Citations

Abstract Alveolar macrophages (AMs) are critical for host defense and immunity against Pneumocystis. Through surface-associated receptors, AMs recognize, phagocytose, and kill Pneumocystis organisms, producing cytokines that promote pathogen clearance but also contribute to lung inflammation and injury. The EphA2 receptor, a member of the receptor tyrosine kinase (RTK) family, contains an extracellular region that binds activating ligands (Ephrins) and, more recently, has been shown to bind fungal β-glucans, leading to activation of its intracellular tyrosine kinase domain. The EphA2 signaling pathway has important roles in cancer, angiogenesis, and inflammatory diseases such as atherosclerosis. EphA2 is highly expressed in epithelial and endothelial cells, and emerging evidence indicates that it serves as a critical regulator of host defense against bacterial, viral, and fungal pathogens. In oropharyngeal candidiasis, EphA2 was identified as a receptor for fungal β-glucans in oral epithelial cells, mediating proinflammatory signaling through signal transducer and activator of transcription 3 (STAT3) and mitogen-activated protein kinase (MAPK) pathways. Our laboratory recently demonstrated that EphA2 on lung epithelial cells binds Pneumocystis organisms, and that β-glucans from these organisms activate downstream signaling leading to IL-6 release. However, the role of EphA2 signaling in AM immune responses during Pneumocystis pneumonia (PCP) remains unknown. To address this, we examined EphA2-deficient (EphA2−/−) AMs stimulated with Pneumocystis β-glucans. Compared to wild-type AMs, EphA2−/− AMs exhibited significantly decreased production of proinflammatory cytokines, including IL-6 and TNF-α. In CD4-depleted mouse models of PCP, EphA2−/− lungs displayed markedly reduced levels of inflammatory cytokines and increased organism burdens, indicating impaired immune activation but preserved fungal persistence. Importantly, in CD4-depleted mice undergoing standard trimethoprim-sulfamethoxazole (TMP/SMX) therapy, concurrent administration of the EphA2 small-molecule inhibitor ALW-II-41-27 significantly reduced lung TNF-α responses without evidence of toxicity. These findings demonstrate that EphA2 receptor signaling plays a critical role in regulating macrophage-mediated inflammatory responses to Pneumocystis. While EphA2 deficiency dampens pulmonary inflammation, it also compromises fungal clearance, highlighting a delicate balance between host defense and immunopathology. Our data suggest that targeted modulation of EphA2 signaling during anti-Pneumocystis therapy may provide a novel adjunctive approach to reduce detrimental lung inflammation associated with PCP, potentially improving outcomes in affected patients. This abstract is funded by: NIH R01 HL-62150

Abstract Rationale Pneumocystis pneumonia (PJP) remains a major opportunistic infection in immunocompromised hosts, with limited therapeutic options. Previous studies identified a conserved cysteine residue (C353) in Pneumocystisphosphoglucomutase (PGM), analogous to that found in other pathogenic fungi but absent in mammalian PGMs, suggesting an exploitable antifungal target. We aimed to define the functional significance and therapeutic potential of targeting Pneumocystis PGM. Methods Full-length P. jirovecii and P. murina PGM cDNAs were cloned into S. cerevisiae pgm2Δ yeast (deficient in PGM activity) using the pYES2.1 TOPO vector for heterologous expression. Recombinant yeast lysates were tested for PGM activity in the presence of the thiol-reactive inhibitor ISFP10 (0-200 μM). Parallel assays were performed using recombinant human PGM lacking C353. Yeast growth inhibition in these strains in the presence of ISFP10 was also measured over 72 hours. For in vitro and ex vivo assays, P. murina organisms were treated with ISFP10, and viability was quantified via qPCR of the mitochondrial 16S ribosomal copy number. Precision-cut lung slice (PCLS) ex vivo cultures from a murine PCP model and in vivo CD4-depleted mice were used to assess ISFP10 efficacy and toxicity. Results Both P. jirovecii and P. murina PGMs showed potent, dose-dependent inhibition of enzymatic activity by ISFP10 up to 12.5 μM, with no inhibition of human PGM at concentrations up to 200 μM. Yeast complementation assays confirmed that ISFP10 significantly reduced the growth of pgm2Δ yeast expressing Pneumocystis spp. PGM. In vitro incubation of P. murina with ISFP10 resulted in significant reductions in organismal 16S mitochondrial copy number, comparable to pentamidine. In ex vivo PCLS models, ISFP10 decreased P. murina burden without affecting uninfected tissue viability. In vivo, ISFP10 administration for seven days significantly reduced fungal burden in CD4-depleted mice with an acceptable safety profile. Conclusions These findings demonstrate that Pneumocystis PGM activity depends on a conserved cysteine residue that is selectively targetable by ISFP10. Pharmacologic inhibition of PGM impairs P. murina viability in vitro, ex vivo, and in vivo with minimal host toxicity. Collectively, these data establish PGM as a promising, fungus-specific antifungal target and support the further development of small-molecule inhibitors targeting Pneumocystis PGM. This abstract is funded by: NIH R01 HL-62150, R21 AI-181542

