulch (182). A total of 59%of the nursery workers who had at least 10 yearsof exposure to soil and sphagnum moss in Ari-zona had positive skin test responses to sporo-trichin, compared with 11% in the control pop-ulation (69). An outbreak of 14 cases of sporo-trichosis in a Vermont tree nursery was alsoatributed to contaminated sphagnum moss,which was introduced by one shipment fromWisconsin, an endemic area (32). It is unclearwhether sphagnum moss is contaminated withthe fungus in the bogs, during harvesting, orafter harvesting (154). The straw used for pack-ing a variety of articles, such as earthenware andceramics, is a common source of infection by S.schenckii (45, 50, 180). In Uruguay, almost one-half of the patients with sporotrichosis wereinfected during armadillo hunting (106). Re-cently, Mayorga et al. (122) described an en-demic area of sporotrichosis around Lake Ayarzain Guatemala. In 45% of the cases infection was

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correlated with handling fish (Tilapia mozam-

bica). The main source of human contaminationwith S. schenckii is the soil, where the funguscan live as a saprophyte and be transported bya suitable carrier (plant elements [17, 115], in-sects, or mammals [95, 99]). The possibility ofhuman carriers of S. schenckii is unlikely ac-

cording to Padilha-Gon,alves and Pereira (un-published data). These workers cultured nasaland pharyngeal specimens from 200 individuals,including patients with active sporotrichosis, pa-

tients cured of sporotrichosis, relatives of pa-

tients, and healthy persons in endemic areas; allcultures were negative.Primary pulmonary infections by S. schenckii

are also possible (100). Isolation of this fungusfrom sputum, bronchial washings, or tissue inassociation with a positive serology or skin testwas regarded as evidence for this kind of con-

tamination (170, 172, 177). Experiments in micehave also shown that primary lung infections are

possible. As reviewed by Lynch (100), humanprimary pulmonary sporotrichosis is character-ized by cavitation of the upper lobes. Michelson(128) observed that 6 of 15 reported cases were

associated with chronic alcoholism, suggestingthat bronchial aspiration is a likely way offunguspenetration.As calculated by Mariat (113), the incidence

of sporotrichosis in 414 patients described in theliterature showed a sex distribution of 53.6%females and 46.4% males. Most patients wereunder 30 years of age, and children less than 10years old were infected frequently. In anothersample involving 541 cases, the sex distributionwas 71% males and 29% females (159). The in-cidence in different age groups showed a rela-tively uniform distribution, with 18.8% of thecases in children 0 to 10 years old.

MORPHOLOGICAL DIVERSITY ANDULTRASTRUCTURE

The morphological characteristics of S. schen-ckii and C. stenoceras have been studied byMariat and co-workers (119, 137). The cell typesformed in vitro and in vivo are compared below.

Early ultrastructural studies on the yeast andmycelial phases of S. schenckii (84, 85) showedthat yeast-phase cells formed a halo about 0.7 to1.0 ,Am wide which was absent in the mycelialphase. The yeast cell walls were 100 to 300 nmthick and had two distinct electron-dense layers.Electron-dense microfibrils were associated withthe yeast-phase walls but were absent in themycelial-phase walls. Each yeast cell containedseveral storage granules and intracytoplasmicmembranes. Bud scars were detected, and mul-tiple budding was interpreted in part as being a

reflection of abortive development of the myce-lial phase (167). The cell wall thickness in my-celial-phase cells was 80 to 140 nm. Each sessileconidium had degenerative changes in its inter-nal organization, and a thickened plate-likestructure was present at the point of hyphalattachment (85). The cell walls of pigmentedconidia were 330 nm thick and had two layers;these were 210 nm thick (inner layer) and 120nm thick (outer layer). On the external layer,irregular electron-opaque granules representingpigment particles were observed (120). Theywere apparently included in a matrix of electron-transparent material. The conidial cytoplasmcontained large lipid storage bodies which werenot found in yeast cells or hyphae (46, 206).Young hyphal cells of S. schenckii, C. steno-ceras, Ceratocystis pluriannulata, C. ulmi, andC. minor had unusual osmiophilic structures(48). Such structures reacted strongly with thio-carbohydrazide and were partially solubilizedwith sodium methoxide. A lipoprotein complexhas been suggested (48). Other intracellularstructures described previously as microendo-spores (148), endogenous spores (137), and as-cospores (200) could represent osmiophilic inclu-sions in cells of C. ulmi and S. schenckii (48).

S. schenckii Cell Types In Vitro and InVivo

Slide cultures of S. schenckii show hyaline,regularly septated mycelial filaments (diameter,1 to 2 ,um) and oval, pyriform, or elongatedconidia (1.5 to 3 by 3 to 6 tm), which are bornsingly or in groups and are inserted directly onsmall denticles around each conidiophore (rad-ulaspores) or, more characteristically, emerge onapical sympodia (sympodulospores). Conidia areusually hyaline, but pathogenic strains of S.schenckii can also form spherical or conical pig-mented conidia (diameter, 2.4 to 3.7 ,um) withthick walls, which are inserted on larger pedicels(137). The yeast form of S. schenckii was firstobtained in vitro by Lutz and Splendore (99).Fusiform and ovoid cells (2.5 to 5 by 3.5 to 6.5pim) form at 370C, and their multiplication in-volves single or multiple budding. Yeast formsoriginate from the sides and tips of hyphae, andtheir primordia can be located on hyphae bystaining with fluorescent concanavalin A (ConA)or by indirect immunofluorescence with a serumdirected against yeast antigens (93, 206). Thetransformation from mycelium to yeast in tissueculture involves formation of yeast cells directlyfrom the mycelium. Conidia germinate intoshort hyphae, which in turn give rise to yeastcells. Yeast cells can also arise by fragmentationof chains of oidia, which often occur inside hy-phae (46, 66).

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Observation of the fungal morphology in vivoalso shows a diversity of cell types according tothe fungal strain and the host reaction to infec-tion. Yeast cells predominate in vivo, but othercell types are occasionally present. Asteroid bod-ies occur in lesions with extensive necrosis, par-

ticularly in hamsters (121). In organs with inva-sive micronodular lesions, few yeasts are present.They are round, but can become cigar-shaped as

the lesions increase in size. Cigar-shaped cellsare dominant around large suppurative lesionsand multiply actively. Asteroid bodies consist ofa central chlamydospore (diameter, 5 to 8 /Lm)with a thick wall, surrounded by radiating pro-

jections (1 to 12 ,um) of eosinophilic material.Such structures may result in part from precip-itations of antigen-antibody complexes on thespore surfaces (98). Lurie (96) observed asteroidbodies in 17 of 32 primary lesions and in 22 of 31secondary lesions. Mycelial hyphae are not usu-

ally seen in vivo in biological specimens, in partbecause of their shortness, their small number,and the difficulty of resolving them from cellularinfiltrates, nuclear fragments, and stain particles(90). However, their presence in tissue sectionsin human sporotrichosis has been reported oc-

casionally (90, 103, 146). In a biopsy specimenfrom human tissue which was fixed in 10% form-aldehyde, embedded in paraffin, sectioned, andstained in sequence with periodic acid-Schiffreagent and hematoxylin plus eosin, the follow-ing fungal elements were detected (90): solitaryspores (oval or spherical bodies), a spore with a

germ tube, cigar-shaped cells which were free or

inside giant cells, asteroid bodies which stainedbetter with hematoxylin plus eosin than withperiodic acid-Schiff reagent, and aggregated my-celial hyphae (length, 3 to 15 ,um), which some-

times had a spore attached terminally andstained with periodic acid-Schiff reagent but notwith hematoxylin plus eosin. Lavalle et al. (86)suggested an in vivo biological cycle to explainthe origin of the asteroid bodies. These authorsobserved that cigar-shaped cells gave rise tospherical forms by gemulation and that thesecells further differentiated and synthesized a

double membrane. The cells then became sur-

rounded by an acid-resistant material (thickness,1 to 2 ,um), from which thin radiating projectionsemerged and became variably voluminous insize, thus forming asteroid bodies.Round forms of S. schenckii may be common

in human infections, as demonstrated in a pa-tient from the French Antilles (118). The lesionsof this patient contained unusual numbers ofspherical cells; however, these spherical cellsyielded a typical conidial morphology when cul-tured in vitro (Fig. 1).

Dimorphism in S. schenckii

Studies on S. schenckii dimorphism have fo-cused on the nutritional factors influencingphase transition, as well as some differentialproperties of the yeast and mycelial phases. Theyeast phase of S. schenckii is obtained when thisorganism is grown in a medium containing caseinhydrolysate or amino acids, glucose, thiamine,and biotin (35). Mechanical agitation of the cul-ture and a temperature of 370C are conditionswhich favor the mycelium-to-yeast transition.Arginine and glycine are as effective as caseinhydrolysate. A medium containing ammoniumsulfate, asparagine, or arginine as the N sourcesupports the mycelium-to-yeast transition, pro-vided that the culture is incubated at 370C witha stream of air-CO2 (95:5). Thiamine is an essen-tial requirement; the pyrimidine moiety is theeffective structure (34). A synthetic medium wasdevised to grow S. schenckii in the yeast phaseat both 37 and 250C (125); this medium con-tained ammonium carbonate and three aminoacids, including L-cysteine. However, Drouhetand Mariat (35) found no evidence that sub-stances with SH groups affected the mycelium-to-yeast transition in S. schenckii.There have been limited studies to compare

the biochemical differences between the yeastand mycelial phases. In one strain, the ratio ofribonucleic acid to DNA in the mycelial phasevaried with the culture age fom 17.3:1 to 7.4:1; inthe yeast phase this ratio varied from 1.4:1 to0.7:1 (117). However, these ratios were straindependent and were not confirmed for other S.schenckii isolates (121). Oxygen consumption,as measured in a regular Warburg respirometer(112) with cells after different incubation times,was less in yeast cells than in mycelial hyphae,which always consumed more oxygen; this con-sumption decreased sharply with time. Such arelationship was inverse to that observed inBlastomyces dermatitidis, another dimorphicfungus. A comparison of the proteins from theyeast and mycelial phases was attempted byusing acrylamide gel electrophoresis (20). Theyeast phase had two extra proteins, as inferredby the appearance of two extra bands, but therelevance of these proteins to dimorphism wasnot explored. Walbaum et al. (215) did not detectchymotrypsin A activity in yeast extracts,whereas this activity was present in mycelialforms (S. Massamba, Ph.D. thesis, Faculty ofLille, Lille, France, 1970).

Variations in cell wall constituents have oftenbeen associated with dimorphism in fungi '(33,76), but a complete picture of the sequence ofevents in morphogenesis is still not possible.

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Insoluble glucans and proteins have been asso-ciated with cell-shaping processes and are con-sidered to be important in determining the rigid-ity of the cell wall (75). However, few observa-tions apply to different species or even differentstrains of dimorphic fungi. In S. schenckii, yeastforms contain thick layers of peptido-polysac-charide complexes at the cell surface, unlike themycelial phase, which has a structure that ismuch less developed. However, these layers are

easily sloughed off into the medium by attrition(14), and their role in cell morphogenesis isunclear. The compositions of the washed wallsof three different cell types of S. schenckii, freeof the loosely attached surface layers, have beendetermined (157). The main constituents areindicated in Table 1. The yeast cell walls differedfrom the mycelial cell walls in that they hadhigher carbohydrate contents and lower proteinand lipid contents. There were characteristicproportions of saturated and unsaturated fattyacids in the yeast walls, and these differed fromthe proportions in the mycelial and conidialwalls. A C183 lipid, possibly linolenic acid, was

present in the yeast walls and absent in the wallsof the other cell types. This was probably a realcomponent and not a contaminant because a

synthetic medium (125) was used to grow theyeast phase. The proteins from yeast walls haddifferent proportions ofamino acids, particularlythreonine, serine, lysine, arginine, and glutamicacid, which may suggest the presence of specificpeptides. Cysteine was not detected. The con-tent of insoluble polysaccharides was slightly

lower in the yeast walls than in the hyphal walls,but the proportion of water- and alkali-solublepolysaccharides was higher (157). Chitin waspresent in similar concentrations in all types.Conidial and mycelial cell walls had similar pro-portions of the main constituents.These results do not indicate large quantita-

tive differences between the cell wall compo-nents of yeast forms on the one hand and hyphaland conidial forms on the other, but the differ-ences detected may well be sufficient to accountfor the morphological diversity. Studies whichdescribe variations in the structures of the cellwall rhamnomannans of the different cell typesare discussed below. It seems that complex in-teractions of several constituents must takeplace to determine cell wall elasticity or rigidity.In S. schenckii, the role of soluble and insolubleglucans in dimorphism, as suggested for otherfungi, is doubtful, since the structures of thesepolysaccharides were very similar in all celltypes (156).

Ultrastructure and the Mycelium-to-YeastTransition

Yeast cells of S. schenckii, Paracoccidioidesbrasiliensis, B. dermatitidis, and Histoplasmacapsulatum undergo a phase conversion whichis characterized by a transitional cell type thatcontains mixed structures (84). The appearanceof discrete intracytoplasmic membranes pre-

cedes the appearance of the transitional celltype. Transitional forms arising from hyphae bydirect budding or by oidial cell formation (Fig.

