Abstract

Vaccines developing immune responses toward surface carbohydrates conjugated to proteins are effective in preventing infection and death by bacterial pathogens. Traditional production of these vaccines utilizes complex synthetic chemistry to acquire and conjugate the glycan to a protein. However, glycoproteins produced by bacterial protein glycosylation systems are significantly easier to produce, and could possible be used as vaccine candidates. In this work, we functionally expressed the Burkholderia pseudomallei O polysaccharide (OPS II), the Campylobacter jejuni oligosaccharyltransferase (OTase), and a suitable glycoprotein (AcrA) in a designer E. coli strain with a higher efficiency for production of glycoconjugates. We were able to produce and purify the OPS II-AcrA glycoconjugate, and MS analysis confirmed correct glycan was produced and attached. We observed the attachment of the O-acetylated deoxyhexose directly to the acceptor protein, which expands the range of substrates utilized by the OTase PglB. Injection of the glycoprotein into mice generated an IgG immune response against B. pseudomallei, and this response was partially protective against an intranasal challenge. Our experiments show that bacterial engineered glycoconjugates can be utilized as vaccine candidates against B. pseudomallei. Additionally, our new E. coli strain SDB1 is more efficient in glycoprotein production, and could have additional applications in the future.

Highlights

  • Burkholderia pseudomallei, a Gram-negative saprophyte, is the causative agent for melioidosis and is endemic in Southeast Asia and Northern Australia (Cheng and Currie, 2005)

  • We demonstrate that the biosynthesis of the B. pseudomallei O ANTIGEN POLYSACCHARIDE II (OPS II) can be reconstituted in Escherichia coli (E. coli)

  • CLONING AND EXPRESSION OF THE B. pseudomallei K96243 O ANTIGEN POLYSACCHARIDE II (OPS II) LOCUS IN E. coli Previous work identified a region consisting of 21 potential open reading frames, and further investigation identified a cluster of 15 genes required for the biosynthesis of B. pseudomallei K96243 OPS II (Figure 1A; DeShazer et al, 1998)

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Summary

Introduction

Burkholderia pseudomallei, a Gram-negative saprophyte, is the causative agent for melioidosis and is endemic in Southeast Asia and Northern Australia (Cheng and Currie, 2005). It is highly resistant to harsh environmental pressures, and it is classified as a potential class B bioterrorism weapon due to its high infectivity when aerosolized (Silva and Dow, 2013). Two different LPS structures named O-polysaccharide (OPS) I and II are present in B. pseudomallei, and OPS II was shown to be required for serum resistance and virulence (Knirel et al, 1992; Perry et al, 1995; DeShazer et al, 1998). B. pseudomallei has an intrinsically high resistance to several different classes of antibiotics, which increases the potential danger of this organism. Due to the increasing prevalence of new resistance genes, and the increasing number of cases, new alternatives to treat and prevent melioidosis are required

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