Journal:  Journal of Medical Microbiology

Title: Quantification of cholix exotoxin, an ADP-ribosylating factor in Vibrio cholerae strains by developed sandwich bead-ELISA.

Authors:  Sharda Prasad Awasthi, Nityananda Chowdhury, Noritoshi Hatanaka, Atsushi Hinenoya, Thandavarayan Ramamurthy, Masahiro Asakura and Shinji Yamasaki*

Abstract:
Introduction. Cholix toxin (ChxA) is an ADP-ribosylating exotoxin produced by Vibrio cholerae. However, to date, there is no quantitative assay available for ChxA, which makes it difficult to detect and estimate the level of ChxA produced by V. cholerae.
Hypothesis/Gap Statement. It is important to develop a reliable and specific quantitative assay to measure the production level of ChxA, which will help us to understand the role of ChxA in V. cholerae pathogenesis.
Aim. The aim of this study was to develop a bead-based sandwich ELISA (bead-ELISA) for the quantification of ChxA and to evaluate the importance of ChxA in the pathogenesis of V. cholerae infection.
Methodology. Anti-rChxA was raised in New Zealand white rabbits, and Fab-horse radish peroxidase conjugate was prepared by the maleimide method to use in the bead-ELISA. This anti-ChxA bead-ELISA was applied to quantify the ChxA produced by various V. cholerae strains. The production of ChxA was examined in different growth media such as alkaline peptone water (APW), Luria-Bertani broth and AKI. Finally, the assay was evaluated using a mouse lethality assay with representative V. cholerae strains categorized as low to high ChxA-producers based on anti-ChxA bead-ELISA.
Results. A sensitive bead-ELISA assay, which can quantify from 0.6 to 60 ng/ml of ChxA, was developed. ChxA was mostly detected in the extracellular cell-free supernatant and its production level varied from 1.2 ng/ml to 1.6 µg/ml. The highest ChxA production was observed when V. cholerae strains were cultured in LB broth, but not in APW or AKI medium. The ChxA-producer V. cholerae strains showed 20–80 % lethality and only the high ChxA II-producer was statistically more lethal than a non-ChxA-producer, in the mice model assay. ChxA I and II production levels were not well correlated with mice lethality, and this could be due to the heterogeneity of the strains tested.
Conclusion. ChxA I to III was produced mostly extracellularly at various levels depending on strains and culture conditions. The bead-ELISA developed in this study is useful for the detection and quantification of ChxA in V. cholerae strains.

　コレラ菌は水系感染症コレラの原因菌であり、これまで発展途上国を中心に7回の世界的流行を引き起こしてきた。主な病原因子としては、コレラ毒素や腸管定着に関わるTCP線毛が知られる。しかし、コレラを引き起こすのは、200以上あるO血清型の内O1とO139のみである。残りの血清型のコレラ菌は、non-O1/non-O139コレラ菌と呼ばれ、これらも散発性下痢や旅行者下痢症の他、心内膜炎、関節炎、髄膜炎、敗血症等の様々な腸管外感染症を引き起こす重要な病原菌である。non-O1/non-O139コレラ菌にはコレラ毒素やTCP線毛を持つものは殆どなく、別の病原因子として3型および6型分泌装置、ヘモリシン、耐熱性毒素 (Stn) 等が同定され、non-O1/non-O139コレラ菌が多様な病原性を有することが病原因子プロファイリングからも示されてきた。そんな中、2010年新たにコリックス毒素 (ChxA) が発見された。ChxAはnon-O1/non-O139コレラ菌の47%が保有するとも報告されており (Environ Microbiol Rep 2010;2:198-207)、コレラ菌の病態発現に重要な役割を持つと考えられる。
　本研究ではChxA特異的サンドイッチbead-ELISAを構築し、これによりコレラ菌が産生するChxAを定量することができるようになりました。