Genetic divergence at the SODA locus of six different formae

Medical Mycology 2000, 38, 289–300 Accepted 9 March 1998

Genetic divergence at the SODA locus of six differentformae speciales of Pneumocystis carinii

C. M. DENIS*,†, E. MAZARS‡,§, K. GUYOT‡, C. OÚ DBERG-FERRAGUT¶, E. VISCOGLIOSI$, E. DEI-CAS‡, &A. E. WAKEFIELD**Molecular Infectious Diseases Group, Department o f Paediatrics, Institute of Mo lecular Medicine, University o f O xford,UK; †Faculte Libre des Sciences, 13, rue de Toul, 59000 Lille, France; ‡Departement de Microbiologie desEcosystemes, Institut Pasteur de Lille, 1, rue du Pr Calmette, BP 245, 59019 Lille, France; §Hopital de Valenciennes,59322 Valenciennes, France; ¶Laborato ire de Chimie Biologique, UMR 8576 CNRS -USTL , Bat. C9, 59655 Villeneuved’A scq Cedex, France; $Labo ratoire de Biologie des Protistes, URA CNRS 1944, Universite de C lermont -Ferrand,63177 Aubieres Cedex, France; Faculte de Medecine, Centre Hospitalier Regional Universitaire, 1, place de Verdun,59850 Lille, France

Genetic divergence at the SODA (manganese-dependent superoxide dismutase, Mn-SOD) locus were compared in six Pneumocystis carinii formae speciales isolated frommouse, rabbit, human, macaque and pig. A degenerate oligonucleotide primerstrategy was designed to amplify 85–90% of the full-length SODA gene from P.carinii genomic DNA isolates. DNA sequence analysis revealed an A:T bias in thenucleotide composition (71–77·2%) and the presence of seven small introns (41–142bp), interrupting each P. carinii open reading frame (ORF) at the same position. TheMnSOD deduced amino acid sequences from all P. carinii isolates shared residueswhich were conserved within the MnSOD family and which are required forenzymatic activity and binding of the cofactor metal. Phylogenetic analysis includingMnSOD sequences from representatives of the fungal phyla Basidiomycota andAscomycota indicated that the P. carinii formae speciales form a monophyletic groupthat is related to the budding yeasts (subphylum Saccharomycotina, previously calledclass Hemiascomycetes) in the Ascomycota. In the whole Pneumocystis group, P.carinii f. sp. hominis, P. carinii f. sp. macacae and P. carinii f. sp. oryctolagi MnSODsequences clustered together, as did the rat-derived P. carinii and P. carinii f. sp.muris sequences.

Keywords fungi, Pneumocystis carinii, polymorphism, superoxide dismutase

Introduction

Pneumocystis carinii is a major pathogen causing life-threatening disease in acquired immune de�ciencysyndrome (AIDS) patients and also in other immuno-

suppressed patients. Originally classi�ed as a protozoan,P. carinii has been found to be a member of the kingdomFungi according to molecular studies at the 18S riboso-mal (r)RNA locus [1]. Subsequent data from a largenumber of loci have all con�rmed the fungal nature of P.carinii [2–4]. Nevertheless, its exact position within thefungal kingdom has yet to be established, some datasuggesting that P. carinii is either included as a memberof the Basidiomycota [5] or as an early branch of theAscomycota [6–9]. Other authors have suggested a close

Correpondence: C. M. Denis, Molecular Infectious Disease Group,Department of Paediatrics, Institute of Molecular Medicine, Uni-versity of Oxford, John Radcliffe Hospital, Headington, OxfordOX3 9DS, UK. Tel.: »44 1865 222344; fax: »44 1865 222626;e-mail: [email protected]

© 2000 ISHAM

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290 Denis et al.

association with the ‘Rhizopoda:Myxomycota:Zygomy-cota’ group [10], but that was based on analyses of the 5SrRNA sequences, which are unreliable for assessing rela-tionships between higher taxa [11,12].

