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Mycologia, 96(3), 2004, pp. 429–438. q 2004 by The Mycological Society of America, Lawrence, KS 66044-8897 Issued 4 June 2004

Molecular and phenotypic description of Pneumocystis wakefieldiae sp. nov., a new species in rats Melanie T. Cushion1

The cumulative genotypic and phenotypic data support a species distinction between these two organisms. Key words: genetic comparisons, karyotypic profiles, phylogenetic species recognition, Pneumocystis pneumonia, systematics

University of Cincinnati College of Medicine, Department of Internal Medicine, Division of Infectious Diseases, 231 Albert Sabin Way, Cincinnati, Ohio 45267-0560

Scott P. Keely James R. Stringer University of Cincinnati College of Medicine, Department of Molecular Genetics, Biochemistry and Microbiology, 231 Albert Sabin Way, Cincinnati, Ohio 45267-0554

INTRODUCTION

Pneumocystis fungi reside in the lungs of mammals and can cause lethal pneumonia if the host loses immune function as a result of malnourishment, disease, congenital defects or chemically induced immunosuppression (Walzer et al 1973, Walzer et al 1976). Chagas first observed novel developmental forms in the lungs of guinea pigs, nonhuman primates (marmosets) and a human examined post mortem that were infected with Trypanosoma cruzi (Chagas 1909). He believed the Pneumocystis cysts found in their lungs represented schizogonic stages in a hypothetical sexual cycle of the trypanosomes. The Delanoe¨s showed that the lung forms described by Chagas were present in sewer rats not infected with trypanosomes (Delanoe¨ P and Delanoe¨ Mme. 1912) and concluded that a new protozoan organism had been discovered and named it Pneumocystis carinii. Chagas concurred and withdrew his description of the forms as stages in the trypanosome life cycle. The presumption that Pneumocystis was a protozoan was not breached definitively until analysis of the SSU rDNA (small-subunit ribosomal RNA) sequence showed that the Pneumocystis from rat lung was a fungus (Edman et al 1988, Stringer et al 1989). Subsequent analyses of many other genes of Pneumocystis carinii have confirmed its placement in the fungal kingdom(Cushion et al 2004). Homology searches of more than 5000 expressed sequence tags showed that the majority (;70%) were most similar to fungal genes of Schizosaccharomyces pombe and Neurospora crassa (Cushion et al 2004). Pneumocystis found in the lungs of a wide spectrum of mammalian species (Hughes 1987, Cushion MT 1998) are morphologically similar, impeding clear species distinction using light microscopic techniques. All have asci (spherical cysts, 5–8 mm diam) with up to eight ascospores (daughter forms, 1–2 mm) and clusters of yeast-like cells (trophic forms, 1–

Abstract: Organisms in the genus Pneumocystis are fungi that reside in the lungs of mammals that can cause a lethal pneumonia once the hosts lose immune function. The genus Pneumocystis contains many members, but only two species have been described formally to date, P. carinii, the type species found in rats, and P. jirovecii, resident in human beings. Rats have been shown to harbor another organism in addition to P. carinii, Pneumocystis wakefieldiae sp. nov., formerly known as Pneumocystis carinii f. sp. ratti, which is described here. Although often found together and morphologically similar, P. carinii and P. wakefieldiae are phenotypically and genetically divergent. We used the phylogenetic species recognition approach to distinguish these organisms as two distinct species and estimated the evolutionary time of their separation. Nucleotide sequence comparisons of seven homologous genes showed 4–7% divergence between the P. wakefieldiae and P. carinii sequences, which was in contrast to the 0–0.8% divergence observed within P. carinii species. Even greater divergence (30%) occurred in sequences located between genes. The MSG (major surface glycoprotein) gene families of P. carinii and P. wakefieldiae are 35% divergent from one another and differ with respect to sequence elements associated with regulation of their transcription. Differences in reactivity of monoclonal antibodies and polyclonal antisera reflected these genetically distinct surface antigens. Karyotypic analysis of P. wakefieldiae produced a single profile that was distinct from all 12 profiles known for P. carinii. Eight homologous genes were localized to chromosomes of different sizes in the two species. Accepted for publication October 20, 2003. 1 Corresponding author. E-mail: [email protected]

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5 mm) (Cushion 1998b). The cell walls of P. carinii asci are stained with methenamine silver as are other fungal cell walls, but the ascospores or trophic forms do not stain. Ultrastructural studies have revealed subtle differences in the sizes of trophic form filopodia, filopodia morphology and density of membrane-limited cytoplasmic granules among Pneumocystis from rats, mice and rabbits (Nielsen et al 1998). Likewise, the doubling times of the organism varied in mice, rats and rabbits, indicating genetic and phenotypic differences (Aliouat et al 1999). The dramatic differences at the gene sequence level have established that there are many distinct organisms in the genus (The Pneumocystis Workshop 1994). These observations were joined by demonstrations of host specificity (Aliouat et al 1994, Gigliotti et al 1993). Pneumocystis organisms from different mammals were named subsequently as formae specialis using host affiliation (e.g., Pneumocystis carinii f. sp. hominis from humans, P. carinii f. sp. muris from mouse and P. carinii f. sp. oryctolagi from rabbits, etc.) (The Pneurocystis workshop 1994, Stringer et al 1997). Frenkel took the step of elevating formae specialis from rats and humans to the species level, P. carinii and P. jirovecii, respectively (Frenkel 1999). The description as species of Pneumocystis from different hosts has met with approval, but the same host can harbor more than one species. A second Pneumocystis from rat was identified and originally called ‘‘variant’’ P. carinii (Liu et al 1992, Cushion et al 1993b) and later renamed P. carinii f. sp. ratti (Frenkel 1999, Stringer et al 1997). In addition, three other formae specialis of P. carinii have been described from the lungs of wild rats, but these have been identified solely by differences in a specific region of DNA in the mitochondrial large-subunit rRNA (Palmer et al 2000). Their provisional status must remain until further genetic and phenotypic analyses are conducted. Here, using the phylogenetic approach to species recognition, we describe the taxon Pneumocystis wakefieldiae sp. nov., based on phenotype and genotype. We have undertaken this effort for the following reasons. First, it is necessary to make a distinction between the two species to understand the origin of genes cloned from organisms derived from rat lungs. For example, if two genes were cloned from a rat carrying an infection caused by both organisms, the 4–7% difference observed between the two sequences may be misconstrued as being from paralogs within a single genome. Second, there is little information concerning the biological consequences of infections by either or both species. To begin to explore potential phenotypes, a clear understanding of each species is necessary. Third, the rat model of

