Comprehensive genetic analysis of adhesin proteins and their role in virulence ofCandida albicans

Abstract

Candida albicans is a microbial fungus that exists as a commensal member of the human microbiome and an opportunistic pathogen. Cell surface-associated adhesin proteins play a crucial role in C. albicans’ ability to undergo cellular morphogenesis, develop robust biofilms, colonize, and cause infection in a host. However, a comprehensive analysis of the role and relationships between these adhesins has not been explored. We previously established a CRISPR-based platform for efficient generation of single- and double-gene deletions in C. albicans, which was used to construct a library of 144 mutants, comprising 12 unique adhesin genes deleted singly, and every possible combination of double deletions. Here, we exploit this adhesin mutant library to explore the role of adhesin proteins in C. albicans virulence. We perform a comprehensive, high-throughput screen of this library, using Caenorhabditis elegans as a simplified model host system, which identified mutants critical for virulence and significant genetic interactions. We perform follow-up analysis to assess the ability of high- and low-virulence strains to undergo cellular morphogenesis and form biofilms in vitro, as well as to colonize the C. elegans host. We further perform genetic interaction analysis to identify novel significant negative genetic interactions between adhesin mutants, whereby combinatorial perturbation of these genes significantly impairs virulence, more than expected based on virulence of the single mutant constituent strains. Together, this study yields important new insight into the role of adhesins, singly and in combinations, in mediating diverse facets of virulence of this critical fungal pathogen.

Unconventional Animal Models in Infectious Disease Research

“…In contrast, our previously reported liquid-based P. aeruginosa assay, which is based on intoxication rather than infection, showed relatively few bacteria in the intestine, more consistent with transit of undigested bacteria, rather than intestinal colonization (Kirienko et al, 2013). However, in liquidbased C. elegans -Candida albicans model, host colonization was observed (Rosiana et al, 2021). To test whether colonization is related to virulence in C. elegans-E. faecium liquid assay, we selected six strains from Clade A1, including the three with the highest (TX2029, TX2051, and TX2416) and the lowest (S447, TX2022, and TX2025) virulence, and infected C. elegans with them in the liquid assay (Figure 6A).…”

“…In liquid, growth is limited, quorum sensing has no role, and production of the siderophore pyoverdine drives a lethal host intoxication (Kirienko et al, 2015;Kang et al, 2018;Kang and Kirienko, 2020). A recently published analysis of the role of Candida albicans adhesins in virulence using a liquid-based C. elegans model (Rosiana et al, 2021) reveals a third possibility. In this model, like the P. aeruginosa and E. faecium models, greater colonization is observed in an agar-based assay than a liquid assay, although colonization still occurs in liquid, like with E. faecium.…”

Development and Characterization of High-Throughput Caenorhabditis elegans – Enterococcus faecium Infection Model

“…In contrast, our previously reported liquid-based P. aeruginosa assay, which is based on intoxication rather than infection, showed relatively few bacteria in the intestine, more consistent with transit of undigested bacteria, rather than intestinal colonization ( Kirienko et al., 2013 ). However, in liquid-based C. elegans – Candida albicans model, host colonization was observed ( Rosiana et al., 2021 ). To test whether colonization is related to virulence in C. elegans- E .…”

“…A recently published analysis of the role of Candida albicans adhesins in virulence using a liquid-based C. elegans model ( Rosiana et al., 2021 ) reveals a third possibility. In this model, like the P. aeruginosa and E. faecium models, greater colonization is observed in an agar-based assay than a liquid assay, although colonization still occurs in liquid, like with E. faecium .…”

Adhesins in the virulence of opportunistic fungal pathogens of human

“…Als1ΔIff4Δ, Hyr1ΔAls5Δ, Als7ΔAls5Δ double mutants have been found to be low virulence strains and showed higher percentage worm survival in liquid killing assay upon infection with these mutant stains compared with wild type C. albicans stain. Upon genetic interaction analysis, the double mutant strains of adhesins Als5Δ Eap1Δ, or Als5ΔHwp2Δ conferred significant low virulence as compared to single mutant strains of these adhesin genes showing the synergistic effect of these mutations on virulence (Rosiana et al 2021 ). Virulence in Paracoccidioides involve increased expression of multiple adhesin gene at different phases of infection.…”