Revisiting the Pneumocystis host specificity paradigm and transmission ecology in wild Southeast Asian rodents.

Pneumocystis fungi are opportunistic parasites of mammalian lungs whose evolution, ecology and host specificity in natural host populations remain poorly understood and controversial. Using an extensive collection of 731 lung samples from 27 rodent species sampled in five Southeast Asian countries, and nested PCR amplification of mitochondrial and nuclear genes, we investigated the host specificity and genetic structure of Pneumocystis lineages infecting wild rodents. We also identified the rodent species playing a central role in the transmission of these parasites using network analysis and centrality measurement and we characterized the environmental conditions allowing Pneumocystis infection in Southeast Asia using generalized linear mixed models. Building upon an unprecedented Pneumocystis sampling from numerous rodent species belonging to closely related genera, our findings provide compelling evidence that the host specificity of Pneumocystis lineages infecting rodents is not restricted to a single host species or genus as often presented in the literature but it encompasses much higher taxonomic levels and more distantly related rodent host species. The phylogenetic species status at both mitochondrial and nuclear genetic markers of at least three new Pneumocystis lineages, highly divergent from Pneumocystis species currently described, is also suggested by our data. Our models show that the probability of Pneumocystis infection in rodent hosts is positively correlated to environmental variables reflecting habitat fragmentation and landscape patchiness. Synanthropic and habitat-generalist rodents belonging to the Rattus, Sundamys and Bandicota genera played a role of bridge host species for Pneumocystis spreading in these heterogeneous habitats, where they can reach high population densities. These are critical findings improving our understanding of the ecology of these enigmatic parasites and the role played by cospeciation and host switches in their evolution. Our results also confirmed the role of land-use change and habitat fragmentation in parasite amplification and spillover in rodents.

Evolutionary history of Pneumocystis fungi in their African rodent hosts.

Pneumocystis is a genus of parasitic fungi infecting lung tissues in a wide range of mammal species, displaying a strong host specificity and patterns of co-speciation with their hosts. However, a recent study on Asiatic murids challenged these patterns reporting several Pneumocystis lineages/species shared by different host species or even genera in the Rattini and Murini tribes. Here we screened lung samples of 27 species of African rodents from five families for the presence of Pneumocystis DNA. Using reconstructed multi-locus phylogenies of both hosts and parasites, we tested the hypothesis of their co-evolution. We found that Pneumocystis is widespread in African rodents, detected in all but seven screened host species, with species-level prevalence ranging from 5.9 to 100%. Several host species carry pairs of highly divergent Pneumocystis lineages/species. The retrieved co-phylogenetic signal was highly significant (p = .0017). We found multiple co-speciations, sorting events and two host-shift events, which occurred between Murinae and Deomyinae hosts. Comparison of genetic distances suggests higher substitution rates for Pneumocystis relative to the rodent hosts on neutral loci and slower rates on selected ones. We discuss life-history traits and population dynamics factors which could explain the observed results.

A Molecular Window into the Biology and Epidemiology of Pneumocystis spp.

Pneumocystis, a unique atypical fungus with an elusive lifestyle, has had an important medical history. It came to prominence as an opportunistic pathogen that not only can cause life-threatening pneumonia in patients with HIV infection and other immunodeficiencies but also can colonize the lungs of healthy individuals from a very early age. The genus Pneumocystis includes a group of closely related but heterogeneous organisms that have a worldwide distribution, have been detected in multiple mammalian species, are highly host species specific, inhabit the lungs almost exclusively, and have never convincingly been cultured in vitro, making Pneumocystis a fascinating but difficult-to-study organism. Improved molecular biologic methodologies have opened a new window into the biology and epidemiology of Pneumocystis. Advances include an improved taxonomic classification, identification of an extremely reduced genome and concomitant inability to metabolize and grow independent of the host lungs, insights into its transmission mode, recognition of its widespread colonization in both immunocompetent and immunodeficient hosts, and utilization of strain variation to study drug resistance, epidemiology, and outbreaks of infection among transplant patients. This review summarizes these advances and also identifies some major questions and challenges that need to be addressed to better understand Pneumocystis biology and its relevance to clinical care.

Pneumocystosis in dogs: meta-analysis of 43 published cases including clinical signs, diagnostic procedures, and treatment.

