Biology subjects

Krysan, D. J.

Publications and source records attributed to Krysan, D. J..

At least 19 recordsLinked to original sources

bioRxiv2025

Cryptococcus neoformans adapts to host CO2 concentrations through the coordinated remodeling of central carbon metabolism, oxidative stress resistance, and membrane homeostasis.

Cryptococcus neoformans is an environmental pathogen that remodels its cellular physiology to survive within mammals and, in susceptible hosts, cause life-threatening meningoencephalitis. Of the many distinctions between the external environment and mammalian tissues, CO2 concentration in the host is 2 orders of magnitude higher than in the environment and represent a critical stress for C. neoformans. C. neoformans strains that do not replicate at host CO2 concentrations are less virulent in mouse models of infection, further supporting CO2 tolerance as a virulence trait. To further understand the genetic determinants of C. neoformans CO2 tolerance, we performed a near genome-wide screen for deletion mutants with altered CO2 fitness using a competitive growth assay. A total of 301 of 4698 deletion mutants showed altered CO2 tolerance (245 reduced fitness; 51 increased fitness) demonstrating the global effect of host CO2 on C. neoformans physiology. Based on this data set as well as a metabolomic analysis of C. neoformans adaptation to host CO2, we show that remodeling of central carbon metabolism, oxidative stress buffering and membrane homeostasis represent an integrated response to CO2 stress that is mediated in part by the TOR-Ypk1 signaling axis. We propose that CO2-induced capsule formation leads to reduced cellular glucose which, in turn, triggers remodeling of central carbon metabolism toward utilization of alternative carbon sources and increased mitochondrial respiration/reactive oxygen generation. Thus, these data provide a near genome-wide profile of the genetic determinants of C. neoformans CO2 tolerance as well as a model for how this important environmental human fungal pathogen alters its physiology to proliferate in the host.

microbiology↗

bioRxiv2024

Similarities and distinctions in the activation of the Candida glabrata Pdr1 regulatory pathway by azole and non-azole drugs

Incidences of fluconazole (FLC) resistance among Candida glabrata clinical isolates is a growing issue in clinics. The pleiotropic drug response (PDR) network in C. glabrata confers azole resistance and is defined primarily by the Zn2Cys6 zinc cluster-containing transcription factor Pdr1 and target genes such as CDR1, that encodes an ATP-binding cassette transporter protein thought to act as a FLC efflux pump. Mutations in the PDR1 gene that render the transcription factor hyperactive are the most common cause of fluconazole resistance among clinical isolates. The phenothiazine class drug fluphenazine and a molecular derivative, CWHM-974, which both exhibit antifungal properties, have been shown to induce the expression of Cdr1 in Candida spp. We have used a firefly luciferase reporter gene driven by the CDR1 promoter to demonstrate two distinct patterns of CDR1 promoter activation kinetics: gradual promoter activation kinetics that occur in response to ergosterol limitations imposed by exposure to azole and polyene class antifungals and a robust and rapid CDR1 induction occurring in response to the stress imposed by fluphenazines. We can attribute these different patterns of CDR1 induction as proceeding through the promoter region of this gene since this is the only segment of the gene included in the luciferase reporter construct. Genetic analysis indicates that the signaling pathways responsible for phenothiazine and azole induction of CDR1 overlap but are not identical. The short time course of phenothiazine induction suggests that these compounds may act more directly on the Pdr1 protein to stimulate its activity. ImportanceCandida glabrata has emerged as the second-leading cause of candidiasis due in part to its ability to acquire high level resistance to azole drugs, a major class of antifungal, that acts to block the biosynthesis of the fungal sterol ergosterol. The presence of azole drugs causes the induction of a variety of genes involved in controlling susceptibility to this drug class including drug transporters and ergosterol biosynthetic genes such as ERG11. We found that the presence of azole drugs leads to an induction of genes encoding drug transporters and ERG11, while exposure of C. glabrata cells to antifungals of the phenothiazine class of drugs caused a much faster and larger induction of drug transporters but not ERG11. Coupled with further genetic analyses of the effects of azole and phenothiazine drugs, our data indicate that these compounds are sensed and responded to differentially in the yeast cell.

molecular biology↗

bioRxiv2024

Analogues of the anti-malaria drug mefloquine have broad spectrum antifungal activity and are efficacious in a model of disseminated Candida auris infection.

