Supplementary Material for: Lactic Acid Bacteria as a Surface Display Platform for Campylobacter jejuni Antigens

<p><b><i>Background:</i></b> Food poisoning and diarrheal diseases continue to pose serious health care and socioeconomic problems worldwide. <i>Campylobacter</i> spp. is a very widespread cause of gastroenteritis. Over the past decade there has been increasing interest in the use of lactic acid bacteria (LAB) as mucosal delivery vehicles. They represent an attractive opportunity for vaccination in addition to vaccination with attenuated bacterial pathogens. <b><i>Methods:</i></b> We examined the binding ability of hybrid proteins to nontreated or trichloroacetic acid (TCA)-pretreated LAB cells by immunofluorescence and Western blot analysis. <b><i>Results:</i></b> In this study we evaluated the possibility of using GEM (Gram-positive enhancer matrix) particles of <i>Lactobacillus salivarius</i> as a binding platform for 2 conserved, immunodominant, extracytoplasmic <i>Campylobacter jejuni</i> proteins: CjaA and CjaD. We analyzed the binding ability of recombinant proteins that contain <i>C. jejuni</i> antigens (CjaA or CjaD) fused with the protein anchor (PA) of the <i>L. lactis </i>peptidoglycan hydrolase AcmA, which comprises 3 LysM motifs and determines noncovalent binding to the cell wall peptidoglycan. Both fused proteins, i.e. 6HisxCjaAx3LysM and 6HisxCjaDx3LysM, were able to bind to nontreated or TCA-pretreated <i>L. salivarius</i> cells. <b><i>Conclusion:</i></b> Our results documented that the LysM-mediated binding system allows us to construct GEM particles that present 2 <i>C. jejuni</i> antigens.</p

Interplay between DsbA1, DsbA2 and C8J1298 Periplasmic Oxidoreductases of Campylobacter jejuni and Their Impact on Bacterial Physiology and Pathogenesis

The bacterial proteins of the Dsb family catalyze the formation of disulfide bridges between cysteine residues that stabilize protein structures and ensure their proper functioning. Here, we report the detailed analysis of the Dsb pathway of Campylobacter jejuni. The oxidizing Dsb system of this pathogen is unique because it consists of two monomeric DsbAs (DsbA1 and DsbA2) and one dimeric bifunctional protein (C8J_1298). Previously, we showed that DsbA1 and C8J_1298 are redundant. Here, we unraveled the interaction between the two monomeric DsbAs by in vitro and in vivo experiments and by solving their structures and found that both monomeric DsbAs are dispensable proteins. Their structures confirmed that they are homologs of EcDsbL. The slight differences seen in the surface charge of the proteins do not affect the interaction with their redox partner. Comparative proteomics showed that several respiratory proteins, as well as periplasmic transport proteins, are targets of the Dsb system. Some of these, both donors and electron acceptors, are essential elements of the C. jejuni respiratory process under oxygen-limiting conditions in the host intestine. The data presented provide detailed information on the function of the C. jejuni Dsb system, identifying it as a potential target for novel antibacterial molecules