Interaction of Mycoplasmas With Host Cells

Interaction of Mycoplasmas With Host Cells

SHLOMO ROTTEM

Department of Membrane and Ultrastructure Research,The Hebrew University-Hadassah Medical School, Jerusalem, Israel

I. Introduction 418II. Adherence to Host Tissues 418

A. Adhesins 418B. Accessory proteins 419C. Receptors 420

III. Invasion Into Host Cells 420A. Invasins and receptors 421B. Changes in the host cell cytoskeleton 421C. Signal transduction 421D. Survival and multiplication within host cells 422

IV. Fusion With Host Cells 422A. Factors mediating fusion 423B. Molecules implicated in fusion 423

V. Possible Mechanisms of Damage to Host Cells 424A. Competition for precursors 424B. Damage induced by adherence 424C. Damage induced by fusion 424D. Cytopathic effects 424

VI. Circumventing the Host Immune System 425VII. Modulating the Immune System 426

A. Modulatory effects on monocytes and macrophages 426B. Characterization of modulins 426C. Lipoproteins 426D. Superantigens 427E. Membrane lipids 428F. Signaling pathways by modulins 428G. Mitogenic activity 428H. Oncogenic activity 428

VIII. Conclusions 429

Rottem, Shlomo. Interaction of Mycoplasmas With Host Cells. Physiol Rev 83: 417432, 2003; 10.1152/phys-rev.00030.2002.-The mycoplasmas form a large group of prokaryotic microorganisms with over 190 speciesdistinguished from ordinary bacteria by their small size, minute genome, and total lack of a cell wall. Owing to theirlimited biosynthetic capabilities, most mycoplasmas are parasites exhibiting strict host and tissue specificities. Theaim of this review is to collate present knowledge on the strategies employed by mycoplasmas while interacting withtheir host eukaryotic cells. Prominant among these strategies is the adherence of mycoplasma to host cells,identifying the mycoplasmal adhesins as well as the mammalian membrane receptors; the invasion of mycoplasmasinto host cells including studies on the role of mycoplasmal surface molecules and signaling mechanisms in theinvasion; the fusion of mycoplasmas with host cells, a novel process that raises intriguing questions of howmicroinjection of mycoplasma components into eukaryotic cells subvert and damage the host cells. The observa-tions of diverse interactions of mycoplasmas with cells of the immune system and their immunomodulatory effectsand the discovery of genetic systems that enable mycoplasmas to rapidly change their surface antigenic compositionhave been important developments in mycoplasma research over the past decade, showing that mycoplasmaspossess an impressive capability of maintaining a dynamic surface architecture that is antigenically and functionallyversatile, contributing to the capability of the mycoplasmas to adapt to a large range of habitats and cause diseasesthat are often chronic in nature.

Physiol Rev83: 417432, 2003; 10.1152/physrev.00030.2002.

www.prv.org 4170031-9333/03 $15.00 Copyright 2003 the American Physiological Society

on Septem

ber 15, 2014D

ownloaded from

I. INTRODUCTION

Mycoplasmas (class Mollicutes) are the smallest andsimplest self-replicating bacteria (82). These microorgan-isms lack a rigid cell wall and are bound by a singlemembrane, the plasma membrane. Wall-less bacteriawere first described 100 years ago, and now over 190species, widely distributed among humans, animals, in-sects and plants, are known (88). The lack of a cell wall isused to distinguish these microorganisms from ordinarybacteria and to include them in a separate class namedMollicutes. Most human and animal mollicutes are Myco-plasma and Ureaplasma species of the family Mycoplas-mataceae. Because mycoplasmas have an extremely smallgenome (0.582.20 Mb compared with the 4.64 Mb ofEscherichia coli), these organisms have limited metabolicoptions for replication and survival. Phylogenetically, themollicutes are related to Gram-positive bacteria fromwhich they developed by genome reduction (43). There-fore, the mollicutes are not at the root of the phylogenetictree but are most probably late evolutionary products (43,61). The smallest genome of a self-replicating organismknown at present is the genome of Mycoplasma geni-talium (0.58 Mb; Ref. 34). Comparative genomic studiessuggested that the genome of this organism still carriesalmost double the number of genes included in the mini-mal gene set essential for cellular function (61, 74).

Owing to their limited biosynthetic capabilities, mostmycoplasmas are parasites exhibiting strict host and tis-sue specificities (82, 89). The mycoplasmas enter an ap-propriate host in which they multiply and survive for longperiods of time. These microorganisms have evolved mo-lecular mechanisms needed to deal with the host immuneresponse and the transfer and colonization in a new host.These mechanisms include mimicry of host antigens, sur-vival within phagocytic and nonphagocytic cells, and gen-eration of phenotypic plasticity. The major question iswhether mycoplasmas cause damage to the host cells andto what extent the damage is clinically apparent. Myco-plasmas have long resisted detailed analyses because ofcomplex nutritional requirements, poor growth yields,and a paucity of useful genetic tools. Although questionsstill far outnumber answers, significant progress has beenmade in identifying the mechanisms by which mycoplas-mas interact and damage eukaryotic host cells.

II. ADHERENCE TO HOST TISSUES

Many animal mycoplasmas depend on adhesion tohost tissues for colonization and infection. In these my-coplasmas adherence is the major virulence factor, andadherence-deficient mutants are avirulent (6, 81). Thebest studied adherence systems are those of M. pneu-moniae, the causative agent of primary atypical pneumo-

nia in humans, which inhabits the respiratory tract, andM. genitalium, which preferentially colonize the urogen-ital tract. These organisms exhibit the typical polymor-phism of mycoplasmas, with the most common flask andfilamentous shaped (Fig. 1). Cytadherence of these organ-isms to cells in the respiratory or urogenital epithelium isan initial and essential step in tissue colonization andsubsequent disease pathogenesis (5).

A. Adhesins

M. pneumoniae is the most extensively studied sys-tem with respect to the adhesins and receptors. Thisorganism has a polar, tapered cell extension at one of thepoles containing an electron-dense core in the cytoplasma(Fig. 1). This structure, termed the tip organelle, functionsboth as an attachment organelle and as the leading end ingliding-type motility. A surface 169-kDa protein desig-nated P1 (47) and a 30-kDa protein designated P30 (19)are densely clustered at the tip organelle of virulent M.pneumoniae (Fig. 2), providing polarity to the cytadher-ence event. Both proteins elicit a strong immunologicalresponse in convalescent-phase sera from humans andexperimentally infected hamsters, and anti-P1 or anti-P30monoclonal antibodies block this cytadherence (4, 81).Neither P1 nor P30 mutants cytadhere (50, 103). Atpresent, it is apparent that P1 is the adhesin of M. pneu-moniae and P30 is a protein associated with the adher-ence process. Nonetheless, the exact role of P30 in ad-

FIG. 1. Adherence of Mycoplasma pneumoniae to host cells. A:scanning electron microscopy of filamentous M. pneumoniae grown inbroth medium. (Micrograph courtesy of S. Razin, The Hebrew Universi-ty-Hadassah Medical School.) B: transmission electron microscopy offlask-shaped M. pneumoniae (M) attached by the terminal tip organelle(arrow) to ciliated mucosal cells. (Micrograph courtesy of A. M. Collier,The University of North Carolina School of Medicine.) Magnification: A,10,000; B, 36,000.

418 SHLOMO ROTTEM

Physiol Rev VOL 83 APRIL 2003 www.prv.org

on Septem

ber 15, 2014D

ownloaded from

herence is not yet fully understood. The P30 mutantsappear to localize the adhesin P1 to the tip organelle,suggesting that P30 is required for P1 function rather thantrafficking to the tip. Recently, it has been shown that P30plays a role in cell development because its absence leadsto morphological abnormalities, notably ovoid or multi-lobed cells (85). These cells, having poorly defined tiporganelles, were associated with the loss of P30 (85). It isreasonable to assume that mutations in P30 will confer adefect in cell development, motility, and the potential tocytadhere to respiratory epithelium. Most importantly,the homologies predicted to be shared between P30 andmammalian structural proteins are implicating molecularmimicry as a basis for mycoplasma-mediated postinfec-tious autoimmunity (20). Recent findings demonstratethat M. pneumoniae interacts in the respiratory systemalso with the surfactant protein D (SP-D, Ref. 15), a majorconstituent of the alveolar environment that acts to keepalveoli from collapsing during the expiratory phase of therespiratory cycle. The primary mycoplasmal determinantsparticipating in the interaction of M. pneumoniae withhuman SP-D were membrane glycolipids (15), suggestingthat there is more than one type of adhesin on the cellsurface of M. pneumoniae.

B. Accessory Proteins

The isolation and characterization of M. pneumoniaemutants that possess P1 and P30, yet fail to cytadhere,suggest that the tip-mediated adherence of M. pneu-moniae to eukaryotic target cells is more complex (50,81). Two groups of accessory proteins, the first includinga 40-kDa and a 90-kDa proteins (P40 and P90), and the

second, proteins HMW1-HMW3, were described (25, 50,51). These proteins are not adhesins, but the loss of one ofthem is associated with the inability of M. pneumoniae tocytadhere, whereas its regain results in reversion to acytadherence-positive phenotype (81). P40 and P90 werefound to be surface proteins localized mainly at the tiporganelle (Fig. 2). These proteins are closely associatedwith P1 (53, 54) but are not directly involved in receptorbinding. Although P1 is present on the surface of strainslacking P40 and/or P90, it is not clustered at the tiporganelle, but scattered on the surface of the mycoplasma(50). Since P40 and P90 were responsible for the associ-ation of P1 with M. pneumoniae Triton shell, it has beensuggested that the P1 is kept in the membrane by P40 andP90 in a way that allows it to closely interact with thecytoskeleton forming or associated proteins.

In certain noncytadhering mutant strains anothergroup of proteins, designated HMW1, HMW2, and HMW3,is missing, while in the corresponding revertants, cytad-herence was regained. These proteins are coded by twounlinked genetic loci in the M. pneumoniae chromosome.HMW1 (112 kDa) and HMW3 (74 kDa) are members of afamily of mycoplasma proteins that share acidic residuesand an internal proline-rich domain in repeated motifs(25, 50). The proline-rich domains probably impart anextended conformation to the protein backbone, whilethe hydrophobic surface provided by the proline residuesmay contribute to interactions with other mycoplasmaproteins. Similar to their counterparts lacking P40 andP90, mutants devoid of HMW1-HMW3 are avirulent, cytad-here very poorly, and fail to cluster P1 at the tip organelle(40, 79). Furthermore, their tip organelle lacks the char-acteristic truncated appearance seen in wild-type M.

FIG. 2. Schematic diagram of the location of themajor cytadherence and accessory proteins in M.pneumoniae. [Modified from Balish and Krause (4).]