Pneumocystis is widely used by investigators. Reports from different laboratories have shown varying results for drug effects, immunological responses, etc. and increased standardization of the model by identification of the Pneumocystis species causing the infection might lead to a clearer understanding of the diversity of biological responses. MATERIALS AND METHODS

Source of Pneumocystis.—Organisms were obtained from the lungs of individual immunosuppressed rats by homogenization and purification processes described in detail within each publication (see Cushion et al 1993a). Lung tissues were homogenized in a Stomacher Lab Blender 80 (Fisher Scientific, Cincinnati, Ohio). Erythrocytes in the homogenate were lysed by treatment with aqueous ammonium chloride (0.85%). Pneumocystis organisms were separated from remaining intact host cells by low- and high-speed centrifugations, followed by filtration through 10 mm filters. Gene sequences.—Sequences used for comparisons were obtained from the literature and from the GenBank database (http://www.ncbi.nlm.nih.gov/Genbank/GenbankOverview. html). These sequences were used: thymidylate synthase: M25415 (P. carinii), (Keely et al 1994); TATA binding protein: U14410 (P. carinii), U14411 (P. wakefieldiae); alphasubunit of a guanine nucleotide binding protein: U30792 (P. carinii), U30790 (P. wakefieldiae); BiP precursor: L46790 (P. carinii), U40994 (P. wakefieldiae); heat shock protein 70: U80968 (P. carinii), U80969 (P. wakefieldiae); mitochondrial large-subunit rRNA regions: M58604 (P. carinii), U20173 (P. wakefieldiae); nuclear small-subunit rRNA: M86760, X12708 (P. carinii); L13614, L11279, L27658 (P. wakefieldiae); and internal transcribed spacer regions (ITS 1 and ITS2) within the nuclear ribosomal RNA locus (Palmer et al 2000). Mitogen-activated protein kinase 2 of P. carinii (Mkp2), F077548, was used to demonstrate the chromosomal localization differences between the two species. The number of each sequence type analyzed and the number of rats used in these analyses are found in each reference. In our own studies of the mtLSU, a minimum of three organism populations from three different rats were used. Electrophoretic karyotyping.—After Pneumocystis organisms were passed through two 10 mm pore filters (Mitex, Millipore Corp., Bedford, Massachusetts), the preparations were treated with DNAase I (Boehringer-Mannheim Biochemicals, Indianapolis, Indiana) at 10 mg/mL in a solution of 150 mM NaCl-10 mM MgCl2-10 mM Tris at pH 7.2 for 30 min at 37 C to digest extracellular DNA. DNAase was inhibited by removing magnesium ions by washing once with 250 mM EDTA and twice with 125 mM EDTA (Cushion et al 1993a, b). Organisms were embedded in 0.8% low-melt agarose (Boehringer-Mannheim). Gel-embedded organisms were digested with 0.25 mg proteinase K (BoehringerMannheim) per mL in a solution of 1% N-lauroylsarcosine (Sigma Chemical Co., St. Louis, Missouri) 20.45 M EDTA0.01 M Tris at 55 C for 24–48 h. Digested samples were stored at 4 C in 0.5 M EDTA. Gels for contour clamped