Only three classes of antifungal drugs are currently in clinical use. Here, we report that derivatives of the malarial drug mefloquine have broad spectrum antifungal activity including difficult to treat molds and endemic fungi. Pharmacokinetic and efficacy studies of NSC-4377 indicate it penetrates the central nervous system and is active against Candida auris in vivo. These data strongly support the further development of mefloquine analogs as a potentially new class of antifungal molecules.

microbiology↗

bioRxiv2024

Temporal dynamics of Candida albicans morphogenesis and gene expression reveals distinctions between in vitro and in vivo filamentation.

Candida albicans is a common human fungal pathogen that is also a commensal of the oral cavity and gastrointestinal tract. C. albicans pathogenesis is linked to its transition from budding yeast to filamentous morphologies including hyphae and pseudohyphae. The centrality of this virulence trait to C. albicans pathobiology has resulted in extensive characterization a wide range factors associated with filamentation with a strong focus on transcriptional regulation. The vast majority of these experiments have used in vitro conditions to induce the yeast-to-filament transition. Taking advantage of in vivo approaches to quantitatively characterize both morphology and gene expression during filamentation during mammalian infection, we have investigated the dynamics of these two aspects of filamentation in vivo and compared them to in vitro filament induction with "host-like" tissue culture media supplemented with serum at mammalian body temperature. Although filamentation shares many common features in the two conditions, we have found two significant differences. First, alternative carbon metabolism genes are expressed early during in vitro filamentation and late in vivo, suggesting significant differences in glucose availability. Second, C. albicans begins a hyphae-to-yeast transition after 4hr incubation while we find little evidence of hyphae-to-yeast transition in vivo up to 24hr post infection. We show that the low rate of in vivo hyphae-to-yeast transition is likely due to very low expression of PES1, a key driver of lateral yeast in vitro, and that heterologous expression of PES1 is sufficient to trigger lateral yeast formation in vivo. ImportanceCandida albicans filamentation is correlated with virulence and is an intensively studied aspect of C. albicans biology. The vast majority of studies on C. albicans filamentation are based on in vitro induction of hyphae and pseudohyphae. Here we used an in vivo filamentation assay and in vivo expression profiling to compare the tempo of morphogenesis and gene expression between in vitro and in vivo filamentation. Although the hyphal gene expression profile is induced rapidly in both conditions, it remains stably expressed over a 12hr time course in vivo while it peaks after 4hr in vitro and is reduced. This reduced hyphal gene expression in vitro correlates with reduced hyphae and increased hyphae-to-yeast transition. In contrast, there is little evidence of evidence of hyphae-to-yeast transition in vivo.

microbiology↗

bioRxiv2023

The role of the C. albicans transcriptional repressor NRG1 during filamentation and disseminated candidiasis is strain-dependent

Candida albicans is one of the most common causes of superficial and invasive fungal disease in humans. Its ability to cause disease has been closely linked to its ability to undergo a morphological transition from budding yeast to filamentous forms (hyphae and pseudohyphae). The ability of C. albicans strains isolated from patients to undergo filamentation varies significantly. In addition, the filamentation phenotypes of mutants involving transcription factors that positively regulate hyphal morphogenesis can also vary from strain to strain. Here, we characterized the virulence, in vitro and in vivo filamentation, and in vitro and in vivo hypha-associated gene expression profiles of four poorly filamenting C. albicans isolates and their corresponding deletion mutants of the repressor of filamentation NRG1. The two most virulent strains, 57055 and 78048, show robust in vivo filamentation while remaining predominately yeast phase exposed to RPMI+10% bovine calf serum at 37{degrees}C; the two low virulence strains (94015 and 78042) do not filament well under either condition. Deletion of NRG1 increases hyphae formation in the SC5314 derivative SN250 but only pseudohyphae are formed in the clinical isolates in vivo. Deletion of NRG1 modestly increased the virulence of 78042 which was accompanied by increased expression of hyphae-associated genes without an increase in filamentation. Strikingly, deletion of NRG1 in 78048 reduced filamentation, expression of candidalysin (ECE1) and virulence in vivo without dramatically altering establishment of infection. Thus, the function of NRG1 varies significantly within this set of C. albicans isolates and can actually suppress filamentation in vivo. ImportanceClinical isolates of the human fungal pathogen Candida albicans show significant variation in their ability to undergo in vitro filamentation and in the function of well-characterized transcriptional regulators of filamentation. Here, we show that Nrg1, a key repressor of filamentation and filament specific gene expression in standard reference strains, has strain dependent functions, particularly during infection. Most strikingly, loss of NRG1 function can reduce filamentation, hypha-specific gene expression such as the toxin candidalysin, and virulence in some strains. Our data emphasize that the functions of seemingly fundamental and well-conserved transcriptional regulators such as Nrg1 are contextual with respect to both environment and genetic background.