Abstract Background Alveolar macrophages (AMs) are central to host defense against Pneumocystis, mediating organism clearance while also contributing to lung inflammation. EphA2, a transmembrane receptor that binds fungal β-glucans, has emerged as a regulator of host–pathogen interactions, but its role in AM responses during Pneumocystis pneumonia (PCP) is unknown. Methods We evaluated AM inflammatory responses to Pneumocystis β-glucans in the presence and absence of EphA2. In vivo studies used a CD4-depleted mouse model of PCP comparing wild-type and EphA2-deficient (EphA2−/−) mice. Lung cytokine responses and organism burden were assessed. Additionally, EphA2 signaling was pharmacologically inhibited using ALW-II-41-27 during trimethoprim-sulfamethoxazole treatment. Results EphA2 deficiency significantly reduced AM proinflammatory cytokine responses to Pneumocystis β-glucans. In CD4-depleted PCP mice, EphA2−/− animals demonstrated decreased lung inflammatory cytokines, but increased organism burden compared to controls. Pharmacologic inhibition of EphA2 during antimicrobial therapy significantly suppressed lung inflammation. Conclusions EphA2 signaling plays a critical role in AM-mediated host defense against Pneumocystis by promoting inflammatory responses necessary for organism control. However, this pathway also contributes to lung inflammation. Targeting EphA2 may represent a novel strategy to modulate inflammation during severe PCP while preserving antimicrobial defense.

Introduction. Aspergillus fumigatus phosphoglucomutase ( Af PGM) is a central enzyme in fungal carbohydrate metabolism that catalyses the interconversion of glucose-1-phosphate and glucose-6-phosphate, generating precursors such as uridine diphosphate glucose required for the synthesis of key cell wall polysaccharides. Hypothesis/Gap Statement. Although Af PGM has been implicated in fungal viability and cell wall integrity, further functional and molecular characterization is required to better define its role in cell wall-associated processes and to evaluate its potential as a selective antifungal target. Aim. This study aimed to further characterize the functional contribution of Af PGM to fungal growth, cell wall-related phenotypes, biofilm formation and enzymatic activity. Methodology. A conditional A. fumigatus P alcA ::pgm mutant was used to assess growth, soluble β -glucan levels and biofilm formation. Functional complementation was evaluated by heterologous expression of Afpgm in a Saccharomyces cerevisiae pgm2Δ strain. Enzymatic inhibition was examined using the isothiazolone compound ISFP10. Results. Reduced Af PGM activity was associated with impaired growth, altered cell wall-related phenotypes, decreased soluble β -glucan levels and diminished biofilm biomass. Expression of Afpgm restored growth and sedimentation defects in the yeast pgm2Δ mutant to WT levels, confirming functional conservation. ISFP10 inhibited Af PGM activity and disrupted associated fungal phenotypes. Conclusion. These findings further define the functional role of Af PGM in fungal carbohydrate metabolism and cell wall-associated biology. The selective inhibition of Af PGM relative to human phosphoglucomutase supports its potential as a promising target for the development of novel antifungal therapies.