TABLE 1. Analyses of cell walls from different S. schenckii cell typesa% Recovery in:

Component(s)Yeast forms Mycelium Conidia

Neutral sugars 61 ± 1.3 (12)b 44 ± 0.9 (12) 47 ± 1.0 (8)Hexosamine 7.0 ± 0.4 (9) 7.0 ± 0.3 (9) 8.3 ± 0.6 (6)Glucose as amylose 1.8 ± 0.0 (3) 1.6 ± 0.0 (3) 1.7 ± 0.0 (2)Glucosec 48.1 ± 0.2 (3) 54.9 ± 0.9 (3) 57.9 ± 0.4 (2)Mannosec 36.0 ± 0.8 (3) 28.8 ± 0.5 (3) 25.1 ± 0.5 (2)Rhamnosec 15.7 ± 0.7 (3) 16.2 ± 0.9 (3) 17.0 ± 0.2 (2)Galactose Trace Trace TraceLipids 18.0 + 0.9 (9) 26.0 ± 1.3 (9) 19.6 ± 1.2 (6)Phosphate 0.7 ± 0.0 (6) 1.2 ± 0.0 (6) 0.8 ± 0.0 (4)Total N 2.9 ± 0.1 (6) 3.9 ± 0.1 (6) 4.5 ± 0.1 (4)N of chitin 0.5 0.5 0.6N as proteind 14.3 21.2 24.3Protein' 14.4 ± 0.7 (3) 21.7 ± 1.0 (3) 24.2 ± 0.5 (2)Totalf 101.1 99.9 99.9

a Unless otherwise indicated, values are average percentages of cell wall weight ± standard error from severaldeterminations, using two (conidia) or three (yeast or mycelium) different cell wall preparations.

b Values in parentheses are numbers of determinations.'Values are percentages of total carbohydrate.d (Total N - N of chitin) x 6.25.'Determined by amino acid analysis.fTotal for neutral sugar, hexosamine, lipid, phosphate, and protein analyses. From Previato et al. (157).

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2) have been observed in S. schenckii (46, 84).The claim that yeast cells could also arise bybudding from conidia (217) has not been sub-stantiated. Under conditions favoring phase con-version, lateral, budlike protrusions emerge fromnhyphae. The cell wall of each bud appears t;oarise from the inner part of the hyphal wall. Itsouter surface has prominent, electron-dense rr i-crofibrillar structures, which contrast with thefinely roughened surface of the hyphal wall (Fig.3). Microfibrillar structures, which are charac-teristic of yeast forms and their primordia onhyphae, can be visualized better by stainingglutaraldehyde-fixed cells with dialyzed iron(46). The formation of yeast cells by fragmen-tation of chains of oidia (66) has been confirmed6iy electron microscopy of thin sections of S.schenckii hyphae. The oidial yeast cells alsohave dense microfibrillar material at the cellsurface. The cell wall of each oidial cell appar-ently arises from the inner layer of the degen-erate parent hypha. According to Garrison et al.(46), oidial cell formation in S. schenckii resem-bles the yeast cell ontogeny described in P.brasiliensis (25). Yeast cells with microfibrillarstructures at the cell walls, which arose by budformation or by oidial chain fragmentation, werealso observed in C. stenoceras (47).

Cytochemistry of Cell Wall StructuresThe cell surface components of S. schenckii

yeast- and mycelial-phase cells were able to bindConA, and reactivity with this lectin was visu-alized by a cytochemical method in which horse-radish peroxidase and diaminobenzidine wereused (206). By staining with fluorescein-labeledConA, only yeasts and their primordia on hy-phae were strongly reactive (Fig. 4). This prob-ably reflected a greater concentration of ConA-reactive components in yeast walls comparedwith hyphal walls. With the peroxidase method,two cell wall layers of ConA-reactive compo-nents could be demonstrated in yeasts, in con-trast to a single layer in hyphae and conidia.The outer sublayer of the yeast-phase cell wallsof a human strain of S. schenckii (206) showeda variable degree of reactivity with ConA (Fig.5), but a correlation between reactivity and ageof cells was not evident. It was hypothesized thatthe basal sublayer of the yeast cell walls wascomposed mainly of ConA-reactive peptidosac-charides and that the outer sublayer consistedof long-chain rhamnomannans, which are un-reactive with ConA. A likely candidate for ConAreactivity seemed to be a peptido-rhamnoman-nan complex. It had been shown that prepara-tions of peptido-polysaccharides isolated fromtwo strains of S. schenckii reacted with ConA

FIG. 2. Chain ofoidia inside an S. schenckii hyphastained externally by the ConA-horseradish peroxi-dase-diaminobenzidine method. x4,362.

when a double-diffusion technique was used,although relatively high concentrations of thelectin (5 mg/mnl) were required. No precipitationoccurred with the corresponding rhamnoman-nans dissociated from the peptido complexes byhot alkali extraction. ConA-reactive, threonine-or serine-linked, short oligosaccharide chainsthat occur in parallel with the long polysaccha-ride chains are known to be structural featuresof yeast mannans (10). The presence of suchchains in S. schenckii has not been investigateddirectly, although peptido-polysaccharides of S.schenckii have a high proportion of hydroxyl-ated amino acids, especially serine (92, 206).However, such residues could be linked to pol-ysaccharide chains, as suggested for the peptidocomplexes of Cladosporium werneckii (87) andC. ulmi (193). ConA reacts with residues havinga 3,4,6-arabino-a-D-glycopyranosyl structure,which include nonreducing end units, 2-0-sub-stituted a-D-mannopyranose units, and a-D-glu-copyranose units (184). The reaction of S. schen-ckii cell walls with ConA probably involves man-nose residues. This hypothesis is supported bythe ability ofS. schenckii yeast forms or peptido-

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V....HSt

Mi

B

WI..HCW[

r..-FIG. 3. Mycelium-to-yeast transformnation in S. schenckii. (A) Hyphal cell (HC) and bud cell (BC) after

septum (S) formation. Note the mitochondria (Mi), vacuoles (v), and the difference in thickness of themicrofibrillar material (FM) of the bud cell wall (BCW) and hyphal cell wall (HCW). Glutaraldehyde-dialyzed iron-osmium staining. (B) Budlike (B) protrusion communicating with a hypha by microfibrillarmateriaL Permnanganate staining Bar = 0.25 pm. (From Garrison et al [46]; used with permission.)

polysaccharides to agglutinate mannose-sensi-tive Escherichia coli K-12 (206).The ConA-reactive outer layer of the cell wall

of S. schenckii yeast forms probably correspondsto the acidic microfibrillar material described byGarrison et al. (46). To study the distribution ofanionic groups at the cell surface further, label-ing by colloidal iron hydroxide and labeling by

cationized ferritin were explored (14). When thecolloidal iron hydroxide method was used, tworeactive layers were clearly observed in yeastforms, and the heterogeneity of labeling of dif-ferent cells of the same population was similarto the heterogeneity observed with ConA label-ing (Fig. 6). Mycelial and conidial forms reactedpoorly with colloidal iron hydroxide. Consider-

A

--FM

1- .1 I

.. .4q.

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ing the localization of the colloidal iron hydrox-ide-reactive components, it is possible that thesecomponents also correspond to the peptido-pol-ysaccharide complexes, which are more abun-dant in yeast forms. Isolated peptido-rhamno-mannans contained several aspartic and glu-tamic acid residues, and some, but not all, pol-ymers had glucuronic acid residues (205). Al-though in the absence of other recognized acidresidues acidic amino acids were tentativelyidentified as the carriers of the anionic groupsreactive with colloidal iron hydroxide (14), thepresence of other strongly acidic moleculesshould be investigated further. When the ferritinmethod was used, the same bilayer of reactivecomponents was observed in S. schenckii yeastforms (14). In one case, detachment of the outerlayer was observed clearly and was a directdemonstration (Fig. 6) of the release of surfacecomponents into the medium in the form of acontinuous ribbon-like material. Release intothe medium of microtubules or vesicles arisingby the association of several units ofceratoulmin(the peptido-rhamnomannan of C. ulmi) has alsobeen reported (195).

BIOCHEMICAL ACTIVITIES ANDMAJOR CONSTITUENTS

S. schenckii grows in a simple medium con-taining inorganic salts, asparagine, glucose, andthiamine (35). Other amino acids and vitaminsare stimulatory but not essential. Carbon aux-onograms of 13 strains (121) showed that glu-cose, galactose, maltose, xylose, and glycerolwere carbon sources for all strains, whereas or-ganic acids were not utilized. Other saccharideswere assimilated by some, but not all strains. Allstrains of S. schenckii were able to hydrolyzestarch (121, 198).Other enzymatic activities were determined in

S. schenckii yeast extracts by direct analysis bygel electrophoresis in agarose (215). About 30bands of migrating proteins had enzymatic ac-tivities, comprising mainly the following: dehy-drogenases, particularly for malate, 8l-hydroxy-butyrate, and glutamate; catalase and peroxi-dases; and several hydrolases, such as acid andalkaline phosphatases, ,B-glucosidase, leucyl-aminopeptidase, aldolase, f8-amylase, ribonucle-ase, and deoxyribonuclease. The properties oftwo esterases were studied in more detail. Oneof these, which was able to hydrolyze butyryl-thiocholine, differed from known cholinesterasesby being heat (60°C) stable and resistant toeserine and diisopropyl fluorophosphate. Theesterases of ,B-naphthyl acetate were also notaffected by eserine and diisopropyl fluorophos-phate, but they were inactivated by heating at

FIG. 4. Staining of S. schenckii yeast forms (blackarrows) and their primordia on hyphae (white ar-rows) by the fluorescent ConA method. (From Tra-vassos et al. [205]; used with permission)

60°C. Lipase, a-amylase, ,8-galactosidase, ,8-glu-curonidase, and proteolytic activities, as well assome other dehydrogenase activities, were notdetected by this method. Several constituents ofS. schenckii yeast extract were immunogenic inrabbits, as determined by an immunoelectropho-retic analysis of the resulting antisera. The mainenzymatic activity located on the immunoelec-trophoretogram was ,B-glucosidase, but amy-lases, acid phosphatases, aldolases, esterases,malate and glucose-6-phosphate dehydrogenase,and peroxidase were also detected. Steric hin-drance might have interfered with detection onthe immunoelectrophoretograms of a number ofother activities, including ribonuclease and de-oxyribonuclease.Both pathogenic and nonpathogenic strains of

S. schenckii assimilate creatine and creatinineas sole nitrogen sources (187). Pathogenic strainsof S. schenckii, as well as the wild-type strain ofC. stenoceras isolated by Mariat (114), assimi-lated guanidoacetic acid, creatine, and creatinineonly in the presence of thiamine (188). A non-related pathogen, such as C. neoformans, assim-ilated creatinine but not creatine or guanido-acetic acid (187). All strains of S. schenckii thatgrew at 370C and assimilated creatine and cre-atinine caused visible muscle swelling and abcessformation when injected into mice (186). Inoc-ulation with as few as 10 conidia of one strainwas sufficient to produce intramuscular ab-scesses.The presence of neuraminidase in two strains

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A

0

B

FIG. 5. Reactivity of yeast cells of S schenckii with ConA. (A and C) Strain 1099.12 was strongly anduniformly stained, and two layers of reactive material (inner sublayer [OI] and outer fibrillar sublayer[OF]) were observed. (D) Strain 1099.18 reacted heterogenously with ConA, with variation in the staining ofthe outer fibrillar sublayer. (B) Unstained control cell. (A) x16,385. (B) x14,154. (C) 22,308. (D) x16,923. (FromTravassos et al. [2056; used with permission)

of S. schenckii and in one strain of C. stenoceraswas shown by the thiobarbituric acid assay, us-ing a number of human glycoproteins, lipopro-teins, and immunoglobulins, as well as fibrino-gen, as substrates (132). Lipolytic and fibrino-lytic activities were also detected in both species.Neuraminidase may be an important pathogenicfactor in sporotrichosis in association with otherenzymes which metabolize animal substrates. S.schenckii strains failed to produce neuramini-dase after prolonged cultivation at 37°C (132).The presence of neuraminidase in S. schenckiiand the strong reactivity of cell surface compo-

nents with colloidal iron hydroxide in acid (14)suggest that sialic acid residues might be presentin S. schenckii cells (C. Alviano, unpublisheddata).Of the substances used to inhibit the growth

of S. schenckii, potassium iodide and antibodieshave been studied in most detail, because oftheir therapeutic significance. KI had very littlefungistatic activity in vitro since growth of themycelial phase was suppressed only at 20% KIand growth of the yeast phase was suppressedat 6 to 7% KI (210). However, with moleculariodine, mycelia were killed with a solution con-

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taining 10 mg of iodine per 100 ml after 10 min,and killing of yeast cells required only 2 mg ofiodine per 100 ml. The miniimal inhibitory con-centration of iodine was between 0.4 and 0.6 mgof iodine per 100 ml. A direct activity of iodineplus KI on S. schenckii was also reported byWada (214). However, it is possible that theeffects of iodine and iodides in vivo are due inpart to stimulation of phagocytosis (173), in-

creases in inflammation (191), or enhancementof proteolysis with resolution ofgranulomas (89).There is no parallel between the in vitro and

in vivo actions of griseofulvin (from Penicilliumgriseofulvum) on S. schenckii, as this substanceis not inhibitory in vitro but was reported to beas active as KI in the treatment of two cases oflymphocutaneous sporotrichosis (51). Antibiot-ics active against S. schenckii in vitro include

FIG. 6. Staining ofanionic groups in S. schenckii yeast forms. (A) Cell wall bilayer reactivity with colloidaliron hydroxide. x22,556. (B) Detachment of the outer layer of cell components reacting with ferritin. X22,556.(From Benchimol et al. [14]; used with permission.)

