Rapid Identification of Novel Immunodominant Proteins and Characterization of a Specific Linear Epitope of Campylobacter jejuni

Abstract

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.

Identification of Antigenic Proteins of the Nosocomial Pathogen Klebsiella pneumoniae

“…The screening of a cDNA based expression library of K. pneumoniae has successfully identified a number of novel, previously unknown immunogenic proteins. This is well in accordance to previous achievements of this method, which we have employed for the identification of both C. jejuni [5] and S. enterica [6] antigens. Consequently, in this current study we aimed to detect suitable proteins for the specific identification of K. pneumoniae.…”

“…Recently, we have successfully employed this approach to unveil immunogenic proteins for both Campylobacter jejuni [5] and Salmonella enterica [6].…”

A 3q gene signature associated with triple negative breast cancer organ specific metastasis and response to neoadjuvant chemotherapy

“…Raw CEL files of GSE25066 (n = 508) were downloaded from the NCBI Gene Expression Omnibus (GEO) database and normalized using frozen Robust Multi-Array Analysis (fRMA) method, a procedure that allows one to pre-process microarrays individually or in small batches and to then combine the data into a single comparable dataset for further analyses3031. The other 4,164 breast cancer gene expression profiles from 25 breast cancer datasets (GSE11121, GSE12093, GSE12276, GSE1456, GSE16391, GSE16446, GSE17705, GSE19615, GSE20194,GSE20271, GSE2034, GSE20685, GSE20711, GSE21653, GSE25066,GSE2603, GSE26971, GSE31519, GSE3494, GSE42568, GSE45255, GSE4922, GSE5327, GSE6532, GSE7390 and GSE9195) were obtained on the Affymetrix U133A or U133 Plus 2.0 expression array1130313233343536373839404142434445464748495051525354555657. These samples were collected from InsilicoDB database58 and normalized using fRMA method.…”

Epitope mapping of Campylobacter jejuni flagellar capping protein (FliD) by chicken (Gallus gallus domesticus) sera

“…[25][26][27]. Two recent studies further explored the specificity of immune responses to the Campylobacter FlaA and cj0669 proteins [28,29]. However, little information about the immune responses of chickens to other flagellar proteins, including FliD, is available.…”