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homogeneous electrical field (CHEF) electrophoresis contained 1% FMC SeaKem GTG-agarose (SeaKem, Rockland, Maine) prepared in 0.53 TBE (45 mM Tris HCl, 45 mM boric acid, 1.25 mM EDTA) for a total volume of 200 mL and final dimensions of 14 3 21 cm. Electrophoresis was performed using either a Bio-Rad CHEF DR II or CHEF DR III apparatus. Gels were run for 104–144 h, at 14 C, in 0.53 TBE at 3.8V/cm with a 50 s initial pulse that gradually was increased to 100 s. Chromosome size DNA bands were transferred by capillary action to positively charged nylon blots under neutral conditions, UV cross-linked to the membranes and hybridized with gene probes made radioactive with a32P, as described previously (Cushion et al 1993a, b). The karyotypes used for the current study were drawn from a total of 715 organism populations from lungs of individual immunosuppressed rat that were analyzed by CHEF from 1997 through 1999; 460 of the rats had infections that included both P. carinii and P. wakefieldiae; 242 infection were comprised only of P. carini; while 13 rats appeared only to be infected with P. wakefieldiae (Icenhour 2001a). Microscopic analysis.—Analysis of the spatial distribution of the two species in rat lungs by immunofluorescent assay was performed as a part of a dissertation in the laboratory of M. Cushion and briefly described below (Icenhour 2001b). Histological sections of paraformaldehyde-fixed and inflated lungs from rats harboring karyotype-verified mixed P. carinii-P. wakefieldiae first were incubated overnight in a humid chamber with a dilution of 1:60 polyclonal antisera generated from immunized rabbits with purified preparations of P. wakefieldiae (Cocalico Biologicals Inc., Reamstown, Pennsylvania) and labeled with Texas Red (Texas Red-X Protein Labeling Kit, Molecular Probes, Eugene, Oregon). The slides then were washed with 0.25 M Tris-HCl buffer, blocked with 1%BSA/3% FCS for 5 min and incubated 1 h with a 1:20 dilution of monoclonal antibody specific for P. carinii major surface glycoprotein (RA-E7) (Linke et al 1998), which was conjugated to FITC by use of a FluoroTag FITC Conjugation Kit (Sigma, St. Louis, Missouri). The slides were washed in Tris/0.1% Tween for 2 min and in buffer twice for 5 min each wash. The slides were examined by confocal microscopy using a Nikon PCM2000 confocal microscope. Control slides without antibodies were used to establish background fluorescence. Histological sections of one of the rats inoculated with 107 organisms containing equal numbers of P. carinii and P. wakefieldiae are shown in the Results section. The remaining five rats showed the same distribution patterns.

RESULTS

Chromosomes.—The existence of P. wakefieldiae was suggested first by rRNA sequence data (Liu et al 1992). That the two different rRNA sequences were from two discrete Pneumocystis genomes was shown in 1993 (Cushion et al 1993b). Although P. wakefieldiae was found only in the presence of P. carinii, the two genomes were recognizable because they are par-

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titioned into chromosomes of different sizes. Our laboratories have used Contour Clamped Homogeneous Electrical Field (CHEF) pulsed field gel electrophoresis to separate chromosomes of Pneumocystis genomes on the basis of size from hundreds of rats obtained from several commercial vendors (Cushion et al 1993a, b, Stringer et al 1993). These studies have established that there are at least 12 distinct electrophoretic karyotypes of P. carinii which we refer to as ‘‘forms’’ (Rebholz and Cushion 2001). By contrast, all 40 P. wakefieldiae preparations analyzed so far have produced a single electrophoretic karyotype. The P. wakefieldiae electrophoretic karyotype clearly is different from all 12 of the P. carinii karyotype patterns. To illustrate its differences, the chromosomes of P. wakefieldiae were separated on the same CHEF gel with eight of the 12 electrophoretic karyotypes of P. carinii (FIG. 1A). Note the difference in numbers of bands between the two genomes. The P. wakefieldiae electrophoretic karyotype has 14 bands (FIG. 1A, lanes 9, 10), while there are 12–16 bands produced by the P. carinii forms (FIG. 1A, lanes 1–8). The sizes of certain chromosomes are also quite distinct between the two species. The first chromosome-sized band is approximately 660 kb in P. wakefieldiae and ;680 kb in the P. carinii patterns. Bands exclusive to P. wakefieldiae appear at 580, 365 and 345 kb, permitting unambiguous detection of this species in a mixed P. carinii-P. wakefieldiae infection (FIG. 1A, lanes 9, 10). In addition to obvious differences in band sizes and profile patterns, gene localization studies using hybridization of radioactive- or chemically labeled gene probes provided further evidence of the diversity between these two genomes. For example, the mitogen-activated protein kinase 2 gene (Mkp2) mapped to a band of 602 kb in P. carinii while the same gene was found on the 410 Kb band in P. wakefieldiae (FIG. 1B). The gene localization data for each species is listed in TABLE I. It should be noted that the pulsed field gel electrophoresis studies would not have detected co-infections in which the minority species numbered less than 10 000 000 organisms, which is the number of organisms required to produce a band pattern in gels stained with Sybr-Gold. Indeed, subsequent studies using species-specific PCR showed that minor populations of either species can be present in rats that produced only a karyotype of the other Pneumocystis species (Palmer et al 1999). In fact, P. wakefieldiae rarely is found as the single infecting population in a rat lung. A single strand polymorphism technique also was shown to differentiate the two species in mixed infections with varying levels of each organism (Nahimana et al 2001).

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FIG. 1. Electrophoretic karyotypes and hybridization of Mkp2 to Pneumocystis carinii and Pneumocystis wakefieldiae. Panel A: Sybr-gold stained CHEF gels: Lanes: (1) P. carinii form 1; (2) P. carinii form 2; (3) P. carinii form 3; (4) P. carinii form 4 ; (5) P. carinii form 5; (6) P. carinii form 6; (7) P. carinii form 7; (8) P. carinii form 8 (barely visible); (9) P. wakefieldiae; (10) P. wakefieldiae ; (11) P. carinii -P. wakefieldiae from a mixed infection of the same rat lung. Panel B: Hybridization of the blotted karyotype with Mkp2. Lanes follow those in Panel A. Note the different size chromosome bands to which the probe hybridized in P. carinii (Lanes 1–8) and P. wakefieldiae (Lanes 9, 10). Both bands are evident in the mixed infection, Lane 11. Chromosome sizes were calculated by linear regression based on the migration of a 48.5 kb lambda ladder (r 2 5 0.9972).

