Mutations in enterobacterial common antigen biosynthesis restore outer membrane barrier function in <i>Escherichia coli tol‐pal</i> mutants

Jiang, Xiang'Er; Tan, Wee Boon; Shrivastava, Rahul; Seow, Deborah Chwee San; Chen, Swaine L.; Guan, Xue Li; Chng, Shu‐Sin

doi:10.1111/mmi.14590

Cited by 12 publications

(15 citation statements)

References 74 publications

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“…Typhimurium, and S . Enteritidis (Gilbreath et al, 2012 ; Jaiswal et al, 2015 ; Jiang et al, 2020 ; Rai et al, 2021 ). ECA consists of trisaccharide repeating unit, GlcNAc, ManNAcA, and 4-acetamido-4,6-dideoxy-D-galactose (Fuc4NAc).…”

Section: Discussionmentioning

confidence: 99%

wecB Gene of Salmonella Gallinarum Plays a Critical Role in Systemic Infection of Fowl Typhoid

et al. 2022

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Salmonella enterica serovar Gallinarum (S. Gallinarum) is a host-specific pathogen causing fowl typhoid, a severe systemic infection in poultry, which leads to substantial economic losses due to high morbidity and mortality in many developing countries. However, less is known about the pathogenic characteristics and mechanism of S. Gallinarum-induced systemic infection in chickens. In this study, we deleted the S. Gallinarum UDP-N-acetylglucosamine-1-phosphate transferase gene, which contributes to the biosynthesis of enterobacterial common antigen (ECA), and studied the pathogenicity of this wecB::Cm strain in a chicken model of systemic infection. The wecB::Cm mutant strain showed comparable growth but lower resistance to bile acid and nalidixic acid than the wild-type strain in vitro. In the oral infection model of chickens, the virulence of the wecB::Cm strain was significantly attenuated in vivo. Chickens infected with wild-type strain showed typical clinical signs and pathological changes of fowl typhoid and died between 6 and 9 days post-infection, and the bacteria rapidly disseminated to systemic organs and increased in the livers and spleens. In contrast, the wecB::Cm mutant strain did not cause chicken death, there were no significant clinical changes, and the bacterial numbers in the liver and spleen of the chickens were significantly lower than those of the chickens infected with the wild-type strain. In addition, the expression of interleukin (IL)-1β, tumor necrosis factor (TNF)-α, and CXCLi1 in the livers of wecB::Cm-infected chickens was significantly lower than that of the chickens infected with the wild-type strain. Furthermore, the attenuated wecB::Cm strain could persistently colonize the liver and spleen at low levels for up to 25 days post-infection and could induce a protective immune response in the chickens. These results indicate that the wecB gene is an important virulence factor of S. Gallinarum in the chicken model of systemic infection, and the avirulent wecB::Cm mutant could possibly be used as a live-attenuated vaccine strain for controlling fowl typhoid.

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Section: Discussionmentioning

confidence: 99%

wecB Gene of Salmonella Gallinarum Plays a Critical Role in Systemic Infection of Fowl Typhoid

et al. 2022

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“…Rcs is also activated by mutants of the Tol-Pal system which has a role in maintaining lipid homeostasis in the outer membrane ( Clavel et al, 1996 ; Shrivastava et al, 2017 ). It has been shown that Tol mutants show enhanced sensitivity to vancomycin, and this is rescued by mutants in the wec operon ( Jiang et al, 2020 ). Thus, the fact that both tolA and wec mutants show enhanced resistance to model honey could be directly related to the defect in ECA biosynthesis or to the induction of the Rcs phosphorelay, and this will repay further investigation.…”

