A marked increase in the number of patients with AIDS and other immunodeficiency disorders in recent years has resulted in the emergence of opportunistic fungi as predominant human pathogens all over the world. Candida species are the leading cause of disseminated fungal infections in neonates (premature infants), immuno-compromised individuals, diabetic and post-operative patients. Although Candida albicans is still the major culprit responsible for about 60% of these infections, the incidence of systemic infections due to the non-albicans species of Candida such as C. glabrata, C. tropicalis etc. has increased significantly in the last two decades. C. glabrata is the second to fourth most common Candida bloodstream pathogen depending upon the geographical region, and accounts for up to 30% of Candida bloodstream infections.

C. glabrata is a haploid budding yeast and is closely related to the model yeast Saccharomyces cerevisiae. Despite its similarity to S. cerevisiae and the lack of common virulence traits such as hyphae formation and secreted proteases, C. glabrata can establish itself as a successful pathogen under specific host conditions. Survival in vivo is a complex multifactorial process requiring the co-ordination of several responses including the ability to survive the nutrient poor host environment, to evade the host immune response, and to develop resistance to anti-fungal drugs. Research in our laboratory is focused on studying multiple facets of C. glabrata-host interaction using a combined approach of genetics, molecular biology, whole genome sequencing, transcriptional profiling, proteomic and host-infection analyses, as outlined below.

Project 1: Characterization of glycosylphosphatidylinositol-linked aspartyl proteases in Candida glabrata: role in pathogenicity

A family of eleven glycosylphosphatidylinositol-linked, cell surface-associated aspartyl proteases is essential for pathogenesis of C. glabrata. These proteases, also referred as yapsins, are encoded by CgYPS1-11 genes. We have previously shown that CgYapsins regulate diverse aspects of C. glabrata pathobiology viz., cell wall remodeling, vacuole homeostasis, intracellular pH maintenance under low pH conditions similar to the acidic host niches (gastrointestinal and vaginal tracts), and survival in host immune cells. Additionally, we have shown that NLRP3 inflammasome- and Syk-dependent production and secretion of IL-1β is deleterious for intracellular survival of C. glabrata, and CgYapsins promote survival in human macrophages by suppressing IL-1β secretion. Our current research is focused on delineating cellular processes that are regulated by the proteolytic activity of CgYapsins, and examine their centrality to Candida virulence. Towards this end, we have identified the interactors of these proteases through IP-MS approach. Characterization of identified CgYapsin-interactors, along with their potential cleavage by CgYps1-11 enzymes, is currently underway. Additionally, we are interested in deciphering how CgYapsins modulate the host innate immune response using macrophage, epithelial and murine model systems.

Project 2: Identification of novel antifungal drug targets and delineation of drug resistance mechanisms in Candida glabrata

Treatment of C. glabrata infections is limited by its inherent low susceptibility towards the most commonly used azole antifungals. A pre-requisite for developing new combinatorial drugs is a deeper understanding of the molecular mechanisms of resistance towards currently available anti-fungal armamentarium. Of existing antifungals, the azole drugs are widely employed owing to their efficacy, oral availability, well-tolerability and cost-effectiveness. Using the forward genetics approach, we have addressed the azole susceptibility of C. glabrata, and demonstrated centrality of Rho1 GTPase-mediated signalling and RNA polymerase II Mediator complex subunits, to survival of azole stress as well as to transcriptional activation of multidrug efflux pumps which is the most common mechanism of antifungal drug resistance in clinical settings. Additionally, we reported a hitherto unknown physiological effect of the fluconazole antifungal on actin organization in C. glabrata. We showed that the CgFab1 kinase (1-phosphatidylinositol-3-phosphate 5-kinase) promotes actin structure remodelling probably through regulating the association of actin-binding protein CgCof1 with phosphatidylinositol 3,5-bisphosphate (PI3,5P2). We have also uncovered a pivotal role for the PI(3,5)P2 synthesis complex proteins in tolerance to echinocandin antifungals, and identified CgVac7 protein (positive regulator of PI(3,5)P2 synthesis) as a new target for antifungal therapy. Currently, we are working on elucidating the molecular basis underlying cross resistance of C. glabrata towards two mainstream antifungals, azoles and echinocandins.

Project 3: Mechanisms of iron acquisition and ion homeostasis in Candida glabrata

An important requirement for survival in vivo is the ability of a pathogen to acquire critical nutrients such as iron from the host tissues. However, free iron is present in very limited amount, with cellular iron largely present either in the insoluble ferric form (oxides and hydroxides) or tightly bound to the high-affinity carrier and storage proteins in the mammalian host. Iron acquisition, therefore, is a constant struggle for microbial pathogens, with pathogens evolving highly specific strategies to scavenge iron, in accordance with the iron availability. Not much is known as to how C. glabrata manages to extract iron from the iron-limiting environment of the host. We are interested in delineating the major iron acquisition and assimilation mechanisms in C. glabrata, with iron also being a key modulator of host-pathogen interaction. We have previously identified components of the high-affinity iron uptake system, and showed their centrality to virulence. Our recent work has implicated CgHog1 [a terminal MAPK of the HOG (high osmolarity glycerol) response pathway] and the sole phosphoinositide 3-kinase CgVps34, in iron homeostasis. Currently, we are working towards gaining mechanistic insights into CgHog1 and CgVps34-mediated regulation of iron acquisition pathways, and the consequent effect/s on intracellular survival, adherence to host and virulence of C. glabrata.

Project 4: Functional genomic analysis of Candida glabrata-macrophage interaction

Neutrophils and macrophages constitute the first line of defense against Candida species. However, little is known about the unique strategies that C. glabrata employs to multiply in, and, avoid recognition by host phagocytic cells since it lacks some of the key fungal virulence traits such as hyphae formation and secreted proteases. Using an in vitro system comprised of the human monocytic cell line THP-1, we had previously demonstrated that wild-type C. glabrata cells possess the ability to impede phagolysosome acidifi-cation, counteract /survive the reactive oxygen species generated, and proliferate in THP-1 macrophages. We had also shown a pivotal role of chromatin remodelling in metabolic adaptation of fungal cells to the nutrient-poor macrophage environment, and survival against oxidative stress-induced DNA damage. Presently, we are extending these findings by studying the role of histone dosage and chromatin architecture in the transcriptional and proteomic responses of C. glabrata to macrophage internalization. Additionally, we are delineating the macrophage response to C. glabrata infection using RNA-Seq and proteomic analyses.