microbiology↗

bioRxiv2023

Comparative dynamics of gene expression during in vitro and in vivo Candida albicans filamentation.

Candida albicans is one of them most common causes of fungal disease in humans and is a commensal member of the human microbiome. The ability of C. albicans to cause disease is tightly correlated with its ability to undergo a morphological transition from budding yeast to a filamentous form (hyphae and pseudohyphae). This morphological transition is accompanied by the induction of a set of well characterized hyphae-associated genes and transcriptional regulators. To date, the vast majority of data regarding this process has been based on in vitro studies of filamentation using a range of inducing conditions. Recently, we developed an in vivo imaging approach that allows the direct characterization of morphological transition during mammalian infection. Here, we couple this imaging assay with in vivo expression profiling to characterize the time course of in vivo filamentation and the accompanying changes in gene expression. We also compare in vivo observations to in vitro filamentation using a medium (RPMI 1640 tissue culture medium with 10% bovine calf serum) widely used to mimic host conditions. From these data, we make the following conclusions regarding in vivo and in vitro filamentation. First, the transcriptional programs regulating filamentation are rapidly induced in vitro and in vivo. Second, the tempo of filamentation in vivo is prolonged relative to in vitro filamentation and the period of high expression of genes associated with that process is also prolonged. Third, hyphae are adapting to changing infection environments after filamentation has reached steady-state. ImportanceCandida albicans filamentation is correlated with virulence and is an intensively studied aspect of C. albicans biology. The vast majority of studies on C. albicans filamentation are based on in vitro induction of hyphae and pseudohyphae. Here we used an in vivo filamentation assay and in vivo expression profiling to compare the tempo of morphogenesis and gene expression between in vitro and in vivo filamentation. Although the hyphal gene expression profile is induced rapidly in both conditions, it remains stably expressed over the 24hr time course in vivo while the expression of other environmentally responsive genes is dynamic. As such, it is important to regard the filamentation process as a separate growth phase of C. albicans that is as adaptable to changing growth conditions as the more familiar yeast phase.

microbiology↗

bioRxiv2023

The Candida albicans reference strain SC5314 contains a rare, dominant allele of the transcription factor Rob1 that modulates biofilm formation and oral commensalism

Candida albicans is a diploid human fungal pathogen that displays significant genomic and phenotypic heterogeneity over a range of virulence traits and in the context of a variety of environmental niches. Here, we show that the effects of Rob1 on biofilm and filamentation virulence traits is dependent on both the specific environmental condition and the clinical strain of C. albicans. The C. albicans reference strain SC5314 is a ROB1 heterozygote with two alleles that differ by a single nucleotide polymorphism at position 946 resulting in a serine or proline containing isoform. An analysis of 224 sequenced C. albicans genomes indicates that SC5314 is the only ROB1 heterozygote documented to date and that the dominant allele contains a proline at position 946. Remarkably, the ROB1 alleles are functionally distinct and the rare ROB1946Sallele supports increased filamentation in vitro and increased biofilm formation in vitro and in vivo, suggesting it is a phenotypic gain-of-function allele. SC5314 is amongst the most highly filamentous and invasive strains characterized to date. Introduction of the ROB1946Sallele into a poorly filamenting clinical isolate increases filamentation and conversion of an SC5314 laboratory strain to a ROB1946S homozygote increases in vitro filamentation and biofilm formation. In a mouse model of oropharyngeal infection, the predominant ROB1946P allele establishes a commensal state while the ROB1946S phenocopies the parent strain and invades into the mucosae. These observations provide an explanation for the distinct phenotypes of SC5314 and highlight the role of heterozygosity as a driver of C. albicans phenotypic heterogeneity. ImportanceCandida albicans is a commensal fungus that colonizes human oral cavity and gastrointestinal tracts but also causes mucosal as well as invasive disease. The expression of virulence traits in C. albicans clinical isolates is heterogenous and the genetic basis of this heterogeneity is of high interest. The C. albicans reference strain SC5314 is highly invasive and expresses robust filamentation and biofilm formation relative to many other clinical isolates. Here, we show that SC5314 derivatives are heterozygous for the transcription factor Rob1 and contain an allele with a rare gain-of-function SNP that drives filamentation, biofilm formation, and virulence in a model of oropharyngeal candidiasis. These finding explain, in part, the outlier phenotype of the reference strain and highlight the role of heterozygosity plays in the strain-to-strain variation of diploid fungal pathogens.