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pimaricin (from Streptomyces natalensis [136]),tennecetin (from Streptomyces chattanoogensis[218]), nystatin (from Streptomyces noursei[110]), and amphotericin B (from Streptomycesnodosus). Amphotericin B is active against theyeast phase at a minimal inhibitory concentra-tion of 0.07 to 0.5 ,g/ml, but the mycelial phaseis much more resistant (minimal inhibitory con-centration, >40 ag/ml) (171). Amphotericin B ata concentration of 50 jig/ml stimulated the 02consumption of yeast forms of S. schenckii at30°C (112). This drug is the drug of choice inthe treatment of extracutaneous sporotrichosis(151). Penicillin is inactive against S. schenckiiin vitro and does not stimulate the yeast-to-mycelium conversion; streptomycin does not en-hance growth (110).The cell surface polysaccharides, peptido-

polysaccharides, and other major components ofS. schenckii are discussed elsewhere in this re-view. The properties of the DNAs from S. schen-ckii and Ceratocystis species are presented be-low. The phospholipids from S. schenckii, C.stenoceras wild type, and a strain regarded as apathogenic mutant of C. stenoceras have beencompared (18). All strains contained cardiolipin,phosphatidylethanolamine, phosphatidylmono-methylethanolamine, phosphatidylserine, leci-thin, lysophosphatidylethanolamine, and lyso-lecithin. Only the wild-type C. stenoceras straincontained phosphatidylinositol; this lipid wasabsent in S. schenckii and also in the pathogenicmutant of C. stenoceras. Bievre and Mariat (19)separated polar and neutral lipids on a silicicacid column and identified methyl esters of fattyacids by gas-liquid chromatography. C16 (pal-mitic acid) and C18:2 (linoleic acid) fatty acidspredominated. S. schenckii and pathogenic mu-tants of C. stenoceras had very similar fatty acidcompositions. The wild-type C. stenoceras strainhad polar lipids with less C16 and more C18:2 andalso contained C2m,1 and C20 fatty acids that werevirtually absent in the polar lipid fractions of S.schenckii. The netural lipids of C. stenocerashad less C08:1 and more C18:3 compared with thelipids of S. schenckii and the pathogenic mu-tants. Fatty acids were also identified in the cellwalls of different cell types of S. schenckii (157).The C16, C18, C18:1, and C18:2 fatty acids weredetected in both lipid fraction I and lipid fractionII extracted by the method of Bartnicki-Garciaand Nickerson (12). The presence of C18:3 (43.6%)in fraction II of yeast walls but not mycelial orconidial walls was noted. Stretton and Dart (192)studied the long-chain fatty acids of 11 strainsof S. schenckii grown on an extensively defattedmedium at 30°C. C16, C18:1, and C18:2 isomerswere predominant; C18 was present at 0.8 to 6.2%,and C18:3 was present at 0.3 to 1.3%. Two strains

isolated from soil had trace amounts of branchedshorter-chain fatty acids; these strains containedpredominantly palmitic acid and less C18:2.

Question of the Perfect Form of S.schenckii

It is generally accepted that the perfect formof S. schenckii is a species of Ceratocystis (137).Several Ceratocystis species produce sympodu-losporogenous conidial states indistinguishablefrom the conidial state of Sporothrix (198).Upadhyay and Kendrick (209) identified 16 im-perfect states of Ceratocystis and also includedSporothrix. Mariat and Diez (114, 119) proposedthat C. stenoceras might be the perfect form ofS. schenckii. This species is morphologicallysimilar to S. schenckii and forms perithecia ofthe Ceratocystis type. Furthermore, somestrains of C. stenoceras require thiamine, growat 370C, and convert to the yeast phase (137).All morphologically related strains of Ceratocys-tis cross-react serologically with S. schenckii,but the antigen similarity can be extended tomorphologically unrelated fungi, such as C. ulmi(72). Taylor (198) observed gross pathologicalsigns in mice after intraperitoneal injection ofseveral Ceratocystis species, but these microor-ganisms were unable to kill the animals. In con-trast, strain G118 (=strain IP-1013.70) of C. sten-oceras, which was isolated from a human scalp,was able to produce lethal infections in mice andhamsters (114). In contrast to the wild type, thefungi recovered from organs of hamsters inocu-lated with strain IP-1013.70 could not form per-ithecia and showed an increased pathogenicityfor hamsters (114). These strains (strains 118AIand 118AII or IP-1020.70 and IP-1021.70) wereregarded as asexual pathogenic mutants of C.stenoceras. Under certain conditions, these mu-tants formed conical pigmented conidia identicalto those of S. schenckii. The wild-type C. sten-oceras strain formed only hyaline spores.

In a study on the properties of the DNAs of S.schenckii and several strains of Ceratocystis(126), both wild-type strain IP-1013.70 of Mariatand the pathogenic mutant strain IP-1021.70were included. The guanine plus cytosine con-tents of the DNAs of three S. schenckii strains,as well as the guanine plus cytosine content ofthe DNA of the mutant, were very similar (av-erage, 54.7 mol%). In contrast, wild-type strainIP-1013.70 and another strain of C. stenoceras(CBS 237.32) had guanine plus cytosine contentsof 52.6 mol%. All other species of Ceratocystishad base compositions differing from the basecomposition of S. schenckii, with the exceptionof C. minor strain CBS 138.36 (guanine pluscytosine content, 54.6 mol%). Cross-hybridiza-tion studies showed that the DNAs of all S.

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schenckii strains had a high degree of relativebinding (range, 70 to 100%). The DNA ofmutantstrain IP-1021.70 bound to a labeled S. schenckiiDNA with a high degree of homology (87%). Incontrast, C. stenoceras wild-type DNA and C.pilifera DNA had percentages of relative bind-ing to the same S. schenckii DNA of only 30 and35%, respectively. Interestingly, C. minor DNAbound with a homology of 75%. Based on theseresults, the mutant strain of C. stenoceras wasreclassified as S. schenckii (126). It probablyarose from a mixed culture of C. stenoceras andS. schenckii, with the latter having been selectedby inoculation into hamsters. The similaritiesamong the DNAs of different S. schenckiistrains suggest that the strains of this fungus arenot the imperfect stages of different Ceratocystisspecies but that S. schenckii is a separate entity,presumably derived from one specific Ceratocys-tis species. C. minor is a better candidate thanC. stenoceras to be the perfect form, based onthe DNA hybridization studies, but it is possiblethat another so-far-unrecognized species of Cer-atocystis may be phylogenetically closer to S.schenckii than C. minor is. Nishimura and Mi-yazi (141) reported a case of cutaneous sporotri-chosis in which a perithecium-like structure waspresent at the surface of the lesion, but it waslarger than the perithecium of C. stenoceras.However, no perithecium could be obtained invitro.A total of 14 monoascosporic cultures derived

from C. stenoceras strain IP-1013.70 (114) wereshown to have variable pathogenicity in mice;almost one-half of the inoculated animals werekilled. This shows that some strains of C. sten-oceras isolated in nature may also cause pro-gressive infections in animals, as S. schenckiidoes. In agreement with this, one strain of C.stenoceras isolated from the soil in Corse,France, was pathogenic in mice (115).Taylor (199) studied the homogeneity of clin-

ical isolates of S. schenckii from all over theworld and their relation to avirulent strains andOphiostoma (Ceratocystis) species by using apyrolysis-gas chromatography method. Hyphaeof different strains were pyrolyzed at 3500C. Thechromatographic patterns for all S. schenckiiclinical isolates were identical. The pyrolysisproducts of Ophiostoma stenoceras includedtwo slow-moving components which were absentin the patterns of S. schenckii. However, twovirulent S. schenckii strains which producedascigerous states similar to C. stenoceras gavepyrograms nearly identical to those of the an-ascigerous isolates. Several differences were ob-served when the pyrograms of other Ophios-toma species or avirulent S. schenckii strainswere compared. A study of the DNAs of these

ascigerous forms of S. schenckii in comparisonwith the DNAs of C. stenoceras and the asexualvirulent strains of S. schenckii would be a crucialstep in attempting to reconcile the availabledata. In evaluating the various reports on thesimilarities and differences between C. steno-ceras and S. schenckii and the DNA base com-position and hybridization studies, one mustbear in mind that some phenotypes, no matterhow easily demonstrated, reflect the expressionof only a limited number of genes, which in turnmay contribute little to the overall homology inDNAs.

CELLULAR AND EXOCELLULARPOLYSACCHARIDES

S. schenckii and Ceratocystis species formsome polysaccharides which are firmly bound tothe cell wall and some polysaccharides whichare released into the medium. These polysac-charides are usually associated with peptidecomponents in the form of covalent complexesin which the carbohydrate part represents about85% of the total. Soluble and insoluble polysac-charides and their complexes participate inbuilding the structural complex of the cell wall.Crude preparations and purified polysaccharidesor peptido-polysaccharides from S. schenckiihave been used as antigens in serological reac-tions and in skin tests for the diagnosis of spo-rotrichosis. A variety of chemical structureshave been characterized, and these are describedbelow.

Methods for Isolation of Alkali-SolublePolysaccharides

Usually, peptido-rhamnomannans from S.schenckii accumulate in the medium and can beisolated directly by ethanol precipitation of thesupernatants of autoclaved cultures or from cellwashings made with hot water. The precipitateis dissolved in water, dialyzed against distilledwater, and reprecipitated with ethanol. Lloydand Bitoon (92) fractionated a peptido-rham-nomannan preparation from a yeast culture byprecipitation with hexadecyltrimethylammo-nium bromide (Cetavlon). One fraction was pre-cipitated by Cetavlon directly and contained44% rhaxnnose, 20% mannose, and 16.2% protein.A second fraction was precipitated by Cetavlonas the borate complex at pH 8.5 and was purifiedfurther on a diethylaminoethyl-Sephadex col-umn. The major product, which was eluted with0.3 M NaCl, contained 33.5% rhamnose, 51%mannose, and 14.2% protein. By using precipi-tation by Cetavlon, peptido-polysaccharidesfrom two other S. schenckii strains (206) wereisolated from cultures grown at 25 and 370C in

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a synthetic medium. The ratios of rhamnose tomannose in these preparations ranged fromroughly 1:1 to 1:1.6; a small amount of galactosewas also precipitated. The amount of protein inthese preparations was 16.4 to 33.6%.

Isolation of immunologically active S. schen-ckii peptido-polysaccharides which were able toinduce both immediate and delayed-type hyper-sensitivity reactions was possible by extractionwith phenol (142). A mycelium was defatted byrepeated extraction with ethanol-ethyl ether(1:1); then it was extracted with 45% aqueousphenol and centrifuged. The water layer wasdialyzed against distilled water and lyophilized.Finally, the crude peptido-polysaccharide frac-tion was isolated in the excluded eluate from aSephadex G-100 column. In one preparation thepartially purified peptido-polysaccharide frac-tion represented 0.6% of the dry mycelium byweight (174).Aoki et al. (6) obtained an S. schenckii poly-

saccharide complex which was effective in elic-iting a sporotrichin reaction (skin test) by dis-rupting the fungus in a French press. However,since this preparation showed cross-reactivitywith an Aspergillus antigen, it may also havecontained some of the galactomannan compo-nent of S. schenckii cell wall along with therhamnomannan. To obtain peptido-polysaccha-rides which are active both serologically and inskin tests, it is not necessary to disrupt the cellssince these complexes are readily isolated fromthe medium.To obtain rhamnomannans free from peptido

complexes, extraction of cells with 2% KOH at100°C for 2 h has been used. If both the exocel-lular and the cellular polysaccharides are to beanalyzed together, after concentration in vacuothe culture supernatant is precipitated withethanol in the presence of 1% sodium acetate.The precipitate is mixed with the cell pellet,suspended in 2% OH, and extracted as before.Alkali extracts are neutralized with acetic acidand centrifuged, and the supernatant fluid isconcentrated and precipitated with ethanol. Theprecipitate, which has been washed with ethanoland dried, is the crude polysaccharide prepara-tion. Purification of rhamnomannans is per-formed by precipitation with Fehling solutionovernight at 4°C. The insoluble copper com-plexes are centrifuged, washed in 2% KOH,washed in ethanol, and dissociated by shakingwith a cation-binding ion-exchange resin. Theresin is decanted, and the supernatant is repre-cipitated with ethanol, washed, and dried. S.schenckii polysaccharides purified in this wayhave ratios of rhamnose to mannose rangingfrom 1:1 to 1:1.2 (202) and contain less than 1%

N and less than 0.4% P. Possibly due to co-precipitation with the rhamnomannans, a smallamount of a galactose-containing polymer (up to5%) is sometimes detected in preparations.When the Fehling solution-purified polysac-

charides include neutral and acidic rhamnoman-nans, they can be fractionated on a column ofdiethylaminoethyl cellulose. Elution is with wa-ter-5% acetic acid-5% formic acid (55). Eluatesare neutralized and reprecipitated with ethanol.

Before purification with Fehling solution, theexocellular polysaccharides may contain mainlygalactose, as was shown with one strain of C.stenoceras (55). The galactose-containing poly-mers of S. schenckii and C. stenoceras are usu-ally not precipitated with Fehling solution andthus can be isolated from the mother liquor ofthe copper complexes. The procedure involvesneutralization of the supernatant of the Fehlingsolution reaction mixture with acetic acid, di-alysis against distilled water, and deionizationwith an ion-exchange resin. The deionized pol-ysaccharide solution is then concentrated invacuo and precipitated with ethanol. Althoughthis procedure enriches for the galactose-con-taining polysaccharides, the preparations usu-ally contain some rhamnose and glucose in ad-dition to galactose and mannose. Part of theglucose is amylose (157), and part is a f8-linkedsoluble glucan (156). Soluble glucans are bestisolated from cell walls rather than from wholecells since during the process of cell rupture andexhaustive washing most of the galactomannansare removed and the remaining rhamnomannansare efficiently precipitated by Fehling solution.

