GUT clearance regulation and its subversion by candida albicans - the CDC reports that fungal infections are responsible for ~9 million outpatient visits, ~75,000 hospitalizations and an estimated…

This federal Project Grant award of $318,554.00 from the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) under the Diabetes, Digestive, and Kidney Diseases Extramural Research program (CFDA 93.847) will support research at Valparaiso University to investigate the regulation of chloride-driven fluid secretion and its subversion by the fungal pathogen Candida albicans.

The key objectives of this 3-year research project are to: 1) determine the role of the hormone 17-β estradiol in regulating the yeast-to-hypha morphological shift in C. albicans, 2) elucidate the post-translational mechanisms leading to internalization of the NKCC1 chloride transporter and its fate in the endocytic pathway, and 3) demonstrate how C. albicans subverts the PKC-mediated NKCC1 internalization to inhibit chloride-based fluid secretion, a key host defense mechanism. This research aims to advance the understanding of the cellular events surrounding C. albicans gut infection and inform the development of improved therapies for invasive candidiasis.

GUT clearance regulation and its subversion by candida albicans - the CDC reports that fungal infections are responsible for ~9 million outpatient visits, ~75,000 hospitalizations and an estimated direct medical cost of ~$7 billion yearly. These numbers urge expansion of our knowledge on fungal infections to improve patient health. Studies strongly support that invasive candidiasis originates from the GUT, but the events encompassing invasive candidiasis (e.g., cues regulating yeast to hypha shift) are unclear. Chloride (CL-)-driven fluid secretion represents a potent host defense mechanism for clearing GUT pathogens. Despite its public health importance, the regulation of fluid secretion is not well studied. We have shown that protein kinase c (PKC) mediated endocytosis of the basolateral NA+-k+-2CL- cotransporter 1 (NKCC1) is probably the main pathway blunting CL- secretion. Our broad objectives are to define the mechanistic details of CL- secretion regulation in normal and in the pathological context of invasive candidiasis. To answer those questions, we have established the following goals: (i) define the role of E2 (at blood level found in critically ILL patients) on the regulation of yeast to hypha shift in c. Albicans, (II) elucidate the posttranslational signal causing internalization of NKCC1 and its fate in the endocytic pathway and (III) demonstrate that c. Albicans subverts the PKC-mediated NKCC1 endocytosis to inhibit CL- secretion. Our central hypothesis is that during sepsis and in ILL patients c. Albicans subverts CL – mediated fluid secretion by increasing NKCC1 internalization to gain an advantage before cell invasion. The rationale of our study is that without a firm understanding of those events IT will be difficult to combat and improve patient health during invasive candidiasis. We will test our hypothesis by addressing the following specific aims: 1) is 17-β estradiol (E2) regulating c. Albicans morphology shift? We will use bright field microscopy to monitor c. Albicans morphology during the exposure of E2 2) is NKCC1 ubiquitination, after PKC activation, responsible for its internalization and what is the fate of internalized NKCC1? We will (i) test for the ubiquitination of NKCC1 after PKC activation by immunoblot and (II) use fluorescence microscopy and endocytic markers to map NKCC1 in the endocytic pathway to check if IT is recycled or degraded. 3) is PKC-mediated NKCC1 internalization the cellular mechanism subverted by c. Albicans to decrease fluid secretion in colonic epithelial cells? We will (i) use biochemical and fluorescence techniques to monitor NKCC1 membrane expression during c. Albicans infection in presence or absence of PKC inhibitors and (II) use the short circuit current to measure CL- secretion, during c. Albicans infection in presence or absence of PKC inhibitors. Our work is innovative because a) IT proposes to establish for the first time that pathological concentration of E2 modulates c. Albicans virulence and b) provides new insight on mechanisms prior cell invasion, which have been long overlooked. Our application is significant because the outcomes will expand our knowledge on the cellular events surrounding c. Albicans infection in the GUT epithelium and help future research to design better drugs/therapies against invasive candidiasis in the GUT and other organs (e.g., vagina, skin).