Identification and Molecular Cloning of a Unique Hyaluronan Synthase from Pasteurella multocida

Paul L Deangelis,Wei Jing,Richard R Drake,Ann Mary Achyuthan

doi:10.1074/jbc.273.14.8454

Paul L Deangelis, Wei Jing + Show 2 more

Open Access

https://doi.org/10.1074/jbc.273.14.8454

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Abstract

Type A Pasteurella multocida, a prevalent animal pathogen, employs a hyaluronan [HA] polysaccharide capsule to avoid host defenses. We utilized transposon insertional mutagenesis to identify the P. multocida HA synthase, the enzyme that polymerizes HA. A DNA fragment from a wild-type genomic library could direct HA production in vivo in Escherichia coli, a bacterium that normally does not produce HA. Analysis of truncated plasmids derived from the original clone indicated that an open reading frame encoding a 972-residue protein was responsible for HA polymerization. This identification was confirmed by expression cloning in E. coli; we observed HA capsule formation in vivo and detected activity in membrane preparations in vitro. The polypeptide size was verified by photoaffinity labeling of the native P. multocida HA synthase with azido-UDP sugar analogs. Overall, the P. multocida sequence is not very similar to the other known HA synthases from streptococci, PBCV-1 virus, or vertebrates. Instead, a portion of the central region of the new enzyme is more homologous to the amino termini of other bacterial glycosyltransferases that produce different capsular polysaccharides or lipopolysaccharides. In summary, we have discovered a unique HA synthase that differs in sequence and predicted topology from the other known enzymes.

Highlights

Type A Pasteurella multocida, a prevalent animal pathogen, employs a hyaluronan [HA] polysaccharide capsule to avoid host defenses
Both UDP-GlcA and UDP-GlcNAc were required for HA synthesis in vitro
As in the case of streptococcal HasA [21] and Xenopus DG42 [11], it appears that one polypeptide species, PmHAS, transfers two distinct sugar groups to the nascent HA chain

Summary

Introduction

Type A Pasteurella multocida, a prevalent animal pathogen, employs a hyaluronan [HA] polysaccharide capsule to avoid host defenses. P. Silver; Ref. 18), an acapsular derivative of a K1 strain (which produces a polysialic acid capsule) that possesses all the same general capsular polysaccharide transport machinery as K5 but not high levels of UDPGlc dehydrogenase.

Results

Conclusion