Candida albicans is the most common invasive human fungal pathogen. We show that C. albicans thioredoxin reductase, Trr1, is an attractive antifungal target: it is essential at human body temperature, and fungal and human thioredoxin reductases are structurally divergent, predicting high selectivity of fungal-targeted inhibitors. TRR1 depletion directly impairs oxidative damage repair, but also triggers cascading disruption of stress signaling and metabolic adaptation. Impaired oxidative stress endurance and consequent amphotericin hypersensitivity are anticipated effects of TRR1 depletion. We unexpectedly find it also sensitizes Candida to cell wall stress and to a first-line echinocandin antifungal agent. TRR1-depleted cells have decreased cell wall glucan content. Driven by demand for NADPH reducing equivalents, these cells increase glucose-6-phosphate flux into the pentose phosphate pathway (PPP) as evinced by sharply elevated activity of the PPP's first, rate-limiting enzyme. Since UDP-glucose-the substrate for cell wall glucan biosynthesis-is also derived from glucose-6-phosphate, we propose that metabolic pathway competition for this shared intermediate between NADPH production and cell wall glucan biosynthesis underlies the cell wall weakness of TRR1-depleted cells. Decreased activity of a key UDP-glucose biosynthetic enzyme supports this mechanism. Trr1 loss of function further drives feed-forward damage cycles: it accelerates respiration which increases reactive oxygen species, reduces gluconeogenesis which further limits glucose-6-phosphate availability, and suppresses oxidative- and cell wall stress signaling pathways. Our findings support Trr1 inhibition as a promising approach to improved treatment of C. albicans infections.

Anti-trypanosomal therapy is generally recommended for individuals under age 50 with the indeterminate form of Chagas disease to prevent disease progression. However, benznidazole and nifurtimox are associated with adverse drug reactions. We performed a retrospective review of treatment tolerability among patients with Chagas disease referred to Boston Medical Center from June 2016 to June 2024. There were 125 individuals evaluated, of whom 32 (25.6%) had contraindications to and 2 (1.6%) declined antiparasitic treatment. Ninety-one started therapy (83 with benznidazole, 8 with nifurtimox) with monitoring co-managed by infectious diseases physicians and pharmacists. Following benznidazole initiation, 70 (84.3%) had at least one adverse event, of which allergic (39/83, 47.0%), gastrointestinal (38/83, 45.8%), and neuropsychiatric (33/83, 39.8%) reactions were most common. Rash led to treatment discontinuation in 19 patients (22.9%) and met criteria for grade 3 severity in 13 (15.7%). Adjunctive therapies for rash included topical and systemic steroids and systemic antihistamines. Peripheral neuropathy led to treatment cessation for 13 patients (15.7%). Gastrointestinal adverse effects occurred in 38 patients (45.8%), were relatively mild, and managed with H2 blockers or proton pump inhibitors. Thirty (36.1%) patients were unable to complete 60 days of benznidazole, of whom 15 switched to nifurtimox. Eight patients started with nifurtimox during a benznidazole shortage. Nifurtimox was more frequently associated with gastrointestinal side effects (21/23, 91.3%) compared to benznidazole. Ultimately, 83 patients (91.2%) received at least 30 days, and 68 patients (74.7%) completed at least 60 days of benznidazole or nifurtimox. Multiple strategies were used to prevent and alleviate adverse events; multi-disciplinary team management was essential. These findings underscore the support needed for individuals with Chagas disease to tolerate and complete therapy and highlight the need for safer and more effective options to facilitate access to treatment.

