Immunogenicity and efficacy of CNA25 as a potential whole-cell vaccine against systemic candidiasis.

Disseminated fungal infections account for ~1.5 million deaths per year worldwide, and mortality may increase further due to a rise in the number of immunocompromised individuals and drug-resistance fungal species. Since an approved antifungal vaccine is yet to be available, this study explored the immunogenicity and vaccine efficacy of a DNA polymerase mutant strain of Candida albicans. CNA25 is a pol32ΔΔ strain that exhibits growth defects and does not cause systemic candidiasis in mice. Immunized mice with live CNA25 were fully protected against C. albicans and C. parapsilosis but partially against C. tropicalis and C. glabrata infections. CNA25 induced steady expression of TLR2 and Dectin-1 receptors leading to a faster recognition and clearance by the immune system associated with the activation of protective immune responses mostly mediated by neutrophils, macrophages, NK cells, B cells, and CD4+ and CD8+ T cells. Molecular blockade of Dectin-1, IL-17, IFNγ, and TNFα abolished resistance to reinfection. Altogether, this study suggested that CNA25 collectively activates innate, adaptive, and trained immunity to be a promising live whole-cell vaccine against systemic candidiasis.

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.

Cell Cycle Analysis of Candida albicans by Flow Cytometry.

The cell cycle is a vital process of cell division that is required to sustain life. Since faithful cell division is critical for the proper growth and development of an organism, the study of the cell cycle becomes a fundamental research objective. Saccharomyces cerevisiae has been an excellent unicellular system for unraveling the secrets of cell division, and the process of synchronization in budding yeast has been standardized. Cell synchronization is a crucial step of cell cycle analysis, where cells in a culture at different stages of the cell cycle are arrested to the same phase and, upon release, they progress synchronously. The cellular synchronization of S. cerevisiae is easily achieved by a pheromone or other chemicals like hydroxyurea treatment; however, such methodologies seem to be ineffective in synchronizing cells of multimorphic fungi such as Candida albicans. C. albicans is a human pathogen that can grow in yeast, pseudohyphal, and hyphal forms; these forms differ in morphology as well as cell cycle progression. More importantly, upon subjecting to DNA replication inhibitors for synchronization, C. albicans develops hyphal structures and grows asynchronously. Therefore, here we describe a simple and easy method to synchronize C. albicans cells in the G1 phase and the subsequent analysis of cell cycle progression by using flow cytometry.

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.

The ubiquitin-binding domain of DNA polymerase η directly binds to DNA clamp PCNA and regulates translesion DNA synthesis.

DNA polymerase eta (Polη) is a unique translesion DNA synthesis (TLS) enzyme required for the error-free bypass of ultraviolet ray (UV)-induced cyclobutane pyrimidine dimers in DNA. Therefore, its deficiency confers cellular sensitivity to UV radiation and an increased rate of UV-induced mutagenesis. Polη possesses a ubiquitin-binding zinc finger (ubz) domain and a PCNA-interacting-protein (pip) motif in the carboxy-terminal region. The role of the Polη pip motif in PCNA interaction required for DNA polymerase recruitment to the stalled replication fork has been demonstrated in earlier studies; however, the function of the ubz domain remains divisive. As per the current notion, the ubz domain of Polη binds to the ubiquitin moiety of the ubiquitinated PCNA, but such interaction is found to be nonessential for Polη's function. In this study, through amino acid sequence alignments, we identify three classes of Polη among different species based on the presence or absence of pip motif or ubz domain and using comprehensive mutational analyses, we show that the ubz domain of Polη, which intrinsically lacks the pip motif directly binds to the interdomain connecting loop (IDCL) of PCNA and regulates Polη's TLS activity. We further propose two distinct modes of PCNA interaction mediated either by pip motif or ubz domain in various Polη homologs. When the pip motif or ubz domain of a given Polη binds to the IDCL of PCNA, such interaction becomes essential, whereas the binding of ubz domain to PCNA through ubiquitin is dispensable for Polη's function.

Structural analyses of PCNA from the fungal pathogen Candida albicans identify three regions with species-specific conformations.

