Opportunistic Human Fungal Pathogen Research Articles

Two Infection Assays to Study Non-Lethal Virulence Phenotypes in C. Albicans using C. Elegans.

While pathogens can be deadly to humans, many of them cause a range of infection types with non-lethal phenotypes. Candida albicans, an opportunistic fungal pathogen of humans, is the fourth most common cause of nosocomial infections which results in ~40% mortality. However, other C. albicans infections are less severe and rarely lethal and include vulvovaginal candidiasis, impacting ~75% of women, as well as oropharyngeal candidiasis, predominantly impacting infants, AIDS patients and cancer patients. While murine models are most frequently used to study C. albicans pathogenesis, these models predominantly assess host survival and are costly, time consuming, and limited in replication. Therefore, several mini-model systems, including Drosophila melanogaster, Danio rerio, Galleria mellonella, and Caenorhabditis elegans, have been developed to study C. albicans. These mini-models are well-suited for screening mutant libraries or diverse genetic backgrounds of C. albicans. Here we describe two approaches to study C. albicans infection using C. elegans. The first is a fecundity assay which measures host reproduction and monitors survival of individual hosts. The second is a lineage expansion assay which measures how C. albicans infection affects host population growth over multiple generations. Together, these assays provide a simple, cost-effective way to quickly assess C. albicans virulence.

Fingolimod Potentiates the Antifungal Activity of Amphotericin B.

Candida albicans (C. albicans) is an opportunistic human fungal pathogen that can cause severe infection in clinic. Its incidence and mortality rate has been increasing rapidly. Amphotericin B (AMB), the clinical golden standard antifungal agent, has severe side effects that limit its clinical application. Thus, lowering the concentration and increasing the efficacy of AMB in a combinatorial antifungal therapy have been pursued by both industry and academia. Here we identify that fingolimod (FTY720), an immunomodulatory drug used for oral treatment of relapsing-remitting multiple sclerosis, can potentiate the efficacy of AMB against C. albicans growth synergistically. Furthermore, we observe an antifungal efficacy of FTY720 in combination with AMB against diverse fungal pathogens. Intriguingly, cells treated with both drugs are hypersensitive to endothelial endocytosis and macrophage killing. This is later found to be due to the hyperaccumulation of reactive oxygen species and the corresponding increase in activities of superoxide dismutase and catalase in the cells that received combinatorial treatment. Therefore, the combination of AMB and FTY720 provides a promising antifungal strategy.

Intraspecies Transcriptional Profiling Reveals Key Regulators of Candida albicans Pathogenic Traits.

The human commensal and opportunistic fungal pathogen Candida albicans displays extensive genetic and phenotypic variation across clinical isolates. Here, we performed RNA sequencing on 21 well-characterized isolates to examine how genetic variation contributes to gene expression differences and to link these differences to phenotypic traits. C. albicans adapts primarily through clonal evolution, and yet hierarchical clustering of gene expression profiles in this set of isolates did not reproduce their phylogenetic relationship. Strikingly, strain-specific gene expression was prevalent in some strain backgrounds. Association of gene expression with phenotypic data by differential analysis, linear correlation, and assembly of gene networks connected both previously characterized and novel genes with 23 C. albicans traits. Construction of de novo gene modules produced a gene atlas incorporating 67% of C. albicans genes and revealed correlations between expression modules and important phenotypes such as systemic virulence. Furthermore, targeted investigation of two modules that have novel roles in growth and filamentation supported our bioinformatic predictions. Together, these studies reveal widespread transcriptional variation across C. albicans isolates and identify genetic and epigenetic links to phenotypic variation based on coexpression network analysis.IMPORTANCE Infectious fungal species are often treated uniformly despite clear evidence of genotypic and phenotypic heterogeneity being widespread across strains. Identifying the genetic basis for this phenotypic diversity is extremely challenging because of the tens or hundreds of thousands of variants that may distinguish two strains. Here, we use transcriptional profiling to determine differences in gene expression that can be linked to phenotypic variation among a set of 21 Candida albicans isolates. Analysis of this transcriptional data set uncovered clear trends in gene expression characteristics for this species and new genes and pathways that were associated with variation in pathogenic processes. Direct investigation confirmed functional predictions for a number of new regulators associated with growth and filamentation, demonstrating the utility of these approaches in linking genes to important phenotypes.

