Cathy Collins

Harnessing Hsp90 function as a powerful, broadly effective therapeutic strategy for fungal infectious disease

Invasive fungal infections are a leading cause of mortality among immunocompromised individuals. Treatment is notoriously difficult with the limited armamentarium of antifungal drugs, whose efficacy is compromised by host toxicity, a limited activity spectrum, or the emergence of drug resistance. We previously established that the molecular chaperone Hsp90 enables the emergence and maintenance of fungal drug resistance. For the most prevalent fungal pathogen of humans, Candida albicans, Hsp90 mediates resistance to azoles, which inhibit ergosterol biosynthesis and are the most widely deployed antifungals in the clinic. For the emerging opportunistic pathogen Aspergillus terreus, Hsp90 is required for basal resistance to echinocandins, which inhibit β(1, 3)-glucan synthesis and are the only new class of antifungals to reach the clinic in decades. Here, we explore the therapeutic potential of Hsp90 inhibitors in fungal disease using a tractable host-model system, larvae of the greater wax moth Galleria mellonella, and a murine model of disseminated disease. Combination therapy with Hsp90 inhibitors that are well tolerated in humans and an azole rescued larvae from lethal C. albicans infections. Combination therapy with an Hsp90 inhibitor and an echinocandin rescued larvae from infections with the most lethal mold, Aspergillus fumigatus. In a murine model of disseminated candidiasis, genetic compromise of C. albicans HSP90 expression enhanced the therapeutic efficacy of an azole. Thus, harnessing Hsp90 provides a much-needed strategy for improving the treatment of fungal disease because it enhances the efficacy of existing antifungals, blocks the emergence of drug resistance, and exerts broad-spectrum activity against diverse fungal pathogens.

Hsp90 Orchestrates Temperature-Dependent Candida albicans Morphogenesis Via Ras1-PKA Signaling

BACKGROUND Hsp90 is an environmentally contingent molecular chaperone that influences the form and function of diverse regulators of cellular signaling. Hsp90 potentiates the evolution of fungal drug resistance by enabling crucial cellular stress responses. Here we demonstrate that in the leading fungal pathogen of humans, Candida albicans, Hsp90 governs cellular circuitry required not only for drug resistance but also for the key morphogenetic transition from yeast to filamentous growth that is crucial for virulence. This transition is normally regulated by environmental cues, such as exposure to serum, that are contingent upon elevated temperature to induce morphogenesis. The basis for this temperature dependence has remained enigmatic. RESULTS We show that compromising Hsp90 function pharmacologically or genetically induces a transition from yeast to filamentous growth in the absence of external cues. Elevated temperature relieves Hsp90-mediated repression of the morphogenetic program. Hsp90 regulates morphogenetic circuitry by repressing Ras1-PKA signaling. Modest Hsp90 compromise enhances the phenotypic effects of activated Ras1 signaling whereas deletion of positive regulators of the Ras1-PKA cascade blocks the morphogenetic response to Hsp90 inhibition. Consistent with the requirement for morphogenetic flexibility for virulence, depletion of C. albicans Hsp90 attenuates virulence in a murine model of systemic disease. CONCLUSIONS Hsp90 governs the integration of environmental cues with cellular signaling to orchestrate fungal morphogenesis and virulence, suggesting new therapeutic strategies for life-threatening infectious disease. Hsp90s capacity to govern a key developmental program in response to temperature change provides a new mechanism that complements the elegant repertoire that organisms utilize to sense temperature.

PKC Signaling Regulates Drug Resistance of the Fungal Pathogen Candida albicans via Circuitry Comprised of Mkc1, Calcineurin, and Hsp90

Fungal pathogens exploit diverse mechanisms to survive exposure to antifungal drugs. This poses concern given the limited number of clinically useful antifungals and the growing population of immunocompromised individuals vulnerable to life-threatening fungal infection. To identify molecules that abrogate resistance to the most widely deployed class of antifungals, the azoles, we conducted a screen of 1,280 pharmacologically active compounds. Three out of seven hits that abolished azole resistance of a resistant mutant of the model yeast Saccharomyces cerevisiae and a clinical isolate of the leading human fungal pathogen Candida albicans were inhibitors of protein kinase C (PKC), which regulates cell wall integrity during growth, morphogenesis, and response to cell wall stress. Pharmacological or genetic impairment of Pkc1 conferred hypersensitivity to multiple drugs that target synthesis of the key cell membrane sterol ergosterol, including azoles, allylamines, and morpholines. Pkc1 enabled survival of cell membrane stress at least in part via the mitogen activated protein kinase (MAPK) cascade in both species, though through distinct downstream effectors. Strikingly, inhibition of Pkc1 phenocopied inhibition of the molecular chaperone Hsp90 or its client protein calcineurin. PKC signaling was required for calcineurin activation in response to drug exposure in S. cerevisiae. In contrast, Pkc1 and calcineurin independently regulate drug resistance via a common target in C. albicans. We identified an additional level of regulatory control in the C. albicans circuitry linking PKC signaling, Hsp90, and calcineurin as genetic reduction of Hsp90 led to depletion of the terminal MAPK, Mkc1. Deletion of C. albicans PKC1 rendered fungistatic ergosterol biosynthesis inhibitors fungicidal and attenuated virulence in a murine model of systemic candidiasis. This work establishes a new role for PKC signaling in drug resistance, novel circuitry through which Hsp90 regulates drug resistance, and that targeting stress response signaling provides a promising strategy for treating life-threatening fungal infections.

