Pol32, an accessory subunit of DNA polymerase delta, plays an essential role in genome stability and pathogenesis of Candida albicans

Abstract

Candida albicans is a pathobiont that inflicts serious bloodstream fungal infections in individuals with compromised immunity and gut dysbiosis. Genomic diversity in the form of copy number alteration, ploidy variation, and loss of heterozygosity as an adaptive mechanism to adverse environments is frequently observed in C. albicans . Such genomic variations also confer a varied degree of fungal virulence and drug resistance, yet the factors propelling these are not completely understood. DNA polymerase delta (Polδ) is an essential replicative DNA polymerase in the eukaryotic cell and is yet to be characterized in C. albicans . Therefore, this study was designed to gain insights into the role of Polδ, especially its non-essential subunit Pol32, in the genome plasticity and life cycle of C. albicans . PCNA, the DNA clamp, recruits Polδ to the replication fork for processive DNA replication. Unlike in Saccharomyces cerevisiae , the PCNA interaction protein (PIP) motif of CaPol32 is critical for Polδ’s activity during DNA replication. Our comparative genetic analyses and whole-genome sequencing of POL32 proficient and deficient C. albicans cells revealed a critical role of Pol32 in DNA replication, cell cycle progression, and genome stability as SNPs, indels, and repeat variations were largely accumulated in pol32 null strain. The loss of pol32 in C. albicans conferred cell wall deformity; Hsp90 mediated azoles resistance, biofilm development, and a complete attenuation of virulence in an animal model of systemic candidiasis. Thus, although Pol32 is dispensable for cell survival, its function is essential for C. albicans pathogenesis; and we discuss its translational implications in antifungal drugs and whole-cell vaccine development.

RAD51–WSS1-dependent genetic pathways are essential for DNA–protein crosslink repair and pathogenesis in Candida albicans

“…The PFGE of chromosomal DNA of various C. albicans strains was performed using a modified protocol ( 60 ). About 6 × 10 8 C. albicans cells were processed to obtain spheroplast.…”

A chemically-induced attenuated strain ofCandida albicansgenerates robust protective immune responses and prevents systemic candidiasis development

“…The systemic challenge of C. albicans cells induces multi-organ failure in mice similar to in humans that eventually kills the host 36,37 . Since we observed a differential virulence of fungal cells when they were co-cultured with immune cells, we determined the disease-causing ability of these fungal cells in vivo.…”

“…The ultrathin section of C. albicans and EDTA-treated C. albicans cells were examined and visualized by TEM. The C. albicans cells were subjected to aldehydes for pre-fixation and osmium tetraoxide for post-fixation followed by contrast staining using uranyl acetate as described before 36 . The hardened sample blocks were sectioned using Leica EM UC7 microtome and the samples on the copper grids were visualized under the JEM-2100Plus JEOL TEM imaging machine.…”

Hsp90-Mediated Multi-Drug Resistance in DNA Polymerase-Defective Strains of Candida albicans

“…Polα-primase provides the RNA–DNA primer required to initiate DNA replication from the origins. In S. cerevisiae and C. albicans , the Polδ holoenzyme consists of Pol3, Pol31, and Pol32 subunits [ 27 ]. Pol32 subunit is dispensable in both the yeasts; however, in its absence, the processivity and fidelity of Polδ become compromised and C. albicans cells exhibit a slow growth phenotype; sensitivity to DNA-damaging agents; an increased rate of heterozygosity loss; the accumulation of genome instability, mostly in the intergenic and repetitive sequence regions of the genome; and resistance to only azole drugs [ 27 ].…”

“…In S. cerevisiae and C. albicans , the Polδ holoenzyme consists of Pol3, Pol31, and Pol32 subunits [ 27 ]. Pol32 subunit is dispensable in both the yeasts; however, in its absence, the processivity and fidelity of Polδ become compromised and C. albicans cells exhibit a slow growth phenotype; sensitivity to DNA-damaging agents; an increased rate of heterozygosity loss; the accumulation of genome instability, mostly in the intergenic and repetitive sequence regions of the genome; and resistance to only azole drugs [ 27 ]. Surprisingly, the azole drug resistance phenotype was not due to acquired mutations that accumulated in pol32 ΔΔ, but rather due to Hsp90’s function and cell wall/membrane deformity [ 27 ].…”