Pneumocystis murina Promotes Inflammasome Formation and NETosis during Pneumocystis Pneumonia.

Pneumocystis jirovecii pneumonia (PjP) poses a serious risk to individuals with compromised immune systems, such as individuals with HIV/AIDS or undergoing immunosuppressive therapies for cancer or solid organ transplants. Severe PjP triggers excessive lung inflammation, resulting in lung function decline and consequential alveolar damage, potentially culminating in acute respiratory distress syndrome. Non-HIV patients face a 30-60%mortality rate, emphasizing the need for a deeper understanding of inflammatory responses in PjP. Prior research emphasized macrophages in Pneumocystis infections, neglecting neutrophils' role in tissue damage. Consequently, the overemphasis on macrophages led to an incomplete understanding of the role of neutrophils and inflammatory responses. In the current investigation, our RNAseq studies on a murine surrogate model of PjP revealed heightened activation of the NLRP3 inflammasome and NETosis cell death pathways in their lungs. Immunofluorescence staining confirmed Neutrophil Extracellular Trap (NET) presence in the lungs of the P. murina -infected mice, validating our findings. Moreover, isolated neutrophils exhibited NETosis when directly stimulated with P. murina . While isolated NETs did not compromise P. murina viability, our data highlight the potential role of neutrophils in promoting inflammation during P. murina pneumonia through NLRP3 inflammasome assembly and NETosis. These pathways, essential for inflammation and pathogen elimination, bear the risk of uncontrolled activation leading to excessive tissue damage and persistent inflammation. This pioneering study is the first to identify the formation of NETs and inflammasomes during Pneumocystis infection, paving the way for comprehensive investigations into treatments aimed at mitigating lung damage and augmenting survival rates for individuals with PjP.

Extracellular vesicles from Pneumocystis carinii-infected rats impair fungal viability but are dispensable for macrophage functions.

Pneumocystis spp. are host obligate fungal pathogens that can cause severe pneumonia in mammals and rely heavily on their host for essential nutrients. The lack of a sustainable in vitro culture system poses challenges in understanding their metabolism, and the acquisition of essential nutrients from host lungs remains unexplored. Transmission electron micrographs show that extracellular vesicles (EVs) are found near Pneumocystis spp. within the lung. We hypothesized that EVs transport essential nutrients to the fungi during infection. To investigate this, EVs from P. carinii- and P. murina-infected rodents were biochemically and functionally characterized. These EVs contained host proteins involved in cellular, metabolic, and immune processes as well as proteins with homologs found in other fungal EV proteomes, indicating that Pneumocystis may release EVs. Notably, EV uptake by P. carinii indicated their potential involvement in nutrient acquisition and a possibility for using engineered EVs for efficient therapeutic delivery. However, EVs added to P. carinii in vitro did not show increased growth or viability, implying that additional nutrients or factors are necessary to support their metabolic requirements. Exposure of macrophages to EVs increased proinflammatory cytokine levels but did not affect macrophages' ability to kill or phagocytose P. carinii. These findings provide vital insights into P. carinii and host EV interactions, yet the mechanisms underlying P. carinii's survival in the lung remain uncertain. These studies are the first to isolate, characterize, and functionally assess EVs from Pneumocystis-infected rodents, promising to enhance our understanding of host-pathogen dynamics and therapeutic potential.IMPORTANCEPneumocystis spp. are fungal pathogens that can cause severe pneumonia in mammals, relying heavily on the host for essential nutrients. The absence of an in vitro culture system poses challenges in understanding their metabolism, and the acquisition of vital nutrients from host lungs remains unexplored. Extracellular vesicles (EVs) are found near Pneumocystis spp., and it is hypothesized that these vesicles transport nutrients to the pathogenic fungi. Pneumocystis proteins within the EVs showed homology to other fungal EV proteomes, suggesting that Pneumocystis spp. release EVs. While EVs did not significantly enhance P. carinii growth in vitro, P. carinii displayed active uptake of these vesicles. Moreover, EVs induced proinflammatory cytokine production in macrophages without compromising their ability to combat P. carinii. These findings provide valuable insights into EV dynamics during host-pathogen interactions in Pneumocystis pneumonia. However, the precise underlying mechanisms remain uncertain. This research also raises the potential for engineered EVs in therapeutic applications.