The growing number of patients susceptible to invasive Candida albicans infections has intensified the need for new antifungal targets in pathways essential for fungal growth and pathogenesis. Among these pathways, phosphate homeostasis has emerged as a significant determinant of virulence, yet how phosphate availability shapes cell wall structure in response to host-derived oxidative stress remains unclear. During commensal growth, C. albicans cells typically enjoy phosphate repletion and a neutral redox environment. Transitioning to invade host tissues, they simultaneously experience phosphate deprivation and intense extrinsic oxidative stress. Here, we employ solid-state NMR to render details of cell wall remodeling in response to oxidative stress, in its dependence on phosphate. Phosphate deprived cells remodel the rigid wall core and reduce hydration and polymer mobility in the absence of oxidative stress. During hydrogen peroxide exposure, highly mobile outer polysaccharides are primary interactors. In wildtype cells, some of these polymers are recruited into the rigid core, reinforcing the wall scaffold, whereas phosphate transport mutants fail to undergo this remodeling. These findings establish phosphate acquisition as a component of oxidative defense and link nutrient sensing and -availability to the mechanical resilience of the fungal cell wall, revealing an architectural vulnerability with relevance for antifungal development.

Candida albicans is the most common invasive human fungal pathogen. Each of the currently available 3 antifungal drug classes is limited by toxicities, drug interactions or inadequate penetration of critical tissues. We discovered a connection between C. albicans TOR and thioredoxin reductase, Trr1. Fungal and human thioredoxin reductases diverge fundamentally and are essential for repair of oxidatively damaged macromolecules, so they may be promising drug targets. We found that Trr1 is required for C. albicans ' growth at human body temperatures, hyphal growth regulation and management of oxidative and cell wall stress. Its depletion sensitizes C. albicans to AmphotericinB and to an echinocandin. TRR1 -depleted cells have lower cell wall glucan content. Metabolomics experiments highlighted their perturbed pentose phosphate pathway (PPP). They have sharply elevated activity of glucose-6-phosphate dehydrogenase, the first, rate-limiting enzyme of the PPP. A key glycolytic, gluconeogenic and UDP-glucose biosynthetic enzyme each show decreased activity. UDP-glucose is the substrate of cell wall glucan-producing enzymes. We propose that Trr1-depleted cells' accelerated glucose-6-phosphate flux into the PPP, driven by demand for NADPH reducing equivalents, diminishes glucose-6-phosphate availability for UDP-glucose production and hence for cell wall construction, weakening the wall. Strikingly dysregulated signaling pathways in Trr1-depleted cells contribute to their stress hypersensitivities.

Abstract Background Chagas disease is a neglected parasitic infection that affects ∼300,000 people in the US, with a prevalence of ∼49 per 100,000 in Massachusetts, USA. Untreated Chagas disease leads to irreversible cardiac morbidity and death in 20-30% of cases, yet < 1% receive antitrypanosomal therapy in the US. We describe Chagas disease epidemiology and the care continuum of the Strong Hearts Program, an initiative centered at the East Boston Neighborhood Health Center (EBNHC) in Boston, MA.Fig. 1.Workflow for Chagas disease care at East Boston Neighborhood Health Center Clinicians receive support from Strong Hearts volunteers and/or care navigators for the confirmatory process at CDC and for ensuring completion of the specialty clinic referral process. Methods Extracted from EBNHC electronic medical records, diagnostic uptake and prevalence were analyzed, stratified by key demographic characteristics. We used chi-squared tests to compare differences in proportions across groups. We abstracted information from the medical records of Chagas patients to identify barriers in the continuum of care, addressed and documented by Strong Hearts’ patient care navigators.Fig. 2.Continuum of care for Chagas disease as supported by the Strong Hearts project.Abbreviated overview of the Chagas disease care continuum of a subset of 90 EBNHC patients diagnosed between 3/2017 - 9/2022. N and % of each step (rectangle) refer to patients having completed the previous step as denominator. Elapsed time between steps is given as median days (on arrows between steps), with IQR in parenthesis. Results Following or concomitant with 10 provider- and > 20 community information sessions in East Boston, 14,354 patients were screened at EBNHC 3/2017 - 5/2023, using a protocol approved by the EBNHC board (Fig. 1). Confirmed Chagas patients were referred to Boston Medical Center (BMC) for further evaluation and treatment if indicated. Per quarter, a median of 572 patients were screened at EBNHC (IQR: 393 – 712). The Chagas prevalence in the population was 0.7% (95% CI: 0.6% – 0.9%) with no sex difference. A significant age gradient showed the lowest prevalence in < 20 year olds (0%) and the highest in 40-49 year olds (0.8%; p=< 0.001). Of a subset of 90 EBNHC patients, 44 (49%) began and 28 (31%) completed antitrypanosomal therapy in the BMC Infectious Disease clinic (Fig. 2). Major barriers to diagnosis and treatment included complexity and delays of confirmatory testing at CDC and barriers within the medical system, the latter rectifiable if prioritized by administrators and clinicians. Outside barriers included patients’ inability to take time off from work, obtain child care or transportation. Conclusion Given the significant prevalence of Chagas disease in the US, increased patient access to diagnostics, therapy and cardiology follow-up are needed. We find that Chagas care by motivated primary care providers is feasible with appropriate support. Disclosures Daniel Bourque, MD, Kephera Diagnostics: Grant/Research Support Natasha Hochberg, MD, MPH, Novartis: Employee|Novartis: Stocks/Bonds (Public Company) Davidson Hamer, MD, CDC, Parasitic Diseases: Grant/Research Support|Kephera: Grant/Research Support