S. schenckii and CeratocystisRhamnomannans

Early studies (5) showed that the polysaccha-rides from S. schenckii contain mannose, rham-nose, and glucose. Ishizaki (70) isolated threeserologically active polysaccharides from a cul-ture medium, which had mannose and rhamnoseas the main constituents but also containedsmaller amounts of galactose and glucose. Aserologically active peptido-rhamnomannan wasisolated from yeast cells of S. schenckii (92),which contained traces of galactose and no glu-cose. Rhamnomannans with trace amounts ofgalactose or glucose or both were also formed by40 strains of Ceratocystis and Graphium (185),by Taphrina deformans (57), and by Hyaloden-dron pyrium (203). Among human pathogenicfungi, however, rhamnomannans seem to be re-stricted to S. schenckii. In a comparison of therhamnomannans extracted with hot dilute alkalifrom S. schenckii, C. stenoceras, and C. ulmi inwhich methylation-fragmentation-mass spec-

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trum analysis was used, it was evident that allpolysaccharides were very similar (202) and con-tained L-rhamnose as 2-0-substituted and non-reducing end units. Mannose was present as 4-0-, 2,4-di-O-, and 3,6-di-O-substituted units anda few 2-0-, 3-0-, and 6-0-substituted units.Trace amounts of 4-0- and 2,4-di-O-substitutedD-mannose units were obtained from the poly-saccharides of C. ulmi and one strain of C.stenoceras. The specific rotations of the rham-nomannans were consistent with predominantlya-linked structures. a-D-Mannopyranosyl non-reducing end units were not detected, except inone strain of S. schenckii studied previously(92), which had a small proportion (3.7%) ofthese units. Methylation analysis of the polysac-charides resulting from mild acid hydrolysis (pH

a-L-Rhap1

13

-* 6)-a-D-Manp-(1 -* 6)-I


1.1 at 10000) to remove the rhamnose end unitsshowed the presence of an a-(1 -- 6)-linkedmannopyranosyl main chain (92), as in the C.ulmi polysaccharide (58). Figure 7 shows themain structures of S. schenckii rhamnomannans(Fig. 7, structures I and II).The proportions of these two structures were

significantly different in polysaccharides thatwere isolated from cultures grown under differ-ent conditions. The dirhamnosyl structures(structure II) predominated in polysaccharidesobtained at 250C, compared with polysaccha-rides obtained at 370C. This could indicate astructural feature determined by temperature ora phenotype restricted to one particular celltype. As discussed above, yeast forms are fa-vored at 370C, whereas at 250C the mycelium

a-L-Rhap1

2a-L-Rhap

1

-- 4)-a-D-Manp-III

3-* 6)-a-D-Manp-(1 -) 6)-

II

a-D-Manp1

42

-- 4)-a-D-Manp-(1 -. 4)-a-D-Manp-IV

a-L-Rha1

4a-D-Manp

1l

a-L-Rha1

4a-D-Manp

1

2 2-* 4)-a-D-Manp-(1 -. 4)-a-D-Manp-(1 -* 4)-a-D-Manp-

VI

a-D-Manp1

a-D-Manp1

a-D-Manp1

4,2 2

-*4)-a-D-Manp-(l 4)-a-D-Manp-(l 1 4)-a-D-Manp-V

a-L-Rhap1

4fi-D-GlcpA

1

2a-L-Rhap

1

3-- 6)-a-D-Manp-(1 -. 6)-

VII

2-- 6)-a-D-Manp-(1 -- 6)-a-D-Manp-(1 -* 6)-

VIII

FIG. 7. Polysaccharide structures. Rhap, Rhamnopyranose; Manp, mannopyranose; Rha, rhamnose;GlcpA, glycopyranosyluronic acid.


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phase predominates. To approach this questionand to distinguish temperature and morpholog-ical effects, S. schenckii yeast forms were grownin a synthetic medium at both 25 and 370C. Toobtain 100% yeast forms, screw-capped 2-literflasks were used, and ammonium carbonate wasadded to shaken cultures. The rhamnomannansformed by these yeast cultures contained onlymonorhamnosyl side chains (structure I), as de-termined by methylation analysis and '3C NMRspectroscopy (125). By using these methods, itwas also determined that the temperature effect(shift from 25 to 3700) was reflected by the lossof 4-0- and 2,4-di-0-substituted a-D-mannopy-ranose units in the rhamnomannan. Thus,monorhamnosyl rhamnomannans were the poly-saccharides synthesized by the yeast forms, ir-respective of the incubation temperature. In an-other medium, which yielded cultures of mixedmorphology at 370C and mycelial cultures at25°C, a dirhamnosyl (structure II) rhamnoman-nan was formed. To determine whether thispolysaccharide was associated with conidia orhyphae, attempts were made to analyze thesetwo cell types separately. A strain of S. schenckiigrown at 250C in the medium described by To-riello and Mariat (201) formed a mycelium cul-ture lacking conidia after a short incubation, andthe polysaccharide synthesized was a typicaldirhamnosyl rhamnomannan (125). The samestrain was grown in another medium favoringsporulation at 250C (202), and conidia were sep-arated from the mycelium by filtration throughgauze. On methylation the polysaccharide ex-tracted from isolated conidia gave only a smallproportion of 3,4-dimethyl-1,2,5-triacetylrham-nitol, representing less than 10% of the propor-tion of 2,3,4-trimethyl-1,5-diacetylrhamnitol andthus indicating a small proportion of (1 -+ 2)-linked rhamnosyl residues. These results, con-firmed by 13C NMR spectroscopy, demonstratedthe formation in conidia of a monorhamnosylrhamnomannan (204) similar to the polysaccha-ride synthesized by the yeast forms. The finalidentification of the dirhamnosyl side chains inthe polysaccharides formed by mycelial culturesof S. schenckii was made by isolating the trisac-charide 0-a-L-rhamnopyranosyl-(l 2)-O-a-L-rhamnopyranosyl-(1 -* 3)-a,,f-D-mannopyra-nose by partial acetolysis of the rhamnomannan(94).As described above, S. schenckii polysaccha-

rides obtained at 250C had a relatively highproportion of 4-0- and 2,4-di-0-substituted a-D-mannopyranose units. Smith degradation of thepolysaccharide containing such units gave eryth-ritol, 2-0-a-D-mannopyranosyl-D-erythritol, and0-a-D-mannopyranosyl-(1 -- 4)-O-a-D-manno-pyranosyl- (1 -- 2)-D-erythritol; these could arise

from structures III, IV, and V (Fig. 7), respec-tively. The products obtained by partial acidhydrolysis and partial acetolysis (55) support thepresence of these structures. However, the sidechains of structures IV and V are probably sub-stituted at position 0-4 in the original polysac-charide by a-L-rhamnopyranose groups since ifsubstitution occurred at position 0-2 or 0-3, theglycosidic linkage would be stable to partial ace-tolysis. Furthermore, no a-D-mannopyranosenonreducing end units were detected (202).Structure VI (Fig. 7) is a probable structure forthis minor feature of the polysaccharide of S.schenckii. A similar structure is presumablypresent in some, but not all, C. stenoceras rham-nomannans.

In summary, it was concluded that yeast formsand conidia of S. schenckii are characterized bythe production of rhamnomannans in whichmonorhamnosyl side chains predominate,whereas hyphae synthesize mainly polysaccha-rides with dirhamnosyl side chains (Fig. 8).These results were generally confirmed whencultures were examined directly by fluorescentantibody techniques, using rabbit antisera spe-cific for mono- and dirhamnosyl structures, asdescribed below.

3C Nuclear Magnetic ResonanceSpectroscopy

Natural abundance "3C NMR spectroscopywas used initially as a method of fingerprintingS. schenckii and C. stenoceras polysaccharides(203). S. schenckii rhamnomannans were clas-sified into three spectral groups (types I, II, andIII), and the three C. stenocera.s rhamnoman-nans gave a fourth group which clearly differedfrom the S. schenckii polysaccharides spectralgroups. A consistent difference was the absenceof signals at &c 103.5 to 103.7, 99.7 to 99.8, and96.5 to 96.6 in the C-1 region. Additional signalsat 8c 75.0, 81.4, and 97.0 and the presence of avery prominent signal at 8c 105.4 were differen-tial features of the spectra of the C. stenocerasrhamnomannans. The type III spectra of S.schenckii (203) rhamnomannans correspondedto structures synthesized at 370C, which weredifferent from the structures synthesized at250C. Type III spectra did not contain signals at8, 105.4, 103.5, 96.6, and 80.0 to 80.2. The spectraof type I and type H rhamnomannans wereseparated by more subtle differences, whichwere related to the absence in the latter of minorpeaks at &. 80.2 to 80.4 and 96.6 to 96.7 and thepresence of minor peaks at 8c 97.0, as in thespectra of C. stenoceras rhamnomannans. Whenpresent, the signals at 8c 105.4 were always minorin the spectra of S. schenckii rhamnomannans.

All of these differences should be regarded as

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S. SCHENCKII AND CERATOCYSTIS

S. schenckiiYeasts and

conidiaMycelia

C. stenocerasmycelia

C. ulmiTnycelia

a -L-Rhap1

l3

-*6)-a-D-Manp-

a-L-Rhap1

I3

--6)-a-D-Manp-

a-L-Rhap1

2

a-L-Rhap1

3--6)-a-D-Manp-

a-L-Rhap1

43.

--i6)-a-D-Manp-

a-L-Rhap1

4

4

f3-D-Gl%ppA1

3a-L-Rhap

1

3--)6)-a-D-Manp-

a-L-Rhap1

4a-D-Manp

1

2-.4-a-D-Manp-

FIG. 8. Summary of the major, well-characterized structures occurring in polysaccharides isolated from S.schenckii and two species of Ceratocystis. The conidia were isolated from an S. schenckii mycelial phase;cultures in the mycelial phase consisted of hyphae and conidia. Rhap, Rhamnopyranose; Manp, mannopy-ranose; GlcpA, glycopyranosyluronic acid.

representing real structural differences in thesepolysaccharides. Spectra are taken from intact,untreated polysaccharide solutions in D20 at70°C (55). Figure 9 shows spectra of S. schenckiiand C. stenoceras rhamnomannans obtained at25 and 370C.

Since the structures of these polysaccharideswere known, it was possible to assign manysignals to specific structural features. Accordingto the methylation data, two structures shouldhave given prominent signals in the "3C NMRspectra, and these were the 3,6-di-O-substituteda-D-mannopyranose units of the main chain andthe a-L-rhamnopyranose nonreducing end unitslinked to the 3-0 positions of the main chain.Indeed, the prominent signals in most spectrawere at 8, 97.9 to 98.1 and 100.9 to 101.1, andthey were readily assigned to the C-1 region ofterminal rhamnopyranosyl residues and the 3,6-di-O-substituted mannopyranose of the mainchain (203). The C-1 signal of the nonreducingunit of 3-0-a-L-rhamnopyranosyl-a,f8-D-man-

nose which was isolated by partial acetolysis ofthe C. ulmi rhamnomannan (58) was at 8, 97.4.A prominent signal at 8, 100.5 was also detectedin the spectrum of an H. pyrium rhamnomannanwith a similar structure. In spectra of S. schen-ckii rhamnomannans in which the side chainscontain mainly a-L-rhamnosyl-(1 2)-a-L-rhamnose sequences, the chemical shift of theC-1 of the internal rhamnose unit would beexpected to move approximately 1 to 6 ppmupfield, analogous to the effect of 2-0 substitu-tion of mannose units (54); simultaneously, thesignal at Sc 98.0 should be proportionally smaqler.This combined picture was in fact observed inseveral type I spectra of S. schenckii rhamno-mannans, in which a more prominent signal at8c 96.5 was accompanied by a reduced signal at&c 98.0 (for examples, see spectra of strains1099.23 and 1099.27 in reference 203).Another signal relationship observed in the

13C NMR spectra of S. schenckii rhamnoman-nans (250C) involved signals at Sc 103.5 to 103.7

pi-L-PR,ha4P1

43

r-a-t),-D-MEIBP-I?

1

2

14a

--+)-q.-p-Maqp.-

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A

I)

aIdS

B

C

FIG. 9. Partial 13C NMR spectra of S. schenckiimonorhamnosyl rhamnomannan (A), dirhamnosylrhamnomannan (B), and C. stenoceras glycurono-rhamnomannan (C). The polysaccharide in (A) wasobtained at 37°C; the polysaccharides in (B) and (C)were obtained at 25°C. (From Gorin et al. [551, Men-donga et al. [125], and Travassos and Mendonga-Previato [2041; used with permission.)

and 96.6. The intensities of these signals alwaysparalleled each other, and the assignment of thesignal at 8, 103.5 to the C-1 of the a-L-rhamno-pyranose nonreducing end unit in the sequencea-L-rhamnosyl-(1 -. 2)-a-L-rhamnose was pos-sible by analysis of the spectrum of the trisac-

charide a-L-rhamnosyl-(l -- 2)-a-L-rhamnosyl-(1 -. 3)-a,,8-D-mannose (125). The absence ofthe signal at &c 103.5 in the spectra of C. steno-ceras rhamnomannans indicated that sidechains containing the dirhamnosyl sequencewere not present. Later studies (55) showed thatthe C. stenoceras polysaccharide contained aside chain with the sequence L-rhamnosyl-(1 --

4)-,8-D-glucuronic acid-(1 -* 2)-a-L-rhamnose.The chemical shifts of the C-1 positions of theseunits were at 8c 102.4, 105.5, and 97.3, respec-tively. A spectrum of an acid-degraded glucu-rono-rhamnomannan from C. stenoceras lackedthe signals at Sc 102.4 and 80.3, which was inter-preted as being due to the removal of the a-L-rhamnopyranose nonreducing end unit, andhence these signals were assigned to the C-1 ofthe terminal a-L-rhamnose linked to the ,8-glu-curonic acid units and to the C-4 of the latterresidues substituted at 0-4. The signals at Sc105.4 to 105.6 and 81.4 to 81.6, which were char-acteristic of C. stenoceras rhamnomannan, arosefrom the C-1 of fB-D-glucopyranosyluronic acidand the C-2 of 2-0-substituted a-L-rhamnopy-ranose since the chemical shifts corresponded tothose of 2-0-fi-D-glucopyranosyluronic acid-a-L-rhamnose (55).The assignment of the signal at Sc 102.3 in the

spectra of S. schenckii rhamnomannans wasmade by comparison with the spectra of theoligosaccharides containing 4-0-substituted a-D-mannopyranose units which were obtained bypartial acid hydrolysis of the polysaccharides.The C-1 of 4-0-a-D-mannopyranosyl-a,fl-D-mannose (reducing unit) was at 8A 102.8, and thesignal at &c 100.1 should have corresponded tothe C-1 of a 2,4-di-O-substituted a-D-mannopy-ranose unit because of the effect of 2-0 substi-tution (55, 203).The simplest rhamnomannan synthesized by

S. schenckii was the rhamnomannan from onehuman strain grown at 37°C (125), which con-tained a (1 -- 6)-linked a-D-mannopyranosemain chain substituted in the C-3 positions bya-L-rhamnopyranose side chains, but had onlytraces of4-0- and 2,4-di-O-substituted a-D-man-nopyranose units and 2-0-substituted a-L-rham-nopyranose units. This polysaccharide produceda 13C NMR spectrum with 12 major peaks anda very small peak at &, 62.8 (C-6 of a-D-manno-pyranose units unsubstituted at 0-6 [54]). Thetwo signals at the C-1 region (8c 101.1 and 98.3)had already been assigned, along with CH3 signalof rhamnose at 8c 18.5 (125, 203). The origin ofthe remaining nine signals was determined bycomparison with the chemical shifts of carbonsfrom methyl-a-L-rhamnopyranoside andmethyl-a-D-mannopyranoside and by compari-son with the spectra of the polysaccharides ob-

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tained by growing S. schenckii with D-[6-2H2]glucose or D-glucose containing twice thenatural amount of 13C in the C-2 position (55).

