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.

Fig. 1.

Gram-negative and Gram-positive bacteria modify their cell wall components with amine-containing substituents (shown in red). Some Gram-negative bacteria (e.g., Salmonella enterica) modify their lipid A phosphate groups with phosphoethanolamine and aminoarabinose. Additionally, glycerophospholipids modified with amino acids, such as lysine, represent major membrane lipids in Gram-positive bacteria (e.g., S. aureus). Gram-positives can further modify lipoteichoic acids and wall teichoic acids with d-alanine. The addition of amine-containing substituents to these cell wall components helps decrease the overall negative charge of the bacterial cell wall and protects the bacterium against attack by CAMPs.

Fig. 2.

MALDI-TOF MS of V. cholerae O1 El Tor and O1 classical lipid A species. Previously, V. cholerae O1 El Tor and O1 classical strains were shown to synthesize a similar hexa-acylated lipid A species (12) (A). This common lipid A species is shown at m/z 1,755.93 and m/z 1,756.1 for the El Tor (B) and classical (C) biotypes, respectively. The El Tor biotype produces additional lipid A species with peaks at m/z 1,813.9 and m/z 1,870.9.

Fig. 3.

V. cholerae O1 El Tor synthesize glycine-modified lipid A species. UVPD MS was used to analyze the V. cholerae ions ([M – H]⁻, m/z 1,756; [M – H]⁻, m/z 1,813; and [M – H]⁻, m/z 1,870 [A–C]). Cleavages are indicated by dashed lines and the m/z values of the resulting fragment ions are shown in Fig. S1. The m/z values and cleavage sites highlighted in red font represent the unique fingerprints associated with the glycine or diglycine modifications. The precursor ion is denoted by an asterisk. The fragmentation patterns support that V. cholerae synthesizes a glycine- and diglycine-modified lipid A structure at the 3′-position of the glucosamine disaccharide. “x55,” “x65,” and “x20” denote a section of the spectrum that has been magnified 55, 65, or 20 times, respectively, to more easily visualize product ions.

Fig. 4.

vc1577 is involved in glycine modification of V. cholerae lipid A and is cotranscribed with vc1578 and vc1579. (A) Lipid A was isolated from O1 El Tor vc1577::kan and analyzed by MALDI-TOF MS. A predominant peak at m/z 1,757.1 was consistent with the loss of a glycine. MALDI-TOF analysis of lipid A isolated from vc1577::kan, vc1577 resulted in major peaks at m/z 1,812.9 and 1,869.9, which is consistent with the addition of glycine and diglycine residues, respectively. Unmodified hexa-acylated lipid A is also present at m/z 1,755.9. (B) A schematic of the genetic organization of vc1577 (almG), vc1578 (almF), and vc1579 (almE) is shown. RT-PCR was done to determine if vc1577, vc1578, and vc1579 are cotranscribed and indicated a read-through transcript (product 1) containing vc1579-77. Additional RT-PCRs confirmed read-through transcripts for vc1579-78 (product 2) and vc1578-77 (product 3). V. cholerae genomic DNA template was used as a positive control for primers and amplified product sizes; however, for the negative control (−RT), cDNA without reverse transcriptase added was used as template, verifying that no DNA contamination had occurred.

Fig. 5.

AlmG (Vc1577), AlmF (Vc1578), and AlmE (Vc1579) confer polymyxin resistance to V. cholerae. Polymyxin MIC for various strains was determined on LB agar using ETest polymyxin B strips. (A) Wild-type V. cholerae O1 El Tor are polymyxin-resistant with a MIC of 96 μg/mL However, the vc1577 mutant, lacking the glycine modification of lipid A, is nearly 100-fold more sensitive to polymyxin B. When O1 El Tor vc1577::kan is complemented in trans, the strain gains polymyxin resistance. (B) Introduction of a plasmid expressing vc1579-77 confers polymyxin resistance to O1 classical strains. (C) MALDI-TOF MS of the lipid A of the classical biotype expressing pW77-79 revealed ions at m/z 1,813.5 and m/z 1,870.5, corresponding to lipid A modified by either one or two glycine residues, respectively. The minor peak at m/z 1,757.5 represents unmodified hexa-acylated V. cholerae lipid A.

Fig. 6.

Proposed model for the synthesis of glycine-modified lipid A species in V. cholerae. The model indicates glycine is ligated to the carrier protein, AlmF-SH (Vc1578), as a thioester. The reaction is catalyzed by the amino acid ligase, AlmE (Vc1579), in the cytoplasm. AlmG (Vc1577) then catalyzes the transfer of glycine from AllmF-S-glycine to the hexa-acylated V. cholerae lipid A species in the inner membrane. Presumably, diglycine would arise from a second addition to the lipid A molecule. Glycine-modified forms of lipid A are then transported to the bacterial surface providing resistance to polymyxin.