Ian Leaves

The emerging fungus Candida auris is a drug resistant global public health threat and WHO critical priority pathogen. Recommended first-line invasive candidiasis treatment is echinocandin monotherapy, but C. auris can develop on-treatment resistance via FKS1/2 gene mutations and additional, previously unexplained mechanisms. To better understand echinocandin failure in C. auris , we sequenced the genomes of echinocandin refractory FKS1/2 wild-type C. auris serial isolates from two critically unwell patients in London, UK. Population analysis profiling revealed echinocandin heteroresistance, and in vitro culture of clinical isolates at supra-MIC concentrations of anidulafungin (8 μg/ml) exhibited morphotypic heterogeneity. Small colony variants (SCVs) and large colony variants (LCVs) showed elevated MICs with polyploidy (to 4n and above) alongside adaptive changes in cell wall β−1,3-glucan content. LCVs contained significantly more mutations in calcineurin-related stress tolerance pathway gene CRZ1 compared to clinical parents and SCVs, associated with further increases in MIC. These findings indicate progressive step-wise accrual of adaptation to echinocandins, including genomic instability, alterations in stress tolerance pathways, and cell wall remodelling, paving the way for resistance emergence.

Dietary supplementation with prebiotics such as inulin has been associated with a broad range of health benefits. However, the effects of inulin on the opportunistic fungal pathogen Candida albicans, which resides as a commensal in the gut, have not been characterized. Here, RNA sequencing revealed that inulin affects the expression of C. albicans genes associated with cell wall construction, adhesion, and yeast-hypha morphogenesis. Consistent with these changes in gene expression, inulin inhibited hyphal development, increased adhesion to human Caco-2 and A431 cells, decreased the thickness of the inner layer of the C. albicans cell wall, reduced the exposure of cell wall pathogen-associated molecular patterns [β-(1,3)-glucan and chitin], and affected antifungal drug sensitivity. These changes impacted host immune recognition and cytokine responses, ultimately attenuating the virulence of C. albicans in an invertebrate infection model. Therefore, dietary supplementation with inulin is likely to influence host-fungus interactions. IMPORTANCE: The benefits of prebiotic dietary supplements, such as inulin (a natural plant dietary fiber), are thought to include a healthier gut microbiome, a reduced risk of colon cancer, and lower cholesterol levels. Unsurprisingly, prebiotic usage is increasing rapidly. However, while the effects of prebiotics upon gut bacteria have been characterized, the impacts upon Candida albicans, an opportunistic fungal pathogen that resides in the human gut, have remained obscure. We show that inulin affects the expression of virulence-related phenotypes and antifungal drug sensitivity in Candida . Furthermore, we show that inulin reduces the virulence of this fungus in an invertebrate model, consistent with the idea that inulin may lower the risk of fungal infection in healthy individuals.

Invasive, high-mortality yeast infections caused by pathogenic Candida species are the most common fungal nosocomial bloodstream infections. The World Health Organization (WHO) has called for improved prevention and diagnostic strategies for fungal pathogens. Here, we demonstrate that Candida -related antibodies can be detected using glycan microarrays containing synthetic glycans to guide the discovery of oligosaccharide epitopes for diagnostics and vaccine design. Pure, synthetic mannans and β -glucans, resembling carbohydrates found on the surface of Candida spp., were employed to screen and compare sera from infected humans and mice with noninfected individuals. IgM antibodies directed toward β -glucans were observed shortly after infection, and after a longer time of infection, IgM and IgG antibodies that preferentially recognize mannans. Phosphodiester-linked mannosides and the β -(1,2)-mannose monomer help to distinguish different Candida spp. The tetrasaccharide antigen β -(1,2)Man- α -(1,2)Man- α -(1,2)Man- α -(1,2)Man, and the pentasaccharide antigens α -(1,2)Man- α -(1,3)Man- α -(1,2)Man- α -(1,2)Man- α -(1,2)Man and β -(1,3)Glc- β -(1,3)Glc- β -(1,3)Glc-[ β -(1,6)Glc]- β -(1,3)Glc were identified as potential epitopes for detection, e.g., through monoclonal antibody lateral flow tests, and the development of glycoconjugate or monoclonal antibody vaccines against Candida.