INTERACTIONS OF MYCOPLASMAS WITH HOST CELLS 419


on Septem

ber 15, 2014D

ownloaded from

pneumoniae, suggesting that one or more of the HMWproteins is required both to anchor P1 at the attachmentorganelle and to maintain the proper architecture of thetip organelle (102). Consistent with this interpretation isthe observation that these proteins are components of theTriton X-100-insoluble mycoplasma cytoskeleton and assuch may have a structural role in the localization of P1(90, 101). The deduced amino acid sequence of HMW3shows that this protein is largely hydrophilic. Results ofwhole cell radioimmunoprecipitation experiments re-vealed that HMW3 is not exposed on the surface of M.pneumoniae cells (50; Fig. 2).

Because the tip organelle in mutants having reducedlevels of HMW3 lacks the truncated tip appearance char-acteristic of wild-type M. pneumoniae (102), and loss ofHMW3 resulted in subtle changes in morphology, inabilityto cluster the adhesion P1 and reduced cytadherence(109), it has been suggested that HMW3 contribute to thearchitecture and stability of the tip organelle (50, 109).Similar to HMW3, HMW1 has an unusual subcellular dis-tribution. This protein is a peripheral membrane proteinthat is antibody accessible on the outer surface of wild-type M. pneumoniae, (3, Fig. 2). The subcellular locationof HMW2 is yet unknown, but there are suggestions thatthis protein is present near the base of the tip organelle(40).

How do the accessory proteins promote adherence?As already mentioned, clustering of P1 at the tip organelleappears to be necessary to attachment providing a criticalconcentration of the adhesion molecule required for se-curing a stable primary association with receptor mole-cules on the host cells. In the nonadhering mutants, P1fails to cluster at the tip organelle (4). It is conceivablethat one or more of the accessory proteins plays a role inthe lateral movement and concentration of the adhesionmolecules at the tip organelle. How this is accomplishedis still not fully understood. A plausible hypothesis is thatto fulfill this role, the accessory proteins must be associ-ated with the mycoplasma cytoskeleton, which is respon-sible not only for the lateral movement and proper orien-tation of P1, but also for changes in cell shape, celldivision, and motility (82, 88). Recent studies showed thatthe loss of HMW2 from wild-type M. pneumoniae cellsresulted in accelerated turnover of HMW1 and othercytadherence-accessory proteins, probably by proteolysis(3). These findings suggest a role for HMW2 in promotingthe export of HMW1 to the cell surface, where it is stableand fully functional (3).

C. Receptors

The role of host cell surface sialoglycoconjugates asreceptors for mycoplasmas has long been established(81). The carbohydrate moiety of the glycoprotein, which

serves as a receptor for M. pneumoniae on human eryth-rocytes, has been identified as having a terminalNeuAc(23)Gal(14)GlcNAc sequence (84). Neverthe-less, neuraminidase treatment has frequently failed toabolish the ability of various eukaryotic cells to bind M.pneumoniae (36). A sialic acid-free glycoprotein, isolatedfrom cultured human lung fibroblasts, which serves as areceptor for M. pneumoniae, has been isolated by Gearyet al. (37). Sulfated glycolipids containing terminalGal(3SO4)1 residues were also found to function as re-ceptors (52). Clearly, there is more than one type ofreceptor for M. pneumoniae and apparently for otheradhering mycoplasmas as well. It is interesting to notethat at the primary site of M. pneumoniae infection, theapical microvillar border and the cilia carry the sialogly-coconjugate-type receptors, whereas the secretory cellsand mucus lack them, favoring attachment of M. pneu-moniae to the ciliated cells (59).

III. INVASION INTO HOST CELLS

Current theory holds that mycoplasmas remain at-tached to the surface of epithelial cells (82), althoughsome mycoplasmas have evolved mechanisms for enter-ing host cells that are not naturally phagocytic. The intra-cellular location is obviously a privileged niche, well pro-tected from the immune system and from the action ofmany antibiotics. The ability of M. penetrans, isolatedfrom the urogenital tract of acquired immunodeficiencysyndrome (AIDS) patients (56, 57), to invade and survivewithin host cells has been intensively studied. This micro-organism has invasive properties and localizes in the cy-toplasm and perinuclear regions (2, 12, 38). Other myco-plasmas known to be surface parasites such as M. fer-mentans (100, 105), M. pneumoniae (4), M. genitalium(48), and M. gallisepticum (110), under certain circum-stances, reside within nonphagocytic cells.

In studying bacterial invasion, it is essential to differ-entiate between microorganisms adhering to a host celland those which have penetrated the cell. The early lightmicroscopic and electron microscopic observations ofmycoplasmas engulfed in membrane vesicles lead to con-flicting interpretations. Are the mycoplasmas intracyto-plasmatic, or are they at the bottom of crypts formed bythe invagination of the cell membrane (117)? A moresophisticated ultrastructural study was based on a com-bined immunochemistry and electron microscopy ap-proach. Staining surface polysaccharides of the host cellwith ruthenium red allows a better differentiation be-tween intracellular and extracellular mycoplasmas (105).Currently, the gentamicin resistance assay is the mostcommon assay to differentiate intracellular from extracel-lular bacteria (28, 94). In this assay, the extracellularbacteria are killed by gentamicin, but the intracellular

420 SHLOMO ROTTEM


on Septem

ber 15, 2014D

ownloaded from

bacteria are shielded from the antibiotic because of thelimited penetration of the gentamicin into eukaryoticcells. The gentamicin procedure was successfully adaptedto mycoplasma systems (2, 110). M. penetrans and M.gallisepticum are relatively susceptible to gentamicin. Inthe case of M. penetrans, the susceptibility to the antibi-otic can be markedly increased by adding low concentra-tions of Triton X-100 to the medium (2). For example, acombination of 200 g gentamicin and 0. 01% Triton X-100resulted in an 8 log decrease in CFU within 1 h of incu-bation at 37C. The low Triton X-100 concentrations af-fected neither the viability of the host cells nor theirpermeability to gentamicin. Low Triton X-100 concentra-tions have only a slight effect on the viability of M. pen-etrans or on the binding of M. penetrans to HeLa cells (2).Usually the number of intracellular bacteria is determinedby washing the host cells free of the antibiotic, lysingthem with mild detergents to release the bacteria andcounting the colonies (31). Because mycoplasmas are assusceptible to detergent lysis as the host cells, dilutions ofthe mycoplasma-infected host cells should be plated di-rectly onto solid mycoplasma media without lysing thembeforehand. Each mycoplasma colony represents oneinfected host cell rather than a single intracellularmycoplasma (28).

Immunofluorescent staining of internalized bacteriaand of those remaining on the cell surface, combined withconfocal laser scanning microscopy, has demonstratedthat M. penetrans penetrates eukaryotic cells (6, 12). Thisnondestructive, high-resolution method allowed infectedhost cells to be optically sectioned after fixation andimmunofluorescent labeling. Imaging single infected HeLacells revealed that invasion is both time and temperaturedependent. Penetration of HeLa cells has been observedas early as 20 min after infection (12), whereas invasion ofcultured HEp-2 cells by M. penetrans has been shown tobegin after 2 h of infection (6).

A. Invasins and Receptors

Bacterial invasion of eukaryotic cells involves com-plex bacterial and host cell processes. Invasion is associ-ated with adhesins as well as host cell receptors thatmediate interaction of the bacteria with the host cell (14,94). It is likely that surface molecules (proteins and lipids)that facilitate the adhesion process will have an effect onthe invasion. Nevertheless, adherence to the surface ofhost cells is not sufficient to trigger events that lead toinvasion. The signals generated by the interaction of hostcells with invasive mycoplasmas have yet to be investi-gated. Bacterial invasion is based on the ability of severalbacteria to bind fibronectin (27) or sulfated polysaccha-rides (26, 27). These compounds form a molecular bridgebetween the bacteria and different types of host cell

surface proteins that enables invasion (14, 26). Fibronec-tin binding activity was detected in M. penetrans. Thisorganism, which contains a 65-kDa fibronectin bindingprotein, binds selectively immobilized fibronectin (38). Anincrease in the invasive capacity of M. fermentans, whicharises from the potential of this organism to bind plas-minogen and activate it by urokinase to plasmin, has beenrecently described (113). Plasmin, a protease with broadsubstrate specificity, may alter M. fermentans-cell surfaceproteins and thereby promotes its internalization. Pro-teolytic modification of bacterial and/or host cell surfaceprotein(s) is an emerging theme in the study of bacterialpathogenicity. For example, the plasminogen activator ofYersinia pestis degrades bacterial outer membrane proteinstriggering virulence (98). Similarly, a secreted protease wasshown to stimulate the fibronectin-dependent uptake ofStreptococcus pyogenes into eukaryotic cells (14).

B. Changes in the Host Cell Cytoskeleton

Almost all invasive bacteria that come into contactwith the host cell surface trigger cytoskeletal rearrange-ments that facilitate bacterial internalization (31, 39, 75).

M. penetrans invasion of HeLa cells depends on thecapacity of the cells to assemble actin microfilaments, astreatment with cytochalasin D has a dramatic effect onthe invasion of HeLa cells by M. penetrans (2). Further-more, both vinblastine, which disrupts microtubules, andtaxol, which freezes microtubules, virtually abolish pene-tration of M. penetrans (12). These findings suggest thatalterations in the polymerization dynamics and stability ofmicrotubules inhibits the invasion of M. penetrans intoHeLa cells. On the other hand, the entry of M. gallisepti-cum into chicken embryo fibroblasts is inhibited by themicrotubule inhibitor nocodazole, but not by cytochalasinD, suggesting that M. gallisepticum may use a differentstrategy from that of M. penetrans for reaching the intra-cellular space (110).

C. Signal Transduction

Involvement of the host cell cytoskeleton in internal-ization is considered to be the result of a host cell signaltransduction cascade induced by the invasive bacterium.As in many signal transduction processes initiated bybacteria, kinases and/or phosphatases are usually in-volved (75, 83). The invading mycoplasmas generate up-take signals that trigger the assembly of highly organizedcytoskeletal structures in the host cells (38). Yet, thenature of these signals and the mechanisms used to trans-duce them are not fully understood. Specific activation ofprotein kinases occurs during the internalization of mostof the bacteria taken up by microtubule-dependent mech-anisms (86). It has been shown that invasion of HeLa cells



on Septem

ber 15, 2014D

ownloaded from

by M. penetrans is associated with tyrosine phosphoryla-tion of a 145-kDa host cell protein (2). Tyrosine phosphor-ylation activates phospholipase C to generate two secondmessengers: phosphatidylinositol metabolites and diacyl-glycerol (DAG). Changes in host cell lipid turnover occuras a result of M. penetrans binding and/or invasion ofMolt-3 lymphocytes (91). These changes include the ac-cumulation of DAG and the release of unsaturated fattyacids, predominantly long-chain polyunsaturated onessuch as docosahexanoic acid (C22:6, Ref. 91). Nonetheless,metabolites of phosphatidylinositol were not detected.These observations support the hypothesis that M. pen-etrans stimulates host phospholipases to cleave mem-brane phospholipids, thereby initiating the signal trans-duction cascade. Because in HeLa cells, which are in-vaded by M. penetrans, DAG is generated, it is likely thatthe protein kinase C is activated in the host cells. Indeed,transient protein kinase C activation was demonstrated ininvaded HeLa cells by several methods, including trans-location to the plasma membrane and enzymatic activity(12). However, activation was weak and transient, peak-ing at 20 min postinfection. How any of these differentsignal transduction events lead to specific microtubuleactivity resulting in mycoplasmal internalization is un-known. The role of these signals in the penetration, sur-vival, and proliferation of mycoplasmas within host cells,as well as the involvement of the lipid intermediates in thepathobiological alterations taking place in the host cells,merit further investigation.

