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Cloning and characterization of sialidases with 2-6' and 2-3' sialyl lactose specificity from Pasteurella multocida
1Department of Medical Microbiology and Parasitology, University of Georgia, Athens, Georgia 30602, USA.
Abstract:
Pasteurella multocida is a mucosal pathogen that colonizes the respiratory system of susceptible hosts. Most isolates of P. multocida produce sialidase activity, which may contribute to colonization of the respiratory tract or the production of lesions in an active infection. We have cloned and sequenced 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 production. However, this mutant exhibited reduced enzyme activity and growth rate on 2-3' sialyl lactose compared to the wild type. Subsequently, we demonstrated the presence of two sialidases by cloning another sialidase gene that differed from nanH in DNA sequence and substrate specificity. NanB demonstrated activity on both 2-3' and 2-6' sialyl lactose, while NanH demonstrated activity only on 2-3' sialyl lactose. Neither enzyme liberated sialic acid from colominic acid (2-8' sialyl lactose). Recombinant E. coli containing the sialidase genes were able to utilize several sialoconjugants when they were provided as sole carbon sources in minimal medium. These data suggest that sialidases have a nutritional function and may contribute to the ability of P. multocida to colonize and persist on vertebrate mucosal surfaces.
Insights
Pasteurella multocida possesses two sialidase enzymes, NanH and NanB, which break down sialic acid. These enzymes are crucial for the bacterium
Area of Science:
- Microbiology
- Bacterial Pathogenesis
- Enzymology
Background:
- Pasteurella multocida is a significant mucosal pathogen, commonly colonizing the respiratory tracts of various hosts.
- Sialidase activity is prevalent in P. multocida isolates and is hypothesized to aid in respiratory tract colonization and lesion development.
Purpose of the Study:
- To investigate the role and characteristics of sialidase enzymes in Pasteurella multocida.
- To clone, sequence, and characterize sialidase genes from P. multocida.
Main Methods:
- Cloning and sequencing of the sialidase gene nanH from a fowl cholera isolate.
- Construction and analysis of an insertional mutant for nanH.
- Cloning and characterization of a second sialidase gene, nanB.
- Assessing enzyme activity on various sialyl lactose substrates and colominic acid.
- Utilizing recombinant Escherichia coli to evaluate sialoconjugant utilization.
Main Results:
- The nanH gene was cloned and sequenced, revealing homology with microbial sialidases.
- A nanH mutant showed reduced sialidase activity and growth on 2-3' sialyl lactose.
- A second sialidase, NanB, was identified with distinct substrate specificity (2-3' and 2-6' sialyl lactose) compared to NanH (2-3' sialyl lactose only).
- Neither NanH nor NanB degraded colominic acid (2-8' sialyl lactose).
- Recombinant E. coli expressing these sialidases could utilize sialoconjugants as sole carbon sources.
Conclusions:
- Pasteurella multocida produces at least two distinct sialidases, NanH and NanB.
- These sialidases likely serve a nutritional function, enabling the utilization of sialoconjugates.
- Sialidase activity may be a key factor in the colonization and persistence of P. multocida on mucosal surfaces.