Campafungins: Inhibitors of Candida albicans and Cryptococcus neoformans Hyphal Growth.
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ABSTRACT:
SUBMITTER: Perlatti B
PROVIDER: S-EPMC7530089 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Autophagy is used by eukaryotes in bulk cellular material recycling and in immunity to intracellular pathogens. We evaluated the role of macrophage autophagy in the response to Cryptococcus neoformans and Candida albicans, two important opportunistic fungal pathogens. The autophagosome marker LC3 (microtubule-associated protein 1 light chain 3 alpha) was present in most macrophage vacuoles containing C. albicans. In contrast, LC3 was found in only a few vacuoles containing C. neoformans previously opsonized with antibody but never after complement-mediated phagocytosis. Disruption of host autophagy in vitro by RNA interference against ATG5 (autophagy-related 5) decreased the phagocytosis of C. albicans and the fungistatic activity of J774.16 macrophage-like cells against both fungi, independent of the opsonin used. ATG5-knockout bone marrow-derived macrophages (BMMs) also had decreased fungistatic activity against C. neoformans when activated. In contrast, nonactivated ATG5-knockout BMMs actually restricted C. neoformans growth more efficiently, suggesting that macrophage autophagy plays different roles against C. neoformans, depending on the macrophage type and activation. Interference with autophagy in J774.16 cells also decreased nonlytic exocytosis of C. neoformans, increased interleukin-6 secretion, and decreased gamma interferon-induced protein 10 secretion. Mice with a conditionally knocked out ATG5 gene in myeloid cells showed increased susceptibility to intravenous C. albicans infection. In contrast, these mice manifested no increased susceptibility to C. neoformans, as measured by survival, but had fewer alternatively activated macrophages and less inflammation in the lungs after intratracheal infection than control mice. These results demonstrate the complex roles of macrophage autophagy in restricting intracellular parasitism by fungi and reveal connections with nonlytic exocytosis, humoral immunity, and cytokine signaling.
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Project description:Candida albicans is an opportunistic pathogen, typically found as a benign commensal yeast living on skin and mucosa, but poised to invade injured tissue to cause local infections. In debilitated and immunocompromised individuals, C. albicans may spread to cause life-threatening systemic infections. Upon contact with serum and at body temperature, C. albicans performs a regulated switch to filamentous morphology, characterized by emergence of a germ tube from the yeast cell followed by mold-like growth of branching hyphae. The ability to switch between growth morphologies is an important virulence factor of C. albicans. To identify compounds able to inhibit hyphal morphogenesis, we screened libraries of existing drugs for inhibition of the hyphal switch under stringent conditions. Several compounds that specifically inhibited hyphal morphogenesis were identified. Chemogenomic analysis suggested an interaction with the endocytic pathway, which was confirmed by direct measurement of fluid-phase endocytosis in the presence of these compounds. These results suggest that the activity of the endocytic pathway, which is known to be particularly important for hyphal growth, represents an effective target for hyphae-inhibiting drugs.
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Project description:Disruption of vacuolar biogenesis in the pathogenic yeast Candida albicans causes profound defects in polarized hyphal growth. However, the precise vacuolar pathways involved in yeast-hypha differentiation have not been determined. Previously we focused on Vps21p, a Rab GTPase involved in directing vacuolar trafficking through the late endosomal prevacuolar compartment (PVC). Herein, we identify two additional Vps21p-related GTPases, Ypt52p and Ypt53p, that colocalize with Vps21p and can suppress the hyphal defects of the vps21?/? mutant. Phenotypic analysis of gene deletion strains revealed that loss of both VPS21 and YPT52 causes synthetic defects in endocytic trafficking to the vacuole, as well as delivery of the virulence-associated vacuolar membrane protein Mlt1p from the Golgi compartment. Transcription of all three GTPase-encoding genes is increased under hyphal growth conditions, and overexpression of the transcription factor Ume6p is sufficient to increase the transcription of these genes. While only the vps21?/? single mutant has hyphal growth defects, these were greatly exacerbated in a vps21?/? ypt52?/? double mutant. On the basis of relative expression levels and phenotypic analysis of gene deletion strains, Vps21p is the most important of the three GTPases, followed by Ypt52p, while Ypt53p has an only marginal impact on C. albicans physiology. Finally, disruption of a nonendosomal AP-3-dependent vacuolar trafficking pathway in the vps21?/? ypt52?/? mutant, further exacerbated the stress and hyphal growth defects. These findings underscore the importance of membrane trafficking through the PVC in sustaining the invasive hyphal growth form of C. albicans.
