Characterization of the Pneumocystis jirovecii and Pneumocystis murina phosphoglucomutases (Pgm2s): a potential target for Pneumocystis therapy.

Pneumocystis cyst life forms contain abundant ß-glucan carbohydrates, synthesized using ß-1,3 and ß-1,6 glucan synthase enzymes and the donor uridine diphosphate (UDP)-glucose. In yeast, phosphoglucomutase (PGM) plays a crucial role in carbohydrate metabolism by interconverting glucose 1-phosphate and glucose 6-phosphate, a vital step in UDP pools for ß-glucan cell wall formation. This pathway has not yet been defined in Pneumocystis. Herein, we surveyed the Pneumocystis jirovecii and Pneumocystis murina genomes, which predicted a homolog of the Saccharomyces cerevisiae major PGM enzyme. Furthermore, we show that PjPgm2p and PmPgm2p function similarly to the yeast counterpart. When both Pneumocystis pgm2 homologs are heterologously expressed in S. cerevisiae pgm2Δ cells, both genes can restore growth and sedimentation rates to wild-type levels. Additionally, we demonstrate that yeast pgm2Δ cell lysates expressing the two Pneumocystis pgm2 transcripts individually can restore PGM activities significantly altered in the yeast pgm2Δ strain. The addition of lithium, a competitive inhibitor of yeast PGM activity, significantly reduces PGM activity. Next, we tested the effects of lithium on P. murina viability ex vivo and found the compound displays significant anti-Pneumocystis activity. Finally, we demonstrate that a para-aryl derivative (ISFP10) with known inhibitory activity against the Aspergillus fumigatus PGM protein and exhibiting 50-fold selectivity over the human PGM enzyme homolog can also significantly reduce Pmpgm2 activity in vitro. Collectively, our data genetically and functionally validate phosphoglucomutases in both P. jirovecii and P. murina and suggest the potential of this protein as a selective therapeutic target for individuals with Pneumocystis pneumonia.

[Progress of researches on developmental processes and reproduction mode of Pneumocystis].

Pneumocystis, an important opportunistic fungal pathogen that parasitizes in multiple mammalian lungs, may cause life-threatening Pneumocystis pneumonia (PCP) and even death among immunocompromised individuals. With the rapid development of high-throughput sequencing and multi-omics technologies, systematic comparative analyses of genome, transcriptome, and whole-genome sequencing results demonstrate that Pneumocystis is a type of obligate biotrophic fungi, and requires obtaining nutrition from hosts. In addition, sexual reproduction is an essential process for Pneumocystis survival, production and transmission, and asexual reproduction facilitates Pneumocystis survival, which provides new insights into understanding of the whole developmental process of Pneumocystis in the host lung and inter-host transmission of Pneumocystis. This review summarizes the advances in the reproduction mode of Pneumocystis and underlying mechanisms, which provides insights into prevention and treatment of PCP, notably for the prophylaxis against nosocomial transmission of PCP.

Integrated multi-omics analyses reveal the altered transcriptomic characteristics of pulmonary macrophages in immunocompromised hosts with Pneumocystis pneumonia.