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.

Extracellular Vesicles from Pneumocystis carinii -Infected Rats Impair Fungal Viability but are Dispensable for Macrophage Functions.

Pneumocystis spp. are host obligate fungal pathogens that can cause severe pneumonia in mammals and rely heavily on their host for essential nutrients. The lack of a sustainable in vitro culture system poses challenges in understanding their metabolism and the acquisition of essential nutrients from host lungs remains unexplored. Transmission electron micrographs show Extracellular Vesicles (EVs) are found near Pneumocystis spp. within the lung. We hypothesized that EVs transport essential nutrients to the fungi during infection. To investigate this, EVs from P. carinii and P. murina infected rodents were biochemically and functionally characterized. These EVs contained host proteins involved in cellular, metabolic, and immune processes as well as proteins with homologs found in other fungal EV proteomes, indicating Pneumocystis may release EVs. Notably, EV uptake by P. carinii indicated their potential involvement in nutrient acquisition and indicate a possibility for using engineered EVs for efficient therapeutic delivery. However, EVs added to P. carinii in vitro , did not show increased growth or viability, implying that additional nutrients or factors are necessary to support their metabolic requirements. Exposure of macrophages to EVs increased proinflammatory cytokine levels, but did not affect macrophages' ability to kill or phagocytose P. carinii . These findings provide vital insights into P. carinii and host EV interactions, yet the mechanisms underlying P. carinii 's survival in the lung remain uncertain. These studies are the first to isolate, characterize, and functionally assess EVs from Pneumocystis -infected rodents, promising to enhance our understanding of host-pathogen dynamics and therapeutic potential.

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.

The Effects of Sex and Strain on Pneumocystis murina Fungal Burdens in Mice.

Many preclinical studies of infectious diseases have neglected experimental designs that evaluate potential differences related to sex with a concomitant over-reliance on male model systems. Hence, the NIH implemented a monitoring system for sex inclusion in preclinical studies. METHODS: Per this mandate, we examined the lung burdens of Pneumocystis murina infection in three mouse strains in both male and female animals at early, mid, and late time points. RESULTS: Females in each strain had higher infection burdens compared to males at the later time points. CONCLUSION: Females should be included in experimental models studying Pneumocystis spp.

The Antifungal and Anti-Pneumocystis Activities of the Novel Compound A3IS (Mycosinate).

A3IS (Mycosinate) is a synthetic product which only contains ingredients found naturally within honey. A3IS is a broad-spectrum antimicrobial product which produces a sustained release of hydrogen peroxide at low but therapeutic levels. The product elicits this release through an enzymatic reaction between glucose oxidase and the substrate glucose once the product is hydrated. As medical uses for different honeys are being re-evaluated, the purpose of this study was to evaluate the in vitro effects of A3IS against a comprehensive panel of human pathogens, including Pneumocystis species, providing a unique assessment against a panel of eukaryotic pathogens. Without exception, A3IS exhibited significant efficacy at 50% and 100% inhibitory concentrations against a broad spectrum of human pathogens including yeasts, molds (both hyaline and dematiaceous), and dimorphic fungi. Notably, A3IS was effective against fungal strains with a high level of resistance to fluconazole or voriconazole. The 50% inhibitory concentrations for Pneumocystis carinii and P. murina (surrogates for P. jirovecii) were considered "Marked" and "Moderate" on an established rank scale, and would be considered for in vivo studies, based on an established in vitro-in vivo pipeline. These results indicate that A3IS is a novel anti-fungal agent against an extensive range of human fungal pathogens.