In Vitro Antifungal Drug Resistance Profiles of Clinically Relevant Members of the Mucorales (Mucoromycota) Especially with the Newer Triazoles.

Mucoromycoses (infections caused by members of the order Mucorales, phylum Mucoromycota [ex-Zygomycota]) are highly destructive, rapidly progressive infections, with dire prognoses especially when they occur in immunocompromised hosts. Current treatment guidelines recommend liposomal formulations of amphotericin B with adjunctive surgery as first line therapy, with the newer triazoles posaconazole or isavuconazole as alternative treatments, or as salvage therapy. Among the many organisms belonging to this order, a limited number of species in the genera Rhizopus, Mucor, Lichtheimia and Rhizomucor are responsible for most cases of human infection. Here, we present the minimum inhibitory concentration data (MICs) for amphotericin B, posaconazole, isavuconazole, itraconazole and voriconazole with a panel of over 300 isolates of the five most common agents of human infection (Lichtheimia corymbifera, Rhizopus arrhizus, R. microsporus, Rhizomucor pusillus and Mucor spp.) determined using the CLSI broth microdilution method. In agreement with previous studies, the most active antifungal drug for all Mucorales was amphotericin B, with MICs within the range that would predict susceptibility with Aspergillus fumigatus. Conversely, MICs for voriconazole against all species tested were high, and above the range associated with clinical efficacy with A. fumigatus. Interestingly, whilst isavuconazole and posaconazole MIC distributions indicated in vitro activity against some members of the Mucorales, activity was species-dependent for both agents. These data underscore the importance of accurate identification of the causative agents of mucoromycosis, coupled with antifungal susceptibility testing of individual isolates, in determining the optimal treatment of infections caused by these aggressive opportunistic human fungal pathogens.

Associations between Cryptococcus Genotypes, Phenotypes, and Clinical Parameters of Human Disease: A Review.

The genus Cryptococcus contains two primary species complexes that are significant opportunistic human fungal pathogens: C. neoformans and C. gattii. In humans, cryptococcosis can manifest in many ways, but most often results in either pulmonary or central nervous system disease. Patients with cryptococcosis can display a variety of symptoms on a spectrum of severity because of the interaction between yeast and host. The bulk of our knowledge regarding Cryptococcus and the mechanisms of disease stem from in vitro experiments and in vivo animal models that make a fair attempt, but do not recapitulate the conditions inside the human host. To better understand the dynamics of initiation and progression in cryptococcal disease, it is important to study the genetic and phenotypic differences in the context of human infection to identify the human and fungal risk factors that contribute to pathogenesis and poor clinical outcomes. In this review, we summarize the current understanding of the different clinical presentations and health outcomes that are associated with pathogenicity and virulence of cryptococcal strains with respect to specific genotypes and phenotypes.

Antifungal activity of Syzygium samarangense leaf extracts against Candida.

Candida species are opportunistic human fungal pathogens that cause acute and chronic infections against which only few antifungal agents are available. Here we have elucidated the antifungal effect of Syzygium samarangense leaf extracts (SSLE). Antifungal activity of SSLE was studied against Candida albicans, C. krusei, C. parapsilosis, C. glabrata, C. auris and C. tropicalis. Following experiments were performed: minimum fungicidal concentration (MFC) determination, agar well disc diffusion assays, fungal morphology analysis using scanning electron microscope (SEM), ex vivo fungal survival assays on porcine tongue and skin and in vivo fungal survival assays using Drosophila melanogaster fly model. Results demonstrated MFC of SSLE ranges between 100 and 125mgml-1 . SEM images showed cell wall degradation of C. albicans when treated with SSLE. Around 75% decrease in C. albicans viability was observed when infected porcine tongue and skin were treated using SSLE. The C. albicans infected D. melanogaster when fed with SSLE showed significant decrease (around 80%) of fungal count than the infected control. Furthermore, agar plate disc diffusion assays demonstrated that the antifungal activity of SSLE could be due to chalcone, which is one of the active constituents in SSLE. Our study demonstrated that SSLE could be used for the topical treatment of Candida infections.

