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

Yu Sun,Xiangkai Zhuge,Fang Tang,Rui Chen,Xuhang Wang,Qianwen Gong,Jianjun Dai,Feng Xue,Bin Xu

doi:10.3389/fcimb.2020.592906

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.

Highlights

Extraintestinal pathogenic Escherichia coli (ExPEC) is an important pathogen that causes extraintestinal infections, including urinary tract infection, bloodstream infection, and meningitis in humans and animals (Russo and Johnson, 2000; Zhuge et al, 2018)
There are three activation pathways in the complement system, namely, the classical pathway (CP) that is mediated by antibodies bound to the surface of the pathogen, the lectin pathway (LP) that is mediated by the recognition of carbohydrates on the surface of the pathogen, and the alternative pathway (AP) that is mediated by the spontaneous cleavage of the C3 component (Harboe and Mollnes, 2008)
The OD450 value of the serum-treated group was significantly higher than that of the BSA group (p < 0.5, Figure 1B). These results indicated that ExPEC possessed the ability to bind Factor H (FH) in human serum

Summary

Introduction

Extraintestinal pathogenic Escherichia coli (ExPEC) is an important pathogen that causes extraintestinal infections, including urinary tract infection, bloodstream infection, and meningitis in humans and animals (Russo and Johnson, 2000; Zhuge et al, 2018). Avoiding the defense of host immune system facilitates ExPEC infections. The mechanism by which ExPEC escapes the immune system in bloodstream infections remains unclear. The complement system is an important component of the serum, serving as the critical line of the immune defense system to control bacterial infections (Dalia and Weiser, 2011). When C3b is deposited on the surface of pathogens, it leads to the formation of membrane attack complexes (MACs) that causes the lysis of pathogens (Jozsi et al, 2015). The C3b deposition plays a key role of in complement activation. Factor H (FH), one of the key negative regulators of complement activation, inhibits the formation of C3 convertase and promotes its degradation, thereby reducing the conversion of C3 to C3b. FH assists Factor I cleaving C3b into inactive C3b (Cabezas et al, 2018)

Methods

Results

Conclusion