Many hospitals have stopped or are considering stopping universal admission testing for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We discuss reasons why admission testing should still be part of a layered system to prevent hospital-acquired SARS-CoV-2 infections during times of significant community transmission. These include the morbidity of SARS-CoV-2 in vulnerable patients, the predominant contribution of presymptomatic and asymptomatic people to transmission, the high rate of transmission between patients in shared rooms, and data suggesting surveillance testing is associated with fewer nosocomial infections. Preferences of diverse patient populations, particularly the hardest-hit communities, should be surveyed and used to inform prevention measures. Hospitals' ethical responsibility to protect patients from serious infections should predominate over concerns about costs, labor, and inconvenience. We call for more rigorous data on the incidence and morbidity of nosocomial SARS-CoV-2 infections and more research to help determine when to start, stop, and restart universal admission testing and other prevention measures.

Phosphorus is essential in all cells’ structural, metabolic and regulatory functions. For fungal cells that import inorganic phosphate (Pi) up a steep concentration gradient, surface Pi transporters are critical capacitators of growth. Fungi must deploy Pi transporters that enable optimal Pi uptake in pH and Pi concentration ranges prevalent in their environments. Single, triple and quadruple mutants were used to characterize the four Pi transporters we identified for the human fungal pathogen Candida albicans, which must adapt to alkaline conditions during invasion of the host bloodstream and deep organs. A high-affinity Pi transporter, Pho84, was most efficient across the widest pH range while another, Pho89, showed high-affinity characteristics only within one pH unit of neutral. Two low-affinity Pi transporters, Pho87 and Fgr2, were active only in acidic conditions. Only Pho84 among the Pi transporters was clearly required in previously identified Pi-related functions including Target of Rapamycin Complex 1 signaling, oxidative stress resistance and hyphal growth. We used in vitro evolution and whole genome sequencing as an unbiased forward genetic approach to probe adaptation to prolonged Pi scarcity of two quadruple mutant lineages lacking all 4 Pi transporters. Lineage-specific genomic changes corresponded to divergent success of the two lineages in fitness recovery during Pi limitation. Initial, large-scale genomic alterations like aneuploidies and loss of heterozygosity eventually resolved, as populations gained small-scale mutations. Severity of some phenotypes linked to Pi starvation, like cell wall stress hypersensitivity, decreased in parallel to evolving populations’ fitness recovery in Pi scarcity, while severity of others like membrane stress responses diverged from Pi scarcity fitness. Among preliminary candidate genes for contributors to fitness recovery, those with links to TORC1 were overrepresented. Since Pi homeostasis differs substantially between fungi and humans, adaptive processes to Pi deprivation may harbor small-molecule targets that impact fungal growth, stress resistance and virulence.

