Studies on the expression and secretion of pyolysin, the cholesterol-dependent cytolysin of Arcanobacterium pyogenes

Persistent Link:
http://hdl.handle.net/10150/280038
Title:
Studies on the expression and secretion of pyolysin, the cholesterol-dependent cytolysin of Arcanobacterium pyogenes
Author:
Gilbert, Stefani
Issue Date:
2002
Publisher:
The University of Arizona.
Rights:
Copyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.
Abstract:
Arcanobacterium pyogenes is a gram-positive, fastidious, facultative anaerobe that can persist in a number of animal species both as a commensal and as a pathogen. The economic impact from A. pyogenes -related bovine mastitis and liver abscess infections makes the characterization of possible targets for vaccine development important. One such target, pyolysin (PLO), is a member of the cholesterol-dependent cytolysin (CDC) family and a major virulence factor and host protective antigen in A. pyogenes pathogenesis. Here, sequence analysis of the chromosomal region surrounding plo, which encodes PLO, indicated that the plo gene and an upstream open reading frame, orf121, constitute a genomic islet conserved across geographically diverse isolates. Two genes downstream of plo, ftsY and ffh, encode homologues of the protein components of the signal recognition particle (SRP) pathway which may be involved in plo expression and secretion. However, the role of ftsY and ffh in plo expression could not be defined as they were found essential in A. pyogenes, and ffh was unable to complement a heterologous system. Analysis of plo mRNA and PLO levels across a growth curve indicated that plo is transcriptionally or post-transcriptionally regulated. Sequence analysis of the plo promoter region identified three 11-mer repeats, R1, R2 and R3, and two putative sigma70-like promoter sequences, P1 and P2. Primer extension experiments suggested active transcription from P1 and P2 in vitro. Analysis of transcriptional fusions of successive truncations of the plo promoter region and site-directed mutations in the promoter motifs to a chloramphenicol acetyl transferase (cat) gene, indicated a role for each motif in the regulation of plo expression in vitro, with R2 required for activation and P2 being the regulated promoter. While a null mutation in orf121 did not have a significant effect on plo expression in vitro, gel shift analysis confirmed the ability of some factor within A. pyogenes soluble cellular extracts to bind specifically to the plo promoter region. The identification of cis- and trans-acting factors involved in plo expression provides a basis for characterizing PLO expression in vivo, and may lead to a better understanding of the global regulation of pathogenesis in A. pyogenes.
Type:
text; Dissertation-Reproduction (electronic)
Keywords:
Biology, Microbiology.; Health Sciences, Pathology.; Biology, Veterinary Science.
Degree Name:
Ph.D.
Degree Level:
doctoral
Degree Program:
Graduate College; Veterinary Science and Microbiology
Degree Grantor:
University of Arizona
Advisor:
Songer, J. Glenn

Full metadata record

DC FieldValue Language
dc.language.isoen_USen_US
dc.titleStudies on the expression and secretion of pyolysin, the cholesterol-dependent cytolysin of Arcanobacterium pyogenesen_US
dc.creatorGilbert, Stefanien_US
dc.contributor.authorGilbert, Stefanien_US
dc.date.issued2002en_US
dc.publisherThe University of Arizona.en_US
dc.rightsCopyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.en_US
dc.description.abstractArcanobacterium pyogenes is a gram-positive, fastidious, facultative anaerobe that can persist in a number of animal species both as a commensal and as a pathogen. The economic impact from A. pyogenes -related bovine mastitis and liver abscess infections makes the characterization of possible targets for vaccine development important. One such target, pyolysin (PLO), is a member of the cholesterol-dependent cytolysin (CDC) family and a major virulence factor and host protective antigen in A. pyogenes pathogenesis. Here, sequence analysis of the chromosomal region surrounding plo, which encodes PLO, indicated that the plo gene and an upstream open reading frame, orf121, constitute a genomic islet conserved across geographically diverse isolates. Two genes downstream of plo, ftsY and ffh, encode homologues of the protein components of the signal recognition particle (SRP) pathway which may be involved in plo expression and secretion. However, the role of ftsY and ffh in plo expression could not be defined as they were found essential in A. pyogenes, and ffh was unable to complement a heterologous system. Analysis of plo mRNA and PLO levels across a growth curve indicated that plo is transcriptionally or post-transcriptionally regulated. Sequence analysis of the plo promoter region identified three 11-mer repeats, R1, R2 and R3, and two putative sigma70-like promoter sequences, P1 and P2. Primer extension experiments suggested active transcription from P1 and P2 in vitro. Analysis of transcriptional fusions of successive truncations of the plo promoter region and site-directed mutations in the promoter motifs to a chloramphenicol acetyl transferase (cat) gene, indicated a role for each motif in the regulation of plo expression in vitro, with R2 required for activation and P2 being the regulated promoter. While a null mutation in orf121 did not have a significant effect on plo expression in vitro, gel shift analysis confirmed the ability of some factor within A. pyogenes soluble cellular extracts to bind specifically to the plo promoter region. The identification of cis- and trans-acting factors involved in plo expression provides a basis for characterizing PLO expression in vivo, and may lead to a better understanding of the global regulation of pathogenesis in A. pyogenes.en_US
dc.typetexten_US
dc.typeDissertation-Reproduction (electronic)en_US
dc.subjectBiology, Microbiology.en_US
dc.subjectHealth Sciences, Pathology.en_US
dc.subjectBiology, Veterinary Science.en_US
thesis.degree.namePh.D.en_US
thesis.degree.leveldoctoralen_US
thesis.degree.disciplineGraduate Collegeen_US
thesis.degree.disciplineVeterinary Science and Microbiologyen_US
thesis.degree.grantorUniversity of Arizonaen_US
dc.contributor.advisorSonger, J. Glennen_US
dc.identifier.proquest3053906en_US
dc.identifier.bibrecord.b42819581en_US
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