Streptococcus dysgalactiae: interactions with host cells and characterizations of GapC
Date
2003
Authors
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Journal ISSN
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Publisher
ORCID
Type
Degree Level
Masters
Abstract
To determine if Streptococcus dysgalactiae GapC plays a role in the
pathogenesis of S. dysgalactiae, an attempt was made to inactivate gapC gene
expression by insertion of a Ω Kmʳ cassette, resulting in a single-crossover
recombination event. However, attempts to obtain a double-crossover recombination
were not successful. The failure to obtain double-crossover mutants together with the
result of this study showing that there is only a single copy of gapC in S. dysgalactiae,
and the impact of iodoacetate, a specific GAPDH inhibitor on viability of bacteria,
suggests that gapC is an essential gene required for basic metabolism.
The role of GapC in the S. dysgalactiae adherence/penetration on/into MAC-T
cells (a bovine mammary epithelial derived cell line) was investigated with a GapC
specific polyclonal antibody. The addition of a molar excess antibody (10 µg/mL) did
not directly inhibit bacterial adherence/penetration on/into MAC-T cells. This suggested
that GapC, presented on the bacterial surface, probably does not directly participate in
adherence/penetration during the pathogenesis.
The effect of S. dysgalactiae on MAC-T cell gene expression was also
investigated using bovine microarrays. The data was analyzed by B-statistic and three
genes that showed the highest possibility of differential expression were (i) succinate
dehydrogenase complex, subunit A, flavoprotein (accession number BF0460 15), (ii)
myosin, light polypeptide kinase (accession number AW465934) and (iii) an open
reading frame (ORF) (accession number AW463818), respectively. None of the
selected genes showed differential expression between infected and non-infected MACT
cells when tested by Northern blot analysis. It is possible that the conditions used did not mimic the pathogenesis of mastitis in vivo since some of the genes we had expected
to detect (i.e. inflammation-associated genes) were not on the list of 25 genes with the
highest possibility to show differential expression. It should also be noted that the data
analysis performed might not be accurate due to the limited number of microarray slides
used.
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Degree
Master of Science (M.Sc.)
Department
Veterinary Microbiology
Program
Veterinary Microbiology