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Structure of an essential inner membrane protein-LPS complex identifies a novel anti-microbial peptide

ABSTRACT: Lipopolysaccharide (LPS) resides in the outer membrane (OM) of Gram-negative bacteria where it is responsible for barrier function and immune modulation. LPS is the target of polymyxins, last-resort antibiotics whose clinical use is threatened by increasing resistance. Steps in LPS biogenesis are known, but how LPS biosynthesis and transport to the OM is coordinated remains poorly understood. Here, we find that depletion of the Escherichia coli inner membrane protein PbgA attenuates pathogenesis and produces defects in the OM. Structural analysis of PbgA identifies an enzyme superfamily known to modify the envelopes of Gram-positive and Gram-negative bacteria; however, PbgA reveals a defunct hydrolase-active site, a distinctive architecture, and an unprecedented lipid A-binding motif along the periplasmic leaflet of the inner membrane. Unlike canonical lipid-binding proteins, PbgA achieves LPS coordination primarily through backbone-mediated interactions. Offensive mutations within the lipid A-binding motif disrupt the OM barrier, suggesting that LPS perception by PbgA plays a key role in maintaining OM homeostasis. PbgA-derived synthetic peptides selectively bind to LPS in vitro and inhibit the growth of diverse Gram-negative bacterial species, including polymyxin-resistant strains. Our studies uncover an essential pseudo-hydrolase with an unexpected link to OM biogenesis, highlight a new structural paradigm in selective lipid recognition, and provide important templates for future antibiotic discovery.

INSTRUMENT(S): Orbitrap Fusion

ORGANISM(S): Escherichia Coli K-12 (ncbitaxon:83333)

SUBMITTER: erik verschueren

PROVIDER: MSV000083754 | MassIVE | Wed May 08 14:46:00 BST 2019

REPOSITORIES: MassIVE

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Structure of the essential inner membrane lipopolysaccharide-PbgA complex.

Clairfeuille Thomas T Buchholz Kerry R KR Li Qingling Q Verschueren Erik E Liu Peter P Sangaraju Dewakar D Park Summer S Noland Cameron L CL Storek Kelly M KM Nickerson Nicholas N NN Martin Lynn L Dela Vega Trisha T Miu Anh A Reeder Janina J Ruiz-Gonzalez Maria M Swem Danielle D Han Guanghui G DePonte Daniel P DP Hunter Mark S MS Gati Cornelius C Shahidi-Latham Sheerin S Xu Min M Skelton Nicholas N Sellers Benjamin D BD Skippington Elizabeth E Sandoval Wendy W Hanan Emily J EJ Payandeh Jian J Rutherford Steven T ST

Nature 20200812 7821

Lipopolysaccharide (LPS) resides in the outer membrane of Gram-negative bacteria where it is responsible for barrier function1,2. LPS can cause death as a result of septic shock, and its lipid A core is the target of polymyxin antibiotics3,4. Despite the clinical importance of polymyxins and the emergence of multidrug resistant strains5, our understanding of the bacterial factors that regulate LPS biogenesis is incomplete. Here we characterize the inner membrane ...[more]

PMID: 32788728

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Project description:In Gram-negative bacteria, lipoproteins are major components of the outer membrane (OM) where they play a variety of roles, from the involvement in β-barrel assembly to virulence. Bacteroidetes, a widespread phylum of Gram-negative bacteria, including free-living organisms, commensals and pathogens, encode an exceptionally high number of outer membrane lipoproteins. These proteins are crucial in this phylum mainly because they are key components of SUS-like nutrient acquisition systems as well as of the Type IX secretion and gliding motility machineries. The transport of lipoproteins to the OM has mainly been studied in E. coli and relies on the Lol system, composed of the inner membrane extraction machinery LolCDE, the periplasmic carrier LolA and the OM lipoprotein LolB. While most Lol proteins are essential and conserved across Gram-negative bacteria, to date, no LolB homologs have been identified outside of γ- and β-proteobacteria. How lipoproteins reach and are inserted in the OM of Bacteroidetes is not known. Here we identified LolB homologs in Bacteroidetes and disclosed the co-existence of several LolA and LolB homologs in several species. We provide evidence that LolA1 and LolB1 of F. johnsoniae are devoted to targeting gliding and T9SS lipoproteins to the OM. A proteomic analysis of the OM composition of the lolA1 and lolB1 deletion strain supports this evidence. Furthermore, we show that, while LolB1 and LolA1 have conserved functions in Bacteroidetes, they are partially functionally different from their E. coli counterparts. We also show that surface lipoprotein transport is LolA and LolB independent. Finally, the finding that, in the absence of LolA and LolB homologs, lipoproteins still localize to the OM, strongly suggests the presence in Bacteroidetes of a yet unidentified Lol-alternative transport pathway. In conclusion, Bacteroidetes seem to have evolved different and probably more complex lipoprotein transport pathways than other Gram-negative bacteria and further research is required to uncover their complexity.