AlmG, responsible for polymyxin resistance in pandemic Vibrio cholerae, is a glycyltransferase distantly related to lipid A late acyltransferases

Abstract

Cationic antimicrobial peptides (CAMPs), such as polymyxins, are used as a last-line defense in treatment of many bacterial infections. However, some bacteria have developed resistance mechanisms to survive these compounds. Current pandemic O1 Vibrio cholerae biotype El Tor is resistant to polymyxins, whereas a previous pandemic strain of the biotype Classical is polymyxin-sensitive. The almEFG operon found in El Tor V. cholerae confers >100-fold resistance to antimicrobial peptides through aminoacylation of lipopolysaccharide (LPS), expected to decrease the negatively charged surface of the V. cholerae outer membrane. This Gram-negative system bears striking resemblance to a related Gram-positive cell-wall remodeling strategy that also promotes CAMP resistance. Mutants defective in AlmEF-dependent LPS modification exhibit reduced fitness in vivo Here, we present investigation of AlmG, the hitherto uncharacterized member of the AlmEFG pathway. Evidence for AlmG glycyl to lipid substrate transferase activity is demonstrated in vivo by heterologous expression of V. cholerae pathway enzymes in a specially engineered Escherichia coli strain. Development of a minimal keto-deoxyoctulosonate (Kdo)-lipid A domain in E. coli was necessary to facilitate chemical structure analysis and to produce a mimetic Kdo-lipid A domain AlmG substrate to that synthesized by V. cholerae. Our biochemical studies support a uniquely nuanced pathway of Gram-negative CAMPs resistance and provide a more detailed description of an enzyme of the pharmacologically relevant lysophosphospholipid acyltransferase (LPLAT) superfamily.

Keywords: LABLAT; LPLAT; Vibrio cholera; acyltransferase; aminoacyltransferase; antibiotic resistance; antimicrobial peptide (AMP); bacterial membrane; bacterial pathogenesis; lipopolysaccharide.

Figure 1.

Multiple differences in the predominant chemical structures of Kdo-lipid A domains of E. coli K-12 compared with V. cholerae biotype El Tor. Inset provides numeric classification legend for describing acyl chain position along the glucosamine disaccharide. A, E. coli possess a bi-functional Kdo transferase that transfers individual Kdo sugars to lipid IV_A and Kdo-lipid IV_A successively. The lipid A secondary acyltransferase LpxL and then LpxM acyltransferases produce the predominantly observed hexaacyl-Kdo₂-lipid A. B, V. cholerae Kdo-lipid A domains contain hydroxylaurate chains at 3- and 3′-positions. V. cholerae expresses a monofunctional KdtA that transfers a single Kdo residue to lipid IV_A and a Kdo kinase that phosphorylates Kdo-lipid IV_A. V. cholerae LpxL (Vc0213) transfers a myristate (C14:0) to the 2′-position hydroxylacyl chain of the glucosamine disaccharide. LpxN (Vc0212) transfers a 3-hydroxylaurate (3-OH C12:0; blue) to the 3′-position hydroxyacyl chain to generate hexaacyl-monophosphoryl-Kdo lipid A. AlmEFG adds glycine to the 3-hydroxylaurate of hexaacyl-monophosphoryl-Kdo lipid A. Glycine and diglycine modified hexaacyl-monophosphoryl-Kdo-lipid A are highly abundant in V. cholerae biotype El Tor under standard growth conditions.

Figure 2.

Electrophoretic separation and ProQ Emerald dye visualization of isolated LPS. Numbered lanes below the LPS gel correspond to strains listed on the right. S. enterica serovar typhimurium LT2 is included as a control (lane 1), to show a typical O-antigen repeat pattern observed in Gram-negative LPS structures. Laboratory E. coli K-12 strains, including W3110 (lane 2), synthesize a truncated LPS molecule due to mutations in O-antigen synthesis genes. Smaller LPS molecules migrate further toward the bottom of these gels. All ΔrfaDFC mutant strains (compare lanes 2–4 with lanes 5–8) synthesize an even more truncated molecule, corresponding to the lack of core oligosaccharide, also known as deep rough mutation. The LPS profile shown is representative of at least three biological replicates.

Figure 3.