D. Survival and Multiplication Within Host Cells

The intracellular fate of invading bacteria can varygreatly. Most invasive bacteria appear to be able to sur-

vive intracellularly for extended periods of time, at least ifthey have reached a suitable host cell (29). Other engulfedbacteria are degraded intracellularly via phagosome-lyso-some fusion. The invasive bacteria either remain andmultiply within the endosomes after invasion or are re-leased via exocytocis and/or the lysis of the endosomeswhich may allow multiplication within the cytoplasm.Most ultrastructural studies performed with engulfed my-coplasmas revealed mycoplasmas within membrane-bound vesicles (48, 100, 105). Persistence of M. penetranswithin NIH/3T3 cells, Vero cells, human endothelial cells,HeLa cells, WI-38 cells, and HEp-2 cells has been observedover a 4896 h postinfection (38, 57). M. gallisepticumremains viable within HeLa cells during 2448 h of intra-cellular residence (110). The observation of vesiclesstuffed with M. penetrans in various host cells was takenas an indication that M. penetrans is able to divide withinintracellular vesicles of the host cells (57). Nonetheless,the intracellular multiplication of mycoplasmas remainsto be convincingly demonstrated.

IV. FUSION WITH HOST CELLS

The lack of a rigid cell wall allows direct and intimatecontact of the mycoplasma membrane with the cytoplas-mic membrane of the host cell. Under appropriate condi-tions, such contact may lead to cell fusion (Fig. 3). Fusionof mycoplasmas with eukaryotic host cells has been firstobserved in electron microscopic studies (88). The devel-opment of energy transfer and fluorescence methods hasenabled investigation of the fusion process on a quantita-tive basis in an experimental cell culture-mycoplasmasystem and has also allowed the identification of fuso-genic mycoplasmas.

FIG. 3. Schematic diagram of the fu-sion of a mycoplasma with a eukaryoticcell.

422 SHLOMO ROTTEM


on Septem

ber 15, 2014D

ownloaded from

A. Factors Mediating Fusion

In all the fusogenic Mycoplasma species tested, fu-sogenicity is dependent on the unesterified cholesterolcontent of the cell membrane (104). Fusogenic activitycan be found only among mycoplasmas requiring unester-ified cholesterol for growth, whereas Acholeplasma spe-cies, which do not require cholesterol, are nonfusogenic.Furthermore, adaptation of M. capricolum to grow in theabsence of cholesterol results in a marked reduction inmembrane-cholesterol content and renders the organismnonfusogenic (104). Fusogenicity of M. fermentans withMolt-3 cells is markedly stimulated by Ca2 and dependson the proton gradient across the mycoplasma cell mem-brane, decreasing markedly when the proton gradient iscollapsed by proton ionophores (24).

B. Molecules Implicated in Fusion

Among the Mycoplasma species, the human myco-plasma, M. fermentans, is highly fusogenic, capable offusing with a variety of cells (33). Furthermore, it hasbeen shown that the polar lipid fraction of this organismis capable of enhancing the fusion of small, unilamellarphosphatidylcholine-cholesterol (1:1 molar ratio) vesicleswith Molt-3 lymphocytes in a dose-dependent manner,suggesting that a lipid component acts as a fusogen (11,

92). In an attempt to identify the fusogen, detailed lipidanalyses of M. fermentans membranes were performed(23, 92, 114), revealing that the polar lipid fraction of thisorganism is dominated by the presence of unusual cho-line-containing phosphoglycolipids (Fig. 4). The majortype (MfGL-II) has been identified as 6-O-(3-phosphoryl-choline-2-amino-1phospho-1,3-propanediol)--D-gluco-pyranosyl (1-3)-1,2-diacyl-glycerol with hexadecanoyl(16:0) and octadecanoyl (18:0) in a molar ratio of 3.6:1constituting the major acyl residues (114). Other choline-containing lipids identified are MfGL-I (65, 66), which issimilar to MfGL-II but without the 2-amino-1,3-propane-diol-1,3-bisphosphate, and the ether lipids 1-O-alkyl-/alk-enyl-2-O-acyl-glycero-3-phosphocholine (MfEL) and its ly-soform 1-O-alkyl-/alkenyl-glycero-3-phosphocholine (lyso-MfEL) (108).

It has been proposed that MfGL-II is the fusogeniccomponent in M. fermentans strain PG18 (92), yet arecent study showed that despite the fact that the respi-ratory isolates of M. fermentans, strains M39 and M52,have no MfGL-II, these strains fused with Molt-3 cells atalmost the same rate and to about the same extent asPG18, suggesting that in these strains MfGL-II is not thefusogenic component (11). It is widely accepted that thereorganization of the membrane structure that occursduring fusion requires that the lipid bilayer is broken upand that other inverted configurations, such as reversed

FIG. 4. Structure of the major choline-containingphospholipids of M. fermentans. The MfEL contains ahexadecyl residue. R, acyl. [From Ben-Menachem et al.(11).]



on Septem

ber 15, 2014D

ownloaded from

nonbilayer aggregates, are being formed (18, 60, 97). Nev-ertheless, analyses of the phase behavior of MfGL-II/H2Omixtures by solid state 31P and pulsed-field gradient dif-fusion NMR spectroscopy revealed that MfGL-II is a bi-layer stabilizing lipid incapable of undergoing a phasetransition from a lamellar to an inverted configuration (8).This property of MfGL-II is difficult to reconcile with arole in membrane fusion. On the other hand, it is wellestablished that lysolipids can substantially enhance therate of fusion in model membranes as well as in biomem-branes (18), and it is plausible that the lyso ether lipidfound in all M. fermentans strains (108) may act as afusogen. Very little is known about the role of membraneproteins in the fusion process. The observation that fu-sion of M. fermentans with Molt-3 cells was inhibited bypretreatment of intact M. fermentans with proteolyticenzymes (24) implies that this organism possesses a pro-teinase-sensitive receptor(s) responsible for bindingand/or the establishment of tight contact with the cellsurface of the host cell involved in fusion.

V. POSSIBLE MECHANISMS OF DAMAGETO HOST CELLS

A. Competition for Precursors

Genomic analyses of mycoplasmas have revealed thelimited biosynthetic capabilities of these microorganisms(45, 46, 78). Mycoplasmas apparently lost almost all thegenes involved in the biosynthesis of amino acids, fattyacids, cofactors, and vitamins and therefore depend onthe host microenvironment to supply the full spectrum ofbiochemical precursors required for the biosynthesis ofmacromolecules (78, 82). Competition for these biosyn-thetic precursors by mycoplasmas may disrupt host cellintegrity and alter host cell function. Nonfermenting My-coplasma spp. utilize the arginine dihydrolase pathwayfor generating ATP (78) and rapidly deplete the hostsarginine reserves affecting protein synthesis, host celldivision, and growth (87). Certain strains of arginine-utilizing Mycoplasma spp. have been shown to inducechromosomal aberrations in host cells, most commonlychromosomal breakage, multiple translocations, a reduc-tion in chromosome number, and the appearance of newand/or additional chromosome varieties (5, 67). Becausehistones are rich in arginine, it has been suggested thatarginine utilization by mycoplasmas inhibits histone syn-thesis and causes chromosomal damage (5, 67, 87). M.fermentans infection of rat astrocytes has been shownrecently to result in a choline-deficient environment andin the induction of apoptosis (9). Choline is an essentialdietary component that ensures the structural integrityand signaling functions of the cell membranes; it is themajor source of methyl groups in the diet, and it directly

affects cholinergic neurotransmission, transmembranesignaling, and lipid transport and metabolism (115).

B. Damage Induced by Adherence

The attachment of mycoplasmas to the surface ofhost cells may interfere with membrane receptors or altertransport mechanisms of the host cell. The disruption ofthe K channels of ciliated bronchial epithelial cells byMycoplasma hyopneumoniae that resulted in ciliostasishas been described (21). The host cell membrane is alsovulnerable to toxic materials released by the adheringmycoplasmas. Although toxins have not been associatedwith mycoplasmas, the production of cytotoxic metabo-lites and the activity of cytolytic enzymes is well estab-lished. Oxidative damage to the host cell membrane byperoxide and superoxide radicals excreted by the adher-ing mycoplasmas appears to be experimentally well-sub-stantiated (1). The intimate contact of the mycoplasmawith the host cell membrane may also result in the hy-drolysis of host cell phospholipids catalyzed by the potentmembrane-bound phospholipases present in many myco-plasma species (96). This could trigger specific signalcascades (86) or release cytolytic lysophospholipids ca-pable of disrupting the integrity of the host cellmembrane (92, 93).

C. Damage Induced by Fusion

During the fusion process, mycoplasma componentsare delivered into the host cell and affect the normalfunctions of the cell. A whole array of potent hydrolyticenzymes has been identified in mycoplasmas (82, 95, 96).Most remarkable are the mycoplasmal nucleases (76, 77,82) that may degrade host cell DNA. It has recently beenshown that M. fermentans contains a potent phosphopro-tein phosphatase (95). Phosphorylation of cellular constit-uents by interacting cascades of serine/threonine and ty-rosine protein kinases and phosphatases is a major meansby which a eukaryotic cell responds to exogenous stimuli(86). The delivery of an active phosphoprotein phospha-tase into the eukaryotic cell upon fusion may interferewith the normal signal transduction cascade of the hostcell. In addition to delivery of the mycoplasmal cell con-tent into the host cell, fusion also allows insertion ofmycoplasmal membrane components into the membraneof the eukaryotic host cell. This could alter receptorrecognition sites as well as affect the induction and ex-pression of cytokines and alter the cross-talk between thevarious cells in an infected tissue.

D. Cytopathic Effects

Contamination of a cell culture by mycoplasmas maygo undetected because mycoplasma infections do not

424 SHLOMO ROTTEM


on Septem

ber 15, 2014D

ownloaded from

produce overt turbid growth commonly associated withbacterial and fungal contamination. The morphologicalcellular changes can be minimal or unapparent. Fre-quently, the cellular changes are similar to those causedby nutritional effects such as the depletion of aminoacids, sugars, or nucleic acid precursors. These morpho-logical effects can be reversed by changing the medium orby replenishing the medium with fresh nutrients.