Abstract Candida auris is an emerging fungal pathogen with high rates of multidrug resistance, posing a significant global health threat. Immunosuppressed patients are particularly vulnerable to multidrug-resistant (MDR) C. auris infections. This systematic review and meta-analysis (SR-MA) aimed to assess the global prevalence of MDR-C. auris isolates in humans and their resistance to antifungal drugs, primarily fluconazole (FLZ), voriconazole (VOR), and amphotericin B (AmB). A comprehensive search was conducted in PubMed, Scopus, and ScienceDirect databases on published studies regarding clinical C. auris isolates and the use of antifungal susceptibility testing assay from 17 March 2015 to 1 February 2024. Meta-analysis was conducted using random-effects models to determine the estimated pooled prevalence of MDR-C. auris isolates and their antifungal resistance profiles from immunosuppressed humans. A total of 81 research studies were included in the final analysis, with the overall pooled prevalence of the three most important antifungal drugs; FLZ (92.5% | 95% CI: 89.5–94.7), VOR (49.0% | 95% CI: 37.6–60.4), and AmB (51.0% | 95% CI: 42.3–59.7). According to the subgroup analyses, FLZ-resistant C. auris was the most prevalent isolate observed in the five continents reporting compared to the VOR- and AmB-resistant C. auris having lower pooled prevalence. Further, higher pooled prevalence of MDR-C. auris isolates were observed from males (68.2%, n = 578) compared to females (37.1%, n = 283). Finally, the global pooled prevalence for mortality in immunosuppressed patients with MDR-C. auris from 13 research studies was 41.5% (95% CI: 29.9–54.2). This SR-MA study establishes the variation of MDR-C. auris resistance patterns observed from different continents and the importance to conduct further research studies using potent antifungal drugs. Accordingly, it is imperative to establish stringent surveillance on the increasing prevalence of antifungal resistance particularly in low-income regions with limited research resources to prevent increasing prevalence of MDR-C. auris.

Abstract Pneumocystis jirovecii pneumonia remains a significant cause of mortality and morbidity in immunocompromised populations worldwide. Mortality rates range from 5% to 30% in HIV-positive individuals and 4% to 76% in those without HIV. Recognizing its public health impact, the World Health Organization (WHO) included Pneumocystis jirovecii in its 2022 “Fungal Priority Pathogens” list. In this review, we will summarize published findings over the last 14 years on the immunopathogenic mechanisms underlying Pneumocystis pneumonia–related lung injury and examine the potential for targeting these pathways in adjunctive immune modulation therapy. Notably, recent studies have identified promising immune-based interventions, including PD-1/PD-L1 blockade and IL-7 therapy, which may enhance pathogen clearance while controlling damaging inflammation. Additionally, small molecule inhibitors such as BRD5529 and ALW-II-41-27 have shown potential in reducing lung injury by modulating proinflammatory signaling pathways in Pneumocystis pneumonia.