Molecular genetic divergence.—Divergence between orthologous gene sequences of P. carinii and P. wakefieldiae is summarized in TABLE II. The percent divergence ranges were 4–31%, depending on whether the sequences were from coding or noncoding regions. The degree of sequence variation is similar to that exhibited by different species in other genera of TABLE I.

fungi (Stringer 1996). For instance, in a region of the nuclear locus encoding small ribosomal-subunit rRNA, the two Pneumocystis species are 6.6% divergent, which is similar to the divergence between the two Candida species, C. albicans and C. tropicalis, which are 5.8% different at this locus (Stringer 1996). Divergence values from portions of the 18S

Locations of genes in P. wakefieldiae and P. carinii form 1

Gene

P. wakefieldiae DNA band size (kb)

P. carinii DNA band size (kb); Chromosome band (K)

Reference

Nuclear small subunit rRNA Alpha-subunit of guanine nucleotide binding protein (Pcg1) Enolase (Eno1) Mitogen activated protein kinase 2 (Mkp2) Inosine monophosphate dehydrogenase (IMPDH) a-factor pheromone receptorlike gene (ste3) UCS

530 437

505 460

K7 K9

(Cushion et al 1993b) (Smulian et al 1996)

340 602

530 425

K6 K10/11

(Fox and Smulian 2001) This report

630

425

K10/11

(Weinberg et al 1994)

345

680

K1

(Smulian et al 2001)

415

450

K9

BiP

380

330

K15

(Schaffzin and Stringer 2000, Sunkin and Stringer 1996) (Stedman and Buck 1996)

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Percent divergence between DNA sequences of P. carinii and P. wakefieldiae DNA sequence

% divergence

Thymidylate synthase TATA Binding Protein (TBP) Alpha-subunit of guanine nucleotide binding protein BiP precursor Heat shock protein 70 Mitochondrial large subunit rRNA Nuclear small subunit rRNA

6 9 17.5 17 18 14 6.6

26S rRNA Internal transcribed spacer regions I and II

5 30,31

5.8S

rRNA gene can vary from region to region. Therefore, we compared the divergence values for entire 18S rRNA genes (data not shown). The difference between P. carinii and P. wakefieldiae is 1.8%, which again is greater than the difference between C. albicans and C. tropicalis (1.4%) and nearly triple the 0.7% difference between Saccharomyces cerevisiae and S. paradoxus 18S rRNA genes. In addition, the divergence between P. wakefieldiae and P. carinii 18S rRNA genes exceeds the divergence between P. carinii and P. carinii f. sp. muris (1.3%), which is the type of Pneumocystis found in mice (unpublished data). Another obvious difference is the absence in P. wakefieldii of the 390 bp intron that is present in 39 region of the 18S rRNA gene of P. carinii (Liu et al 1992, Cushion et al 1993b, Ortiz-Rivera et al 1995). Divergence values for the different Pneumocystis species are much larger in the regions between nuclear rRNA genes (internal transcribed spacers [ITS] [TABLE II]). This effect is expected because fungal ITS are known to accumulate substitutions approximately 10 times faster than the flanking genes (Berbee and Taylor 2001). The substitution rate for the 18S rDNA genes of all fungi is 1.26 3 10210 substitutions/site/year, whereas for the spacers it is 0.8 3 1029 for Histoplasma capsulatum and averages 1.4 3 102 9 for several sister species (Kasuga et al 2002). The divergence in sequences between P. carinii and P. wakefieldiae is in contrast to that observed among the 12 different karyotype forms of P. carinii (FIG. 1A). In sequences from five genetic regions of organism populations of defined karyotypes (purified from 2–3 rats each) the sequence differences range was 0–0.8% within the coding sequences and 2–4% in the ITS regions (Cushion 1998a, Keely et al 1996, Stringer 1996). Phenotypic differences.—Analysis of electrophoretic karyotypes identified rats infected predominantly

4

Reference (Keely et al 1994) (Sunkin and Stringer 1995) (Smulian et al 1996) (Stedman and Buck 1996) (Stedman et al 1998) (Sinclair et al 1991) (Edman et al 1988, Liu and Leibowitz 1993, Stringer et al 1989) (Liu et al 1992) (Ortiz-Rivera et al 1994, Ortiz-Rivera et al 1995) (Liu et al 1992)

with either P. carinii or P. wakefieldiae. Organisms prepared from such rats were used in immunoblotting studies that showed differences in antigenic reactivities between these two species (Vasquez et al 1996). Vasquez et al demonstrated that monoclonal antibodies generated to the major surface glycoprotein family (MSG) from P. carinii did not react with proteins of P. wakefieldiae. Likewise, polyclonal antisera made with P. carinii as the immunogen did not react with P. wakefieldiae proteins. Immunostaining of histological sections of rat lung with differentially labeled antibodies generated to the surface antigens of P. carinii and P. wakefieldiae clearly identified these distinct populations within a co-infected rat lung. As shown in FIG. 2, both species coexist in the same lung alveoli in apposition and can be differentiated by antibodies generated to their distinct surface antigens. P. carinii was stained by FITClabeled antibodies and appeared green, while P. wakefieldiae antisera conjugated to Texas Red stained the organisms red. In addition, the two species can be separated physically by a magnetic-bead technique using these antibodies (Icenhour and Cushion 2001). TAXONOMY

Based on the data presented and summarized herein, a new species of rat-derived Pneumocystis is proposed. Pneumocystis wakefieldiae M.T. Cushion, S.P. Keely, et J.R. Stringer, sp.nov.

