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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.

BackgroundInexpensive high-throughput DNA sequencing has democratized access to genetic information for most organisms so that research utilizing a genome or transcriptome of an organism is not limited to model systems. However, the quality of the assemblies of sampled genomes can vary greatly which hampers utility for comparisons and meaningful interpretation. The uncertainty of the completeness of a given genome sequence can limit feasibility of asserting patterns of high rates of gene loss reported in many lineages.ResultsWe propose a computational framework and sequence resource for assessing completeness of fungal genomes called FGMP (Fungal Genome Mapping Project). Our approach is based on evolutionary conserved sets of proteins and DNA elements and is applicable to various types of genomic data. We present a comparison of FGMP and state-of-the-art methods for genome completeness assessment utilizing 246 genome assemblies of fungi. We discuss genome assembly improvements/degradations in 57 cases where assemblies have been updated, as recorded by NCBI assembly archive.ConclusionFGMP is an accurate tool for quantifying level of completion from fungal genomic data. It is particularly useful for non-model organisms without reference genomes and can be used directly on unassembled reads, which can help reducing genome sequencing costs.

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.

The advent of complex multicellularity (CM) was a pivotal event in the evolution of animals, plants and fungi. In the fungal Ascomycota, CM is based on hyphal filaments and arose in the Pezizomycotina. The genus Neolecta defines an enigma: phylogenetically placed in a related group containing mostly yeasts, Neolecta nevertheless possesses Pezizomycotina-like CM. Here we sequence the Neolecta irregularis genome and identify CM-associated functions by searching for genes conserved in Neolecta and the Pezizomycotina, which are absent or divergent in budding or fission yeasts. This group of 1,050 genes is enriched for functions related to diverse endomembrane systems and their organization. Remarkably, most show evidence for divergence in both yeasts. Using functional genomics, we identify new genes involved in fungal complexification. Together, these data show that rudimentary multicellularity is deeply rooted in the Ascomycota. Extensive parallel gene divergence during simplification and constraint leading to CM suggest a deterministic process where shared modes of cellular organization select for similarly configured organelle- and transport-related machineries.

Background Inexpensive high-throughput DNA sequencing has democratized access to genetic information for most organisms so that research utilizing a genome or transcriptome of an organism is not limited to model systems. However, the quality of the assemblies of sampled genomes can vary greatly which hampers utility for comparisons and meaningful interpretation. The uncertainty of the completeness of a given genome sequence can limit feasibility of asserting patterns of high rates of gene loss reported in many lineages. Results We propose a computational framework and sequence resource for assessing completeness of fungal genomes called FGMP (Fungal Genome Mapping Project). Our approach is based on evolutionary conserved sets of proteins and DNA elements and is applicable to various types of genomic data. We present a comparison of FGMP and state-of-the-art methods for genome completeness assessment utilizing 246 genome assemblies of fungi. We discuss genome assembly improvements/degradations in 57 cases where assemblies have been updated, as recorded by NCBI assembly archive. Conclusion FGMP is an accurate tool for quantifying level of completion from fungal genomic data. It is particularly useful for non-model organisms without reference genomes and can be used directly on unassembled reads, which can help reducing genome sequencing costs.

Background Pneumocystis jirovecii is an opportunistic fungus that causes Pneumocystis pneumonia (PCP) in immunocompromised hosts. Over an 11-month period, we observed a rise in cases of PCP among kidney-transplant recipients (KTR), prompting an outbreak investigation. Methods Clinical and epidemiologic data were collected for KTR diagnosed with PCP between July 2019 and May 2020. Pneumocystis strain typing was performed using restriction fragment length polymorphism analyses and multilocus sequence typing in combination with next-generation sequencing. A transmission map was drawn, and a case-control analysis was performed to determine risk factors associated with PCP. Results Nineteen cases of PCP in KTR were diagnosed at a median of 79 months post-transplantation; 8 received monthly belatacept infusions. Baseline characteristics were similar for KTR on belatacept versus other regimens; the number of clinic visits was numerically higher for the belatacept group during the study period (median 7.5 vs 3). Molecular typing of respiratory specimens from 9 patients revealed coinfection with up to 7 P. jirovecii strains per patient. A transmission map suggested multiple clusters of interhuman transmission. In a case-control univariate analysis, belatacept, lower absolute lymphocyte count, non-White race, and more transplant clinic visits were associated with an increased risk of PCP. In multivariate and prediction power estimate analyses, frequent clinic visits was the strongest risk factor for PCP. Conclusions Increased clinic exposure appeared to facilitate multiple clusters of nosocomial PCP transmission among KTR. Belatacept was a risk factor for PCP, possibly by increasing clinic exposure through the need for frequent visits for monthly infusions. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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-β glucan, an essential constituent of cell walls of most fungi. Besides, inhibitors of the enzyme Kre6 involved in the synthesis of 1,6-β 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. [ABSTRACT FROM AUTHOR]

