Dataset Information

Factor H Is Bound by Outer Membrane-Displayed Carbohydrate Metabolism Enzymes of Extraintestinal Pathogenic Escherichia coli and Contributes to Opsonophagocytosis Resistance in Bacteria.

ABSTRACT: Extraintestinal pathogenic Escherichia coli (ExPEC) causes bloodstream infections in humans and animals. Complement escape is a prerequisite for bacteria to survive in the bloodstream. Factor H (FH) is an important regulatory protein of the complement system. In this study, ExPEC was found to bind FH from serum. However, the mechanisms of ExPEC binding to FH and then resistance to complement-mediated attacks remain unclear. Here, a method that combined desthiobiotin pull-down and liquid chromatography-tandem mass spectrometry was used to identify the FH-binding membrane proteins of ExPEC. Seven identified proteins, which all were carbohydrate metabolic enzymes (CMEs), including acetate kinase, fructose-bisphosphate aldolase, fumarate reductase flavoprotein subunit, L-lactate dehydrogenase, dihydrolipoamide dehydrogenase, phosphoenolpyruvate synthase, and pyruvate dehydrogenase, were verified to recruit FH from serum using GST pull-down and ELISA plate binding assay. The ELISA plate binding assay determined that these seven proteins bind to FH in a dose-dependent manner. Magnetic beads coupled with any one of seven proteins significantly reduced the FH recruitment of ExPEC (p < 0.05) Subsequently, immunofluorescence, colony blotting, and Western blotting targeting outer membrane proteins determined that these seven CMEs were located on the outer membrane of ExPEC. Furthermore, the FH recruitment levels and C3b deposition levels on bacteria were significantly increased and decreased in an FH-concentration-dependent manner, respectively (p < 0.05). The FH recruitment significantly enhanced the ability of ExPEC to resist the opsonophagocytosis of human macrophage THP-1 in an FH-concentration-dependent manner (p < 0.05), which revealed a new mechanism for ExPEC to escape complement-mediated killing. The identification of novel outer membrane-displayed CMEs which played a role in the FH recruitment contributes to the elucidation of the molecular mechanism of ExPEC pathogenicity.

SUBMITTER: Sun Y

PROVIDER: S-EPMC7868385 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Factor H Is Bound by Outer Membrane-Displayed Carbohydrate Metabolism Enzymes of Extraintestinal Pathogenic <i>Escherichia coli</i> and Contributes to Opsonophagocytosis Resistance in Bacteria.

Sun Yu Y Xu Bin B Zhuge Xiangkai X Tang Fang F Wang Xuhang X Gong Qianwen Q Chen Rui R Xue Feng F Dai Jianjun J

Frontiers in cellular and infection microbiology 20210125

Extraintestinal pathogenic <i>Escherichia coli</i> (ExPEC) causes bloodstream infections in humans and animals. Complement escape is a prerequisite for bacteria to survive in the bloodstream. Factor H (FH) is an important regulatory protein of the complement system. In this study, ExPEC was found to bind FH from serum. However, the mechanisms of ExPEC binding to FH and then resistance to complement-mediated attacks remain unclear. Here, a method that combined desthiobiotin pull-down and liquid c ...[more]

PMID: 33569353

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Project description:Flagellum-mediated bacterial motility is important for bacteria to take up nutrients, adapt to environmental changes, and establish infection. The twin-arginine translocation system (Tat) is an important protein export system, playing a critical role in bacterial physiology and pathogenesis. It has been observed for a long time that the Tat system is critical for bacterial motility. However, the underlying mechanism remains unrevealed. In this study, a comparative transcriptomics analysis was performed with extraintestinal pathogenic Escherichia coli (ExPEC), which identified a considerable number of genes differentially expressed when the Tat system was disrupted. Among them, a large proportion of flagellar biosynthesis genes showed downregulation, indicating that transcription regulation plays an important role in mediating the motility defects. We further identified three Tat substrate proteins, MdoD, AmiA, and AmiC, that were responsible for the nonmotile phenotype. The Rcs system was deleted in the Δtat, the ΔmdoD, and the ΔamiAΔamiC strains, which restored the motility of ΔmdoD and partially restored the motility of Δtat and ΔamiAΔamiC. The flagella were also observed in all of the ΔtatΔrcsDB, ΔmdoDΔrcsDB, and ΔamiAΔamiCΔrcsDB strains, but not in the Δtat, ΔmdoD, and ΔamiAΔamiC strains, by using transmission electron microscopy. Quantitative reverse transcription-PCR data revealed that the regulons of the Rcs system displayed differential expression in the tat mutant, indicating that the Rcs signaling was activated. Our results suggest that the Rcs system plays an important role in mediating the motility defects of the tat mutant of ExPEC. IMPORTANCE The Tat system is an important protein export system critical for bacterial physiology and pathogenesis. It has been observed for a long time that the Tat system is critical for bacterial motility. However, the underlying mechanism remains unrevealed. In this study, we combine transcriptomics analysis and bacterial genetics, which reveal that transcription regulation plays an important role in mediating the motility defects of the tat mutant of extraintestinal pathogenic Escherichia coli. The Tat substrate proteins responsible for the motility defects are identified. We further show that the Rcs system contributes to the motility suppression. We for the first time reveal the link between the Tat system and bacterial motility, which is important for understanding the physiological functions of the Tat system.

Dataset Information

Factor H Is Bound by Outer Membrane-Displayed Carbohydrate Metabolism Enzymes of Extraintestinal Pathogenic Escherichia coli and Contributes to Opsonophagocytosis Resistance in Bacteria.

Publications

Factor H Is Bound by Outer Membrane-Displayed Carbohydrate Metabolism Enzymes of Extraintestinal Pathogenic <i>Escherichia coli</i> and Contributes to Opsonophagocytosis Resistance in Bacteria.

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