Amino acid addition to Vibrio cholerae LPS establishes a link between surface remodeling in Gram-positive and Gram-negative bacteria

Abstract

Historically, the O1 El Tor and classical biotypes of Vibrio cholerae have been differentiated by their resistance to the antimicrobial peptide polymyxin B. However, the molecular mechanisms associated with this phenotypic distinction have remained a mystery for 50 y. Both Gram-negative and Gram-positive bacteria modify their cell wall components with amine-containing substituents to reduce the net negative charge of the bacterial surface, thereby promoting cationic antimicrobial peptide resistance. In the present study, we demonstrate that V. cholerae modify the lipid A anchor of LPS with glycine and diglycine residues. This previously uncharacterized lipid A modification confers polymyxin resistance in V. cholerae El Tor, requiring three V. cholerae proteins: Vc1577 (AlmG), Vc1578 (AlmF), and Vc1579 (AlmE). Interestingly, the protein machinery required for glycine addition is reminiscent of the Gram-positive system responsible for D-alanylation of teichoic acids. Such machinery was not thought to be used by Gram-negative organisms. V. cholerae O1 El Tor mutants lacking genes involved in transferring glycine to LPS showed a 100-fold increase in sensitivity to polymyxin B. This work reveals a unique lipid A modification and demonstrates a charge-based remodeling strategy shared between Gram-positive and Gram-negative organisms.outer membrane | cell envelope | endotoxin | ultraviolet photodissociation T he Gram-negative pathogen Vibrio cholerae is the causative agent responsible for ∼300,000 reported cases annually of the severe diarrheal disease cholera. Since 1961, resistance to polymyxin B, a cationic antimicrobial peptide (CAMP), has been used to clinically differentiate between the two V. cholerae O1 biotypes, El Tor and classical (1). Interestingly the O1 classical biotype, which is polymyxin-sensitive, caused the first six cholera pandemics; however, polymyxin-resistant O1 El Tor strains are responsible for the current, seventh pandemic. Despite its importance, the molecular mechanisms accounting for this phenotypic difference have remained unknown for nearly 50 y.During infection, the cells of the innate immune system secrete CAMPs, which are small, positively charged proteins. Much like polymyxin, these peptides bind to and disrupt the bacterial cell membrane, ultimately resulting in death of the invading bacterial cell. Although Gram-negative and Gram-positive bacteria possess different cell wall structures, both have evolved mechanisms to remodel their cell envelope in response to CAMPs. These mechanisms often involve a common theme: neutralizing the negative charge of major cell wall components.The major surface component of Gram-negative bacteria is LPS, which is composed of three distinct regions: lipid A, core oligosaccharide, and O-antigen polysaccharide (2). Lipid A is the bioactive portion of LPS, which activates the human innate immune system through the Toll-like receptor 4 (TLR4)/myeloid differentiation factor 2 complex (3). The negatively charged lipid A domain is synthesized through a well-...

Deciphering the tRNA-dependent lipid aminoacylation systems in bacteria: Novel components and structural advances

“…In Gram-negative bacteria, lipid A in the outer membrane is the substrate for various positively charged modifications. Biosynthesis pathways for these modifications are not shown (For details see [116-118]). In certain species of both Gram-positive and Gram-negative bacteria, aminoacyl-phosphatidylglycerol synthases (aaPGSs) transfer aa from aa-tRNA (pre-formed by cytosolic aa-tRNA synthetases, aaRS) to phosphatidylglycerol (PG) in the membrane.…”

Bilayer Properties of Lipid A from Various Gram-Negative Bacteria

“…S1), which is denoted as VC-A. Hankins et al (35) reported the chemical modifications of a glycine or diglycine residue to the C3 0 position secondary acyl chain, which are denoted as VC-B (glycine) and VC-C (diglycine), respectively.…”

“…Vibrio cholerae is the causative agent of cholera, which is usually transmitted by unclean water and often causes severe diarrhea in humans and animals (35). V. cholerae lipid A consists of the standard headgroup and six acyl chains.…”

Polymyxin B Resistance and Biofilm Formation in Vibrio cholerae Are Controlled by the Response Regulator CarR

“…Both BPI and polymyxin B are thought to interact with the lipid A moiety of lipopolysaccharide (LPS). In fact, lipid acylation catalyzed by MsbA/LpxN (VC0212), the genes for which encode a lipid A secondary hydroxyacyltransferase, and glycine and diglycine modification catalyzed by AlmG (VC1577), AlmF (VC1578), and AlmE (VC1579) were found to be critical for V. cholerae polymyxin B resistance (5,6). It is proposed that a decrease in cell surface negative charge and membrane fluidity resulting from the glycine modification could impact antimicrobial peptide resistance.…”