Identification of the Major Antigenic Protein of Helicobacter cinaedi and the Immunogenicity During Infections in Humans

Department of Microbiology and Department of Gastroenterology and Hepatology, Graduate School of Medical Sciences, Kumamoto University, Kumamoto 860-8556, Japan, Department of Microbiology, School of Pharmacy, Aichi Gakuin University, Nagoya 464-8650, Japan, Chemo-Sero-Therapeutic Research Institute, Kumamoto 860-8568, Japan, Department of Infectious Diseases, Faculty of Medicine, Oita University, Oita 879-5593, Japan, and Kumamoto Orthopedic Hospital, Kumamoto 862-0976, Japan

^* To whom correspondence should be addressed. Email: takakaik{at}gpo.kumamoto-u.ac.jp.

	Abstract

Helicobacter cinaedi infection is now recognized as an increasingly important emerging disease. Its pathogenesis and epidemiological features are not fully understood, however. Here, we investigated the antigenic protein of H. cinaedi and the immunological response to it in H. cinaedi-infected patients. We constructed a genomic library of H. cinaedi from an H. cinaedi clinical isolate, and various H. cinaedi recombinant proteins were expressed. We identified the 30-kDa protein, encoded in an 822-bp H. cinaedi genome, as a major antigen, which was specifically recognized by serum from an H. cinaedi-immunized rabbit and H. cinaedi-infected patients. The gene encoding this 30-kDa antigen had high sequence similarity with genes encoding putative membrane proteins of bacteria. To evaluate whether the 30-kDa protein can be applied in serological testing for H. cinaedi infections, the recombinant protein was expressed in Escherichia coli as an His-tagged fusion protein and purified by Ni²⁺ affinity chromatography. Western blot analysis revealed strong immunoreactivity of the 31-kDa fusion protein with serum antibody from patients infected with H. cinaedi, but such an immunoreaction was absent or was very weak with uninfected control serum. An enzyme-linked immunosorbent assay using this H. cinaedi major antigen showed significantly higher antibody titers for H. cinaedi-infected subjects compared with various control groups. We therefore conclude that the 30-kDa putative membrane protein is a major antigen of H. cinaedi and is useful for immunological and serological testing for clinical diagnosis and for further epidemiological study of H. cinaedi infection in humans.