Genomic insights into the host specific adaptation of the Pneumocystis genus.

Pneumocystis jirovecii, the fungal agent of human Pneumocystis pneumonia, is closely related to macaque Pneumocystis. Little is known about other Pneumocystis species in distantly related mammals, none of which are capable of establishing infection in humans. The molecular basis of host specificity in Pneumocystis remains unknown as experiments are limited due to an inability to culture any species in vitro. To explore Pneumocystis evolutionary adaptations, we have sequenced the genomes of species infecting macaques, rabbits, dogs and rats and compared them to available genomes of species infecting humans, mice and rats. Complete whole genome sequence data enables analysis and robust phylogeny, identification of important genetic features of the host adaptation, and estimation of speciation timing relative to the rise of their mammalian hosts. Our data reveals insights into the evolution of P. jirovecii, the sole member of the genus able to infect humans.

Diversity and Complexity of the Large Surface Protein Family in the Compacted Genomes of Multiple Pneumocystis Species.

Pneumocystis, a major opportunistic pathogen in patients with a broad range of immunodeficiencies, contains abundant surface proteins encoded by a multicopy gene family, termed the major surface glycoprotein (Msg) gene superfamily. This superfamily has been identified in all Pneumocystis species characterized to date, highlighting its important role in Pneumocystis biology. In this report, through a comprehensive and in-depth characterization of 459 msg genes from 7 Pneumocystis species, we demonstrate, for the first time, the phylogeny and evolution of conserved domains in Msg proteins and provide a detailed description of the classification, unique characteristics, and phylogenetic relatedness of five Msg families. We further describe, for the first time, the relative expression levels of individual msg families in two rodent Pneumocystis species, the substantial variability of the msg repertoires in P. carinii from laboratory and wild rats, and the distinct features of the expression site for the classic msg genes in Pneumocystis from 8 mammalian host species. Our analysis suggests multiple functions for this superfamily rather than just conferring antigenic variation to allow immune evasion as previously believed. This study provides a rich source of information that lays the foundation for the continued experimental exploration of the functions of the Msg superfamily in Pneumocystis biology.IMPORTANCEPneumocystis continues to be a major cause of disease in humans with immunodeficiency, especially those with HIV/AIDS and organ transplants, and is being seen with increasing frequency worldwide in patients treated with immunodepleting monoclonal antibodies. Annual health care associated with Pneumocystis pneumonia costs ∼$475 million dollars in the United States alone. In addition to causing overt disease in immunodeficient individuals, Pneumocystis can cause subclinical infection or colonization in healthy individuals, which may play an important role in species preservation and disease transmission. Our work sheds new light on the diversity and complexity of the msg superfamily and strongly suggests that the versatility of this superfamily reflects multiple functions, including antigenic variation to allow immune evasion and optimal adaptation to host environmental conditions to promote efficient infection and transmission. These findings are essential to consider in developing new diagnostic and therapeutic strategies.

MUC1 mediates Pneumocystis murina binding to airway epithelial cells.

Previous studies have shown that Pneumocystis binds to pneumocytes, but the proteins responsible for binding have not been well defined. Mucins are the major glycoproteins present in mucus, which serves as the first line of defence during airway infection. MUC1 is the best characterised membrane-tethered mucin and is expressed on the surface of most airway epithelial cells. Although by electron microscopy Pneumocystis primarily binds to type I pneumocytes, it can also bind to type II pneumocytes. We hypothesized that Pneumocystis organisms can bind to MUC1 expressed by type II pneumocytes. Overexpression of MUC1 in human embryonic kidney HEK293 cells increased Pneumocystis binding, while knockdown of MUC1 expression by siRNA in A549 cells, a human adenocarcinoma-derived alveolar type II epithelial cell line, decreased Pneumocystis binding. Immunofluorescence labelling indicated that MUC1 and Pneumocystis were co-localised in infected mouse lung tissue. Incubation of A549 cells with Pneumocystis led to phosphorylation of ERK1/2 that increased with knockdown of MUC1 expression by siRNA. Pneumocystis caused increased IL-6 and IL-8 secretion by A549 cells, and knockdown of MUC1 further increased their secretion in A549 cells. Taken together, these results suggest that binding of Pneumocystis to MUC1 expressed by airway epithelial cells may facilitate establishment of productive infection.

