Current research projects
Introduction
Microbial pathogens utilize a variety of strategies to facilitate survival in the infected host. One of the most important mechanisms is the ability to respond to stress and adapt to an adverse host environment. Therefore, inhibiting stress response pathways constitutes a promising antimicrobial therapy.
We study a human fungal pathogen Cryptococcus neoformans to understand the mechanistic cellular processes used by pathogenic microorganisms to allow survival in the infected host. C. neoformans is a major opportunistic fungal pathogen worldwide and a leading cause of morbidity and mortality in AIDS patients.
In addition, the sibling species, Cryptococcus gattii is responsible for the recent outbreak of fungal-caused meningitis in the Pacific north-west of the U.S.
Our work with C. neoformans has led us to hypothesize that this pathogen has evolved unique pathways to control cell division in a manner that allows it to survive within a human host. Testing this hypothesis would provide insights into how eukaryotic pathogens adapt to the host environment and could potentially reveal new targets for therapeutic interventions. In addition, our research will lead to an improved understanding of the evolutionary events that have resulted in alternative mechanisms of mitosis.
Key events during mitotic cell cycle in C. neoformans.
A model showing clustering of centromeres, a gradual change in kinetochore architecture, and the nuclear envelope dynamics during the progression of the cell cycle in C. neoformans. The budding index at each stage was calculated by measuring the relative bud sizes of at least 100 cells.
(Kozubowski L, Yadav V. et al. MBio. 2013 Oct 1;4(5):e00614-13)
Mechanisms of fluconazole-induced aneuploidy in Cryptococcus neoformans (currently supported by the NIH grant R15AI119801)
Recent reports indicate a high importance of genome plasticity in the pathogenicity of C. neoformans. For example, changes in chromosomal copy number are a major factor contributing to the resistance to the azole drug fluconazole in vitro and in vivo. The list of key resistance genes whose copy number increases in fluconazole-resistant C. neoformans isolates is well established. However, very little is known about the molecular mechanisms that govern changes in chromosomal copy number in this organism. The main objective of our studies is to elucidate mechanisms responsible for generation of aneuploidy when C. neoformans is exposed to fluconazole. We have recently shown that pleiotropic effects of FLC on growth and mitotic division lead to an increase in DNA content, resulting in cells less sensitive to the drug. Cells with increased DNA content continue to proliferate and therefore increase the chance of forming resistant populations. We are currently elucidating how the non-genetic heterogeneity in C. neoformans population influences survival in the presence of fluconazole. We also study a connection between fluconazole and the metal-mediated DNA damage as a possible mechanism that contributes to chromosomal instability.
The biology of septin proteins and their role in high temperature growth in C. neoformans (currently supported by the NIH grant 1P20GM109094-01A1)
Septins are filament forming GTP-ases that contribute to cytokinesis, exocytosis, cell surface organization, and vesicle fusion by mechanisms that are poorly characterized. While in all animal and fungal cells septins participate in cytokinesis, in some species septins are not essential for this process. Elucidating how septins contribute to cytokinesis is the key to our understanding of how organisms evolved to adapt diverse mechanisms of cell division.
Septin complex in Cryptococcus neoformans is not required for cytokinesis to occur at 24°C, which is in contrast to the well established essential role of septins in Saccharomyces cerevisiae. However, septins become essential at 37°C. The findings that septins are only conditionally essential in C. neoformans suggest that they may primarily contribute to a fidelity mechanism that is needed to assure that cytokinesis works under stressful conditions. Our main objective is to identify and characterize components of cytokinesis in C. neoformans with an emphasis on the role of septins. Our current work focuses on the mechanisms through which an anillin homologue in C. neoformans contributes to the organization and dynamics of the septin complex. We also investigate several other genes that have been found in a genetic screen as essential for growth at 37°C.
Septins form a complex that assembles at the junction between the mother and the daughter cell (mother-bud neck). Here septin Cdc10 was tagged with a fluorescent protein mCherry and observed by the fluorescence microscopy.