BACKGROUND:Invasive fungal disease (IFD) is a frequent complication in pediatric lung transplant recipients, occurring in up to 12% of patients in the first year. Risk factors for infection include impaired lung defenses and intense immunosuppressive regimens. While most IFD occurs from Aspergillus, other fungal conidia are continuously inhaled, and infections with fungi on a spectrum of human pathogenicity can occur. CASE REPORT:We report a case of a 17-year-old lung transplant recipient in whom Irpex lacteus and Rhodotorula species were identified during surveillance bronchoscopy. She was asymptomatic and deemed to be colonized by Irpex lacteus and Rhodotorula species following transplant. 2 years after transplantation, she developed a fever, respiratory symptoms, abnormal lung imaging, and histological evidence of acute and chronic bronchitis on transbronchial biopsy. After developing symptoms concerning for a pulmonary infection and graft dysfunction, she was treated for a presumed IFD. Unfortunately, further diagnostic testing could not be performed at this time given her tenuous clinical status. Despite the initiation of antifungal therapy, her graft function continued to decline resulting in a second lung transplantation. CONCLUSIONS:This case raises the concern for IFD in lung transplant recipients from Irpex species. Further investigation is needed to understand the pathogenicity of this organism, reduce the incidence and mortality of IFD in lung transplant recipients, and refine the approach to diagnosis and manage the colonization and isolation of rare, atypical fungal pathogens in immunocompromised hosts.

ABSTRACTThe fungal pathogen Candida albicans must acquire phosphate to colonize, infect, and proliferate in the human host. C. albicans has four inorganic phosphate (Pi) transporters, Pho84 being the major high-affinity transporter; its cells can also use glycerophosphocholine (GPC) as their sole phosphate source. GPC is a lipid metabolite derived from deacylation of the lipid phosphatidylcholine. GPC is found in multiple human tissues, including the renal medulla, where it acts as an osmolyte. C. albicans imports GPC into the cell via the Git3 and Git4 transporters. Internalized GPC can be hydrolyzed to release Pi. To determine if GPC import and subsequent metabolism affect phosphate homeostasis upon Pi limitation, we monitored growth and phenotypic outputs in cells provided with either Pi or GPC. In pho84∆/∆ mutant cells that exhibit phenotypes associated with Pi limitation, GPC provision rescued sensitivity to osmotic and cell wall stresses. The glycerophosphodiesterase Gde1 was required for phenotypic rescue of osmotic stress by GPC provision. GPC provision, like Pi provision, resulted in repression of the PHO regulon and activation of TORC1 signaling. Pi uptake was similar to GPC uptake when phosphate availability was low (200 µM). While available at lower concentrations than Pi in the human host, GPC is an advantageous Pi source for the fungus because it simultaneously serves as a choline source. In summary, we find GPC is capable of substituting for Pi in C. albicans by many though not all criteria and may contribute to phosphate availability for the fungus in the human host.IMPORTANCECandida albicans is the most commonly isolated species from patients suffering from invasive fungal disease. C. albicans is most commonly a commensal organism colonizing a variety of niches in the human host. The fungus must compete for resources with the host flora to acquire essential nutrients such as phosphate. Phosphate acquisition and homeostasis have been shown to play a key role in C. albicans virulence, with several genes involved in these processes being required for normal virulence and several being upregulated during infection. In addition to inorganic phosphate (Pi), C. albicans can utilize the lipid-derived metabolite glycerophosphocholine (GPC) as a phosphate source. As GPC is available within the human host, we examined the role of GPC in phosphate homeostasis in C. albicans. We find that GPC can substitute for Pi by many though not all criteria and is likely a relevant physiological phosphate source for C. albicans.