In this manner the complete assignments ofthe signals of a spectrum from the monorham-nosyl rhamnomannan of S. schenckii could bemade (Table 2). Assignments of signals arisingfrom side chains of S. schenckii and C. steno-ceras rhamnomannans isolated from culturesgrown at 250C were also determined (Table 3).Signals arising from the a-D-(1 -- 6)-linked D-

mannopyranan main chains of S. schenckii andC. stenoceras rhamnomannans were obtainedon a polysaccharide isolated either by Smithdegradation of the acidic polysaccharide (55) or

by mild acid hydrolysis of the neutral polymer(C. Kubelka, unpublished data). The linear un-

branched mannopyranan gave signals that couldbe assigned as C-1 (8& 101.1), C-2 and C-3 (Sc72.6), C-4 (8, 68.6), C-5 (&c 71.7), and C-6 (8c 67.6)by comparison with the signals of methyl-a-D-mannopyranoside and by taking into accountthe effects of 6-0 substitution on the C-5 and C-6 signals of mannopyranose residues.

Acidic Rhamnomannan of Ceratocystisstenoceras

The rhamnomannan of C. stenoceras was dif-ferentiated from that of S. schenckii on the basisof its NMR spectrum (202). With cultures grownin a synthetic medium at 250C, mixtures ofpolysaccharides isolated from culture superna-

tants and extracted from cells of S. schenckiiand C. stenoceras were analyzed by proton mag-netic resonance (PMR) (202) and by 13C NMR(203) spectroscopy. Consistently, the rhamno-mannans of spectroscopy C. stenoceras pro-

duced PMR spectra in which the H-1 regioncontained all five to six signals described previ-ously for spectra of polysaccharides of the Cer-atocystis clavata group (185), but lacked a signalat 8 5.6 (T 4.40 to 4.42). This signal was a char-acteristic of the PMR spectrum of S. schenckiipolysaccharides obtained at 250C, although itx;as absent or much reduced in the spectra ofpolysaccharides obtained at 35 to 370C (201,203). The existence of structural differences inthe polysaccharides of C. stenoceras and S.schenckii was confirmed by 'C NMR spectros-copy (203) and chemical analysis (55). The exo-

cellular polysaccharides of C. stenoceras CBS237.32 were purified by precipitation with Fehl-ing solution and then fractionated by diethyl-aminoethyl- cellulose chromatography. Whenthe column was washed with water, a mixture ofa rhamnomannan and a galactose-containingpolysaccharide was obtained. Elution with 5%acetic acid gave a fraction whose i3C NMR spec-trum was identical to that of a rhamnomannan

TABLE 2. Complete assignments of signals in the13C NMR spectrum of S. schenckii strain 1099.18

rhamnomannan obtained at 37°Ca

Signal, Sr (700C) Assignment(ppm)b Assigrument

101.1 C-1 of 3,6-di-O-substituted a-D-mannopyranose units

98.3 C-1 of a-L-rhamnopyranose non-reducing end units




72.0-71.9 C-2 and C-3 of a-L-rhamnopyra-nose nonreducing end units





62.8 (Trace)c C-6 of 0-6-unsubstituted a-D-mannopyranose units

18.4 CH3 of a-L-rhamnopyranoseunits

a See Fig. 9A.b Signals in the &c 66.3 to 76.6 region have their

values corrected for 700C (+0.6 ppm), as they wereoriginally obtained at 330C (55).

'This rhamnomannan contains only trace amountsof 4-0-substituted a-D-mannopyranose units.

having a main chain of (1 -- 6)-linked a-D-man-nopyranose units, each unit of which was sub-stituted at 0-3 by an a-L-rhamnopyranosylgroup. Further elution with 5% formic acid pro-vided an acidic polysaccharide containing glu-curonic acid units. Methylation of the C. steno-ceras acidic rhamnomannan and analysis of theneutralized free 0-methylaldoses by paper chro-matography produced a spot with the same Rfas 2,3-di-0-methyl-D-glucuronic acid. The pres-ence of 4-0-substituted glucuronic acid unitswas further confirmed by reduction and iden-tification on gas-liquid chromatography ofmethyl-2,3-di-0-methyl-a,,8-glucopyranosideand by characterization of erythronic acid andthe 1,4-lactone by paper chromatography of theethanol-soluble fraction of a metaperiodate hy-drolysate. Partial hydrolysis of a purified cellwall polysaccharide from C. stenoceras and frac-tionation on a cellulose column provided a sac-charide which, when hydrolyzed, gave rhamnoseand glucuronolactone. A 2-0 linkage was indi-cated since the product was resistant to oxida-tion by lead tetraacetate and the negative spe-cific rotation ([a]R, -18') was consistent with a

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704 TRAVASSOS ANDLLOYDM

TABLE 3. Assignments of low-field 13C NMR signals arising from side chains of S. schenckii and C.stenoceras rhamnomannans obtained at 25°Ca

Species Signal, Oc (700C) Assignment(ppm)S. schenckii 103.7 C-1 of a-L-rhamnopyranose nonreducing end unit of 0-a-L-rhamnopyrano-

syl-(1 -s 2)-0-cs-L-rhamnopyranose102.3 C-1 of 4-0-substituted mannopyranose of 0-a-L-rhamnopyranosyl-(l -. 4)-

0-a-D-mannopyranosyl-(l -- 2)-D-mannopyranoseC-1 of a-L-rhamnopyranose nonreducing end unit of 0-a-L-rhamnopyrano-

syl-(l - 4)-0-a-D-mannopyranose98.2 C-1 of a-L-rhamnopyranose nonreducing end unit of 0-a-L-rhamnopyrano-

syl-(1 -s 3)-D-mannopyranose96.8 C-1 of 2-0-substituted a-L-rhmlopyranose units of 0-a-L-rhamnopyrano-

syl-(l - 2)-0-a-L-rhamnopyranosyl-(1 -. 3)-D-mannopyranose80.3 C-2 of 2-0-substituted a-L-rhamnopyranose units

C. stenocerasb 105.6 C-1 of 4-0-substituted f8-D-glycopyranosyluronic acid residues102.4 C-1 of a-L-rhaxnnopyranose nonreducing end unit of a-L-rhamnopyranosyl-(l

-. 4)-,8-D-glucuronic acid98.3 C-1 of a-L-rhamnopyranose nonreducing end unit of 0-a-L-rhamnopyrano-

syl-(1 -l 3)-D-mannopyranose97.3 C-1 of 2-0-substituted a-L-rhaxnnopyranose unit of 0-a-L-rhaxnnopyranosyl-

(1 - 4)-O-fl-D-glucuronic acid-(I -. 2)-0-a-L-rhamnopyranosyl-(1 3)-D-mannopyranose

81.6 C-2 of 2-0-substituted a-L-rhamnopyranosyl residues of 0-a-L-rhamnopyra-nosyl-(l- 4)-0-j8-D-glucuronic acid-(1 -s 2)-0-a-rhamnopyranosyl-(13)-D-mannopyranose

80.3 C-4 of 4-0-substituted ,B-D-glycopyranosyluronic acid units

a See Fig. 9.b C. stenoceras strain 1099.40 (CBS 237.32).

,B-glycosidic linkage. Because of the absence ofa-D-mannopyranosyl nonreducing end units(202) and the susceptibility of side chains tohydrolysis with dilute acid, it is likely that theglycopyranosyluronic acid residues are substi-tuted by' L-rhamnopyranosyl nonreducing endunits. This work indicates that both structuresI and VII (Fig. 7) occur in the acidic rhamno-mannans of C. stenoceras. It is interesting tonote that a similar sequence [a-L-rhamnosyl-(1 -. 4)-uronic acid-(1 -- 2)-a-L-rhamnose] ispresent in an acidic polysaccharide from cul-tured sycamore tree (Acer pseudoplatanus)cells, but in this case the uronic acid is a-D-galactopyranosyluronic acid (196).The "3C NMR spectrum of the glucurono-

rhamnomannan (see above) gave support to thestructures discussed above. Significantly, thisspectrum lacks signals at Sc 103.7 and 96.6, cor-responding to the C-1 positions of the 0-a-L-rhamnopyranosyl-(1 -* 2)-a--L-rhamnopyranosylside chains of S. schenckii polysaccharide (125).Thus, the main difference between the C. sten-oceras and S. schenckii rhamnomannans ob-tained at 25°C or in the mycelial phase is thepresence ofdirhamnosyl side chains in the latter.Do rhamnomannans from S. schenckii also

contain glucuronic acid units? The 13C NMRspectra of rhamnomannans from 20 differentstains of S. schenckii grown at 25°C showed

minor peaks at &, 105.3 to 105.5 in seven poly-saccharides. Signals at 8, 80.3 were also ob-served, but signals corresponding to the C-1 andC-2 of a-L-rhamnopyranose units substituted at0-2 with ,B-glucuronic acid units (8, 97.3 and81.6) were absent in all spectra of type I poly-saccharides (203). Type II rhamnomannans (twostrains) gave spectra with minor peaks at &,105.4 and 97.0, but the signal at &, 81.6 was notdetected. These results suggest that f8-D-gluco-pyranosyluronic acid units may also be presentin some S. schenckii rhamnomannans, but it isdoubtful that the side chains of these polymershave the same structure as the side chains of theacidic rhamnomannans from C. stenoceras. Amore detailed study (205) was carried out withone strain of S. schenckii (strain 1099.12) whichwas originally described (as strain IP-1021.70) asa pathogenic mutant of C. stenoceras. An earlyreport (126) suggested that this strain mighthave consisted of a mixture of C. stenoceras andS. schenckii and that the latter may have beenselected by inoculation into susceptible animals.Since the strain 1099.12 culture could still con-tain a mixture of both fungi or variants withmixed characteristics, an attempt was made toseparate different types of rhamnomannansfrom the original culture or from clones of theoriginal culture (205). The occurrence of differ-ent structures was monitored by "3C NMR spec-

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troscopy. Rhamnomannans with varying pro-portions 'of mono- and dirhamnosyl side chainswere characterized by groing this strain in ayeast nitrogen base medium at 25 or 370C. How-ever, a culture in Sabouraud medium at 250Cformed a rhamnomannan whose 13C NMR spec-trum was a combination of patterns of the spec-tra of the C. stenoceras and S. schenckii poly-saccharides. This culture was plated onto solidSabouraud agar at 370C, and several clones weresubcultured in liquid medium at 250C. Althoughthe polysaccharides formed by most of thesesubcultures gave spectra similar to the spectrumof the original culture, a rhamnomannan fromone clone was identical to the rhamnomannanof C. stenoceras on the basis of 'C NMR spec-troscopy. Fractionation of cellular rhamnoman-nans from strain 1099.12 on diethylaminoethyl-cellulose provided a neutral monorhamnosylrhamnomannan with a high proportion of 4-0-and 2,4-di-O-substituted a-D-mannopyranoseunits and an acidic rhamnomannan similar tothe rhamnomannan of C. stenoceras except forthe simultaneous presence of dirhamnosyl sidechains, as inferred by 13C NMR spectra. Nofurther attempt was made to determine the ho-mogeneity of the acidic fraction, These resultsseem to indicate that neutral and acidic rham-nomannans are different polymers which areformed concomitantly by C. stenoceras andsome S. schenckii strains and that the acidictype is preferentially synthesized at 25 ratherthan at 370C. The data described above suggestthat S. schenckii strain 1099.12 is heterogeneousand exhibits different phenotypes, which corre-spond to rhamnomannans of varying structures.

Other Ceratocystis polysacoharidesThe polysaccharides of Ceratocystis were clas-

sified by Spencer and Gorin (185) into 11 groups,based on their PMR spectra. C. stenoceras poly-saccharides which have been compared with S.schenckii polysaccharides (203) belong to the C.clavata group, together with the rhamnoman-nans from C. clavata, Ceratocystis catonianum,C. pilifera, and C. pluriannulata (PMR signalsat 8 5.88, 5.74, 5.50, 5.37, 5.28, and 5.20). Therhamnomannans ofthe C. minor group, a specieshaving a relationship to S. schenckii (126), gavePMR spectra with signals at 8 5.88, 5.75, 5.50,and 5.34.Besides C. stenoceras, the only other Cerato-

cystis species whose rhamnomannan was studiedin more detail was C. ulmi. The PMR (H-1region) spectrum of this rhamnomannan wasvery similar to that of the rhamnomannan ofone strain of C. stenoceras (202) in that thesignal at 8 5.88 was absent or very much reduced.

