Aminoarabinose is essential for lipopolysaccharide export and intrinsic antimicrobial peptide resistance in Burkholderia cenocepacia^†

Abstract

Summary One common mechanism of resistance against antimicrobial peptides in Gram‐negative bacteria is the addition of 4‐amino‐4‐deoxy‐l‐arabinose (l‐Ara4N) to the lipopolysaccharide (LPS) molecule. Burkholderia cenocepacia exhibits extraordinary intrinsic resistance to antimicrobial peptides and other antibiotics. We have previously discovered that unlike other bacteria, B. cenocepacia requires l‐Ara4N for viability. Here, we describe the isolation of B. cenocepacia suppressor mutants that remain viable despite the deletion of genes required for l‐Ara4N synthesis and transfer to the LPS. The absence of l‐Ara4N is the only structural difference in the LPS of the mutants compared with that of the parental strain. The mutants also become highly sensitive to polymyxin B and melittin, two different classes of antimicrobial peptides. The suppressor phenotype resulted from a single amino acid replacement (aspartic acid to histidine) at position 31 of LptG, a protein component of the multi‐protein pathway responsible for the export of the LPS molecule from the inner to the outer membrane. We propose that l‐Ara4N modification of LPS provides a molecular signature required for LPS export and proper assembly at the outer membrane of B. cenocepacia, and is the most critical determinant for the intrinsic resistance of this bacterium to antimicrobial peptides.

ArnT proteins that catalyze the glycosylation of lipopolysaccharide share common features with bacterialN-oligosaccharyltransferases

“…The functional data for B. cenocepacia ΔarnT were consistent with the chemical structure of the B. cenocepacia lipid A, as examined by mass spectrometry using matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) in the negative ion mode. The mass spectrum of the wild-type lipid A sample showed the presence of L-Ara4N, as indicated by ion peaks corresponding to tetra-acylated lipid A with L-Ara4N in the monophosphate (at m/z 1495.3) and diphosphate (at m/z 1575.3) forms ( Figure 3D) in agreement with the reported B. cenocepacia lipid A structure (Silipo et al 2005;Hamad et al 2012;Tavares-Carreon et al 2015). These ion peaks were absent in the lipid A spectrum of ΔarnT alone and ΔarnT expressing ArnT D39A Fig.…”

“…This strain is highly sensitive to PmB, but viable due to a suppressor mutation that rescues export of lipid A-core to the outer membrane (Hamad et al 2012). Alanine replacements are commonly used to identify the functional relevance of predicted conserved aspartates in DXD motifs of many glycosyltransferases (Breton and Imberty 1999;Maeda et al 2001;Liu and Mushegian 2003;Korres and Verma 2006;Birch et al 2009;Ramiscal et al 2010;Perrin-Tricaud et al 2011;Korkegian et al 2014).…”

“…The modification of lipid A of B. cenocepacia with L-Ara4N is constitutive and essential for B. cenocepacia cell viability (Ortega et al 2007), as L-Ara4N-modified lipid A is required for LPS export to the outer membrane (Hamad et al 2012;Ortega et al 2007). Further, L-Ara4N ligation to lipid A confers PmB resistance to B. cenocepacia and other bacteria such as Salmonella enterica serovar Typhimurium, Pseudomonas aeruginosa, Bor.detella bronchiseptica, Yersinia enterocolitica, Yersinia pestis and Proteus mirabilis (Kaca et al 1990;Boll et al 1994;Guo et al 1997Guo et al , 1998Ernst et al 1999;Loutet and Valvano 2011;Rolin et al 2014).…”

Identification of the Flagellin Glycosylation System in Burkholderia cenocepacia and the Contribution of Glycosylated Flagellin to Evasion of Human Innate Immune Responses

“…Freshly prepared chemically competent E. coli GT115 cells were transformed by the calcium chloride method. Plasmids were mobilized into B. cenocepacia by triparental mating (14,38).…”

“…Construction of Mutants in B. cenocepacia-Unmarked deletion mutants were constructed as described previously (14,38). Briefly, the target genes were deleted by allelic exchange using the pGPI-SceI-2 plasmid containing the corresponding upstream and downstream fragments.…”