Progress 07/01/05 to 06/30/09
Outputs
OUTPUTS: PI left the University of Arizona. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
PI left the University of Arizona.
Publications
- No publications reported this period
|
Progress 01/01/08 to 12/31/08
Outputs
OUTPUTS: As colonization is often the first step in the pathogenic process, it is important to understand the mechanisms by which Arcanobacterium pyogenes can adhere to, and subsequently colonize the host. It is our hypothesis that, like other mucosal pathogens such as Staphylococcus aureus and the streptococci, A. pyogenes adheres to host epithelial cells through the expression of surface proteins which bind to host extracellular matrix proteins such as fibronectin and collagen. We have also identified four fimbrial biogenesis operons which are required for the expression of fimbriae. Fimbriae are hair-like surface projections which are involved in adhesion to host cells. One of these operon consists of three genes, fimB, fimA and srtC1, with a similar genetic organization to that of the type 2 fimbrial biogenesis operon of Actinomyces naeslundii. A. pyogenes strains express these fimbriae, as determined by transmission electron microscopy (TEM) and Western blotting with an antibody directed to the major subunit protein, FimA. However, fimbrial expression is confined to only a small percentage of cells. A. pyogenes carries a second fimbrial biogenesis operon with a similar genetic organization to that described above. To date, A. naeslundii and A. pyogenes are the only organism to carry these type 2 operons. In addition, this organism carries two operon with a genetic organization more consistent with that of other Gram positive fimbrial biogenesis operons. Mutants were constructed in the fimB, fimA or srtC1 genes. Mutation of the fimA gene completely abolished fimbrial stand synthesis, as measured by TEM and Western blotting, consistent with the hypothesis that FimA is the major structural protein in this fimbrial type. Mutants in fimB and srtC1 displayed reduced adhesion to HeLa epithelial cells, indicating their importance in fimbrial biogenesis and adhesion. A srtC1 mutant was able to produce FimA protein, but was unable to polymerize the subunits into a fimbrial strand. Mutants in these genes also had delayed ability to produce biofilms, but produced mature biofilms within 72 hours. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Our long-term objective is to identify the mechanisms by which A. pyogenes causes disease in domestic animals, in order to develop strategies for prevention of infection. Information about the molecular structure of adhesins and their in vivo function will allow for the rational design of novel vaccines, antibodies and/or adherence agonists for the control or prevention of A. pyogenes colonization, and subsequent infection. For example, passive immunization of calves or piglets with maternal colostrum containing antibodies to adhesins may reduce or prevent colonization with A. pyogenes.
Publications
- Silva, E., Gaivao, M., Leitao, S., Jost, B.H., Carneiro, C., Vilela, C.L., Lopes da Costa, L. and Mateus, L. (2008). Genomic characterization of Arcanobacterium pyogenes isolates recovered from the uterus of dairy cows with normal puerperium or endometritis. Veterinary Microbiology, 132:111 118.
- Miller, A.N., Herath, S., Jost, B.H., Bryant, C.E. and Sheldon, M.I. (2008). Effect of the exotoxin pyolysin of Arcanobacterium pyogenes on the bovine endometrium. American Journal of Reproductive Immunology or Theriogenology, submitted.
|
Progress 01/01/07 to 12/31/07
Outputs
As colonization is often the first step in the pathogenic process, it is important to understand the mechanisms by which Arcanobacterium pyogenes can adhere to, and subsequently colonize the host. It is our hypothesis that, like other mucosal pathogens such as Staphylococcus aureus and the streptococci, A. pyogenes adheres to host epithelial cells through the expression of surface proteins which bind to host extracellular matrix proteins such as fibronectin and collagen. We have also identified four fimbrial biogenesis operons which are required for the expression of fimbriae. Fimbriae are hair-like surface projections which are involved in adhesion to host cells. One of these operon consists of three genes, fimB, fimA and srtC1, with a similar genetic organization to that of the type 2 fimbrial biogenesis operon of Actinomyces naeslundii. A. pyogenes strains express these fimbriae, as determined by transmission electron microscopy (TEM) and Western blotting with an
antibody directed to the major subunit protein, FimA. However, fimbrial expression is confined to only a small percentage of cells. A. pyogenes carries a second fimbrial biogenesis operon with a similar genetic organization to that described above. To date, A. naeslundii and A. pyogenes are the only organism to carry these type 2 operons. In addition, this organism carries two operon with a genetic organization more consistent with that of other Gram positive fimbrial biogenesis operons. Mutants were constructed in the fimB, fimA or srtC1 genes. Mutation of the fimA gene completely abolished fimbrial stand synthesis, as measured by TEM and Western blotting, consistent with the hypothesis that FimA is the major structural protein in this fimbrial type. A mutant in fimB displayed reduced adhesion to HeLa epithelial cells, suggestion that this protein may provide the adhesive function of the fimbriae. A srtC1 mutant was able to produce FimA protein, but was unable to polymerize the
subunits into a fimbrial strand.