Characterization of Pneumocystis murina Bgl2, an Endo-ß-1,3-Glucanase and Glucanosyltransferase.

Glucan is the major cell wall component of Pneumocystis cysts. In the current study, we have characterized Pneumocystis Bgl2 (EC 3.2.1.58), an enzyme with glucanosyltransferase and ß-1,3 endoglucanase activity in other fungi. Pneumocystis murina, Pneumocystis carinii, and Pneumocystis jirovecii bgl2 complementary DNA sequences encode proteins of 437, 447, and 408 amino acids, respectively. Recombinant P. murina Bgl2 expressed in COS-1 cells demonstrated ß-glucanase activity, as shown by degradation of the cell wall of Pneumocystis cysts. It also cleaved reduced laminaripentaose and transferred oligosaccharides, resulting in polymers of 6 and 7 glucan residues, demonstrating glucanosyltransferase activity. Surprisingly, confocal immunofluorescence analysis of P. murina-infected mouse lung sections using an antibody against recombinant Bgl2 showed that the native protein is localized primarily to the trophic form of Pneumocystis in both untreated mice and mice treated with caspofungin, an antifungal drug that inhibits ß-1,3-glucan synthase. Thus, like other fungi, Bgl2 of Pneumocystis has both endoglucanase and glucanosyltransferase activities. Given that it is expressed primarily in trophic forms, further studies are needed to better understand its role in the biology of Pneumocystis.

Genetic diversity of Pneumocystis jirovecii from a cluster of cases of pneumonia in renal transplant patients: Cross-sectional study.

Pneumocystis jirovecii can cause severe potentially life-threatening pneumonia (PCP) in kidney transplant patients. Prophylaxis of patients against PCP in this setting is usually performed during 6 months after transplantation. The aim of this study is to describe the molecular epidemiology of a cluster of PCP in renal transplant recipients in Brazil. Renal transplant patients who developed PCP between May and December 2011 had their formalin-fixed paraffin-embedded (FFPE) lung biopsy samples analysed. Pneumocystis jirovecii 23S mitochondrial large subunit of ribosomal RNA (23S mtLSU-rRNA), 26S rRNA, and dihydropteroate synthase (DHPS) genes were amplified by polymerase chain reaction (PCR), sequenced, and analysed for genetic variation. During the study period, 17 patients developed PCP (only four infections were documented within the first year after transplantation) and six (35.3%) died. Thirty FFPE samples from 11 patients, including one external control HIV-infected patient, had fungal DNA successfully extracted for further amplification and sequencing for all three genes. A total of five genotypes were identified among the 10 infected patients. Of note, four patients were infected by more than one genotype and seven patients were infected by the same genotype. DNA extracted from FFPE samples can be used for genotyping; this approach allowed us to demonstrate that multiple P. jirovecii strains were responsible for this cluster, and one genotype was found infecting seven patients. The knowledge of the causative agents of PCP may help to develop new initiatives for control and prevention of PCP among patients undergoing renal transplant and improve routine PCP prophylaxis.

The Major Surface Glycoprotein of Pneumocystis murina Does Not Activate Dendritic Cells.

