The pathogenesis of the bovine environmental pathogen Streptococcus uberis
has been extensively studied, but it remains incompletely understood. The organism
produces a surface protein capable of binding lactoferrin (Lbp), originally identified as
an M-like streptococcal protein. We hypothesized that Lbp may play a role as a
virulence factor. Structural similarity and amino acid sequence homology of Lbp to Mrelated
proteins of Group A streptococci suggested a possible functional similarity
between Lbp and M-like proteins, which are involved in evasion of host antibacterial
defenses, adhesion to host epithelial cells and intracellular invasion by the bacteria.
Alternatively, high-affinity binding of the abundant iron-chelating component of the
host milk (lactoferrin) suggests that Lbp of S. uberis might play role in iron acquisition
by the bacterium. Finally, Lbp might serve as a receptor for signal transduction in the
bacterial cell or alter host cell signalling during infection, when the bacteria with
surface-bound lactoferrin adhere to or invade the host epithelial cells.
In order to test the hypothesis that Lbp is a virulence factor of S. uberis, a
mutant strain of S. uberis unable to express the lactoferrin-binding protein was
generated and the role of the protein was studied in comparative analyses of the mutant
and the parent strains.
The results of our study indicated that unlike many streptococcal M-like
proteins, the lactoferrin-binding protein of S. uberis did not appear to play a role in
overcoming host innate and acquired immune antibacterial responses. Both S. uberis
and its lbp mutant were ingested by bovine blood neutrophils and were similar in their ability to survive in fresh bovine blood regardless of the presence either of lactoferrin or of anti-Lbp antibodies.
Lbp did not promote bacterial adhesion to host epithelial cells and it was not
essential for the internalization of the bacteria by host epithelial cells, since both S.
uberis and lbp mutant were found capable of adhering, invading, intracellular survival
and intracellular growth when the bacteria were co-cultured with bovine mammary
epithelial cells. No significant differences in numbers of adherent or internalized
bacteria per host cell were found between wild type and lbp mutant cells.
S. uberis requirements for iron were determined to be low and Lbp was not
essential for iron acquisition by the organism from iron-saturated lactoferrin.
To study the role of Lbp in bacterial virulence during infection of bovine
mammary glands, dairy cows in the second half of their lactation periods were
challenged with the wild type S. uberis and with the lbp mutant. The results of in vivo
infection suggested that expression of Lbp by the bacteria was not essential for
colonization of the host mammary gland and that expression of Lbp was not associated
with differences in severity of mastitis or with different levels of shedding of the
bacteria by infected animals.
To study the role of Lbp in signal transduction, differential bacterial cellular
protein phosphorylation in the presence of bovine lactoferrin was analyzed. Since no
differences in protein phosphorylation profiles were detected between S. uberis and the
lbp mutant, it was concluded that Lbp is probably not a part of a classical bacterial twocomponent
signalling pathway. However, we demonstrated that the expression of host
genes potentially involved in cell morphogenesis, motility and signal transduction was
regulated depending on the expression of Lbp by S. uberis. Down-regulation of the
expression of selected host genes was verified by quantitative reverse transcription
PCR. Putative iron responsive elements were identified in mRNA of several of these
genes. Down-regulation of these genes in the cells overloaded with ferric iron was
demonstrated by RT PCR. These results indicate that Lbp of S. uberis may interfere
with host cellular signalling pathways by inducing perturbations in the cell iron status.
This suggests that Lbp of S. uberis may be a virulence factor, playing a role in signal
transduction or in the regulation of gene expression in host cells.
| Identifer | oai:union.ndltd.org:USASK/oai:usask.ca:etd-07142005-140713 |
| Date | 22 July 2005 |
| Creators | Moshynskyy, Igor |
| Contributors | Schryvers, Anthony B., Potter, Andrew A., Polley, Lydden, Perez-Casal, Jose, Gordon, John R., Deneer, Harry, Chirino-Trejo, Manuel, Babiuk, Lorne A. |
| Publisher | University of Saskatchewan |
| Source Sets | University of Saskatchewan Library |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://library.usask.ca/theses/available/etd-07142005-140713/ |
| Rights | unrestricted, I hereby certify that, if appropriate, I have obtained and attached hereto a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted is the same as that approved by my advisory committee. I hereby grant to University of Saskatchewan or its agents the non-exclusive license to archive and make accessible, under the conditions specified below, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report. |
Page generated in 0.0066 seconds