Calcium-dependent ESCRT recruitment and lysosome exocytosis maintain epithelial integrity during Candida albicans invasion.
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ABSTRACT:
SUBMITTER: Westman J
PROVIDER: S-EPMC8755444 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:GH72 family of ?-(1,3)-glucanosyltransferases is unique to fungi and is required for cell wall biogenesis, morphogenesis, virulence, and in some species is essential for life. Candida albicans PHR1 and PHR2 are pH-regulated genes that encode GH72 enzymes highly similar to Gas1p of Saccharomyces cerevisiae. PHR1 is expressed at pH ? 5.5 while PHR2 is transcribed at pH ? 5.5. Both are essential for C. albicans morphogenesis and virulence. During growth at neutral-alkaline pH, Phr1p-GFP preferentially localizes to sites of active cell wall formation as the incipient bud, the mother-daughter neck, the bud periphery, and concentrates in the septum at cytokinesis. We further investigated this latter localization. In chs3? cells, lacking the chitin of the chitin ring and lateral cell wall, Phr1p-GFP still concentrated along the thin line of the primary septum formed by chitin deposited by chitin synthase I (whose catalytic subunit is Chs1p) suggesting that it plays a role during formation of the secondary septa. RO-09-3143, a highly specific inhibitor of Chs1p activity, inhibits septum formation and blocks cell division. However, alternative septa are produced and are crucial for cell survival. Phr1p-GFP is excluded from such aberrant septa. Finally, we determined the effects of RO-09-3143 in cells lacking Phr1p. PHR1 null mutant was more susceptible to the drug than the wild type. The phr1? cells were larger, devoid of septa, and underwent endomitosis and cell death. Phr1p and Chs1p cooperate in maintaining cell integrity and in coupling morphogenesis with nuclear division in C. albicans.
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Project description:Invasive cells use small invadopodia to breach basement membrane (BM), a dense matrix that encases tissues. Following the breach, a large protrusion forms to clear a path for tissue entry by poorly understood mechanisms. Using RNAi screening for defects in Caenorhabditis elegans anchor cell (AC) invasion, we found that UNC-6(netrin)/UNC-40(DCC) signaling at the BM breach site directs exocytosis of lysosomes using the exocyst and SNARE SNAP-29 to form a large protrusion that invades vulval tissue. Live-cell imaging revealed that the protrusion is enriched in the matrix metalloprotease ZMP-1 and transiently expands AC volume by more than 20%, displacing surrounding BM and vulval epithelium. Photobleaching and genetic perturbations showed that the BM receptor dystroglycan forms a membrane diffusion barrier at the neck of the protrusion, which enables protrusion growth. Together these studies define a netrin-dependent pathway that builds an invasive protrusion, an isolated lysosome-derived membrane structure specialized to breach tissue barriers.
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Project description:BACKGROUND:Saccharomyces cerevisiae ScGdt1 and mammalian TMEM165 are two members of the UPF0016 membrane protein family that is likely to form a new group of Ca2+/H+ antiporter and/or a Mn2+ transporter in the Golgi apparatus. We have previously shown that Candida albicans CaGDT1 is a functional ortholog of ScGDT1 in the response of S. cerevisiae to calcium stress. However, how CaGdt1 together with the Golgi calcium pump CaPmr1 regulate calcium homeostasis and cell wall integrity in this fungal pathogen remains unknown. METHODS:Chemical sensitivity was tested by dilution assay. Cell survival was examined by measuring colony-forming units and staining with Annexin V-FITC and propidium iodide. Calcium signaling was examined by expression of downstream target gene CaUTR2, while cell wall integrity signaling was revealed by detection of phosphorylated Mkc1 and Cek1. Subcellular localization of CaGdt1 was examined through direct and indirect immunofluorescent approaches. Transcriptomic analysis was carried out with RNA sequencing. RESULTS:This study shows that Candida albicans CaGDT1 is also a functional ortholog of ScGDT1 in the response of S. cerevisiae to cell wall stress. CaGdt1 is localized in the Golgi apparatus but at distinct sites from CaPmr1 in C. albicans. Loss of CaGDT1 increases the sensitivity of cell lacking CaPMR1 to cell wall and ER stresses. Deletion of CaGDT1 and/or CaPMR1 increases calcium uptake and activates the calcium/calcineurin signaling. Transcriptomic profiling reveals that core functions shared by CaGdt1 and CaPmr1 are involved in the regulation of cellular transport of metal ions and amino acids. However, CaGdt1 has distinct functions from CaPmr1. Chitin synthase gene CHS2 is up regulated in all three mutants, while CHS3 is only up regulated in the pmr1/pmr1 and the gdt1/gdt1 pmr1/pmr1 mutants. Five genes (DIE2, STT3, OST3, PMT1 and PMT4) of glycosylation pathway and one gene (SWI4) of the cell wall integrity (CWI) pathway are upregulated due to deletion of CaGDT1 and/or CaPMR1. Consistently, deletion of either CaPMR1 or CaGDT1 activates the CaCek1-mediated CWI signaling in a cell wall stress-independent fashion. Calcineurin function is required for the integrity of the cell wall and vacuolar compartments of cells lacking both GDT1 and CaPMR1. CONCLUSIONS:CaPmr1 is the major player in the regulation of calcium homeostasis and cell wall stress, while CaGdt1 plays a compensatory role for CaPmr1 in the Golgi compartment in C. albicans.
