Theodore J. Kottom

The fungal cell wall is a dynamic, carbohydrate-rich structure whose composition is closely coordinated with cellular metabolism. Phosphoglucomutase (PGM) interconverts glucose-1- phosphate and glucose-6-phosphate, linking carbon metabolism with UDP-glucose-dependent cell-wall biosynthesis. We previously identified functional Pgm2 homologs from Pneumocystis jirovecii and Pneumocystis murina . Here, using a Saccharomyces cerevisiae pgm2 Δ complementation model, we examined the relationship between Pgm2 activity, β-1,3-glucan, and cell-wall integrity. Pgm2 deficiency reduced β-1,3-glucan content and altered zymolyase sensitivity without major changes in cell-wall thickness. Bulk RNA sequencing revealed that the pgm2 Δ mutant exhibited coordinated metabolic and cell-wall transcriptional responses, including increased SLT2/MPK1 , RLM1 , FKS2/GSC2 , GFA1 , CHS1 , CRH1 , SED1 , and CWP1 expression and decreased FKS1 , GAS1 , GAS3 , and CHS2 expression. Expression of either Pjpgm 2 or Pmpgm2 broadly shifted these responses toward wild-type levels. Selected transcriptional changes were further evaluated by qRT-PCR, which supported the major stress and cell-wall responses identified by RNA sequencing. FKS2/GSC2 showed the same directional pattern, although differences were not statistically significant by qRT-PCR. Despite this compensatory response, Pgm2-deficient cells were markedly more susceptible to sublethal caspofungin, whereas either Pneumocystis Pgm2 homolog restored tolerance toward wild-type levels. These findings link Pgm2-dependent glucose-phosphate metabolism with β-1,3-glucan levels and cell-wall function. Given the importance of β-1,3-glucan to the Pneumocystis cyst wall and its limited cell-wall biosynthetic pathways, these findings further support Pgm2 as an important component of Pneumocystis cell-wall biology and a potential antifungal target. HIGHLIGHTS: Pneumocystis Pgm2 restores β-1,3-glucan cell wall phenotypes in Pgm2-defecient yeast. Pgm2 loss remodels the fungal cell-wall integrity transcriptional program. Pgm2 deficiency induces FKS2 and sensitizes yeast to caspofungin, a phenotype reversed by Pneumocystis Pgm2 homologs.

Background and Objective The novel small molecule ISFP10 inhibits fungal phosphoglucomutase (PGM) activity in Aspergillus fumigatus and Pneumocystis spp. With 50-fold selectivity over the human isoform, conferred by a conserved cysteine residue present in several pathogenic fungi, ISFP10 may represent a promising broad-spectrum antifungal. We evaluated its activity against Candida albicans and Candida glabrata, followed by toxicology assessment in mice. Methods Minimum inhibitory concentration susceptibilities were performed on three strains of C. albicans and C. glabrata and compared with fluconazole. Growth inhibition was further assessed in an 8-h, concentration-dependent growth assay. For toxicology, C57BL6 mice received once-daily intraperitoneal injections of vehicle control (dimethyl sulfoxide) or ISFP10 (10 mg/kg) for 7 days. Body weight, complete blood count (CBC), blood chemistry, histopathology of major organs, and lung extracellular matrix (ECM) and cytokine levels (ELISA, qPCR) were analyzed. Results ISFP10 inhibited all tested Candida strains with significantly greater potency than fluconazole. It also showed concentration-dependent inhibition of growth in liquid culture. In mice, no significant differences in daily or final body weight, ECM transcript levels, or interleukin (IL)-6 expression were observed between groups. Tumor necrosis factor-α (TNFα) levels in lung tissue were significantly reduced in ISFP10-treated animals, suggesting a potential anti-inflammatory effect. Blood chemistry and CBC revealed minor abnormalities (increased neutrophils, decreased potassium), but these were not clinically significant. Histopathologic evaluation showed no evidence of organ toxicity. Conclusions ISFP10 demonstrates potent antifungal properties against C. albicans and C. glabrata and a favorable safety profile in vivo, supporting its further development as a potential broad-spectrum antifungal agent.

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.

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.

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.

