Role of the HefC Efflux Pump in Helicobacter pylori Cholesterol-Dependent Resistance to Ceragenins and Bile Salts

The human gastric pathogen Helicobacter pylori modifies host cholesterol via glycosylation and incorporates the glycosylated cholesterol into its membrane; however, the benefits of cholesterol to H. pylori are largely unknown. We speculated that cholesterol in the H. pylori membrane might alter the susceptibility of these organisms to membrane-disrupting antibacterial compounds. To test this hypothesis, H. pylori strains were cultured in Ham's F-12 chemically defined medium in the presence or absence of cholesterol. The two cultures were subjected to overnight incubations with serial 2-fold dilutions of 10 bile salts and four ceragenins, which are novel bile salt derivatives that mimic membrane-disrupting activity of antimicrobial peptides. H. pylori cultured with cholesterol was substantially more resistant to seven of the bile salts and three ceragenins than H. pylori cultured without cholesterol. In most cases, these cholesterol-dependent differences ranged from 2 to 7 orders of magnitude; this magnitude depended on concentration of the agent. Cholesterol is modified by glycosylation using Cgt, a cholesteryl glycosyltransferase. Surprisingly, a cgt knockout strain still maintained cholesterol-dependent resistance to bile salts and ceragenins, indicating that cholesterol modification was not involved in resistance. We then tested whether three putative, paralogous inner membrane efflux pumps, HefC, HefF, or HefI, played a role. While HefF and HefI appeared unimportant, HefC was shown to play a critical role in the resistance to bile salts and ceragenins by multiple methods in multiple strain backgrounds. Thus, both cholesterol and the putative bile salt efflux pump HefC play important roles in H. pylori resistance to bile salts and ceragenins.