Ultrasensitive SERS platform made via femtosecond laser micromachining for biomedical applications

Surface-enhanced Raman spectroscopy (SERS) is a research method in which a lack of cost-effective, versatile platforms with high enhancement factor (EF) is still a major obstacle to its widespread use. The platforms should be also easy to manufacture, stable in time (for weeks or even for months) and manufactured with a highly reproducible method.We demonstrate SERS platforms based on silicon modified on the surface by laser ablation and covered with SERS-active metal. The substrates were fabricated by a femtosecond laser, thus the method is simple, very fast and creates highly uniform SERS platforms in a large number. The platform was tested with para-mercaptobenzoic acid (p-MBA) in terms of sensitivity and reproducibility. The calculated EF was at the level of 108 and the standard deviation (SD) gives 7% for 10−6 M solution of p-MBA based on the intensity of the band at 1073 cm−1. Optimized SERS substrate also exhibits excellent stability for up to six months.We also give the proof-of-concept of using our platform and, for the first time, the SERS analysis of the most important human opportunistic fungal pathogen Candida spp. (Candida glabrata, Candida albicans SN148 and C. albicans BWP17). Finally, the chemometric analysis in the form of Principal Component Analysis (PCA) allowed to strain differentiation of Candida spp., and to distinguish the studied Candida species from Gram-positive bacterial samples with Staphylococcus aureus. Our results demonstrate that the proposed SERS platform is a perfect substrate for detection, identification and differentiation between fungal and bacterial pathogens using SERS technique.

TSC1 Suppresses Macrophage Necroptosis for the Control of Infection by Fungal Pathogen Candida albicans.

Candida albicans is the most common, opportunistic human fungal pathogen whose complex interplay with the host innate immune system remains incompletely understood. In this study, we revealed that infection macrophages with C. albicans triggers prominent cell death, which is largely attributed to the RIPK3/MLKL-mediated necroptosis. Our results further demonstrated that the TSC1-mTOR pathway plays a pivotal role in the control of macrophage necroptosis upon engaging the Dectin-1/2 and TLR-2/4 pathways through fungal components β-glucan/α-mannan or Sel1, respectively. Notably, the rapamycin-sensitive mTORC1 pathway, rather than the rapamycin-insensitive mTORC2 pathway, was responsible for elevated activation of RIPK1, RIPK3, and MLKL in TSC1-deficient macrophages. Following systemic infection with C. albicans, mice with macrophage/neutrophil-specific deletion of Tsc1 (Tsc1 M/N-/-) showed heightened fungal burden in multiple organs, such as the kidney, liver, and spleen, severe morbidity, and mortality. Notably, Tsc1 M/N-/- kidneys exhibited prominent cell death and concomitant loss of tissue-resident macrophages, which likely contributing to a dampened phagocytosis of fungal pathogens. Together, our data demonstrate a crucial role for the TSC1-mTOR pathway in the regulation of macrophage necroptosis and suggest that both Dectin- and TLRs-induced necroptosis may undermine the immune defense effector functions of these innate receptors during C. albicans infection.

The involvement of the Candida glabrata trehalase enzymes in stress resistance and gut colonization

ABSTRACT Candida glabrata is an opportunistic human fungal pathogen and is frequently present in the human microbiome. It has a high relative resistance to environmental stresses and several antifungal drugs. An important component involved in microbial stress tolerance is trehalose. In this work, we characterized the three C. glabrata trehalase enzymes Ath1, Nth1 and Nth2. Single, double and triple deletion strains were constructed and characterized both in vitro and in vivo to determine the role of these enzymes in virulence. Ath1 was found to be located in the periplasm and was essential for growth on trehalose as sole carbon source, while Nth1 on the other hand was important for oxidative stress resistance, an observation which was consistent by the lower survival rate of the NTH1 deletion strain in human macrophages. No significant phenotype was observed for Nth2. The triple deletion strain was unable to establish a stable colonization of the gastrointestinal (GI) tract in mice indicating the importance of having trehalase activity for colonization in the gut.