Introduction: With the extensive use of immunosuppressants, immunosuppression-associated pneumonitis including Pneumocystis jirovecii pneumonia (PCP) has received increasing attention. Though aberrant adaptive immunity has been considered as a key reason for opportunistic infections, the characteristics of innate immunity in these immunocompromised hosts remain unclear. Methods: In this study, wild type C57BL/6 mice or dexamethasone-treated mice were injected with or without Pneumocystis. Bronchoalveolar lavage fluids (BALFs) were harvested for the multiplex cytokine and metabolomics analysis. The single-cell RNA sequencing (scRNA-seq) of indicated lung tissues or BALFs was performed to decipher the macrophages heterogeneity. Mice lung tissues were further analyzed via quantitative polymerase chain reaction (qPCR) or immunohistochemical staining. Results: We found that the secretion of both pro-inflammatory cytokines and metabolites in the Pneumocystis-infected mice are impaired by glucocorticoids. By scRNA-seq, we identified seven subpopulations of macrophages in mice lung tissues. Among them, a group of Mmp12+ macrophages is enriched in the immunocompetent mice with Pneumocystis infection. Pseudotime trajectory showed that these Mmp12+ macrophages are differentiated from Ly6c+ classical monocytes, and highly express pro-inflammatory cytokines elevated in BALFs of Pneumocystis-infected mice. In vitro, we confirmed that dexamethasone impairs the expression of Lif, Il1b, Il6 and Tnf, as well as the fungal killing capacity of alveolar macrophage (AM)-like cells. Moreover, in patients with PCP, we found a group of macrophages resembled the aforementioned Mmp12+ macrophages, and these macrophages are inhibited in the patient receiving glucocorticoid treatment. Additionally, dexamethasone simultaneously impaired the functional integrity of resident AMs and downregulated the level of lysophosphatidylcholine, leading to the suppressed antifungal capacities. Conclusion: We reported a group of Mmp12+ macrophages conferring protection during Pneumocystis infection, which can be dampened by glucocorticoids. This study provides multiple resources for understanding the heterogeneity and metabolic changes of innate immunity in immunocompromised hosts, and also suggests that the loss of Mmp12+ macrophages population contributes to the pathogenesis of immunosuppression-associated pneumonitis.

CLEC4A and CLEC12B C-type lectin receptors mediate interactions with Pneumocystis cell wall components.

Introduction. C-type lectin receptors (CLRs) are prominently expressed on myeloid cells where they perform multiple functions including serving as pattern recognition receptors (PRRs) to drive innate as well as adaptive immunity to pathogens. Depending on the presence of a tyrosine-based signalling motif, CLR-microbial pathogen engagement may result in either anti- or pro-inflammatory signalling.Impact statement. In this manuscript, we report our laboratory study of two novel CLRs that recognize Pneumocystis murina cell wall homogenates (CWH) and a purified Pneumocystis carinii cell wall fraction (CWF).Aim. To study the potential of newly generated hFc-CLR fusions on binding to Pneumocystis murina CWHs and P. carinii CWFs and subsequent downstream inflammatory signalling analysis.Methods. Newly generated hFc-CLR fusion CLEC4A and CLEC12B were screened against P. murina CWHs and P. carinii CWFs preparations via modified ELISA. Immunofluorescence assay (IFA) was utilized to visualize hFc-CLR fusion binding against intact fixed fungal life forms to verify results. Quantitative PCR (q-PCR) analysis of lung mRNA from the mouse immunosuppressed Pneumocystis pneumonia (PCP) model versus uninfected mice was employed to detect possible changes in the respective Clec4a and Clec12b transcripts. Lastly, siRNA technology of both CLRs was conducted to determine effects on downstream inflammatory events in mouse macrophages stimulated in the presence of P. carinii CWFs.Results. We determined that both CLEC4A and CLEC12B hFc-CLRs displayed significant binding with P. murina CWHs and P. carinii CWFs. Binding events showed significant binding to both curdlan and laminarin, both polysaccharides containing ß-(1,3) glucans as well as N-acetylglucosamine (GlcNAc) residues and modest yet non-significant binding to the negative control carbohydrate dextran. IFA with both CLR hFc-fusions against whole P. murina life forms corroborated these findings. Lastly, we surveyed the mRNA expression profiles of both CLRs tested above in the mouse immunosuppressed Pneumocystis pneumonia (PCP) model and determined that both CLRs were significantly up regulated during infection. Lastly, siRNA of both CLRs in the mouse RAW macrophage cell line was conducted and results demonstrated that silencing of Clec4a resulted in no significant changes in TNF-alpha generation in P. carinii CWF stimulated macrophages. On the contrary, silencing of Clec12b CLR resulted in significant decreases in TNF-alpha in RAW cells stimulated with the same CWF.Conclusion. The data presented here provide new members of the CLRs family recognizing Pneumocystis. Future studies using CLEC4A and/or CLEC12B deficient mice in the PCP mouse model should provide further insights into the host immunological response to Pneumocystis.

Evaluation of the PneumoGenius® PCR assay for the diagnosis of Pneumocystis pneumonia and the detection of Pneumocystis dihydropteroate synthase mutations in respiratory samples.

