Fungal antigenic variation using mosaicism and reassortment of subtelomeric genes' repertoires.

Surface antigenic variation is crucial for major pathogens that infect humans. To escape the immune system, they exploit various mechanisms. Understanding these mechanisms is important to better prevent and fight the deadly diseases caused. Those used by the fungus Pneumocystis jirovecii that causes life-threatening pneumonia in immunocompromised individuals remain poorly understood. Here, though this fungus is currently not cultivable, our detailed analysis of the subtelomeric sequence motifs and genes encoding surface proteins suggests that the system involves the reassortment of the repertoire of ca. 80 non-expressed genes present in each strain, from which single genes are retrieved for mutually exclusive expression. Dispersion of the new repertoires, supposedly by healthy carrier individuals, appears very efficient because identical alleles are observed in patients from different countries. Our observations reveal a unique strategy of antigenic variation. They also highlight the possible role in genome rearrangements of small imperfect mirror sequences forming DNA triplexes.

Anidulafungin Treatment Blocks the Sexual Cycle of Pneumocystis murina and Prevents Growth and Survival without Rescue by an Alternative Mode of Replication.

The proposed life cycle of fungi in the genus Pneumocystis has typically included both an asexual cycle via binary fission and a sexual cycle. Until recently, the strategy used for sexual replication was largely unknown, but genomic and functional assays now support a mode known as primary homothallism (self-fertilization). The question of whether an asexual cycle contributes to the growth of these fungi remains. Treatment of Pneumocystis pneumonia in immunosuppressed rodent models with the class of drugs known as echinocandins is challenging the historical concept of asexual replication. The echinocandins target 1,3-ß-D-glucan (BG) synthesis resulting in death for most fungi. Because Pneumocystis species have both non-BG expressing life cycle stages (trophic forms) and BG-expressing asci, treatment with anidulafungin and caspofungin resulted in elimination of asci, with large numbers of non-BG expressing organisms remaining in the lungs. Transcriptional analyses of anidulafungin treated Pneumocystis murina-infected lungs indicated that these agents were blocking the sexual cycle. In the present study, we explored whether there was an asexual or alternative method of replication that could rescue P. murina survival and growth in the context of anidulafungin treatment. The effects of anidulafungin treatment on early events in the sexual cycle were investigated by RT-qPCR targeting specific mating genes, including mam2, map3, matMi, matPi, and matMc. Results from the in vivo and gene expression studies clearly indicated there was no rescue by an asexual cycle, supporting these fungi's reliance on the sexual cycle for survival and growth. Dysregulation of mating-associated genes showed that anidulafungin induced effects early in the mating process. IMPORTANCE The concept of a sexually obligate fungus is unique among human fungal pathogens. This reliance can be exploited for drug development and here we show a proof of principle for this unusual target. Most human fungal pathogens eschew the mammalian environment with its battery of immune responses. Pneumocystis appear to have evolved to survive in such an environment, perhaps by using sexual replication to help in DNA repair and to introduce genetic variation in its major surface antigen family because the lung is the primary environment of these pathogens. The concept of primary homothallism fits well into its chosen ecosystem, with ready mating partners expressing both mating type receptors, and a sexual cycle that can introduce beneficial genetic variation without the need for outbreeding.

Cell Fusion May Be Involved in the Homothallic Mating of Pneumocystis Species.

Pneumocystis species are obligate fungal biotrophs that colonize the lungs of mammals. They cause deadly pneumonia in immunocompromised hosts. The sexual phase seems obligate during their life cycle and essential for survival because it is believed to ensure proliferation and transmission between hosts. Here, we consider if the sexual phase is initiated by the fusion of two cells or by nucleus duplication in order to generate diploid cells that can undergo meiosis. The juxtaposition of the nucleus-associated organelles of pairs of cells with fused cytoplasmic membranes demonstrated that cell fusion can occur. Nevertheless, the frequency of cell fusion remains to be determined, and it cannot be excluded that both cell fusion and nucleus duplication are used to ensure the occurrence of the essential sexual phase. In vitro culturing of these fungi is a major milestone that could clarify the issue.

Pneumocystis Mating-Type Locus and Sexual Cycle during Infection.