5 Pneumocystis carinii f. sp. ratti (The Pneumocystis Workshop 1994) 5 Pneumocystis carinii ‘‘variant’’ (Cushion et al 1993b) Descriptio Latinum nova speciae: Non filiosae similes fermento formae quae inhabitant in alveolis pulmoneis rattorum (Rattus norvegicus). Extracellulares et haestae in Typi Primi pneumocytis alveolaris luminis cum coryumbis admixtarum progredientum formarum extendentibus in al-

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FIG. 2. Distribution of Pneumocystis carinii and P. wakefieldiae within rat lung tissue sections. Each panel is a digital image from a rat lung section stained with antibodies specific for P. carinii and P. wakefieldiae. Panel A, negative control (no antibodies); Panels B–E, incubated with both antibodies; Panels B–C, 103 objective; Panels D–E, 1003 objective. P. wakefieldiae organisms appear red and P. carinii organisms are green. veolae lumen. Cellae holitariae (formae trophicae), unusquinque mm, sunt uninucleatae, irregulares, tenuitunicatae, consistae de membranis plasmaticis et dividunt se fissione binaria. Asci (cysti), quinque-octo mm, sunt crassitunicati, globosi, cum duabus membranis plasmaticis, et continent octo rotundos ad ovatiles ascospores, quisque unus-duo mm; vacui, apparent falciformi aut irregulares. P. wakefieldiae est morphologiciter inspectandus a P. carinii, sed duo species sunt valde varii DNA ordine. Differentiae ordinibus orthologosarum genium (differentiae expressae ut per centum) sequunt: thymidylatum synthosum (sex per centum) (Keely et al 1994), TATA-colligans proteinus (novem per centum) (Sunkin and Stringer 1995), alpha-subunitas guanini nucleotidi colligantis proteini (septendecim per centum) (Smulian et al 1996), BiP praenuntius (septendecim per centum) (Stedman and Buck 1996), aestus-offensio proteinus septuaginta (duodeviginti per centum) (Stedman et al 1998), internae transscriptae seperantes regiones intra nuclearem ribosomalem RNA locum (triginta et duo per centum) (Edman et al 1988, Ortiz-Rivera et al 1995). Altera clara DNA differentia est quod P. carinii habet 360 bp intronum in 39 regione 18S rRNA genis, sed P. wakefieldi caret hunc intronum (Edman et al 1989, Liu and Leibowitz 1993).

Typus: Consociatae Civitates Americae, Cincinnatensis, Ohio. Extractae e pulmone duodecim-hebdomacum immunosuppressi Longorum Evanorum ratti (Rattus norvegicus).

Pneumocystis wakefieldiae M.T. Cushion, S.P. Keely and J.R. Stringer, sp. nov. Nonfilamentous yeast-like organisms resident in the pulmonary alveoli of rats (Rattus norvegicus). Extracellular and adhered to Type I pneumocytes of the alveolar lumen with clusters of admixed presumptive developmental stages extending into the alveolar lumen. The vegetative cells (trophic forms) measuring 1–5 mm, are uninucleate, of irregular shape, thinwalled, composed of two plasma membranes and replicate by binary fission. Asci (cysts), measuring 5–8 mm, are thick-walled, globose, with two plasma membranes and contain eight round to ovoid ascospores, each 1–2 mm; when empty, they appear falciform or irregular. P. wakefieldiae is indistinguishable morphologically from P. carinii, but the two species are very different as revealed by DNA sequence. Differences in se-

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quences of orthologous genes (differences expressed as percents) are: thymidylate synthase (6%) (Keely et al 1994), TATA-binding protein (9%) (Sunkin and Stringer 1995), alpha-subunit of a guanine nucleotide binding protein (17%) (Smulian et al 1996), BiP precursor (17%) (Stedman and Buck 1996), heat shock protein 70 (18%) (Stedman et al 1998), ITS regions within the nuclear ribosomal RNA locus (32%) (Edman et al 1989, Ortiz-Rivera et al 1995). Another obvious DNA difference is that P. carinii has a 360 bp intron in the 39 region of the 18S rRNA gene, while P. wakefieldiae lacks this intron (Edman et al 1988, Liu and Leibowitz 1993) TY PE: United States of America. Cincinnati, Ohio. Extracted from lungs of a 12 wk immunosuppressed Long Evans rat (Rattus norvegicus). Organism preparations have been submitted as cryopreserved samples to American Type Culture Collection, accession number pending. Cryopreserved samples are stored at the Cincinnati Veterans Affairs Medical Center, Cincinnati, Ohio. Etymology.—wakefieldiae; after our departed colleague, Ann E. Wakefield, who developed a PCRbased assay to differentiate the two species and whose contributions to the general understanding of the molecular biology, epidemiology and evolution of Pneumocystis have greatly advanced our knowledge of this genus. DISCUSSION

Based on both phenotypic and genetic criteria, the organism formerly named P. carinii f. sp. ratti now is recognized as the distinct species Pneumocystis wakefieldiae. Recognition of Pneumocystis wakefieldiae is based on the following observations. First, P. wakefieldiae is specific to rats. This specificity separates it from all but one other Pneumocystis species, P. carinii. Second, P. wakefieldiae and P. carinii can be separated and analysis of each isolated organism has shown that they exhibit obvious phenotypic and global genetic differences. Such differences are difficult to reconcile with the view that they are the same species. If P. carinii and P. wakefieldiae were members of the same sexual species, their proximity would foster genetic exchange, which would work against the accretion of differences. TABLE II shows that P. wakefieldiae exhibits numerous genetic differences when compared to P. carinii. The existence of these differences is consistent with the notion that P. wakefieldiae is species based on an operational phylogenetic species recognition (PSR) approach, which has been developed for and applied to other fungi (Berbee and Taylor 2001, Fisher et al 2002). PSR relies on DNA sequence data and there-