Abstract INTRODUCTION Candida auris has emerged as a global pathogenic yeast that disproportionately affects patients in healthcare settings. Infection ranges from localized skin infections to severe bloodstream infections. Multidrug resistance, hardiness on environmental surfaces, and the ability to persist as a colonizer in both sick and healthy patients enables it to spread rapidly in healthcare settings leading to significant morbidity and mortality. Multidrug resistance has led to limited effective therapies and necessitates the need for novel therapeutic options. Here we evaluate the efficacy of ISFP10, an antifungal compound with a novel mechanism of action, against C. auris. ISFP10 inhibits fungal phosphoglucomutase (PGM), an enzyme required for production of substrates for fungal cell wall synthesis (Figure 1). METHODSMIC Testing The compound ISFP10 was submitted to the National Institute of Allergy and Infectious Diseases Therapeutic Development Services for half-maximal and maximal inhibitory concentration (IC50/IC100) testing. C. auris isolates were tested using typical drug-dilution methods. Saccharomyces cerevisiae – C. auris PGM Growth Curve Testing This proof of mechanism assay was instituted to isolate PGM in Saccharomyces for growth curve testing. The Candida auris PGM was cloned into pYES2.1 downstream of an inducible galactose promoter. This plasmid was transformed into S. cerevisiae. C. auris PGM plasmid-containing S. cerevisiae was incubated for 24 hours in 2% glucose minimal media. Using absorbance readings, the inoculum was normalized to 5x105 cells/mL in galactose-containing media. The inoculum was plated onto a 96-well plate in a drug-dilution scheme with ISFP10 at concentrations ranging from 200uM to 6.25uM along with DMSO control wells. Plates were incubated at 30° C for 72 hours in a spectrophotometer with absorbance readings every hour. Growth curves were plotted using GraphPad Prism software. RESULTS The half-maximal inhibitory concentrations (IC50) and maximal inhibitory concentrations (IC100) were consistently low across the three C. auris isolates. The IC50 ranged from 0.5 µg/mL to 1.0 µg/mL, while the IC100 was 1.0 µg/mL for each of the three isolates (Table 1). Growth curves demonstrated concentration-dependent inhibition of C. auris PGM plasmid-containing S. cerevisiae. Figure 2 shows full inhibition of fungal growth at 25µM with dose-dependent delayed growth at lower concentrations. CONCLUSIONS ISFP10 shows promise for inhibiting C. auris PGM in a dose-dependent pattern. It's high in vitro activity and novel mechanism of action necessitates further investigation as a potential therapeutic for C. auris.

ABSTRACT Background and Objective Previously, the novel small molecule ISFP10 has been shown to inhibit fungal phosphoglucomutase (PGM) activity in Aspergillus fumigatus and Pneumocystis spp. With 50-fold selectivity over the human PGM molecule due to the presence of a unique yet conserved cysteine residue present in a number pathogenic fungal PGMs, use of this compound may provide a novel broad-spectrum approach to treating fungal infections. Accordingly, we sought to determine the tolerability in test animals receiving this compound, as well as the potential antifungal activity of ISFP10 on cultures of the common fungal pathogens Candida albicans and Candida glabrata . Methods C57BL6 mice received once daily intraperitoneal (IP) injections of 100 mL of vehicle control (DMSO) or ISFP10 at a concentration of 10 mg/kg. Body weights were recorded daily for 7 days of treatment. On the final day, mice were weighed and euthanized. Postmortem blood collection was conducted via cardiac puncture and distributed to EDTA and lithium heparin tubes for complete blood count (CBC) and comprehensive blood chemistry panels, respectively. Liver, kidney, and lung tissue were also harvested and placed in 10% formalin for H&E staining and blinded histopathologic scoring. Lung samples were further analyzed for proinflammatory cytokines using enzyme-linked immunosorbent assays (ELISA) and quantitative PCR (qPCR). Furthermore, ISPF10 was tested for antifungal activity via 8-hour growth curve analysis in a concentration-dependent fashion against Candida albicans and Candida glabrata . Results There was no significant difference in the daily or final body weights of the mice receiving 10 mg/kg of ISFP10 compared to those of the vehicle control group. Extracellular matrix (ECM) transcripts for IL-6 and TNFα were statistically similar via qPCR. ELISA results of proinflammatory cytokines for IL-6 was not significant whereas TNFα levels in lung tissue from the ISFP10 treatment group were significantly reduced, indicating a potential anti-inflammatory effect of ISFP10 at this dosage. Overall, blood chemistry and CBC analysis revealed no overall significant differences between the two groups, except for increased neutrophil counts and decreased potassium levels in samples collected from ISFP10 treated animals compared to the vehicle control group. These laboratory abnormalities were not of clinical significance to the test animals. Blinded histopathological examination revealed no abnormalities or evidence of critical organ toxicity from all groups. Inhibition of C. albicans and C. glabrata culture growth by ISFP10 was concentration-dependent in YPD liquid media containing the ISFP10 compared to vehicle control. Conclusions Our preliminary testing of ISFP10 revealed no inherent safety or toxicology concerns within the observed parameters. These data further support significant culture suppressive activity against C. albicans and C. glabrata . Taken together, these observations of ISFP10 further indicate that targeting PGM might be a novel and viable therapeutic strategy for serious fungal infections. Key points An inhibitor specific to fungal PGM enzymes, termed ISFP10, was generally well tolerated when administered via intraperitoneal (IP) injection in mice. ISFP10 displays antifungal activity in a concentration-dependent manner against C. albicans and C. glabrata .