microbiology↗

bioRxiv2023

CWHM-974 is a fluphenazine derivative with improved antifungal activity against Candida albicans due to reduced susceptibility to multidrug transporter-mediated resistance mechanisms

Multidrug resistance (MDR) transporters such as ATP Binding Cassette (ABC) and Major Facilitator Superfamily (MFS) proteins are important mediators of antifungal drug resistance, particularly with respect to azole class drugs. Consequently, identifying molecules that are not susceptible to this mechanism of resistance is an important goal for new antifungal drug discovery. As part of a project to optimize the antifungal activity of clinically used phenothiazines, we synthesized a fluphenazine derivative (CWHM-974) with 8-fold higher activity against Candida spp. compared to the fluphenazine and with activity against Candida spp. with reduced fluconazole susceptibility due to increased multidrug resistance transporters. Here, we show that the improved C. albicans activity is because fluphenazine induces its own resistance by triggering expression of CDR transporters while CWHM-974 induces expression but does not appear to be a substrate for the transporters or is insensitive to their effects through other mechanisms. We also found that fluphenazine and CWHM-974 are antagonistic with fluconazole in C. albicans but not in C. glabrata, despite inducing CDR1 expression to high levels. Overall, CWHM-974 represents a unique example of a medicinal chemistry-based conversion of chemical scaffold from MDR-sensitive to MDR-resistant and, hence, active against fungi that have developed resistance to clinically used antifungals such as the azoles.

microbiology↗

bioRxiv2023

Novel keto-alkyl-pyridinium antifungal molecules active in models of in vivo Candida albicans vascular catheter infection and ex vivo Candida auris skin colonization

New antifungal therapies are needed for both systemic, invasive infections as well as superficial infections of mucosal and skin surfaces as well as biofilms associated with medical devices. The resistance of biofilm and biofilm-like growth phases of fungi contributes to the poor efficacy of systemic therapies to non-systemic infections. Here, we describe the identification and characterization of a novel keto-alkyl-pyridinium scaffold with broad spectrum activity (2-16 {micro}g/mL) against medically important yeasts and moulds, including clinical isolates resistant to azoles and/or echinocandins. Furthermore, these keto-alkyl-pyridinium agents retain substantial activity against biofilm phase yeast and have direct activity against hyphal A. fumigatus. Although their toxicity precludes use in systemic infections, we found that the keto-alkyl-pyridinium molecules reduce C. albicans fungal burden in a rat model of vascular catheter infection and reduce Candida auris colonization in a porcine ex vivo model. These initial pre-clinical data suggest that molecules of this class may warrant further study and development.

microbiology↗

bioRxiv2023

Candida albicans oropharyngeal infection is an exception to iron-based nutritional immunity

Candida albicans is a commensal of the human gastrointestinal tract and one of the most causes of human fungal disease, including mucosal infections such as oropharyngeal candidiasis and disseminated infections of the bloodstream and deep organs. We directly compared the in vivo transcriptional profile of C. albicans during oral infection and disseminated infection of the kidney to identify niche specific features. Although the expression of a set of environmentally responsive genes were correlated in the two infection sites (Pearson R2, 0.6), XXX genes were differentially expressed. Virulence associated genes such as hyphae-specific transcripts were expressed similarly in the two sites. Genes expressed during growth in a poor carbon source (ACS1 and PCK1) were upregulated in oral tissue relative to kidney. Most strikingly, C. albicans in oral tissue shows the transcriptional hallmarks of an iron-replete state while in the kidney it is in the expected iron starved state. Interestingly, C. albicans expresses genes associated with a low zinc environment in both niches. Consistent with these expression data, deletion of two transcription factors that activate iron uptake genes (SEF1, HAP5) have no effect on virulence in a mouse model of oral candidiasis. During microbial infection, the host sequesters iron and other metal nutrients to suppress growth of the pathogen in a process called nutritional immunity. Our results indicate that C. albicans is subject to iron and zinc nutritional immunity during disseminated infection but is exempted from iron nutritional immunity during oral infection.

