H. somni is capable of forming a prominent biofilm, and luxS is known to play an important role in biofilm formation through quorum sensing, but has also been postulated to function in gene regulation. In order to further study the function of H. somni LuxS, mutants 2336::TnluxS and 2336::TnuspE were identified from a bank of mutants generated with EZ-Tn5 <KAN-2>Tnp transposome (EpiCentre). The 2336::TnluxS and 2336::TnuspE mutants were highly attenuated in mice, but only 2336::TnuspE was deficient in biofilm formation. However, the electrophoretic profiles of the LOS and serum sensitivity of both mutants were substantially altered compared to the parent strain, but exopolysaccharide production during biofilm formation also only decreased in 2336::TnuspE. The altered phenotypes were partially restored in complemented recombinant clones obtained using shuttle vector pHS649S. To clarify whether luxS regulates the expression of various virulence genes, mRNA from both the parent strain and 2336::TnluxS was sequenced. It was determined that the transcription level of 53 genes in 2336::TnluxS and 42 genes in 2336::TnuspE in planktonic form were changed.
In biofilm, 320 genes in 2336::TnluxS and 230 genes in 2336::TnuspE were differentially regulated compared to biofilm formed by strain 2336.
The immunogloblin binding protein A (IbpA) of H. somni is known to be cytotoxic to phagocytic cells. In this study, we found that strains with a mutation in ibpA were less capable of early replication in monocytes. The IbpA protein concentrated from the culture supernatant of strain 2336 facilitated the intracellular survival of strain 129Pt, which lacks IbpA. However, the ability of several ibpA mutants to resist intracellular killing was not significantly impaired by 48 h post-infection. Two transposon mutants 2336::TnluxS and 2336::TnuspE replicated in monocytes in a similar manner as the ibpA mutants. Confocal microscopy revealed that the intracellular-replicable strains (2336, 64Vc, 2336::TnluxS, 2336::TnuspE and the ibpA mutants) prevented the acidification of the bacterial-containing phagosome and the expression of lysosome marker LAMP-2, which may facilitate survival of H. somni in monocytes.
An enzyme-linked immunosorbent assay was developed to detect bovine antibodies to the H. somni exopolysaccharide that is formed during biofilm formation. When an index value of 0.268 was used the sensitivity of the assay for experimentally- and naturally-infected calves was 90.5% at 3 weeks post-infection, and the specificity of the assay for healthy calves was 92.5%. The EPS ELISA may aid in identifying calves with diseases due to H. somni. / Ph. D.
| Identifer | oai:union.ndltd.org:VTETD/oai:vtechworks.lib.vt.edu:10919/52585 |
| Date | 13 October 2014 |
| Creators | Pan, Yu |
| Contributors | Veterinary Medicine, Inzana, Thomas J., Witonsky, Sharon G., Boyle, Stephen M., Melville, Stephen B. |
| Publisher | Virginia Tech |
| Source Sets | Virginia Tech Theses and Dissertation |
| Detected Language | English |
| Type | Dissertation |
| Format | ETD, application/pdf, application/pdf |
| Rights | In Copyright, http://rightsstatements.org/vocab/InC/1.0/ |
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