Pneumocystis pneumonia (PCP) is the most frequent fungal opportunistic infection defining AIDS in HIV-infected patients, and is of growing importance in HIV-negative patients. In this latter category of patients, the diagnosis mainly relies on real-time polymerase chain reaction (qPCR) detection of Pneumocystis jirovecii (Pj) on respiratory samples. The PneumoGenius® kit (PathoNostics) allows the simultaneous detection of Pj mitochondrial large subunit (mtLSU) and dihydropteroate synthase (DHPS) polymorphisms, which could be of interest to anticipate therapeutic failure. This study aimed at evaluating its clinical performance on 251 respiratory specimens (239 patients), (i) for P. jirovecii detection in clinical samples, and (ii) for DHPS polymorphisms detection in circulating strains. Patients were classified according to modified European Organization for Research and Treatment of Cancer/Invasive Fungal Infections Cooperative Group and the National Institute of Allergy and Infectious Diseases Mycoses Study Group (EORTC/MSG) criteria, as having proven PCP (n = 62), probable PCP (n = 87), Pneumocystis colonization (n = 37), and no PCP (n = 53). Compared with in-house qPCR, the sensitivity of PneumoGenius® assay for P. jirovecii detection reached 91.9% (182/198), the specificity was excellent (100%, 53/53) and the global concordance was 93.6% (235/253). A total of four diagnoses of proven/probable PCP were missed by the PneumoGenius® assay, reaching a 97.5% sensitivity (157/161) in this sub-group. The 12 other 'false-negative' results were obtained in patients diagnosed as colonized using the in-house PCR. DHPS genotyping was successful for 147/182 samples with PneumoGenius® and revealed dhps mutation in 8 samples, which were all confirmed by sequencing. In conclusion, PneumoGenius® assay missed the detection of low-burden PCP. This lower sensitivity for PCP diagnosis can be balanced by a higher specificity (P. jirovecii colonization less frequently detected) and the efficient detection of DHPS hot spot mutations.

The diagnosis of Pneumocystis pneumonia (PCP) relies on DNA detection of P. jirovecii in respiratory samples. In this study, we show that the commercial assay PneumoGenius® has a lower sensitivity than our in-house qPCR for PCP diagnosis, but provides accurate results for DHPS genotyping.

Analysis of Pneumocystis Transcription Factor Evolution and Implications for Biology and Lifestyle.

Pneumocystis jirovecii kills hundreds of thousands of immunocompromised patients each year. Yet many aspects of the biology of this obligate pathogen remain obscure because it is not possible to culture the fungus in vitro independently of its host. Consequently, our understanding of Pneumocystis pathobiology is heavily reliant upon bioinformatic inferences. We have exploited a powerful combination of genomic and phylogenetic approaches to examine the evolution of transcription factors in Pneumocystis species. We selected protein families (Pfam families) that correspond to transcriptional regulators and used bioinformatic approaches to compare these families in the seven Pneumocystis species that have been sequenced to date with those from other yeasts, other human and plant pathogens, and other obligate parasites. Some Pfam families of transcription factors have undergone significant reduction during their evolution in the Pneumocystis genus, and other Pfam families have been lost or appear to be in the process of being lost. Meanwhile, other transcription factor families have been retained in Pneumocystis species, and some even appear to have undergone expansion. On this basis, Pneumocystis species seem to have retained transcriptional regulators that control chromosome maintenance, ribosomal gene regulation, RNA processing and modification, and respiration. Meanwhile, regulators that promote the assimilation of alternative carbon sources, amino acid, lipid, and sterol biosynthesis, and iron sensing and homeostasis appear to have been lost. Our analyses of transcription factor retention, loss, and gain provide important insights into the biology and lifestyle of Pneumocystis. IMPORTANCE Pneumocystis jirovecii is a major fungal pathogen of humans that infects healthy individuals, colonizing the lungs of infants. In immunocompromised and transplant patients, this fungus causes life-threatening pneumonia, and these Pneumocystis infections remain among the most common and serious infections in HIV/AIDS patients. Yet we remain remarkably ignorant about the biology and epidemiology of Pneumocystis due to the inability to culture this fungus in vitro. Our analyses of transcription factor retentions, losses, and gains in sequenced Pneumocystis species provide valuable new views of their specialized biology, suggesting the retention of many metabolic and stress regulators and the loss of others that are essential in free-living fungi. Given the lack of in vitro culture methods for Pneumocystis, this powerful bioinformatic approach has advanced our understanding of the lifestyle of P. jirovecii and the nature of its dependence on the host for survival.