microbiology↗

bioRxiv2022

Intravital imaging-based genetic screen reveals the transcriptional network governing Candida albicans filamentation during mammalian infection

Candida albicans is one of the most common human fungal pathogens. C. albicans pathogenesis is tightly linked to its ability to under a morphogenetic transition from typically budding yeast to filamentous forms of hyphae and pseudohyphae. Filamentous morphogenesis is the most intensively studied C. albicans virulence traits; however, nearly all of these studies have been based on in vitro induction of filamentation. Using an intravital imaging assay of filamentation during mammalian infection, we have screened a library of transcription factor mutants to identify those that modulate both the initiation and maintenance of filamentation in vivo. We coupled this initial screen with genetic interaction analysis and in vivo transcription profiling to characterize the transcription factor network governing filamentation in infected mammalian tissue. Three core positive (Efg1, Brg1, and Rob1) and two core negative regulators (Nrg1 and Tup1) of filament initiation were identified. No previous systematic analysis of genes affecting the elongation step has been reported and we found that large set of transcription factors affect filament elongation in vivo including four (Hms1, Lys14, War1, Dal81) with no effect on in vitro elongation. We also show that the gene targets of initiation and elongation regulators are distinct. Genetic interaction analysis of the core positive and negative regulators revealed that the master regulator Efg1 primarily functions to mediate relief of Nrg1 repression and is dispensable for expression of hypha-associated genes in vitro and in vivo. Thus, our analysis not only provide the first characterization of the transcriptional network governing C. albicans filamentation in vivo but also revealed a fundamentally new mode of function for Efg1, one of the most widely studied C. albicans transcription factors.

microbiology↗

bioRxiv2022

Candida albicans filamentation does not require the cAMP-PKA pathway in vivo

Candida albicans is one of the most prevalent human fungal pathogens. Its ability to transition between budding yeast and filamentous morphological forms (pseudohyphae and hyphae) is tightly associated with its pathogenesis. Based on in vitro studies, the cAMP-Protein Kinase A (PKA) pathway is a key regulator of C. albicans morphogenesis. Using an intravital imaging approach, we investigated the role of the cAMP-PKA pathway during infection. Consistent with their roles in vitro, the downstream effectors of the cAMP-PKA pathway Efg1 and Nrg1 function, respectively, as an activator and a repressor of in vivo filamentation. Surprisingly, strains lacking the adenylyl cyclase, CYR1, showed only slightly reduced filamentation in vivo despite being completely unable to filament in RPMI+10% serum at 37{degrees}C. Consistent with these findings, deletion of the catalytic subunits of PKA (Tpk1 and Tpk2), either singly or in combination, generated strains that also filamented in vivo but not in vitro. In vivo transcription profiling of C. albicans isolated from both ear and kidney tissue showed that the expression of a set of 184 environmentally responsive correlated well with in vitro filamentation (R2 0.62-0.68) genes. This concordance suggests that the in vivo and in vitro transcriptional responses are similar but that the upstream regulatory mechanisms are distinct. As such, these data emphatically emphasize that C. albicans filamentation is a complex phenotype that occurs in different environments through an intricate network of distinct regulatory mechanisms. ImportanceThe fungus Candida albicans causes a wide range of disease in humans from common diaper rash to life-threatening infections in patients with compromised immune systems. As such, the mechanisms for its ability to cause disease are of wide interest. An intensely studied virulence property of C. albicans is its ability to switch from a round yeast form to filament-like forms (hyphae and pseudohyphae). Surprisingly, we have found that a key signaling pathway that regulates this transition in vitro, the protein kinase A pathway, is not required for filamentation during infection of the host. Our work not only demonstrates that the regulation of filamentation depends upon the specific environment C. albicans inhabits but also underscores the importance of studying these mechanisms during infection.

microbiology↗

bioRxiv2022

FKS1 is required for Cryptococcus neoformans fitness in vivo: application of copper-regulated gene expression to mouse models of cryptococcosis

