Progress 07/01/01 to 09/30/05
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. The fimbrial biogenesis operon consists of three genes, fimB, fimA and srtA, with a similar genetic organization to that of the type 2 fimbrial biogenesis operon of Actinomyces naeslundii. fimA encodes a protein with 27% amino acid identity and 41% similarity to the A. naeslundii type 2 fimbrial subunit, and the 45.7kDa FimA protein
contains the pilin motif and E box, required for Gram positive fimbrial biogenesis. The C terminus of the 90.5kDa FimB has 42% amino acid similarity to Orf977, the putative adhesin from A. naeslundii type 2 fimbriae, and like that protein contains a fibronectin binding domain and an E box. srtA encodes a 32.5kDa sortase with 68% amino acid similarity to Orf365 from A. naeslundii, and this protein is probably responsible for fimbrial assembly, as described for Corynebacterium diphtheriae. 94.7% of A. pyogenes isolates carry all three genes (n=53), as determined by DNA hybridization with gene specific probes. A. pyogenes expresses fimbriae, as determined by transmission electron microscopy and Western blotting with an antibody directed to the putative major subunit, FimA. However, fimbrial expression is confined to only a small percentage of cells, at least under standard growth conditions, consisting of tryptic soy agar with 5% bovine blood, 38oC and 5% CO2. Fimbrial expression appears
to increase with higher CO2 concentrations and longer incubation times. Like A. naeslundii fimbriae, A. pyogenes fimbriae may also play a role in adhesion to host tissue.
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.
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Progress 01/01/04 to 12/31/04
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). Downstream of cbpA we identified several genes which may also be involved in adhesion; fimB, encoding a protein with similarity to a fibronectin-binding, fimbrial biogenesis gene from Actinomyces naeslundii; fimA, encoding a putative fimbrial subunit protein; and srtA, a gene encoding sortase, an enzyme involved in fimbrial assembly in Gram positive bacteria.
Knockout mutants have been constructed in each of these genes and we are in the process of characterizing their phenotypes with respect to fimbrial expression and adhesion to host cells. Rabbit antiserum to FimA was prepared and we are able to identify fimbriae on the surface of A. pyogenes cells by immunogold electron microscopy. In addition, we have identified growth conditions which stimulate fimbrial expression.
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. (2004). Corynebacterium and Arcanobacterium. In: Pathogenesis of bacterial infections of animals. C.L. Gyles, C.O. Thoen, J.F. Prescott and J.G. Songer (eds). 3rd Edition. Blackwell Publishing, Ames, IA.
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Progress 01/01/03 to 12/31/03
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). Downstream of cbpA we identified several genes which may also be involved in adhesion; orf887, encoding a protein with similarity to the Streptococcus pyogenes fibronectin-binding proteins SOF/SfbII; fimA, encoding a fimbrial subunit protein; and orf301, a gene encoding sortase, an enzyme involved in fimbrial assembly in Gram positive bacteria. Knockout mutants
have been constructed in each of these genes and we are in the process of characterizing their phenotypes with respect to fimbrial expression and adhesion to host cells.
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
- Esmay, P.A., Billington, S.J., Link, M.A., Songer, J.G. and Jost, B.H. (2003). The Arcanobacterium pyogenes collagen binding protein, CbpA, promotes adhesion to host cells. Infection and Immunity, 71:4368 4374.
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Progress 01/01/02 to 12/31/02
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 clone containing the C-terminal two-thirds of a gene, cbpA (formerly mbpA). The entire gene has been cloned and sequenced. The 3.4kb cbpA gene encodes a protein with a predicted molecular mass of 125.3kD. The protein has an MSCRAMM domain structure and is located in the bacterial cell wall. Furthermore, we have shown that an A. pyogenes cell wall protein can bind collagen, as determined by Far-Western blotting using bovine type I collagen (Sigma) as the ligand.
The size of this protein is consistent with the size of mature CbpA at 121.9kD. Not all strains of A. pyogenes carried the cbpA gene. Only 47% of strains tested were positive when hybridized with a cbpA-specific probe (n=75). An E. coli strain expressing recombinant HIS-tagged CbpA was constructed and protein was purified by metal affinity chromatography. Purified protein was able to bind to collagens type I, II and IV, but not fibronectin. An A. pyogenes cbpA mutant was constructed by allelic exchange. This strain is unable to bind collagen in a Far Western blot. Furthermore, this strain has an approximately 50% reduction in its ability to bind to the collagen expressing cell lines, HeLa and 3T6, indicating that CbpA is indeed important in host cell adhesion. Upstream of cbpA, we identified two open reading frames, orf302, encoding a protein with similarity to a putative regulatory protein from Streptomyces coelicolor, and orf309', a partial ORF encoding a gene with similarity to a
choline transport protein from Erwinia amylovora. Downstream of cbpA was orf887, encoding a protein with similarity to the Streptococcus pyogenes fibronectin-binding proteins SOF/SfbII, suggesting that this region may encode an adhesion gene cluster.
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., Post, K.W., Songer, J.G. and Billington, S.J. (2002). Isolation of Arcanobacterium pyogenes from the porcine gastric mucosa. Veterinary Research Communications, 26:419-425.
- Jost, B.H., Songer, J.G., Billington, S.J. (2002). Identification of a second Arcanobacterium pyogenes neuraminidase, and involvement of neuraminidase activity in host cell adhesion. Infection and Immunity, 70:1106-1112.
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Progress 07/01/01 to 12/31/01
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 clone containing the C-terminal two-thirds of a gene, cbpA (formerly mbpA). The partial CbpA had 25.9% amino acid identity and 56.5% amino acid similarity to the Staphylococcus aureus collagen adhesion, Cna. The objectives of this study were to clone and sequence the remainder of cbpA; to knock out cbpA by allelic exchange and determine whether CbpA plays a role in adhesion to HeLa cells; and to purify recombinant CbpA and determine the types of extracellular
matrix proteins to which CbpA binds. To date, the entire gene has been cloned and sequenced. The 3.4kb cbpA gene encodes a protein with a predicted molecular mass of 125.3kD. A putative rho independent terminator (deltaG=-19.2kcal/mole) was identified downstream of cbpA. CbpA contains an N-terminal signal sequence and a C-terminal cell wall-sorting signal, suggesting cell wall localization. Furthermore, we have shown that A. pyogenes can bind to collagen, as cell wall extract contains an 120kD collagen binding protein, as determined by Far-Western blotting using bovine type I collagen (Sigma) as the ligand. The size of this protein is consistent with the size of mature CbpA at 121.9kD. Upstream of cbpA, we identified two open reading frames, orf221, encoding a protein with similarity to a putative regulatory protein from Streptomyces coelicolor, and orf309', a partial ORF encoding a gene with similarity to a choline transport protein from Erwinia amylovora. Downstream of cbpA was
orf887, encoding a protein with similarity to the Streptococcus pyogenes fibronectin-binding proteins SOF/SfbII. During the next year we plan to construct allelic exchange plasmids and use them to construct a cbpA mutant of A. pyogenes, and test this strain for the ability to a) bind collagen in a Far-Western blot, and b) determine whether this mutant has reduced ability to adhere to cultured host epithelial cells.
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
- Link, M.A, Jost, B.H., Songer, J.G. and Billington, S.J. (2001). The role of an Arcanobacterium pyogenes extracellular matrix binding protein in adhesion to host epithelial cells. Proceedings, Arizona-Nevada Branch American Society for Microbiology Annual Meeting, Tempe AZ, USA.
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