Development of zebrafish infection and screening model for Clostridium difficile

Abstract

Clostridium difficile (C. difficile) infection (CDI) is an antibiotic-associated intestinal disease and is considered to be the main cause of healthcare-associated infections. The typical symptom of CDI manifests as mild to moderate diarrhea and pseudomembranous colitis. The hallmarks of the disease include neutrophil infiltration, and lesions in the colonic epithelium. C. difficile produces two major virulence factors, named TcdA and TcdB. Both toxins translocate the N-terminal glucosyltransferase domain (GTD) into the cytosol of target cells and inactivate Rho GTPases by glucosylation. This inactivation causes cell rounding, a redistribution of the actin cytoskeleton, and further an apoptotic cytotoxic effect. Although metronidazole and vancomycin remain the main drugs to treat CDI in the last three decades, new treatments or non-antibiotics agents are needed due to the emergence of hypervirulent strains. In the first part of this study, a stable transgenic zebrafish that expresses the GTD of TcdA/B under the control of a hatching gland specific enhancer was established. This model would be used for screening and identifying pharmacological substances against Tcd toxicity. Meanwhile, this zebrafish model was co-expressed a fluorescence-based apoptosis biosensor with the GTD of TcdA/B. Thus, the physiological condition of hatching gland cells was monitored by fluorescent signal. The innate immune system plays a crucial role in CDI onset and progression. In the second part of this study, the response of macrophages and neutrophils against C. difficile was analyzed. Using the Gal4/UAS system, a stable transgenic zebrafish line with expression of yellow Citrine fluorescent protein in neutrophils was generated. To combine this line for monitoring macrophages simultaneously, the binary transcriptional LexA/lexAop system was applied to express the fluorescent red tagRFP-T protein in macrophages. Both macrophages and neutrophils were able to phagocytose C. difficile. Furthermore, to unravel the function of macrophages in C. difficile elimination, Tamoxifen-inducible Caspase was co-expressed in the macrophage transgenic strain to trigger apoptosis (ATTACTM). The ablation and regeneration kinetics of macrophages were characterized by counting the number of RFP-positive cells. This model represents a tool to analyze the progression of CDI in the presence or absence of macrophages.