P. carinii infection has been reported in many mam-malian hosts, such as animals with acquired immunode�-ciency or malnourishment, including foals [13,14], piglets[15], dogs [16] and monkeys [17]. P. carinii has also beenfound in the lungs of a wide variety of mammalian hostspecies which include laboratory animals, zoo animalsand wild animals [18–21].

In recent years, the proposition that the genus Pneu-mocystis comprises a heterogeneous group of organismshas been strengthened through phenotypic and genotypicdata. Heterogeneity among different P. carinii isolateshas been reported at the antigenic level [22–24], at themorphological level [25,26] and at the isoenzymatic level[27]. Differences in karyotypic pro�les [2,28] and DNAsequence variation at informative loci [29] support thebiodiversity observed among the different host-derived P.carinii isolates. Finally, cross-infection experiments havedemonstrated a strong speci�city of a particular P. cariniiisolate for the host species from which it originates [30–32]. A trinomial nomenclature using the term formaespeciales has been adopted to distinguish between thedifferent isolates of P. carinii, based on the host-speciesspeci�city of the organism [33,34].

Superoxide dismutases (SOD; EC 1.15.1.1) are ubiqui-tous enzymes essential in cellular protection against reac-tive oxygen species [35]. Three major classes of SOD havebeen described according to the cofactor metal present inthe active site [36]: copper:zinc-containing SODs (Cu:Zn-SODs), found in eukaryotes and bacteria; manganese-containing SODs (MnSODs), present in prokaryotes andmitochondria; and iron-containing SODs (FeSODs), de-tected in prokaryotes, chloroplasts and protists. Mn-SODs and FeSODs share a high degree of sequence andstructural homology whereas Cu:ZnSODs appear to beunrelated to the Mn:Fe types of SOD [37]. MnSODs andFeSODs have been successfully used in phylogeneticanalysis in Metazoa, Gram-positive bacteria, Archaebac-teria and protists [37–40].

We have recently reported the molecular characteriza-tion of a gene (SODA) encoding a manganese-dependentsuperoxide dismutase in rat-derived P. carinii [41]. Thecurrent study is the �rst attempt to use a single copynuclear gene, SODA, as a tool to analyse the geneticdiversity between six different host-derived Pneumocystisisolates, from rat, mouse, rabbit, human, monkey andpig. Since SODs are considered useful phylogeneticmarkers, the relationships between Pneumocystis and 11fungal species which are representative of the Ascomy-cota and the Basidiomycota phyla have been examined.

Materials and methods

Samples

P. carinii organisms were obtained from two cortico-steroid-treated outbred mice (Institut Pasteur of Lille,Lille, France), from a rabbit at weaning (Vasseur,Prouzel, France), from a simian immunode�ciency virus(SIV)-infected Rhesus monkey (Macaca mulatta) kindlyprovided by Dr Durand-Joly [42], from a pig sponta-neously infected with P. carinii kindly provided by Dr V.Bille-Hansen and Dr O. P. Settnes [43]. Human broncho-scopic alveolar lavages (BALs) from two human im-munode�ciency virus (HIV)-infected patients (D503,D798) with P. carinii pneumonia (PCP), were kindlyprovided by Dr R. F. Miller (Royal Free and UniversityCollege Medical School, London, UK). Post-mortemlung tissue from a patient with Hodgkin’s disease (C180)was kindly provided by Dr B. Lundgren (University ofCopenhagen, Copenhagen, Denmark). Uninfected hostlung tissues and BAL samples were processed in parallelto be used as negative controls.

Pneumocystis nomenclature

The trinomial nomenclature for different isolates of P.carinii, based on the host of origin, is used in this paper.Pneumocystis organisms isolated from infected mouselungs are known as Pneumocystis carinii f. sp. muris ;rabbit-derived as Pneumocystis carinii f. sp. oryctolagi ;human-derived as Pneumocystis carinii f. sp. hominis ;macaque-derived as Pneumocystis carinii f. sp. macacae ;and pig-derived as Pneumocystis carinii f. sp. suis [33,34].Genes coding for all the P. carinii formae speciales Mn-SODs described in this study are named SODA in agree-ment with the nomenclature used in Saccharomycescere×isiae, Leishmania and Trypanosoma [44].