The major surface glycoprotein (Msg) is the most abundant surface protein among Pneumocystis species. Given that Msg is present on both the cyst and trophic forms of Pneumocystis and that dendritic cells play a critical role in initiating host immune responses, we undertook studies to examine activation of bone marrow-derived myeloid dendritic cells by Msg purified from Pneumocystis murina. Incubation of dendritic cells with Msg did not lead to increased expression of CD40, CD80, CD86, or major histocompatibility complex class II or to increased secretion of any of 10 cytokines. Microarray analysis identified very few differentially expressed genes. In contrast, lipopolysaccharide-activated dendritic cells had positive results of all of these assays. However, Msg did bind to mouse mannose macrophage receptor and human DC-SIGN, 2 C-type lectins expressed by dendritic cells that are important in recognition of pathogen-associated high-mannose glycoproteins. Deglycosylation of Msg demonstrated that this binding was dependent on glycosylation. These studies suggest that Pneumocystis has developed a mechanism to avoid activation of dendritic cells, potentially by the previously identified loss of genes that are responsible for the high level of protein mannosylation found in other fungi.

Comparative Population Genomics Analysis of the Mammalian Fungal Pathogen Pneumocystis.

Pneumocystis species are opportunistic mammalian pathogens that cause severe pneumonia in immunocompromised individuals. These fungi are highly host specific and uncultivable in vitro Human Pneumocystis infections present major challenges because of a limited therapeutic arsenal and the rise of drug resistance. To investigate the diversity and demographic history of natural populations of Pneumocystis infecting humans, rats, and mice, we performed whole-genome and large-scale multilocus sequencing of infected tissues collected in various geographic locations. Here, we detected reduced levels of recombination and variations in historical demography, which shape the global population structures. We report estimates of evolutionary rates, levels of genetic diversity, and population sizes. Molecular clock estimates indicate that Pneumocystis species diverged before their hosts, while the asynchronous timing of population declines suggests host shifts. Our results have uncovered complex patterns of genetic variation influenced by multiple factors that shaped the adaptation of Pneumocystis populations during their spread across mammals.IMPORTANCE Understanding how natural pathogen populations evolve and identifying the determinants of genetic variation are central issues in evolutionary biology. Pneumocystis, a fungal pathogen which infects mammals exclusively, provides opportunities to explore these issues. In humans, Pneumocystis can cause a life-threatening pneumonia in immunosuppressed individuals. In analysis of different Pneumocystis species infecting humans, rats, and mice, we found that there are high infection rates and that natural populations maintain a high level of genetic variation despite low levels of recombination. We found no evidence of population structuring by geography. Our comparisons of the times of divergence of these species to their respective hosts suggest that Pneumocystis may have undergone recent host shifts. The results demonstrate that Pneumocystis strains are widely disseminated geographically and provide a new understanding of the evolution of these pathogens.

ß-Glucans Are Masked but Contribute to Pulmonary Inflammation During Pneumocystis Pneumonia.

ß-glucans, which can activate innate immune responses, are a major component in the cell wall of the cyst form of Pneumocystis In the current study, we examined whether ß-1,3-glucans are masked by surface proteins in Pneumocystis and what role ß-glucans play in Pneumocystis-associated inflammation. For 3 species, including Pneumocystis jirovecii, which causes Pneumocystis pneumonia in humans, Pneumocystis carinii, and Pneumocystis murina, ß-1,3-glucans were masked in most organisms, as demonstrated by increased exposure following trypsin treatment. Using quantitative polymerase chain reaction and microarray techniques, we demonstrated in a mouse model of Pneumocystis pneumonia that treatment with caspofungin, an inhibitor of ß-1,3-glucan synthesis, for 21 days decreased expression of a broad panel of inflammatory markers, including interferon γ, tumor necrosis factor α, interleukin 1ß, interleukin 6, and multiple chemokines/chemokine ligands. Thus, ß-glucans in Pneumocystis cysts are largely masked, which likely decreases innate immune activation; this mechanism presumably was developed for interactions with immunocompetent hosts, in whom organism loads are substantially lower. In immunosuppressed hosts with a high organism burden, organism death and release of glucans appears to be an important contributor to deleterious host inflammatory responses.

Distinguishing highly similar gene isoforms with a clustering-based bioinformatics analysis of PacBio single-molecule long reads.

