Final Report Summary - CAMPY SECRETION (Comparative Proteomics and Function of Secreted Proteins of Campylobacter jejuni) Background The bacterial pathogen Campylobacter jejuni (C. jejuni) is the principal cause of bacterial foodborne infections and is estimated to cause > 100 million episodes of enteritis worldwide each year. Despite the high incidence of infection, the bacterial factors that cause the disease are still largely unknown. Many enteropathogenic bacteria cause diarrhea. The goal of this project was to identify toxic factors that are secreted by C. jejuni with the ultimate goal to facilitate rational drug / vaccine design. Main results Analysis of Campylobacter secretion profiles Protein profiles of wild type C. jejuni were compared with those of mutants with defects in different protein export systems. Secretion deficient strains included mutants with defects in type II, flagellar (type III) and Tat secretion systems. This analysis yielded over 20 different proteins that were actively secreted into the medium dependent on the growth conditions. Mass spectrometry was used to identify the nature of the proteins. More than 80 % of identified proteins were secreted through the flagellar export apparatus. This group of proteins contained flagellar proteins such as FlaA, FlaB, FlaC as well as FspA and several proteins with unknown function. Functional analysis of secreted proteins Several of the secreted proteins were expressed in E. coli. Antibodies were raised against the purified proteins for identification and tracking in biological assays. Proteins purified in this way included the putative adhesin FlaC. In a complementary approach targeted mutagenesis was employed to construct genetically defined mutants. Both the mutants and purified FlaC protein were tested for interaction with cultured epithelial cells. Purified FlaC supposed to bind to the hsp90 receptor on host cells, showed no interaction with epithelial cells. Binding assays for FspA are in progess. The mutant strains showed unaltered invasion in the cell culture infection model. These results suggest that under the in vitro conditions employed, the tested secreted proteins were not essential for cell damage or to establish cellular infection. Identification of a novel type VI secretion system (T6SS) One of the secreted hypothetical C. jejuni proteins showed some similarity with components of type VI secretion system, thus far not reported to be present in C. jejuni. T6SS proteins often modulate host cell function and/or facilitate bacterial replication in a complex microbiological environment. We discovered a fully functional T6SS in C. jejuni. Constructed mutants defective in distinct part of the T6SS failed to secrete the T6SS proteins. The secreted T6SS proteins influence the function of eukaryotic cells. The exact molecular mechanism of secretion is currently under investigation. Inhibitors of protein secretion To better understand the protein secretion process, we tested candidate drugs that influence the function of type IV secretion systems in other pathogens. Testing of C. jejuni showed altered protein secretion in the presence of distinct drugs. Infection assays showed a drug-dependent reduction of C. jejuni infection of epithelial cells, suggesting that the affected proteins are important for cellular infection. Electrophoresis and mass spectrometry confirmed that the main alterations in protein expression profiles. Infection assays in the presence and absence of these drugs showed a drug-dependent reduction of C. jejuni infection of epithelial cells, suggesting that the affected proteins are important for cellular infection. Drug-affinity chromatography in conjunction with mass spectrometry will be applied to identify the drug target. These experiments are beyond the timeframe of the current project and will be a topic of future research. Conclusions and impact Our results indicate that the major foodborne pathogen C. jejuni secretes a series of proteins into the environment via different export systems. For the first time, a functional type VI secretion system was identified and shown to affect the function of eukaryotic cells. Drug inhibitors showed to be effective tools to manipulate the secretion of proteins identification of the drug target may facilitate development of a novel class of bacterial virulence blocking compounds that can be used against bacterial pathogens.