DNA extraction

DNA was extracted from P. carinii-infected mouse andrabbit lung tissues as described by Mazars et al. [45]. P.carinii-infected pig, monkey and human lung tissues andBAL samples were processed as described by Tsolaki etal. [46]. Genomic DNA was also extracted from unin-fected host samples in order to exclude ampli�cationproducts due to host DNA.

DNA ampli�cation

Single round polymerase chain reactions (PCR), usingboth degenerate primers and speci�c primers for con-served regions of the rat-derived P. carinii SODA gene

© 2000 ISHAM, Medical Mycology, 38, 289–300


SO DA in six Pneumocystis carinii formae speciales 291

[41], were carried out on the different host-derived P.carinii samples (Table 1). PCR was performed using0·025 U ml¼ 1 of Biotaq DNA polymerase (Bioline, Lon-don, UK) in reaction mixtures containing a �nal concen-tration of 67 mM Tris-HCl (pH 8·8), 16 mM (NH4)2SO4 ,0·01% Tween 20 (w:v), 3 mM MgCl2, 200 mM (each) de-oxynucleoside triphosphate (dNTP; Roche DiagnosticFrance S.A., Meylan, France), 2 mM (each) oligonucle-otide primer. PCRs performed on human samples werecarried out using 0·05 U ml¼ 1 of Taq DNA polymerase(Promega, Lyon, France) in 10 mM Tris-HCl (pH 9·0),50 mM KCl, 0·1% Triton X-100, 3 mM MgCl2 , with iden-tical �nal concentrations of dNTP and primers as de-scribed above. The PCR thermocycling conditions wereas follows: 40 cycles consisting of denaturation at 94 °Cfor 90 s, annealing at 40 °C for 90 s, and extension at72 °C for 90 s.

C loning and sequencing of ampli�cation products

After puri�cation from 1·5% agarose gels using Gene-clean II (Bio101; Stratech Scienti�c, UK), the PCR prod-ucts were cloned into pGEM®-T easy vector system(Promega) according to the manufacturer’s instructions.The recombinant plasmids were transformed in Es-cherichia coli strain DH5a. DNA was prepared fromovernight cultures using the Wizard® Plus Miniprep kit(Promega). A minimum of four recombinant clones perP. carinii isolate were sequenced using M13 universalprimers or internal primers on both strands. Sequencingwas performed either manually using the Sequenase 2·0kit (Amersham Pharmacia Biotech Europe GmbH, Oray,France) or automatically using the Big Dye™ Terminator

Cycle Sequencing Ready Reaction kit (Perkin ElmerS.A., Courtaboeuf, France) and the ABI Prism™ 377DNA Sequencer running the Data Collection SoftwareVersion 2·1 (PE Applied Biosystems, Inc., Foster City,CA, USA). Sequence data analysis was performed usingChromas 1·44 software (C. McCarthy, Grif�th Univer-sity, Australia).

Sequence and phylogenetic analysis

Nucleotide and derived amino acid sequences werealigned visually and compared with sequences from otherorganisms with the use of the MUST package [47]. Acces-sion numbers of the sequences retrieved from GenBank(www.ncbi.nlm.nih:gov:genbank:index.html) and EMBL(www.embl-heidelberg.de:externalinfo :generalinfo :ser-vices.html) databases are as follows. MnSODs: Rat-derived P. carinii Z79785, Saccharomyces cere×isiaeP00447, Candida albicans AF031478, Candida sp.Y11598, Aspergillus fumigatus U53561, Penicilliumchrysogenum AF026790, Ganoderma microsporumU56403, Fomitopsis cf. rosea U56108, Amauroderma rudeU56109, Homo sapiens P04179, He×ea brasiliensisP35017, E. coli A P00448; FeSOD: E. coli B P09157,Legionella pneumophila P31108, Entamoeba histolyticaP34107. The GenBank accession numbers for new SODAsequences reported here are AF146751 for P. carinii f. sp.muris, AF146752 for P. carinii f. sp. oryctolagi,AF146753 for P. carinii f. sp. hominis, AF146754 for P.carinii f. sp. macacae and AF146755 for P. carinii f. sp.suis.