BACKGROUND:Chagas disease is a potentially life-threatening neglected disease of poverty that is endemic in continental Latin America. Caused by Trypanosoma cruzi (T. cruzi), it is one of six parasitic diseases in the United States targeted by the Centers for Disease Control as a public health problem in need of action. An estimated 300,000 people are infected with T. cruzi in the United States (US). Although its morbidity, mortality and economic burden are high, awareness of Chagas disease is lacking among many healthcare providers in the US. The purpose of this analysis is to determine if the number of diagnostic tests performed at a community health center serving an at-risk population for Chagas disease increased after information sessions. A secondary aim was to determine if there was a difference by provider type, i.e., nurse practitioner vs. physician, or by specialty in the number of patients screened.METHODOLOGY/PRINCIPAL FINDINGS:We conducted a retrospective data analysis of the number of Chagas serology tests performed at a community health center before and after information sessions for clinicians. A time series analysis was conducted focusing on the Adult and Family Medicine Departments at East Boston Neighborhood Health Center (EBNHC). Across all departments there were 1,957 T. cruzi tests performed before the sessions vs. 2,623 after the sessions. Interrupted time series analysis across departments indicated that testing volume was stable over time prior to the sessions (pre-period slope = +4.1 per month; p = 0.12), followed by an immediate shift after the session (+51.6; p = 0.03), while testing volume remained stable over time after the session (post-period slope = -6.0 per month; p = 0.11).CONCLUSION/SIGNIFICANCE:In this study, Chagas testing increased after information sessions. Clinicians who began testing their patients for Chagas disease after learning of the importance of this intervention added an extra, potentially time-consuming task to their already busy workdays without external incentives or recognition.

Whether to commit limited cellular resources toward growth and proliferation, or toward survival and stress responses, is an essential determination made by Target of Rapamycin Complex 1 (TORC1) for a eukaryotic cell in response to favorable or adverse conditions. Loss of TORC1 function is lethal. The TORC1 inhibitor rapamycin that targets the highly conserved Tor kinase domain kills fungal pathogens like Candida albicans , but is also severely toxic to human cells. The least conserved region of fungal and human Tor kinases are the N-terminal HEAT domains. We examined the role of the 8 most N-terminal HEAT repeats of C . albicans Tor1. We compared nutritional- and stress responses of cells that express a message for N-terminally truncated Tor1 from repressible tetO , with cells expressing wild type TOR1 from tetO or from the native promoter. Some but not all stress responses were significantly impaired by loss of Tor1 N-terminal HEAT repeats, including those to oxidative-, cell wall-, and heat stress; in contrast, plasma membrane stress and antifungal agents that disrupt plasma membrane function were tolerated by cells lacking this Tor1 region. Translation was inappropriately upregulated during oxidative stress in cells lacking N-terminal Tor1 HEAT repeats despite simultaneously elevated Gcn2 activity, while activation of the oxidative stress response MAP kinase Hog1 was weak. Conversely, these cells were unable to take advantage of favorable nutritional conditions by accelerating their growth. Consuming oxygen more slowly than cells containing wild type TOR1 alleles during growth in glucose, cells lacking N-terminal Tor1 HEAT repeats additionally were incapable of utilizing non-fermentable carbon sources. They were also hypersensitive to inhibitors of specific complexes within the respiratory electron transport chain, suggesting that inefficient ATP generation and a resulting dearth of nucleotide sugar building blocks for cell wall polysaccharides causes cell wall integrity defects in these mutants. Genome-wide expression analysis of cells lacking N-terminal HEAT repeats showed dysregulation of carbon metabolism, cell wall biosynthetic enzymes, translational machinery biosynthesis, oxidative stress responses, and hyphal- as well as white-opaque cell type-associated genes. Targeting fungal-specific Tor1 N-terminal HEAT repeats with small molecules might selectively abrogate fungal viability, especially when during infection multiple stresses are imposed by the host immune system.

