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.

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.

Thesis (M.Sc. in Microbiol.) -- Mahidol University, 1970.

Thesis (M. Med. Sc.)--University of Hong Kong, 1999. / Includes bibliographical references (leaves 25-28).

Thesis (M.Med.Sc.)--University of Hong Kong, 1999. / Includes bibliographical references (leaves 25-28). Also available in print.

No description available.

Thesis (Ph. D.)--Marshall University, 2002. / Title from document title page. Document formatted into pages; contains x, 147 p. Includes abstract. References at the end of each chapter.

No description available.

Lic.-avh. (sammanfattning) Stockholm : Karol. inst., 2005. / Härtill 3 uppsatser.

The aim of this project was to examine the Streptococcal IgA1 proteases, with particular interest on the Streptococcus pneumoniae enzyme. IgA1 protease of S. pneumoniae was identified and characterised. A non-reducing polyacrylamide gel system was employed to screen clinical isolates for IgA1 protease activity. Of 187 isolates tested 18% were found to be IgA1 protease negative, there was no correlation with the site of isolation of the organism and its ability to produce the enzyme. Attempts were made to clone the pneumococcal IgA1 protease gene using the cosmid pEMBLcos4, the plasmid vector pLG339 and the ? replacement vector ?EMB4. Libraries were screened for pneumolysin and IgA1 protease activity. Clones that expressed pneumolysin were identified by overlaying with sheep red blood cells. One haemolytic clone was not inhibited in the presence of cholesterol. Screening for IgA1 protease activity identified clones with IgA1 protease-ike activity but this activity was not stably expressed. Closer analysis of the libraries suggested that pneumococcal DNA was highly unstable when cloned into E. coil plasmid and cosmid vectors.