Andrew E. Scott

Flagellar glycosylation in Burkholderia pseudomallei and Burkholderia thailandensis.

Glycosylation of proteins is known to impart novel physical properties and biological roles to proteins from both eukaryotes and prokaryotes. In this study, gel-based glycoproteomics were used to identify glycoproteins of the potential biothreat agent Burkholderia pseudomallei and the closely related but nonpathogenic B. thailandensis. Top-down and bottom-up mass spectrometry (MS) analyses identified that the flagellin proteins of both species were posttranslationally modified by novel glycans. Analysis of proteins from two strains of each species demonstrated that B. pseudomallei flagellin proteins were modified with a glycan with a mass of 291 Da, while B. thailandensis flagellin protein was modified with related glycans with a mass of 300 or 342 Da. Structural characterization of the B. thailandensis carbohydrate moiety suggests that it is an acetylated hexuronic acid. In addition, we have identified through mutagenesis a gene from the lipopolysaccharide (LPS) O-antigen biosynthetic cluster which is involved in flagellar glycosylation, and inactivation of this gene eliminates flagellar glycosylation and motility in B. pseudomallei. This is the first report to conclusively demonstrate the presence of a carbohydrate covalently linked to a protein in B. pseudomallei and B. thailandensis, and it suggests new avenues to explore in order to examine the marked differences in virulence between these two species.

Characterization of the Burkholderia pseudomallei K96243 Capsular Polysaccharide I Coding Region

ABSTRACT Burkholderia pseudomallei is the causative agent of melioidosis, a disease endemic to regions of Southeast Asia and Northern Australia. Both humans and a range of other animal species are susceptible to melioidosis, and the production of a group 3 polysaccharide capsule in B. pseudomallei is essential for virulence. B. pseudomallei capsular polysaccharide (CPS) I comprises unbranched manno-heptopyranose residues and is encoded by a 34.5-kb locus on chromosome 1. Despite the importance of this locus, the role of all of the genes within this region is unclear. We inactivated 18 of these genes and analyzed their phenotype using Western blotting and immunofluorescence staining. Furthermore, by combining this approach with bioinformatic analysis, we were able to develop a model for CPS I biosynthesis and export. We report that inactivating gmhA, wcbJ, and wcbN in B. pseudomallei K96243 retains the immunogenic integrity of the polysaccharide despite causing attenuation in the BALB/c murine infection model. Mice immunized with the B. pseudomallei K96243 mutants lacking a functional copy of either gmhA or wcbJ were afforded significant levels of protection against a wild-type B. pseudomallei K96243 challenge.

Burkholderia pseudomallei Capsular Polysaccharide Conjugates Provide Protection against Acute Melioidosis

ABSTRACT Burkholderia pseudomallei, the etiologic agent of melioidosis, is a CDC tier 1 select agent that causes severe disease in both humans and animals. Diagnosis and treatment of melioidosis can be challenging, and in the absence of optimal chemotherapeutic intervention, acute disease is frequently fatal. Melioidosis is an emerging infectious disease for which there are currently no licensed vaccines. Due to the potential malicious use of B. pseudomallei as well as its impact on public health in regions where the disease is endemic, there is significant interest in developing vaccines for immunization against this disease. In the present study, type A O-polysaccharide (OPS) and manno-heptose capsular polysaccharide (CPS) antigens were isolated from nonpathogenic, select-agent-excluded strains of B. pseudomallei and covalently linked to carrier proteins. By using these conjugates (OPS2B1 and CPS2B1, respectively), it was shown that although high-titer IgG responses against the OPS or CPS component of the glycoconjugates could be raised in BALB/c mice, only those animals immunized with CPS2B1 were protected against intraperitoneal challenge with B. pseudomallei. Extending upon these studies, it was also demonstrated that when the mice were immunized with a combination of CPS2B1 and recombinant B. pseudomallei LolC, rather than with CPS2B1 or LolC individually, they exhibited higher survival rates when challenged with a lethal dose of B. pseudomallei. Collectively, these results suggest that CPS-based glycoconjugates are promising candidates for the development of subunit vaccines for immunization against melioidosis.

Protection against Experimental Melioidosis following Immunization with Live Burkholderia thailandensis Expressing a manno-Heptose Capsule

ABSTRACT Melioidosis is a severe infectious disease caused by Burkholderia pseudomallei. It is highly resistant to antibiotic treatment, and there is currently no licensed vaccine. Burkholderia thailandensis is a close relative of Burkholderia pseudomallei but is essentially avirulent in mammals. In this report, we detail the protective efficacy of immunization with live B. thailandensis E555, a strain which has been shown to express an antigenic capsule similar to that of B. pseudomallei. Immunization with E555 induced significant protection against a lethal intraperitoneal B. pseudomallei challenge in a mouse model of infection, with no mice succumbing to infection over the course of the study, even with challenges of up to 6,000 median lethal doses. By comparison, mice immunized with B. thailandensis not expressing a B. pseudomallei-like capsule had significantly decreased levels of protection. E555-immunized mice had significantly higher levels of IgG than mice immunized with noncapsulated B. thailandensis, and these antibody responses were primarily directed against the capsule.

