Polymyxin B Resistance in El Tor Vibrio cholerae Requires Lipid Acylation Catalyzed by MsbB

Antimicrobial peptides are critical for innate antibacterial defense. Both Gram-negative and Gram-positive microbes have mechanisms to alter their surfaces and resist killing by antimicrobial peptides. In Vibrio cholerae, two natural epidemic biotypes, classical and El Tor, exhibit distinct phenotypes with respect to sensitivity to the peptide antibiotic polymyxin B: classical strains are sensitive and El Tor strains are relatively resistant. We carried out mutant screens of both biotypes, aiming to identify classical V. cholerae mutants resistant to polymyxin B and El Tor V. cholerae mutants sensitive to polymyxin B. Insertions in a gene annotated msbB (encoding a predicted lipid A secondary acyltransferase) answered both screens, implicating its activity in antimicrobial peptide resistance of V. cholerae. Analysis of a defined mutation in the El Tor biotype demonstrated that msbB is required for resistance to all antimicrobial peptides tested. Mutation of msbB in a classical strain resulted in reduced resistance to several antimicrobial peptides but in no significant change in resistance to polymyxin B. msbB mutants of both biotypes showed decreased colonization of infant mice, with a more pronounced defect observed for the El Tor mutant. Mass spectrometry analysis showed that lipid A of the msbB mutant for both biotypes was underacylated compared to lipid A of the wild-type isolates, confirming that MsbB is a functional acyltransferase in V. cholerae.Pathogenic bacteria that colonize the digestive tract must overcome a variety of stresses imposed upon them by the host. Epithelial cells in the crypts of the intestinal lumen (Paneth cells and enterocytes) produce large amounts of antimicrobial peptides called defensins (16). Defensins, like most antimicrobial peptides, are thought to act by associating with the lipopolysaccharide (LPS) on the bacterial surface (through electrostatic interactions) and then permeabilizing the membranes, leading to cell death (37, 48). Gram-negative bacteria have developed a wide range of strategies to overcome the antimicrobial activity of these peptides, including production of proteases that degrade the peptides (41), production of secretory proteins that bind the peptides and prevent them from accessing their target (21), production of efflux systems that actively pump antimicrobial peptides back into the environment if they access the bacterial cytoplasm (36), and incorporation of positively charged groups into lipid A, which reduces the net anionic charge of the bacterial surface and decreases the affinity of the peptides for the membrane (10, 13, 14).LPS of Gram-negative bacteria is composed of three main parts: (i) the O-antigen polysaccharide (O-PS); (ii) the relatively conserved core polysaccharide (core-PS); and (iii) lipid A, the hydrophobic lipid component responsible for biological activities within the host (9, 25). The lipid A region of the LPS is anchored in the bacterial outer membrane, and the hydrophilic core-PS and O-PS project outward into the environment. LPS comprises 70% of the bacterial outer membrane and is the main surface-associated antigen recognized by the innate immune system. Toll-like receptors in the host recognize the lipid A portion of the LPS in association with MD2 and CD14 and stimulate inflammation to attract immune cells and clear bacterial infections (5, 27). The strong immune response to lipid A is the reason that LPS has historically been referred to as “endotoxin” (20). Some pathogens regulate the structure of their lipid A and its acylation patterns in order to adapt to the host environment, thereby contributing to greater fitness within the host (12, 31).Vibrio cholerae causes cholera, an epidemic diarrheal disease. Disease occurs when contaminated food or water is ingested, resulting in a voluminous secretory diarrhea that can lead to dehydration and death if left untreated. The V. cholerae species is not homogeneous, with distinctions made on the basis of serogroup, serotype, biotype, production of cholera toxin, and potential for epidemic spread. While more than 200 serogroups have been identified, only two of these, O1 and O139, are associated with epidemic cholera (33). V. cholerae O1 strains can be subdivided into two biotypes, classical and El Tor, which differ biochemically and clinically (3). The first six cholera pandemics were caused by the classical biotype, but the current (seventh) pandemic has been caused by the El Tor biotype (33). Classical strains typically cause a more severe disease, while El Tor strains cause less severe and sometimes even asymptomatic cases. However, El Tor strains appear to have increased fitness in the environment, which may be why they have largely replaced classical strains as the cause of disease in recent years (49).The subdivision into the classical or El Tor biotype is based on several laboratory tests (3). One of the commonly used tests is assessing sensitivity of the strain to the antimicrobial peptide polymyxin B. Classical strains are very sensitive to polymyxin B, while El Tor strains are relatively resistant. We hypothesize that differences in surface structures of the two biotypes are responsible for differential sensitivity. To test this and to determine the genetic basis of antimicrobial peptide resistance in V. cholerae, we carried out genetic screens to identify genes associated with resistance and sensitivity to polymyxin B in El Tor and classical V. cholerae, respectively. As a result of these screens, we chose to further characterize the role of msbB, a lipid IVA acyltransferase gene, with regard to antimicrobial peptide resistance and virulence in V. cholerae. We report that msbB contributes to resistance of El Tor strains to all antimicrobial peptides tested. Mutation of msbB in a classical strain led to significantly reduced innate resistance to several antimicrobial peptides, not including polymyxin B. While msbB mutants of both biotypes exhibit decreased colonization of infant mice, a more significant decrease was observed for the El Tor mutant. Mass spectrometry analysis confirmed that deletion of msbB from either biotype resulted in loss of an acyl chain, as expected. These results suggest that msbB from V. cholerae is required for wild-type antimicrobial peptide resistance and colonization. However, some biotype-specific phenotypes imply that the role of msbB may be different in each biotype.