Reinforcing Lipid A Acylation on the Cell Surface of Acinetobacter baumannii Promotes Cationic Antimicrobial Peptide Resistance and Desiccation Survival

Abstract

Acinetobacter baumannii is an emerging Gram-negative pathogen found in hospitals and intensive care units. In order to persist in hospital environments, A. baumannii withstands desiccative conditions and can rapidly develop multidrug resistance to conventional antibiotics. Cationic antimicrobial peptides (CAMPs) have served as therapeutic alternatives because they target the conserved lipid A component of the Gram-negative outer membrane to lyse the bacterial cell. However, many Gram-negative pathogenic bacteria, including A. baumannii, fortify their outer membrane with hepta-acylated lipid A to protect the cell from CAMP-dependent cell lysis. Whereas in Escherichia coli and Salmonella, increased production of the outer membrane acyltransferase PagP results in formation of protective hepta-acylated lipid A, which reinforces the lipopolysaccharide portion of the outer membrane barrier, A. baumannii does not carry a gene that encodes a PagP homolog. Instead, A. baumannii has evolved a PagP-independent mechanism to synthesize protective hepta-acylated lipid A. Taking advantage of a recently adapted A. baumannii genetic recombineering system, we characterized two putative acyltransferases in A. baumannii designated LpxLAb (A. baumannii LpxL) and LpxMAb (A. baumannii LpxM), which transfer one and two lauroyl (C12:0) acyl chains, respectively, during lipid A biosynthesis. Hepta-acylation of A. baumannii lipid A promoted resistance to vertebrate and polymyxin CAMPs, which are prescribed as last-resort treatment options. Intriguingly, our analysis also showed that LpxMAb-dependent acylation of lipid A is essential for A. baumannii desiccation survival, a key resistance mechanism for survival in hospital environments. Compounds that inhibit LpxMAb-dependent hepta-acylation of lipid A could act synergistically with CAMPs to provide innovative transmission prevention strategies and treat multidrug-resistant infections.

Structure-guided enzymology of the lipid A acyltransferase LpxM reveals a dual activity mechanism

“…As the acyl chain length deviated from 12 carbons in either direction, the K M increased and the specific activity decreased. A strong preference by AbLpxM for a lauryl-ACP donor is also consistent with careful measurements of the mass of lipid A produced by A. baumannii with and without LpxM (8).…”

“…Similarly, P. aeruginosa can produce fully acylated lipid A even when CMP-Kdo synthase (KdsB) is inhibited or when acting upon purified lipid IV A (29,30), suggesting that the presence of the Kdo moieties on the lipid acceptor is dispensable for some LpxL/LpxM orthologs. In A. baumannii, LpxM adds lauryl groups to the R-3-hydroxyacyl chains at both the 3′-and 2-positions of lipid A precursors, producing hepta-acylated lipid A (8). This secondary activity may necessitate flexibility in the active site to accommodate multiple conformations of lipid A, so as to position the substrate properly.…”

“…In the nosocomial pathogen A. baumannii, LpxM is required for virulence in the Galleria mellonella (greater wax moth) infection model, and for protection from desiccation, which increases infection and transmission rates (8). Additionally, in E. coli clinical isolate H16, deletion of chromosomal lpxM results in a drastic decrease in virulence after i. p. injection into BALB/c mice (12), and deletion of the gene in the E058 strain of avian pathogenic E. coli (APEC) results in defects in avian macrophage invasion and decreased bacterial loads in several organs following inoculation into the left thoracic air sac in chickens (13).…”

“…In this work, we present the X-ray crystal structure of a bacterial LPLAT protein, the LABLAT LpxM from the pathogenic bacterium A. baumannii (AbLpxM), which possesses two known acyltransferase activities (8) (Fig. 1).…”

Pathogenic Acinetobacter: from the Cell Surface to Infinity and Beyond

“…Specifically, these modifications occur on the lipid A structure of Acinetobacter spp. and lead to decreased susceptibility to antibiotic and antimicrobial peptides and increased survival during desiccation (37)(38)(39). Acinetobacter spp.…”