Mycoplasmal attachment to eukaryotic cells maysometimes lead to a pronounced cytopathic effect. At-tachment permits the mycoplasma contaminant to releasenoxious enzymatic and cytolytic metabolites directly ontothe tissue cell membrane. Some mycoplasmas selectivelycolonize defined areas of the cell culture. This results inmicrocolony formation producing microlesions and smallfoci of necrosis, e.g., M. pulmonis, or form plaques, e.g., M.gallisepticum, in an agar overlay system (5). Microcoloniza-tion suggests that mycoplasma-specific receptors are local-ized in defined areas of the cell monolayer. However, otherfermenting mycoplasmas, e.g., M. hyorhinis, attach to everycell and destroy the entire monolayer, producing a general-ized cytopathic effect. With HeLa cells infected by the inva-sive M. penetrans, the most pronounced effect was thevacuolation of the host cells (12). The vacuoles appeared tobe empty, differing from the described membrane-boundvesicles containing clusters of bacteria (57). The numberand size of the vacuoles depended on duration of infection.Because vacuolation is not obtained with M. penetrans cellfractions (12), it is unlikely that a necrotizing cytotoxin isinvolved in the generation of the cellular lesions. A possiblemechanism that leads to vacuolation may be associated withthe accumulation of organic peroxides upon invasion ofHeLa cells by M. penetrans. Indeed, when HeLa cells weregrown with the antioxidant -tocopherol, the level of accu-mulated organic peroxides was extremely low, and vacuo-lation was almost completely abolished (12).

Being unable to synthesize nucleotides, mycoplas-mas developed potent nucleases, either soluble ones se-creted into the extracellular medium or membrane-boundnucleases (7, 68, 82) apparently as a means of producingnucleic acid precursors required for metabolism. It hasbeen shown that, occasionally, secreted mycoplasmalnucleases are taken up by the host cells (76, 77). Thus itwas suggested that the cytotoxicity of M. penetrans ismediated at least in part to a secreted mycoplasmal en-donuclease that is cleaving DNA and/or RNA of the hostcells (7), and the endonuclease activity of M. bovis wasimplicated in the increased sensitivity of lymphocytic celllines to various inducers of apoptosis (99).

VI. CIRCUMVENTING THE HOST IMMUNESYSTEM

Circumvention of the host immune system is of ut-most importance to the survival of a mycoplasma within

its host. The major mechanisms that have been studied atlength are molecular mimicry and phenotypic plasticity,which ensure that the mycoplasmas are not fully or effi-ciently recognized by the hosts immune system. Molecu-lar mimicry refers to antigenic epitopes that have beenshown to be shared by different mycoplasmas and hostcells and were proposed as putative factors involved inevasion of host defense mechanisms and/or induction ofautoantibodies observed during infections with certainmycoplasmas. Mycoplasmas are also endowed with phe-notypic plasticity defined as the ability of a single geno-type to change its antigenic make-up producing more thanone alternative form of morphology, physiological state,and/or behavior in response to environmental condition.Phenotypic plasticity can be accomplished by two distinctmechanisms: a response to environmental signals or ran-dom changes of expression of single or multiple genes.The use of signal transduction pathways to sense signalsin the host environment and respond accordingly by ex-pressing gene products is necessary for survival in thehost (107, 112). The apparent scarcity in mycoplasmas ofregulatory genes functioning as sensors to environmentalstimuli and of genes encoding transcriptional factors sug-gests, but does not rule out, that adaptation of mycoplas-mas to the changing environment is not per se a responseto signals. The common way to achieve phenotypic plas-ticity in mycoplasmas is by antigenic variation, a termthat refers to the reversible high-frequency gain or loss ofsurface components that is a common survival strategyused by bacterial pathogens (62, 112). These surface com-ponents that in microorganisms include components offlagella, pili, outer membrane, or capsules are the majortargets for the host antibody response. Therefore, theability of a microorganism to rapidly change the surfaceantigenic repertoire and consequently vary the immuno-genicity of these components allows the microorganismto avoid detection or to outpace a hosts immune system.Lacking a cell wall, locomotive organelles, or pili, most ofthe variable surface components of mycoplasmas aremembrane proteins. These proteins are mature, pro-cessed prokaryotic lipoproteins anchored to the mem-brane by DAG and amide-linked fatty acids (13). A myco-plasma population may spontaneously and randomly gen-erate distinct lipoprotein populations with a variety ofantigenic phenotypes, heterotypes, that will survive thespecific host response capable of eliminating the predom-inant homotypes. Notably, the molecular switchingevents leading to the generation of these heterotypes arereversible, and the escape variants produced through ran-dom genetic variation must inherit the ability to produce,at a high frequency, a wide range of antigenic phenotypes.A considerable evolutionary dividend to the microbialpathogen of such random phenotypic switching can beachieved even before the onset of a specific immuneresponse. For example, by fine tuning of the specificities



on Septem

ber 15, 2014D

ownloaded from

of variant receptors or adhesion factors throughout thecell population, there is a better chance that a givenvariant will succeed in finding the preferred receptors onthe mosaic of different tissues displayed by the host. Itmay also provide the mycoplasma, during the course ofparasitic life, the flexibility to reach and adapt to differentniches within the host where distinctive receptors may berequired for colonization. Despite the very limited geneticinformation that mycoplasmas contain, the number ofmycoplasmal genes involved in diversifying the antigenicnature of their cell surface is unexpectedly high. Theutilization of multiple variable genes organized as genefamilies, allowing the generation of an extensive reper-toire of antigenic variants, is a common theme in patho-genic bacteria and parasites for maintaining surface vari-ability (107, 112). By oscillating at a high frequency, thesegenes allow numerous combinatorial antigenic reper-toires to be generated. Genetic mechanisms of antigenicvariation emerging from the mycoplasma studies can bebroadly divided into three categories: 1) variation by ho-mopolymeric repeats, 2) variation by chromosomal re-arrangements, and 3) variation by reiterated codingsequence domains. Comprehensive and comparativereviews on the genetic mechanisms generating antigenicvariation of surface proteins in mycoplasmas and otherbacteria and on the role of antigenic variation in bacteria-host cell interactions have been recently published (13,82, 89, 107, 111, 112)

VII. MODULATING THE IMMUNE SYSTEM

A. Modulatory Effects on Monocytesand Macrophages

It is increasingly recognized that for many bacteriainduction of cytokines is a major virulence mechanism(41, 112). The induced cytokines have a wide range ofeffects on the eukaryotic host cell and are recognized asimportant mediators of tissue pathology in infectious dis-eases. It appears that although mycoplasmas circumventphagocytosis, they interact with mononuclear and poly-morphonuclear phagocytes stimulating the synthesis ofcytokines with proinflammatory action (88, 92). Theseimmunomodulatory influences depend on both the im-mune cells and the Mycoplasma spp. involved. Macro-phage-mediated cytolysis of fibrosarcoma A9HT inducedby whole cells of M. orale was first described by Lowen-stein et al. (58). Cytolysis of the neoplastic cells wasobtained even with macrophages from the lipopolysac-charide (LPS)-unresponsive C3H/HeJ mice, suggestingthat the mechanism of activation is different from that ofLPS (53). Since then over 20 Mycoplasma spp. have beenshown to activate monocytes, macrophages, and brainastrocytes and induce secretion of the proinflammatory

cytokines tumor necrosis factor (TNF)-, interleukin(IL)-1 and IL-6, chemokines, such as IL-8, monocyte che-moattractant protein 1 (MCP-1), macrophage inflamatoryprotein 1 (MIP-1), granulocyte-monocyte colony stimu-lating factors (GM-CSFs), as well as prostaglandins andnitric oxide (82, 92). More recent observations suggestthat the mechanisms underlying macrophage activationby whole cells are in many cases identical to those em-ployed by their purified membrane lipoproteins, support-ing the notion that lipoproteins are the principle compo-nent of intact mycoplasmas activating monocytes/macro-phages and playing an important role in the inflammatoryresponse during infection (13, 41).

The potent molecules and mediators released bycells responding to mycoplasmas and mycoplasma-de-rived cell components enhance expression of major his-tocompatibility complex (MHC) class I and class II anti-gens and of costimulatory end cell adhesion molecules inleukocytes and endothelial cells, induce recruitment andextravasation of leukocytes to the site of infection andcause local tissue damage (41, 82, 90). It is interesting tonote that mycoplasmal infections are not necessarily as-sociated with a strong inflammatory response, and somemycoplasmas colonize the respiratory and urogenitaltracts with no apparent clinical symptoms. It is thereforetempting to speculate that in addition to triggering theproduction of proinflammatory cytokines, certain organ-isms have the capacity to downregulate NFB or to in-duce anti-inflammatory cytokines such as IL-4, IL-10,IL-13, or transforming growth factor-, contributing tothe complex network of synergistic and antagonistic in-fluences induced by mycoplasmas on cells of theimmune system.

B. Characterization of Modulins

The term modulin has been proposed to describecomponents and products of bacteria that have the ca-pacity to stimulate cytokine synthesis. The first and mostwidely studied modulin was the LPS of Gram-negativebacteria (41). During the past decade it has been shownthat in addition to LPS, other bacterial components,mainly those associated with the cell wall, such as pep-tidoglycan fragments, lipoteichoic acid, and mureinlipoproteins can stimulate mammalian cells to producecytokines (41). Recent attempts to identify mycoplasmalcytokine-inducing moieties have targeted membrane li-poproteins (13, 70, 90), superantigens (16, 17), and cho-line-containing phosphoglycolipids (10, 92).