Background: The newly identified small molecule ISFP10 has demonstrated the ability to inhibit fungal phosphoglucomutases (PGM), leading to decreased fungal growth and survival. Exhibiting 50 times greater selectivity for fungal PGM compared to the human homolog, ISFP10 shows promise as a broad-spectrum antifungal and a candidate for preliminary testing in rodent models of fungal infections, including Pneumocystis pneumonia (PCP). In preparation for targeted and relevant administration of the drug in fungal mouse models of infection, the current study describes the maximum-tolerated-dose (MTD) and pharmacokinetics (PK) of ISFP10 in mice. Methods: For the MTD study, 24 C57BL/6 mice were randomly distributed into 6 groups (2M/2F per group): Placebo (vehicle; 10% DMSO with 90% of 0.5% methylcellulose in 0.9% NaCl) at various doses (12.5, 25, 50, 100, 200 mg/kg), were administered twice daily by intraperitoneally (IP) for 7 consecutive days. For the PK study, 108 mice were administered either vehicle or ISFP10 in single-dose intraperitoneal (IP) injections twice daily for 7 consecutive days at concentrations of 0, 12.5, 25, 50, and 100 mg/kg. Pharmacokinetics were assessed in plasma, and epithelial lining fluid (ELF). Analysis of this test compound was performed using LC-MS/MS for PK evaluation on blood samples drawn from mice at multiple time points. Results: IP administered, ISFP10 did not produce significant changes in body weight, food consumption or adverse events in the MTD up to 100 mg/kg dosing. The plasma PK study demonstrated T _max values ranging from 2 to 8 h. Non-compartmental analysis (NCA) yielded elimination half-life values, t _1/2 , of 4.39 and 5.31 h for the 12.5 and 50 mg/kg dose groups. Concentration dependent peak-blood concentration (C _max ) at the dosing and length tested ranged from 3.450-5.140 ug/ml. Conclusions: In conclusion, ISFP10, administered intraperitoneally to mice twice daily at doses up to 100 mg/kg, exhibited no inherent safety concerns based on the analyzed parameters. Pharmacokinetic analysis revealed slow absorption and distribution kinetics, with plasma T _max values ranging from 2 to 8 hours and elimination half-lives of approximately 4–5 hours at lower doses. These data support broader in vivo testing of the inhibitor as potential novel antifungal in fungal diseases including PCP.

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 .

Background and Objective The EphA2 receptor inhibitor ALW-II-41-27 has proven to be an effective in vitro antagonist of Pneumocystis β-glucan-induced proinflammatory signaling. This suggests its potential as a candidate for initial anti-inflammatory drug testing in the rodent model of Pneumocystis pneumonia (PCP). Methods Initially, single-dose intraperitoneal (IP) injections of ALW-II-41-27 were administered at concentrations of 0, 10, 15, 20, and 30 mg/kg over a 24-h treatment period. Pharmacokinetics were assessed in plasma, bronchoalveolar lavage fluid (BALF), and epithelial lining fluid (ELF). Following these assessments, a final single mg/kg dosing was determined. Mice received daily IP injections of either vehicle or 20.0 mg/kg of ALW-II-41-27 for 10 days, with their weights recorded daily. On day 11, mice were weighed and euthanized. Lungs, liver, and kidneys were harvested for H&E staining and pathology scoring. Lung samples were further analyzed for proinflammatory cytokines using enzyme-linked immunosorbent assay (ELISA) and extracellular matrix production using quantitative PCR (qPCR). Postmortem blood collection was conducted for complete blood count (CBC) blood chemistry analysis. Lastly, ALW-II-41-27 was administered to mice prior to fungal β-glucans challenge to determine in vivo effects on lung inflammation. Results This report describes the PK assessment of ALW-II-41-27 given via IP in C57BL/6 mice. After PK data were generated, we tested ALW-II-41-27 at 20 mg/kg IP in mice and noted no significant changes in daily or final weight gain. ELISA results of proinflammatory cytokines from lung tissues showed no major differences in the respective groups. qPCR analysis of extracellular matrix transcripts were statistically similar. Examination and pathology scoring of H&E slides from lung, liver, and kidney in all groups and subsequent pathology scoring showed no significant toxicity. Blood chemistry and CBC analyses revealed no major abnormalities. Additionally, administering ALW-II-41-27 before intratracheal inoculation of fungal β-glucans, known to induce a strong proinflammatory response in the lungs, significantly reduced lung tissue IL-1β levels. Conclusions In our initial general safety and toxicology assessments, ALW-II-41-27 displayed no inherent safety concerns in the analyzed parameters. These data support broader in vivo testing of the inhibitor as a timed adjunct therapy to the deleterious proinflammatory host immune response often associated with anti-Pneumocystis therapy.