Protein interactome analysis of iduronic acid-containing glycosaminoglycans reveals a novel flagellar invasion factor MbhA

Pathogens are able to exploit specific glycosaminoglycans (GAGs), especially iduronic acid (IdoA)-containing GAGs, to invade the host. By analyzing Escherichia coli proteome chip data, we identified the interactomes of three IdoA-containing GAGs: heparin, heparin sulfate (HS), and chondroitin sulfate B (CSB). Using non-IdoA-containing GAG, chondroitin sulfate C, as a negative control, 157 proteins specifically binding with IdoA-containing GAGs were revealed in the present study. These proteins showed functional enrichment in protein synthesis and metabolism. Fifteen proteins which commonly interacts with three IdoA-containing GAGs were further examined. The regular expression for motif showed these common IdoA interactome shared a conserved sequence. Among them, we identified a second flagellar system outer membrane protein, MbhA. The MbhA has Kd values of 8.9 × 10⁻⁸ M, 5.3 × 10⁻⁷ M, and 1.79 × 10⁻⁷ M to interact with heparin, HS, and CSB, respectively. Using flow cytometry, we confirmed that the MbhA protein can bind to human epithelial cells HCT-8. Overexpression of mbhA increased the percentage of invasion in E. coli which lacks a second flagellar system. Moreover, pre-blocking of HCT-8 cells with MbhA inhibited the bacterial invasion, implying the importance of the direct interaction of MbhA and the host cell surface on bacterial invasion. Significance: We analyzed the Escherichia coli proteomic data to elucidate the interactomes of three different IdoA-containing GAGs (heparin, HS, and CSB) because these IdoA-containing GAGs can mediate bacterial invasion to the host. Through proteomic and systematic analysis, a second flagellar system outer membrane protein, MbhA, was also identified in the present study. Affinity assay confirmed that MbhA can bind to three IdoA-containing GAGs heparin, HS, and CSB. The result of flow cytometry also showed MbhA can interact with human epithelial cells HCT-8. Results of bacteria invasion assay showed overexpression of mbhA promoted the bacterial invasion. Moreover, pre-blocking of HCT-8 cells with MbhA also reduced the percentage of bacterial invasion. These findings correspond well that MbhA is one of invasion factors.