C. Lipoproteins

Lipoproteins are found in the cytoplasmic membraneand in the outer membrane of many Gram-positive and

426 SHLOMO ROTTEM


on Septem

ber 15, 2014D

ownloaded from

Gram-negative bacteria. All membrane-anchored bacteriallipoproteins contain a lipoylated amino-terminal cysteinylresidue which, in some cases, is N-acylated (Fig. 5). Li-poproteins are extremely abundant in the cell membraneof mycoplasmas. In M. pneumoniae, for example, of anestimated number of 150 membrane proteins, 46 openreading frames encoding putative lipoprotein genes havebeen identified (46). Chemical analyses of mycoplasmallipoproteins have revealed that their lipoylation mecha-nism is similar to that of Gram-negative and Gram-posi-tive bacteria (13). However, in many mycoplasmas, thelipoproteins are not N-acylated (Fig. 3), nor has an N-acyltransferase gene been found in the genome (34, 45,46). The first reports on the cytokine-inducing ability ofmycoplasmal lipoproteins showed that a lipoprotein fromM. fermentans (49, 70) or M. arginini (44) is capable ofstimulating the release of proinflammatory cytokinessuch as IL-1, IL-6, and TNF- from human peripheralblood monocytes in a dose-dependent manner. Compari-son of the effects of intact lipoproteins with those ofproteinase-K-treated lipoproteins reveals that the lipoy-lated amino terminus is responsible for the immunostimu-lating properties of the lipoproteins (13, 29). However, itis not certain whether all naturally occurring mycoplas-mal membrane lipoproteins are potent macrophage acti-vators. The importance of the lipid residue has beenemphasized by the isolation and characterization of nat-urally occurring lipopeptides with macrophage-activatingpotential from two mycoplasmas. A macrophage-activat-ing lipopeptide with a molecular mass of 2 kDa (MALP-2)has been identified in the cell membrane of M. fermen-tans (73), and two lipopeptides, derived from the variablelipoproteins VlpA and VlpC, respectively, were character-ized in M. hyorhinis (72). Further information on thefunctionally important lipopeptide moieties has been ob-

tained by analyzing the cytokine-inducing potency of anumber of synthetic peptides, which are analogs of theMALP-2 of M. fermentans (35, 71). These studies clearlydemonstrated that the macrophage-activating agentscarry a fatty acid-substituted amino-terminal S-(2,3-bisacyloxypropyl) cysteinyl group, but lack the N-acyllong-chain fatty acid present in bacterial lipoproteins.This feature renders these compounds exceptionally ac-tive as macrophage stimulators in vitro (72, 73).

D. Superantigens

Superantigens (SAg) are potent immunoregulatoryproteins produced by bacteria, viruses, and mycoplasmas(32, 63, 69). These proteins are able to activate largeproportions of the peripheral T-lymphocyte population,inducing them to secrete a large panel of cytokines bothin vivo and in vitro (32, 42). The SAg are presenteddirectly to T cells in association with various class IImajor histocompatibility complex molecules on acces-sory cell surfaces, usually without the need for processing(22, 32), and are recognized predominantly by T cellsbearing specific V-chain segments of the T-cell receptorfor antigen (TCR) (42, 63). Because recognition is depen-dent on fewer restricting elements that are required fortraditional antigens, large numbers of naive T cells may beactivated. This response contributes to the marked in-flammation seen after in vivo administration of SAg,which has clear implications for disease pathogenesis(69). The superantigen of M. arthritidis (MAM) was mostthoroughly studied (16). MAM has been cloned and se-quenced, and the functional regions of the molecule weredescribed (17). There is currently no evidence that othermycoplasmas have SAg.

FIG. 5. Schematic structure of the lipoylated aminoterminus of bacterial lipoproteins. I: mycoplasmal lipopro-tein with the amino-terminal cysteinyl residue lipoylatedby a diacylglyceryl (DAG) but not N-acylated. II: lipopro-tein of Escherichia coli lipoylated by both a DAG and afatty acyl moiety. a.a., amino acid.



on Septem

ber 15, 2014D

ownloaded from

E. Membrane Lipids

Very few mycoplasmal lipids have been investigatedfor cytokine stimulating activity. MfGL-II, the major cho-line-containing phosphoglycolipid of M. fermentans (Fig.4), has been found to be associated with the secretion ofinflammatory mediators by human monocytes (92) and byrat cells of the central nervous system (10). Stimulation ofprimary rat astrocytes by MfGL-II caused activation ofprotein kinase C, secretion of nitric oxide and prostaga-landine E2, in a dose-dependent manner (10). Deacylationof MfGL-II or treatment with monoclonal anti-phospho-choline antibodies pronouncedly reduced the stimulatoryactivity, suggesting that the fatty acyl residues are es-sential for activity and that the terminal phosphocho-line moiety plays an important role in MfGL-IIsstimulation (10).

F. Signaling Pathways by Modulins

The monocyte surface molecule CD14 is part of thereceptor complex for several microbial products. This 50-to 55-kDa glycoprotein is expressed predominantly onmyeloid cells, and in addition to being a high-affinityreceptor for LPS, it has been implicated in the response topeptidoglycan and other bacterial cell wall components(41). The failure of anti-CD14 monoclonal antibodies toblock cytokine induction by M. fermentans lipoproteins(49, 80) indicates that cytokine induction by M. fermen-tans lipoproteins does not proceed through CD14 (49).This was further supported by the finding that cytokinesynthesis by a human monocyte cell line induced by M.fermentans lipoproteins was not affected by pretreat-ment of the cells with vitamin D3, known to increaseCD14 expression on the cell surface (80). In contrast toCD14, the toll-like receptors (TLRs) contain all of thecharacteristics that one would expect from a true patternrecognition receptor, including the presence of a truesignal-transducing intracellular domain (35). Althoughonly recently described, the list of putative ligands forTLR2 and TLR4 is already impressively large. Both TLR2and TLR4 have been reported to function as LPS sig-nal transducers (35). However, lipoproteins/lipopeptidesfrom M. fermentans activated cells expressing TLR2 butnot those expressing TLR4, suggesting that TLR2 is acentral pattern recognition receptor in host response toM. fermentans invasion (35).

The downstream signaling events that follow theTLR-mediated activation by mycoplasmal lipoprotreinsand lead to cytokine synthesis seem to be similar to theintracellular events induced by LPS. The TLRs have acytoplasmic domain that is homologous to the IL-1 recep-tor. Thus it is likely that TLR2 activates the NF-B path-way, and perhaps other proinflammatory pathways as

well, via their interactions with IL-1 receptor signalinggenes (55). Indeed, M. fermentans lipoproteins or thelipoprotein-derived MALP-2 lipopeptide activate NF-Band AP-1, the two transcription factors playing a centralrole in the induction of proinflammatory cytokines, aswell as the mitogen-activated protein kinase family mem-bers including extracellular signal-regulated kinases 1 and2, c-Jun amino-terminal kinase, and p38 (35, 80).

G. Mitogenic Activity

Numerous reports describe the mitogenic stimula-tion of lymphocytes by mycoplasmas in a nonspecificpolyclonal manner both in vitro and in vivo (90), yet manyof these mitogenic effects were not immunologically wellcharacterized. These effects may be indirect and dueprimarily to cytokine release by macrophages and mono-cytes. Different Mycoplasma spp. have been shown tostimulate B cells, T cells, or both nonspecifically. Antibod-ies of different specificities, with no affinity for mycoplas-mal antigens, are generated both in vitro and in vivo,following exposure of lymphoid cells to different myco-plasmas possessing B cell mitogens (82, 88, 90). It isworth noting that there is a clear distinction betweenmycoplasma-induced DNA synthesis and differentiationof activated B cells into antibody-producing cells. Thus,on the one hand, stimulation of DNA synthesis is notalways a prerequisite for the induction of polyclonalimmunoglobulin secretion by B cells exposed to myco-plasmas. On the other hand, stimulation of mitosis bymycoplasmas does not necessarily trigger subsequentdifferentiation of the dividing lymphocytes into plasma cells(92). Various Mycoplasma spp. are potent stimulators ofT-cell-derived cytokines such as IL-2, interferon-, or IL-4.They exert multiple amplifying effects on phagocytes andlymphocytes and affect the balance between Th1 and Th2populations of CD4 T cells, thereby influencing the direc-tion of the subsequent effector phases of the immune re-sponse, and augmenting natural killer cell activity (82, 88).

Our present knowledge of the biochemical nature ofmycoplasma mitogens is very limited. It appears thatmycoplasmal B-cell mitogens differ from bacterial LPS intheir ability to activate lymphocytes from C3H/HeJ micethat are poor responders to LPS and their inducing poten-tial is unaffected by polymyxin B (82). Furthermore, thereare indications that a single mycoplasma may carry morethan one cell constituent interacting with lymphocytes.Thus the AIDS-associated M. penetrans mitogens appearto include a glycolipid as well as a major membranelipoprotein (88).

H. Oncogenic Activity

Mycoplasmas can grow in close interaction withmammalian cells, often silently for a long period of time.

428 SHLOMO ROTTEM


on Septem

ber 15, 2014D

ownloaded from

However, prolonged interactions with mycoplasmas withseemingly low virulence could, through a gradual andprogressive course, induce chromosomal instability aswell as malignant transformation, promoting tumorousgrowth of mammalian cells (30, 106, 116). This mycoplasma-mediated transformation of cells has a long latency anddemonstrates distinct multistage progression (106). Over-expression of H-ras and c-myc oncogenes was found tobe closely associated with both the initial reversible andthe subsequent irreversible states of the mycoplasma-mediated transformation of cells (116).

VIII. CONCLUSIONS

Over the last decade, intensive studies have beencarried out to understand the strategy employed by amycoplasma pathogen to interact with host cells and toavoid or subvert host protective measures. The identifi-cation of mycoplasmal membrane components that par-ticipate in the adhesion process will shape and directfuture efforts toward understanding the molecular orga-nization of adhesion-associated proteins and to furtheridentify mammalian membrane receptors for mycoplas-mas and mycoplasma products. The finding that somemycoplasmas can reside intracellularly opens up newhorizons to study the role of mycoplasma and host sur-face molecules in invasion. Although the ability of inter-nalized mycoplasmas to multiply within the host cell re-mains to be convincingly demonstrated, the reports ofinvasive mycoplasmas offer new insights into the poten-tial virulence strategies employed by mycoplasmas. Inva-sion of nonphagocytic host cells, if only for a short periodof time, may provide mycoplasmas with the ability tocross mucosal barrier and gain access to internal tissues.The intracellular organisms are also resistant to the hostdefense mechanisms as well as to antibiotic treatmentand may account for the difficulty to eradicate mycoplas-mas from cell cultures. The fusion of mycoplasmas witheukaryotic host cells raises exciting questions on howmicroinjection of mycoplasmal components into eukary-otic cells affects host cells. The fusion process as well asthe invasion of host cells by mycoplasmas brings up anemerging theme in mycoplasma research: the subversionby mycoplasmas of host cell functions mainly in signal-transduction pathways and cytoskeleton organization.Nevertheless, as the invasion and fusion studies rely ex-clusively on in vitro experiments done with culturedmammalian cells rather than organ cultures of the actualtarget tissue or intact animals, the interpretation of theresults should be cautious. The discovery of genetic sys-tems that enable the mycoplasma cell to rapidly changeits antigenic characteristics has been one of the majordevelopments in mycoplasma research over the past de-cade. It is now clear that these minute wall-less microor-

ganisms possess an impressive capability of maintaining asurface architecture that is antigenically and functionallyversatile. These variable surface antigens undoubtedlycontribute to the capability of the mycoplasmas to adaptto a large range of habitats and to cause diseases, whichare often chronic in nature. Although mycoplasmas cir-cumvent phagocytosis, they interact with mononuclearand polymorphonuclear phagocytes, suppressing or stim-ulating them by a combination of direct and indirectcytokine-mediated effects. It appears that for many my-coplasmas, induction of cytokines is a major virulencemechanism. The induced cytokines have a wide range ofeffects on the eukaryotic host cell and are recognizedas important mediators of tissue pathology in infectiousdiseases.

