Structural characterisation of the capsular polysaccharide expressed by Burkholderia thailandensis strain E555:: wbiI (pKnock-KmR) and assessment of the significance of the 2-O-acetyl group in immune protection

Abstract

Burkholderia pseudomallei and its close relative B. mallei are human pathogens that are classified as Tier 1 bio-threat agents. Both organisms have previously been shown to constitutively produce a capsular polysaccharide (CPS) that is both a virulence determinant and protective antigen. Extraction and purification of CPS for use as a potential vaccine candidate requires containment level 3 laboratories which is expensive and time-consuming. B. thailandensis strain E555 is closely related to B. pseudomallei and B. mallei, but is non-pathogenic to humans and based on immunological cross-reactivity has previously been shown to express a B. pseudomallei-like CPS. In this study, capsular polysaccharide isolated from an O-antigen deficient strain of B. thailandensis E555 was identified by 1H and 13C NMR spectroscopy as -3-)-2-O-acetyl-6-deoxy-β-d-manno-heptopyranose-(-1, and identical to that produced by B. pseudomallei. This was further substantiated by anti-CPS monoclonal antibody binding. In connection with the production of CPS fragments for use in glycoconjugate vaccines, we set out to assess the importance or otherwise of the CPS 2-OAc groups in immune protection. To this end conjugates of the native and de-O-acetylated CPS with the Hc fragment of tetanus toxin (TetHc) were used as vaccines in a mouse model of melioidosis. The level of protection provided by deacetylated CPS was significantly lower than that from native, acetylated CPS. In addition, sera from mice vaccinated with the deacetylated CPS conjugate did not recognise native CPS. This suggests that CPS extracted from B. thailandensis can be used as antigen and that the acetyl group is essential for protection.

Presence of B. thailandensis and B. thailandensis expressing B. pseudomallei-like capsular polysaccharide in Thailand, and their associations with serological response to B. pseudomallei

“…pseudomallei are highly similar (94.4% and 96% nucleotide and protein similarity, respectively) [ 17 ], and nuclear magnetic resonance spectroscopy has shown that the structures of CPS produced by E555 and that of B . pseudomallei are identical [ 23 ]. Previously, BTCV has been isolated from human blood in the USA in 2003 (strain CDC3015869; ST101, USA [ 24 ]) and from environmental samples in Cambodia in 2010 (strain E555; ST696), Gabon in 2013 (strain D50; ST1126 [ 25 ]) and Laos in 2015 (strain ST_10; ST696 [ 26 ]).…”

Assessments of hepatitis B virus-like particles and Crm197 as carrier proteins in melioidosis glycoconjugate vaccines

“…To construct the glycoconjugates, extracted CPS from B. thailandensis strain E555 [32] was activated with sodium periodate and covalently linked to Crm197 or GD3K6D3G TCVLPs via reductive amination. Conjugation was confirmed by SDS PAGE and agarose gel with Coomassie staining (Figure 2A and 2B, respectively), which confirms the shift in molecular weight from unconjugated carrier protein to conjugate; and immunogold staining TEM (Figure 3A and 3B), which confirmed presence of CPS immunogenic epitope integrity through binding of an anti-CPS monoclonal.…”

“…The O-PS deficient mutant of B. thailandensis E555 harbouring a kanamycin-resistance marked, in-frame deletion of its wbiI gene ( B. thailandensis E555 :: wbiI (p-Knock KmR)) [31] was grown in 2 L of LB broth overnight at 37°C with shaking. The CPS was extracted via a modified hot-phenol method and purified as described previously [32].…”

Outer Membrane Vesicle Vaccines from Biosafe Surrogates Prevent Acute Lethal Glanders in Mice

“…In this study, strong antibody responses were observed following vaccination with OMVs particularly towards the LPS O-antigen and CPS. Other works have shown high IgG and IgM responses are generated by mice vaccinated with the CPS from B. thailandensis E555 [ 30 ]. The higher immunogenicity of lipid A in B. thailandensis LPS may contribute to better memory responses by inducing stronger local inflammation that attracts more antigen presenting cells.…”

“…Exceptions to the rule have been discovered, where environmental isolates of B. thailandensis strains with a capsule have been identified but remain highly attenuated [ 29 ]. A recent paper has demonstrated that CPS isolated from one such strain, ( B. thailandensis strain E555) can afford significant protection to mice challenged intraperitoneally with B. pseudomallei K96243 [ 30 ]. For the most part, close genetic and antigenic homology means that material from any of the three species can be used to vaccinate against glanders or melioidosis but does not discount the possibility that species-specific antigens might be absent [ 31 , 32 , 33 ].…”