Pneumocystis species colonize mammalian lungs and cause deadly pneumonia if the immune system of the host weakens. Each species presents a specificity for a single mammalian host species. Pneumocystis jirovecii infects humans and provokes pneumonia, which is among the most frequent invasive fungal infections. The lack of in vitro culture methods for these fungi complicates their study. Recently, high-throughput sequencing technologies followed by comparative genomics have allowed a better understanding of the mechanisms involved in the sexuality of Pneumocystis organisms. The structure of their mating-type locus corresponding to a fusion of two loci, Plus and Minus, and the concomitant expression of the three mating-type genes revealed that their mode of sexual reproduction is primarily homothallism. This mode is favored by microbial pathogens and involves a single self-compatible mating type that can enter into the sexual cycle on its own. Pneumocystis sexuality is obligatory within the host's lungs during pneumonia in adults, primary infection in children, and possibly colonization. This sexuality participates in cell proliferation, airborne transmission to new hosts, and probably antigenic variation, processes that are crucial to ensure the survival of the fungus. Thus, sexuality is central in the Pneumocystis life cycle. The obligate biotrophic parasitism with obligate sexuality of Pneumocystis is unique among fungi pathogenic to humans. Pneumocystis organisms are similar to the plant fungal obligate biotrophs that complete their entire life cycle within their hosts, including sex, and that are also difficult to grow in vitro.

Expression Pattern of the Pneumocystis jirovecii Major Surface Glycoprotein Superfamily in Patients with Pneumonia.

BACKGROUND: The human pathogen Pneumocystis jirovecii harbors 6 families of major surface glycoproteins (MSGs) encoded by a single gene superfamily. MSGs are presumably responsible for antigenic variation and adhesion to host cells. The genomic organization suggests that a single member of family I is expressed at a given time per cell, whereas members of the other families are simultaneously expressed. METHODS: We analyzed RNA sequences expressed in several clinical samples, using specific weighted profiles for sorting of reads and calling of single-nucleotide variants to estimate the diversity of the expressed genes. RESULTS: A number of different isoforms of at least 4 MSG families were expressed simultaneously, including isoforms of family I, for which confirmation was obtained in the wet laboratory. CONCLUSION: These observations suggest that every single P. jirovecii population is made of individual cells with distinct surface properties. Our results enhance our understanding of the unique antigenic variation system and cell surface structure of P. jirovecii.

Ability of quantitative PCR to discriminate Pneumocystis jirovecii pneumonia from colonization.

Introduction. Pneumocystis jirovecii pneumonia (PCP) is a severe disease affecting immunocompromised patients. Diagnosis is difficult due to the low sensitivity of direct examination and inability to grow the pathogen in culture. Quantitative PCR in bronchoalveolar lavage fluid (BAL) has high sensitivity, but limited specificity for distinguishing PCP from colonization.Aim. To assess the performance of an in-house quantitative PCR to discriminate between PCP and colonization.Methodology. This was a single-centre retrospective study including all patients with a positive PCR result for P. jirovecii in BAL between 2009 and 2017. Irrespective of PCR results, PCP was defined as the presence of host factors and clinical/radiological criteria consistent with PCP and (i) the presence of asci at direct examination of respiratory sample or (ii) anti-PCP treatment initiated with clinical response and absence of alternative diagnosis. Colonization was considered for cases who did not receive anti-PCP therapy with a favourable outcome or an alternative diagnosis. Cases who did not meet the above mentioned criteria were classified as 'undetermined'.Results. Seventy-one patients with positive P. jirovecii PCR were included (90 % non-HIV patients). Cases were classified as follows: 37 PCP, 22 colonization and 12 undetermined. Quantitative PCR values in BAL were significantly higher in patients with PCP versus colonization or undetermined (P<0.0001). The cut-off of 5×103 copies/ml was able to discriminate PCP cases from colonization with 97 % sensitivity, 82 % specificity, 90 % positive predictive value and 95 % negative predictive value.Conclusions. Our quantitative PCR for P. jirovecii in BAL was reliable to distinguish PCP cases from colonization in this predominantly non-HIV population.

Genomics and evolution of Pneumocystis species.

The genus Pneumocystis comprises highly diversified fungal species that cause severe pneumonia in individuals with a deficient immune system. These fungi infect exclusively mammals and present a strict host species specificity. These species have co-diverged with their hosts for long periods of time (> 100 MYA). Details of their biology and evolution are fragmentary mainly because of a lack of an established long-term culture system. Recent genomic advances have unlocked new areas of research and allow new hypotheses to be tested. We review here new findings of the genomic studies in relation with the evolutionary trajectory of these fungi and discuss the impact of genomic data analysis in the context of the population genetics. The combination of slow genome decay and limited expansion of specific gene families and introns reflect intimate interactions of these species with their hosts. The evolutionary adaptation of these organisms is profoundly influenced by their population structure, which in turn is determined by intrinsic features such as their self-fertilizing mating system, high host specificity, long generation times, and transmission mode. Essential key questions concerning their adaptation and speciation remain to be answered. The next cornerstone will consist in the establishment of a long-term culture system and genetic manipulation that should allow unravelling the driving forces of Pneumocystis species evolution.

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.