Section: Discussionmentioning

confidence: 99%

Use of Transposon Directed Insertion-Site Sequencing to Probe the Antibacterial Mechanism of a Model Honey on E. coli K-12

et al. 2022

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Antimicrobial resistance is an ever-growing health concern worldwide that has created renewed interest in the use of traditional anti-microbial treatments, including honey. However, understanding the underlying mechanism of the anti-microbial action of honey has been hampered due to the complexity of its composition. High throughput genetic tools could assist in understanding this mechanism. In this study, the anti-bacterial mechanism of a model honey, made of sugars, hydrogen peroxide, and gluconic acid, was investigated using genome-wide transposon mutagenesis combined with high-throughput sequencing (TraDIS), with the strain Escherichia coli K-12 MG1655 as the target organism. We identified a number of genes which when mutated caused a severe loss of fitness when cells were exposed to the model honey. These genes encode membrane proteins including those involved in uptake of essential molecules, and components of the electron transport chain. They are enriched for pathways involved in intracellular homeostasis and redox activity. Genes involved in assembly and activity of formate dehydrogenase O (FDH-O) were of particular note. The phenotypes of mutants in a subset of the genes identified were confirmed by phenotypic screening of deletion strains. We also found some genes which when mutated led to enhanced resistance to treatment with the model honey. This study identifies potential synergies between the main honey stressors and provides insights into the global antibacterial mechanism of this natural product.

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“…Experiments to determine the phospholipid distribution between IM and OM and the ratio of phospholipid to LPS at the OM were repeated exactly as described previously [127], with one addition, cell cultures were harvested at stationary phase (18 h growth) in addition to midexponential phase of growth (OD 600 = 0.5). Briefly, cultures were grown in triplicate in either 5 ml (stationary) or 10 ml LB (mid-exponential) with 1 μCi/ml [ 32 P]-disodiumphosphate (Perkin Elmer, NEX011001MC) to label phospholipids and LPS.…”

Section: Membrane Lipid Composition Analysesmentioning

confidence: 99%

“…Cells were harvested by centrifugation, washed, and resuspended in 5 ml of 20% sucrose in 10mM Tris-HCl (pH 8.0 (w/w) containing 1 mM PMSF and 50 μg/ml DNase I), and lysed using a high-pressure French press. The cell lysate was centrifuged and the supernatant subjected to sucrose density fractionation over 16 h, as described previously [127]. IM (7)(8)(9) and OM (12)(13)(14) fractions were collected and concentrated into 320 μl TBS and LPS and/or phospholipid extracted and dried as described previously [128].…”

Section: Membrane Lipid Composition Analysesmentioning

confidence: 99%

Loss of YhcB results in dysregulation of coordinated peptidoglycan, LPS and phospholipid synthesis during Escherichia coli cell growth

et al. 2021

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The cell envelope is essential for viability in all domains of life. It retains enzymes and substrates within a confined space while providing a protective barrier to the external environment. Destabilising the envelope of bacterial pathogens is a common strategy employed by antimicrobial treatment. However, even in one of the best studied organisms, Escherichia coli, there remain gaps in our understanding of how the synthesis of the successive layers of the cell envelope are coordinated during growth and cell division. Here, we used a whole-genome phenotypic screen to identify mutants with a defective cell envelope. We report that loss of yhcB, a conserved gene of unknown function, results in loss of envelope stability, increased cell permeability and dysregulated control of cell size. Using whole genome transposon mutagenesis strategies, we report the comprehensive genetic interaction network of yhcB, revealing all genes with a synthetic negative and a synthetic positive relationship. These genes include those previously reported to have a role in cell envelope biogenesis. Surprisingly, we identified genes previously annotated as essential that became non-essential in a ΔyhcB background. Subsequent analyses suggest that YhcB functions at the junction of several envelope biosynthetic pathways coordinating the spatiotemporal growth of the cell, highlighting YhcB as an as yet unexplored antimicrobial target.

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Mutations in enterobacterial common antigen biosynthesis restore outer membrane barrier function in Escherichia coli tol‐pal mutants

Cited by 12 publications

References 74 publications

wecB Gene of Salmonella Gallinarum Plays a Critical Role in Systemic Infection of Fowl Typhoid

wecB Gene of Salmonella Gallinarum Plays a Critical Role in Systemic Infection of Fowl Typhoid

Use of Transposon Directed Insertion-Site Sequencing to Probe the Antibacterial Mechanism of a Model Honey on E. coli K-12

Loss of YhcB results in dysregulation of coordinated peptidoglycan, LPS and phospholipid synthesis during Escherichia coli cell growth

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