BACKGROUND: Gene isoforms are commonly found in both prokaryotes and eukaryotes. Since each isoform may perform a specific function in response to changing environmental conditions, studying the dynamics of gene isoforms is important in understanding biological processes and disease conditions. However, genome-wide identification of gene isoforms is technically challenging due to the high degree of sequence identity among isoforms. Traditional targeted sequencing approach, involving Sanger sequencing of plasmid-cloned PCR products, has low throughput and is very tedious and time-consuming. Next-generation sequencing technologies such as Illumina and 454 achieve high throughput but their short read lengths are a critical barrier to accurate assembly of highly similar gene isoforms, and may result in ambiguities and false joining during sequence assembly. More recently, the third generation sequencer represented by the PacBio platform offers sufficient throughput and long reads covering the full length of typical genes, thus providing a potential to reliably profile gene isoforms. However, the PacBio long reads are error-prone and cannot be effectively analyzed by traditional assembly programs. RESULTS: We present a clustering-based analysis pipeline integrated with PacBio sequencing data for profiling highly similar gene isoforms. This approach was first evaluated in comparison to de novo assembly of 454 reads using a benchmark admixture containing 10 known, cloned msg genes encoding the major surface glycoprotein of Pneumocystis jirovecii. All 10 msg isoforms were successfully reconstructed with the expected length (~1.5 kb) and correct sequence by the new approach, while 454 reads could not be correctly assembled using various assembly programs. When using an additional benchmark admixture containing 22 known P. jirovecii msg isoforms, this approach accurately reconstructed all but 4 these isoforms in their full-length (~3 kb); these 4 isoforms were present in low concentrations in the admixture. Finally, when applied to the original clinical sample from which the 22 known msg isoforms were cloned, this approach successfully identified not only all known isoforms accurately (~3 kb each) but also 48 novel isoforms. CONCLUSIONS: PacBio sequencing integrated with the clustering-based analysis pipeline achieves high-throughput and high-resolution discrimination of highly similar sequences, and can serve as a new approach for genome-wide characterization of gene isoforms and other highly repetitive sequences.

Genome analysis of three Pneumocystis species reveals adaptation mechanisms to life exclusively in mammalian hosts.

Pneumocystis jirovecii is a major cause of life-threatening pneumonia in immunosuppressed patients including transplant recipients and those with HIV/AIDS, yet surprisingly little is known about the biology of this fungal pathogen. Here we report near complete genome assemblies for three Pneumocystis species that infect humans, rats and mice. Pneumocystis genomes are highly compact relative to other fungi, with substantial reductions of ribosomal RNA genes, transporters, transcription factors and many metabolic pathways, but contain expansions of surface proteins, especially a unique and complex surface glycoprotein superfamily, as well as proteases and RNA processing proteins. Unexpectedly, the key fungal cell wall components chitin and outer chain N-mannans are absent, based on genome content and experimental validation. Our findings suggest that Pneumocystis has developed unique mechanisms of adaptation to life exclusively in mammalian hosts, including dependence on the lungs for gas and nutrients and highly efficient strategies to escape both host innate and acquired immune defenses.

Pneumocystis encodes a functional endo-ß-1,3-glucanase that is expressed exclusively in cysts.

ß-1,3-glucan is a major cell wall component of Pneumocystis cysts. We have characterized endo-ß-1,3-glucanase (Eng) from 3 species of Pneumocystis. The gene eng is a single-copy gene that encodes a protein containing 786 amino acids in P. carinii and P. murina, and 788 amino acids in P. jirovecii, including a signal peptide for the former 2 but not the latter. Recombinant Eng expressed in Escherichia coli was able to solubilize the major surface glycoprotein of Pneumocystis, thus potentially facilitating switching of the expressed major surface glycoprotein (Msg) variant. Confocal immunofluorescence analysis of P. murina-infected mouse lung sections localized Eng exclusively to the cyst form of Pneumocystis. No Eng was detected after mice were treated with caspofungin, a ß-1,3-glucan synthase inhibitor that is known to reduce the number of cysts. Thus, Eng is a cyst-specific protein that may play a role in Msg variant expression in Pneumocystis.