Candida albicans (C. albicans)is a leading source of fungal infections in humans, resulting in over 250,000 deaths annually. C. albicanscolonizes multiple host niches and causes invasive disease, requiring its adaptation to wide variations in nutrient availability. Phosphate is an essential nutrient, and several genes associated with phosphate acquisition are differentially expressed during infection. In addition to inorganic phosphate (Pi), C. albicans utilizes the abundant lipid metabolite, glycerophosphocholine (GPC), as a phosphate source. GPC is transported into the cell via the Git3 and Git4 transporters. The Git transporters are required for full virulence in a mouse model of disseminated candidiasis, but the mechanism for that virulence defect is unknown. Here, we probe the importance of GPC uptake to cellular metabolism by examining two aspects of its metabolic fate: i) its catabolism to liberate Pi), and ii) its potential conversion to phosphatidylcholine (PC) via reacylation. To query its role in phosphate homeostasis, we employed a knockout strain of the high affinity phosphate transporter, Pho84. Cells lacking Pho84 display sensitivity to several host‐related stressors and decreased virulence. Our results show that provision of cells with GPC as phosphate source alleviates hypersensitivity of pho84∆/∆ mutants to osmotic, oxidative, and cell wall stress. The rate at which GPC and Pi are transported into the cell varies depending upon the total level of phosphate provided, with Pitransport exceeding GPC transport by a maximum of two‐fold at 500 µM total phosphate. The effect of exogenous GPC on induction of the PHO regulon, the cellular system of phosphate homeostasis, is complex and concentration dependent. Beyond its role in phosphate metabolism, GPC has other physiologically relevant metabolic fates, namely its reacylation to form a phosphatidylcholine (PC) molecule. In Saccharomyces cerevisiae, the first step of this novel reacylation pathway is catalyzed by the acyltransferase, Gpc1, which converts GPC to lyso‐PC. Initial in vivo labeling studies under phosphate replete conditions indicate that the C. albicans homolog of Gpc1 is a GPC acyltransferase and that the reacylation pathway is functional in C. albicans. In vitro assays in strains lacking or containing Gpc1 are underway. Taken together, our results indicate that C. albicans can utilize the abundant lipid metabolite, GPC, via at least two metabolic pathways to promote its growth and survival in the human host.

Candida bloodstream infections (CBSIs) have decreased among pediatric populations in the United States, but remain an important cause of morbidity and mortality. Species distributions and susceptibility patterns of CBSI isolates diverge widely between children and adults. The awareness of these patterns can inform clinical decision-making for empiric or pre-emptive therapy of children at risk for candidemia. CBSIs occurring from 2006–2016 among patients in a large children’s hospital were analyzed for age specific trends in incidence rate, risk factors for breakthrough-CBSI, and death, as well as underlying conditions. Candida species distributions and susceptibility patterns were evaluated in addition to the anti-fungal agent use. The overall incidence rate of CBSI among this complex patient population was 1.97/1000 patient-days. About half of CBSI episodes occurred in immunocompetent children and 14% in neonatal intensive care unit (NICU) patients. Anti-fungal resistance was minimal: 96.7% of isolates were fluconazole, 99% were micafungin, and all were amphotericin susceptible. Liposomal amphotericin was the most commonly prescribed anti-fungal agent included for NICU patients. Overall, CBSI-associated mortality was 13.7%; there were no deaths associated with CBSI among NICU patients after 2011. Pediatric CBSI characteristics differ substantially from those in adults. The improved management of underlying diseases and antimicrobial stewardship may further decrease morbidity and mortality from CBSI, while continuing to maintain low resistance rates among Candida isolates.

Candida albicans is the most common cause of fungal sepsis. Inhibition of inflammasome activity confers resistance to polymicrobial and LPS-induced sepsis; however, inflammasome signaling appears to protect against C. albicans infection, so inflammasome inhibitors are not clinically useful for candidiasis. Here we show disruption of GSDMD, a known inflammasome target and key pyroptotic cell death mediator, paradoxically alleviates candidiasis, improving outcomes and survival of Candida -infected mice. Mechanistically, C. albicans hijacked the canonical inflammasome-GSDMD axis-mediated pyroptosis to promote their escape from macrophages, deploying hyphae and candidalysin, a pore-forming toxin expressed by hyphae. GSDMD inhibition alleviated candidiasis by preventing C. albicans escape from macrophages while maintaining inflammasome-dependent but GSDMD-independent IL-1β production for anti-fungal host defenses. This study demonstrates key functions for GSDMD in Candida ’s escape from host immunity in vitro and in vivo and suggests that GSDMD may be a potential therapeutic target in C. albicans -induced sepsis.