Address for reprint requests and other correspondence: S.Rottem, Dept. of Membrane and Ultrastructure Research, TheHebrew University-Hadassah Medical School, PO Box 12272,Jerusalem 91120, Israel (E-mail: [email protected]).

REFERENCES

1. ALMAGOR M, KAHANE I, GILON C, AND YATZIV S. Protective effects ofthe glutathione redox cycle and vitamin E on cultured fibroblastsinfected by Mycoplasma pneumoniae. Infect Immun 52: 240244,1986.

2. ANDREEV J, BOROVSKY Z, ROSENSHINE I, AND ROTTEM S. Invasion ofHeLa cells by Mycoplasma penetrans and the induction of tyrosinephosphorylation of a 145 kDa host cell protein. FEMS MicrobiolLett 132: 189194, 1995.

3. BALISH MF, HAHN TW, POPHAM PL, AND KRAUSE DC. Stability ofMycoplasma pneumoniae cytadherence-accessory protein HMW1correlates with its association with the Triton shell. J Bacteriol 183:36803688, 2001.

4. BALISH MF AND KRAUSE DC. Cytadherence and the cytoskeleton. In:Molecular Biology and Pathogenicity of Mycoplasmas, edited byRazin S and Herrmann R. New York: Plenum, 2002, p. 491518.

5. BARILE MF AND ROTTEM S. Mycoplasmas in cell cultures. In: RapidDiagnosis of Mycoplasmas, edited by Kahane I and Adoni A. NewYork: Plenum, 1993, p. 155193.

6. BASEMAN JB AND TULLY JG. Mycoplasmas: sophisticated, reemerg-ing, and burdened by their notoriety. Emerg Infect Dis 3: 2132,1997.

7. BENDJENNAT M, BLANCHARD A, LOUTFI M, MONTAGNIER L, AND BAH-RAOUI E. Role of Mycoplasma penetrans endonuclase P40 as apotential pathogenic determinant. Infect Immun 67: 44564462,1999.

8. BEN-MENACHEM G, BYSTROM T, RECHNITZER H, ROTTEM S, RILFORS L,AND LINDBLOM G. The physico-chemical characteristics of the phos-phocholine containing glycoglycerolipid MfGL-II govern the perme-ability properties of Mycoplasma fermentans. Eur J Biochem 268:36943701, 2001.

9. BEN-MENACHEM G, MOUSA A, BRENNER T, PINTO F, ZAHRINGER U, ANDROTTEM S. Choline-deficiency induced by Mycoplasma fermentansenhances apoptosis of rat astrocytes. FEMS Microbiol Lett 201:157162, 2001.

10. BEN-MENACHEM G, ROTTEM S, TARSHIS M, BARASH V, AND BRENNER T.Mycoplasma fermentans glycolipid (MfGL-II) triggers inflamma-tory response in rat astrocytes. Brain Res 803: 3438, 1998.

11. BEN-MENACHEM G, ZAHRINGER U, AND ROTTEM S. The phosphocholinemotif in membranes of Mycoplasma fermentans strains. FEMSMicrobiol Lett 199: 137141, 2001.

12. BOROVSKY Z, TARSHIS M, ZHANG P, AND ROTTEM S. Mycoplasma pene-trans invasion of HeLa cells induces protein kinase C activationand vacuolation in the host cells. J Med Microbiol 47: 915922,1998.



on Septem

ber 15, 2014D

ownloaded from

13. CHAMBAUD I, WROBLEWSKI H, AND BLANCHARD A. Interactions betweenmycoplasma lipoproteins and the host immune system. TrendsMicrobiol 7: 493499, 1999.

14. CHAUSEE MS, COLE R, AND VAN PUTTEN JPM. Streptococcal erythro-genic toxin B abrogates fibronectin dependent internalization ofStreptococcus pyogenes by cutured mammalian cells. Infect Im-mun 68: 32263232, 2000.

15. CHIBA H, PATTANAJITVILAI S, EVANS AJ, HARBECK RA, AND VOELKER DR.Human surfactant protein D binds Mycoplasma pneumoniae byhigh affinity interactions with lipids. J Biol Chem 277: 2037920385, 2002.

16. COLE BC. The immunobiology of Mycoplasma arthritidis and itssuperantigen MAM. Curr Top Microbiol Immunol 174: 107119,1991.

17. COLE BC, KNUDTSON KL, OLIPHANT A, SAWITZKE AD, POLE A, MANOHARM, BENSON LS, AHMED E, AND ATKIN CL. The sequence of the Myco-plasma arthritidis superantigen, MAM: identification of functionaldomains and comparison with microbial superantigens and plantlectin mitogens. J Exp Med 183: 11051110, 1996.

18. CULLIS PR AND HOPE MJ. Lipid polymorphism, lipid asymmetry andmembrane fusion. In: Molecular Mechanisms of Membrane Fusionedited by Ohki S, Doyle D, Flanagan TD, Hui SW, and Mayhew E.New York: Plenum, 1988, p. 3751.

19. DALLO SF, CHAVOYA A, AND BASEMAN JB. Characterization of the genefor a 30-kDa adhesin-related protein of Mycoplasma pneumoniae.Infect Immun 58: 41634165, 1990.

20. DALLO SF, LAZZELL AL, CHAVOYA A, REDDY SP, AND BASEMAN JB.Biofunctional domains of the Mycoplasma pneumoniae P30 adhe-sin. Infect Immun 64: 25952601, 1996.

21. DEBEY MC AND ROSS RF. Ciliostasis and loss of cilia induced byMycoplasma hyopneumoniae in porcine tracheal organ cultures.Infect Immun 62: 53125318, 1994.

22. DELLABONA P, PECCOUD J, KAPPLER J, MARRACK P, BENOIST C, ANDMATHIS D. Superantigens interact with MHC class II moleculesoutside of the antigen groove. Cell 62: 11151121, 1990.

23. DEUTSCH J, SALMAN M, AND ROTTEM S. An unusual polar lipid fromthe cell membrane of Mycoplasma fermentans. Eur J Biochem227: 897902, 1995.

24. DIMITROV DS, FRANZOSO G, SALMAN M, BLUMENTHAL R, TARSHIS M,BARILE MF, AND ROTTEM S. Mycoplasma fermentans, incognitusstrain, cells are able to fuse with T-lymphocytes. Clin Infect Dis 17:S305S308, 1993.

25. DIRKSEN LB, PROFT T, HILBERT H, PLAGENS H, HERRMANN R, ANDKRAUSE DC. Nucleotide sequence analysis and characteriztion ofthe hmw gene cluster of Mycoplasma pneumoniae. Gene 171:1925, 1996.

26. DUENSING TD, WING JS, AND VAN PUTTEN JPM. Sulfated polysaccha-ride-directed recruitment of mammalian host proteins: a novelstrategy in microbial pathogenesis. Infect Immun 67: 44634468,1999.

27. DZIEWANOWSKA KJ, PLATT M, DEOBALD CFK, BALES W, TRUMBLE WR,AND BOHACH GA. Fibronectin binding protein and host cell tyrosinekinase are required for internalization of Staphylococcus aureus byepithelial cells. Infect Immun 67: 46734678, 1999.

28. ELSINGHORST EA. Measurement of invasion by gentamicin resis-tance. Methods Enzymol 236: 405420, 1994.

29. FENG SH AND LO SC. Lipid extract of Mycoplasma penetrans pro-teinase K-digested lipid associated membrane proteins rapidly ac-tivates NF-kappa B and activator protein 1. Infect Immun 67:29512956, 1999.

30. FENG SH, TSAM S, RODRIGUEZ J, AND LO SC. Mycoplasmal infectionsprevent apoptosis and induce malignant transformation of interleu-kin-3-dependent 32D hematopoietic cells. Mol Cell Biol 19: 79958002, 1999.

31. FINLAY BB, RUSCHKOWSKI S, AND DEDHAR S. Cytoskeletal rearrange-ments accompanying Salmonella entry into epithelial cells. J CellSci 99: 283296, 1991.

32. FLEISCHER B. Stimulation of human T cells by microbial superan-tigens. Immunol Res 10: 349355, 1991.

33. FRANZOSO G, DIMITROV DS, BLUMENTHAL R, BARILE MF, AND ROTTEM S.Fusion of M fermentans, strain incognitus, with T lymphocytes.FEBS Lett 303: 251254, 1992.

34. FRASER CM, GOCAYNE JD, WHITE O, ADAMS MD, CLAYTON RA, FLEISCH-

MANN RD, BULT CJ, KERLAVAGE AR, SUTTON G, KELLY JM, FRITCHMANJL, WEIDMAN JF, SMALL KV, SANDUSKY M, FUHRMANN J, NGUYEN D,UTTERBACK TR, SAUDEK DM, PHILLIPS CA, MERRICK JM, TOMB JF,DOUGHERTY BA, BOTT KF, HU PC, LUCIER TS, PETTERSON SN, SMITHHO, HUTCHISON CA III, AND VENTER JC. The minimal gene comple-ment of Mycoplasma genitalium. Science 270: 397403, 1995.

35. GARCIA J, LEMERCIER B, ROMAN-ROMAN S, AND RWADI G. A Myoplasmafermentans-derived synthetic lipopeptide induces AP-1 and NF-Bactivity and cytokine secretion in macrophages via the activation ofmitogen-activated protein kinase pathways. J Biol Chem 273:3439134398, 1998.

36. GEARY SJ AND GABRIDGE MG. Characterization of a human lungfibroblast receptor site for Mycoplasma pneumoniae. Isr J Med Sci23: 462468, 1987.

37. GEARY SJ, GABRIDGE MG, INTRES R, DRAPER DL, AND GLADD MF.Identification of mycoplasma binding proteins utilizing a 100 kilo-dalton lung fibroblast receptor. J Receptor Res 9: 465478, 1990.

38. GIRON JA, LANGE M, AND BASEMAN JB. Adherence, fibronectin bind-ing, and induction of cytoskeleton reorganization in cultured hu-man cells by Mycoplasma penetrans. Infect Immun 64: 197208,1996.

39. GUZMAN CA, ROHDE M, AND TIMMIS KN. Mechanisms involved inuptake of Bordetella bronchiseptica by mouse dendritic cells. In-fect Immun 62: 55385544, 1994.

40. HAHN TW, WILLBY MJ, AND KRAUSE DC. Hmw1 is required for cytad-hesin P1 trafficking to the attachment organelle in Mycoplasmapneumoniae. J Bacteriol 180: 12701276, 1998.

41. HENDERSON B, POOLE S, AND WILSON M. Bacterial modulins: a novelclass of virulence factors which cause host tissue pathology byinducing cytokine synthesis. Microbiol Rev 60: 316341, 1996.

