Identification of Novel Glycosyltransferases Required for Assembly of the Pasteurella multocida A:1 Lipopolysaccharide and Their Involvement in Virulence

Abstract

We previously determined the structure of the Pasteurella multocida Heddleston type 1 lipopolysaccharide (LPS) molecule and characterized some of the transferases essential for LPS biosynthesis. We also showed that P. multocida strains expressing truncated LPS display reduced virulence. Here, we have identified all of the remaining glycosyltransferases required for synthesis of the oligosaccharide extension of the P. multocida Heddleston type 1 LPS, including a novel ␣-1,6 glucosyltransferase, a ␤-1,4 glucosyltransferase, a putative bifunctional galactosyltransferase, and two heptosyltransferases. In addition, we identified a novel oligosaccharide extension expressed only in a heptosyltransferase (hptE) mutant background. All of the analyzed mutants expressing LPS with a truncated main oligosaccharide extension displayed reduced virulence, but those expressing LPS with an intact heptose side chain were able to persist for long periods in muscle tissue. The hptC mutant, which expressed LPS with the shortest oligosaccharide extension and no heptose side chain, was unable to persist on the muscle or cause any disease. Furthermore, all of the mutants displayed increased sensitivity to the chicken antimicrobial peptide fowlicidin 1, with mutants expressing highly truncated LPS being the most sensitive.

Development of a Rapid Multiplex PCR Assay To Genotype Pasteurella multocida Strains by Use of the Lipopolysaccharide Outer Core Biosynthesis Locus

“…Our studies on the L1/serovar 1 strain VP161 have shown that any shortening of the LPS outer core structure in this strain results in attenuation of virulence in chickens (24), but it is possible that isolates expressing truncated LPS may be able to persist in some host niches but are not as virulent as parent strains expressing fulllength LPS. Indeed, infection of chickens with a VP161 hptE LPS mutant (which produces a highly truncated outer core) showed that this mutant could persist at the site of muscle injection but could not be recovered from the blood, thus supporting this hypothesis (20). Importantly, many of the structures expressed by L3 and L6 genotype strains mimic host glycosphingolipids, and this may allow the bacteria to avoid recognition by the components of the innate immune system (10,11).…”

“…We have shown previously that the 16 unique LPS outer core structures produced by the P. multocida Heddleston type strains are PM993 H8 2002 Duck PM995 H3 2002 Chicken PM1075 H16 2004 Chicken PM1098 H15 2004 Unknown PM1099 H10 2004 Unknown PM1103 H10 2004 Unknown PM1113 NT 2004 Avian PM1120 NT 2005 Chicken PM1124 H1, H4, H12 2005 Unknown PM1128 H10 2005 Bovine PM1132 H1, H3, H4, H10, H14 2005 Pig PM1153 H1, H3, H7 2005 Avian PM1165 H1 2006 Duck PM1193 H3 2006 Duck PM1205 H1 2007 Emu PM1258 NT 2010 Chicken PM1268 NT 2010 Chicken PM1300 H4 2009 Turkey PM1304 H1 2009 Chicken PM1315 H1 2009 Chicken PM1316 H4 2009 Unknown PM1317 H3 2009 Unknown PM1320 H10, H13, H14 2010 Chicken PM1369 H1 2010 Chicken PM1396 H1, H3 2010 Unknown generated from only eight distinct genetic loci, which we have named L1 to L8 ( Fig. 1) (9)(10)(11)(12)(13)(14)(15)20)<...>…”

“…For compositional analysis of LPS produced by the Australian field isolates, small quantities of LPS were isolated from plate-grown cells as described previously (20). O-deacylated LPS (LPS-OH), core oligosaccharide (OS), and completely deacylated LPS were all isolated and purified from LPS as described previously (21).…”

Structural and Genetic Basis for the Serological Differentiation of Pasteurella multocida Heddleston Serotypes 2 and 5

“…For complementation experiments using lpt-3, plasmids pAL99 or pAL284 were used to transform P. multocida M1404. For heterologous expression experiments, the plasmids pAL99, pAL580, pAL581, and pAL588 were separately used to transform the VP161 gatA mutant (AL725) ( Table 1), which has a truncated LPS structure terminating at the Hep IV residue (4).…”

“…For culture on solid media, 1.5% agar was added to either BHI or nutrient broth (Oxoid) containing 0.3% yeast extract or strains were grown on chocolate agar. For structural studies of LPS, strains were ultimately grown in 24 liters of BHI in a 28-liter NBS fermentor as described previously (4).…”