Mechanisms of Surface Antigenic Variation in the Human Pathogenic Fungus Pneumocystis jirovecii.

Microbial pathogens commonly escape the human immune system by varying surface proteins. We investigated the mechanisms used for that purpose by Pneumocystis jirovecii This uncultivable fungus is an obligate pulmonary pathogen that in immunocompromised individuals causes pneumonia, a major life-threatening infection. Long-read PacBio sequencing was used to assemble a core of subtelomeres of a single P. jirovecii strain from a bronchoalveolar lavage fluid specimen from a single patient. A total of 113 genes encoding surface proteins were identified, including 28 pseudogenes. These genes formed a subtelomeric gene superfamily, which included five families encoding adhesive glycosylphosphatidylinositol (GPI)-anchored glycoproteins and one family encoding excreted glycoproteins. Numerical analyses suggested that diversification of the glycoproteins relies on mosaic genes created by ectopic recombination and occurs only within each family. DNA motifs suggested that all genes are expressed independently, except those of the family encoding the most abundant surface glycoproteins, which are subject to mutually exclusive expression. PCR analyses showed that exchange of the expressed gene of the latter family occurs frequently, possibly favored by the location of the genes proximal to the telomere because this allows concomitant telomere exchange. Our observations suggest that (i) the P. jirovecii cell surface is made of a complex mixture of different surface proteins, with a majority of a single isoform of the most abundant glycoprotein, (ii) genetic mosaicism within each family ensures variation of the glycoproteins, and (iii) the strategy of the fungus consists of the continuous production of new subpopulations composed of cells that are antigenically different.IMPORTANCEPneumocystis jirovecii is a fungus causing severe pneumonia in immunocompromised individuals. It is the second most frequent life-threatening invasive fungal infection. We have studied the mechanisms of antigenic variation used by this pathogen to escape the human immune system, a strategy commonly used by pathogenic microorganisms. Using a new DNA sequencing technology generating long reads, we could characterize the highly repetitive gene families encoding the proteins that are present on the cellular surface of this pest. These gene families are localized in the regions close to the ends of all chromosomes, the subtelomeres. Such chromosomal localization was found to favor genetic recombinations between members of each gene family and to allow diversification of these proteins continuously over time. This pathogen seems to use a strategy of antigenic variation consisting of the continuous production of new subpopulations composed of cells that are antigenically different. Such a strategy is unique among human pathogens.

Diversity of Pneumocystis jirovecii Across Europe: A Multicentre Observational Study.

Pneumocystis jirovecii is an airborne human-specific ascomycetous fungus responsible for Pneumocystis pneumonia (PCP) in immunocompromised patients, affecting >500,000 patients per year (www.gaffi.org). The understanding of its epidemiology is limited by the lack of standardised culture. Recent genotyping data suggests a limited genetic diversity of P. jirovecii. The objective of the study was to assess the diversity of P. jirovecii across European hospitals and analyse P. jirovecii diversity in respect to clinical data obtained from the patients. Genotyping was performed using six already validated short tandem repeat (STR) markers on 249 samples (median: 17 per centre interquartile range [11-20]) from PCP patients of 16 European centres. Mixtures of STR markers (i.e., ≥2 alleles for ≥1 locus) were detected in 67.6% (interquartile range [61.4; 76.5]) of the samples. Mixture was significantly associated with the underlying disease of the patient, with an increased proportion in HIV patients (78.3%) and a decreased proportion in renal transplant recipients (33.3%) (p<0.001). The distribution of the alleles was significantly different (p<0.001) according to the centres in three out of six markers. In analysable samples, 201 combinations were observed corresponding to 137 genotypes: 116 genotypes were country-specific; 12 in two; six in three; and two in four and one in five countries. Nine genotypes were recorded more than once in a given country. Genotype 123 (Gt123) was significantly associated with France (14/15, p<0.001) and Gt16 with Belgium (5/5, p<0.001). More specifically, Gt123 was observed mainly in France (14/15/16 patients) and in renal transplant patient (13/15). Our study showed the wide population diversity across Europe, with evidence of local clusters of patients harbouring a given genotype. These data suggest a specific association between genotype and underlying disease, with evidence of a different natural history of PCP in HIV patients and renal transplant recipients.

Identification and Functional Ascertainment of the Pneumocystis jirovecii Potential Drug Targets Gsc1 and Kre6 Involved in Glucan Synthesis.