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Project description:Candida albicans is an opportunistic and polymorphic fungal pathogen that causes mucosal, disseminated and invasive infections in humans. Transition from the yeast form to the hyphal form is one of the key virulence factors in C. albicans contributing to macrophage evasion, tissue invasion and biofilm formation. Nontoxic small molecules that inhibit C. albicans yeast-to-hypha conversion and hyphal growth could represent a valuable source for understanding pathogenic fungal morphogenesis, identifying drug targets and serving as templates for the development of novel antifungal agents. Here, we have identified the triterpenoid saponin family of gymnemic acids (GAs) as inhibitor of C. albicans morphogenesis. GAs were isolated and purified from Gymnema sylvestre leaves, the Ayurvedic traditional medicinal plant used to treat diabetes. Purified GAs had no effect on the growth and viability of C. albicans yeast cells but inhibited its yeast-to-hypha conversion under several hypha-inducing conditions, including the presence of serum. Moreover, GAs promoted the conversion of C. albicans hyphae into yeast cells under hypha inducing conditions. They also inhibited conidial germination and hyphal growth of Aspergillus sp. Finally, GAs inhibited the formation of invasive hyphae from C. albicans-infected Caenorhabditis elegans worms and rescued them from killing by C. albicans. Hence, GAs could be useful for various antifungal applications due to their traditional use in herbal medicine.
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Project description:Various stimuli, including N-acetylglucosamine (GlcNAc), induce the fungal pathogen Candida albicans to switch from budding to hyphal growth. Previous studies suggested that hyphal morphogenesis is stimulated by transcriptional induction of a set of genes that includes known virulence factors. To better understand hyphal development, we examined the role of GlcNAc metabolism using a triple mutant lacking the genes required to metabolize exogenous GlcNAc (hxk1? nag1? dac1?). Surprisingly, at low ambient pH (?pH 4), GlcNAc stimulated this mutant to form hyphae without obvious induction of hyphal genes. This indicates that GlcNAc can stimulate a separate signal to induce hyphae that is independent of transcriptional responses. Of interest, GlcNAc could induce the triple mutant to express hyphal genes when the medium was buffered to a higher pH (>pH 5), which normally occurs after GlcNAc catabolism. Catabolism of GlcNAc raises the ambient pH rather than acidifying it, as occurs after dextrose catabolism. This synergy between alkalinization and GlcNAc to induce hyphal genes involves the Rim101 pH-sensing pathway; GlcNAc induced rim101? and dfg16? mutants to form hyphae, but hyphal gene expression was partially defective. These results demonstrate that hyphal morphogenesis and gene expression can be regulated independently, which likely contributes to pathogenesis at different host sites.
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Project description:Heteroresistance to fluconazole (FLC) in Cryptococcus is a transient adaptive resistance which is lost upon release from the drug pressure. It is known that clones heteroresistant to FLC invariably contain disomic chromosomes, but how disomy is formed remains unclear. Previous reports suggested that the aneuploid heteroresistant colonies in Cryptococcus emerge from multinucleated cells, resembling the case in Candida albicans Although a small number of cells containing multiple nuclei appear in a short time after FLC treatment, we provide evidence that the heteroresistant colonies in the presence of FLC arise from uninucleate cells without involving multinuclear/multimeric stages. We found that fidelity of chromosome segregation in mitosis plays an important role in regulation of FLC heteroresistance frequency in C. neoformans Although FLC-resistant colonies occurred at a very low frequency, we were able to modulate the frequency of heteroresistance by overexpressing SMC1, which encodes a protein containing an SMC domain in chromosome segregation. Using time-lapse microscopy, we captured the entire process of colony formation from a single cell in the presence of FLC. All the multinucleated cells formed within a few hours of FLC exposure failed to multiply after a few cell divisions, and the cells able to proliferate to form colonies were all uninucleate without exception. Furthermore, no nuclear fusion event or asymmetric survival between mother and daughter cells, a hallmark of chromosome nondisjunction in haploid organisms, was observed. Therefore, the mechanisms of aneuploidy formation in C. neoformans appear different from most common categories of aneuploid formation known for yeasts.IMPORTANCE The gold standard of cryptococcosis treatment consists of induction therapy with amphotericin B followed by lifelong maintenance therapy with fluconazole (FLC). However, prolonged exposure to FLC induces the emergence of clones heteroresistant to azoles. All the heteroresistant clones thus far analyzed have been shown to be aneuploids, but how the aneuploid is formed remains unclear. Aneuploidy in fungi and other eukaryotic cells is known to result most commonly from chromosome missegregation during cell division due to defects in any one of the multiple components and processes that are required for the formation of two genetically identical daughter cells. Although formation of multinucleated cells has been observed in cells exposed to FLC, evidence for the emergence of drug-resistant aneuploid populations directly from such cells has been lacking. We show the evidence that the aneuploid in fluconazole-heteroresistant clones of Cryptococcus neoformans is derived neither from multinucleated cells nor from chromosome missegregation.
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Project description:Localization of specific mRNAs is an important mechanism through which cells achieve polarity and direct asymmetric growth. Based on a framework established in Saccharomyces cerevisiae, we describe a She3-dependent RNA transport system in Candida albicans, a fungal pathogen of humans that grows as both budding (yeast) and filamentous (hyphal and pseudohyphal) forms. We identify a set of 40 mRNAs that are selectively transported to the buds of yeast-form cells and to the tips of hyphae, and we show that many of the genes encoded by these mRNAs contribute to hyphal development, as does the transport system itself. Although the basic system of mRNA transport is conserved between S. cerevisiae and C. albicans, we find that the cargo mRNAs have diverged considerably, implying that specific mRNAs can easily move in and out of transport control over evolutionary timescales. The differences in mRNA cargos likely reflect the distinct selective pressures acting on the two species.
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