Novel Biological Functions of the NsdC Transcription Factor in Aspergillus fumigatus.

ABSTRACTThe fungal zinc finger transcription factor NsdC is named after, and is best known for, its essential role in sexual reproduction (never in sexual development). In previous studies with Aspergillus nidulans, it was also shown to have roles in promotion of vegetative growth and suppression of asexual conidiation. In this study, the function of the nsdC homologue in the opportunistic human pathogen A. fumigatus was investigated. NsdC was again found to be essential for sexual development, with deletion of the nsdC gene in both MAT1-1 and MAT1-2 mating partners of a cross leading to complete loss of fertility. However, a functional copy of nsdC in one mating partner was sufficient to allow sexual reproduction. Deletion of nsdC also led to decreased vegetative growth and allowed conidiation in liquid cultures, again consistent with previous findings. However, NsdC in A. fumigatus was shown to have additional biological functions including response to calcium stress, correct organization of cell wall structure, and response to the cell wall stressors. Furthermore, virulence and host immune recognition were affected. Gene expression studies involving chromatin immunoprecipitation (ChIP) of RNA polymerase II (PolII) coupled to next-generation sequencing (Seq) revealed that deletion of nsdC resulted in changes in expression of over 620 genes under basal growth conditions. This demonstrated that this transcription factor mediates the activity of a wide variety of signaling and metabolic pathways and indicates that despite the naming of the gene, the promotion of sexual reproduction is just one among multiple roles of NsdC.

Human Pathogenic Candida Species Respond Distinctively to Lactic Acid Stress

Several Candida species are opportunistic human fungal pathogens and thrive in various environmental niches in and on the human body. In this study we focus on the conditions of the vaginal tract, which is acidic, hypoxic, glucose-deprived, and contains lactic acid. We quantitatively analyze the lactic acid tolerance in glucose-rich and glucose-deprived environment of five Candida species: Candida albicans, Candida glabrata, Candida parapsilosis, Candida krusei and Candida tropicalis. To characterize the phenotypic space, we analyzed 40–100 clinical isolates of each species. Each Candida species had a very distinct response pattern to lactic acid stress and characteristic phenotypic variability. C. glabrata and C. parapsilosis were best to withstand high concentrations of lactic acid with glucose as carbon source. A glucose-deprived environment induced lactic acid stress tolerance in all species. With lactate as carbon source the growth rate of C. krusei is even higher compared to glucose, whereas the other species grow slower. C. krusei may use lactic acid as carbon source in the vaginal tract. Stress resistance variability was highest among C. parapsilosis strains. In conclusion, each Candida spp. is adapted differently to cope with lactic acid stress and resistant to physiological concentrations.

Galactomannan Produced by Aspergillus fumigatus: An Update on the Structure, Biosynthesis and Biological Functions of an Emblematic Fungal Biomarker.

The galactomannan (GM) that is produced by the human fungal pathogen Aspergillus fumigatus is an emblematic biomarker in medical mycology. The GM is composed of two monosaccharides: mannose and galactofuranose. The furanic configuration of galactose residues, absent in mammals, is responsible for the antigenicity of the GM and has favoured the development of ELISA tests to diagnose aspergillosis in immunocompromised patients. The GM that is produced by A. fumigatus is a unique fungal polysaccharide containing a tetramannoside repeat unit and having three different forms: (i) membrane bound through a glycosylphosphatidylinositol (GPI)-anchor, (ii) covalently linked to β-1,3-glucans in the cell wall, or (iii) released in the culture medium as a free polymer. Recent studies have revealed the crucial role of the GM during vegetative and polarized fungal growth. This review highlights these recent data on its biosynthetic pathway and its biological functions during the saprophytic and pathogenic life of this opportunistic human fungal pathogen.