TLC and MALDI-MS structural analysis of biosynthetically engineered deep-rough E. coli that produce minimal Kdo-lipid A domains. Text labels along the plate depict origin where lipid material was spotted before TLC separation, as well as proposed lipid species. A, each lane represents lipid material from E. coli K12 W3110 ΔlpxMΔlpxT (lane 1), ΔlpxMΔlpxTΔrfaDFC (lane 2), ΔlpxMΔlpxTΔrfaDFC harboring pQlink (lane 3), or pQlink::LpxN (lane 4). B, MALDI-MS analysis of non-radiolabeled material in corresponding lanes in A. Right y axis denotes total counts, and the x axis is of the m/z range analyzed. C, each lane represents lipid material isolated from E. coli K12 W3110 ΔlpxMΔlpxTΔrfaDFCΔeptA (lane 5), ΔlpxMΔlpxTΔrfaDFCΔeptB (lane 6), or ΔlpxMΔlpxTΔrfaDFCΔeptAΔeptB (lane 7). D, MALDI-MS analysis of non-radiolabeled material in corresponding lanes in C. Right y axis denotes total counts, and the x axis is of the m/z range analyzed. E, major structures of penta-acylated or LpxN modified Kdo₂-lipid A species in synthetically engineered E. coli. Other important structures can be found in supplemental Fig. S3.

Figure 4.

TLC analysis of isolated ³²P-lipids from deep-rough E. coli that express V. cholerae glycine modification components. Each lane represents lipid material from E. coli K12 W3110 ΔlpxMΔlpxTΔrfaDFCΔeptAΔeptB harboring pQlink plasmids that heterologously express V. cholerae proteins indicated alphabetically below the plate (N, E, F, G, or S) and as detailed in the box legend (bottom right).

Figure 5.

MALDI-MS structural analysis of deep-rough E. coli that express V. cholerae glycine modification components. All spectra are from the same background strain of E. coli W3110 ΔlpxMΔlpxTΔrfaDFCΔeptAΔeptB. Lipids analyzed here correspond to strains from Fig. 4: W3110 ΔlpxMΔlpxTΔrfaDFCΔeptAΔeptB strains harboring pQlink plasmids expressing lpxN or lpxN, almEFG as indicated. MALDI-MS analyzed lipids were isolated from cultures grown without ³²P. Left y axis denotes relative intensity and the x axis is of the m/z range analyzed.

Figure 6.

Phylogram of experimentally verified representative members of the LABLAT subgroup. Maximum likelihood confidence values are reported for each branch. Branch lengths represent rate of difference in amino acid sequence, a distance legend is provided at the bottom. See under “Experimental procedures” and supplemental Fig. S4 for sequence information used to generate the phylogram.

Figure 7.

Expression of single site AlmG alanine variants in V. cholerae ΔalmG strains. The determined MIC value is reported below each image. Strains tested include E7946 wild type (WT), a positive control, and E7946 ΔalmG strains harboring pQlink expression plasmids that contain no gene (negative control), wild-type copy (complemented), or single-site AlmG alanine variants as indicated above each image. Representative plates imaged from experiments performed in triplicate are shown.

Figure 8.

Unusual AlmG catalytic residues suggest an alternative mechanism for synthesis of glycine-modified Kdo-lipid A domains. Both hypothetical mechanisms begin with phosphorylated VprA directly promoting expression of the almEFG operon. In a two-step catalytic mechanism, AlmE (Vc1579) generates glycyl-AMP from glycine and ATP to activate glycine for transfer onto carrier protein holo-AlmF (Vc1578). Holo-AlmF is generated after 4′-phosphopantetheinyl of coenzyme A is transferred onto apo-AlmF by the phosphopantetheinyl transferase AcpS (Vc2457). In mechanism I (left), AlmG at the inner membrane uses glycyl-AlmF as the aminoacyl donor for transfer onto the secondary hydroxylauryl acyl chain of V. cholerae hexaacylated-monophosphoryl-Kdo-lipid A. At least two rounds of glycine transfer can occur. In mechanism II (right), AlmG uses glycyl-AlmF to transfer glycine onto hydroxyl-lauryl acyl carrier protein (AcpP; Vc2020). Two rounds of glycine transfer can occur. LpxN then transfers glycyl-3-hydroxylaurate from AcpP onto pentaacylated-monophosphoryl-Kdo-lipid A. In either mechanism, diglycine or glycine-modified lipid A is then transported to the bacterial surface to provide resistance against cationic antimicrobial peptides such as polymyxin. OM, outer membrane; P, periplasm; IM, inner membrane.