First Molecular Detection of Pneumocystis spp. in the Golden Jackal (Canis aureus).

Forty-six golden jackals (Canis aureus) were collected between November 2020 and February 2021 in five counties of Serbia. Lung samples were screened for the presence of Pneumocystis DNA by pan-Pneumocystis PCR on the mtLSU rRNA gene obtaining PCR products of 370 bp in length. Pneumocystis DNA was detected in the lungs from 6/46 (13.04%) golden jackals. Four were females and two were males; four were classified as adults and two as subadults. Positive samples were confirmed in 4/5 investigated counties. No gross pathologic lung lesions were observed in this study. The sequences of Pneumocystis spp. from golden jackals were identical to one another and showed the highest similarity with Pneumocystis spp. sequences of dogs (98% nucleotide identity). The genetic variation was comparable to Pneumocystis spp. of raccoon dogs (95-97% nucleotide identity), red foxes (91-95% nucleotide identity), ferrets (86% nucleotide identity), and another Pneumocystis type in dogs (P. canis Ck2, 81% nucleotide identity) was higher. Golden jackals may be carriers and may play a nonnegligible role in the spread of Pneumocystis spp. Although this finding cannot be directly related to any clinical manifestation or pathologic lesions, a possible role in the exacerbation of different pulmonary disorders should be considered.

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.

Analysis of lncRNA and mRNA Repertoires in Lung From BAFF-R-Deficient Pneumocystis-Infected Mice.

Background: Pneumocystis pneumonia (PCP) is a common medical issue in immunosuppressive patients. Increasing evidence supports that B cells may play an essential role in PCP individuals. The present study aims to integrate lncRNA and mRNA expression profiles and further investigate the molecular function of mature B cells in PCP. Methods: The lung tissue of wild-type (WT) mice and B-cell-activating factor receptor-deficient (mature B-cell deficiency, BAFF-R-/-) mice were harvested at 3 weeks after being infected with pneumocystis. After total RNAs were extracted, transcriptome profiling was performed following the Illumina HiSeq 3000 protocol. lncRNA-targeted miRNA pairs were predicted using the online databases. The Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) enrichment pathways were analyzed to functionally annotate these differentially expressed genes. Additionally, the immune-related lncRNA-miRNA-mRNA-ceRNA network was subsequently performed. The quantitative real-time PCR (RT-PCR) analysis was conducted to evaluate the lncRNA and mRNA expression profiles in WT-PCP mice and BAFF-R-/- PCP mice. Results: Compared with the control group, 166 mRNAs were observed to be aberrantly expressed (fold change value ≥2; P <0.05) in the BAFF-R-/- PCP group, including 39 upregulated and 127 downregulated genes, while there were 69 lncRNAs differently expressed in the BAFF-R-/- PCP group, including 15 upregulated and 54 downregulated genes. In addition, GO and KEGG pathway analyses showed that BAFF-R deficiency played an important role in the primary and adaptive immune responses in PCP. Furthermore, the lncRNA and mRNA co-expression network was established. We noted that the core network of lncRNA-TF (transcription factor) pairs could be classified into the categories including infection and immunity pathways. Conclusion: In summary, in this study, we further explored the role of mature B cells in the pathogenesis and progression of PCP and the data demonstrated that BAFF-R deficiency could play a significant role in immune regulation in the PCP population.

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.

RNA Polymerase II Transcription in Pneumocystis: TFIIB from Pneumocystis carinii Can Replace the Transcriptional Functions of Fission Yeast Schizosaccharomyces pombe TFIIB In Vivo and In Vitro.