Consensus Multilocus Sequence Typing Scheme for Pneumocystis jirovecii.

Pneumocystis jirovecii is an opportunistic human pathogenic fungus causing severe pneumonia mainly in immunocompromised hosts. Multilocus sequence typing (MLST) remains the gold standard for genotyping of this unculturable fungus. However, the lack of a consensus scheme impedes a global comparison, large scale population studies and the development of a global MLST database. To overcome this problem this study compared all genetic regions (19 loci) currently used in 31 different published Pneumocystis MLST schemes. The most diverse/commonly used eight loci, β-TUB, CYB, DHPS, ITS1, ITS1/2, mt26S and SOD, were further assess for their ability to be successfully amplified and sequenced, and for their discriminatory power. The most successful loci were tested to identify genetically related and unrelated cases. A new consensus MLST scheme consisting of four genetically independent loci: β-TUB, CYB, mt26S and SOD, is herein proposed for standardised P. jirovecii typing, successfully amplifying low and high fungal burden specimens, showing adequate discriminatory power, and correctly identifying suspected related and unrelated isolates. The new consensus MLST scheme, if accepted, will for the first time provide a powerful tool to investigate outbreak settings and undertake global epidemiological studies shedding light on the spread of this important human fungal pathogen.

Interactions of FK506 and Rapamycin With FK506 Binding Protein 12 in Opportunistic Human Fungal Pathogens.

Over the past few decades advances in modern medicine have resulted in a global increase in the prevalence of fungal infections. Particularly people undergoing organ transplants or cancer treatments with a compromised immune system are at an elevated risk for lethal fungal infections such as invasive candidiasis, aspergillosis, cryptococcosis, etc. The emergence of drug resistance in fungal pathogens poses a serious threat to mankind and it is critical to identify new targets for the development of antifungals. Calcineurin and TOR proteins are conserved across eukaryotes including pathogenic fungi. Two small molecules FK506 and rapamycin bind to FKBP12 immunophilin and the resulting complexes (FK506-FKBP12 and rapamycin-FKBP12) target calcineurin and TOR, respectively in both humans and fungi. However, due to their immunosuppressive nature these drugs in the current form cannot be used as an antifungal. To overcome this, it is important to identify key differences between human and fungal FKBP12, calcineurin, and TOR proteins which will facilitate the development of new small molecules with higher affinity toward fungal components. The current review highlights FK506/rapamycin-FKBP12 interactions with calcineurin/TOR kinase in human and fungi, and development of non-immunosuppressive analogs of FK506, rapamycin, and novel small molecules in inhibition of fungal calcineurin and TOR kinase.

Insights into the Multi-Azole Resistance Profile in Candida haemulonii Species Complex

The Candida haemulonii complex (C. duobushaemulonii, C. haemulonii, and C. haemulonii var. vulnera) is composed of emerging, opportunistic human fungal pathogens able to cause invasive infections with high rates of clinical treatment failure. This fungal complex typically demonstrates resistance to first-line antifungals, including fluconazole. In the present work, we have investigated the azole resistance mechanisms expressed in Brazilian clinical isolates forming the C. haemulonii complex. Initially, 12 isolates were subjected to an antifungal susceptibility test, and azole cross-resistance was detected in almost all isolates (91.7%). In order to understand the azole resistance mechanistic basis, the efflux pump activity was assessed by rhodamine-6G. The C. haemulonii complex exhibited a significantly higher rhodamine-6G efflux than the other non-albicans Candida species tested (C. tropicalis, C. krusei, and C. lusitaneae). Notably, the efflux pump inhibitors (Phe-Arg and FK506) reversed the fluconazole and voricolazole resistance phenotypes in the C. haemulonii species complex. Expression analysis indicated that the efflux pump (ChCDR1, ChCDR2, and ChMDR1) and ERG11 genes were not modulated by either fluconazole or voriconazole treatments. Further, ERG11 gene sequencing revealed several mutations, some of which culminated in amino acid polymorphisms, as previously reported in azole-resistant Candida spp. Collectively, these data point out the relevance of drug efflux pumps in mediating azole resistance in the C. haemulonii complex, and mutations in ERG11p may contribute to this resistance profile.