This signal, which is associated with the H-1 of2,4-di-O-substituted a-D-mannopyranose units,is virtually absent in these rhamnomannans(202). The structure of the C. ulmi rhamnoman-nan was first determined by Gorin and Spencer(58). Based on methylation data and PMR spec-troscopy, this polysaccharide has a (1 -- 6)-linked a-D-mannopyranose main chain substi-tuted at C-3 by nonreducing a-L-rhamnopyra-nose end units. Side chains with more than onerhamnose unit were also suggested, but an a-(1

4) linkage was thought to occur, based on aperiodate oxidation method. This structure wasreexamined by methylation analysis (202) andwas shown to contain (1 -e 2)-linked a-L-rham-nopyranose residues, as do S. schenckii and C.stenoceras rhamnomannans. However, an a-D-rhamnopyranosyl-(1 -- 2)-a-D-rhamnopyrano-syl side chain characteristic of S. schenckii isunlikely to be present in C. ulmi because the 2-0-linked rhamnosyl residues are stable to partialacid hydrolysis (193), suggesting a substituentother than rhamnose. There is no evidence sofar for the occurrence of a rhamnose side chainwhich also contains ,B-D-glucuronopyranosyl-uronic acid residues, as in C. stenoceras. Uron-osyl residues were not detected in the C. ulmipolysaccharide when a carbazole reagent wasused (193).The peptido-polysaccharide of C. ulmi has

been studied in more detai in view of its impor-tance in plant pathogenicity. Salemink et al.(166) obtained a partially purified toxin fromculture filtrates of C. ulmi and showed that itwas a glycopeptide. Later, the partially purifiedglycopeptide was shown to consist of a range ofcomponents of varying molecular weights (H.Rebel, Ph.D. thesis, State University of Utrecht,Utrecht, The Netherlands, 1969). A glycopeptidethat was isolated from the supernatant of a C.ulmi culture grown in ao defied medium and waspurified by passg through an ion-exchange col-urmn and then by affinity chromatography onConA-Sepharose consisted of two major com-ponents; the larger of these components had amolecular weight of 2.7 x 105, and the smallercomponent had a molecular weight of 7 x 10i(193). Binding of this glycopeptide to ConA can-not be explained on the basis of the reactivity ofC. ulmi rhamnomannan because this polysac-charide has very few, if any, a-D-mannopyranoseend units (202). As described above, a parallelsituation was observed for the ConA reactivityof S. schenckii peptido-polysaccharide com-plexes; in this case the peptido-rhamnomannansbut not the rhamnomannans obtained from thepeptido-rhamnomannans were precipitated byConA by double diffusion in agarose (206).

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Whether this reactivity was due to oligosaccha-ride chains linked to serine or threonine residuesof the peptide (10) was not determined. How-ever, Strobel et al. (193) suggested that in C.ulmi glycopeptides carbohydrate chains, whichhave an average length of 25 sugar residues, areattached to either the threonine or the serine ofthe peptide. This conclusion was based on thefact that 70 to 85% of these amino acids werelost upon mild alkaline treatment of the glyco-peptide.As is the case with S. schenckii (14), the

glycopeptide from C. ulmi is sloughed off intothe culture medium and reassociates to formvesicles or tubular bodies (ceratoulmin). Theseorganized masses of glycopeptides are probablyresponsible for some of the disease symptomsobserved in plants. Zentmyer (224) observedwilting in young elm seedlings after injection ofa fungus-free culture filtrate. This observationwas confirmed by Takai (195), who studied theeffect of ceratoulmin on young elm seedlings incomparison with the effects of glycopeptidesfrom other Ceratocystis species. Ceratoulminwas not synthesized by Ceratocystis dryocoeti-dis, Ceratocystis fagacearum, Ceratocystis ma-jor, C. minor, or Ceratocystis piceae since thecorresponding glycopeptides were not toxicwhen injected into elm seedlings. According toStrobel et al. (193), the C. ulmi toxin (glycopep-tide) caused wilting in alfafa, petunias, gera-niums, and crab apples, indicating a lack ofspecies specificity. A high-molecular-weight dex-tran (5 x 105) had the same activity as the toxinon the flow of fluids in elm cuttings.The polysaccharides from Ceratocystis brun-

nea, Ceratocystis fimbriata, and Ceratocystisparadoxa, comprising mannans, glucomannans,and galactomannans (4, 58), have also been stud-ied, but their structures are not discussed heresince they apparently are not related to S. schen-ckii polysaccharides.

Galactose-Containing PolysaccharidesGalactose-containing polysaccharides are usu-

ally found in the culture media of S. schenckiiand C. stenoceras. The original surface locationof these galactose-containing polysaccharideswas shown by the immunofluorescent stainingof S. schenckii yeast forms with an anti-Hor-modendrum serum, which was specific for ga-lactofuranose residues (Lloyd, unpublisheddata). The presence of,B-D-galactofuranose unitsin galactomannans is suggested by "3C NMRspectroscopy and methylation-fragmentationdata (56, 125).The NMR signals at de 109.3, 82.5, 78.5, 84.8,

and 64.5 corresponded to the C-1, C-2, C-3, C-4,and C-6, respectively, of 8l-galactofuranose units,

as determined by analogy with the signals ofmethyl-13-D-galactofuranoside (56). Other sig-nals in the spectrum were at &, 103.7, 101.1, 99.8,and 62.7, corresponding to the C-1 positions ofa-D-mannopyranose units (54).The first galactomannan identified was from

a conidialess mycelium culture of S. schenckiistrain 1099.12 grown in brain heart infusion me-dium at 25°C for a short incubation period. Thegalactomannan was preciptated with Fehling so-lution and contained 10% galactose. Methyla-tion-fragmentation analysis showed the pres-ence of a-D-mannopyranose nonreducing endunits, 6-0- and 2,6-di-O-substituted a-D-man-nopyranose units, and a few 2-0- and 3-0-sub-stituted a-D-mannopyranose units (125). A prob-able structure for the mannan portion is shownin Fig. 7, structure VIII. The structure of themain chain was confirmed by removal of theside chains with exo-D-mannosidase, but thelinkages of f,-D-galactofuranose to a-D-manno-pyranose units are not clear. A (1 -* 2) linkageshould give a C-1 signal in the 13C NMR spec-trum at 8, 107 to 107.5, but the C-1 signal presentwas at &, 109.3, suggesting a (1 -+ 6) linkage. Thepossibility of the presence of a longer side chaincontaining the sequence fl-D-galactofuranosyl-(1

6)-a-D-mannopyranosyl-(1 -- 2)-D-manno-pyranose was not investigated. It is doubtfulthat such a galactomannan structure is the onewhich is generally present in S. schenckii poly-saccharides isolated after prolonged incubationsbecause such polysaccharides are precipitatedwith Fehling solution and most preparations ofrhamnomannans purified by Fehling solutionprecipitation contain only traces of galactose(202). The homogeneity of strain 1099.12 hasbeen questioned (205). The mycelium which pro-duced the galactomannan after a short incuba-tion did not form the typical rhamnomannanuntil sporulation occurred. One possible expla-nation is that a fast-growing fungal variant wasselected during the short incubation period.A search for galactose-containing polysaccha-

rides which were not precipitable by Fehlingsolution led to the isolation of additional galac-tomannans from four strains of S. schenckii andC. stenoceras (L. Mendonca-Previato, P. A. J.Gorin, and L. R. Travassos, manuscript in prep-aration). These polysaccharides contained 2-0-and 2,6-di-O-substituted a-D-mannopyranoseunits, as well as terminal nonreducing and 6-0-substituted f8-D-galactofuranose units. It seemsthat galactomannans with high ratios of galac-tose to mannose (0.4:1 to 2.0:1) that are notprecipitated by Fehling solution are commonconstituents of S. schenckii and C. stenoceras.The possible existence of a separate galactan inS. schenckii and C. stenoceras, as mentioned in

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some publications (55, 206), was not substan-tiated. A galactan structure was identified in C.paradoxa polysaccharides (4). Recently, Naka-mura (133) reported the isolation of a biologi-cally active peptido-rhamnogalactan from theyeast form of S. schenckii after deoxycholateextraction and purification by diethylamino-ethyl-Sephadex chromatography. This complexstructure cross-reacted' with the peptido-rham-nomannan and induced positive skin tests. Ifconfirmed, the presence of this additional anti-gen will make the general picture of S. schenckiisurface constituents even more complex.

S. schenckii Cell Wall GlucansThe cell walls of different cell types of S.

schenckii contain 44 to 61% neutral sugars, andglucose is the major constituent (157). Previatoet al. (156) fractionated cell wall preparationsfrom one strain to isolate soluble and insolubleglucans. By methylation-fragmentation analysisand a quantitative periodate oxidation method,the proportions of ,B-(1 -. 3), ,8-(1 -+ 4), and ,8-(1 -+ 6) linkages in the soluble and insolubleglucans were determined (Table 4). Both solubleand insoluble glucans of S. schenckii gave riseto a fi-(1 -* 3)-linked polysaccharide, along withglycerol and erythritol, when Smith degradationand mild hydrolytic conditions were used. Hy-drolysis with the exo- and endo-f8-(l -* 3)-glu-canase of Arthrobacter luteus and a mixture ofthe ,B-(1 -- 3)- and,-(1 -) 6)-endoglucanases ofBacillus circulans (42) produced only glucoseand laminaribose and traces of other oligosac-charides. The solubility properties of these glu-cans probably depend on the ratio of ,B-(1 -* 3)-linked glycopyranose units to fl-(1 -) 6)- and f8-(1 --* 4)-linked glycopyranose units. Whereas ablock-type structure of ,B-(1 -- 3)-linked unitshas been suggested in both soluble and insolubleglucans, it seems that a similar sequence of 8B-(1.-- 4)-linked units is unlikely in the soluble pol-ymers, as it would resemble cellulose and beinsoluble. The presence in the same polymer ofmixed 13-(1 -- 4), f-(1 -- 3), and fB-(1 -* 6)linkages in these S. schenckii glucans is a struc-tural feature which is uncommon in other fungalglucans. The shiitake mushroom Lentinusedodes contains a 8-glucan with all three ofthese linkages (211), but their ratio is differentfrom the ratio in S. schenckii glucans.

It has been reported that linear ,8-D-(1 -* 3)-linked fungal glucans protect against tumors im-planted in animals (165, 221). Glucans with longsequences of 8-(1 -- 6)-linked units are generallyless effective. The antitumor activity depends onthe molecular weight of the polysaccharide(145), but the influence of other structural fea-tures, such as branching (30, 183), is still unclear.

TABLE 4. Proportions of linkages in soluble andinsoluble glucans from different S. schenckii cell

typesa

% In:Linkage

Yeasts Conidia Hyphae

Insoluble glucans,8-(1 -3) 66 65 65,B-(1 4) 5 10 9,-(1 6) 29 25 26

Soluble glucansi-(1 -3) 44 45 45,8-(1 4) 28 31 29,B-(1 - 6) 28 24 26a See reference 156.

Steric factors, rather than the primary struc-tures of polysaccharides, may be critical for an-titumor activity (130). The insoluble linear glu-can of S. schenckii caused complete regressionof sarcoma 180 implanted in mice at a dose of 10mg/kg per day (157) when a standard therapyschedule was used (197).

S. schenckii glucan has not been used so farto protect against infections by the fungus itself,but the effectiveness of bakers' yeast glucan hasbeen studied (190). Mice which were inoculatedsubcutaneously with S. schenckii developed skinnodules and liver lesions. Yeast glucan givenintralesionally or intravenously limited thespread of the infection.

IMMUNOCHEMISTRY OF S. SCHENCKIISURFACE ANTIGENS

The nature of the antigenic determinants insome S. schenckii polysaccharides has been elu-cidated. However, since several other polymershave now been characterized in both S. schen-ckii and C. stenoceras, further studies will benecessary to comprehend the full extent of theantigenic reactivities in these species.

Several precipitin arcs are usually obtained bydouble-diffusion tests in agar with hyperimmunesera raised against whole cells. Up to six to eightprecipitin arcs were observed with antigens pres-ent in an S. schenckii culture filtrate, but onlytwo arcs were observed after purification of thepolysaccharides with Fehling solution (201). Hu-man sera from patients with sporotrichosis alsoprecipitate purified polysaccharides or peptido-polysaccharides (72, 94), but their specificitiesare still little understood.

Antigenic Determinants and Cross-Reactivities

Surface antigens of S. schenckii include therhamnomannans and their peptido complexes.-Other than a report by Nakamura (133), therehave been no studies to characterize the anti-

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708 TRAVASSOS ANDl LLO'YD

genic deterninants presenit hi other sWit4ce cmtn-ponents (either proteins or carbohydrates);That different Atrain6 and even difterent bell

types of one strafin ean express differefit anti'enswas shown by Nishigawa et al. (139). Sera wereprepared in rabbits agaihst yeast cells from ahuman iportti'ihosis strain ahd an S. schenckiistrain Isolated tofri tho §oil in Japan. Thesestraing were indistinguishable by several criteria.When the serum raised agaihst the strain fobmsoil (Kurme 23) Was absorbed with yeast celsof the human strain, the absorbed serum wasunreactive with both strains by agglutination orby indirect immunofluorescence tests. However,when the seruf against the human strain wasabsorbed with yeast celLs of the Kururhe 23strain, the absorbed seruinm reacted with the ho-mologous antigen but not with the soil strain;the conclusion of the authors was that the hu-man strain had a unique antigen. Hyphae onslide cultures did hot react with this absorbedserum, but conidia were reactive. Yeast cellsfrom clinical isolates showed variable responsesto the absorbed serum, ranging from positive orweakly positive to negative. The same rabbitsera were tested against cells of C. stenocerasand C. ulmi (61). Mycelial and conidial forms ofCeratocystis strains reacted with the unab-sorbed sera but not with the serum absorbedwith the soil strain. It is interesting to note thatC. stenoceras perithecia did not react with eitherabsorbed or unabsorbed serum (61).From the structure of the rhamnomannans of

S. schenckii isolated at 25 and 37°C or from themycelial and yeast phases (94), it was possibleto determine the specificities of rabbit and hu-man sera to this organism. Sera were raised inrabbits against acetone-dried cel of S. schen-ckii strain 1099.12, which was known to synthe-size rhamnomannans with high proportions ofdirhamnosyl side chains when grown in a sup-plemented yeast nitrogen base medium (202) at250C (-85% dirhamnosyl side chains [202], seealso "3C NMR spectrum in reference 205). Thesesera were precipitated very efficiently by thehomologous rhamnomannan and the rhamno-mannan obtained from another strain of S.schenckii grown at 250C, but very poorly by thecorresponding rhamnomannans obtained at370C (94). The specificity of the serum raisedagainst cells grown at 370C (producing mainly amonorhamnosyl rhamnomannan) was wider, asit precipitated rhamnomannans obtained at both25 and 370C; the homologous polysaccharidewas a slightly better antigen. The serum raisedagainst S. schenckii cells grown at 25°C reactedpoorly with the rhamnomannans of C. steno-ceras, C. pilifera, C. ulmi, and C. minor also

