Current Research: Defining the functions of novel genes involved in membrane modification in Salmonella
The Gram-negative bacterial envelope has a unique structure that is a permeability barrier promoting resistance to antimicrobials and innate immunity. Gram-negative bacteria are enveloped by two lipid bilayers. The inner membrane (IM) is a phospholipid bilayer made of membrane proteins and glycerophospholipids such as phosphatidylglycerol (PG), phosphatidylethanalomine (PE) and cardiolipin (CL). In contrast, the outer membrane (OM) is asymmetrical, composed of a phospholipid inner leaflet and a lipopolysaccharide (LPS) based outer leaflet. Lipoproteins and other membrane proteins span this asymmetrical bilayer and play important role in creating and maintaining the bilayer. Between the two membranes lies the peptidoglycan cell wall. LPS consists of three distinct structural regions: the carbohydrate repeating O-antigen, the carbohydrate core, and lipid A which makes up the outer leaflet of the OM. Lipid A, an amphipathic molecule made of glucosamine disaccharide and acyl chains, creates a very hydrophobic environment and diffusion barrier to both hydrophobic and hydrophilic compounds. Strongly cationic chemicals such as cationic antimicrobial peptides (CAMPs) including polymyxin bind to negatively charged phosphate residues on LPS sugars, disrupting LPS structures and increasing the permeability of the OM. Upon breaching the OM CAMPs ultimately permeablize the IM eventually disrupting the cell. For almost all Gram-negative bacteria, lipid A and the core components of LPS are essential, and the O-antigen is dispensable. Another key set of factors controlling the permeability of the OM are porins which control the influx of hydrophilic molecules. Nutrients as well as some antimicrobials enter the periplasmic space through porin channels. Consistent with its overall increased resistance to antimicrobials Ab contains a relatively slow porin with a small channel. In contrast to the OM, the IM is a symmetrical bilayer composed of phospholipids and proteins that is more permeable to hydrophobic compounds than the OM. The IM also contains efflux pumps that extrude antimicrobials . Mutations in OM porins and increased expressions of efflux proteins are known to cause increased antibiotic resistance.
Regulated alterations of the Gram-negative bacterial envelope are important for antimicrobial peptide resistance and disease pathogenesis. A wide-variety of Gram-negative bacteria regulate their surface structures in response to environmental conditions to promote pathogenic mechanisms including resistance to host innate immune molecules such as antimicrobial peptides. Antimicrobial peptide resistance mechanisms involve general principles applicable to the pathogenesis and environmental survival of many microbes. The principles of regulated antimicrobial peptide resistance in Gram-negative bacterial pathogens were originally discovered for S. typhimurium. The PhoPQ regulatory system senses specific environmental cues to increase membrane hydrophobicity and neutralize negative charges on the bacterial surface. The PhoQ sensor kinase is activated by cationic antimicrobial peptides (CAMP), acidic pH, and low divalent cationic concentrations. PhoPQ and PmrAB, activate transcription of genes encoding enzymes that alter the acylation state of lipid A
, or neutralize lipid A phosphates with cationic constituents like aminoarabinose and ethanolamine. Specific lipid A modifications include addition of 4-aminoarabinose (L-Ara4N) to the 1’ phosphate of the sugar backbone of lipid A, and addition of ethanolamine phosphates to the 1-phosphate to promote to promote resistance to polymyxin. Additional enzymes modify lipid A in response to specific OM damage including the OM enzyme PagP, which transfers fatty acids (palmitate in enteric organisms) from phospholipids in the inner leaflet of the OM to lipid A, which makes up the outer leaflet. In contrast, the OM PagL enzyme can deacylate lipid A. Additional enzymes change the hydroxylation status of the 3-OH myristic acid acyl chain. Many of these genes and proteins have been implicated in either colonization, antibiotic resistance, or disease pathogenesis of a wide variety of pathogens including: Vibrio cholera, Legionella pneumophila, P. aeruginosa, Rhizobia spp, Yersinia spp, Francisella spp, Bordetella spp., Helicobacter pylori, Klebsiella spp., Burkholderia spp, Shigella spp., and pathogenic E. coli. Ab lacks the PhoPQ system but encodes the PmrAB system known to regulate aminoarabinose and phosphoethanalomine addition to lipid A phosphates.
Papers defining that bacteria environmentally regulate lipid A structure and defing genes as important to lipid A acylation, deacylation, and phosphate modification.