Background Chagas disease is a vector borne infection of poverty endemic to Latin America which affects an estimated 40,000 women of child-bearing age in the United States (US). In the US Chagas disease is concentrated among individuals who have lived in endemic areas. Prenatal diagnosis and treatment are needed to prevent congenital transmission. The objective of this study was to assess perceived barriers to Chagas disease screening among prenatal care providers in Obstetrics/Gynecology and Family Medicine Departments of a tertiary care safety-net hospital caring for a significant at-risk population. Methodology/Principal findings An anonymous survey was distributed to 178 Obstetrics/Gynecology and Family Medicine practitioners. Of the 66 respondents, 39% thought Chagas screening was very important, and 48% somewhat important as a public health initiative. One third judged screening patients during clinic visits as very important. Most respondents (64%) reported being familiar with Chagas disease. However, only 32% knew how to order a test and only 22% reported knowing what to do if a test was positive. Conclusions/Significance These findings will be incorporated into measures to facilitate full implementation of Chagas screening, and can inform initiatives at other centers who wish to address this deeply neglected infection among their patient families. Greater integration of information on Chagas disease screening and treatment in medical and nursing education curricula can contribute to addressing this disease with the focus that its potentially fatal sequelae merit.

The Candida albicans high-affinity phosphate transporter Pho84 is required for normal Target of Rapamycin signaling, oxidative stress resistance and virulence of this fungal pathogen. It also contributes to C. albicans’ tolerance of two antifungal drug classes, polyenes and echinocandins. Echinocandins inhibit biosynthesis of a major cell wall component, beta-1,3-glucan. Cells lacking Pho84 were hypersensitive to other forms of cell wall stress beyond echinocandin exposure, while their cell wall integrity signaling response was weak. Metabolomics experiments showed that levels of phosphoric intermediates, including nucleotides like ATP and nucleotide sugars, were low in pho84 mutant compared to wild type cells recovering from phosphate starvation. Non-phosphoric precursors like nucleobases and nucleosides were elevated. Outer cell wall phosphomannan biosynthesis requires a nucleotide sugar,GDP-mannose. The nucleotide sugar UDP-glucose is the substrate of enzymes that synthesize two major structural cell wall polysaccharides, beta-1,3- and beta-1,6-glucan. Another nucleotide sugar, UDP-N-acetylglucosamine, is the substrate of chitin synthases which produce a stabilizing component of the intercellular septum and of lateral cell walls. Lack of Pho84 activity, and phosphate starvation, potentiated pharmacological or genetic perturbation of these enzymes. Our model is that low substrate concentrations of beta-D-glucan- and chitin synthases diminish enzymatic reaction rates and potentiate pharmacologic inhibitors to decrease the yield of their cell wall-stabilizing products. Phosphate import is not conserved between fungal and human cells, and humans do not synthesize beta-D-glucans or chitin. Hence inhibiting these processes simultaneously could yield potent antifungal effects with low toxicity to humans.