The major fungal pathogen, Candida albicans, exists as a commensal in the gastrointestinal tract of healthy humans. Fungal colonisation levels increase during gut dysbiosis, when the local microbiota and short-chain fatty acid (SCFA) concentrations become perturbed. Individually, acetic, propionic and butyric acids are reported to exert differential effects on C. albicans. In this study, we tested whether combinations of these SCFAs, at concentrations that broadly reflect healthy and dysbiotic gut profiles, influence virulence-related phenotypes. The selected healthy and dysbiotic SCFA mixes slowed the growth of C. albicans SC5314, increased resistance to cell wall stresses (Calcofluor White, SDS, caspofungin), differentially affected the exposure of the key cell surface pathogen-associated molecular patterns (PAMPs) β-1,3-glucan, chitin and mannan, and influenced total chitin content compared with non-SCFA controls. However, few differences were observed between the healthy and dysbiotic mixes. Furthermore, comparison of isolates from other epidemiological clades revealed that most effects of the SCFA mixes were strain-specific, reflecting the high degree of phenotypic variation reported previously between clinical isolates. Interestingly, the healthy SCFA mix inhibited hyphal development to a greater extent than the dysbiotic mix in some C. albicans isolates including SC5314. This was not reflected in differential adhesion to Caco-2 cells or in altered virulence in the Galleria model of systemic candidiasis. We conclude that SCFA mixtures reflecting those present in the human gut subtly influence some virulence-related phenotypes in C. albicans in a strain-specific manner.

Sporothrix brasiliensis is an emerging fungal pathogen frequently associated with zoonotic transmission of sporotrichosis by contaminated cats. Within 25 years, the disease has spread not only throughout Brazil but now to neighboring countries in Latin America. Thermo-dimorphism, melanin, glycans, adhesins, and secreted vesicles have been associated with the ability of Sporothrix species to cause disease in the mammalian host. Although certain virulence factors have been proposed as potential determinants for sporotrichosis, the scarcity of molecular tools for performing reverse genetics in Sporothrix has significantly impeded the dissection of mechanisms underlying the disease. Here, we demonstrate that PEG-mediated protoplast transformation is a powerful method for heterologous gene expression in S. brasiliensis, S. schenckii, and S. chilensis . Combined with CRISPR/Cas9 gene editing, this transformation protocol enabled the deletion of the putative DHN-melanin synthase gene pks1 , which is a proposed virulence factor of Sporothrix species. To improve in locus integration of deletion constructs, we deleted the KU ⁸⁰ homolog that is critical for non-homologous end-joining DNA repair. The use of Δ ku80 strains from S. brasiliensis enhanced homologous-directed repair during transformation resulting in increased targeted gene deletion in combination with CRISPR/Cas9. In conclusion, our CRISPR/Cas9-based transformation protocol provides an efficient tool for targeted gene manipulation in Sporothrix species. IMPORTANCE: Sporotrichosis caused by Sporothrix brasiliensis is a disease that requires long periods of treatment and is rapidly spreading across Latin America. The virulence of this fungus and the surge of atypical and more severe presentations of the disease raise the need for an understanding of the molecular mechanisms underlying sporotrichosis, as well as the development of better diagnostics and antifungal therapies. By developing molecular tools for accurate genetic manipulation in Sporothrix, this study addresses the paucity of reliable and reproducible tools for stable genetic engineering of Sporothrix species, which has represented a major obstacle for studying the virulence determinants and their roles in the establishment of sporotrichosis.

Actin-related protein (ARP2/3) complex is a heteroheptameric protein complex, evolutionary conserved in all eukaryotic organisms. Its conserved role is based on the induction of actin polymerization at the interface between membranes and the cytoplasm. Plant ARP2/3 has been reported to participate in actin reorganization at the plasma membrane during polarized growth of trichomes and at the plasma membrane–endoplasmic reticulum contact sites. Here we demonstrate that individual plant subunits of ARP2/3 fused to fluorescent proteins form motile spot-like structures in the cytoplasm that are associated with peroxisomes in Arabidopsis and tobacco. ARP2/3 is found at the peroxisome periphery and contains the assembled ARP2/3 complex and the WAVE/SCAR complex subunit NAP1. This ARP2/3-positive peroxisomal domain colocalizes with the autophagosome and, under conditions that affect the autophagy, colocalization between ARP2/3 and the autophagosome increases. ARP2/3 subunits co-immunoprecipitate with ATG8f and peroxisome-associated ARP2/3 interact in vivo with the ATG8f marker. Since mutants lacking functional ARP2/3 complex have more peroxisomes than wild type, we suggest that ARP2/3 has a novel role in the process of peroxisome degradation by autophagy, called pexophagy.

Summary: Sporothrix brasiliensis is an emerging fungal pathogen frequently associated with zoonotic transmission of sporotrichosis. Although certain virulence factors have been proposed as potential sporotrichosis determinants, the scarcity of molecular tools for reverse genetics studies on Sporothrix has significantly impeded the dissection of mechanisms underlying the disease. Here, we demonstrate that PEG-mediated protoplast transformation is a powerful method for heterologous expression in S. brasiliensis, S. schenckii and S. chilensis . Combined with CRISPR/Cas9 gene editing, this transformation protocol allowed the deletion of the putative DHN-melanin synthase gene pks1 , which is a proposed virulence factor of Sporothrix species. To improve in locus integration of deletion constructs, we deleted the KU ⁸⁰ homologue that is critical for non-homologous end-joining DNA repair. The use of S. brasiliensis Δ ku80 strains enhanced homologous-directed repair during transformation resulting in increased targeted gene deletion. In conclusion, our CRISPR/Cas9-based transformation protocol provides an efficient tool for targeted gene manipulation in Sporothrix species.