The Long-Acting Echinocandin, Rezafungin, Prevents Pneumocystis Pneumonia and Eliminates Pneumocystis from the Lungs in Prophylaxis and Murine Treatment Models.

Rezafungin is a novel echinocandin in Phase 3 development for prevention of invasive fungal disease caused by Candida spp., Aspergillus spp. and Pneumocystis jirovecii in blood and marrow transplantation patients. For such patients, standard antifungal prophylaxis currently comprises an azole for Candida and Aspergillus plus trimethoprim-sulfamethoxazole (TMP-SMX) for Pneumocystis pneumonia (PCP) despite drug-drug-interactions and intolerability that may limit their use, thus, alternatives are desirable. Rezafungin demonstrates a favorable safety profile and pharmacokinetic properties that allow for once-weekly dosing in addition, to antifungal activity against these predominant pathogens. Herein, the in vivo effects of rezafungin against Pneumocystis murina pneumonia were evaluated in immunosuppressed mouse models of prophylaxis and treatment using microscopy and qPCR assessments. In the prophylaxis model, immunosuppressed mice inoculated with P. murina were administered TMP-SMX (50/250 mg/kg 1×/week or 3×/week), caspofungin (5 mg/kg 3×/week), rezafungin (20 mg/kg, 1×/week or 3×/week; 5 mg/kg, 3×/week) intraperitoneally for 2, 4, 6 and 8 weeks, then immunosuppressed for an additional 6 weeks. Rezafungin administered for 4 weeks prevented P. murina from developing infection after rezafungin was discontinued. In the treatment model, immunosuppressed mice with P. murina pneumonia were treated with rezafungin 20 mg/kg 3×/week intraperitoneally for 2, 4, 6 and 8 weeks. Treatment with rezafungin for 8 weeks resulted in elimination of P. murina. Collectively, these studies showed that rezafungin could both prevent infection and eliminate P. murina from the lungs of mice. These findings support the obligate role of sexual reproduction for survival and growth of Pneumocystis spp. and warrant further investigation for treatment of P. jirovecii pneumonia in humans.

The Persistent Challenge of Pneumocystis Growth Outside the Mammalian Lung: Past and Future Approaches.

The pathogenic fungi in the genus, Pneumocystis, have eluded attempts to continuously grow them in an ex vivo cultivation system. New data from transcriptomic and genomic sequencing studies have identified a myriad of absent metabolic pathways, helping to define their host obligate nature. These nutrients, factors, and co-factors are acquired from their mammalian host and provide clues to further supplementation of existing media formulations. Likewise, a new appreciation of the pivotal role for the sexual cycle in the survival and dissemination of the infection suggests that Pneumocystis species are obligated to undergo mating and sexual reproduction in their life cycle with a questionable role for an asexual cycle. The lack of ascus formation in any previous cultivation attempts may explain the failure to identify a sustainable system. Many characteristics of these ascomycetes suggest a biotrophic existence within the lungs of the mammalian hosts. In the present review, previous attempts at growing these fungi ex vivo are summarized. The significance of their life cycle is considered, and a list of potential supplements based on the genomic and transcriptomic studies is presented. State of the art technologies such as metabolomics, organoids, lung-on-a chip, and air lift cultures are discussed as potential growth systems.

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

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

The Promise of Lung Organoids for Growth and Investigation of Pneumocystis Species.

Pneumocystis species (spp.) are host-obligate fungal parasites that colonize and propagate almost exclusively in the alveolar lumen within the lungs of mammals where they can cause a lethal pneumonia. The emergence of this pneumonia in non-HIV infected persons caused by Pneumocystis jirovecii (PjP), illustrates the continued importance of and the need to understand its associated pathologies and to develop new therapies and preventative strategies. In the proposed life cycle, Pneumocystis spp. attach to alveolar type 1 epithelial cells (AEC1) and prevent gas exchange. This process among other mechanisms of Pneumocystis spp. pathogenesis is challenging to observe in real time due to the absence of a continuous ex vivo or in vitro culture system. The study presented here provides a proof-of-concept for the development of murine lung organoids that mimic the lung alveolar sacs expressing alveolar epithelial type 1 cells (AEC1) and alveolar type 2 epithelial cells (AEC2). Use of these 3-dimensional organoids should facilitate studies of a multitude of unanswered questions and serve as an improved means to screen new anti- PjP agents.