Multidrug resistant Candida glabrata in an oral lichen planus patient

Oral lichen planus (OLP) is a mucocutaneous disease characterized by inflammation leading to severe damage to the epithelial basal layer. This report describes a case of a 65-year-old Iranian female with a complaint of multifocal white lesions on the buccal and tongue mucosa with a reticular pattern and a focal pseudomembranous appearance (similar to thrush), together with burning symptoms for fourteen months. The patient was using topical fluocinolone acetonide 0.1%, two to three times a day at least for six months to control the ulcers and erythema. Molecular analysis of the thrush sample revealed Candida glabrata as the causative agent. Histopathologic examination confirmed the diagnosis of OLP. The minimum inhibitory concentration analysis was performed according to CLSI-M60 and revealed that this C. glabrata isolate was multidrug-resistant (resistant to nystatin, fluconazole, micafungin, and caspofungin). Finally, this patient was treated with amphotericin B oral suspension (0.5 g/5 ml) thrice daily for 14 days. The thrush disappeared completely and the patient no longer had any pain or burning sensations. Candida glabrata, the second most common cause of candidemia, is a major opportunistic fungal pathogen of humans that causes systemic as well as, mucosal and superficial infections. This case is the first case of OLP accompanied by multidrug-resistant C. glabrata.

An intergenic “safe haven” region in Cryptococcus neoformans serotype D genomes

Cryptococcus neoformans is an opportunistic human fungal pathogen and serves as a model organism for studies of eukaryotic microbiology and microbial pathogenesis. C. neoformans species complex is classified into serotype A, serotype D, and AD hybrids, which are currently considered different subspecies. Different serotype strains display varied phenotypes, virulence, and gene regulation. Genetic investigation of important pathways is often performed in both serotype A and D reference strains in order to identify diversification or conservation of the interrogated signaling network. Many genetic tools have been developed for C. neoformans serotype A reference strain H99, including the gene free “safe haven” (SH) regions for DNA integration identified based on genomic features. However, no such a genomic safe haven region has been identified in serotype D strains. Here, capitalizing on the available genomic, transcriptomic, and chromatin data, we identified an intergenic region named as SH3 for the serotype D reference strains JEC21 and XL280. We also designed a sgRNA and a vector facilitating any alien gene integration into SH3 through a CRISPR-Cas9 system. We found that gene inserted in this region complemented the corresponding gene deletion mutant. Fluorescent reporter gene inserted in SH3 can also be expressed efficiently. Insertion in SH3 itself did not alter the expression of adjacent genes and did not affect the growth or mating of C. neoformans. Thus, SH3 provides a resource for genetic manipulations in serotype D strains and will facilitate comparative analyses of gene functions in this species complex. In addition, the incorporation of the multi-omic data in our selection of the safe haven region could help similar studies in other organisms.

Interactions of microorganisms with host mucins: a focus on Candida albicans.

Mucus is an important host innate defense factor that lines most epithelial cell layers of the body and provides crucial physical and biological protection against pathogenic microorganisms. Mucins are the main glycoproteins of mucus that are responsible for interacting with microorganisms and are critical for the antimicrobial properties of mucus. The mechanisms by which microorganisms interact with mucins are poorly understood, especially in terms of fungi, and these interactions are continually evolving. Work in bacterial pathogens has shown that mucins inhibit bacterial virulence traits, including quorum sensing, toxin secretion and biofilm formation. Among the fungal clade, the common opportunistic human fungal pathogen and commensal Candida albicans engages in constant battle with the host innate immune system. This battle creates strong selective pressures for C. albicans to evolve in response to the host. Recent work in C. albicans found that mucins inhibit specific virulence traits, such as surface adherence, filamentation, biofilm formation and the production of secreted proteases. Here we review the current knowledge of microbial interactions with mucins, with a special emphasis on the interactions between C. albicans and mucins.

