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Characterization and localization of the Campylobacter jejuni transformation system proteins CtsE, CtsP, and CtsX.

ABSTRACT: The human pathogen Campylobacter jejuni is naturally competent for transformation with its own DNA. Genes required for efficient transformation in C. jejuni include those similar to components of type II secretion systems found in many Gram-negative bacteria (R. S. Wiesner, D. R. Hendrixson, and V. J. DiRita, J Bacteriol 185:5408-5418, 2003, http://dx.doi.org/10.1128/JB.185.18.5408-5418.2003). Two of these, ctsE and ctsP, encode proteins annotated as putative nucleotide binding nucleoside triphosphatases (NTPases) or nucleoside triphosphate (NTP) binding proteins. Here we demonstrate that the nucleotide binding motifs of both proteins are essential for their function in transformation of C. jejuni. Localization experiments demonstrated that CtsE is a soluble protein while CtsP is membrane associated in C. jejuni. A bacterial two-hybrid screen identified an interaction between CtsP and CtsX, an integral membrane protein also required for transformation. Topological analysis of CtsX by the use of LacZ and PhoA fusions demonstrated it to be a bitopic, integral membrane protein with a cytoplasmic amino terminus and a periplasmic carboxyl terminus. Notwithstanding its interaction with membrane-localized CtsX, CtsP inherently associates with the membrane, requiring neither CtsX nor several other Cts proteins for this association.

SUBMITTER: Beauchamp JM

PROVIDER: S-EPMC4285969 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Campylobacter jejuni remains one of the major gut pathogens of our time. Its zoonotic nature and wide-spread distribution in industrialized countries calls for a quick and reliable diagnostic tool. Antibody-based detection presents a suitable means to identify pathogenic bacteria. However, the knowledge about immunodominant targets is limited. Thus, an approach is presented, which allows for the rapid screening of numerous cDNA derived expression clones to identify novel antigens. The deeper understanding of immunodominant proteins assists in the design of diagnostic tools and furthers the insight into the bacterium's pathogenicity as well as revealing potential candidates for vaccination. We have successfully screened 1536 clones of an expression library to identify 22 proteins that have not been described as immunodominant before. After subcloning the corresponding 22 genes and expression of full-length proteins, we investigated the immunodominant character by microarrays and ELISA. Subsequently, seven proteins were selected for epitope mapping. For cj0669 and cj0920c linear epitopes were identified. For cj0669, specificity assays revealed a specific linear epitope site. Consequently, an eleven amino acid residue sequence TLIKELKRLGI was analyzed via alanine scan, which revealed the glycine residue to be significant for binding of the antibody. The innovative approach presented herein of generating cDNAs of prokaryotes in combination with a microarray platform rendering time-consuming purification steps obsolete has helped to illuminate novel immunodominant proteins of C.jejuni. The findings of a specific linear epitope pave the way for a plethora of future research and the potential use in diagnostic applications such as serological screenings. Moreover, the current approach is easily adaptable to other highly relevant bacteria making it a formidable tool for the future discovery of antigens and potential biomarkers. Consequently, it is desirable to simplify the identification of structural epitopes, as this would extend the spectrum of novel epitopes to be detected.

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Characterization and localization of the Campylobacter jejuni transformation system proteins CtsE, CtsP, and CtsX.

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