Characterization of HcaA, a novel autotransporter protein inHelicobacter cinaedi, and its role in host cell adhesion

Abstract

ABSTRACTHelicobacter cinaediinfects the human gut and causes invasive infections such as bacteremia and cellulitis by bacterial translocation. However, howH. cinaediattaches to host cells and establishes infection has not been elucidated. In this study, we focused on a novel autotransporter protein, i.e.,H. cinaediautotransporter protein A (HcaA) to examine its relationship withH. cinaedipathogenicity. The cytotoxicity ofH. cinaediinfection in the colon epithelial cell lines (Caco-2 and HT-29) by lactate dehydrogenase assay showed a significant reduction in cytotoxicity by HcaA knockout. Adhesion assays further revealed that the HcaA-knockout strain showed significantly reduced attachment ofH. cinaedito the human epithelial colorectal adenocarcinoma cell line (Caco-2) compared to the wild-type strain. To determine the role of HcaA inH. cinaediinfectionin vivo, H. cinaediwild-type and HcaA-knockout strains were orally infected C57BL/6 mice. The colonized bacteria were then measured 7, 14, and 28-days post-infection. The number of colonizedH. cinaedicells was significantly lower in HcaA-knockout strain infections than in wild-type strain infections at 7 days post-infection. Recombinant HcaA protein showed strong adhesion characteristics to the human monocytic cell line (U937) by adhesion assay used recombinant purified HcaA protein. The adherent activity was diminished by the replacement of the RGD motif in HcaA with RAD, indicating the contribution of the RGD motif in HcaA to host cell adherence. These results suggest that HcaA, a novel autotransporter protein inH. cinaedi, plays a significant role in establishing infection as an adhesin.IMPORTANCEHelicobacterspecies are classified as gastric or enterohepatic according to their habitat. EnterohepaticHelicobacterspecies inhabit the intestine, colon and liver; among them,H. cinaedihas been most frequently isolated from humans.H. cinaedicommonly causes bacteremia and cellulitis in immunocompromised hosts. Here, we focused on theH. cinaediautotransporter protein A (HcaA), a novel virulence factor inH. cinaedi. We discovered that HcaA contributed to cell adhesion via its RGD motif. Furthermore, in animal experiments, the number of colonized bacteria was decreased in HcaA knockout strain infections, supporting the hypothesis that HcaA contributes to the adhesion ofH. cinaedito host cells. Our study provides a novel mechanism for the establishment ofH. cinaediinfections and is expected to provide new insights into the role of autotransporter proteins in the establishment ofHelicobacterspecies infection.