42. HERMAN A, KAPPLER JW, MARRACK P, AND PULLEN AM. Superantigens:mechanism of T-cell stimulation and role in immune responses.Annu Rev Immunol 9: 745772, 1991.

43. HERRMANN R, GOHLMANN HWH, REGULA JT, WEINER J III, PIRKL E,UEBERLE B, AND FRANK R. Mycoplasmas, the smallest known bacte-ria. In: Microbial Evolution and Infection, edited by Gobel UB andRuf BR. Reinbeck, Germany: Einhorn-Presse Verlag, 1999, p. 7179.

44. HERBELIN A, RUUTH E, DELORME D, MICHEL-HERBELIN C, AND PRAZ F.Mycoplasma arginini TUH-14 membrane lipoproteins induce pro-duction of interleukin-1, interleukin-6, and tumor necrosis factoralpha by human monocytes. Infect Immun 62: 46904694, 1994.

45. HIMMELREICH RH, HILBERT H, PLAGENS H, PIRKI E, LI BC, AND HERR-MANN R. Complete sequence analysis of the genome of the bacte-rium Mycoplasma pneumoniae. Nucleic Acids Res 24: 44204449,1996.

46. HIMMELREICH RH, PLAGENS H, HILBERT H, REINER B, AND HERRMAN R.Comparative analysis of the genomes of the bacteria Mycoplasmapneumoniae and Mycoplasma genitalium. Nucleic Acids Res 25:701712, 1997.

47. INAMINE JM, DENNY TP, LOECHEL S, SCHAPER U, HUANG CH, BOTT KF,AND HU PC. Nucleotide sequence of the P1 attachment-protein geneof Mycoplasma pneumoniae. Gene 64: 217229, 1988.

48. JENSEN JG, BLOM J, AND LIND K. Intracellular location of Myco-plasma genitalium in cultured Vero cells as demonstrated byelectron microscopy. Int J Pathol 75: 9198, 1993.

49. KOSTYAL DA, BUTLER GH, AND BEEZHOLD DH. A 48-kilodalton Myco-plasma fermentans membrane protein induces cytokine secretionby human monocytes. Infect Immun 62: 37933800, 1994.

50. KRAUSE DC. Mycoplasma pneumoniae cytadherence: unravellingthe tie that binds. Mol Microbiol 20: 247253, 1996.

51. KRAUSE DC, LEITH DK, WILSON RM, AND BASEMAN JB. Identification ofMycoplasma pneumoniae proteins associated with hemadsorptionand virulence. Infect Immun 35: 809817, 1982.

52. KRIVAN HC, OLSON LD, BARILE MF, GINSBURG V, AND ROBERTS DD.Adhesion of Mycoplasma pneumoniae to sulfated glycolipids andinhibition by dextran sulfate. J Biol Chem 264: 92839288, 1989.

53. LAYH-SCHMITT G AND HARKENTHAL M. The 40- and 90-kDa membraneproteins (ORF6 gene product) of Mycoplasma pneumoniae areresponsible for the tip structure formation and P1 (adhesin) asso-ciation with the Triton shell. FEMS Microbiol Lett 174: 143149,1999.

54. LAYH-SCHMITT G AND HERRMANN R. Spatial arrangement of gene

430 SHLOMO ROTTEM


on Septem

ber 15, 2014D

ownloaded from

products of the P1 operon in the membrane of Mycoplasma pneu-moniae. Infect Immun 62: 974979, 1994.

55. LIEN E, SELATI TJ, YOSHIMURA A, FLO TH, RAWADI G, FINBERG RW,CARROLL JD, ESPEVIK E, INGALIS RR, RADOLF JD, AND GOLENBOCK DT.Toll-like receptor 2 functions as a pattern recognition receptor fordiverse bacterial products. J Biol Chem 274: 3341933425, 1999.

56. LO SC. Mycoplasmas in AIDS. In: Mycoplasmas: Molecular Biologyand Pathogenesis, edited by Maniloff J, McElhaney RN, Finch LR,and Baseman JB. Washington, DC: Am Soc Microbiol, 1992, p.525548.

57. LO SC, HAYES MM, KOTANI H, PIERCE PF, WEAR DJ, NEWTON PB III,TULLY JG, AND SHIH JW. Adhesion onto and invasion into mamma-lian cells by Mycoplasma penetrans: a newly isolated mycoplasmafrom patients with AIDS. Mod Pathol 6: 276280, 1993.

58. LOEWENSTEIN J, ROTTEM S, AND GALLILY R. Induction of macrophage-mediated cytolysis of neoplastic cells by mycoplasmas. Cell Immu-nol 77: 290297, 1983.

59. LOVELESS RW AND FEIZI T. Sialo-oligosaccharide receptors for My-coplasma pneumoniae and related oligosaccharides of poly-N-acetyl lactosamine series are polarized at the cilia and apicalmi-crovillar domains of the ciliated cells in human bronchial epithe-lium. Infect Immun 57: 12851289, 1989.

60. LUCY JA. The fusion of biological membranes. Nature 227: 814817,1970.

61. MANILOFF J. The minimal gene genome: on being the right size.Proc Natl Acad Sci USA 93: 1000410006, 1996.

62. MARKHAM PF, GLEW MD, SYKES JE, BOWDEN TR, POLLOCKS TD,BROWNING GF, WHITHEAR KG, AND WALKER ID. The organization ofthe multigene family which encodes the major cell surface protein,pMGA, of Mycoplasma gallisepticum. FEBS Lett 352: 347352,1994.

63. MARRACK P AND KAPPLER J. The staphylococcal enterotoxins andtheir relatives. Science 248: 705711, 1990.

64. MARSHALL AJ, MILES RJ, AND RICHARDS L. The phagocytosis of my-coplasmas. J Med Microbiol 43: 239250, 1995.

65. MATSUDA K, KASAMA T, ISHIZUKA I, HANDA S, YAMAMOTO N, AND TAKI T.Structure of a novel phosphocholine-containing glycoglycerolipidsfrom Mycoplasma fermentans. J Biol Chem 269: 3312333128,1994.

66. MATSUDA K, LI JL, HARASAWA R, AND YAMAMOTO N. Phosphocholine-containing glycoglycerolipids (GGPL-I and GPL-III) are species-specific major immunodeterminants of Mycoplasma fermentans.Biochim Biophys Acta 1349: 112, 1997.

67. MCGARRITY GJ, KOTANI H, AND BUTLER GH. Mycoplasmas and tissueculture cells. In: Mycoplasmas: Molecular Biology and Pathogen-esis, edited by Maniloff J, McElhaney RN, Finch LR, and BasemanJB. Washington, DC: Am Soc Microbiol, 1992, p. 445454.

68. MINION FC, JARVILL-TAYLOR KJ, BILLINGS DE, AND TIGGES E. Mem-brane-associated nuclease activities in mycoplasmas. J Bacteriol175: 78427847, 1993.

69. MU HH, SAWITZKE AD, AND COLE BC. Modulation of cytokine profilesby the mycoplasma superantigen Mycoplasma arthritidis mitogenparallels susceptibility to arthritis induced by M. arthritidis. InfectImmun 68: 11421149, 2000.

70. MUHLRADT PF AND FRISCH M. Purification and partial biochemicalcharacterization of a Mycoplasma fermentans-derived substancethat activates macrophages to release nitric oxide, tumor necrosisfactor, and interleukin-6. Infect Immun 62: 38013807, 1994.

71. MUHLRADT PF, KIESS M, MEYER H, SUSSMUTH R, AND JUNG G. Isolation,structure elucidation, and synthesis of a macrophage stimulatorylipopeptide from Mycoplasma fermentans acting at picomolar con-centration. J Exp Med 185: 19511958, 1997.

72. MUHLRADT PF, KIESS M, MEYER H, SUSSMUTH R, AND JUNG G. Structureand specific activity of macrophage-stimulating lipopeptides fromMycoplasma hyorhinis. Infect Immun 66: 48044810, 1998.

73. MUHLRADT PF, MEYER H, AND JANSEN R. Identification of S-(2,3-dihydroxypropyl) cystein in a macrophage-activating lipopeptidefrom Mycoplasma fermentans. Biochemistry 35: 77817786, 1996.

74. MUSHEIGIAN A AND KOONIN EV. A minimal gene set for cellular lifederived by comparison of complete bacterial genomes. Proc NatlAcad Sci USA 9: 1026810273, 1996.

75. OELSCHLAEGER TA AND KOPECKO DJ. Microtubule dependent invasionpathways to bacteria. In: Bacterial Invasion Into Eukaryotic Cells.

Subcellular Biochemistry, edited by Oelschlaeger TA and HackerJ. New York: Plenum, 2000, vol. 33, p. 319.

76. PADDENBERG R, WEBER A, WULF S, AND MANNHERZ HG. Mycoplasmanucleases able to induce internucleosomal DNA degradation incultured cells possess many characteristics of eukaryotic apoptoticnucleases. Cell Death Differ 5: 517528, 1998.

77. PADDENBERG R, WULF S, WEBER A, HEIMANN P, BECK LA, ANDMANNHERZ HG. Internucleosomal DNA fragmentation in culturedcells under conditions reported to induce apoptosis may be causedby mycoplasma endonucleases. Eur J Cell Biol 71: 105119, 1996.

78. POLLACK JD, WILLIAMS MV, AND MCELHANEY RN. The comparativemetabolism of the mollicutes (mycoplasmas): the utility for taxo-nomic classification and the relationship of putative gene annota-tion and phylogeny to enzymatic function. Crit Rev Microbiol 23:269354, 1997.

79. POPHAM PL, HAHN TW, KREBES K, AND KRAUSE D. Loss of HMW1 andHMW3 in noncytadhering mutants of Mycoplasma pneumoniaeoccurs posttranslationally. Proc Natl Acad Sci USA 94: 1397913984, 1997.

80. RAWADI G, GARCIA J, LEMERCIER B, AND ROMAN-ROMAN S. Signaltransduction pathways involved in the activation of NF-B, AP-1,and c-fos by Mycoplasma fermentans membrane lipoproteins inmacrophages. J Immunol 162: 21932203, 1999.

81. RAZIN S AND JACOBS E. Mycoplasma adhesion. J Gen Microbiol 138:407422, 1992.

82. RAZIN S, YOGEV D, AND NAOT Y. Molecular biology and pathogenicityof mycoplasmas. Microbiol Rev 63: 10941156, 1998.

83. RING A AND TUOMANEN E. Host cell invasion by Streptococcus pneu-moniae. In: Bacterial Invasion Into Eukaryotic Cells. SubcellularBiochemistry, edited by Oelschlaeger TA and Hacker J. New York:Plenum, 2000, vol. 33, p. 125135.