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Project description:The exocyst plays a crucial role in the targeting of secretory vesicles to the plasma membrane during exocytosis. It has been shown to be involved in diverse cellular processes including yeast budding. However, the mechanism of the exocyst regulating yeast budding has not been fully elucidated. Here we report a novel interaction between the exocyst component Sec15 and the Ras-family GTPase Rsr1, a master regulator of bud-site-selection system, in the fungus Candida albicans. We present several lines of evidence indicating physical and genetic interaction of Sec15 with Rsr1. In vitro binding assays and co-immunoprecipitation studies showed that Sec15 associated physically with Rsr1. Deletion of RSR1 completely abolished the polarised localisation of Sec15 as well as all the other exocyst components in both yeast and hyphal cells, suggesting a functional interaction between Sec15 and Rsr1. We also show that C. albicans Sec15 interacts directly with the polarity determinant Bem1 and the type V myosin, Myo2. Disruption of the interaction by shutting off SEC15 results in mislocaliztion of Bem1-GFP. These findings highlight the important role of Sec15 in polarised cell growth by providing a direct functional link between bud-site-selection and exocytosis.
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Project description:The ability of pathogenic microorganisms to assimilate essential nutrients from their hosts is critical for pathogenesis. Here we report endothelial zinc sequestration by the major human fungal pathogen, Candida albicans. We hypothesised that, analogous to siderophore-mediated iron acquisition, C. albicans utilises an extracellular zinc scavenger for acquiring this essential metal. We postulated that such a "zincophore" system would consist of a secreted factor with zinc-binding properties, which can specifically reassociate with the fungal cell surface. In silico analysis of the C. albicans secretome for proteins with zinc binding motifs identified the pH-regulated antigen 1 (Pra1). Three-dimensional modelling of Pra1 indicated the presence of at least two zinc coordination sites. Indeed, recombinantly expressed Pra1 exhibited zinc binding properties in vitro. Deletion of PRA1 in C. albicans prevented fungal sequestration and utilisation of host zinc, and specifically blocked host cell damage in the absence of exogenous zinc. Phylogenetic analysis revealed that PRA1 arose in an ancient fungal lineage and developed synteny with ZRT1 (encoding a zinc transporter) before divergence of the Ascomycota and Basidiomycota. Structural modelling indicated physical interaction between Pra1 and Zrt1 and we confirmed this experimentally by demonstrating that Zrt1 was essential for binding of soluble Pra1 to the cell surface of C. albicans. Therefore, we have identified a novel metal acquisition system consisting of a secreted zinc scavenger ("zincophore"), which reassociates with the fungal cell. Furthermore, functional similarities with phylogenetically unrelated prokaryotic systems indicate that syntenic zinc acquisition loci have been independently selected during evolution.
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Project description:In Candida albicans, calcium ions (Ca2+) regulate the activity of several signaling pathways, especially the calcineurin signaling pathway. Ca2+ homeostasis is also important for cell polarization, hyphal extension, and plays a role in contact sensing. It is therefore important to obtain accurate tools with which Ca2+ homeostasis can be addressed in this fungal pathogen. Aequorin from Aequorea victoria has been used in eukaryotic cells for detecting intracellular Ca2+. A codon-adapted aequorin Ca2+-sensing expression system was therefore designed for probing cytosolic Ca2+ flux in C. albicans. The availability of a novel water-soluble formulation of coelenterazine, which is required as a co-factor, made it possible to measure bioluminescence as a readout of intracellular Ca2+ levels in C. albicans. Alkaline stress resulted in an immediate influx of Ca2+ from the extracellular medium. This increase was exacerbated in a mutant lacking the vacuolar Ca2+ transporter VCX1, thus confirming its role in Ca2+ homeostasis. Using mutants in components of a principal Ca2+ channel (MID1, CCH1), the alkaline-dependent Ca2+ spike was greatly reduced, thus highlighting the crucial role of this channel complex in Ca2+ uptake and homeostasis. Exposure to the antiarrhythmic drug amiodarone, known to perturb Ca2+ trafficking, resulted in increased cytoplasmic Ca2+ within seconds that was abrogated by the chelation of Ca2+ in the external medium. Ca2+ import was also dependent on the Cch1/Mid1 Ca2+ channel in amiodarone-exposed cells. In conclusion, the aequorin Ca2+ sensing reporter developed here is an adequate tool with which Ca2+ homeostasis can be investigated in C. albicans.
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