Homologous recombination in hybridoma cells: dependence on time and fragment length.

Mutant hybridoma-myeloma cell lines that are defective in immunoglobulin production are expected to be useful for defining the molecular requirements of immunoglobulin gene expression. The analysis of such mutants would be greatly facilitated if they could be mapped by marker rescue, i.e., by identifying the segments of wild-type DNA that can restore the normal phenotype by homologous recombination with the mutant chromosomal immunoglobulin gene. To assess the feasibility of this type of mapping, we have measured the efficiency with which fragments of wild-type DNA recombine with a mutant hybridoma immunoglobulin gene and restore normal immunoglobulin production. We found that most if not all recombinants were detectable 2 days after DNA transfer and that the frequency of gene restoration increased with increasing length of the transferred mu gene fragments, between 1.2 and 9.5 kilobases. These results indicate that the available technology should be adequate to map mutations in the mu gene to within approximately 1 kilobase.

Differential activation of human and guinea pig complement by pentameric and hexameric IgM

Human and mouse IgM can be polymerized as a hexamer in addition to a pentamer. Our previous work with mouse IgM measured activation of guinea pig complement by highly enriched preparations of hexamer and pentamer and showed that hexamer is >100‐fold more active than pentamer. In this report pentamer and hexamer were compared for their capacity to activate complement in a homogeneic system, i.e. chimeric mouse V/human Cμ IgM pentamer and hexamer were assayed separately for their capacity to activate human (and guinea pig) complement. In both the homogeneic and the xenogeneic systems hexamer was more active than pentamer, but the magnitude of the difference between hexamer and pentamer depended on the complement source. Whereas chimeric hexamer activated guinea pig complement >100‐fold more efficiently than did chimeric pentamer, this hexamer was only 4–13‐fold more active than pentamer when assayed with human complement. Similarly, mouse hexamer, which was >100‐fold more active than mouse pentamer with guinea pig complement, was only ∼2‐fold more active than mouse pentamer with human complement. Mouse hexameric and pentameric IgM were each ∼20‐fold more active with human complement than were the corresponding chimeric isoforms of IgM.

The concerted action of Msh2 and UNG stimulates somatic hypermutation at A . T base pairs.

ABSTRACT Mismatch repair plays an essential role in reducing the cellular mutation load. Paradoxically, proteins in this pathway produce A·T mutations during the somatic hypermutation of immunoglobulin genes. Although recent evidence implicates the translesional DNA polymerase η in producing these mutations, it is unknown how this or other translesional polymerases are recruited to immunoglobulin genes, since these enzymes are not normally utilized in conventional mismatch repair. In this report, we demonstrate that A·T mutations were closely associated with transversion mutations at a deoxycytidine. Furthermore, deficiency in uracil-N-glycolase (UNG) or mismatch repair reduced this association. These data reveal a previously unknown interaction between the base excision and mismatch repair pathways and indicate that an abasic site generated by UNG within the mismatch repair tract recruits an error-prone polymerase, which then introduces A·T mutations. Our analysis further indicates that repair tracts typically are ∼200 nucleotides long and that polymerase η makes ∼1 error per 300 T nucleotides. The concerted action of Msh2 and UNG in stimulating A·T mutations also may have implications for mutagenesis at sites of spontaneous cytidine deamination.

Global Analysis of the Fungal Microbiome in Cystic Fibrosis Patients Reveals Loss of Function of the Transcriptional Repressor Nrg1 as a Mechanism of Pathogen Adaptation.