ABSTRACT Pneumocystis jirovecii pneumonia (PJP) remains a significant cause of morbidity and mortality during AIDS. In AIDS, the absence of CD4 immunity results in exuberant and often fatal PJP. In addition, organism clearance requires a balanced macrophage response since excessive inflammation promotes lung injury and respiratory failure. Corticosteroids given in addition to antibiotics significantly improve outcomes during PJP. However, concerns exist that corticosteroids further suppress immunity and increase co-infections. New strategies to promote killing and clearance of Pneumocystis while balancing lung inflammation are required. Prior studies have shown that innate immunity to Pneumocystis is mediated by C-type lectin receptors (CLRs) on macrophages and involves downstream CARD9 activation. CARD9 can be targeted by a novel specific small molecule inhibitor (BRD5529) that significantly reduces inflammatory signaling by macrophages. CARD9 serves as the central intracellular molecule through which Dectin-1, Dectin-2, Mincle, and other CLRs signal. Dectin-1 CLR is activated through its own intracytoplasmic domain, whereas other innate CLRs (e.g., Dectin-2 and Mincle) require interactions with a common Fc-gamma receptor (FcγR) accessory chain to mediate responses. We now observe that mice double deficient in both Dectin-1 and Fcer1g (which lack the FcγR gamma chain) exhibit markedly reduced organism clearance compared with Card9 −/− infected animals. These mice also possess deficiencies in immunoglobulin (Ig) Fc receptors directly mediating antibody responses, further implicating altered humoral responses in Pneumocystis killing. We further demonstrate in the Pneumocystis pneumonia (PCP) mouse model that BRD5529 administration successfully suppresses inflammatory cytokines. Our data support that innate immune responses through the CLR-CARD9 axis and humoral response act together to mediate effective responses resulting in optimal organism killing and generation of host inflammatory responses. Furthermore, host lung inflammation during PCP may be successfully reduced with a novel CARD9 small molecule inhibitor. IMPORTANCE Pneumocystis pneumonia (PCP) causes severe respiratory impairment in hosts with suppressed immunity, particularly those with CD4 deficiencies, such as HIV. In addition to lymphocytic immunity, both innate and humoral immunities also participate in host defense against Pneumocystis . In the current studies, we defined the relative roles of CLR receptor-mediated inflammation, as well as FcgR-related inflammation and clearance of Pneumocystis organisms. Our studies reveal important roles for CLR activities for inducing lung inflammation, which can be ameliorated with a novel small molecule inhibitor of the CARD9 adaptor protein that is necessary for CLR signaling. In contrast, FcgR has a dominant role in organism clearance, underscoring an integral role of humoral responses for the elimination of this infection.

Abstract Overgrowth of the fungus Wallemia mellicola in the intestines of mice enhances the severity of asthma. Wallemia mellicola interacts with the immune system through Dectin-2 expressed on the surface of myeloid and intestinal epithelial cells. Using Dectin-2-deficient mice, we show that the interaction of W. mellicola with Dectin-2 is essential for the gut-lung pathways, enhancing the severity of asthma in mice with W. mellicola intestinal dysbiosis. These findings offer better insight into dysbiosis-associated inflammation and highlight the role pattern recognition receptors have in immune recognition of commensal fungi in the gut, leading to alterations in immune function in the lungs.