We evaluated 43 published cases of dogs with confirmed Pneumocystis infection regarding the value of clinical parameters indicating the presence of the disease as well as tools for the detection of the pathogen. The assessed parameters included clinical signs, laboratory findings, results of thoracic radiography, autopsy, histopathology, methods for the detection of Pneumocystis, as well as medical therapy. Pneumocystosis was diagnosed most often in certain breeds (Cavalier King Charles Spaniel, Miniature Dachshund) with a predisposition for impaired immunity. The median age of the dogs was 1 y. Chronic therapy-resistant respiratory signs, such as tachypnea, dyspnea, and cough, along with leukocytosis, neutrophilia, and hypogammaglobulinemia, were the most frequently described clinical and clinicopathologic abnormalities. Pneumocystosis can be masked by coinfections with other respiratory pathogens, and the successful detection of Pneumocystis organisms is of major relevance. Several detection methods have been used in the past, but only a few provide reliable results. In 2017, the cytologic evaluation of Giemsa-stained bronchoalveolar lavage samples is generally used, even if sensitivity is only moderate. More reliable results can be achieved using special stains or sensitive molecular techniques. Fast and reliable detection of Pneumocystis is the essential basis for appropriate treatment and higher survival chances for dogs.

Comparison of the Clinical Characteristics of Pneumocystis Pneumonia between Patients with Rheumatoid Arthritis Being Treated with Biologics and Those Being Treated without Biologics.

OBJECTIVE: The aim of this study was to compare the clinical characteristics of pneumocystis pneumonia (PCP) between patients with rheumatoid arthritis (RA) being treated with biologics and those being treated without biologics. METHODS: From 8,630 patients with RA in our institution, we enrolled 24 patients who had developed PCP during the course of their treatment. They were divided into two groups according to the treatment they were receiving for RA: the biologics group (n = 12) and the nonbiologics group (n = 12). Clinical characteristics of PCP were compared between the two groups. RESULTS: At PCP diagnosis, the biologics group showed significantly lower serum levels of ß-D-glucan and C-reactive protein than the nonbiologics group, while the biologics group had significantly higher lymphocyte counts than the nonbiologics group. In the nonbiologics group, lower lymphocyte counts were associated with higher ß-D-glucan levels; however, this was not observed in the biologics group. CONCLUSION: The finding that RA patients being treated with biologics developed PCP with relatively normal lymphocyte counts and lower ß-D-glucan levels suggests that the pathophysiology of PCP in those patients is different from that in patients being treated with other antirheumatic drugs.

Pneumocystis-Driven Inducible Bronchus-Associated Lymphoid Tissue Formation Requires Th2 and Th17 Immunity.

Inducible bronchus-associated lymphoid tissue (iBALT) is an ectopic lymphoid structure composed of highly organized T cell and B cell zones that forms in the lung in response to infectious or inflammatory stimuli. Here, we develop a model for fungal-mediated iBALT formation, using infection with Pneumocystis that induces development of pulmonary lymphoid follicles. Pneumocystis-dependent iBALT structure formation and organization required CXCL13 signaling. Cxcl13 expression was regulated by interleukin (IL)-17 family members, as Il17ra-/-, Il17rb-/-, and Il17rc-/- mice failed to develop iBALT. Interestingly, Il17rb-/- mice have intact Th17 responses, but failed to generate an anti-Pneumocystis Th2 response. Given a role for Th2 and Th17 immunity in iBALT formation, we demonstrated that primary pulmonary fibroblasts synergistically upregulated Cxcl13 transcription following dual stimulation with IL-13 and IL-17A in a STAT3/GATA3-dependent manner. Together, these findings uncover a role for Th2/Th17 cells in regulating Cxcl13 expression and provide an experimental model for fungal-driven iBALT formation.

Is Aerosolized Pentamidine for Pneumocystis Pneumonia Prophylaxis in Renal Transplant Recipients Not as Safe as We Might Think?

Outbreaks ofPneumocystispneumonia have been described in renal transplant recipients. Aerosolized pentamidine is frequently used for prophylaxis in this setting. We report our experience with aerosolized pentamidine use in 56 renal transplant recipients. We found high rates of adverse reactions in patients with chronic respiratory disease.

Pathobiology of Pneumocystis pneumonia: life cycle, cell wall and cell signal transduction.

Pneumocystis is a genus of ascomycetous fungi that are highly morbid pathogens in immunosuppressed humans and other mammals. Pneumocystis cannot easily be propagated in culture, which has greatly hindered understanding of its pathobiology. The Pneumocystis life cycle is intimately associated with its mammalian host lung environment, and life cycle progression is dependent on complex interactions with host alveolar epithelial cells and the extracellular matrix. The Pneumocystis cell wall is a varied and dynamic structure containing a dominant major surface glycoprotein, ß-glucans and chitins that are important for evasion of host defenses and stimulation of the host immune system. Understanding of Pneumocystis cell signaling pathways is incomplete, but much has been deduced by comparison of the Pneumocystis genome with homologous genes and proteins in related fungi. In this mini-review, the pathobiology of Pneumocystis is reviewed, with particular focus on the life cycle, cell wall components and cell signal transduction.