Databases of aligned sequences were assembled withthe ED program of the M UST package. A distance matrixbased on the number of identical residues in alignedpositions was calculated with the NET program of MUST.The distance matrix was analysed by the neighbour-join-ing method [48] implemented in the MUST package. Boot-strap proportions were obtained from 1000 replicates.

Phylogenetic trees were also constructed according tothe maximum parsimony method, using the PROTPARS,or DNAPARS programs of the PHYLIP package (PhylogenyInference Package, version 3·5c) [49]. Alignment andboundaries will be sent upon request.

Results

Ampli�ed products of the SODA genes from �veP. carinii iso lates

The primer design strategy was based on oligonucleotidesequences speci�c for the rat-derived P. carinii SODAgene which was recently characterized [41]. PCR wasperformed using genomic (g)DNA from P. carinii f. sp.

Table 1 Oligonucleotide primer sequences

Location on P. carinii f.sp. carinii SODA

Name Nucleotide sequence sequence*

6–35SOD1F 5Æ-TGT TTT ACC AAGTCT TCC TTA TGATTA TCA-3Æ5Æ-TGA TTC ATA ACT 993–1012SOD2RTTC CAA TT-3Æ5Æ-AAR CAY GTWSOD3F 1–32YTW CCW WSW YTWCCW TAY GAY TA-3Æ

SOD4F 82–1015Æ-GCA YTN GAACCW TAT CTT TC-3Æ

SOD5R 5Æ-TGR TTC ATN ACY 993–1012TTC CAR TT-3Æ

R¾[A, G]; Y¾[C, T]; W¾[A, T]; S¾ [G, C]; N ¾[A, G, C, T];F, Forward primer; R, Reverse primer.



292 Denis et al.

muris, P. carinii f. sp. oryctolagi, P. carinii f. sp. hominis,P. carinii f. sp. macacae and P. carinii f. sp. suis. Negativecontrols consisted of gDNA extracted from BAL of anuninfected patient, from lungs of a germ-free rabbit andan uninfected mouse and from blood of an uninfectedmacaque. Porcine P. carinii-free gDNA was not avail-able. Ampli�ed products were retained as candidate frag-ments for cloning if the predicted size was observed andif no product of the same size was ampli�ed in theuninfected host gDNA. Oligonucleotide primer sequenceand localization on the rat-derived P. carinii SODA se-quence are shown in Table 1. Speci�c oligonucleotideprimer sets were used to amplify P. carinii f. sp. muris andP. carinii f. sp. oryctolagi SODA sequences while degener-ate primer sets were annealed on P. carinii f. sp. suis, P.carinii f. sp. hominis and P. carinii f. sp. macacae gDNA(Table 2). About 90% of the gene sequence was charac-terized for P. carinii f. sp. muris, P. carinii f. sp. orycto-lagi, P. carinii f. sp. hominis, P. carinii f. sp. suis as well as85% for P. carinii f. sp. macacae.

Fragments of 938, 840, 829, 764 and 846 bp wereampli�ed at the SODA locus for P. carinii f. sp. muris, P.carinii f. sp. oryctolagi, P. carinii f. sp. hominis, P. cariniif. sp. macacae and P. carinii f. sp. suis, respectively, usingdifferent pairs of primers as indicated in Table 2. ThePCR products were cloned and sequenced.

Genomic DNA of P. carinii f. sp. oryctolagi, P. cariniif. sp. suis and P. carinii f. sp. macacae were extractedfrom a single host individual, whereas three and twoindividuals were available as gDNA sources for P. cariniif. sp. hominis and P. carinii f. sp. muris, respectively. Nopolymorphism was found while comparing the two P.carinii f. sp. muris samples. Single base pair polymor-phisms were observed between the three human P. cariniiisolates (Table 3). The �rst base variation was located in

the second intron. Two nucleotide changes occurred inthe third exon and remained synonymous. The D503sample showed a combination of polymorphisms occur-ring in the C180 and D798 samples (Table 3).

