Endotoxin of Neisseria meningitidis Composed Only of Intact Lipid A: Inactivation of the Meningococcal 3-Deoxy-d-Manno-Octulosonic Acid Transferase

ABSTRACT Lipopolysaccharide, lipooligosaccharide (LOS), or endotoxin is important in bacterial survival and the pathogenesis of gram-negative bacteria. A necessary step in endotoxin biosynthesis is 3-deoxy-d-manno-octulosonic acid (Kdo) glycosylation of lipid A, catalyzed by the Kdo transferase KdtA (WaaA). In enteric gram-negative bacteria, this step is essential for survival. A nonpolar kdtA::aphA-3 mutation was created in Neisseria meningitidis via allelic exchange, and the mutant was viable. Detailed structural analysis demonstrated that the endotoxin of the kdtA::aphA-3 mutant was composed of fully acylated lipid A with variable phosphorylation but without Kdo glycosylation. In contrast to what happens in other gram-negative bacteria, tetra-acylated lipid IVA did not accumulate. The LOS structure of the kdtA::aphA-3 mutant was restored to the wild-type structure by complementation with kdtA from N. meningitidis or Escherichia coli. The expression of a fully acylated, unglycosylated lipid A indicates that lipid A biosynthesis in N. meningitidis can proceed without the addition of Kdo and that KdtA is not essential for survival of the meningococcus.

Download Full-text

Yersinia pestis Is Viable with Endotoxin Composed of Only Lipid A

Journal of Bacteriology ◽

10.1128/jb.187.18.6599-6600.2005 ◽

2005 ◽

Vol 187 (18) ◽

pp. 6599-6600 ◽

Cited By ~ 22

Author(s):

Li Tan ◽

Creg Darby

Keyword(s):

Escherichia Coli ◽

Yersinia Pestis ◽

Outer Membrane ◽

Salmonella Enterica ◽

Lipid A ◽

Gram Negative Bacteria ◽

Membrane Component ◽

Gram Negative ◽

Serovar Typhimurium

ABSTRACT Lipopolysaccharide (LPS) is the major outer membrane component of gram-negative bacteria. The minimal LPS structure for viability of Escherichia coli and Salmonella enterica serovar Typhimurium is lipid A glycosylated with 3-deoxy-D-manno-octulosonic acid (Kdo) residues. Here we show that another member of the Enterobacteriaceae, Yersinia pestis, can survive without Kdo in its LPS.

Download Full-text

Metabolic phospholipid labeling of intact bacteria enables a fluorescence assay that detects compromised outer membranes

The Journal of Lipid Research ◽

10.1194/jlr.ra120000654 ◽

2020 ◽

Vol 61 (6) ◽

pp. 870-883 ◽

Cited By ~ 2

Author(s):

Inga Nilsson ◽

Sheng Y. Lee ◽

William S. Sawyer ◽

Christopher M. Baxter Rath ◽

Guillaume Lapointe ◽

...

Keyword(s):

Escherichia Coli ◽

Gram Negative Bacteria ◽

Metabolic Labeling ◽

Wild Type ◽

Transport Pathways ◽

Gram Negative ◽

E Coli ◽

Outer Membranes ◽

Outer Leaflet ◽

Promising Tool

Gram-negative bacteria possess an asymmetric outer membrane (OM) composed primarily of lipopolysaccharides (LPSs) on the outer leaflet and phospholipids (PLs) on the inner leaflet. The loss of this asymmetry due to mutations in the LPS biosynthesis or transport pathways causes the externalization of PLs to the outer leaflet of the OM and leads to OM permeability defects. Here, we used metabolic labeling to detect a compromised OM in intact bacteria. Phosphatidylcholine synthase expression in Escherichia coli allowed for the incorporation of exogenous propargylcholine into phosphatidyl(propargyl)choline and exogenous 1-azidoethyl-choline (AECho) into phosphatidyl(azidoethyl)choline (AEPC), as confirmed by LC/MS analyses. A fluorescent copper-free click reagent poorly labeled AEPC in intact wild-type cells but readily labeled AEPC from lysed cells. Fluorescence microscopy and flow cytometry analyses confirmed the absence of significant AEPC labeling from intact wild-type E. coli strains and revealed significant AEPC labeling in an E. coli LPS transport mutant (lptD4213) and an LPS biosynthesis mutant (E. coli lpxC101). Our results suggest that metabolic PL labeling with AECho is a promising tool for detecting a compromised bacterial OM, revealing aberrant PL externalization, and identifying or characterizing novel cell-active inhibitors of LPS biosynthesis or transport.