The Pneumocystis genus is an opportunistic fungal pathogen that infects patients with AIDS and immunocompromised individuals. The study of this fungus has been hampered due to the inability to grow it in a (defined media/pure) culture. However, the use of modern molecular techniques and genomic analysis has helped researchers to understand its complex cell biology. The transcriptional process in the Pneumocystis genus has not been studied yet, although it is assumed that it has conventional transcriptional machinery. In this work, we have characterized the function of the RNA polymerase II (RNAPII) general transcription factor TFIIB from Pneumocystis carinii using the phylogenetically related biological model Schizosaccharomyces pombe. The results of this work show that Pneumocystis carinii TFIIB is able to replace the essential function of S. pombe TFIIB both in in vivo and in vitro assays. The S. pombe strain harboring the P carinii TFIIB grew slower than the parental wild-type S. pombe strain in complete media and in minimal media. The S. pombe cells carrying out the P. carinii TFIIB are larger than the wild-type cells, indicating that the TFIIB gene replacement confers a phenotype, most likely due to defects in transcription. P. carinii TFIIB forms very weak complexes with S. pombe TATA-binding protein on a TATA box promoter but it is able to form stable complexes in vitro when S. pombe TFIIF/RNAPII are added. P. carinii TFIIB can also replace the transcriptional function of S. pombe TFIIB in an in vitro assay. The transcription start sites (TSS) of the endogenous adh gene do not change when P. carinii TFIIB replaces S. pombe TFIIB, and neither does the TSS of the nmt1 gene, although this last gene is poorly transcribed in vivo in the presence of P. carinii TFIIB. Since transcription by RNA polymerase II in Pneumocystis is poorly understood, the results described in this study are promising and indicate that TFIIB from P. carinii can replace the transcriptional functions of S. pombe TFIIB, although the cells expressing the P. carini TFIIB show an altered phenotype. However, performing studies using a heterologous approach, like this one, could be relevant to understanding the basic molecular processes of Pneumocystis such as transcription and replication.

Pneumocystis Colonization in Dogs Is as in Humans.

Pneumocystis is an atypical fungus that resides in the pulmonary parenchyma of many mammals, including humans and dogs. Immunocompetent human hosts are usually asymptomatically colonised or show subtle clinical signs, but some immunocompromised people can develop florid life-threatening Pneumocystis pneumonia (PCP). Since much less is known concerning Pneumocystis in dogs, we posit the question: can Pneumocystis colonization be present in dogs with inflammatory airway or lung disease caused by other pathogens or disease processes? In this study, Pneumocystis DNA was detected in bronchoalveolar lavage fluid (BALF) of 22/255 dogs (9%) with respiratory distress and/or chronic cough. Although young dogs (<1 year-of-age) and pedigree breeds were more often Pneumocystis-qPCR positive than older dogs and crossbreds, adult dogs with other infectious conditions and/or a history of therapy-resistant pulmonary disease could also be qPCR-positive, including two patients with suppression of the immune system. Absence of pathognomonic clinical or radiographic signs render it impossible to convincingly discriminate between overt PCP versus other lung/airway disease processes colonised by P. canis. It is possible that colonisation with P. canis might play a certain role as a co-pathogen in some canine patients with lower respiratory disease.

Gene expression in lung epithelial cells following interaction with Pneumocystis carinii and its specific life forms yields insights into host gene responses to infection.

Pneumocystis spp. interacts with epithelial cells in the alveolar spaces of the lung. It is thought that the binding of Pneumocystis to host cell epithelium is needed for life cycle completion and proliferation. The effect of this interaction on lung epithelial cells has previously shown that the trophic form of this organism greatly inhibits p34cdc2 activity, a serine/threonine kinase required for transition from the G2 to M phase in the cell cycle. To gain further insight into the host response during Pneumocystis pneumonia infection, we used microarray technology to profile epithelial cell (A549) gene expression patterns following Pneumocystis carinii interaction. Furthermore, we isolated separate populations of cyst and trophic forms of P. carinii, which were then applied to the lung epithelial cells. Differential expression of genes involved in various cellular functions dependent on the specific P. carinii life form in contact with the A549 cell was identified. The reliability of our data was further confirmed by Northern blot analysis on a number of selected upregulated or downregulated transcripts. The transcriptional response to P. carinii was dominated by cytokines, apoptotic, and antiapoptosis-related genes. These results reveal several previously unknown effects of P. carinii on the lung epithelial cell and provide insight into the complex interactions of host and pathogen.

Additional C-type lectin receptors mediate interactions with Pneumocystis organisms and major surface glycoprotein.

