|Current Research Interests|
A marked increase in the number of patients with AIDS and other immuno-compromise in recent years has resulted in the emergence of fungi as predominant human pathogens all over the world. Candida spp. are the leading cause of disseminated fungal infections in neonates (premature infants), immune-compromised patients, diabetic and post-operative patients. Although Candida albicans is still the major culprit responsible for about 60% of these infections, systemic infections due to the non-albicans species of Candida such as C. glabrata, C. krusei, C. tropicalis etc. have increased significantly in the last two decades. In fact C. glabrata is now the second most common yeast pathogen found in BSIs (Blood Stream Infections) after C. albicans accounting for about 12-15% of total Candida BSIs.
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 secretion of proteolytic activity, C. glabrata can establish itself as a successful pathogen under appropriate 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 lab is focused on some of these responses using a combination of genetics, molecular biology, transcriptional profiling, biochemical and cell culture screens as outlined below.
Project 1: Functional genomic analysis of Candida glabrata-macrophage interactions
Neutrophils and macrophages constitute the first line of defense against Candida spp. However, little is known about the unique strategies that C. glabrata has developed to multiply and avoid recognition by host phagocytic cells since it lacks the key virulence traits of C. albicans such as hyphal formation, secretion of proteolytic activity. To better understand the host-pathogen interaction in C. glabrata, we have established an in vitro system comprised of a human monocytic cell line THP-1 and showed that wild-type cells undergo replication while a C. glabrata protease mutant was rapidly killed by activated THP-1 cells. Using signature-tagged mutagenesis approach, we have screened a library of 18,350 Tn7 insertion mutants, representing 50% of C. glabrata genome, for altered survival profiles in THP-1 macrophages and identified a set of 56 genes, belonging to diverse functional classes, which contributes to its survival/replication in the intracellular milieu of macrophages. Molecular characterization of these mutants is currently underway.
Project 2: Investigating the molecular basis of intrinsic resistance to azole antifungals in Candida glabrata
A major clinical challenge in treating C. glabrata infections is the innate resistance of this yeast towards the anti-fungal drug, fluconazole. 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 anti-fungals, azole class of drugs represents the first line therapy for the treatment and prophylaxis of invasive Candidiasis with fluconaolze being the most widely employed drug owing to its efficacy, oral availability, well-tolerability and cost-effectiveness.
Although low inherent susceptibility / high intrinsic resistance of C. glabrata to the azole class of drugs is well-documented, genetic and molecular basis of this resistance remains poorly understood. In this regard, using a genetic mutant screen, we have demonstrated a pivotal role for Rho1 GTPase-regulated protein kinase C (Pkc) signaling in the survival of azole stress and in the transcriptional activation of multidrug efflux pumps upon drug exposure. We have also identified two components of RNA polymerase II mediator complex and two proteins implicated in actin cytoskeleton biogenesis and ergosterol biosynthesis that are required to sustain viability during fluconazole stress.
Project 3: Role of aspartyl proteases in the patho-biology of C. glabrata
Known virulence factors in C. glabrata include adherence, phenotypic switching and a family of eleven glycosylphosphatidylinositol (GPI)-linked aspartyl proteases called yapsins (encoded by CgYPS1-11 genes). We have previously shown that C. glabrata YPS genes have important roles in activation of, and survival within, macrophages, cell wall maintenance, adherence to mammalian cells and virulence. A part of our research is aimed at delineating the functions of eleven yapsins and to elucidate the role/s, they play, in the physiology and the pathogenesis of C. glabrata.
Towards this end, we have uncovered a novel role for CgYps1 in the regulation of pH homeostasis under acidic environmental conditions wherein an ability to survive the diverse pH environments, via a good pH adaptation response, is a prerequisite to establish successful infections in mammalian host. We showed that CgYps1 is the sole yapsin that is required to survive low external pH environment and weak acid stress. Identification of physiological substrates of CgYps1 through proteomic approaches is currently ongoing.
Project 4: Iron homeostasis mechanisms in C. 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 either in the insoluble ferric form (oxides and hydroxides) or tightly bound to the high affinity carrier proteins (transferrin) in the serum of mammalian hosts. Thus iron acquisition inside host is a constant struggle for the pathogenic organisms and pathogens have evolved 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. In the current study, we are trying to elucidate the mechanisms that C. glabrata has developed to acquire, transport, utilize and store iron in response to various environmental cues via a combination of mutant screens, promoter-fusion and biochemical assays.