There is an urgent need for new antifungals to treat cryptococcal meningoencephalitis, a leading cause of mortality in people living with HIV/AIDS. An important aspect of antifungal drug development is the validation of targets to determine whether they are required for the survival of the organism in animal models of disease. In Cryptococcus neoformans, a copper-regulated promoter (pCTR4-2) has been used to modulate gene expression in vivo previously. The premise for these experiments is that copper concentrations vary depending on the host niche. Here, we directly test this premise and confirm that the expression of CTR4, the promoter used to regulate gene expression, is much lower in the mouse lung compared to the brain. To further explore this approach, we applied to the gene encoding 1,3-{beta}-glucan synthase, FKS1. In vitro, reduced expression of FKS1 has little effect on growth but does activate the cell wall integrity stress response and increase susceptibility to caspofungin, a direct inhibitor of Fks1. These data suggest that compensatory pathways that reduce C. neoformans resistance do so through post-transcriptional effects. In vivo, however, a less pronounced reduction in FKS1 expression leads to a much more significant reduction in lung fungal burden (~1 log10 CFU), indicating that the compensatory responses to a reduction in FKS1 expression are not as effective in vivo as they are in vitro. In summary, use of copper-regulated expression of putative drug targets in vitro and in vivo can provide insights into the biological consequences of reduced activity of the target during infection. ImportanceConditional expression systems are widely used to genetically validate antifungal drug targets in mouse models of infection. Copper-regulated expression using the promoter of the CTR4 gene has been sporadically used for this purpose in C. neoformans. Here, we show that CTR4 expression is low in the lung and high in the brain, establishing the basic premise behind this approach. We applied it to the study of FKS1, the gene encoding for the target of the echinocandin class of 1,3-{beta}-glucan synthase inhibitors. Our in vitro and in vivo studies indicate that C. neoformans tolerates extremely low levels of FKS1 expression. This observation provides a potential explanation for the poor activity of 1,3-{beta}-glucan synthase inhibitors toward C. neoformans.

microbiology↗

bioRxiv2022

Genetic interaction analysis reveals that Cryptococcus neoformans utilizes multiple acetyl-CoA-generating pathways during infection

Cryptococcus neoformans is an important human fungal pathogen for which the external environment is its primary niche. To cause infection and disease, C. neoformans must adapt to a plethora of conditions and stresses inherent to the host environment. Of these stresses, the role of central carbon metabolism has been of interest not only at the level of fundamental pathobiology but also as a potential target for new antifungal drug therapy. Previous work has shown that two non-essential acetyl-CoA metabolism enzymes, ATP-citrate lyase (ACL1) and acetyl-CoA synthetase (ACS1), play important roles in C. neoformans infection. Here, we took a genetic interaction approach to studying the interplay between these two enzymes along with an enzyme initially called ACS2 but which we have found is an acetoacetyl-CoA synthetase; we have renamed the gene 2-ketobutyryl CoA synthetase 1 (KBC1) based on its biochemical activity and the systematic name of its substrate. ACL1 and ACS1 represent a synthetic lethal pair of genes based on our genetic interaction studies. Double mutants of KBC1 with either ACS1 or ACL1 do not have significant synthetic phenotypes in vitro, although we find that deletion of any one of these enzymes reduces fitness within macrophages. Importantly, the acs1{Delta} kbc1{Delta} double mutant has significantly reduced fitness in the CNS relative to either single mutant as well as WT (~2 log10 CFU reduction in fungal burden), indicating the important role these enzymes play during infection. The expression of both ACS1 and KBC1 is increased in vivo relative to in vitro conditions. The acs1{Delta} mutant is hypersusceptible to fluconazole in vivo despite its minimal in vitro phenotypes. These data not only provide insights into the in vivo mechanism of action for a new class of antifungal Acs inhibitors but also into metabolic adaptations of C. neoformans to the host environment. Author SummaryThe adaptation of environmental fungal pathogens to the mammalian host is critical to pathogenesis. Of these adaptations, the remodeling of carbon metabolism is particularly important. Here, we generated a focused set of double mutants of non-essential genes (ACL1, ACS1, KBC1) involved in acetyl-CoA metabolism and examined their fitness in vitro and during CNS infection. From these studies, we found that all three enzymes play important roles during infection but that the role of ACS1/KBC1 was minimal in vitro. Consistent with these observations, the expression of ACS1 and KBC1 was increased in vivo relative to standard in vitro conditions. Furthermore, strains lacking both ACL1 and ACS1 were not viable. These data clearly show that C. neoformans employs multiple carbon metabolism pathways to adapt to the host environment. They also provide insights into the potential mechanism of action for anti-cryptococcal Acs inhibitors.