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fore provides a means to recognize species when the organisms in question have similar morphology and are either asexual or not amenable to analysis based on mating experiments, which is the case for both P. carinii and P. wakefieldiae. Detecting species using PSR, however, requires more than knowledge of sequence divergence because variation can occur within a species. We have used three procedures to gauge the significance of sequence divergence exhibited by P. wakefieldiae. First, we compared the divergence values in TABLE II to those exhibited by species in other genera and within the P. carinii species, as described above. In addition, we have estimated the time elapsed since divergence and tested for concordance of gene genealogies. Time since divergence.—To estimate the time elapsed since divergence, two methods were employed. Method 1 used the rates of change provided by Taylor’s study of 18S rRNA genes and ITS of fungi (Berbee and Taylor 2001). Taylor reported that the average substitution rate for the 18S rDNA gene of fungi is 1.26 3 10210 substitutions/site/year. Because there are 1780 bp in the 18S gene, one expects 2.243 3 1027 changes per gene per year, or 10 000 000 years to reach 2.24 changes. Fifteen million years would be needed to produce the 32 changes seen between P. carinii and P. wakefieldiae. The average substitution rate for the ITS of fungi is 1.4 3 1029. Since there are 600 bp in the ITS region, one expects 8.4 3 1027 changes per gene per year, or 10 000 000 to produce 8.4 changes. Twenty-two million years would be needed to produce the 200 changes seen between P. carinii and P. wakefieldiae. Hence, these calculations indicate that P. carinii and P. wakefieldiae diverged 22– 15 million years BP. In contrast with these data, isolates of P. jirovecii, the human pathogen, exhibit no more than 15 ITS changes when compared one to another, suggesting that strains of P. jirovecii diverged about 2 000 000 years BP. No differences are seen in the 18S genes of P. jirovecii as would be expected if they are this young. The second way to estimate the times of divergence of Pneumocystis species is to examine the timing of divergence among the mammals that host these different organisms. Rats and mice diverged about 14 000 000 years BP, according to fossil records. Molecular data suggest an older split, 30 000 000–40 000 000 years BP. P. carinii is slightly less similar to mouse Pneumocystis than to P. wakefieldiae, by sequence comparisons (Keely et al 1996, Stringer 1996). These data are consistent with those obtained by Method 1 and indicate that P. carinii and P. wakefieldiae have been separate entities for tens of millions years.

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Concordance of gene genealogies.—Another way to test the significance of sequence divergence vis a vis species formation is to test for genealogical concordance by assessing the relationships between presumptive homologous genes. Genealogical concordance means that a tree made from one gene has the same topology as a tree made from another gene. Such concordance is expected if the organisms in question do not exchange genes. At this point, testing for genealogical concordance is limited to two loci, 18S rRNA and thymidylate synthase. These are the only loci for which there are data from P. carinii, P. wakefieldiae and at least two other Pneumocystis species. Sequences are available of the 18S rRNA genes from P. carinii, P. wakefieldiae, P. carinii f. sp. mustelae, P. jirovecii and P. carinii f. sp. muris. Sequences are available of the thymidylate synthase genes from five species, P. carinii. f. sp. mustelae, P. jirovecii, P. wakefieldiae and P. carinii f. sp. muris. The 18S rRNA and thymidylate synthase trees had the same topology. The bootstrap values for the thymidylate synthase tree, represented in Newick Standard notation, were (fungal root, (P. carinii f. sp. mustelae, (P. jirovecii,(P. wakefieldiae, (P. carinii, P. carinii f. sp. muris)98)98)57)99). These data suggest that P. wakefieldiae is the oldest of the three Pneumocystis species found in laboratory rats and mice. Another indication of genealogical concordance is provided by previous studies on a gene family, MSG, members of which constitute nearly 10% of the P. carinii genome. P. wakefieldiae has a similar family of genes, but these differ in sequence by 35% from those in P. carinii. This difference is sufficiently great that an MSG gene probe from P. carinii failed to hybridize to chromosomes from P. wakefieldiae (Cushion et al 1993b). Sequence analysis showed that the MSG genes from the two species form two discrete groups (data not shown). An MSG gene tree made from sequences from both species had two major branches, one of which carried all of the P. carinii genes and the other of which carried all of the P. wakefieldiae MSG genes. These data are consistent with the genealogical concordance data described above and indicate that the two gene families are evolving independently, which would be expected for families in two related species. MSG genes from the two species also differ with respect to sequence elements associated with regulation of transcription (Schaffzin et al 1999, Schaffzin and Stringer 2000). The presence of the genetic and phenotypic divergence between these two species indicates that other biological differences likely exist. Such differences might extend to pathogenicity factors, ability to circumvent the host immune responses, or even growth rates and life cycle differences. The coexistence of

these two species within the same apparent environmental niche raises questions concerning the relationship between them and potential consequences to the host. Recognizing P. wakefieldiae as a species distinct from P. carinii represents the first step toward addressing these intriguing questions. ACKNOWLEDGMENTS

We thank Brian Sebastian, The Seven Hills Schools, Cincinnati, Ohio, for providing the Latin description; Dr. J. Lee Crane, Illinois Natural History Survey, University of Illinois, for advice on the requirements of the International Code of Botanical Nomenclature; and Dr. A. George Smulian for the Mkp2 hybridization data. These studies were supported by the National Institutes of Health, grant numbers R01AI29839. The authors would like to dedicate this publication to the memory of our colleague, Ann E. Wakefield.