What do Pneumocystis organisms tell us about the phylogeography of their hosts? The case of the woodmouse Apodemus sylvaticus in continental Europe and western Mediterranean islands.

Pneumocystis fungi represent a highly diversified biological group with numerous species, which display a strong host-specificity suggesting a long co-speciation process. In the present study, the presence and genetic diversity of Pneumocystis organisms was investigated in 203 lung samples from woodmice (Apodemus sylvaticus) collected on western continental Europe and Mediterranean islands. The presence of Pneumocystis DNA was assessed by nested PCR at both large and small mitochondrial subunit (mtLSU and mtSSU) rRNA loci. Direct sequencing of nested PCR products demonstrated a very high variability among woodmouse-derived Pneumocystis organisms with a total number of 30 distinct combined mtLSU and mtSSU sequence types. However, the genetic divergence among these sequence types was very low (up to 3.87%) and the presence of several Pneumocystis species within Apodemus sylvaticus was considered unlikely. The analysis of the genetic structure of woodmouse-derived Pneumocystis revealed two distinct groups. The first one comprised Pneumocystis from woodmice collected in continental Spain, France and Balearic islands. The second one included Pneumocystis from woodmice collected in continental Italy, Corsica and Sicily. These two genetic groups were in accordance with the two lineages currently described within the host species Apodemus sylvaticus. Pneumocystis organisms are emerging as powerful tools for phylogeographic studies in mammals.

Comparative genomics suggests primary homothallism of Pneumocystis species.

UNLABELLED: Pneumocystis species are fungal parasites of mammal lungs showing host specificity. Pneumocystis jirovecii colonizes humans and causes severe pneumonia in immunosuppressed individuals. In the absence of in vitro cultures, the life cycle of these fungi remains poorly known. Sexual reproduction probably occurs, but the system of this process and the mating type (MAT) genes involved are not characterized. In the present study, we used comparative genomics to investigate the issue in P. jirovecii and Pneumocystis carinii, the species infecting rats, as well as in their relative Taphrina deformans. We searched sex-related genes using 103 sequences from the relative Schizosaccharomyces pombe as queries. Genes homologous to several sex-related role categories were identified in all species investigated, further supporting sexuality in these organisms. Extensive in silico searches identified only three putative MAT genes in each species investigated (matMc, matMi, and matPi). In P. jirovecii, these genes clustered on the same contig, proving their contiguity in the genome. This organization seems compatible neither with heterothallism, because two different MAT loci on separate DNA molecules would have been detected, nor with secondary homothallism, because the latter involves generally more MAT genes. Consistently, we did not detect cis-acting sequences for mating type switching in secondary homothallism, and PCR revealed identical MAT genes in P. jirovecii isolates from six patients. A strong synteny of the genomic region surrounding the putative MAT genes exists between the two Pneumocystis species. Our results suggest the hypothesis that primary homothallism is the system of reproduction of Pneumocystis species and T. deformans. IMPORTANCE: Sexual reproduction among fungi can involve a single partner (homothallism) or two compatible partners (heterothallism). We investigated the issue in three pathogenic fungal relatives: Pneumocystis jirovecii, which causes severe pneumonia in immunocompromised humans; Pneumocystis carinii, which infects rats; and the plant pathogen Taphrina deformans. The nature, the number, and the organization within the genome of the genes involved in sexual reproduction were determined. The three species appeared to harbor a single genomic region gathering only three genes involved in sexual differentiation, an organization which is compatible with sexual reproduction involving a single partner. These findings illuminate the strategy adopted by fungal pathogens to infect their hosts.

Pneumocystis.

Since its initial misidentification as a trypanosome some 100 years ago, Pneumocystis has remained recalcitrant to study. Although we have learned much, we still do not have definitive answers to such basic questions as, where is the reservoir of infection, how does Pneumocystis reproduce, what is the mechanism of infection, and are there true species of Pneumocystis? The goal of this review is to provide the reader the most up to date information available about the biology of Pneumocystis and the disease it produces.

CD4+ T cells and IFN-γ are required for the development of Pneumocystis-associated pulmonary hypertension.

