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

The incidence of infections caused by Candida species, specifically by drug-resistant isolates, is a major health concern as they can disseminate to and colonize most vital organs, enhancing morbidity and mortality. Several molecular mechanisms have been reported to be involved in drug resistance. These are mostly drug- and isolate-specific. Here, we characterized three different genetically modified strains of C. albicans that were multi-drug-resistant (MDR) and deciphered a uniform mechanism responsible for resistance. DNA polymerase epsilon (Polε) is a leading strand-specific polymerase consisting of four subunits, namely, Pol2, Dpb2, Dpb3, and Dpb4. The deletion of one or both of the Dpb3 and Dpb4 subunits in C. albicans rendered multi-drug resistance. A detailed characterization of these strains revealed that acquired mutagenesis, drug efflux pumps, and other known mechanisms did not play a significant role because the complemented strain showed drug sensitivity. More importantly, the function of heat shock protein 90 (Hsp90) in these knockout strains is critical for reducing susceptibility to several antifungal drugs. Cell wall deformity and composition in these strains can add to such a phenotype. The inhibition of Hsp90 function by geldanamycin and tricostatin A sensitized the MDR strains to antifungals. Considering our earlier research and this report, we suggest that replication stress induces Hsp90 expression and activity in order to orchestrate a cellular stress response circuit and thus develop fungal drug resistance. Thus, Hsp90 is an important drug target for use in combinatorial therapy.

Spot Assay and Colony Forming Unit (CFU) Analyses-based sensitivity test for Candida albicans and Saccharomyces cerevisiae.

Cellular sensitivity is an approach to inhibit the growth of certain cells in response to any non-permissible conditions, as the presence of a cytotoxic agent or due to changes in growth parameters such as temperature, salt, or media components. Sensitivity tests are easy and informative assays to get insight into essential gene functions in various cellular processes. For example, cells having any functionally defective genes involved in DNA replication exhibit sensitivity to non-permissive temperatures and to chemical agents that block DNA replication fork movement. Here, we describe a sensitivity test for multiple strains of Saccharomyces cerevisiae and Candida albicans of diverged genetic backgrounds subjected to several genotoxic chemicals simultaneously. We demonstrate it by testing the sensitivity of DNA polymerase defective yeast mutants by using spot analysis combined with colony forming unit (CFU) efficiency estimation. The method is very simple and inexpensive, does not require any sophisticated equipment, can be completed in 2-3 days, and provides both qualitative and quantitative data. We also recommend the use of this reliable methodology for assaying the sensitivity of these and other fungal species to antifungal drugs and xenobiotic factors.

4-nitroquinoline 1-oxide induces immune cells death to onset early immunosuppression during oral squamous cell carcinoma development.

4-Nitroquinoline N-oxide (4-NQO) and its derivatives react with genomic DNA to form stable quinolone monoadducts, which are highly mutagenic and genotoxic. While the chronic high-dose exposure of epithelial cells to a carcinogen such as 4-NQO leads to tumor development, its effect on other cells has not been explored yet. Since the immunosuppression due to aberrant immunological profile is recognized as a significant cause in tumors, here we determine the interaction between 4-NQO and immune cells both in vivo and in vitro, and its effect on oral squamous cell carcinoma (OSCC) progression in a murine model. Immune cell profiling of the spleen and peripheral blood revealed a significant decrease in the B-cell population in 4-NQO-exposed mice than the untreated group. Additionally, γδ T and CD5+ B lymphocyte populations decreased at both pre- and post-cancerous stages of OSCC. These results suggested that 4-NQO induced tumor transition from pre-malignant lesions to OSCC by altering certain immune cells systemically. Next, to establish the effect of 4-NQO on immune cells, human B- and T-cell lines were subjected to 4-NQO; the reduction in cell viability, increase in DNA damage response marker, and induction of apoptosis were more pronounced in B than T cells. Altogether, our results indicated that in addition to the genotoxicity of oral epithelial cells, 4-NQO potentiates long-range effects on specific immune cells to induce cell death to cause very-early immunosuppressive response during oral carcinogenesis, and thus immunosuppression and tumor development are coevolved.

