A penicillin-binding protein inhibits selection of colistin-resistant, lipooligosaccharide-deficient Acinetobacter baumannii

Abstract

The Gram-negative bacterial outer membrane fortifies the cell against environmental toxins including antibiotics. Unique glycolipids called lipopolysaccharide/lipooligosaccharide (LPS/LOS) are enriched in the cell-surface monolayer of the outer membrane and promote antimicrobial resistance. Colistin, which targets the lipid A domain of LPS/LOS to lyse the cell, is the last-line treatment for multidrug-resistant Gram-negative infections. Lipid A is essential for the survival of most Gram-negative bacteria, but colistin-resistant Acinetobacter baumannii lacking lipid A were isolated after colistin exposure. Previously, strain ATCC 19606 was the only A. baumannii strain demonstrated to subsist without lipid A. Here, we show that other A. baumannii strains can also survive without lipid A, but some cannot, affording a unique model to study endotoxin essentiality. We assessed the capacity of 15 clinical A. baumannii isolates including 9 recent clinical isolates to develop colistin resistance through inactivation of the lipid A biosynthetic pathway, the products of which assemble the LOS precursor. Our investigation determined that expression of the well-conserved penicillin-binding protein (PBP) 1A, prevented LOS-deficient colony isolation. The glycosyltransferase activity of PBP1A, which aids in the polymerization of the peptidoglycan cell wall, was lethal to LOS-deficient A. baumannii Global transcriptomic analysis of a PBP1A-deficient mutant and four LOS-deficient A. baumannii strains showed a concomitant increase in transcription of lipoproteins and their transporters. Examination of the LOS-deficient A. baumannii cell surface demonstrated that specific lipoproteins were overexpressed and decorated the cell surface, potentially compensating for LOS removal. This work expands our knowledge of lipid A essentiality and elucidates a drug resistance mechanism.

Keywords: Acinetobacter; colistin; lipopolysaccharide; lipoprotein; peptidoglycan.

Fig. 1.

A. baumannii inactivates lipooligosaccharide biosynthesis to alter immune recognition and susceptibility to clinically relevant antibiotics. (A) ³²P-radiolabeled lipid A was isolated from ATCC 19606, 5075, and AYE parent A. baumannii strains and their LOS-deficient progeny and separated based on hydrophobicity using TLC. (B) Stimulation of human TLR-4/MD2 complex following incubation of bacterial cells (cfu/mL) with HEK blue cells expressing the TLR-4 receptor complex is depicted. (C) MICs for parent and LOS-deficient A. baumannii strains.

Fig. 2.

Inactivation of ponA promotes complete loss of LOS in ATCC 17978 A. baumannii. (A) Illustration depicting PBP1A and its domains with respective catalytic residues (red). (B) ³²P-radiolabeled lipid A isolated from ATCC 17978 wild-type and ponA mutant A. baumannii was separated based on hydrophobicity using TLC. (C) Stimulation of human TLR-4/MD-2 following incubation of bacterial cells (cfu/mL) with HEK blue cells expressing the TLR-4 receptor complex is depicted. (D) Percentage recovery of LOS-deficient A. baumannii after selection of plating either 10⁹ cfu ATCC 17978 (Left) or ATCC19606 (Right) on colistin.

Fig. 3.

Differential expression of ponA and PBP1A in ATCC 17978 and ATCC 19606 A. baumannii. (A) Relative concentration of ponA mRNA in ATCC 17978 and ATCC 19606 wild-type A. baumannii. (B) Immunoblot analysis of PBP1A and NADH chain L proteins in whole-cell lysates from wild-type and mutant ATCC 17978 and ATCC 19606 A. baumannii strains. Each protein was detected using specific polyclonal antiserum.

Fig. 4.

Inactivation of ponA in ATCC 17978 A. baumannii increases expression of lipoprotein transport genes. (A) Venn diagram showing altered pathway expression in ATCC 17978 ΔponA LOS-deficient and ATCC 17978 ΔponA A. baumannii strains (P value ≤0.05). Gene expression was calculated relative to the wild-type parent ATCC 17978 A. baumannii. (B) Relative concentration of lolA mRNA in wild-type and mutant ATCC 17978 and ATCC 19606 A. baumannii strains.

Fig. 5.

Global transcriptional analysis from multiple A. baumannii strains highlights conserved pathways important for loss of LOS. Venn diagrams showing up-regulated (A) and down-regulated (B) pathways in four LOS-deficient A. baumannii strains (P value ≤ 0.05). Gene expression was calculated relative to the parent wild-type A. baumannii strains. The five conserved significantly up-regulated pathways in LOS-deficient A. baumannii included lipoprotein transport, putative lipoproteins, retrograde phospholipid transport, multidrug efflux pumps/CAMP resistance proteins, and the BaeSR two-component system. (C) Heat map illustrating the altered expression of each gene in the five conserved pathways and for PNAG biosynthesis. Our statistical analysis did not highlight the PNAG biosynthesis as conserved responses to loss of LOS, but genes were included in the heat map because this pathway was previously thought to be important for A. baumannii survival without LOS.

Fig. 6.

Lipoproteins are overexpressed and surface-displayed in LOS-deficient A. baumannii. Intact whole cells (A) or cell lysates (B) were incubated with sulfo-NHS-LC-LC-biotin to biotinylate accessible proteins. After labeling, proteins from the soluble (S) and membrane (M) fractions were subjected to SDS/PAGE and Western blotted using streptavidin-HRP. Intact whole cells containing chromosomal His₆-tag fusions including (C) HMPREF0010_1944 (1944-His₆) (11.52 kDa), (D) HMPREF0010_1945 (1945-His₆) (11.83 kDa), and (E) HMPREF0010_2739 (2739-His₆) (14.76 kDa) were treated with sulfo-NHS-LC-LC-biotin, and proteins were separated using SDS/PAGE. Wild-type, LOS-deficient for each respective mutant, and overexpression strains were blotted using a streptavidin-HRP conjugate (Top), anti-his antibody (Middle), or a polyclonal NADH chain L antibody (Bottom).