84. ROBERTS DD, OLSON LD, BARILE MF, GINSBURG V, AND KRIVAN HC.Sialic acid-dependent adhesion of Mycoplasma pneumoniae topurified glycoproteins. J Biol Chem 264: 92899293, 1989.

85. ROMERO-ARROYO CE, JORDAN J, PEACOCK SJ, WILLBY MJ, FARMER MA,AND KRAUSE DC. Mycoplasma pneumoniae protein P30 is requiredfor cytadherence and associated with proper cell development. JBacteriol 181: 10791087, 1999.

86. ROSENSHINE I AND FINLAY BB. Exploitation of host signal transduc-tion pathways and cytoskeletal functions by invasive bacteria.Bioessays 15: 1724, 1993.

87. ROTTEM S AND BARILE MF. Beware of mycoplasmas. Trends Bio-technol 11: 143151, 1993.

88. ROTTEM S AND NAOT Y. Subversion and exploitation of host cells bymycoplasmas. Trends Microbiol 6: 436440, 1998.

89. ROTTEM S AND YOGEV D. Mycoplasma interaction with host eukary-otic cells. In: Bacterial Invasion Into Eukaryotic Cells. SubcellularBiochemistry, edited by Oelschlaeger TA and Hacker J. New York:Plenum, 2000, vol. 33, p. 199228,.

90. RUUTH E AND PRAZ F. Interactions between mycoplasmas and theimmune system. Immunol Rev 112: 133160, 1989.

91. SALMAN M, BOROVSKY Z, AND ROTTEM S. Mycoplasma penetransinvasion of Molt-3 lymphocytes induces changes in the lipid com-position of host cells. Microbiology 144: 34473454, 1998.

92. SALMAN M, DEUTSCH J, TARSHIS M, NAOT Y, AND ROTTEM S. Membranelipids of Mycoplasma fermentans. FEMS Microbiol Lett 123: 255260, 1994.

93. SALMAN M AND ROTTEM S. The cell membrane of Mycoplasma pene-trans: lipid composition and phospholipase A1 activity. BiochimBiophys Acta 1235: 369377, 1995.

94. SHAW JH AND FALKOW S. Model for invasion of human tissue culturecells by Neisseria gonorrhoeae. Infect Immun 56: 16251632, 1988.

95. SHIBATA KI, MAMORU N, YOSHIHIKO S, AND TSUGUO W. Acid phospha-tase purified from Mycoplasma fermentans has protein tyrosinephosphatase-like activity. Infect Immun 62: 313315, 1994.

96. SHIBATA KI, SASAKI T, AND WATANABE T. AIDS-associated mycoplas-mas possess phospholipases C in the membrane. Infect Immun 63:41744177, 1995.

97. SIEGEL DP. Energetics of intermediates in membrane fusion: com-parison of stalk and inverted micellar intermediate structures.Biophys J 76: 291313, 1999.

98. SODEINDE OA, SUBRAHMANYAM YVBK, STARK K, QUAN T, BAO Y, AND



on Septem

ber 15, 2014D

ownloaded from

GOGUEN JD. A surface protease and the invasive character ofplague. Science 258: 10041007, 1992.

99. SOKOLOVA IA, VAUGHAN AT, AND KHODAREV NN. Mycoplasma infectioncan sensitize host cells to apoptosis through contribution of apoptotic-like endonuclease(s). Immunol Cell Biol 76: 526534, 1998.

100. STADTLANDER CT, WATSON HL, SIMECKA JW, AND CASSELL GH. Cyto-pathogenicity of Mycoplasma fermentans (including strain incog-nitos). Clin Infect Dis 17: 289301, 1993.

101. STEVENS MK AND KRAUSE DC. Localization of the Mycoplasma pneu-moniae cytadherence-accessory proteins HMW1 and HMW4 in thecytoskeleton-like Triton shell. J Bacteriol 173: 10411050, 1991.

102. STEVENS MK AND KRAUSE DC. Mycoplasma pneumoniae cytadher-ence phase-variable protein HMW3 is a component of the attach-ment organelle. J Bacteriol 174: 42654274, 1992.

103. SU CJ, CHAVOYA A, AND BASEMAN JB. Spontaneous mutation resultsin loss of the cytadhesin (P1) of Mycoplasma pneumoniae. InfectImmun 57: 32373239, 1989.

104. TARSHIS M, SALMAN M, AND ROTTEM S. Cholesterol is required for thefusion of single unilamellar vesicles with M. capricolum. BiophysJ 64: 709715, 1993.

105. TAYLOR-ROBINSON D, DAVIES HA, SARATHCHANDRA P, AND FURR PM.Intracellular location of mycoplasmas in cultured cells demon-strated by immunocytochemistry and electron microscopy. Int JExp Pathol 72: 705714, 1991.

106. TSAI S, WEAR DJ, SHIH JW, AND LO SC. Mycoplasma and oncogenesispersistent infection and multistage malignant transformation. ProcNatl Acad Sci USA 92: 1019710201, 1995.

107. WASSENAAR TM AND GAASTRA W. Bacterial virulence: can we drawthe line? FEMS Microbiol Lett 201: 17, 2001.

108. WAGNER F, ROTTEM S, HELD HD, UHLIG S, AND ZAHRINGER U. Etherlipids in the cell membrane of Mycoplasma fermentans. Eur J Bio-chem 267: 62766286, 2000.

109. WILLBY MJ AND KRAUSE DC. Characterization of a Mycoplasmapneumoniae hmw3 mutant: implications for attachment organelleassembly. J Bacteriol 184: 30613068, 2002.

110. WINNER F, ROSENGARTEN R, AND CITTI C. In vitro cell invasion ofMycoplasma gallisepticum. Infect Immun 68: 42384244, 2000.

111. WISE KS. Adaptive surface variation in mycoplasmas. Trends Mi-crobiol 1: 59163, 1993.

112. WREN BW. Microbial genome analysis: insights into virulence, hostadaptation and evolution. Nat Rev Genet 1: 3039, 2000.

113. YAVLOVICH A, HIGAZI AR, AND ROTTEM S. Plasminogen binding andactivation by Mycoplasma fermentans. Infect Immun 69: 19771982, 2001.

114. ZAHRINGER U, WAGNER F, RIETSCHEL ETH, BEN-MENACHEM G, DEUTSCHJ, AND ROTTEM S. Primary structure of a new phosphocholine-containing glycoglycerolipid of Mycoplasma fermentans. J BiolChem 272: 2626226270, 1997.

115. ZEISEL SH AND BLUSZTAJN JK. Choline and human nutrition. AnnuRev Nutr 14: 269296, 1994.

116. ZHANG B, SHIH JW, WEAR DJ, TSAI S, AND LO SC. High-level expres-sion of H-ras and c-myc onconogenes in mycoplasma-mediatedmalignant cell tranformation. Proc Soc Exp Biol Med 214: 359366,1997.

117. ZUCKER-FRANKLIN D, DAVIDSON M, AND THOMAS L. The interaction ofmycoplasmas with mammalian cells, HeLa cells, neutrophiles, andeosinophils. J Exp Med 124: 521532, 1966.

432 SHLOMO ROTTEM


on Septem

ber 15, 2014D

ownloaded from

doi:10.1152/physrev.00030.2002 83:417-432, 2003.Physiol RevShlomo RottemInteraction of Mycoplasmas With Host Cells

You might find this additional info useful...

108 articles, 60 of which can be accessed free at:This article cites /content/83/2/417.full.html#ref-list-1

36 other HighWire hosted articles, the first 5 are:This article has been cited by

[PDF] [Full Text] [Abstract], October , 2013; 81 (10): 3742-3749.Infect. Immun.

Hagai Rechnitzer, Shlomo Rottem and Richard HerrmannM129Reconstitution of an Active Arginine Deiminase Pathway in Mycoplasma pneumoniae

[PDF] [Full Text] [Abstract], October , 2013; 41 (5): 1655-1670.Journal of International Medical Research

Min Fu, Li Hui Chen, Guangtao Xia and Yuanchao Zhangarthritis synovial fibroblasts

lipid-associated membrane proteins on rheumatoidUreaplasma urealyticumEffects of

[PDF] [Full Text] [Abstract], February 7, 2014; 343 (6171): 656-661.Science

LernerA. Kyle, Richard B. Pyles, John I. Glass, Andrew B. Ward, Ian A. Wilson and Richard A. Keehwan Kwon, Marta Chesi, Vaughn V. Smider, Daniel R. Salomon, Diane F. Jelinek, RobertMacLeod, Adam Mark, Sherry Niessen, Helen J. Kim, Leopold Kong, Nacyra Assad-Garcia, Rajesh K. Grover, Xueyong Zhu, Travis Nieusma, Teresa Jones, Isabel Boero, Amanda S.Antigen-Antibody UnionA Structurally Distinct Human Mycoplasma Protein that Generically Blocks

[PDF] [Full Text] [Abstract], May , 2014; 52 (5): 1622-1628.J. Clin. Microbiol.

Jonathan D. Kornspan, Shlomo Rottem and Ran Nir-PazPhenomenon) of Mycoplasma Species with Staphylococcus aureus Beta-HemolysisCardiolipin Synthetase Is Involved in Antagonistic Interaction (Reverse CAMP

[PDF] [Full Text] [Abstract] 2014; 68 (3): 281-296.PDA J Pharm Sci and Tech

Pawar and Helena WindsorDilip Ashtekar, Brian Beck, Nick Hutchison, Laura Okhio-Seaman, Barbara Potts, Vinayak Martha Folmsbee, Kerry Roche Lentine, Christine Wright, Gerhard Haake, Leesa Mcburnie,Mycoplasma-Retentive Filters by Filter Manufacturers

To RateAcholeplasma laidlawiiThe Development of a Microbial Challenge Test with

including high resolution figures, can be found at:Updated information and services /content/83/2/417.full.html

can be found at:Physiological Reviewsabout Additional material and information http://www.the-aps.org/publications/prv

website at http://www.the-aps.org/.MD 20814-3991. Copyright 2003 by the American Physiological Society. ISSN: 0031-9333, ESSN: 1522-1210. Visit ourpublished quarterly in January, April, July, and October by the American Physiological Society, 9650 Rockville Pike, Bethesda

provides state of the art coverage of timely issues in the physiological and biomedical sciences. It isPhysiological Reviews

on Septem

ber 15, 2014D

ownloaded from

This information is current as of September 15, 2014.

website at http://www.the-aps.org/.MD 20814-3991. Copyright 2003 by the American Physiological Society. ISSN: 0031-9333, ESSN: 1522-1210. Visit ourpublished quarterly in January, April, July, and October by the American Physiological Society, 9650 Rockville Pike, Bethesda

provides state of the art coverage of timely issues in the physiological and biomedical sciences. It isPhysiological Reviews

on Septem

ber 15, 2014D

ownloaded from

Documents

Interaction of Mycoplasmas With Host Cells