Download Full-text

Identification of Two New Proteins in Spermidine Nucleoids Isolated from Escherichia coli

Journal of Bacteriology ◽

10.1128/jb.181.12.3842-3844.1999 ◽

1999 ◽

Vol 181 (12) ◽

pp. 3842-3844 ◽

Cited By ~ 12

Author(s):

Lizabeth D. Murphy ◽

Judah L. Rosner ◽

Steven B. Zimmerman ◽

Dominic Esposito

Keyword(s):

Escherichia Coli ◽

Functional Form ◽

Dnase I ◽

Gram Negative Bacteria ◽

Wild Type ◽

Gram Negative ◽

Form Analysis ◽

E Coli ◽

Amino Terminal

ABSTRACT The Escherichia coli nucleoid contains DNA in a condensed but functional form. Analysis of proteins released from isolated spermidine nucleoids after treatment with DNase I reveals significant amounts of two proteins not previously detected in wild-type E. coli. Partial amino-terminal sequencing has identified them as the products of rdgC andyejK. These proteins are strongly conserved in gram-negative bacteria, suggesting that they have important cellular roles.

Download Full-text

Biosynthesis of lipopolysaccharides with different length of the O-specific region as a virulence factor of Gram-negative bacteria

Postępy Higieny i Medycyny Doświadczalnej ◽

10.5604/01.3001.0012.1735 ◽

2018 ◽

Vol 72 ◽

pp. 573-586

Author(s):

Eva Krzyżewska ◽

Jacek Rybka

Keyword(s):

Outer Membrane ◽

Lipid A ◽

Specific Antigen ◽

Gram Negative Bacteria ◽

Bacterial Survival ◽

Core Oligosaccharide ◽

Gram Negative ◽

O Antigen ◽

Length Regulation ◽

External Part

The outer membrane of Gram-negative bacteria is a biological structure with a unique composition that significantly contributes to the survival of bacteria in the unfavorable conditions of the host organism. The lipopolysaccharide constitutes about 70% of the external part of the outer membrane. The LPS molecule is composed of three different parts: lipid A, core oligosaccharide and O antigen. Despite the O-specific antigen being one of the most intensely studied surface structures of bacterial polysaccharides, a number of questions regarding the mechanism of the O antigen biosynthesis and its transport to the cell surface are still unanswered. The paper describes the biosynthesis of the lipopolysaccharide molecule, with particular emphasis on the O-specific chain biosynthesis, the mechanism of lipopolysaccharide length regulation and the influence of the type of synthesized O-specific chains on bacterial survival in adverse host organisms.

Download Full-text

Nonclonal Emergence of Colistin Resistance Associated with Mutations in the BasRS Two-Component System in Escherichia coli Bloodstream Isolates

mSphere ◽

10.1128/msphere.00143-20 ◽

2020 ◽

Vol 5 (2) ◽

Cited By ~ 2

Author(s):

Axel B. Janssen ◽

Toby L. Bartholomew ◽

Natalia P. Marciszewska ◽

Marc J. M. Bonten ◽

Rob J. L. Willems ◽

...

Keyword(s):

Escherichia Coli ◽

Lipid A ◽

Gram Negative Bacteria ◽

Colistin Resistance ◽

Anionic Lipid ◽

Gram Negative ◽

Content Type ◽

E Coli ◽

Two Component ◽

Resistant Strains

ABSTRACT Infections by multidrug-resistant Gram-negative bacteria are increasingly common, prompting the renewed interest in the use of colistin. Colistin specifically targets Gram-negative bacteria by interacting with the anionic lipid A moieties of lipopolysaccharides, leading to membrane destabilization and cell death. Here, we aimed to uncover the mechanisms of colistin resistance in nine colistin-resistant Escherichia coli strains and one Escherichia albertii strain. These were the only colistin-resistant strains of 1,140 bloodstream Escherichia isolates collected in a tertiary hospital over a 10-year period (2006 to 2015). Core-genome phylogenetic analysis showed that each patient was colonized by a unique strain, suggesting that colistin resistance was acquired independently in each strain. All colistin-resistant strains had lipid A that was modified with phosphoethanolamine. In addition, two E. coli strains had hepta-acylated lipid A species, containing an additional palmitate compared to the canonical hexa-acylated E. coli lipid A. One E. coli strain carried the mobile colistin resistance (mcr) gene mcr-1.1 on an IncX4-type plasmid. Through construction of chromosomal transgene integration mutants, we experimentally determined that mutations in basRS, encoding a two-component signal transduction system, contributed to colistin resistance in four strains. We confirmed these observations by reversing the mutations in basRS to the sequences found in reference strains, resulting in loss of colistin resistance. While the mcr genes have become a widely studied mechanism of colistin resistance in E. coli, sequence variation in basRS is another, potentially more prevalent but relatively underexplored, cause of colistin resistance in this important nosocomial pathogen. IMPORTANCE Multidrug resistance among Gram-negative bacteria has led to the use of colistin as a last-resort drug. The cationic colistin kills Gram-negative bacteria through electrostatic interaction with the anionic lipid A moiety of lipopolysaccharides. Due to increased use in clinical and agricultural settings, colistin resistance has recently started to emerge. In this study, we used a combination of whole-genome sequence analysis and experimental validation to characterize the mechanisms through which Escherichia coli strains from bloodstream infections can develop colistin resistance. We found no evidence of direct transfer of colistin-resistant isolates between patients. The lipid A of all isolates was modified by the addition of phosphoethanolamine. In four isolates, colistin resistance was experimentally verified to be caused by mutations in the basRS genes, encoding a two-component regulatory system. Our data show that chromosomal mutations are an important cause of colistin resistance among clinical E. coli isolates.

