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).

Grant Number: 1R15DK139519-01
NIH Institute/Center: NIH
Principal Investigator: Patrice Bouyer