Protection against Experimental Melioidosis following Immunisation with a Lipopolysaccharide-Protein Conjugate

Melioidosis is a severe infectious disease caused by Burkholderia pseudomallei. It is refractory to antibiotic treatment and there is currently no licensed vaccine. In this report we detail the construction and protective efficacy of a polysaccharide-protein conjugate composed of B. pseudomallei lipopolysaccharide and the Hc fragment of tetanus toxin. Immunisation of mice with the lipopolysaccharide-conjugate led to significantly reduced bacterial burdens in the spleen 48 hours after challenge and afforded significant protection against a lethal challenge with B. pseudomallei. The conjugate generated significantly higher levels of antigen-specific IgG1 and IgG2a than in lipopolysaccharide-immunised mice. Immunisation with the conjugate also demonstrated a bias towards Th1 type responses, evidenced by high levels of IgG2a. In contrast, immunisation with unconjugated lipopolysaccharide evoked almost no IgG2a demonstrating a bias towards Th2 type responses. This study demonstrates the effectiveness of this approach in the development of an efficacious and protective vaccine against melioidosis.

Immunisation with proteins expressed during chronic murine melioidosis provides enhanced protection against disease.

There is an urgent need for an effective vaccine against human disease caused by Burkholderia pseudomallei, and although a wide range of candidates have been tested in mice none provide high level protection. We considered this might reflect the inability of these vaccine candidates to protect against chronic disease. Using Q-RT PCR we have identified 6 genes which are expressed in bacteria colonising spleens and lungs of chronically infected mice. Three of the genes (BPSL1897, BPSL3369 and BPSL2287) have been expressed in Escherichia coli and the encoded proteins purified. We have also included BPSL2765, a protein known to induce immune responses associated with a reduced incidence of chronic/recurrent disease in humans. Immunisation of mice with a combination of these antigens resulted in the induction of antibody responses against all of the proteins. Compared with mice immunised with capsular polysaccharide or LolC protein, mice immunised with the combination of chronic stage antigens showed enhanced protection against experimental disease in mice.

Protection against Experimental Melioidosis with a Synthetic manno-Heptopyranose Hexasaccharide Glycoconjugate

Melioidosis is an emerging infectious disease caused by Burkholderia pseudomallei and is associated with high morbidity and mortality rates in endemic areas. Antibiotic treatment is protracted and not always successful; even with appropriate therapy, up to 40% of individuals presenting with melioidosis in Thailand succumb to infection. In these circumstances, an effective vaccine has the potential to have a dramatic impact on both the scale and the severity of disease. Currently, no vaccines are licensed for human use. A leading vaccine candidate is the capsular polysaccharide consisting of a homopolymer of unbranched 1→3 linked 2-O-acetyl-6-deoxy-β-d-manno-heptopyranose. Here, we present the chemical synthesis of this challenging antigen using a novel modular disaccharide assembly approach. The resulting hexasaccharide was coupled to the nontoxic Hc domain of tetanus toxin as a carrier protein to promote recruitment of T-cell help and provide a scaffold for antigen display. Mice immunized with the glycoconjugate developed IgM and IgG responses capable of recognizing native capsule, and were protected against infection with over 120 × LD50 of B. pseudomallei strain K96243. This is the first report of the chemical synthesis of an immunologically relevant and protective hexasaccharide fragment of the capsular polysaccharide of B. pseudomallei and serves as the rational starting point for the development of an effective licensed vaccine for this emerging infectious disease.

Characterization of secreted sphingosine-1-phosphate lyases required for virulence and intracellular survival of Burkholderia pseudomallei

Sphingosine‐1‐phosphate (S1P), a bioactive sphingolipid metabolite, plays a critical role in the orchestration of immune responses. S1P levels within the mammalian host are tightly regulated, in part through the activity of S1P lyase (S1PL) which catalyses its irreversible degradation. Herein, we describe the identification and characterization of secreted S1PL orthologues encoded by the facultative intracellular bacteria Burkholderia pseudomallei and Burkholderia thailandensis. These bacterial orthologues exhibited S1PL enzymatic activity, functionally complemented an S1PL‐deficient yeast strain and conferred resistance to the antimicrobial sphingolipid D‐erythro‐sphingosine. We report that secretion of these bacterial S1PLs is pH‐dependent, and is observed during intracellular infection. S1PL‐deficient mutants displayed impaired intracellular replication in murine macrophages (associated with an inability to evade the maturing phagosome) and were significantly attenuated in murine and larval infection models. Furthermore, treatment of Burkholderia‐infected macrophages with either S1P or a selective agonist of S1P receptor 1 enhanced bacterial colocalisation with LAMP‐1 and reduced their intracellular survival. In summary, our studies confirm bacterial‐encoded S1PL as a critical virulence determinant of B. pseudomallei and B. thailandensis, further highlighting the pivotal role of S1P in host‐pathogen interactions. In addition, our data suggest that S1P pathway modulators have potential for the treatment of intracellular infection.

Survival protein A is essential for virulence in Yersinia pestis

Plague is a highly pathogenic disease caused by the bacterium Yersinia pestis. There is currently no vaccine available for prophylaxis and antibiotic resistant strains have been isolated, thus there is a need for the development of new countermeasures to treat this disease. Survival protein A (SurA) is a chaperone that has been linked to virulence in several species of bacteria, including the close relative Yersinia pseudotuberculosis. In this study, we aimed to evaluate the role of SurA in virulence of the highly pathogenic Y. pestis by creating an unmarked surA deletion mutant. The Y. pestis ΔsurA mutant was found to be more susceptible to membrane perturbing agents and was completely avirulent in a mouse infection model when delivered up to 2.1 × 10(5) CFU by the subcutaneous route. This provides strong evidence that SurA would make a promising antimicrobial target.

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

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.