Download Full-text

Pathogenic Neisseriae Can Use Hemoglobin, Transferrin, and Lactoferrin Independently of the tonBLocus

Journal of Bacteriology ◽

10.1128/jb.182.19.5586-5591.2000 ◽

2000 ◽

Vol 182 (19) ◽

pp. 5586-5591 ◽

Cited By ~ 15

Author(s):

Pragnya Jasvantrai Desai ◽

Eric Garges ◽

Caroline Attardo Genco

Keyword(s):

Neisseria Gonorrhoeae ◽

Neisseria Meningitidis ◽

Outer Membrane ◽

Deletion Mutant ◽

Iron Transport ◽

Gram Negative Bacteria ◽

Wild Type ◽

Chromosomal Dna ◽

Gram Negative ◽

Type Strains

ABSTRACT Redundant TonB systems which function in iron transport from TonB-dependent ligands have recently been identified in several gram-negative bacteria. We demonstrate here that in addition to the previously described tonB locus, an alternative system exists for the utilization of iron from hemoglobin, transferrin, or lactoferrin in Neisseria meningitidis andNeisseria gonorrhoeae. Following incubation on media containing hemoglobin, N. meningitidis IR3436 (tonB exbB exbD deletion mutant) and N. gonorrhoeae PD3401 (tonB insertional mutant) give rise to colonies which can grow with hemoglobin. Transfer of Hb+ variants (PD3437 or PD3402) to media containing hemoglobin, transferrin, and/or lactoferrin as sole iron sources resulted in growth comparable to that observed for the wild-type strains. Transformation of N. meningitidis IR3436 or N. gonorrhoeae PD3401 with chromosomal DNA from the Hb+ variants yielded transformants capable of growth with hemoglobin. When we inactivated the TonB-dependent outer membrane hemoglobin receptors (HmbR or HpuB) in the NeisseriaHb+ variants, these strains could not grow with hemoglobin; however, growth was observed with transferrin and/or lactoferrin. These results demonstrate that accumulation of iron from hemoglobin, transferrin, and lactoferrin in the pathogenic neisseriae can occur via a system that is independent of the previously describedtonB locus.

Download Full-text

LpxT-Dependent Phosphorylation of Lipid A in Escherichia coli Increases Resistance to Deoxycholate and Enhances Gut Colonization

Frontiers in Microbiology ◽

10.3389/fmicb.2021.676596 ◽

2021 ◽

Vol 12 ◽

Author(s):

Xudong Tian ◽

Guillaume Manat ◽

Elise Gasiorowski ◽

Rodolphe Auger ◽

Samia Hicham ◽

...

Keyword(s):

Escherichia Coli ◽

Cell Surface ◽

Lipid A ◽

Negative Charge ◽

Polymyxin B ◽

Gram Negative Bacteria ◽

Bacterial Cells ◽

Gram Negative ◽

E Coli ◽

Gut Colonization

The cell surface of Gram-negative bacteria usually exhibits a net negative charge mostly conferred by lipopolysaccharides (LPS). This property sensitizes bacterial cells to cationic antimicrobial peptides, such as polymyxin B, by favoring their binding to the cell surface. Gram-negative bacteria can modify their surface to counteract these compounds such as the decoration of their LPS by positively charged groups. For example, in Escherichia coli and Salmonella, EptA and ArnT add amine-containing groups to the lipid A moiety. In contrast, LpxT enhances the net negative charge by catalyzing the synthesis of tri-phosphorylated lipid A, whose function is yet unknown. Here, we report that E. coli has the intrinsic ability to resist polymyxin B upon the simultaneous activation of the two component regulatory systems PhoPQ and PmrAB by intricate environmental cues. Among many LPS modifications, only EptA- and ArnT-dependent decorations were required for polymyxin B resistance. Conversely, the acquisition of polymyxin B resistance compromised the innate resistance of E. coli to deoxycholate, a major component of bile. The inhibition of LpxT by PmrR, under PmrAB-inducing conditions, specifically accounted for the acquired susceptibility to deoxycholate. We also report that the kinetics of intestinal colonization by the E. coli lpxT mutant was impaired as compared to wild-type in a mouse model of infection and that lpxT was upregulated at the temperature of the host. Together, these findings highlight an important function of LpxT and suggest that a tight equilibrium between EptA- and LpxT-dependent decorations, which occur at the same position of lipid A, is critical for the life style of E. coli.