A Benchmark Study on Error Assessment and Quality Control of CCS Reads Derived from the PacBio RS.

PacBio RS, a newly emerging third-generation DNA sequencing platform, is based on a real-time, single-molecule, nano-nitch sequencing technology that can generate very long reads (up to 20-kb) in contrast to the shorter reads produced by the first and second generation sequencing technologies. As a new platform, it is important to assess the sequencing error rate, as well as the quality control (QC) parameters associated with the PacBio sequence data. In this study, a mixture of 10 prior known, closely related DNA amplicons were sequenced using the PacBio RS sequencing platform. After aligning Circular Consensus Sequence (CCS) reads derived from the above sequencing experiment to the known reference sequences, we found that the median error rate was 2.5% without read QC, and improved to 1.3% with an SVM based multi-parameter QC method. In addition, a De Novo assembly was used as a downstream application to evaluate the effects of different QC approaches. This benchmark study indicates that even though CCS reads are post error-corrected it is still necessary to perform appropriate QC on CCS reads in order to produce successful downstream bioinformatics analytical results.

Outbreak of pneumocystis pneumonia in renal and liver transplant patients caused by genotypically distinct strains of Pneumocystis jirovecii.

BACKGROUND: An outbreak of 29 cases of Pneumocystis jirovecii pneumonia (PCP) occurred among renal and liver transplant recipients (RTR and LTR) in the largest Danish transplantation centre between 2007 and 2010, when routine PCP prophylaxis was not used. METHODS: P. jirovecii isolates from 22 transplant cases, 2 colonized RTRs, and 19 Pneumocystis control samples were genotyped by restriction fragment length polymorphism and multilocus sequence typing analysis. Contact tracing was used to investigate transmission. Potential risk factors were compared between PCP cases and matched non-PCP transplant patients. RESULTS: Three unique Pneumocystis genotypes were shared among 19 of the RTRs, LTRs, and a colonized RTR in three distinct clusters, two of which overlapped temporally. In contrast, Pneumocystis control samples harbored a wide range of genotypes. Evidence of possible nosocomial transmission was observed. Among several potential risk factors, only cytomegalovirus viremia was consistently associated with PCP (P=0.03; P=0.009). Mycophenolate mofetil was associated with PCP risk only in the RTR population (P=0.04). CONCLUSION: We identified three large groups infected with unique strains of Pneumocystis and provide evidence of an outbreak profile and nosocomial transmission. LTRs may be infected in PCP outbreaks simultaneously with RTRs and by the same strains, most likely by interhuman transmission. Patients are at risk several years after transplantation, but the risk is highest during the first 6 months after transplantation. Because patients at risk cannot be identified clinically and outbreaks cannot be predicted, 6 months of PCP chemoprophylaxis should be considered for all RTRs and LTRs.

Characterization of pneumocystis major surface glycoprotein gene (msg) promoter activity in Saccharomyces cerevisiae.

Major surface glycoprotein (Msg), the most abundant cell surface protein of Pneumocystis, plays an important role in the interaction of this opportunistic pathogen with host cells, and its potential for antigenic variation may facilitate evasion of host immune responses. In the present study, we have identified and characterized the promoter region of msg in 3 species of Pneumocystis: P. carinii, P. jirovecii, and P. murina. Because Pneumocystis cannot be cultured, promoter activity was measured in Saccharomyces cerevisiae, a related fungus, using a yeast vector modified to utilize the gene coding for Renilla luciferase as a reporter gene. The 5'-flanking sequences of msg from all three Pneumocystis species showed considerable promoter activity, with increases in luciferase activity up to 15- to 44-fold above baseline. Progressive deletions helped define an ∼13-bp sequence in each Pneumocystis species that appears to be critical for promoter activity. Electrophoretic mobility shift analysis using P. carinii-specific msg promoter sequences demonstrated binding of nuclear proteins of S. cerevisiae. The 144-bp 5'-flanking region of P. murina msg showed 72% identity to that of P. carinii. The 5'-flanking region of P. jirovecii msg showed 58 and 61% identity to those of P. murina and P. carinii, respectively. The msg promoter is a good candidate for inclusion in a construct designed for genetic manipulation of Pneumocystis species.