Rebeil R, Ernst RK, Jarrett CO, Adams KN, Miller SI, Hinnebusch BJ. Characterization of late acyltransferase genes of Yersinia pestis and their role in temperature-dependent lipid A variation. J Bacteriol. 2006 Feb;188(4):1381-8. PubMed PMID: 16452420; PubMed Central PMCID: PMC1367257.
Kawasaki K, Ernst RK, Miller SI. Inhibition of Salmonella enterica serovar Typhimurium lipopolysaccharide deacylation by aminoarabinose membrane modification. J Bacteriol. 2005 Apr;187(7):2448-57. PubMed Central PMCID: PMC1065228.
Zhou Z, Ribeiro AA, Lin S, Cotter RJ, Miller SI, Raetz CR. Lipid A modifications in polymyxin-resistant Salmonella typhimurium: PMRA-dependent 4-amino-4-deoxy-L-arabinose, and phosphoethanolamine incorporation. J Biol Chem. 2001 Nov 16;276(46):43111-21. PubMed PMID: 11535603.
5: Trent MS, Pabich W, Raetz CR, Miller SI. A PhoP/PhoQ-induced Lipase (PagL) that catalyzes 3-O-deacylation of lipid A precursors in membranes of Salmonella typhimurium. J Biol Chem. 2001 Mar 23;276(12):9083-92. PubMed PMID: 11108722.
Gunn JS, Ryan SS, Van Velkinburgh JC, Ernst RK, Miller SI. Genetic and functional analysis of a PmrA-PmrB-regulated locus necessary for lipopolysaccharide modification, antimicrobial peptide resistance, and oral virulence of Salmonella enterica serovar typhimurium. Infect Immun. 2000 Nov;68(11):6139-46. PubMed Central PMCID: PMC97691.
Bishop RE, Gibbons HS, Guina T, Trent MS, Miller SI, Raetz CR. Transfer of palmitate from phospholipids to lipid A in outer membranes of gram-negative bacteria. EMBO J. 2000 Oct 2;19(19):5071-80. PubMed Central PMCID: PMC302101.
Ernst RK, Yi EC, Guo L, Lim KB, Burns JL, Hackett M, Miller SI. Specific lipopolysaccharide found in cystic fibrosis airway Pseudomonas aeruginosa. Science. 1999 Nov 19;286(5444):1561-5. PubMed PMID: 10567263.
Guo L, Lim KB, Poduje CM, Daniel M, Gunn JS, Hackett M, Miller SI. Lipid A acylation and bacterial resistance against vertebrate antimicrobial peptides. Cell. 1998 Oct 16;95(2):189-98. PubMed PMID: 9790526.
Gunn JS, Lim KB, Krueger J, Kim K, Guo L, Hackett M, Miller SI. PmrA-PmrB-regulated genes necessary for 4-aminoarabinose lipid A modification and polymyxin resistance. Mol Microbiol. 1998 Mar;27(6):1171-82. PubMed PMID: 9570402.
Papers defining function of novel outer membrane proteins in S. typhimurium and measurement of the permeability barrier.
Farris C, Sanowar S, Bader MW, Pfuetzner R, Miller SI. Antimicrobial peptides activate the Rcs regulon through the outer membrane lipoprotein RcsF. J Bacteriol. 2010 Oct;192(19):4894-903. PubMed Central PMCID: PMC2944553.
Murata T, Tseng W, Guina T, Miller SI, Nikaido H. PhoPQ-mediated regulation produces a more robust permeability barrier in the outer membrane of Salmonella enterica serovar typhimurium. J Bacteriol. 2007 Oct;189(20):7213-22. Epub 2007 Aug 10. PubMed Central PMCID: PMC2168427.
Brodsky IE, Ernst RK, Miller SI, Falkow S. mig-14 is a Salmonella gene that plays a role in bacterial resistance to antimicrobial peptides. J Bacteriol. 2002 Jun;184(12):3203-13. PubMed Central PMCID: PMC135090.
Guina T, Yi EC, Wang H, Hackett M, Miller SI. A PhoP-regulated outer membrane protease of Salmonella enterica serovar typhimurium promotes resistance to alpha-helical antimicrobial peptides. J Bacteriol. 2000 Jul;182(14):4077-86. PubMed Central PMCID: PMC94595.