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

Genetic analyses in Saccharomyces cerevisiae suggest that nucleotide excision repair (NER), homologous recombination (HR), and protease-dependent repair pathways coordinately function to remove DNA-protein crosslinks (DPCs) from the genome. DPCs are genomic cytotoxic lesions generated because of the covalent linkage of proteins with DNA. Although NER and HR processes have been studied in pathogenic Candida albicans, their roles in DPC repair (DPCR) are yet to be explored. Proteases like Wss1 and Tdp1 (tyrosyl-DNA phosphodiesterase-1) are known to be involved in DPCR; however, Tdp1 that selectively removes topoisomerase-DNA complexes is intrinsically absent in C. albicans. Therefore, the mechanism of DPCR might have evolved differently in C. albicans. Herein, we investigated the interplay of three genetic pathways and found that RAD51-WSS1-dependent HR and protease-dependent repair pathways are essential for DPC removal, and their absence caused an increased rate of loss of heterozygosity in C. albicans. RAD1 but not RAD2 of NER is critical for DPCR. In addition, we observed truncation of chromosome #6 in the cells defective in both RAD51 and WSS1 genes. While the protease and DNA-binding activities are essential, a direct interaction of Wss1 with the eukaryotic DNA clamp proliferating cell nuclear antigen is not a requisite for the function of Wss1. DPCR-defective C. albicans cells exhibited filamentous morphology, reduced immune cell evasion, and attenuation in virulence. Thus, we concluded that RAD51-WSS1-dependent DPCR pathways are essential for genome stability and candidiasis development. Since no vaccine against candidiasis is available for human use yet, we propose to explore DPCR-defective attenuated strains (rad51ΔΔwss1ΔΔ and rad2ΔΔrad51ΔΔwss1ΔΔ) for whole-cell vaccine development.

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

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.

Vaccines against candidiasis: Status, challenges and emerging opportunity.

Candidiasis is a mycosis caused by opportunistic Candida species. The occurrence of fungal infections has considerably increased in the last few years primarily due to an increase in the number of immune-suppressed individuals. Alarming bloodstream infections due to Candida sp. are associated with a higher rate of morbidity and mortality, and are emerged as major healthcare concerns worldwide. Currently, chemotherapy is the sole available option for combating fungal diseases. Moreover, the emergence of resistance to these limited available anti-fungal drugs has further accentuated the concern and highlighted the need for early detection of fungal infections, identification of novel antifungal drug targets, and development of effective therapeutics and prophylactics. Thus, there is an increasing interest in developing safe and potent immune-based therapeutics to tackle fungal diseases. In this context, vaccine design and its development have a priority. Nonetheless, despite significant advances in immune and vaccine biology over time, a viable commercialized vaccine remains awaited against fungal infections. In this minireview, we enumerate various concerted efforts made till date towards the development of anti-Candida vaccines, an option with pan-fugal vaccine, vaccines in the clinical trial, challenges, and future opportunities.

Commensal Fungus Candida albicans Maintains a Long-Term Mutualistic Relationship with the Host To Modulate Gut Microbiota and Metabolism.

Candida albicans survives as a commensal fungus in the gastrointestinal tract, and that its excessive growth causes infections in immunosuppressed individuals is widely accepted. However, any mutualistic relationship that may exist between C. albicans and the host remains undetermined. Here, we showed that a long-term feeding of C. albicans does not cause any noticeable infections in the mouse model. Our 16S and 18S ribosomal DNA (rDNA) sequence analyses suggested that C. albicans colonizes in the gut and modulates microbiome dynamics, which in turn mitigates high-fat-diet-induced uncontrolled body weight gain and metabolic hormonal imbalances. Interestingly, adding C. albicans to a nonobesogenic diet stimulated the appetite-regulated hormones and helped the mice maintain a healthy body weight. In concert, our results suggest a mutualism between C. albicans and the host, contrary to the notion that C. albicans is always an adversary and indicating it can instead be a bona fide admirable companion of the host. Finally, we discuss its potential translational implication as a probiotic, especially in obese people or people dependent on high-fat calorie intakes to manage obesity associated complications. IMPORTANCE Candida albicans is mostly considered an opportunistic pathogen that causes fetal systemic infections. However, this study demonstrates that in its commensal state, it maintains a long-term mutualistic relationship with the host and regulates microbial dynamics in the gut and host physiology. Thus, we concluded that C. albicans is not always an adversary but rather can be a bona fide admirable companion of the host. More importantly, as several genomic knockout strains of C. albicans were shown to be avirulent, such candidate strains may be explored further as preferable probiotic isolates to control obesity.

Quaternary structural diversity in eukaryotic DNA polymerases: monomeric to multimeric form.

Sixteen eukaryotic DNA polymerases have been identified and studied so far. Based on the sequence similarity of the catalytic subunits of DNA polymerases, these have been classified into four A, B, X and Y families except PrimPol, which belongs to the AEP family. The quaternary structure of these polymerases also varies depending upon whether they are composed of one or more subunits. Therefore, in this review, we used a quaternary structure-based classification approach to group DNA polymerases as either monomeric or multimeric and highlighted functional significance of their accessory subunits. Additionally, we have briefly summarized various DNA polymerase discoveries from a historical perspective, emphasized unique catalytic mechanism of each DNA polymerase and highlighted recent advances in understanding their cellular functions.