The microbiome shapes diverse facets of human biology and disease, with the importance of fungi only beginning to be appreciated. Microbial communities infiltrate diverse anatomical sites as with the respiratory tract of healthy humans and those with diseases such as cystic fibrosis, where chronic colonization and infection lead to clinical decline. Although fungi are frequently recovered from cystic fibrosis patient sputum samples and have been associated with deterioration of lung function, understanding of species and population dynamics remains in its infancy. Here, we coupled high-throughput sequencing of the ribosomal RNA internal transcribed spacer 1 (ITS1) with phenotypic and genotypic analyses of fungi from 89 sputum samples from 28 cystic fibrosis patients. Fungal communities defined by sequencing were concordant with those defined by culture-based analyses of 1,603 isolates from the same samples. Different patients harbored distinct fungal communities. There were detectable trends, however, including colonization with Candida and Aspergillus species, which was not perturbed by clinical exacerbation or treatment. We identified considerable inter- and intra-species phenotypic variation in traits important for host adaptation, including antifungal drug resistance and morphogenesis. While variation in drug resistance was largely between species, striking variation in morphogenesis emerged within Candida species. Filamentation was uncoupled from inducing cues in 28 Candida isolates recovered from six patients. The filamentous isolates were resistant to the filamentation-repressive effects of Pseudomonas aeruginosa, implicating inter-kingdom interactions as the selective force. Genome sequencing revealed that all but one of the filamentous isolates harbored mutations in the transcriptional repressor NRG1; such mutations were necessary and sufficient for the filamentous phenotype. Six independent nrg1 mutations arose in Candida isolates from different patients, providing a poignant example of parallel evolution. Together, this combined clinical-genomic approach provides a high-resolution portrait of the fungal microbiome of cystic fibrosis patient lungs and identifies a genetic basis of pathogen adaptation.

Metabolic control of antifungal drug resistance

Fungi have evolved an elegant repertoire of mechanisms to survive the cellular stress exerted by antifungal drugs such as azoles, which inhibit ergosterol biosynthesis inducing cell membrane stress. The evolution and maintenance of diverse resistance phenotypes is contingent upon cellular circuitry regulated by the molecular chaperone Hsp90 and its client protein calcineurin. Here, we establish a novel role for nutrients and nutrient signaling in azole resistance. The vulnerability of Saccharomyces cerevisiae azole resistance phenotypes to perturbation was contingent upon specific auxotrophies. Using strains that acquired azole resistance by Erg3 loss of function as a model for resistance that depends on cellular stress responses, we delineated genetic and environmental factors that mitigate the translation of genotype into resistance phenotype. Compromising a global regulator that couples growth and metabolism to environmental cues, Tor kinase, provides a powerful strategy to abrogate drug resistance of S. cerevisiae and Candida albicans with broad therapeutic potential.

Differential glycosylation of polymeric and monomeric IGM

A small fraction of normal IgM is secreted as monomers rather than polymers. We show here that the mu chains of monomeric IgM are glycosylated differently from the mu chains of polymeric IgM and are comparable in their glycosylation to the mu chains from mutant hybridoma cell lines which produce predominantly monomeric IgM. The difference in glycosylation between monomer and polymer mu chains is due to differences in the terminal processing of their oligosaccharides. The glycosylation of the mutant mu chains is not itself responsible for the block in IgM polymer formation.

A weakened transcriptional enhancer yields variegated gene expression.

Identical genes in the same cellular environment are sometimes expressed differently. In some cases, including the immunoglobulin heavy chain (IgH) locus, this type of differential gene expression has been related to the absence of a transcriptional enhancer. To gain additional information on the role of the IgH enhancer, we examined expression driven by enhancers that were merely weakened, rather than fully deleted, using both mutations and insulators to impair enhancer activity. For this purpose we used a LoxP/Cre system to place a reporter gene at the same genomic site of a stable cell line. Whereas expression of the reporter gene was uniformly high in the presence of the normal, uninsulated enhancer and undetectable in its absence, weakened enhancers yielded variegated expression of the reporter gene; i.e., the average level of expression of the same gene differed in different clones, and expression varied significantly among cells within individual clones. These results indicate that the weakened enhancer allows the reporter gene to exist in at least two states. Subtle aspects of the variegation suggest that the IgH enhancer decreases the average duration (half-life) of the silent state. This analysis has also tested the conventional wisdom that enhancer activity is independent of distance and orientation. Thus, our analysis of mutant (truncated) forms of the IgH enhancer revealed that the 250 bp core enhancer was active in its normal position, ∼1.4 kb 3′ of the promoter, but inactive ∼6 kb 3′, indicating that the activity of the core enhancer was distance-dependent. A longer segment – the core enhancer plus ∼1 kb of 3′ flanking material, including the 3′ matrix attachment region – was active, and the activity of this longer segment was orientation-dependent. Our data suggest that this 3′ flank includes binding sites for at least two activators.