Character istics of the six Pneumocystis MnSO DO RFs

The putative Pneumocystis MnSOD open reading frames(ORFs) differed from one host-derived P. carinii isolateto another (Fig. 1). However, all of them contained theconserved amino acid residues and motifs characteristicof MnSODs and are likely to encode manganese-depen-dent SOD enzymes. The deduced partial amino acidsequences of the coding region of the SODA gene fromP. carinii f. sp. muris, P. carinii f. sp. oryctolagi, P. cariniif. sp. hominis, P. carinii f. sp. macacae and P. carinii f. sp.suis contain 174, 172, 172, 165 and 173 residues, respec-tively. The P. carinii sequences show the presence of allthe conserved SOD motifs and characteristic amino acidresidues known in MnSODs (numbering as in the rat-derived P. carinii MnSOD sequence [41]): the highlyconserved residues that serve as metal ligands His-49,His-99, Asp-184 and His-188; the amino acid residuesGly-94, Gly-95 and Gln-171 allowing the distinction be-tween MnSODs and FeSODs; the aliphatic residue atposition 97 (Ile-97 in P. carinii isolates) proposed to beMnSOD speci�c and conferring H2O2 resistence (versusaromatic amino acid residues in H2O2 sensitive FeSODs).

In a variable domain between MnSOD sequences, twoextra amino acid residues, Tyr-68 and Ser-78, were ob-served in both of the murine sequences compared to theother P. carinii sequences (Fig. 2). Genetic divergencewas calculated by pair-wise comparison on the alignmentshown in Figure 1 including the six P. carinii formaespeciales (nucleotides 104 –992). The rat-derived P. cariniiis closely related to P. carinii f. sp. muris with 9·5 and4·3% divergence at the nucleotide and amino acid levels,respectively. Low levels of divergence were also observedbetween P. carinii f. sp. hominis and P. carinii f. sp.macacae with 11·1% and 1·8% at the nucleotide and

Table 2 Speci�c and degenerate oligonucleotide primers used toamplify genomic DNA extracted from different P. carinii formaespeciales

P. carinii formae Size* of ampli�ed andOligonucleotidesequenced products (bp)primer pairsspeciales

P. carinii f. sp. 938SOD1F:SOD2Rmuris

P. carinii f. sp. 840SOD1F:SOD2Roryctolagi

846P. carinii f. sp. SOD3F:SOD2Rsuis

P. carinii f. sp. 764SOD4F:SOD2Rmacacae

P. carinii f. sp. 829SOD1F:SOD5Rhominis

F, Forward primer; R, Reverse primer; *, Primers omitted.

Table 3 Single base polymorphisms in three samples (D503, C180and D798) of P. carinii f. sp. hominis at the SODA locus

Positions* & Intron:Exon

Sample 110 bp, intron 2 179 bp, exon 3 215 bp, exon 3

D503 C:TC:T A:TT TC180 CT CD798 T

*, Nucleotide position in Fig. 1.



SO DA in six Pneumocystis carinii fo rmae speciales 293

Fig. 1.

© 2000 ISHAM, Medical Myco logy, 38, 289–300


Denis et al.294

Fig. 1 Alignment of consensus nucleotide sequences of the SODA gene from six P. carinii formae speciales. PcRat, Rat-derived P. carinii;PcMu, P. carinii f. sp. muris; PcRb, P. carinii f. sp. oryctolagi; PcHu, P. carinii f. sp. hominis; PcMo, P. carinii f. sp. macacae; PcPi, P. cariniif. sp. suis. Identical nucleotides are indicated by dashes (-). Gaps are symbolized by asterisks (*). Primers used to amplify P. carinii SODAare localized with arrows. Introns are shown in lower case, and the consensus branch point element YYRAY is underlined and italicized.

Intronic sequence analysisThe MnSOD ORFs are apparently interrupted by sevenintrons located at the same position for all of the P.carinii isolates studied (Figs. 1 and 2).