microbiology↗

bioRxiv2021

Loss-of-function ROX1 mutations suppress the fluconazole susceptibility of upc2AΔ mutation in Candida glabrata, implicating additional positive regulators of ergosterol biosynthesis

Two of the major classes of antifungal drugs in clinical use target ergosterol biosynthesis. Despite its importance, our understanding of the transcriptional regulation of ergosterol biosynthesis genes in pathogenic fungi is essentially limited to the role of hypoxia and sterol-stress induced transcription factors such as Upc2 and Upc2A as well as homologs of Sterol Response Element Binding (SREB) factors. To identify additional regulators of ergosterol biosynthesis in Candida glabrata, an important human fungal pathogen with reduced susceptibility to ergosterol biosynthesis inhibitors relative to other Candida spp., we used a serial passaging strategy to isolate suppressors of the fluconazole hypersusceptibility of a upc2A{Delta} deletion mutant. This led to the identification of loss of function mutants in two genes: ROX1, the homolog of a hypoxia gene transcriptional suppressor in Saccharomyces cerevisiae, and CST6, a transcription factor that is involved in the regulation of carbon dioxide response in C. glabrata. Here, we describe a detailed analysis of the genetic interaction of ROX1 and UPC2A. In the presence of fluconazole, loss of Rox1 function restores ERG11 expression to the upc2A{Delta} mutant and inhibits the expression of ERG3 and ERG6, leading to increased levels or ergosterol and decreased levels of the toxic sterol, 14 methyl-ergosta-8,24(28)-dien-3{beta}, 6-diol, relative to upc2A{Delta}. Our observations establish that Rox1 is a negative regulator of ERG gene biosynthesis and indicate that a least one additional positive transcriptional regulator of ERG gene biosynthesis must be present in C. glabrata. ImportanceCandida glabrata is one of the most important human fungal pathogens and has reduced susceptibility to azole class inhibitors of ergosterol biosynthesis. Although ergosterol is the target of two of the three classes of antifungal drugs, relatively little is known about the regulation of this critical cellular pathway. Sterols are both essential components of the eukaryotic plasma membrane and potential toxins; therefore, sterol homeostasis is critical for cell function. Here, we identified two new negative regulators of C. glabrata of ergosterol (ERG) biosynthesis gene expression. Our results also indicate that in addition to Upc2A, the only known activator of ERG genes, additional positive regulators of this pathway must exist.s

microbiology↗

bioRxiv2021

In vivo complex haploinsufficiency-based genetic analysis identifies a transcription factor circuit regulating Candida albicans oropharyngeal infection and epithelial cell endocytosis

Oropharyngeal candidiasis (OPC) is a common infection that complicates a wide range of medical conditions which can cause either mild or severe disease depending on the patient. The pathobiology of OPC shares many features with candidal biofilms of abiotic surfaces. The transcriptional regulation of C. albicans formation of biofilms on abiotic surfaces has been extensively characterized and involves six key transcription factors (Efg1, Ndt80, Rob1, Bcr1, Brg1, and Tec1). To determine whether this same in vitro biofilm transcriptional regulatory network played a role in OPC, we have carried out the first systematic genetic interaction analysis in a mouse model of C. albicans infection. Whereas all six transcription factors are required for in vitro biofilm formation, only three homozygous deletion mutants (tec1{Delta}{Delta}, bcr1{Delta}{Delta}, and rob1{Delta}{Delta}) and one heterozygous mutant (tec1{Delta}/TEC1) have reduced infectivity in a mouse model of OPC, indicating the network is more robust in vivo than in vitro. Although single mutants (heterozygous or homozygous) of BRG1 and EFG1 have no effect on fungal burden, the double heterozygous and homozygous mutants have dramatically reduced infectivity, indicating a critical genetic interaction between these two transcription factors. Using epistasis analysis, we have formulated a genetic circuit [EFG1+BRG1][->]TEC1[->]BCR1 that is required for OPC infectivity and oral epithelial cell endocytosis. Surprisingly, we also found transcription factor mutants with in vitro defects in filamentation such as efg1{Delta}{Delta} and brg1{Delta}{Delta} filament during oral infection and that decreased filamentation did not correlate with decreased infectivity. Taken together, these data indicate that key in vitro biofilm transcription factors are involved in OPC but that the network characteristics and functional connections are remodeled significantly during interactions with tissues. Author SummaryThe pathology of oral candidiasis has features of biofilm formation, a well-studied process in vitro. Based on that analogy, we hypothesized that network of transcription factors that regulates in vitro biofilm formation might have similarities and differences in during oral infection. To test this, we employed the first systematic genetic interaction analysis of C. albicans in a mouse model of oropharyngeal infection. This revealed that the six regulators involved in in vitro biofilm formation played roles in vivo but that the functional connections between factors were quite distinct. Surprisingly, we also found that, while many of the factors are required for filamentation in vitro, none of the transcription factor deletion mutants was deficient for this key virulence trait in vivo. These observations clearly demonstrate that C. albicans regulates key aspects of its biology differently in vitro and in vivo.