An assembly of multiprotein complexes achieves chromosomal DNA replication at the replication fork. In eukaryotes, proliferating cell nuclear antigen (PCNA) plays a vital role in the assembly of multiprotein complexes at the replication fork and is essential for cell viability. PCNA from several organisms, including Saccharomyces cerevisiae, has been structurally characterised. However, the structural analyses of PCNA from fungal pathogens are limited. Recently, we have reported that PCNA from the opportunistic fungal pathogen Candida albicans complements the essential functions of ScPCNA in S. cerevisiae. Still, it only partially rescues the loss of ScPCNA when the yeast cells are under genotoxic stress. To understand this further, herein, we have determined the crystal structure of CaPCNA and compared that with the existing structures of other fungal and human PCNA. Our comparative structural and in-solution small-angle X-ray scattering (SAXS) analyses reveal that CaPCNA forms a stable homotrimer, both in crystal and in solution. It displays noticeable structural alterations in the oligomerisation interface, P-loop and hydrophobic pocket regions, suggesting its differential function in a heterologous system and avenues for developing specific therapeutics. DATABASES: The PDB and SASBDB accession codes for CaPCNA are 7BUP and SASDHQ9, respectively.

'PIPs' in DNA polymerase: PCNA interaction affairs.

Interaction of PCNA with DNA polymerase is vital to efficient and processive DNA synthesis. PCNA being a homotrimeric ring possesses three hydrophobic pockets mostly involved in an interaction with its binding partners. PCNA interacting proteins contain a short sequence of eight amino acids, popularly coined as PIP motif, which snuggly fits into the hydrophobic pocket of PCNA to stabilize the interaction. In the last two decades, several PIP motifs have been mapped or predicted in eukaryotic DNA polymerases. In this review, we summarize our understandings of DNA polymerase-PCNA interaction, the function of such interaction during DNA synthesis, and emphasize the lacunae that persist. Because of the presence of multiple ligands in the replisome complex and due to many interaction sites in DNA polymerases, we also propose two modes of DNA polymerase positioning on PCNA required for DNA synthesis to rationalize the tool-belt model of DNA replication.

Virulence and pathogenicity of a Candida albicans mutant with reduced filamentation.

Deletion of DNA polymerase eta (Rad30/Polη) in pathogenic yeast Candida albicans is known to reduce filamentation induced by serum, ultraviolet, and cisplatin. Because nonfilamentous C. albicans is widely accepted as avirulent form, here we explored the virulence and pathogenicity of a rad30Δ strain of C. albicans in cell-based and animal systems. Flow cytometry of cocultured fungal and differentiated macrophage cells revealed that comparatively higher percentage of macrophages was associated with the wild-type than rad30Δ cells. In contrast, higher number of Polη-deficient C. albicans adhered per macrophage membrane. Imaging flow cytometry showed that the wild-type C. albicans developed hyphae after phagocytosis that caused necrotic death of macrophages to evade their clearance. Conversely, phagosomes kill the fungal cells as estimated by increased metacaspase activity in wild-type C. albicans. Despite the morphological differences, both wild-type and rad30∆ C. albicans were virulent with a varying degree of pathogenicity in mice models. Notably, mice with Th1 immunity were comparatively less susceptible to systemic fungal infection than Th2 type. Thus, our study clearly suggests that the modes of interaction of morphologically different C. albicans strains with the host immune cells are diverged, and host genetic background and several other attributing factors of the fungus could additionally determine their virulence.

Multifaceted activities of DNA polymerase η: beyond translesion DNA synthesis.

DNA polymerases are evolved to extend the 3'-OH of a growing primer annealed to a template DNA substrate. Since replicative DNA polymerases have a limited role while replicating structurally distorted template, translesion DNA polymerases mostly from Y-family come to the rescue of stalled replication fork and maintain genome stability. DNA polymerase eta is one such specialized enzyme whose function is directly associated with casual development of certain skin cancers and chemo-resistance. More than 20 years of extensive studies are available to support TLS activities of Polη in bypassing various DNA lesions, in addition, limited but crucial growing evidence also exist to suggest Polη possessing TLS-independent cellular functions. In this review, we have mostly focused on non-TLS activities of Polη from different organisms including our recent findings from pathogenic yeast Candida albicans.