SummaryClostridium difficile expresses a surface layer (S-layer) which coats the surface of the bacterium and acts as an adhesin facilitating interaction of the bacterium with host enteric cells. The S-layer contains a high-molecular-weight S-layer protein (HMW SLP) and its low-molecular-weight partner protein (LMW SLP). We show that these proteins form a tightly associated non-covalent complex, the H/L complex, and we identify the regions of both proteins responsible for complex formation. The 2.4 Å X-ray crystal structure of a truncated derivative of the LMW SLP reveals two domains. Domain 1 has a two-layer sandwich architecture while domain 2, predicted to orientate towards the external environment, contains a novel fold. Small-angle X-ray scattering analysis of the H/L complex shows an elongated molecule, with the two SLPs arranged 'end-to-end' interacting with each other through a small contact area. Alignment of LMW SLPs, which exhibit high sequence diversity, reveals a core of conserved residues that could reflect functional conservation, while allowing for immune evasion through sequence variation. These structures are the first described for the S-layer of a bacterial pathogen, and provide insights into the assembly and biogenesis of the S-layer.

B. pseudomallei, and other members of the Burkholderia, are among the most antibiotic-resistant bacterial species encountered in human infection. Mortality rates associated with severe B. pseudomallei infection approach 50% despite therapeutic treatment. A protective vaccine against B. pseudomallei would dramatically reduce morbidity and mortality in endemic areas and provide a safeguard for the U.S. and other countries against biological attack with this organism. In this study, we investigated the immunogenicity and protective efficacy of B. pseudomallei-derived outer membrane vesicles (OMVs). Vesicles are produced by Gram-negative and Gram-positive bacteria and contain many of the bacterial products recognized by the host immune system during infection. We demonstrate that subcutaneous (SC) immunization with OMVs provides significant protection against an otherwise lethal B. pseudomallei aerosol challenge in BALB/c mice. Mice immunized with B. pseudomallei OMVs displayed OMV-specific serum antibody and T-cell memory responses. Furthermore, OMV-mediated immunity appears species-specific as cross-reactive antibody and T cells were not generated in mice immunized with E. coli-derived OMVs. These results provide the first compelling evidence that OMVs represent a non-living vaccine formulation that is able to produce protective humoral and cellular immunity against an aerosolized intracellular bacterium. This vaccine platform constitutes a safe and inexpensive immunization strategy against B. pseudomallei that can be exploited for other intracellular respiratory pathogens, including other Burkholderia and bacteria capable of establishing persistent infection.

Burkholderia mallei are Gram-negative bacteria, responsible for the disease glanders. B. mallei has recently been classified as a Tier 1 agent owing to the fact that this bacterial species can be weaponised for aerosol release, has a high mortality rate and demonstrates multi-drug resistance. Furthermore, there is no licensed vaccine available against this pathogen. Lipopolysaccharide (LPS) has previously been identified as playing an important role in generating host protection against Burkholderia infection. In this study, we present gold nanoparticles (AuNPs) functionalised with a glycoconjugate vaccine against glanders. AuNPs were covalently coupled with one of three different protein carriers (TetHc, Hcp1 and FliC) followed by conjugation to LPS purified from a non-virulent clonal relative, B. thailandensis. Glycoconjugated LPS generated significantly higher antibody titres and compared with LPS alone. Further, they improved protection against a lethal inhalation challenge of B. mallei in the murine model of infection.

Burkholderia mallei and B. pseudomallei are Gram-negative pathogenic bacteria, responsible for the diseases glanders and melioidosis, respectively. Furthermore, there is currently no vaccine available against these Burkholderia species. In this study, we aimed to identify protective proteins against these pathogens. Immunization with recombinant B. mallei Hcp1 (type VI secreted/structural protein), BimA (autotransporter protein), BopA (type III secreted protein), and B. pseudomallei LolC (ABC transporter protein) generated significant protection against lethal inhaled B. mallei ATCC23344 and B. pseudomallei 1026b challenge. Immunization with BopA elicited the greatest protective activity, resulting in 100% and 60% survival against B. mallei and B. pseudomallei challenge, respectively. Moreover, sera from recovered mice demonstrated reactivity with the recombinant proteins. Dendritic cells stimulated with each of the different recombinant proteins showed distinct cytokine patterns. In addition, T cells from immunized mice produced IFN-γ following in vitro re-stimulation. These results indicated therefore that it was possible to elicit cross-protective immunity against both B. mallei and B. pseudomallei by vaccinating animals with one or more novel recombinant proteins identified in B. mallei.