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Cloning, Expression, Purification, Regulation, and Subcellular Localization of a Mini-protein from Campylobacter jejuni

Significance Statement

Campylobacter is among the leading causes of bacterial gastroenteritis worldwide. The related disease named campylobacteriosis, affects about 200,000 humans in the European Union each year, which costs about EUR 2.4 billion to public health systems. Despite its impact on human health, this bacteria remains poorly studied and its proteome has not been fully characterized.

The goal of our study was to characterize Cj1169c, a mini-protein from Campylobacter that have never been studied. For this purpose, we cloned the gene Cj1169c in an expression vector in Escherichia coli. Next, we purified the protein which served to produce Cj1169c-specific antibodies that have been used to detect the protein.

Our study has revealed for the first time that Cj1169c is really produced in Campylobacter. We studied the prevalence of this protein in several species of Campylobacter. We found that this protein is only produced in C. jejuni and in C. lari which preferentially colonize birds but not in C. coli (colonizes preferentially pigs). Then, we identified the best growing conditions for Cj1169c protein production. Besides, we demonstrated by several methods that this protein is located in the periplasmic compartment of the bacteria, and could establish via its two cysteines intra- or inter- chain disulfide bridges and may interact with one or more putative partners. According to these data, the Cj1169c protein may be an essential determinant for host colonization.

Cloning, Expression, Purification, Regulation, and Subcellular Localization of a Mini-protein from Campylobacter jejuni. Global Medical Discovery

About The Author

Soumeya Aliouane is a Ph.D student, in Human Pathology; Vascular Pathology and Nutrition at the UMR-MD1, school of medicine, Aix Marseille University, Marseille, France.

About The Author

Dr. Jean-Marie Pagès defended his Doctorat Es-Sciences in 1983. He is Research Director at the French National Institute of Health. JM Pagès has chaired the COST Action BM0701 “Antibiotic transport and efflux” (2008-2012). Heading the UMR_MD1 in Aix-Marseille University, he actively participates in IMI-ND4BB “Molecular basis of the bacterial cell wall permeability” and ITN-Marie Sklodowska-Curie, “Translocation”. He is Member of the SAC of Synchrotron Soleil (Gif/Yvette, France), of the SAC of Instituto de Higiene e Medicina Tropical (Lisboa, Portugal), expert for NSF-USA, NIH-USA, NSERC-Canada, NMRC-Singapour, 7th PCRDT, etc. He has published over 300 scientific papers, abstracts, patents, and presented lectures in bacteriology-biochemistry-chemistry field.

About The Author

Dr. Jean-Michel Bolla received his Ph.D degree in 1990 from Aix-Marseille University in France, working on the assembly of porins in E. coli. Then, he worked at the Necker Faculty of Medicine in Paris-France, for 2 years as a postdoctoral fellow and studied the iron transport by Listeria monocytogenes. He obtained a permanent position in 1992 at the French National Institute of Health as a research assistant. JM Bolla works on Campylobacter since more than 20 years. His main research interest is the envelope of Gram Negative Bacteria, and he recently focused his activity on antibiotic transport through bacterial envelope. He is now head of the team « New Molecules and new targets » of the UMR-MD1.

Journal Reference

Curr Microbiol. 2016 Jan 11.

Soumeya Aliouane, Jean-Marie Pagès, and Jean-Michel Bolla

Aix-Marseille Université, IRBA, TMCD2 UMR-MD1, Faculté de Médecine, 13385 Marseille, France.

e-mail: [email protected]

Abstract: 

The Cj1169c-encoded putative protein of Campylobacter was expressed and purified from E. coli after sequence optimization. The purified protein allowed production of a specific rabbit antiserum that was used to study the protein expression in vitro and its subcellular localization in the bacterial cell and putative interactions with other proteins. This protein is produced in Campylobacter and it clearly localizes into the periplasmic space. The level of protein production depends on factors, including pH, temperature, osmolarity and growth phase suggesting a role in the Campylobacter environmental adaptation. The cysteine residues present in the sequence are probably involved in disulfide bridges, which may promote covalent interactions with other proteins of the Campylobacter envelope..

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