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obtained at 250C. In contrast, the rabbit seruiiagaist cells grown at 370C precipitated quiteWell with these rhamnomannans. These resultssuggest that S. schenckii has a specific antigenicdeterminant when grown at 250C, which is prob-ably the (1 -- 2)-linked dirhamnosyl side chain.this antigenic determinant is not present inrhamnoriannans from Ceratocystis species alsogrbwn at 250C or in S. schenckii rhamnoman-haris obtained at 370C, which is in accordancewith the chemical data described above. Cross-reactions observed among Ceratocystis and S.gthenckii rhamnomannans obtained at 25 and370C with the serum raised against cells grownat 370C Were due to the presence in all cases ofvariable proportions of monorhamnosyl sidechains. These conclusions were confirmed byinhibition studies in which oligosaccharides wereused. The serum, which recognized dirhamnosylside chains had its precipitin reaction with thehomologous antigen completely inhibited by 2X 102 ,umol of O-a-L-rhamnopyranosyl-(l -+ 2)-O-a-L-rhamnopyranosyl-(l -- 3)-D-mannopyra-nose, whereas only 50% inhibition was obtainedwith O-a-L-rhamnopyranosyl-(l -- 3)-D-man-nose and 25% inhibition was obtained with 0-a-L-rhamnopyranosyl-(1 -. 3)-O-a-L-rhamno-pyranosyl-(1 -- 6)-D-galactose (from Rhamnuscathartica [155]) at similar concentrations. D-Mannose or mannose disaccharides were notinhibitory. The homologous precipitin reactionwith the serum recognizing monorhamnosyl sidechains was inhibited at comparable concentra-tions by 0-a-L-rhamnopyranosyl-(l 3)-D-mannose, by the rhamnose trisaccharides, andeven by L-rhamnose (94). Surprisingly, a humanserum (Lop) from a patient with sporotrichosisshowed a strong specificity for the dirhamnosyldeterminants and reacted very poorly with thepolysaccharide obtained at 370C (Fig. 10). Sinceyeast forms (125), which are thought to be thepredominant forms in vivo, synthesize mono-thamnosyl rhamnomannans, this is an unex-pected finding. It is possible that hyphae formin vivo but are not easily demonstrated (seeabove). After further absorptions to increasetheir specificities, these sera were then used tolocate polysaccharides in different S. schenckiicell types by immunofluorescent staining. Inmost cases the predicted reactivities were ob-served; that is, yeasts and conidia were stainedpreferentially by the serum which recognizedmonorhamnosyl determinants, whereas hyphaewere stained by the serum specific for dirham-nosyl determinants. However, some unexpectedreactions were also observed, and these weretentatively explained by the presence of bothside chains in one particular cell type, by the


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F 2

03 4

FIG. 10. Double-diffusion patterns ofrabbit and human sera with S. schenckii L-rhamno- D-mannans. Thecenter wells contained rabbit antiserum prepared against S. schenckii cells grown at 25°C (A), rabbitantiserum prepared against S. schenckii cells grown at 37°C (B), human serum Riv (C), human serum Lop(D), and human serum Guil (E). Outer wells 1 and 3, S. schenckii rhamnomannans from strains 1099.12 and1099.10 obtained at 25°C; outer wells 2 and 4, rhamnomannans from the same strains grown at 37°C. (FromLloyd and Travassos [94]; used with pernission)

cross-reactivity of both sera with antigens ade-quately presented at the cell surface, and byadditional unrecognized specificities.

Cross-reactivities among S. schenckii antigensand rhamnose-containing soluble antigens fromseveral strains of Ceratocystis, Graphium, andEurophium have also been demonstrated byimmunodiffusion tests (72). Since crude culturefiltrate antigens were used, it is not clearwhether these cross-reactions were due to rham-nose-containing determinants other than thosealready recognized or to sugars other than rham-nose. The antigen preparations contained glu-cose or galactose or both, in addition to rham-nose and mannose.

Cross-reactivities between antigens containingnonreducing end units ofL-rhamnose from groupB Streptococcus and S. schenckii have beensuggested (134). A rabbit antiserum against agroup B Streptococcus gave broad immunodif-fusion lines against the homologous antigen andtwo or three lines against S. schenckii andagainst 36 of 39 Ceratocystis and Graphiumantigenic preparations. However, an antiserumagainst S. schenckii gave two precipitin lineswith the homologous antigen and with each ofthe 36 Ceratocystis and Graphium antigens butno lines with the Streptococcus antigen. Previ-ously, Neil et al. (135) had reported cross-reac-tions between S. schenckii and several serologi-cal types of Streptococcus (Diplococcus) pneu-moniae, Leuconostoc mesenteroides, and strep-

tococci of human origin in precipitin and agglu-tination tests. If nonreducing end units of L-rhamnose are the common antigenic determi-nants in all of these microorganisms, bacteria ofthe normal flora, such as group B and group Gstreptococci and Aerobacter aerogenes, shouldbe constant stinuli for the synthesis of anti-bodies that may cross-react with S. schenckiiantigens. Low agglutination titers against S.schenckii are demonstrable in normal humansera (140).

Determinants for Delayed-TypeHypersensitivity

Components eliciting positive skin tests in pa-tients with sporotrichosis are usually present inthe media of S. schenckii cultures (53, 201). Anactive peptido-polysaccharide complex was iso-lated from this source, purified, and shown toinduce both immediate and delayed-type hyper-sensitivity reactions (142). Several reports haveindicated that pure fungal polysaccharides orpeptido-polysaccharides treated with proteasesdo not elicit delayed-type reactions (8, 88, 144),which suggests that the determinants of de-layed-type hypersensitivity are located in thepeptides. Lee and Lloyd (88) found that thepeptide determinants of delayed-type hypersen-sitivity in Exophiala (Cladosporium) werneckiipeptido-galactomannan were stable to even rig-orous acid conditions. By utilizing the alkalilability of the carbohydrate-protein linkages,

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several peptides were isolated, and two of themwere active in skin tests.

In other studies on S. schenckii, workers iso-lated a peptido-polysaccharide complex whichelicited immediate and delayed-type hypersen-sitivity reactions. This polymer contained rham-nose (21%), mannose (55.9%), galactose (10%),and a peptide moiety (12.5%), which was rich inthreonine, serine, and alanine residues (174).Treatment of this preparation with 16 mM per-iodate at 4°C for 100 h did not abolish thedelayed-type reaction, but the product was nolonger active in the passive cutaneous anaphy-laxis test. By treating the peptido-polysaccha-ride with 0.01% papain at 370C for 48 h, a car-bohydrate-rich glycopeptide fraction was iso-lated. The papain-treated product and the orig-inal antigen showed identical reactivities with arabbit antiserum. However, delayed-type reac-tivities, including both inhibition of peritonealcell migrations and delayed skin tests, were di-minished (174). Lloyd and Silva-Hutner (88; un-published data) also found that whereas a pep-tido-rhamnomannan from S. schenckii was skintest positive, the corresponding rhamnomannanderived by hot dilute KOH extraction was inac-tive. In agreement with this, an unpurified pep-tido-polysaccharide prepared by autoclaving aculture and precipitating the supernatant withethanol was very active in skin tests in patientswith sporotrichosis, whereas the polysaccharideisolated from cells ofthe same S. schenckii strainby KOH extraction and methanol precipitationwas inactive (201). Purification by precipitationwith Fehling solution or treatment with 45%phenol at 600C did not affect the skin test reac-tivity. Some positive skin tests in human pa-tients were produced by peptido-polysaccha-rides from culture filtrates of C. stenoceras (116)and, to a lesser degree, by peptido-polysaccha-rides from Ceratocystis ips, C. minor, and C.ulmi (71). In conclusion, it seems that delayed-type determinants in S. schenckii are present inthe peptide moieties of peptido-polysaccharidecomplexes. These determinants are shared bysome antigens from species of Ceratocystis butare rather specific for S. schenckii comparedwith the determinants from other pathogenicfungi. For instance, antigens such as histoplas-min, coccidioidin, and yeast forms of Blasto-myces and Candida do not induce a delayed-type reaction when tested in animals sensitizedto S. schenckii (138).

NATURAL RESISTANCE ANDIMMUNOLOGY IN SPOROTRICHOSISReports of positive skin tests in individuals

without previous histories of sporotrichosis (69,152, 169) suggest that a state of sporotrichosis

infection may commonly exist (28, 152, 178) andthat in many cases the infection is halted beforeany noticeable symptons appear. The lungs maybe a portal of entry in asymptomatic infections(100, 138, 172). Active immunological responseis probably the cause of resistance in healthyindividuals, although S. schenckii is only weaklypathogenic (158) and should be regarded as anopportunistic fungus (113). Favorable conditionsfor the appearance of the disease are important.Beurmann and Gougerot (17) observed that inmost cases another disease condition (e.g., tu-berculosis, diabetes, gout, or alcoholism) pre-ceded sporotrichosis. An association of sporotri-chosis and sarcoidosis has also been reported(38, 102). Inoculation of a great number of S.schenckii conidia is another factor that shoulddetermine the onset of disease. In an endemicarea, frequent contact .with S. schenckii mayoccur by inhalation and ingestion of cell ele-ments which are not in themselves particularlyvirulent. These can sensitize a host by therelease of peptido-polysaccharide antigens fromcells transiently present or saprophytically im-planted. Concomitant immunity is produced inindividuals who have repeated contact with S.schenckii (52). Miranda et al. (129) and Padilha-Goncalves (149) cured several cases by intrader-mal injection of sporotrichin. That an early sen-sitization to S. schenckii peptido-polysaccha-rides occurs in all localized cutaneous infectionsis shown by the high frequency of positive skintests with sporotrichin (28, 53, 162). Apparently,intradermal reactions remain positive for severalyears after the cure of sporotrichosis (123). Incontrast, several patients with disseminated spo-rotrichosis did not give positive skin tests withsporotrichin.The soluble sporotrichin prepared by Gonza-

lez-Ochoa and Figueroa (53), which has strongactivity and a high specificity, has some differentproperties when compared with cellular sporo-trichin. The cellular antigen elicits positive re-actions for prolonged periods after an immuno-sensitizing contact with S. schenckii. Reactionsare not necessarily related to ongoing disease(122). On the other hand, the intradermal reac-tions elicited by soluble sporotrichin can becomenegative 1 to 3 years after cure (122) and aregenerally negative in healthy individuals. Sen-sitivity is greater with the yeast-phase antigensthan with the mycelium-phase antigens. Froman epidemiological point of view, cellular sporo-trichin is the antigen of choice (113). The pep-tido-polysaccharides of the soluble sporotrichin(53) are preferred, antigens for the diagnosis ofsporotrichosis. However, Carrada-Bravo (27)tested 359 pulmonary patients by skin testingwith heat-killed yeast-phase cells and the solu-

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ble antigen of Gonzalez-Ochoa and Figueroa (53)and observed 38.7% positive reactions with theyeast-phase antigen, compared with 9.1% posi-tive reactions with the soluble antigen. Theseresults suggested some degree of cross-reactivitybetween S. schenckii and other fungal antigens.Nielsen (138) used soluble cytoplasmic compo-nents and defatted and detergent-extractedwalls from S. schenckii cells prepared by me-chanical rupture. Cell walls were fmuther treatedwith pronase and ribonuclease. The soluble an-tigen was less active intradermally and in somecases, did not react in patients with proven dis-ease. Only the various preparations containingtreated or untreated cell walls had reactivitiescomparable to the reactivity of standard wholeyeast antigen.Another evaluation of cell-mediated immu-

nity in patients with sporotrichosis was per-formed by a conventional lymphocyte transfor-mation method (153). Lymphocyte stimulationwith phytohemagglutinin was abnormally low ina group of patients with systemic disease. Someof these patients were anergic to a battery ofskin tests. Patients with cutaneous disease ap-peared to have normal cell-mediated immunity.Lymphocytes from seven of eight patients withactive sporotrichosis showed measurable re-sponses to an S. schenckii yeast antigen in vitro(153). Blastogenic responses in vitro were ob-tained with antigens extracted from the myce-lium phase (189), but in this case healthy lym-phocyte donors with positive blastogenic indexesfor S. schenckii antigen also showed high blas-togenic responses to Candida albicans (189).No cross-reaction with Coccidioides immitis wasobserved. In one patient with articular sporotri-chosis the blastogenic index to S. schenckii washigher than the blastogenic index to C. albicansantigen.

Experimental Infections in AnimalsExperimental sporotrichosis in laboratory an-

imals has been reviewed by Lurie (97). By intra-peritoneal inoculation of S. schenckii into ham-sters, a chronic form of sporotrichosis with gumlesions and orchitis and a fatal disseminatedform were obtained. Cigar-shaped, ovoid, andround forms, as well as asteroid bodies, occurredin vivo. When inoculated intraperitoneally, ratsare the most susceptible animals to S. schenckiiinfections. Mice and dogs are also susceptible;rabbits appear to be the most resistant of alllaboratory animals (23). In mice, the disease isprogressive, with destructive lesions of bonesand other organs; death occurs within 3 weeks(208). Adult cats are more resistant to infectionthan newborn cats (17). Barbee et al. (11) inoc-ulated S. schenckii into the rear footpads of

normal cats and cats preinfected with Brugiamalayi, a nematode which produces lymphaticdysfunction. Wartlike lesions were followed bylymphatic involvement from the feet to the pop-liteal lymph nodes. Nodular lesions softened andulcerated. Organisms frequently disseminated tothe viscera. Infections involving the lymphaticsystem were also produced in monkeys (15).