LITERATURE CITED

Aliouat EM, Mazars E, Dei-Cas E, Delcourt P, Billaut P, Camus D. 1994. Pneumocystis cross infection experiments using SCID mice and nude rats as recipient host, showed strong host-species specificity. J Eukaryot Microbiol 41:71S. Aliouat E, Dujardin L, Martinez A, Duriez T, Ricard I, DeiCas E. 1999. Pneumocystis carinii growth kinetics in culture systems and in hosts: involvement of each life cycle parasite stage. J Eukaryot Microbiol 46:116S–117S. Berbee ML, Taylor JW. 2001. Fungal molecular evolution: gene trees and geologic time. In: The Mycota: a comprehensive treatise on fungi as experimental systems for basic and applied research. p 229–245. Chagas C. Nova tripanozomiaze humana. 1909. Mem.Inst. Oswaldo.Cruz. 1, 159–218. Cushion MT. 1998. Pneumocystis carinii. In: Collier L, Balows A, Sussman M, eds. Topley and Wilson’s microbiology and microbial infections. New York: Arnold and Oxford Press. p 645–683. . 1998a. Genetic heterogeneity of rat-derived Pneumocystis. FEMS Immunol Med Microbiol 22:51–58. . 1998b. Taxonomy, genetic organization, and life cycle of Pneumocystis carinii. Semin Respir Infect 13:304– 312. , Kaselis M, Stringer SL, Stringer JR. 1993a. Genetic stability and diversity of Pneumocystis carinii infecting rat colonies. Infect Immun 61:4801–4813. , Slaven BE, Smulian AG. 2004. Pneumocystis genome project and genomic organization. In: Walzer PD, Cushion MT, eds. Pneumocystis pneumonia. New York: Marcel-Dekker Inc. (In press) , Zhang J, Kaselis M, Giuntoli D, Stringer SL, Stringer JR. 1993b. Evidence for two genetic variants of Pneumocystis carinii coinfecting laboratory rats. J Clin Microbiol 31:1217–1223. Delanoe¨ P, Delanoe¨ Mme. 1912. Sur les rapports des kystes

CUSHION

ET AL:

PNEUMOCYSTIS WAKEFIELDIAE :

des Carinii des poumon des rats avec le Trypanosoma lewisii. C.R.Acad.Sci. (Paris) 155:658–660. Edman JC, Kovacs JA, Masur H, Santi DV, Elwood HJ, Sogin ML. 1988. Ribosomal RNA sequence shows Pneumocystis carinii to be a member of the fungi. Nature 334: 519–522. , , , , , . 1989. Ribosomal RNA genes of Pneumocystis carinii. J Protozool 36:18S–20S. Fisher MC, Koenig GL, White TJ, Taylor JW. 2002. Molecular and phenotypic description of Coccidioides posadasii sp. nov., previously recognized as the non-California population of Coccidioides immitis. Mycologia 94: 73–84. Fox D, Smulian AG. 2001. Plasminogen-binding activity of enolase in the opportunistic pathogen Pneumocystis carinii. Medical Mycology 39:495–507. Frenkel JK. 1999. Pneurocystis pneumonia, an immunodeficiency-dependent disease (IDD): a critical historical overview. J. Eukaryot. Microbiol. 46:89S–92S. Gigliotti F, Harmsen AG, Haidaris CG, Haidaris PJ. 1993. Pneumocystis carinii is not universally transmissible between mammalian species. Infect Immun 61:2886– 2890. Hughes WT. 1987. Natural occurrence in animals. In: Hughes WT, ed. Pneumocystis carinii pneumonitis. Boca Raton, Florida: CRC Press Inc. p 57–70. Icenhour CRP. 2001a. Experimental evidence for competitive coexistence of two species of Pneumocystis within rat lungs [Doctoral dissertation]. Cincinnati, Ohio; University of Cincinnati. pp. 127–138. Icenhour CRP. 2001b. Experimental evidence for competitive coexistence of two species of Pneumocystis within rat lungs [Doctoral dissertation]. Cincinnati, Ohio; University of Cincinnati. pp. 44–47, 59. Icenhour CR, Cushion MT. 2001. Putative transmissive form of Pneumocystis carinii f. sp. carinii. J Eukaryot Microbiol Suppl:139S–140S. Kasuga T, White TJ, Taylor JW. 2002. Estimation of nucleotide substitution rates in Eurotiomycete fungi. Mol Biol Evol 19:2318–2324. Keely S, Pai HJ, Baughman R, Sidman C, Sunkin SM, Stringer JR, Stringer SL. 1994. Pneumocystis species inferred from analysis of multiple genes. J Eukaryot Microbiol 41:94S. , Cushion MT, Stringer JR. 1996. Stability of four genetic loci in Pneumocystis carinii sp. f. carinii. J Eukaryot Microbiol 43:49S. Linke MJ, Sunkin SM, Andrews RP, Stringer JR, Walzer PD. 1998. Expression, structure, and location of epitopes of the major surface glycoprotein of Pneumocystis carinii f. sp. carinii. Clin Diagn Lab Immunol 5:50–57. Liu Y, Leibowitz MJ. 1993. Variation and in vitro splicing of group I introns in rRNA genes of Pneumocystis carinii. Nucleic Acids Res 21:2415–2421. , Rocourt M, Pan S, Liu C, Leibowitz MJ. 1992. Sequence and variability of the 5.8S and 26S rRNA genes of Pneumocystis carinii. Nucleic Acids Res 20:3763– 3772.