Introduction. Pathogen-associated molecular patterns' (PAMPs) are microbial signatures that are recognized by host myeloid C-type lectin receptors (CLRs). These CLRs interact with micro-organisms via their carbohydrate recognition domains (CRDs) and engage signalling pathways within the cell resulting in pro-inflammatory and microbicidal responses.Gap statement. In this article, we extend our laboratory study of additional CLRs that recognize fungal ligands against Pneumocystis murina and Pneumocystis carinii and their purified major surface glycoproteins (Msgs).Aim. To study the potential of newly synthesized hFc-CLR fusions on binding to Pneumocystis and its Msg.Methods. A library of new synthesized hFc-CLR fusions was screened against Pneumocystis murina and Pneumocystis carinii organisms and their purified major surface glycoproteins (Msgs) found on the respective fungi via modified ELISA. Immunofluorescence assay (IFA) was implemented and quantified to verify results. mRNA expression analysis by quantitative PCR (q-PCR) was employed to detect respective CLRs found to bind fungal organisms in the ELISA and determine their expression levels in the mouse immunosuppressed Pneumocystis pneumonia (PCP) model.Results. We detected a number of the CLR hFc-fusions displayed significant binding with P. murina and P. carinii organisms, and similarly to their respective Msgs. Significant organism and Msg binding was observed for CLR members C-type lectin domain family 12 member A (CLEC12A), Langerin, macrophage galactose-type lectin-1 (MGL-1), and specific intracellular adhesion molecule-3 grabbing non-integrin homologue-related 3 (SIGNR3). Immunofluorescence assay (IFA) with the respective CLR hFc-fusions against whole P. murina life forms corroborated these findings. Lastly, we surveyed the mRNA expression profiles of the respective CLRs tested above in the mouse immunosuppressed Pneumocystis pneumonia (PCP) model and determined that macrophage galactose type C-type lectin (Mgl-1), implicated in recognizing terminal N-acetylgalactosamine (GalNAc) found in the glycoproteins of microbial pathogens was significantly up-regulated during infection.Conclusion. The data herein add to the growing list of CLRs recognizing Pneumocystis and provide insights for further study of organism/host immune cell interactions.

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.

Performance of real-time PCR and immunofluorescence assay for diagnosis of Pneumocystis pneumonia in real-world clinical practice.

BACKGROUND: PCR is more sensitive than immunofluorescence assay (IFA) for detection of Pneumocystis jirovecii. However, PCR cannot always distinguish infection from colonization. This study aimed to compare the performance of real-time PCR and IFA for diagnosis of P. jirovecii pneumonia (PJP) in a real-world clinical setting. METHODS: A retrospective cohort study was conducted at a 1,300-bed hospital between April 2017 and December 2018. Patients whose respiratory sample (bronchoalveolar lavage or sputum) were tested by both Pneumocystis PCR and IFA were included. Diagnosis of PJP was classified based on multicomponent criteria. Sensitivity, specificity, 95% confidence intervals (CI), and Cohen's kappa coefficient were calculated. RESULTS: There were 222 eligible patients. The sensitivity and specificity of PCR was 91.9% (95% CI, 84.0%-96.7%) and 89.7% (95% CI, 83.3%-94.3%), respectively. The sensitivity and specificity of IFA was 7.0% (95% CI, 2.6%-14.6%) and 99.2% (95% CI, 95.6%-100.0%), respectively. The percent agreement between PCR and IFA was 56.7% (Cohen's kappa -0.02). Among discordant PCR-positive and IFA-negative samples, 78% were collected after PJP treatment. Clinical management would have changed in 14% of patients using diagnostic information, mainly based on PCR results. CONCLUSIONS: PCR is highly sensitive compared with IFA for detection of PJP. Combining clinical, and radiological features with PCR is useful for diagnosis of PJP, particularly when respiratory specimens cannot be promptly collected before initiation of PJP treatment.

First molecular detection of Pneumocystis spp. in red foxes (Vulpes vulpeslinnaeus, 1758) and raccoon dogs (Nyctereutes procyonoidesgray, 1834).

