Lipid Biosynthetic Genes Affect Candida albicans Extracellular Vesicle Morphology, Cargo, and Immunostimulatory Properties

Abstract

dMicrobial secretion is integral for regulating cell homeostasis as well as releasing virulence factors during infection. The genes encoding phosphatidylserine synthase (CHO1) and phosphatidylserine decarboxylase (PSD1 and PSD2) are Candida albicans genes involved in phospholipid biosynthesis, and mutations in these genes affect mitochondrial function, cell wall thickness, and virulence in mice. We tested the roles of these genes in several agar-based secretion assays and observed that the cho1⌬/⌬ and psd1⌬/⌬ psd2⌬/⌬ strains manifested less protease and phospholipase activity. Since extracellular vesicles (EVs) are surrounded by a lipid membrane, we investigated the effects of these mutations on EV structure, composition, and biological activity. The cho1⌬/⌬ mutant releases EVs comparable in size to wild-type EVs, but EVs from the psd1⌬/⌬ psd2⌬/⌬ strain are much larger than those from the wild type, including a population of >100-nm EVs not observed in the EVs from the wild type. Proteomic analysis revealed that EVs from both mutants had a significantly different protein cargo than that of EVs from the wild type. EVs were tested for their ability to activate NF-B in bone marrow-derived macrophage cells. While wild-type and psd1⌬/⌬ psd2⌬/⌬ mutant-derived EVs activated NF-B, the cho1⌬/⌬ mutant-derived EV did not. These studies indicate that the presence and absence of these C. albicans genes have qualitative and quantitative effects on EV size, composition, and immunostimulatory phenotypes that highlight a complex interplay between lipid metabolism and vesicle production.

Fungal Extracellular Vesicles with a Focus on Proteomic Analysis

“…One of the first proteomic analyses of EVs from the yeast form of C. albicans identified 75 proteins that fell into nine functional groups, the largest of which was cell wall related proteins (41%). Lipid analyses of C. albicans EVs found phosphatidyl serine, glucosylceramide and ergosterol, similar to the lipids reported for C. neoformans EVs . The RNA composition of C. albicans EVs was also comparable to that of C. neoformans EVs.…”

“…As observed with C. neoformans EVs, EVs from C. albicans are immunostimulatory. Incubation of C. albicans EVs with murine macrophages or dendritic cells led to the activation of the innate immune cells and production of NF‐κB as well as both pro‐ and anti‐inflammatory cytokines . The immunostimulatory activity of C. albicans EVs was further supported by the decreased fungal burden in wax moth larvae ( Galleria mellonella ) that had been pretreated with EVs prior to infection with C. albicans…”

“…Initial isolation and characterization by transmission election microscopy (TEM) of EVs from C. albicans revealed a population of vesicles that were generally smaller than 50 nm in diameter . Conversely, subsequent analyses using TEM and dynamic light scattering revealed two populations of EVs, those with diameters from 50–200 nm and those with diameters from 200–1000 nm …”

Pathogenic factors inCandidabiofilm-related infectious diseases

“…Wolf et al . () described how nontraditional secretion systems provide a mechanism for microbes to release a protein cargo in C. albicans ‐produced EVs instead of the requirement for signal peptides for protein secretion (Albuquerque et al . ).…”

Concise Review: Developing Best-Practice Models for the Therapeutic Use of Extracellular Vesicles

“…EVs are released by antigen‐presenting cells to modulate immune responses . They are also released by some microbes during infection, delivering virulence factors and eliciting immune responses . Since EVs are not replication‐competent, they are attractive vaccine candidates.…”