A NEW SPECIES OF

PNEUMOCYSTIS

IN RATS

437

Nahimana A, Cushion MT, Blanc DS, Hauser PM. 2001. Rapid PCR-single-strand conformation polymorphism method to differentiate and estimate relative abundance of Pneumocystis carinii special forms infecting rats. J Clin Microbiol 39:4563–4565. Nielsen MH, Settnes OP, Aliouat EM, Cailliez JC, Dei-Cas E. 1998. Different ultrastructural morphology of Pneumocystis carinii derived from mice, rats, and rabbits. APMIS 106:771–779. Ortiz-Rivera M, Liu Y, Felder R, Leibowitz MJ. 1994. Sequence differences in the rRNA ITS regions identify specific isolates of P. carinii. J Eukaryot Microbiol 41: 107S. Ortiz-Rivera M, Liu Y, Felder R, Leibowitz MJ. 1995. Comparison of coding and spacer region sequences of chromosomal rRNA-coding genes of two sequevars of Pneumocystis carinii. J Eukaryot Microbiol 42:44–49. Palmer RJ, Cushion MT, Wakefield AE. 1999. Discrimination of rat-derived Pneumocystis carinii f. sp. carinii and Pneumocystis carinii f. sp. ratti using the polymerase chain reaction. Mol Cell Probes 13:147–155. , Settnes OP, Lodal J, Wakefield AE. 2000. Population structure of rat-derived Pneumocystis carinii in Danish wild rats. Appl Environ Microbiol 66:4954–4961. Rebholz SL, Cushion MT. 2001. Three new karyotype forms of Pneumocystis carinii f. sp. carinii identified by contoured clamped homogeneous electrical field (CHEF) electrophoresis. J Eukaryot Microbiol Suppl:109S– 110S. Schaffzin JK, Garbe TR, Stringer JR. 1999. Major surface glycoprotein genes from Pneumocystis carinii f. sp. ratti. Fungal Genet Biol 28:214–226. , Stringer JR. 2000. The major surface glycoprotein expression sites of two special forms of rat Pneumocystis carinii differ in structure. J Infect Dis 181:1729–1739. Sinclair K, Wakefield AE, Banerji S, Hopkin JM. 1991. Pneumocystis carinii organisms derived from rat and human hosts are genetically distinct. Mol Biochem Parasitol 45: 183–184. Smulian AG, Ryan M, Staben C, Cushion M. 1996. Signal transduction in Pneumocystis carinii: characterization of the genes (pcg1) encoding the alpha subunit of the G protein (PCG1) of Pneumocystis carinii carinii and Pneumocystis carinii ratti. Infect Immun 64:691–701. , Sesterhenn T, Tanaka R, Cushion MT. 2001. The ste3 pheromone receptor gene of Pneumocystis carinii is surrounded by a cluster of signal transduction genes. Genetics 157:991–1002. Stedman TT, Buck GA. 1996. Identification, characterization and expression of the BiP endoplasmic reticulum resident chaperonins in Pneumocystis carinii. Infect Immun 64:4463–4471. , Butler DR, Buck GA. 1998. The HSP70 gene family in Pneumocystis carinii: molecular and phylogenetic characterization of cytoplasmic members. J Eukaryot Microbiol 45:589–599. Stringer JR. 1996. Pneumocystis carinii: what is it, exactly? Clin Microbiol Rev 9:489–498.

438

MYCOLOGIA

, Stringer SL, Zhang J, Baughman R, Smulian AG, Cushion MT. 1993. Molecular genetic distinction of Pneumocystis carinii from rats and humans. J Eukaryot Microbiol 40:733–741. , Wakefield AE, Cushion MT, Dei-Cas E. 1997. Pneumocystis taxonomy and nomenclature: an update. J Eukaryot Microbiol 44:5S–6S. Stringer SL, Stringer JR, Blase MA, Walzer PD, Cushion MT. 1989. Pneumocystis carinii: sequence from ribosomal RNA implies a close relationship with fungi. Exp Parasitol 68:450–461. Sunkin SM, Stringer JR. 1995. Transcription factor genes from rat Pneumocystis carinii. J Eukaryot Microbiol 42: 12–19. , . 1996. Translocation of surface antigen genes to a unique telomeric expression site in Pneumocystis carinii. Mol Microbiol 19:283–295. The Pneumocystis Workshop. 1994. Revised nomenclature

for Pneumocystis carinii. J Eukaryot Microbiol 41:121S– 122S. Vasquez J, Smulian AG, Linke MJ, Cushion MT. 1996. Antigenic differences associated with genetically distinct Pneumocystis carinii from rats. Infect Immun 64:290– 297. Walzer PD, Schultz MG, Western KA, Robbins JB. 1973. Pneumocystis carinii pneumonia and primary immune deficiency diseases of infancy and childhood. J Pediatr 82:416–422. , , , . 1976. Pneumocystis carinii pneumonia and primary immune deficiency diseases. Natl Cancer Inst Monogr 43:65–74. Weinberg GA, O’Gara MJ, Cushion MT. 1994. Coinfection of rats with genetically diverse forms of Pneumocystis carinii demonstrated by P. carinii inosine monophosphate dehydrogenase gene polymorphism. J Eukaryot Microbiol 41:119S.