Research Overview

The primary goal of my laboratory research is to investigate the host defense mechanism known as gut clearance—its regulation under normal physiological conditions, and the strategies by which the gut fungus Candida albicans (C. albicans) evades this clearance to invade the intestinal epithelium. To dissect these cellular and molecular processes, we employ a combination of biochemical, fluorescence-based, and electrophysiological techniques, using human colonic cell lines such as T84 and Caco-2 as experimental models.

In a parallel line of inquiry, we examine how simulated microgravity influences drug resistance in C. albicans when cultured on various solid media.

Gut clearance and its subversion by Candida albicans (NIH sponsored research)

The human intestine is home of one of the largest and most diverse microbiomes. Among its many microorganisms, some are opportunistic pathogens, including the fungus Candida albicans (C. albicans). Under certain environmental conditions within the gut, C. albicans can transition from its yeast form to a filamentous morphology, becoming virulent and capable of invading host tissues, which may lead to life-threatening infections.

In the colon, fluid secretion—often referred to as gut clearance—serves as a key defense mechanism, protecting epithelial cells by flushing away toxins and pathogens. The basolateral Na-K-2Cl cotransporter 1 (NKCC1) plays a critical role in regulating this fluid secretion. Previous studies have demonstrated that activation of protein kinase C (PKC) induces internalization of NKCC1, thereby reducing fluid secretion. However, the physiological regulation of NKCC1 by PKCs remains incompletely understood.

Interestingly, several pathogens can suppress fluid secretion during intestinal infection through mechanisms that are still poorly characterized. While some pathogens are known to activate host PKCs, it is not yet clear whether C. albicans reduces gut clearance via a PKC-dependent pathway.

Project #1: Effect of estrogen on C. albicans morphology

In Project #1, students are testing the effect of estrogen on C. albicans filamentation using bright/fluorescent microscopy and qPCR grown in different liquid media and in presence of a co-culture of intestinal epithelial cells. The picture on the left shows the different C. albicans morphologies: yeast (round), budding (“snowman shape”) and filamentous.

Project #2: Regulation of NKCC1 membrane expression by PKC

In Project #2, students study the posttranslational signal (e.g., ubiquitin) responsible for NKCC1 endocytosis during PKC action and the fate of the vesicles in the endocytic pathway (degradation versus recycling) Students are using biochemical and fluorescence microscopy to study PKC-mediated endocytosis of NKCC1. The picture on the left shows NKCC1 in endocytic vesicles (red arrows) after PKC activation in Madin Darby Canine Kidney Cells expressing eGFP-NKCC1.

Project #3: Subversion of fluid secretion by C. albicans

In Project #3, students are defining the cellular and molecular mechanism of C. albicans subversion (i.e., inhibition) of the Cl–-driven fluid secretion. We hypothesize it is mediated by causing NKCC1 internalization. Students use fluorescence microscopy, biochemical and electrophysiology techniques to tease apart the cellular and molecular events leading to NKCC1 internalization, thus inhibition of fluid secretion.. The picture on the left shows NKCC1 endocytic vesicles (green arrows) in the colonic T84 epithelial cells after 4 hours exposure to C. albicans.

Effect of simulated microgravity on C. albicans drug resistance

(Indiana Space Grant Consortium sponsored project)

Microorganisms exposed to environmental stressors often activate resistance genes. For instance, Candida albicans cultured aboard the International Space Station (ISS) under microgravity conditions and subsequently returned to Earth has been shown to develop resistance to the antifungal drug amphotericin B. Likewise, other microorganisms isolated from the ISS exhibited upregulation of virulence-associated genes.

Testing the resistance of C. albicans to antifugal drugs

Building on this line of research, students investigate the resistance of three C. albicans strains to fluconazole and caspofungin (common antifungal drugs) when grown on different solid media following exposure to either normal gravity or simulated microgravity using a clinostat. Drug resistance is assessed using the Kirby-Bauer disk diffusion assay.

In the accompanying image, the left side illustrates a resistance test for caspofungin in C. albicans after five days of microgravity exposure. The two quadrants on the left (top and bottom) contain control discs (solvent only), while the two quadrants on the right contain caspofungin discs, where clear zones of inhibition are visible.

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