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

Helicobacter cinaedi infects the human gut and causes invasive infections such as bacteremia and cellulitis through bacterial translocation. However, the mechanism by which H. cinaedi attaches to host cells and establishes infection remains unclear. This study aimed to investigate the relationship between a novel putative autotransporter protein, H. cinaedi autotransporter protein A (HcaA), and its role in pathogenicity. The cytotoxicity of H. cinaedi infection in colon epithelial cell lines (Caco-2 and HT-29) was assessed using a lactate dehydrogenase assay, and it was found that cytotoxicity significantly decreased upon HcaA knockout. Adhesion assays further revealed that the HcaA-knockout strain showed significantly reduced attachment to the human epithelial colorectal adenocarcinoma cell line (Caco-2) compared to that of the wild-type strain. Moreover, the recombinant HcaA protein demonstrated strong adhesion properties to the human monocytic cell line (U937). The adhesive activity was diminished when the RGD motif in HcaA was replaced with RAD, indicating that the RGD motif in HcaA is crucial for host cell adhesion. To determine the role of HcaA in H. cinaedi infection in vivo, C57BL/6 mice were orally infected with wild-type and HcaA-knockout H. cinaedi strains. Bacterial colonization was assessed 7, 14, and 28 days post-infection. At 7 days post-infection, colonization was significantly lower in mice infected with the HcaA-knockout strain compared to those infected with the wild-type strain. In conclusion, our findings suggest that HcaA, a novel putative autotransporter protein in H. cinaedi, plays a significant role as an adhesin in establishing colonization. IMPORTANCEHelicobacter species are classified as gastric or enterohepatic according to their habitat. Among enterohepatic Helicobacter species, which inhabit the intestine, colon and liver, H. cinaedi has been most frequently isolated from humans. H. cinaedi often causes bacteremia and cellulitis in immunocompromised hosts. Here, we focused on the H. cinaedi autotransporter protein A (HcaA), a novel virulence factor in H. cinaedi. We discovered that HcaA contributes to cell adhesion via its RGD motif. Furthermore, in animal experiments, bacterial colonization was reduced in mice infected with HcaA-knockout strains, supporting the hypothesis that HcaA contributes to H. cinaedi adhesion to host cells. Our study provides a novel mechanism for the establishment of H. cinaedi infections and provides new insights into the role of autotransporter proteins in the establishment of Helicobacter infection.