Roles of Cholesteryl-α-Glucoside Transferase and Cholesteryl Glucosides in Maintenance of Helicobacter pylori Morphology, Cell Wall Integrity, and Resistance to Antibiotics.

Infection of the human stomach caused by is very common, as the pathogen colonizes more than half of the world's population. It is associated with varied outcomes of infection, such as peptic ulcer disease, gastric ulcers, and mucosa-associated lymphoid tissue lymphoma, and is generally considered a risk factor for the development of gastric adenocarcinoma. Cholesteryl glucosides (CGs) constitute a vital component of the cell wall of and contribute to its pathogenicity and virulence. The gene, which encodes cholesteryl-α-glucoside transferase (CGT), appears critical for the enzymatic function of integrating unique CGs into the cell wall of , and deletion of this gene leads to depletion of CGs and their variants. Herein, we report that the deletion of and consequent deficiency of cholesterol alter the morphology, shape, and cell wall composition of cells, as demonstrated by high-resolution confocal microscopy and flow cytometry analyses of two different type strains of , their isogenic knockouts as well as a reconstituted strain. Moreover, measurement of ethidium bromide (EtBr) influx by flow cytometry showed that lack of CGs increased cell wall permeability. Antimicrobial susceptibility testing revealed that the isogenic knockout strains ( and ) were sensitive to antibiotics, such as fosfomycin, polymyxin B, colistin, tetracycline, and ciprofloxacin, in contrast to the wild-type strains that were resistant to the above antibiotics and tended to form denser biofilms. Lipid profile analysis of both and strains showed an aberrant profile of lipopolysaccharides (LPS) in the strain. Taken together, we herein provide a set of mechanistic evidences to demonstrate that CGs play critical roles in the maintenance of the typical spiral morphology of and its cell wall integrity, and any alteration in CG content affects the characteristic morphological features and renders the susceptible to various antibiotics. is an important cause of chronic gastritis leading to peptic ulcer and is a major risk factor for gastric malignancies. Failure in the eradication of infection and increasing antibiotic resistance are two major problems in preventing colonization. Hence, a deeper understanding of the bacterial survival strategies is needed to tackle the increasing burden of infection by an appropriate intervention. Our study demonstrated that the lack of cholesteryl glucosides (CGs) remarkably altered the morphology of and increased permeability of the bacterial cell wall. Further, this study highlighted the substantial role of CGs in maintaining the typical morphology that is essential for retaining its pathogenic potential. We also demonstrated that the loss of CGs in renders the bacterium susceptible to different antibiotics.