Sugar Phosphorylation Controls Carbon Source Utilization and Virulence of Candida albicans.

Candida albicans is an opportunistic human fungal pathogen that relies upon different virulence traits, including morphogenesis, invasion, biofilm formation, and nutrient acquisition from host sources as well as metabolic adaptations during host invasion. In this study, we show how sugar kinases at the start of glycolysis modulate virulence of C. albicans. Sequence comparison with Saccharomyces cerevisiae identified four enzymes (Hxk1, Hxk2, Glk1, and Glk4) in C. albicans with putative roles in sugar phosphorylation. Hxk2, Glk1, and Glk4 demonstrate a critical role in glucose metabolism, while Hxk2 is the only kinase important for fructose metabolism. Additionally, we show that Hxk1 controls HXK2, GLK1, and GLK4 expression in the presence of fermentable as well as non-fermentable carbon sources, thereby indirectly controlling glycolysis. Moreover, these sugar kinases are important during virulence. Disabling the glycolytic pathway reduces adhesion capacity, while deletion of HXK1 decreases biofilm formation. Finally, we demonstrate that hxk2Δ/Δ glk1Δ/Δ glk4Δ/Δ and hxk1Δ/Δ hxk2Δ/Δ glk1Δ/Δ glk4Δ/Δ have attenuated virulence upon systemic infections in mice. These results indicate a regulatory role for Hxk1 during sugar phosphorylation. Furthermore, these kinases are essential during growth on glucose or fructose, and C. albicans relies on a functional glycolytic pathway for maximal virulence.

Sterol-Response Pathways Mediate Alkaline Survival in Diverse Fungi.

The ability for cells to maintain homeostasis in the presence of extracellular stress is essential for their survival. Stress adaptations are especially important for microbial pathogens to respond to rapidly changing conditions, such as those encountered during the transition from the environment to the infected host. Many fungal pathogens have acquired the ability to quickly adapt to changes in extracellular pH to promote their survival in the various microenvironments encountered during a host infection. For example, the fungus-specific Rim/Pal alkaline response pathway has been well characterized in many fungal pathogens, including Cryptococcus neoformans However, alternative mechanisms for sensing and responding to host pH have yet to be extensively studied. Recent observations from a genetic screen suggest that the C. neoformans sterol homeostasis pathway is required for growth at elevated pH. This work explores interactions among mechanisms of membrane homeostasis, alkaline pH tolerance, and Rim pathway activation. We find that the sterol homeostasis pathway is necessary for growth in an alkaline environment and that an elevated pH is sufficient to induce Sre1 activation. This pH-mediated activation of the Sre1 transcription factor is linked to the biosynthesis of ergosterol but is not dependent on Rim pathway signaling, suggesting that these two pathways are responding to alkaline pH independently. Furthermore, we discover that C. neoformans is more susceptible to membrane-targeting antifungals under alkaline conditions, highlighting the impact of microenvironmental pH on the treatment of invasive fungal infections. Together, these findings further connect membrane integrity and composition with the fungal pH response and pathogenesis.IMPORTANCE The work described here further elucidates how microorganisms sense and adapt to changes in their environment to establish infections in the human host. Specifically, we uncover a novel mechanism by which an opportunistic human fungal pathogen, Cryptococcus neoformans, responds to increases in extracellular pH in order to survive and thrive within the relatively alkaline environment of the human lung. This mechanism, which is intimately linked with fungal membrane sterol homeostasis, is independent of the previously well-studied alkaline response Rim pathway. Furthermore, this ergosterol-dependent alkaline pH response is present in Candida albicans, indicating that this mechanism spans diverse fungal species. These results are also relevant for novel antimicrobial drug development as we show that currently used ergosterol-targeting antifungals are more active in alkaline environments.