The Pneumocystis intron length ranged 41–54 bp, ex-

amino acid levels, respectively. Higher levels of diver-gence were found between the murine P. carinii isolatesand both P. carinii f. sp. oryctolagi and P. carinii f. sp.suis (29·6–31·3% at the nucleotide level, 36·8–38·8% atthe amino acid level).




cept for the third intron which was 144 bp and 142 bplong for rat-derived P. carinii and P. carinii f. sp. muris,respectively, but only 49, 47, 48 and 50 bp for P. carinii f.sp. oryctolagi, P. carinii f. sp. hominis, P. carinii f. sp.macacae and P. carinii f. sp. suis, respectively. The intronsshowed a higher A:T composition than the exons. ThePneumocystis SODA sequences which had a higher intronA:T percentage were also found to show higher A:Tpercentage of the full length sequence, i.e., the murine P.carinii SODA sequences showed the highest intron A:Tcompositions, (77·2% for rat-derived P. carinii and 76·2%for P. carinii f. sp. muris) and P. carinii f. sp. hominis(71·0%) the lowest (Table 4).

The introns in the P. carinii SODA sequences showedhigh percentages of similarity between the two murineisolates (86·2%) and between P. carinii f. sp. hominis andP. carinii f. sp. macacae (83·3%). Introns from rat-derivedP. carinii and P. carinii f. sp. suis were more distant fromeach other than from any other P. carinii isolates (51·7%similarity).

The 5Æ- splice site donor GT- and the 3Æ- splice siteacceptor -AG were conserved for all of the putativeintrons except for intron 2 where a 5Æ- splice site GC- wasfound in the P. carinii f. sp. oryctolagi sequence (Fig. 1).At the 5Æ- splice site, the third base was conserved as an Ain introns 3, 5, 6 and 7, and, at the 3Æ- splice site, as a T inintrons 1, 3, 4, 5 and 6. However, variation was found atthe third base of both splice sites in intron 2 as well as inintrons 1 and 4 at the 5Æ- splice site. Interestingly, in intron7 of all of the P. carinii samples, an A was found insteadof a T at the third base of 3Æ- splice site (Fig. 1).

The branch point eukaryotic consensus element(YYRAY) was present in most of the introns but a fewexceptions should be mentioned (Fig. 1). The YYRAYsequence was missing in intron 6 for all of the P. cariniiisolates. It was also missing in intron 5 of the P. carinii f.sp. suis sequence. Finally, both the murine sequences werelacking YYRAY in intron 1 whereas both the primatesequences lacked it in intron 2.

Molecular phylogeny at the SODA locus

MnSODs and FeSODs are fairly conserved amongst eu-karyotes and prokaryotes, and are useful tools for phylo-genetic analysis as shown by several authors [38–40].Thus, P. carinii SOD amino acid sequences were used in aphylogenetic analysis to examine the relationships amongthe different P. carinii isolates with respect to each otherand to other Fungi. P. carinii sequences were aligned with11 related MnSODs (eight fungal MnSODs, Homo sapi-ens, He×ea brasiliens and E. coli MnSODs) and E. coli, E.histolytica and L. pneumophila FeSODs (Fig. 2).


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296 Denis et al.

Fig. 2.




An unrooted tree was constructed using the neighbourjoining method (Fig. 3). The different P. carinii MnSODsequences formed a monophyletic group which wasstrongly supported by bootstrap analysis (100%). Withinthe P. carinii group, a bootstrap value of 100% sup-ported the monophyly of a group consisting of P. cariniif. sp. muris and rat-derived P. carinii. In contrast, theposition of P. carinii f. sp. suis was poorly resolved(bootstrap value of 40%), whereas P. carinii f. sp. ho-minis, P. carinii f. sp. macacae and P. carinii f. sp.oryctolagi grouped together in 95% of the replicates.

In an overall description of the phylogenetic tree,clades corresponding to well-established major fungalgroups could be recognized. These were two subphyla inAscomycota, that is the Saccharomycotina (previously‘Hemiascomycetes’), represented by S. cere×isiae, C. albi -cans and Candida sp., and the Pezizomycotina (‘Euas-comycetes’), by P. chrysogenum and A. fumigatus. TheBasidiomycota were represented by A. rude, F. cf. roseaand G. microsporum. The Basidiomycota MnSOD se-quences grouped consistently with the PezizomycotinaMnSODs in 84% of the replicates, whereas the host-derived P. carinii isolates grouped together with theSaccharomycotina sequences (bootstrap value of 78%).Currently, Pneumocystis is usually placed with theSchizosaccharomycetales in Basal Ascomycetes orTaphrinomycotina (‘Archiascomycetes’) [50–53].