Download Full-text

Synergistic Interactions of Vancomycin with Different Antibiotics against Escherichia coli: Trimethoprim and Nitrofurantoin Display Strong Synergies with Vancomycin against Wild-Type E. coli

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.03502-14 ◽

2014 ◽

Vol 59 (1) ◽

pp. 276-281 ◽

Cited By ~ 38

Author(s):

Alice Zhou ◽

Tina Manzhu Kang ◽

Jessica Yuan ◽

Casey Beppler ◽

Caroline Nguyen ◽

...

Keyword(s):

Escherichia Coli ◽

The Other ◽

Gram Negative Bacteria ◽

Functional Categories ◽

Wild Type ◽

Gram Negative ◽

Content Type ◽

E Coli ◽

Synergistic Interactions ◽

Pairwise Interactions

ABSTRACTGram-negative bacteria are normally resistant to the antibiotic vancomycin (VAN), which cannot significantly penetrate the outer membrane. We usedEscherichia colimutants that are partially sensitive to VAN to study synergies between VAN and 10 other antibiotics representing six different functional categories. We detected strong synergies with VAN and nitrofurantoin (NTR) and with VAN and trimethoprim (TMP) and moderate synergies with other drugs, such as aminoglycosides. These synergies are powerful enough to show the activity of VAN against wild-typeE. coliat concentrations of VAN as low as 6.25 μg/ml. This suggests that a very small percentage of exogenous VAN does enterE. colibut normally has insignificant effects on growth inhibition or cell killing. We used the results of pairwise interactions with VAN and the other 10 antibiotics tested to place VAN into a functional category of its own, as previously defined by Yeh et al. (P. Yeh, A. I. Tschumi, and R. Kishony, Nat Genet 28:489–494, 2006,http://dx.doi.org/10.1038/ng1755).

Download Full-text

The galE Gene of Campylobacter jejuni Is Involved in Lipopolysaccharide Synthesis and Virulence

Infection and Immunity ◽

10.1128/iai.68.5.2594-2601.2000 ◽

2000 ◽

Vol 68 (5) ◽

pp. 2594-2601 ◽

Cited By ~ 92

Author(s):

Benjamin N. Fry ◽

Shi Feng ◽

Yuen-Yuen Chen ◽

Diane G. Newell ◽

Peter J. Coloe ◽

...

Keyword(s):

Campylobacter Jejuni ◽

Parent Strain ◽

Type Strain ◽

Essential Role ◽

Lipid A ◽

Wild Type Strain ◽

Gram Negative Bacteria ◽

Wild Type ◽

Gram Negative ◽

Important Virulence Factor

ABSTRACT Lipopolysaccharide (LPS) is one of the main virulence factors of gram-negative bacteria. The LPS from Campylobacter spp. has endotoxic properties and has been shown to play a role in adhesion. We previously cloned a gene cluster (wla) which is involved in the synthesis of the Campylobacter jejuni 81116 LPS molecule. Sequence alignment of the first gene in this cluster indicated similarity with galE genes. These genes encode a UDP-glucose 4-epimerase, which catalyzes the interconversion of UDP-galactose and UDP-glucose. A Salmonella galE mutant was transformed with the galE gene from C. jejuni. The LPS analysis of wild-type, galE, and complementedgalE Salmonella strains showed that the C. jejuni galE gene could restore the smooth wild-typeSalmonella LPS. A UDP-glucose 4-epimerase assay was used to demonstrate that the galE gene from C. jejuniencoded this epimerase. We constructed a C. jejuni galEmutant which expressed a lipid A-core molecule of reduced molecular weight that did not react with antiserum raised against the parental strain. These results show an essential role for the galEgene in the synthesis of C. jejuni LPS. ThegalE mutant also showed a reduction in its ability to adhere to and invade INT407 cells. However, it was still able to colonize chickens to the same level as the wild-type strain. The serum resistance and hemolytic activity of this mutant were not changed compared to the parent strain. The ability of the mutant to take up DNA and integrate it in its genome was reduced 20-fold. These results show that LPS of C. jejuni is an important virulence factor.

Download Full-text