A Novel Encochleated Formulation Improves Atovaquone Activity in a Murine Model of Pneumocystis Pneumonia.

Although atovaquone is effective in treating and preventing Pneumocystis pneumonia (PCP), its use is limited by nonlinear absorption and adverse events. The current study was undertaken to examine the activity of encochleated atovaquone (eATQ), a novel lipid-crystal nanoparticle formulation, in a mouse model of PCP. eATQ 100-200 mg was superior to commercially available atovaquone at 14 days in decreasing total Pneumocystis nuclei and asci. eATQ plus anidulafungin reduced nuclei significantly better than commercial atovaquone plus anidulafungin. eATQ is a novel formulation of atovaquone that warrants further evaluation for treatment and prevention of PCP.

Efficacy of Rezafungin in Prophylactic Mouse Models of Invasive Candidiasis, Aspergillosis, and Pneumocystis Pneumonia.

Antifungal prophylaxis is recommended to prevent invasive fungal disease caused by Candida spp., Aspergillus spp., and Pneumocystis jirovecii in patients at risk for opportunistic infections, such as allogeneic blood or marrow transplant recipients, patients with hematological disease undergoing chemotherapy, or patients on immunosuppressive therapies. Current approaches to antifungal prophylaxis require multiple agents to cover these key fungi. Rezafungin, a novel echinocandin designed for next-generation properties (e.g., greater stability and long-acting pharmacokinetics for once-weekly dosing), has demonstrated in vitro activity against Candida and Aspergillus spp. and efficacy against Pneumocystis spp. biofilms. Rezafungin was evaluated in in vivo studies of prophylactic efficacy using immunosuppressed mouse models of invasive candidiasis, aspergillosis, and Pneumocystis pneumonia. Rezafungin reduction of Candida CFU burden was generally greater with increasing drug concentrations (5, 10, or 20 mg/kg) and when rezafungin was administered closer to the time of fungal challenge (day -1, -3, or -5). Similarly, in the aspergillosis model, survival rates increased with drug concentrations and when rezafungin was administered closer to the time of fungal challenge. Against Pneumocystismurina, rezafungin significantly reduced trophic nuclei and asci counts at all doses tested. Rezafungin prevented infection at the two higher doses compared to vehicle and had comparable activity to the active control trimethoprim-sulfamethoxazole at human equivalent doses for prevention. These findings support phase 3 development of rezafungin and the potential for single-agent prophylaxis against invasive fungal disease caused by Candida spp., Aspergillus spp., and Pneumocystis jirovecii.

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.

Correction to: A quantitative systems pharmacology (QSP) model for Pneumocystis treatment in mice.

It was highlighted that the original article [1] contained errors in the figures and their legends and by extension the in-text figure citations. This Corrections article shows the correct figures and correct figure legends.

Chloroquine Analogues as Leads against Pneumocystis Lung Pathogens.

The impact of Pneumocystis pneumonia (PcP) on morbidity and mortality remains substantial for immunocompromised individuals, including those afflicted by HIV infection, organ transplantation, cancer, autoimmune diseases, or subject to chemotherapy or corticosteroid-based therapies. Previous work from our group has shown that repurposing antimalarial compounds for PcP holds promise for treatment of this opportunistic infection. Following our previous discovery of chloroquine analogues with dual-stage antimalarial action both in vitro and in vivo, we now report the potent action of these compounds on Pneumocystis carinii in vitro Identification of chloroquine analogues as anti-PcP leads is an unprecedented finding.