Identification and functional characterization of putative ligand binding domain(s) of JlpA protein of Campylobacter jejuni.

Among the major Surface Exposed Colonization Proteins (SECPs) of Campylobacter jejuni (C. jejuni), Jejuni lipoprotein A (JlpA) plays a crucial role in host cell adhesion specifically by binding to the N-terminal domain of the human heat shock protein 90α (Hsp90α-NTD). Although the JlpA binding to Hsp90α activates NF-κB and p38 MAP kinase pathways, the underlying mechanism of JlpA association with the cellular receptor remains unclear. To this end, we predicted two potential receptor binding sites within the C-terminal domain of JlpA: one spanning from amino acid residues Q332-A354 and the other from S258-T295; however, the latter exhibited weaker binding. To assess the functional attributes of these predicted sequences, we generated two JlpA mutants (JlpAΔ1: S258-T295; JlpAΔ2: Q332-A354) and assessed the Hsp90α-binding affinity-kinetics by in vitro and ex vivo experiments. Our findings confirmed that the residues Q332-A354 are of greater importance in host cell adhesion with a measurable impact on cellular responses. Further, thermal denaturation by circular dichroism (CD) confirmed that the reduced binding affinity of the JlpAΔ2 to Hsp90α is not associated with protein folding or stability. Together, this study provides a possible framework for determining the molecular function of designing rational inhibitors efficiently targeting JlpA.

Bioengineering of LAB vector expressing Haemolysin co-regulated protein (Hcp): a strategic approach to control gut colonization of Campylobacter jejuni in a murine model.

BACKGROUND: Campylobacter jejuni (C. jejuni) is accountable for more than 400 million cases of gastroenteritis each year and is listed as a high-priority gut pathogen by the World Health Organization (WHO). Although the acute infection of C. jejuni (campylobacteriosis) is commonly treated with macrolides and fluoroquinolones, the emergence of antibiotic resistance among C. jejuni warrants the need for an alternative approach to control campylobacteriosis in humans. To this end, vaccines remain a safe, effective, and widely accepted strategy for controlling emerging and re-emerging infectious diseases. In search of a suitable vaccine against campylobacteriosis, recently, we demonstrated the potential of recombinant Haemolysin co-regulated protein (Hcp) of C. jejuni Type VI secretion system (T6SS) in imparting significant immune-protection against cecal colonization of C. jejuni; however, in the avian model. Since clinical features of human campylobacteriosis are more complicated than the avians, we explored the potential of Hcp as a T6SS targeted vaccine in a murine model as a more reliable and reproducible experimental host to study vaccine-induced immune-protection against C. jejuni. Because C. jejuni primarily utilizes the mucosal route for host pathogenesis, we analyzed the immunogenicity of a mucosally deliverable bioengineered Lactic acid bacteria (LAB), Lactococcus lactis (L. lactis), expressing Hcp. Considering the role of Hcp in both structural (membrane-bound) and functional (effector protein) exhibition of C. jejuni T6SS, a head-to-head comparison of two different forms of recombinant LAB vectors (cell wall anchored and secreted form of Hcp) were tested and assessed for the immune phenotypes of each modality in BALB/c mice. RESULTS: We show that regardless of the Hcp protein localization, mucosal delivery of bioengineered LAB vector expressing Hcp induced high-level production of antigen-specific neutralizing antibody (sIgA) in the gut with the potential to reduce the cecal load of C. jejuni in mice. CONCLUSION: Together with the non-commensal nature of L. lactis, short gut transit time in humans, and the ability to express the heterologous protein in the gut, the present study highlights the benefits of bioengineered LAB vectors based mucosal vaccine modality against C. jejuni without the risk of immunotolerance.

Mucosal delivery of live Lactococcus lactis expressing functionally active JlpA antigen induces potent local immune response and prevent enteric colonization of Campylobacter jejuni in chickens.

Successful colonization of the mucosal epithelial cells is the key early step for Campylobacter jejuni (C. jejuni) pathogenesis in humans. A set of Surface Exposed Colonization Proteins (SECPs) are known to take leading role in bacterial adhesion and subsequent host pathogenesis. Among the major SECPs, the constitutively expressed C. jejuni surface lipoprotein Jejuni lipoprotein A (JlpA), interacts with intestinal heat shock protein 90α (Hsp90α) and contributes in disease progression by triggering pro-inflammatory responses via activation of NF-κB and p38 MAP kinase pathways. In addition to its ability to express on the surface, high sequence conservation of JlpA protein among different Campylobacter spp make it a suitable vaccine target against C. jejuni. Given that chickens are the primary source for C. jejuni infection in humans and persistent cecal colonization significantly contribute in pathogen transmission, we explicitly used chickens as a model to test the immune-protective efficacy of JlpA protein. Taking into account that gastro-intestinal tract is the major site for C. jejuni colonization, we chose to use mucosal (intragastric) route as mode for JlpA antigen delivery. To deliver JlpA via mucosal route, we engineered a food grade Lactic acid producing bacteria, Lactococcus lactis (L. lactis) to express functionally active JlpA protein in the surface. Further, we demonstrated its ability to substantially improve the antigen specific local immune responses in the intestine along with significant immune-protection against enteric colonization of C. jejuni in chickens.