ARP2/3 is a heteroheptameric protein complex evolutionary conserved in all eukaryotic organisms. Its conserved role is based on the induction of actin polymerization at the interface between membranes and the cytoplasm. Plant ARP2/3 has been reported to participate in actin reorganization at the plasma membrane during polarized growth of trichomes and at the plasma membrane-endoplasmic reticulum contact sites. We demonstrate here that individual plant subunits of ARP2/3 fused to fluorescent proteins form motile dot-like structures in the cytoplasm that are associated with plant peroxisomes. ARP2/3 dot structure is found at the peroxisome periphery and contains assembled ARP2/3 complex and WAVE/SCAR complex subunit NAP1. This dot occasionally colocalizes with the autophagosome, and under conditions that affect the autophagy, colocalization between ARP2/3 and the autophagosome increases. ARP2/3 subunits co-immunoprecipitate with ATG8f marker. Since mutants lacking functional ARP2/3 complex have more peroxisomes than WT, we link the ARP2/3 complex on peroxisomes to the process of peroxisome degradation by autophagy called pexophagy. Additionally, several other peroxisomal proteins colocalize with ARP2/3 dot on plant peroxisomes. Our results suggest a specific role of ARP2/3 and actin in the peroxisome periphery, presumably in membrane remodelling. We hypothesize that this role of ARP2/3 aids processes at the peroxisome periphery such as peroxisome degradation through autophagy or regulation of peroxisomal proteins localization or function.

ARP2/3 is a heteroheptameric protein complex evolutionary conserved in all eukaryotic organisms. Its conserved role is based on the induction of actin polymerization at the interface between membranes and the cytoplasm. Plant ARP2/3 has been reported to participate in actin reorganization at the plasma membrane during polarized growth of trichomes and at the plasma membrane-endoplasmic reticulum contact sites. We demonstrate here that individual plant subunits of ARP2/3 fused to fluorescent proteins form motile dot-like structures in the cytoplasm that are associated with plant peroxisomes. ARP2/3 dot structure is found at the peroxisome periphery and contains assembled ARP2/3 complex and WAVE/SCAR complex subunit NAP1. This dot occasionally colocalizes with the autophagosome, and under conditions that affect the autophagy, colocalization between ARP2/3 and the autophagosome increases. ARP2/3 subunits co-immunoprecipitate with ATG8f marker. Since mutants lacking functional ARP2/3 complex have more peroxisomes than WT, we link the ARP2/3 complex on peroxisomes to the process of peroxisome degradation by autophagy called pexophagy. Additionally, several other peroxisomal proteins colocalize with ARP2/3 dot on plant peroxisomes. Our results suggest a specific role of ARP2/3 and actin in the peroxisome periphery, presumably in membrane remodelling. We hypothesize that this role of ARP2/3 aids processes at the peroxisome periphery such as peroxisome degradation through autophagy or regulation of peroxisomal proteins localization or function. Significance statement ARP2/3 complex-positive dots associate exclusively with peroxisomes in plant cells, where it colocalizes with autophagosome marker ATG8f and several other proteins. Our experiments link ARP2/3 to pexophagy: colocalization between ARP2/3 dots and autophagosome increases when autophagy processes are induced or inhibited; ARP2/3 and ATG8f colocalize and co-immunoprecipitate, and finally, ARP2/3 mutants’ cells contain more peroxisomes than WT. Our results suggest a novel role of ARP2/3 in peroxisome structure and function regulation.

Mitochondria are highly pleomorphic, undergoing rounds of fission and fusion. Mitochondria are essential for energy conversion, with fusion favouring higher energy demand. Unlike fission, the molecular components involved in mitochondrial fusion in plants are unknown. Here, we show a role for the GTPase Miro2 in mitochondria interaction with the ER and its impacts on mitochondria fusion and motility. Mutations in AtMiro2’s GTPase domain indicate that the active variant results in larger, fewer mitochondria which are attached more readily to the ER when compared with the inactive variant. These results are contrary to those in metazoans where Miro predominantly controls mitochondrial motility, with additional GTPases affecting fusion. Synthetically controlling mitochondrial fusion rates could fundamentally change plant physiology by altering the energy status of the cell. Furthermore, altering tethering to the ER could have profound effects on subcellular communication through altering the exchange required for pathogen defence.