Arnab Pradhan

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.

Fungal pathogens infect billions and kill millions of people each year. Many of these pathogens have evolved strategies to evade our antifungal immune defenses. Candida albicans, for example, masks the proinflammatory pathogen-associated molecular pattern (PAMP) β-1,3-glucan, in response to specific host signals such as lactate. In C. albicans , most β-1,3-glucan lies in the inner cell wall shielded, by the outer mannan layer, from recognition by certain immune cells such as macrophages. β-1,3-glucan that becomes exposed at the cell surface can be shaved off by secreted enzymes. By integrating mathematical modeling with experimentation, we show that the dynamics of this shaving, together with the dynamics of β-1,3-glucan exposure during growth, can account for a range of β-1,3-glucan masking phenotypes. The mathematical model accurately simulates the dynamics of β-1,3-glucan exposure during growth and predicts levels of β-1,3-glucan shaving under a variety of conditions, revealing how subtle differences in growth contribute to observed variabilities in lactate-induced β-1,3-glucan masking. For example, clinical isolates previously thought to display minimal lactate-induced masking are shown to mask robustly. Using a range of C. albicans mutants, we confirm the importance of Gpr1/Gpa2-protein kinase A signaling for lactate-induced β-1,3-glucan shaving and define the contributions of the Xog1 and Eng1 glucanases to this shaving. Furthermore, examination of a shielding-defective C. albicans mnn2 x6 mutant confirms that both β-1,3-glucan shaving and shielding contribute to the dynamism of β-1,3-glucan masking at the fungal cell surface. Dynamism in PAMP masking is likely to be relevant to other fungal pathogens of humans.

Significance: Macrophages protect against microbial infection, in part by engulfing and killing invading microbes. Fungal pathogens such as Candida albicans are known to evade phagocytic killing by forming hyphae that are physically challenging to engulf because of their length. We now find that macrophages can respond by folding the hyphae of C. albicans (and other fungal species). Hyphal folding implies that immune cells can continue to apply mechanical force after their cargo has been internalized. The involvement of Dectin-1, β2-integrin, and actin–myosin polymerization provides initial mechanistic insight. Folding damages hyphae, inhibits their growth, and facilitates their complete engulfment. Therefore, hyphal folding represents an additional weapon in the immune cell armory that presumably contributes to fungal clearance.