The phylogenetic tree inferred from the alignment ofthe nucleotide sequences from the same organisms was inagreement with all these observations (data not shown).In addition, the topology of phylogenetic trees, whichwere built from both protein and nucleotide alignments,was found to be similar when the maximum parsimonymethod was used (data not shown).

Discussion

The rat-derived P. carinii SODA gene was used to designthe ampli�cation strategy of SODA sequences from �veother P. carinii formae speciales.

Primary structures of the Pneumocystis deduced aminoacid SOD sequences contain conserved peptide motifsand four amino acid residues involved in the binding of

the cofactor metal in the catalytic site. Some amino acidmotifs, known to be signatures of MnSODs [54], coincidewith the motifs present in the newly described Pneumo-cystis sequences. Consequently, the Pneumocystis se-quences are highly likely to encode manganese-dependentSOD enzymes. The general similarity of the gene struc-ture (length and position of introns) and the comparableamino acid primary structure between the rat P. cariniiSODA gene and the other P. carinii formae speciales alsosupport this view. The occurrence of seven short intronswithin the P. carinii MnSOD ORFs as well as the A:Tbiased codon usage agree with features already reportedfor P. carinii genes [55,56]. The frequency of intron in the1097 bp P. carinii SODA gene is comparable to that ofthe catalytic domain of the P. carinii protease (PRT1)gene [57] whereas it is higher in comparison to genescontaining a substantial number of introns such as celldivision cycle (cdc2), a-tubulin, b-tubulin and thymidy-late synthase genes [56,58 –60]. The P. carinii SODAintron frequency is also high if compared to the SODAloci of Basidiomycota and P. chrysogenum where onlytwo introns are present [61,62]. The �rst introns of theBasidiomycota and P. chrysogenum are localized at thesame position as introns 3 and 1 predicted in the Pneu-mocystis sequences, respectively (Fig. 2). Interestingly, inBasidiomycota and P. chrysogenum, intron 2 occurs attwo different positions which do not correspond to anyof the Pneumocystis intronic regions (Fig. 2).

Genetic diversity observed at the SODA level strength-ened the suggestion that P. carinii infection is host-spe-cies speci�c and agrees with the provisional trinomialnomenclature of formae speciales adopted recently[33,34]. P. carinii of murine origin appear as a well-sup-ported monophyletic group. This clustering is consistentwith: i) the use of rat-derived P. carinii speci�c primersenabling the ampli�cation of the P. carinii f. sp. murissequence; ii) the presence of a longer third intron in themurine SODA sequences; and iii) the insertion of twoextra amino acid residues which are missing in all theother P. carinii sequences. Murine P. carinii formae spe-ciales were also found to be grouped together with astrong bootstrap con�dence at the mitochondrial largeand small subunit ribosomal RNA loci [29,42] and at the

Fig. 2 Alignment of the deduced amino acid sequences of the SODA gene from six P. carinii formae speciales with a set of MnSODs andFeSODs. PcRat, Rat-derived P. carinii; PcMu, P. carinii f. sp. muris; PcRb, P. carinii f. sp. oryctolagi; PcHu, P. carinii f. sp. hominis; PcMo,P. carinii f. sp. macacae; PcPi, P. carinii f. sp. suis ; Sc, S. cere×isiae ; Ca, C. albicans; Csp, Candida sp.; Af, A. fumigatus; Pch, P. chrysogenum;Gm, G. microsporum; Fr, F. cf. rosea ; Ar, A. rude; Hum, Homo sapiens; Hb, H. brasiliensis; EcA, E. coli A; EcB, E. coli B; Lp, L.pneumophila; Eh, E. histolytica. Residues identical to those in the rat-derived P. carinii sequence are represented by dashes (-). Gaps aresymbolized by asterisks (*). Forward arrows (\) indicate the omitted amino-terminal extensions of several enzymes. Residues crucial for thedistinction between MnSODs and FeSODs are shown in shaded columns. Metal binding ligands are represented by ( ) whereas the residueinvolved in the sensibility of SODs to H2O2 is indicated by ( ß ). The amino acid residue preceding the beginning of Pneumocystis intronsare indicated by (» ) as are those of Basidiomycota ( ) and P. chrysogenum ( ).