Expression of Pneumocystis jirovecii major surface glycoprotein in Saccharomyces cerevisiae.

The major surface glycoprotein (Msg), which is the most abundant protein expressed on the cell surface of Pneumocystis organisms, plays an important role in the attachment of this organism to epithelial cells and macrophages. In the present study, we expressed Pneumocystis jirovecii Msg in Saccharomyces cerevisiae, a phylogenetically related organism. Full-length P. jirovecii Msg was expressed with a DNA construct that used codons optimized for expression in yeast. Unlike in Pneumocystis organisms, recombinant Msg localized to the plasma membrane of yeast rather than to the cell wall. Msg expression was targeted to the yeast cell wall by replacing its signal peptide, serine-threonine-rich region, and glycophosphatidylinositol anchor signal region with the signal peptide of cell wall protein α-agglutinin of S. cerevisiae, the serine-threonine-rich region of epithelial adhesin (Epa1) of Candida glabrata, and the carboxyl region of the cell wall protein (Cwp2) of S. cerevisiae, respectively. Immunofluorescence analysis and treatment with ß-1,3 glucanase demonstrated that the expressed Msg fusion protein localized to the yeast cell wall. Surface expression of Msg protein resulted in increased adherence of yeast to A549 alveolar epithelial cells. Heterologous expression of Msg in yeast will facilitate studies of the biologic properties of Pneumocystis Msg.

Sequencing and characterization of the complete mitochondrial genomes of three Pneumocystis species provide new insights into divergence between human and rodent Pneumocystis.

Pneumocystis jirovecii is an important opportunistic pathogen associated with AIDS and other immunodeficient conditions. Currently, very little is known about its nuclear and mitochondrial genomes. In this study, we sequenced the complete mitochondrial genome (mtDNA) of this organism and its closely related species Pneumocystis carinii and Pneumocystis murina by a combination of sequencing technologies. Our study shows that P. carinii and P. murina mtDNA share a nearly identical number and order of genes in a linear configuration, whereas P. jirovecii has a circular mtDNA containing nearly the same set of genes but in a different order. Detailed studies of the mtDNA terminal structures of P. murina and P. carinii suggest a unique replication mechanism for linear mtDNA. Phylogenetic analysis supports a close association of Pneumocystis species with Taphrina, Saitoella, and Schizosaccharomyces, and divergence within Pneumocystis species, with P. murina and P. carinii being more closely related to each other than either is to P. jirovecii. Comparative analysis of four complete P. jirovecii mtDNA sequences in this study and previously reported mtDNA sequences for diagnosing and genotyping suggests that the current diagnostic and typing methods can be improved using the complete mtDNA data. The availability of the complete P. jirovecii mtDNA also opens the possibility of identifying new therapeutic targets.

Outbreaks of Pneumocystis pneumonia in 2 renal transplant centers linked to a single strain of Pneumocystis: implications for transmission and virulence.

