Cloning and characterization of sialidases with 2-6 ' and 2-3 ' sialyl lactose specificity from Pasteurella multocida

Pasteurella multocida is a mucosal pathogen that colonizes the respiratory system of susceptible hosts. Most isolates of P. multocida produce sialidas e activity, which may contribute to colonization of the respiratory tract o r the production of lesions in an active infection. We have cloned and sequ enced a sialidase gene, nanH, from a fowl cholera isolate of P, multocida. Sequence analysis of NanH revealed that it exhibited significant amino acid sequence homology with many microbial sialidases, Insertional inactivation of nanH resulted in a mutant strain that was not deficient in sialidase pr oduction. However, this mutant exhibited reduced enzyme activity and growth rate on 2-3' sialyl lactose compared to the wild type. Subsequently, we de monstrated the presence of two sialidases by cloning another sialidase gene that differed from nanH in DNA sequence and substrate specificity. NanB de monstrated activity on both 2-3' and 2-6' sialyl lactose, while NanH demons trated activity only on 2-3' sialyl lactose, Neither enzyme liberated siali c acid from colominic acid (2-8' sialyl lactose), Recombinant E, coli conta ining the sialidase genes were able to utilize several sialoconjugants when they were provided as sole carbon sources in minimal medium. These data su ggest that sialidases have a nutritional function and may contribute to the ability of P, multocida to colonize and persist on vertebrate mucosal surf aces.