The most efficient drug against the human pathogenic fungus Pneumocystis jirovecii is cotrimoxazole targeting the folate biosynthesis. However, resistance toward it is emerging and adverse effects occur in some patients. Studies in rodent models suggested that echinocandins could be useful to treat Pneumocystis pneumonia. Echinocandins inhibit the catalytic subunit Gsc1 of the enzymatic complex ensuring the synthesis of 1,3-ß glucan, an essential constituent of cell walls of most fungi. Besides, inhibitors of the enzyme Kre6 involved in the synthesis of 1,6-ß glucan, another essential component of fungal walls, were recently described. We identified and functionally characterized these two potential drug targets in the human pathogen P. jirovecii by rescue of the null allele of the orthologous gene in Saccharomyces cerevisiae. The P. jirovecii proteins Gsc1 and Kre6 identified using those of the relative Pneumocystis carinii as the query sequence showed high sequence identity to the putative fungal orthologs (53-97% in conserved functional domains). The expression of their encoding genes on plasmid rescued the increased sensitivity to, respectively, caspofungin or calcofluor white of the corresponding S. cerevisiae null allele. The uniqueness and likely essentiality of these proteins suggest that they are potential good drug targets.

Pneumocystis jirovecii pneumonia: still a concern in patients with haematological malignancies and stem cell transplant recipients.

Pneumocystis jirovecii can cause life-threatening pneumonia following treatment for haematological malignancies or after HSCT. The mortality rate of P. jirovecii pneumonia (PCP) in these patients is 30%-60%, especially after HSCT. The clinical presentation of PCP in haematology differs from that associated with HIV infection, with the disease being acute and more often severe, having a lower fungal burden and being more frequently linked to treatment with corticosteroids. Most cases occur in patients not receiving adequate prophylaxis. The development of new therapies, including targeted treatments and monoclonal antibodies in various haematological diseases, justifies constant vigilance in order to identify new at-risk populations and give prophylaxis accordingly. The fifth and sixth European Conferences on Infections in Leukaemia (ECIL-5 and ECIL-6) aimed to review risk factors for PCP in haematology patients and to establish evidence-based recommendations for PCP diagnosis, prophylaxis and treatment. This article focuses on the magnitude of the problem, the main differences in clinical presentation between haematology patients and other immunocompromised populations, especially HIV-infected patients, and the main risk factors.

ECIL guidelines for the diagnosis of Pneumocystis jirovecii pneumonia in patients with haematological malignancies and stem cell transplant recipients.

The Fifth European Conference on Infections in Leukaemia (ECIL-5) convened a meeting to establish evidence-based recommendations for using tests to diagnose Pneumocystis jirovecii pneumonia (PCP) in adult patients with haematological malignancies. Immunofluorescence assays are recommended as the most sensitive microscopic method (recommendation A-II: ). Real-time PCR is recommended for the routine diagnosis of PCP ( A-II: ). Bronchoalveolar lavage (BAL) fluid is recommended as the best specimen as it yields good negative predictive value ( A-II: ). Non-invasive specimens can be suitable alternatives ( B-II: ), acknowledging that PCP cannot be ruled out in case of a negative PCR result ( A-II: ). Detecting ß-d-glucan in serum can contribute to the diagnosis but not the follow-up of PCP ( A-II: ). A negative serum ß-d-glucan result can exclude PCP in a patient at risk ( A-II: ), whereas a positive test result may indicate other fungal infections. Genotyping using multilocus sequence markers can be used to investigate suspected outbreaks ( A-II: ). The routine detection of dihydropteroate synthase mutations in cases of treatment failure is not recommended ( B-II: ) since these mutations do not affect response to high-dose co-trimoxazole. The clinical utility of these diagnostic tests for the early management of PCP should be further assessed in prospective, randomized interventional studies.

ECIL guidelines for preventing Pneumocystis jirovecii pneumonia in patients with haematological malignancies and stem cell transplant recipients.

The 5th European Conference on Infections in Leukaemia (ECIL-5) meeting aimed to establish evidence-based recommendations for the prophylaxis of Pneumocystis jirovecii pneumonia (PCP) in non-HIV-infected patients with an underlying haematological condition, including allogeneic HSCT recipients. Recommendations were based on the grading system of the IDSA. Trimethoprim/sulfamethoxazole given 2-3 times weekly is the drug of choice for the primary prophylaxis of PCP in adults ( A-II: ) and children ( A-I: ) and should be given during the entire period at risk. Recent data indicate that children may benefit equally from a once-weekly regimen ( B-II: ). All other drugs, including pentamidine, atovaquone and dapsone, are considered second-line alternatives when trimethoprim/sulfamethoxazole is poorly tolerated or contraindicated. The main indications of PCP prophylaxis are ALL, allogeneic HSCT, treatment with alemtuzumab, fludarabine/cyclophosphamide/rituximab combinations, >4 weeks of treatment with corticosteroids and well-defined primary immune deficiencies in children. Additional indications are proposed depending on the treatment regimen.