BACKGROUND: There have been numerous reports of clustered outbreaks of Pneumocystis pneumonia (PCP) at renal transplant centers over the past 2 decades. It has been unclear whether these outbreaks were linked epidemiologically to 1 or several unique strains, which could have implications for transmission patterns or strain virulence. METHODS: Restriction fragment length polymorphism (RFLP) analysis was used to compare Pneumocystis isolates from 3 outbreaks of PCP in renal transplant patients in Germany, Switzerland, and Japan, as well as nontransplant isolates from both human immunodeficiency virus (HIV)-infected and uninfected patients. RESULTS: Based on RFLP analysis, a single Pneumocystis strain caused pneumonia in transplant patients in Switzerland (7 patients) and Germany (14 patients). This strain was different from the strain that caused an outbreak in transplant patients in Japan, as well as strains causing sporadic cases of PCP in nontransplant patients with or without HIV infection. CONCLUSIONS: Two geographically distinct clusters of PCP in Europe were due to a single strain of Pneumocystis. This suggests either enhanced virulence of this strain in transplant patients or a common, but unidentified, source of transmission. Outbreaks of PCP can be better understood by enhanced knowledge of transmission patterns and strain variation.

Characterization of the meiosis-specific recombinase Dmc1 of pneumocystis.

The life cycle of Pneumocystis, which causes life-threatening pneumonia in immunosuppressed patients, remains poorly defined. In the present study, we have identified and characterized an orthologue of dmc1, a gene specific for meiotic recombination in yeast, in 3 species of Pneumocystis. dmc1 is a single-copy gene that is transcribed as ∼1.2-kb messenger RNA, which encodes a protein of 336-337 amino acids. Pneumocystis Dmc1 was 61%-70% identical to those from yeast. Confocal microscopy results indicated that the expression of Dmc1 is primarily confined to the cyst form of Pneumocystis. By sequence analysis of 2 single-copy regions of the human Pneumocystis jirovecii genome, we can infer multiple recombination events, which are consistent with meiotic recombination in this primarily haploid organism. Taken together, these studies support the occurrence of a sexual phase in the life cycle of Pneumocystis.

Restriction fragment length polymorphism typing demonstrates substantial diversity among Pneumocystis jirovecii isolates.

Better understanding of the epidemiology and transmission patterns of human Pneumocystis should lead to improved strategies for preventing Pneumocystis pneumonia (PCP). We have developed a typing method for Pneumocystis jirovecii that is based on restriction fragment length polymorphism (RFLP) analysis after polymerase chain reaction amplification of an approximately 1300 base-pair region of the msg gene family, which comprises an estimated 50-100 genes/genome. The RFLP pattern was reproducible in samples containing >1000 msg copies/reaction and was stable over time, based on analysis of serial samples from the same patient. In our initial analysis of 48 samples, we found that samples obtained from different individuals showed distinct banding patterns; only samples obtained from the same patient showed an identical RFLP pattern. Despite this substantial diversity, samples tended to cluster on the basis of country of origin. In an evaluation of samples obtained from an outbreak of PCP in kidney transplant recipients in Germany, RFLP analysis demonstrated identical patterns in samples that were from 12 patients previously linked to this outbreak, as well as from 2 additional patients. Our results highlight the presence of a remarkable diversity in human Pneumocystis strains. RFLP may be very useful for studying clusters of PCP in immunosuppressed patients, to determine whether there is a common source of infection.

Variation in the major surface glycoprotein genes in Pneumocystis jirovecii.

The genome of Pneumocystis, which causes life-threatening pneumonia in immunosuppressed patients, contains a multicopy gene family that encodes the major surface glycoprotein (Msg). Pneumocystis can vary the expressed Msg, presumably as a mechanism to avoid host immune responses. Analysis of 24 msg-gene sequences obtained from a single human isolate of Pneumocystis demonstrated that the sequences segregate into 2 branches. Results of a number of analyses suggest that recombination between msg genes is an important mechanism for generating msg diversity. Intrabranch recombination occurred more frequently than interbranch recombination. Restriction-fragment length polymorphism analysis of human isolates of Pneumocystis demonstrated substantial variation in the repertoire of the msg-gene family, variation that was not observed in laboratory isolates of Pneumocystis in rats or mice; this may be the result of examining outbred versus captive populations. Increased diversity in the Msg repertoire, generated in part by recombination, increases the potential for antigenic variation in this abundant surface protein.