Fungal organisms of the genus Pneumocystis may cause Pneumocystis pneumonia (PCP) in humans, but also domestic and wild mammals. Almost every animal species hosts its own genetically distinct Pneumocystis species, however information is sparse. In this study, 62 red foxes (Vulpes vulpes) and 37 raccoon dogs (Nyctereutes procyonoides) were collected in North-East Germany. The lung tissues of the animals were analysed by a new designed specific pan-Pneumocystis mtLSU rRNA gene PCR and sequencing. With this PCR, detection and discrimination of all known Pneumocystis spp. in a single step should be possible. This first detection of Pneumocystis spp. in 29/62 (46.8%) red foxes and 29/37 (78.4%) raccoon dogs indicated, that they harbour two dissimilar strains, as seen by specific single nucleotide position changes (SNPs). Nevertheless, five samples with contrary SNPs showed a probable inter-species transmission.

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.

Transcriptomic and Proteomic Approaches to Finding Novel Diagnostic and Immunogenic Candidates in Pneumocystis.

Pneumocystis pneumonia is the most common serious opportunistic infection in patients with HIV/AIDS. Furthermore, Pneumocystis pneumonia is a feared complication of the immunosuppressive drug regimens used to treat autoimmunity, malignancy, and posttransplantation rejection. With an increasing at-risk population, there is a strong need for novel approaches to discover diagnostic and vaccine targets. There are multiple challenges to finding these targets, however. First, Pneumocystis has a largely unannotated genome. To address this, we evaluated each protein encoded within the Pneumocystis genome by comparisons to proteins encoded within the genomes of other fungi using NCBI BLAST. Second, Pneumocystis relies on a multiphasic life cycle, as both the transmissible form (the ascus) and the replicative form (the trophozoite [troph]) reside within the alveolar space of the host. To that end, we purified asci and trophs from Pneumocystis murina and utilized transcriptomics to identify differentially regulated genes. Two such genes, Arp9 and Sp, are differentially regulated in the ascus and the troph, respectively, and can be utilized to characterize the state of the Pneumocystis life cycle in vivoGsc1, encoding a ß-1,3-glucan synthase with a large extracellular domain previously identified using surface proteomics, was more highly expressed on the ascus form of Pneumocystis GSC-1 ectodomain immunization generated a strong antibody response that demonstrated the ability to recognize the surface of the Pneumocystis asci. GSC-1 ectodomain immunization was also capable of reducing ascus burden following primary challenge with Pneumocystis murina Finally, mice immunized with the GSC-1 ectodomain had limited fungal burden following natural transmission of Pneumocystis using a cohousing model.IMPORTANCE The current report enhances our understanding of Pneumocystis biology in a number of ways. First, the current study provided a preliminary annotation of the Pneumocystis murina genome, addressing a long-standing issue in the field. Second, this study validated two novel transcripts enriched in the two predominant life forms of Pneumocystis These findings allow better characterization of the Pneumocystis life cycle in vivo and could be valuable diagnostic tools. Furthermore, this study outlined a novel pipeline of -omics techniques capable of revealing novel antigens (e.g., GSC-1) for the development of vaccines against Pneumocystis.

Inhibition of polymerase chain reaction: Pathogen-specific controls are better than human gene amplification.

PCR inhibition is frequent in medical microbiology routine practice and may lead to false-negative results; however there is no consensus on how to detect it. Pathogen-specific and human gene amplifications are widely used to detect PCR inhibition. We aimed at comparing the value of PCR inhibitor detection using these two methods. We analysed Cp shifts (ΔCp) obtained from qPCRs targeting either the albumin gene or the pathogen-specific sequence used in two laboratory-developed microbiological qPCR assays. 3152 samples including various matrixes were included. Pathogen-specific amplification and albumin qPCR identified 62/3152 samples (2.0%), and 409/3152 (13.0%) samples, respectively, as inhibited. Only 16 samples were detected using both methods. In addition, the use of the Youden's index failed to determine adequate Cp thresholds for albumin qPCR, even when we distinguished among the different sample matrixes. qPCR targeting the albumin gene therefore appears not adequate to identify the presence of PCR inhibitors in microbiological PCR assays. Our data may be extrapolated to other heterologous targets and should discourage their use to assess the presence of PCR inhibition in microbiological PCR assays.