The type II O‐antigenic polysaccharide moiety of Burkholderia pseudomallei lipopolysaccharide is required for serum resistance and virulence

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Abstract
Melioidosis, an infection caused by the Gram‐negative bacterial pathogen Burkholderia pseudomallei, is endemic in south‐east Asia and northern Australia. Acute septicaemic melioidosis is a major cause of morbidity and mortality, especially in north‐east Thailand. B. pseudomallei is highly resistant to the bactericidal activity of normal human serum (NHS), and we have found that B. pseudomallei 1026b multiplies in 10–30% NHS. We developed a simple screen for the identification of serum‐sensitive mutants based on this novel phenotype. Approximately 1200 Tn5‐OT182 mutants were screened, and three serum‐sensitive mutants were identified. The type II O‐antigenic polysaccharide (O‐PS) moiety of lipopolysaccharide was not present in the serum‐sensitive mutants. A representative serum‐sensitive mutant, SRM117, was killed by the alternative pathway of complement and was less virulent than 1026b in three animal models of melioidosis. The Tn5‐OT182 integrations in the serum‐sensitive mutants were physically linked on the B. pseudomallei chromosome, and further genetic analysis of this locus revealed a cluster of 15 genes required for type II O‐PS production. The proteins encoded by these genes were similar to proteins involved in bacterial polysaccharide biosynthesis. The results presented here demonstrate that type II O‐PS is essential for B. pseudomallei serum resistance and virulence.