Several S. schenckii strains isolated in Mexico(121) were pathogenic for hamsters and mice.Necrotic tissues of mice contained filamentousforms, whereas in hamsters asteroid bodies wereformed preferentially. Induction of filamentousforms in mice was favored by treatment withgriseofulvin or amphotericin B (146).

Simulation of the lymphatic disease of hu-mans was attempted in Syrian hamsters by cu-taneous footpad inoculation (29). The infectionwas of a self-limited, lymphatic form, or, if theinoculum was increased 1,000-fold, a systemic,nonfatal form developed, involving the liver andspleen. Development of increased resistance toreinfection after the primary subcutaneous in-fection was also demonstrated. Resistance todisseminated infection was increased by subcu-taneous inoculation of crude ribosomal prepa-rations or trypsinized cell walls from S. schenckiiyeast forms. Sera from infected animals hadpersistent agglutinin titers against the yeastphase.Guinea pigs are resistant to progressive spo-

rotrichosis; an increased susceptibility to infec-tion was obtained by keeping the animals at10°C (160). Mackinnon and Conti-Diaz (107)obtained lesions in 13 of 15 rats inoculated intra-cardially and kept at 5 to 15°C, but no lesionsoccurred at 31°C. After intrascrotal inoculationof guinea pigs, most animals gave positive skin,agglutination, and immunocytoadherence tests(160). After 4 weeks there was complete resto-ration of the normal tssue and reduced immu-nological reactivity.Experimental sporotrichosis was also induced

in congenitally athymic (nude) mice (175). Nu/nu mice were much more susceptible to intra-venously inoculated S. schenckii than nu/+mice, thus suggesting that the thymus plays arole in host defense. However, elimination of thefingus was initiated on day 8, whereas appear-ance of delayed-type hypersensitivity to a solu-ble S. schenckii antigen developed on day 17after infection in the nu/+ mice. This suggeststhat resistance to the fungus cannot be explainedsolely on the basis of cellular immunity.

Serological Tests for Diagnosis ofSporotrichosis

Among the serological tests for the diagnosisof sporotrichosis, agglutination, gel precipita-

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tion, and complement fixation are used mostwidely. Tube agglutination was introduced early(222) and is regarded as the most sensitive of alltests (21, 161). In a group of 80 patients, Blumeret al. (21) obtained 96% positive agglutinationtests, and a slide latex agglutination test gave94% positive reactions. Complement fixation andimmunodiffusion tests were less sensitive, giving68 and 55% positive reactions, respectively. Theindirect fluorescent antibody technique was alsoused in the diagnosis of 61 cases of localized anddisseminated sporotrichosis and gave positivereactions in 90% of the cases. Nonspecific reac-tions were minimal with all tests (21).

In the agglutination test with a yeast-phaseantigen, serum titers lower than 1:80 should beinterpreted with caution, as they can reflectnonspecific reactions (219). A small percentageof the sera from normal individuals have agglu-tinins to S. schenckii; these could indicate cross-reactivity with bacterial or mycotic antigens orthe previous occurrence of subclinical sporotri-chosis (B. M. Ashbrook, Ph.D. thesis, Duke Uni-versity, Durham, N.C., 1964). For routine use,the agglutination tests should be carried outwith autoclaved suspensions of yeast forms tominimize cross-reactions (124). It appears thatheating aqueous suspensions of yeast forms in-activates or extracts the cross-reacting antigensand leaves behind cells expressing only the an-tigens specific for S. schenckii.The complement fixation test described by

Jones et al. (73) was effective in diagnosingextracutaneous sporotrichosis, but was insensi-tive in diagnosing the cutaneous disease (73,143). This test was used to follow the clinicalresponses of patients to amphotericin B treat-ment (73). Sera from 78 patients with othermycoses and sera from 47 healthy subjects wereunreactive in the complement fixation test. Byusing an antigen from a filtrate of a yeast-phaseculture, precipitin reactions were also tested, butthey were considered nonspecific because in agroup of patients with histoplasmosis one-half ofthe sera contained precipitins to S. schenckiiantigen (73). In contrast, McMillen (124) usedimmunodiffusion and complement fixation testsand observed that in 1,400 sera tested for fungalantibodies, only 1% were positive with a solubleS. schenckii antigen. In 34 lymphocutaneousand extracutaneous cases of sporotrichosis, spe-cific reactions in these tests were obtained in allbut 4 cases. Serological tests in the cutaneousdisease became negative after treatment. In con-trast, positive complement fixation and gel pre-cipitin reactions stayed positive in the extracu-taneous disease for as long as 7 years after cure(124). Negative precipitin reactions are not in-

frequent in some patients with only cutaneouslesions (124, 143). They are of value when thesporotrichotic lesions are large and active (143).

Karlin and Nielsen (81) used filtrates fromboth yeast and hyphal cultures as antigens inimmunodiffusion tests with rabbit and humansera. Antigens from yeast cultures produced dis-tinctive precipitin arcs in the gels. Most serafrom patients gave one precipitin line; occasion-ally sera gave two lines. As many as four differ-ent bands were observed with the yeast antigenand a rabbit serum raised against yeast cells. Nocross-reactivity was observed in agglutinationand precipitin tests with antigens from Histo-plasma capsulatum, C. immitis, B. dermatitidis,C. neoformans, and C. albicans (81). Patientswith articular involvement and with lung diseasehad sera with elevated titers against the S.schenckii antigen. Highly specific reactions werealso observed by Roberts and Larsh (161), whoused a soluble antigen prepared from sonicatedyeast cells; 1,000 sera examined for routine fun-gus serology were negative. No false-positivereactions were obtained with sera from patientswith other mycoses.

In general, therefore, serological tests are spe-cific for S. schenckii antigens, as would be ex-pected considering the nature of their principalconstituents, the pepto-rhamnomannans. Someof the cross-reactions observed may be relatedto antigen preparation or to the presence ofminor components with more common deter-minants. For instance, some antigen prepara-tions were filtrates of cultures containing bakers'yeast extract, which was either used withoutconcentration or concentrated 5- or 10-fold. Thepresence of bakers' yeast mannan in these prep-arations would render the antigen less specificfor S. schenckii, as cross-reactions would occurwith other polymers also containing determi-nants, such as a-D-mannopyranose nonreducingend units and (1 -. 2)-linked mannose oligosac-charides. Real cross-reactions with S. schenckiiantigens may involve the minor galactomannancomponents since they contain antigenic f8-ga-lactofuranose end units that are also present ina variety of other fungi (125). As discussedabove, yeast forms of S. schenckii react with ananti-Hormodendrum serum, and this serumcross-reacts with galactomannans from Asper-gillus fumigatus, Exophiala (Cladosporium)werneckii, and Trichophyton rubrum (194). Ga-lactomannans from S. schenckii and C. steno-ceras are loosely attached to the cell wall (55,157) and are usually found in the culture filtrate.Heating aqueous suspensions of S. schenckiimay completely extract the galactomannan com-ponent and render the remaining cells more

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specific for agglutination tests. As describedabove, a Fehling solution-precipitated rhamno-mannan (125, 202) should be the antigen ofchoice for specific precipitin reactions becausegalactomannans are excluded from the purifiedpolysaccharide. Nielsen (138) observed that thenondialyzable portion of an S. schenckii culturebroth was not capable of detecting hypersensi-tivity in all sensitized subjects and that theintradermal activity was increased slightly byethanol precipitation. Apparently, in this case

the strain of S. schenckii used accumulated fewpeptido-polysaccharide molecules in the me-

dium, so that an incubation period longer thanthat used (72 h) was probably necessary. Inanother S. schenckii strain grown at 250C, littleaccumulation of rhamnomannans was observedafter 2 days of culture, but thereafter the poly-saccharide concentration in the medium in-creased rapidly with time (125). The possibilitythat there may be protein antigens other thanthe peptido-polysaccharide complexes that maybe reactive in the serological tests has not beenexcluded. Surface antigens sensitive to proteasesbut not to periodate have been identified in A.fumigatus (62).

SUNMMARY AND FUTURE PROSPECTSRecent studies with S. schenckii and related

species of Ceratocystis have provided new ex-

perimental systems and new methods that willundoubtedly be useful in further studies on theseand other fungi. Ecological reports defining pref-erential biological associations, such as the as-

sociation of C. stenoceras with Eucalyptus andthe association of S. schenckii with Pinus (115),should stimulate further study on the distribu-tion in nature of these species, as well as of otherrelated Ceratocystis species, including C. minor.Expanded surveys on healthy individuals reac-

tive to sporotrichin should indicate the degreeof human exposure to S. schenckii in endemicand nonendemic areas. New methods for thevisualization of S. schenckii in biological speci-mens in addition to immunofluorescence stain-ing should be used to obtain a better descriptionof cell types present in human infections incomparisons with infected animals. Dimorphismof S. schenckii has been studied from the pointof view of ultrastructure of transitional cells andcell wall constituents. With the available data,no model can be put forward to explain morpho-logical differentiation. However, certain chemi-cal characteristics, such as cell wall rhamnoman-nans, are clearly associated with cell types ofone or the other morphological phase. Very fewother biochemical activities of S. schenckii havebeen recognized. The role of enzymes, such as

neuraminidase and other hydrolases, in the ev-olution of infection should be investigated fur-ther.DNA base composition and hybridization

studies have shown that S. schenckii is homog-enous and bears little relation to several Cera-tocystis species, including C. stenoceras. This israther unexpected in view of the morphologicalsimilarity of the S. schenckii and C. stenocerasconidial forms; however, several biochemical dif-ferences between these two species have alsobeen reported. The DNA of one strain of C.minor has the same base composition as S.schenckii DNA, and a high percentage of rela-tive binding has been obtained between theDNAs of these two species. DNAs from morestrains of C. minor need to be examined toconfirm the phylogenetic relationship of C. mi-nor to S. schenckii. However, the search forother species of Ceratocystis that may be evencloser to S. schenckii in their DNA homologiesshould continue.

Considerable emphasis has been placed on thedetermination of the fine structures of the poly-saccharides from S. schenckii, C. stenoceras,and C. ulmi. PMR spectroscopy mid 13C NMRspectroscopy have been important aids in thisstudy. Typical side chains were recognized in S.schenckii rnamnomannans, which were absentin C. stenoceras polysaccharides. The latter spe-cies formed acidic rhamnomannans containingglucuronic acid units. Simpler side chains con-taining only one residue of L-rhamnose are pres-ent in most rhamnomannans of S. schenckii andCeratocystis spp. and are responsible for theimmunological cross-reactivities of these anti-gens. The regular occurrence in S. schenckii ofgalactomannan, an antigen that is potentiallycross-reactive with polysaccharides containing,8-galactofuranose residues from other patho-genic fungi, may pose an additional difficulty inthe preparation of specific soluble and insolubleS. schenckii antigens. Galactose-free, Fehlingsolution-precipitable rhamnomannans obtainedat 37 as well as 250C should be used in immu-nodiffusion tests for sporotrichosis. Some of theproperties of S. schenckii peptido-polysaccha-rides have been studied, but additional investi-gations are needed to define the nature of theConA-reactive structures, the linkages betweensaccharide chains and peptides, and the chemi-cal structures of the determinants which inducedelayed-type hypersensitivity reactions. Severalinvestigators have described models of experi-mental sporotrichosis in animals. By using these,it should be possible to study the influence ofdifferent cell types, cellular subfractions, andpurified components on the evolution and course

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of infection. With our increased knowledge ofthe distributions and chemical structures of S.schenckii antigens, it should be possible toachieve standardization of antigens for use inthe diagnosis of sporotrichosis.

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2. Ajello, L., and W. Kaplan. 1969. A new variantof Sporothrix schenckii. Mykosen 12:633-644.

3. Altner, P. C., and R. R. Turner. 1970. Sporo-trichosis of bones and joints. Review of theliterature and report of six cases. Clin. Orthop.68:138-148.

4. Alviano, C. S., P. A. J. Gorin, and L. R.Travassos. 1979. Surface polysaccharides ofphytopathogenic strains of Ceratocystis para-doxa and Ceratocystis fimbriata isolated fromdifferent hosts. Exp. Mycol. 3:174-187.

5. Aoki, Y., H. Nakayoshi, S. Asaga, and M.Ono. 1967. Studies on the immunologicallyactive substances in fungi. Jpn. J. Med. Mycol.8:284-291.

6. Aoki, Y., H. Nakayoshi, and M. Ono. 1968.Studies on the immunologically active sub-stances of fungi. III. The chemical propertiesof skin test antigens from organisms of Sporo-trichum schenckii, Cryptococcus neoformans,Aspergillus fumigastus and of serologically ac-tive compounds in several mycotic antigens.Jpn. J. Allergy 17:56-61.

7. Asada, Y., and M. Sofue. 1968. Sporotrichosisin Osaka residents with statistics of 261 casesin Japan. Hifu To Hinyo 10:488-496.

8. Azuma, I., H. Kimura, F. Hirao, E. Tsubura,Y. Yamamura, and A. Misaki. 1971. Bio-chemical and immunological studies on Asper-gillus. III. Chemical and immunological prop-erties of glycopeptide obtained from Aspergil-lus fumigatus. Jpn. J. Microbiol. 15:237-246.

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10. Ballou, C. E. 1976. Structure and biosynthesis ofthe mannan component of the yeast cell enve-lope. Adv. Microb. Physiol. 14:93-158.

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13. Baum, G. L., R. L. Donnerberg, D. Stewart,W. J. Mulligan, and L. R. Putnam. 1969.Pulmonary sporotrichosis. N. Engl. J. Med.280:410-413.

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19. Bievre, C., and F. Mariat. 1975. Compositionen acides gras des lipides polaries et neutres deSporothrix schenckii et de Ceratocystis sten-oceras. Sabouraudia 13:226-230.

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21. Blumer, S. O., L. Kaufman, W. Kaplan, D. W.McLaughlin, and D. E. Kraft. 1973. Compar-ative evaluation of five serological methods forthe diagnosis of sporotrichosis. Appl. Micro-biol. 26:4-8.

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Documents

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