298 Denis et al.

Fig. 3 Unrooted phylogenetic tree ofMnSOD deduced amino acid sequences.The tree is based on 122 positions afterexclusion of all gaps and ambiguousparts of the alignment shown in Fig. 2was constructed using theneighbour-joining method. Bootstrapvalues are given as percentages abovethe individual nodes. The horizontallength of each branch is proportional tothe estimated number of substitutionswhereas the vertical length is arbitrary;Pc f. sp., P. carinii formae speciales.

arom locus [63]. Similarly, the grouping of P. carinii f. sp.hominis and P. carinii f. sp. macacae appears unequivocaland agrees with the phylogenetic analyses at the mito-chondrial large and small subunit rRNA loci [42]. Thephylogenetic relationship between P. carinii f. sp. orycto-lagi and the primate and human P. carinii isolates isclearer at the SODA locus than at the mitochondrialsubunit rRNA loci. The position of P. carinii f. sp. suis isnot better resolved. The branching of the porcine P.carinii may vary slightly according to the alignment (nu-cleotide or amino acid) and to the method (distance orparsimony) used to build the phylogenetic trees. Positionsof the other branches, which are strongly supported bybootstrap values, stay congruent in both methods used.

Lower levels of genetic heterogeneity have been ob-served within each P. carinii forma specialis, and particu-larly in human-derived P. carinii [63,64]. At the SODAlocus, three nucleotides were found to vary from onehuman P. carinii sample to another and also from oneclone to another within the same human P. carinii isolate,suggesting the occurrence of a mixture of two P. cariniitypes in the same individual. Further experimental inves-tigations are needed to ascertain this hypothesis as theintra-forma specialis genetic heterogeneity was beyondthe scope of this study.

On the basis of molecular comparison of a number ofgene sequences (reviewed in [2–4]), P. carinii has beenconsidered to be a member of the kingdom Fungi. Never-theless, its exact taxonomic position within this groupremains controversial with assignment to the Basidiomy-cota [5] or to the Taphrinomycotina in Ascomycota [7,8]

or as a sister group to these two taxa [9]. According toour phylogenetic tree based on the comparison of Mn-SOD sequences, the P. carinii f. spp. compose amonophyletic group closely associated to Saccharomy-cotina (Fig. 3). This result would favour the assignmentof P. carinii f. spp. within the Ascomycota. Nevertheless,more SODA sequence data, especially sequences fromthe polyphyletic Taphrinomycotina group, are requiredto determine accurately the position of P. carinii f. spp.relative to the fungal kingdom.

In conclusion, the DNA and amino acid heterogeneityseen at the SODA locus, a nuclear encoded gene, con-�rmed that the P. carinii group is composed of similarbut distinct formae speciales related to each host species.Considering each host-derived P. carinii isolate as adistinct entity has crucial implications in terms of drugdesign, antigenicity and epidemiology.

Acknowledgements

We would like to acknowledge Dr I. Durand-Joly forproviding the P. carinii f. sp. macacae sample, Dr O. P.Settnes and Dr V. Bille-Hansen for providing the P.carinii f. sp. suis sample, Dr R. F. Miller and Dr B.Lundgren for providing the human samples. This re-search was supported by the Medical Research Council(MRC) and the ‘Institut National de la Sante et de laRecherche Medicale’ (CMD), the Royal Society and theANRS (contract No 98016). This work was developed inthe framework of the PRFMMIP project (‘Programmede Recherche Fondamentale en Microbiologie et Mal-




adies Infectieuses et Parasitaires’, French Ministery ofEducation, Research and Technology).

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Genetic divergence at the SODA locus of six different formae