microbiology↗

bioRxiv2021

Intravital imaging of Candida albicans identifies differential in vitro and in vivo filamentation phenotypes for transcription factor deletion mutants

Candida albicans is an important cause of human fungal infections. A widely studied virulence trait of C. albicans is its ability to undergo filamentation to hyphae and pseudohyphae. Although yeast, pseudohyphae and hyphae are present in pathological samples of infected mammalian tissue, it has been challenging to characterize the role of regulatory networks and specific genes during in vivo filamentation. In addition, the phenotypic heterogeneity of C. albicans clinical isolates is becoming increasingly recognized and correlating this heterogeneity with pathogenesis remains an important goal. Here, we describe the use of an intravital imaging approach to characterize C. albicans filamentation in a mammalian model of infection by taking advantage of the translucence of mouse pinna (ears). Using this model, we have found that the in vitro and in vivo filamentation phenotypes of different C. albicans isolates can vary significantly, particularly when in vivo filamentation is compared to solid agar-based assays. We also show that the well-characterized transcriptional regulators Efg1 and Brg1 appear to play important roles both in vivo and in vitro. In contrast, Ume6 is much more important in vitro than in vivo. Finally, strains that are dependent on Bcr1 for in vitro filamentation are able to form filaments in vivo. This intravital imaging approach provides a new approach to the systematic characterization of this important virulence trait during mammalian infection. Our initial studies provide support for the notion that the regulation and initiation of C. albicans filamentation in vivo is distinct from in vitro induction. ImportanceCandida albicans is one of the most common causes of fungal infections in humans. C. albicans undergoes a transition from a round yeast form to a filamentous form during infection which is critical for its ability to cause disease. Although this transition has been studied in the laboratory for years, methods to do so in an animal model of infection have been limited. We have developed a microscopy method to visualize fluorescently labeled C. albicans undergoing this transition in the subcutaneous tissue of mice. Our studies indicate that the regulation of C. albicans filamentation during infection is distinct from that observed in laboratory conditions.

microbiology↗

bioRxiv2019

Derivatives of the antimalarial drug mefloquine are broad spectrum antifungal molecules with activity against drug-resistant clinical isolates

The antifungal pharmacopeia is critically small, particularly in light of the recent emergence of multi-drug-resistant pathogens such as Candida auris. Herein, we report that derivatives of the anti-malarial drug mefloquine have broad spectrum antifungal activity against pathogenic yeasts and molds. In addition, the mefloquine derivatives have activity against clinical isolates that are resistant to one or more of the three classes of drugs currently used to treat invasive fungal infections, indicating that they have a novel mechanism of action. Importantly, the in vitro toxicity profiles using human cell lines indicate that the mefloquine derivatives are very similar to the parent mefloquine despite being up to 64-fold more active against fungal cells. In addition to direct antifungal activity, sub-inhibitory concentrations of the mefloquine derivatives inhibit the expression of virulence traits including filamentation in C. albicans and capsule formation/melanization in C. neoformans. Mode/mechanism of action experiments indicate that the mefloquine derivatives interfere with both mitochondrial and vacuolar function as part of a multi-target mechanism of action. The broad-spectrum scope of activity, blood-brain-barrier penetration, and large number of previously synthesized analogs available combine to support the further optimization and development of the antifungal activity of this general class of drug-like molecules.

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