The Candida albicans high-affinity phosphate transporter Pho84 is required for normal Target of Rapamycin (TOR) signaling, oxidative stress resistance, and virulence of this fungal pathogen. It also contributes to C. albicans' tolerance of two antifungal drug classes, polyenes and echinocandins. Echinocandins inhibit biosynthesis of a major cell wall component, beta-1,3-glucan. Cells lacking Pho84 were hypersensitive to other forms of cell wall stress beyond echinocandin exposure, while their cell wall integrity signaling response was weak. Metabolomics experiments showed that levels of phosphoric intermediates, including nucleotides like ATP and nucleotide sugars, were low in pho84 mutant compared to wild-type cells recovering from phosphate starvation. Nonphosphoric precursors like nucleobases and nucleosides were elevated. Outer cell wall phosphomannan biosynthesis requires a nucleotide sugar, GDP-mannose. The nucleotide sugar UDP-glucose is the substrate of enzymes that synthesize two major structural cell wall polysaccharides, beta-1,3- and beta-1,6-glucan. Another nucleotide sugar, UDP-N-acetylglucosamine, is the substrate of chitin synthases which produce a stabilizing component of the intercellular septum and of lateral cell walls. Lack of Pho84 activity, and phosphate starvation, potentiated pharmacological or genetic perturbation of these enzymes. We posit that low substrate concentrations of beta-d-glucan- and chitin synthases, together with pharmacologic inhibition of their activity, diminish enzymatic reaction rates as well as the yield of their cell wall-stabilizing products. Phosphate import is not conserved between fungal and human cells, and humans do not synthesize beta-d-glucans or chitin. Hence, inhibiting these processes simultaneously could yield potent antifungal effects with low toxicity to humans.IMPORTANCECandida species cause hundreds of thousands of invasive infections with high mortality each year. Developing novel antifungal agents is challenging due to the many similarities between fungal and human cells. Maintaining phosphate balance is essential for all organisms but is achieved completely differently by fungi and humans. A protein that imports phosphate into fungal cells, Pho84, is not present in humans and is required for normal cell wall stress resistance and cell wall integrity signaling in C. albicans Nucleotide sugars, which are phosphate-containing building block molecules for construction of the cell wall, are diminished in cells lacking Pho84. Cell wall-constructing enzymes may be slowed by lack of these building blocks, in addition to being inhibited by drugs. Combined targeting of Pho84 and cell wall-constructing enzymes may provide a strategy for antifungal therapy by which two sequential steps of cell wall maintenance are blocked for greater potency.

Candida species are the most commonly isolated invasive human fungal pathogens. A role for phosphate acquisition in their growth, resistance against host immune cells, and tolerance of important antifungal medications is becoming apparent. Phosphorus is an essential element in vital components of the cell, including chromosomes and ribosomes. Producing the energy currency of the cell, ATP, requires abundant inorganic phosphate. A comparison of the network of regulators and effectors that controls phosphate acquisition and intracellular distribution, the PHO regulon, between the model yeast Saccharomyces cerevisiae, a plant saprobe, its evolutionarily close relative C. glabrata, and the more distantly related C. albicans, highlights the need to coordinate phosphate homeostasis with adenylate biosynthesis for ATP production. It also suggests that fungi that cope with phosphate starvation as they invade host tissues, may link phosphate acquisition to stress responses as an efficient mechanism of anticipatory regulation. Recent work indicates that connections among the PHO regulon, Target of Rapamycin Complex 1 signaling, oxidative stress management, and cell wall construction are based both in direct signaling links, and in the provision of phosphate for sufficient metabolic intermediates that are substrates in these processes. Fundamental differences in fungal and human phosphate homeostasis may offer novel drug targets.

Despite the availability of potent antifungal agents, acute disseminated candidiasis remains a life-threatening disease in immunocompromised patients. Inhibition of inflammasome activity confers resistance to polymicrobial and LPS-induced sepsis; however, inflammasome signaling appears to protect against C. albicans infection, so inflammasome inhibitors are not clinically useful for candidiasis. Here we discovered that disruption of GSDMD, a known inflammasome target and key pyroptotic cell death mediator, paradoxically alleviated candidiasis, improving outcomes and survival of Candida-infected mice. C. albicans hijacked the canonical inflammasome-GSDMD axis-mediated pyroptosis to promote their escape from macrophages, deploying hyphae and candidalysin, a pore-forming toxin expressed on hyphae. GSDMD inhibition alleviated candidiasis by preventing C. albicans escape from macrophages while maintaining inflammasome-dependent but GSDMD-independent IL-1β production for anti-fungal host defenses. This study demonstrates key functions for GSDMD in Candida’s escape from host immunity and suggests that GSDMD may be a therapeutic target in C. albicans-induced sepsis.