Pulmonary hypertension (PH) is a disease of diverse etiology. Although primary PH can develop in the absence of prior disease, PH more commonly develops in conjunction with other pulmonary pathologies. We previously reported a mouse model in which PH occurs as a sequela of Pneumocystis infection in the context of transient CD4 depletion. Here, we report that instead of the expected Th2 pathways, the Th1 cytokine IFN-γ is essential for the development of PH, as wild-type mice developed PH but IFN-γ knockout mice did not. Because gene expression analysis showed few strain differences that were not immune-function related, we focused on those responses as potential pathologic mechanisms. In addition to dependence on IFN-γ, we found that when CD4 cells were continuously depleted, but infection was limited by antibiotic treatment, PH did not occur, confirming that CD4 T cells are required for PH development. Also, although CD8 T-cells are implicated in the pathology of Pneumocystis pneumonia, they did not have a role in the onset of PH. Finally, we found differences in immune cell phenotypes that correlated with PH, including elevated CD204 expression in lung CD11c(+) cells, but their role remains unclear. Overall, we demonstrate that a transient, localized, immune response requiring IFN-γ and CD4-T cells can disrupt pulmonary vascular function and promote lingering PH.

The ecology of pneumocystis: perspectives, personal recollections, and future research opportunities.

I am honored to receive the second Lifetime Achievement Award by International Workshops on Opportunistic Protists and to give this lecture. My research involves Pneumocystis, an opportunistic pulmonary fungus that is a major cause of pneumonia ("PcP") in the immunocompromised host. I decided to focus on Pneumocystis ecology here because it has not attracted much interest. Pneumocystis infection is acquired by inhalation, and the cyst stage appears to be the infective form. Several fungal lung infections, such as coccidiomycosis, are not communicable, but occur by inhaling < 5 µm spores from environmental sources (buildings, parks), and can be affected by environmental factors. PcP risk factors include environmental constituents (temperature, humidity, SO2 , CO) and outdoor activities (camping). Clusters of PcP have occurred, but no environmental source has been found. Pneumocystis is communicable and outbreaks of PcP, especially in renal transplant patients, are an ongoing problem. Recent evidence suggests that most viable Pneumocystis organisms detected in the air are confined to a patient's room. Further efforts are needed to define the risk of Pneumocystis transmission in health care facilities; to develop more robust preventive measures; and to characterize the effects of climatological and air pollutant factors on Pneumocystis transmission in animal models similar to those used for respiratory viruses.

All-trans retinoic acid in combination with primaquine clears pneumocystis infection.

Pneumocystis pneumonia (PcP) develops in immunocompromised patients. Alveolar macrophages play a key role in the recognition, phagocytosis, and degradation of Pneumocystis, but their number is decreased in PcP. Our study of various inflammatory components during PcP found that myeloid-derived suppressor cells (MDSCs) accumulate in the lungs of mice and rats with Pneumocystis pneumonia (PcP). We hypothesized that treatment with all-trans retinoic acid (ATRA), a metabolite of vitamin A, may effectively control Pneumocystis (Pc) infection by inducing MDSCs to differentiate to AMs. In rodent models of PcP, we found that 5 weeks of ATRA treatment reduced the number of MDSCs in the lungs and increased the number of AMs which cleared Pc infection. We also found that ATRA in combination with primaquine was as effective as the combination of trimethoprim and sulfamethaxazole for treatment of PcP and completely eliminated MDSCs and Pc organisms in the lungs in two weeks. No relapse of PcP was seen after three weeks of the ATRA-primaquine combination treatment. Prolonged survival of Pc-infected animals was also achieved by this regimen. This is the very first successful development of a therapeutic regimen for PcP that combines an immune modulator with an antibiotic, enabling the hosts to effectively defend the infection. Results of our study may serve as a model for development of novel therapies for other infections with MDSC accumulation.

Stealth and opportunism: alternative lifestyles of species in the fungal genus Pneumocystis.

Pneumocystis species are ascomycetous fungi that obligatorily dwell with no apparent ill effect in the lungs of normal mammals, but they become pathogenic when host defenses are compromised. Identified more than 100 years ago, these atypical fungi manifest characteristics that are unique within the Fungi, such as the lack of ergosterol, genetic complexity of surface antigens, and antigenic variation. Thought to be confined to the severely immunocompromised host, Pneumocystis spp. are being associated with new population niches owing to the advent of immunomodulatory therapies and increased numbers of patients suffering from chronic diseases. The inability to grow Pneumocystis spp. outside the mammalian lung has thwarted progress toward understanding their basic biology, but via the use of new genetic tools and other strategies, researchers are beginning to uncover their biological and genetic characteristics including a biphasic life cycle, significant metabolic capacities, and modulation of lifestyles.