Investigation of Host Factors Involved in Bacterial Infection

Abstract

Clostridium difficile infection (CDI) is the leading cause of antibiotic-associated intestinal disease. Antibiotic treatment increases patients’ susceptibility to CDI by disrupting the normal gut microbiota. The disease manifestations present as asymptomatic carrier to diarrhea and pseudomembranous colitis. The main virulence factors of C. difficile are toxins, TcdA and TcdB, and CDI is, thereby, often regarded as a toxin-mediated disease. By inactivating the action of the Rho family through glucosylation, the toxins can lead to actin cytoskeletal rearrangement, tight junction destruction, and further cell death. To systemically investigate host factors involved in C. difficile toxins’ cytotoxic actions, we conducted a RNAi screen using a homemade pro-siRNA library. Cell line-based assay systems for testing toxins’ effects on cell death and apoptosis had been explored in the Caco-2 cell line with purified recombinant TcdB protein. A set of candidate genes that are involved in TcdB-induced cell death were screened out. The functions of some candidates in the host were investigated. As one of top-hit candidates, we demonstrated that HMGB1 was released from the nucleus into the cytosol or even the extracellular environment after exposed to TcdB, consistent with previous studies. Glycyrrhizin as the inhibitor of HMGB1 could significantly reduce TcdB-mediated apoptosis, and thus maintain a higher level of cell viability in cultured cells. In the mice colon ligated loop experiment, glycyrrhizin pretreatment could largely ameliorate TcdB-induced epithelial damage. These data suggested HMGB1 could be a very good therapeutic target for C. difficile infection. We also investigated another prime candidate ‘plakoglobin’, an intracellular constituent of both the adheren junctions and desmosome on epithelial cell surface for maintaining cell adhesion. We demonstrated that plakoglobin was involved in the regulation of mitochondria-dependent apoptotic pathway induced by TcdB, as its silencing blocked TcdB-induced cytochrome c release from the mitochondrial into cytosol. The mechanism that plakoglobin facilitated cytochrome c release during apoptosis could be attributed to the binding of plakoglobin to Bcl-XL and thus leading to the release of Bax/Bak from the sequestration of Bcl-XL, allowing Bax/Bak to form homo-oligodimers on mitochondrial outer membrane (OMM) and subsequent mitochondrial outer membrane permeabilization (MOMP). To my knowledge, this is the first discovery of the relationship between plakoglobin and Bcl-XL against the background of TcdB-induced apoptosis. We also investigated the upstream protein of plakoglobin, E-cadherin on cell surface. We found E-cadherin silencing protected cells from TcdB-induced cell death and reduced the level of TcdB-induced Rac1 glucosylation. However, we did not detect the direct interaction between E-cadherin and TcdB, suggesting E-cadherin may contribute to the entry of TcdB into cells but not as a TcdB direct receptor and the specific function is worth to be further investigated. In another project, I studied the role of bacterial antisense RNAs (asRNAs) and double-stranded RNAs (dsRNAs) in the host immune response and bacterial infection process. It is well known that antisense RNAs are widespread in bacterial cells. By base-pairing with sense RNAs, they can form dsRNAs. In natural conditions, dsRNAs will be cleaved by rnc-encoded RNase III, a dsRNA specific endoribonuclease. The consequence of this process could provide a means for the removal of transcriptional noise and allow the fine-tuning of sense transcript levels by adjusting antisense transcript levels. The depletion of RNase III causes the accumulation of dsRNA, leads to the attenuation of virulence and affects a lot of genes expression, although the mechanism is not clearly understood. To figure out whether accumulated dsRNAs regulated by asRNAs and RNase III could induce host innate immune response, we used rnc mutant E. coli and Salmonella strains as the model system. We found RNase III deficiency affected bacterial growth, viability and virulence, implying asRNAs have a role in regulating bacterial sense gene expression. We also demonstrated bacterial dsRNAs were capable of inducing IFN-β-mediated innate immunity but not the inflammasome pathway. These data demonstrated that bacterial asRNAs and RNase III are involved in regulating genes expression and influencing many aspects of bacteria which could affect bacterial infection process.

Date of Award	11 Mar 2020
Original language	English
Awarding Institution	City University of Hong Kong
Supervisor	Linfeng HUANG (Supervisor) & Yung Fu CHANG (External Co-Supervisor)