Impacts
Our long-term objective is to identify the mechanisms by which A. pyogenes causes disease in domestic animals, in order to develop strategies for prevention of infection. Information about the molecular structure of adhesins and their in vivo function will allow for the rational design of novel vaccines, antibodies and/or adherence agonists for the control or prevention of A. pyogenes colonization, and subsequent infection. For example, passive immunization of calves or piglets with maternal colostrum containing antibodies to adhesins may reduce or prevent colonization with A. pyogenes.
Publications
- Pietrocola, G., Valtulina, V., Rindi, S., Jost, B.H. and Speziale, P. (2007). Functional and structural properties of CbpA, a collagen binding protein from Arcanobacterium pyogenes. Microbiology, 153:3380 3389.
|
Progress 01/01/06 to 12/31/06
Outputs
As colonization is often the first step in the pathogenic process, it is important to understand the mechanisms by which Arcanobacterium pyogenes can adhere to, and subsequently colonize the host. It is our hypothesis that, like other mucosal pathogens such as Staphylococcus aureus and the streptococci, A. pyogenes adheres to host epithelial cells through the expression of surface proteins which bind to host extracellular matrix proteins such as fibronectin and collagen. Previous work identified a 121.9kD collagen binding protein, CbpA, which was present in only 47% of A. pyogenes strains (n=75). The A domain (collagen binding domain) of CbpA was expressed as a HIS-tagged protein to facilitate analysis of this protein. Due to it's smaller size, the A domain was expressed and purified in large quantities. We have shown that CbpA is primarily a collagen binding adhesin of high affinity, which only poorly binds fibrinogen, but not other ECM proteins such as fibronectin,
vitronectin or elastin. CbpA is capable of binding collagens I, II, III, IV, V IX and XI, and showed a propensity to interact with several collagen peptides derived by cyanogen bromide cleavage of type I and II collagens. Furthermore, antibodies raised against CbpA were effective at inhibiting adhesion of A. pyogenes to immobilized collagen, indicating that CbpA is a functional collagen-binding adhesin. Analysis of the immunological cross-reactivity of CbpA with antibodies against other bacterial collagen-binding proteins indicated that CbpA is immunologically related to ACE from Enterococcus. faecalis but not to CNA from Staphylococcus aureus or Acm from Enterococcus faecium.
Impacts
Our long-term objective is to identify the mechanisms by which A. pyogenes causes disease in domestic animals, in order to develop strategies for prevention of infection. Information about the molecular structure of adhesins and their in vivo function will allow for the rational design of novel vaccines, antibodies and/or adherence agonists for the control or prevention of A. pyogenes colonization, and subsequent infection. For example, passive immunization of calves or piglets with maternal colostrum containing antibodies to adhesins may reduce or prevent colonization with A. pyogenes.
Publications
- Valtulina, V., Pietrocola, G., Rindi, S., Jost, B.H. and Speziale, P. (2007). Functional and structural properties of CbpA, a collagen binding protein from Arcanobacterium pyogenes. FEBS Journal, submitted.
|
Progress 01/01/05 to 12/31/05
Outputs
As colonization is often the first step in the pathogenic process, our long term objectives are to understand the mechanisms by which Arcanobacterium pyogenes can adhere to, and subsequently colonize the host. It is our hypothesis that, like other mucosal pathogens such as Staphylococcus aureus and the streptococci, A. pyogenes adheres to host epithelial cells through the expression of surface proteins which bind to host extracellular matrix proteins such as fibronectin and collagen. Previous work identified a 121.9kD collagen binding protein, CbpA, which was present in only 47% of A. pyogenes strains (n=75) and a fimbrial gene operon consisting of three genes, fimB, fimA and srtA, with a similar genetic organization to that of the type 2 fimbrial biogenesis operon of Actinomyces naeslundii. A draft genome of A. pyogenes BBR1 was determined by pyrosequencing to 20X coverage, resulting in 106 contigs. Preliminary analysis of the draft genome indicates that it is
2,288,531 bp in size with a 59.6%G+C. 2187 open reading frames were identified within the genome sequence using the BASys automated genome annotation tool. In addition to previously known host cell adhesins, an additional three fimbrial gene operons and a laminin binding protein were identified. Determination of the draft genome of A. pyogenes allows a more targeted approach of studies aimed at better understanding the pathogenesis of infections caused by this organism.
Impacts
Our long-term objective is to identify the mechanisms by which A. pyogenes causes disease in domestic animals, in order to develop strategies for prevention of infection. Information about the molecular structure of adhesins and their in vivo function will allow for the rational design of novel vaccines, antibodies and/or adherence agonists for the control or prevention of A. pyogenes colonization, and subsequent infection. For example, passive immunization of calves or piglets with maternal colostrum containing antibodies to adhesins may reduce or prevent colonization with A. pyogenes.
Publications
- Jost, B.H. and Billington, S.J. (2005). Arcanobacterium pyogenes: molecular pathogenesis of an animal opportunist. Antonie van Leeuwenhoek, 88:87 102.
|
|