Recently, a new Chlamydia-related organism, Protochlamydia naegleriophila KNic, was discovered within a Naegleria amoeba. To decipher themechanisms at play in the modeling of genomes from the Protochlamydia genus, we sequenced the full genome of Pr. naegleriophila, which includes a 2,885,090 bp chromosome and a 145,285 bp megaplasmid. For the first time within the Chlamydiales order, we describe the presence of a clustered regularly interspaced short palindromic repeats (CRISPR) system, the immune system of bacteria, located on the chromosome. It is composed of a small CRISPR locus comprising eight repeats and associated cas-cse genes of the subtype I-E. A CRISPR locus is also present within Chlamydia sp. Diamant, another Pr. naegleriophila strain, suggesting that the CRISPR system was acquired by a common ancestor of Pr. naegleriophila, after its divergence from Pr. amoebophila. Both nucleotide bias and comparative genomics approaches identified probable horizontal gene acquisitions within two and four genomic islands in Pr. naegleriophila KNic and Diamant genomes, respectively. The plasmid encodes an F-type conjugative system highly similar to 1) that found in the Pam100G genomic island of Pr. amoebophila UWE25 chromosome, as well as on the plasmid of Rubidus massiliensis and 2) to the three genes remaining in the chromosome of Parachlamydia acanthamoebae strains. Therefore, this conjugative system was likely acquired on an ancestral plasmid before the divergence of Parachlamydiaceae. Overall, this new complete Pr. naegleriophila genome sequence enables further investigation of the dynamic processes shaping the genomes of the family Parachlamydiaceae and the genus Protochlamydia. [ABSTRACT FROM AUTHOR]

To examine human gene expression during uncomplicated P. falciparum malaria, we obtained three samples (acute illness, treatment, and recovery) from 10 subjects and utilized each subject's recovery sample as their baseline. At the time of acute illness (day 1), subjects had upregulation of innate immune response, cytokine, and inflammation-related genes (IL-1β, IL-6, TNF, and IFN-γ), which was more frequent with parasitemias >100,000 per μL and body temperatures ≥ 39°C. Apoptosis-related genes (Fas, BAX, and TP53) were upregulated acutely and for several days thereafter (days 1-3). In contrast, the expression of immune-modulatory (transcription factor 7, HLV-DOA, and CD6) and apoptosis inhibitory (c-myc, caspase 8, and Fas Ligand G) genes was downregulated initially and returned to normal with clinical recovery (days 7-10). These results indicate that the innate immune response, cytokine, and apoptosis pathways are upregulated acutely in uncomplicated malaria with concomitant downregulation of immune-modulatory and apoptosis inhibitory genes. [ABSTRACT FROM AUTHOR]

While Pneumocystis has been recognized as both a ubiquitous commensal fungus in immunocompetent mammalian hosts and a major opportunistic pathogen in humans responsible for severe pneumonias in immunocompromised patients, in pigs its epidemiology and association with pulmonary diseases have been rarely reported. Nevertheless, the fungus can be quite abundant in porcine populations with up to 51% of prevalence reported so far. The current study was undertaken to longitudinally quantify Pneumocystis carinii f. sp. suis and other pulmonary pathogens in a cohort of 50 pigs from five Austrian farms (i.e., 10 pigs per farm) with a history of respiratory disease at five time points between the first week and the fourth month of life. The fungus was present as early as the suckling period (16% and 26% of the animals in the first and the third week, respectively), yet not in a high amount. Over time, both the organism load (highest 4.4 × 105 copies/mL) and prevalence (up to 88% of positive animals in the third month) increased in each farm. The relative prevalence of various coinfection patterns was significantly different over time. The current study unravelled a complex co-infection history involving Pneumocystis and other pulmonary pathogens in pigs, suggesting a relevant role of the fungus in the respiratory disease scenario of this host. [ABSTRACT FROM AUTHOR]

SUMMARYEvery human being is presumed to be infected by the fungus Pneumocystis jirovecii at least once in his or her lifetime. This fungus belongs to a large group of species that appear to exclusively infect mammals, with P. jirovecii being the only one known to cause disease in humans. The mystery of P. jirovecii origin and speciation is just beginning to unravel. Here, we provide a review of the major steps of P. jirovecii evolution. The Pneumocystis genus likely originated from soil or plant-associated organisms during the period of Cretaceous ~165 million years ago and successfully shifted to mammals. The transition coincided with a substantial loss of genes, many of which are related to the synthesis of nutrients that can be scavenged from hosts or cell wall components that could be targeted by the mammalian immune system. Following the transition, the Pneumocystis genus cospeciated with mammals. Each species specialized at infecting its own host. Host specialization is presumably built at least partially upon surface glycoproteins, whose protogene was acquired prior to the genus formation. P. jirovecii appeared at ~65 million years ago, overlapping with the emergence of the first primates. P. jirovecii and its sister species P. macacae , which infects macaques nowadays, may have had overlapping host ranges in the distant past. Clues from molecular clocks suggest that P. jirovecii did not cospeciate with humans. Molecular evidence suggests that Pneumocystis speciation involved chromosomal rearrangements and the mounting of genetic barriers that inhibit gene flow among species., Competing Interests: The authors declare no conflict of interest.

Unlabelled: Pneumocystis jirovecii is a fungus that causes severe pneumonia in immunocompromised patients. However, its study is hindered by the lack of an in vitro culture method. We report here the genome of P. jirovecii that was obtained from a single bronchoalveolar lavage fluid specimen from a patient. The major challenge was the in silico sorting of the reads from a mixture representing the different organisms of the lung microbiome. This genome lacks virulence factors and most amino acid biosynthesis enzymes and presents reduced GC content and size. Together with epidemiological observations, these features suggest that P. jirovecii is an obligate parasite specialized in the colonization of human lungs, which causes disease only in immune-deficient individuals. This genome sequence will boost research on this deadly pathogen., Importance: Pneumocystis pneumonia is a major cause of mortality in patients with impaired immune systems. The availability of the P. jirovecii genome sequence allows new analyses to be performed which open avenues to solve critical issues for this deadly human disease. The most important ones are (i) identification of nutritional supplements for development of culture in vitro, which is still lacking 100 years after discovery of the pathogen; (ii) identification of new targets for development of new drugs, given the paucity of present treatments and emerging resistance; and (iii) identification of targets for development of vaccines.