Identification and Characterization of Arcanobacterium haemolyticum Virulence Factors

Persistent Link:
http://hdl.handle.net/10150/193896
Title:
Identification and Characterization of Arcanobacterium haemolyticum Virulence Factors
Author:
Lucas, Erynn Ainslee
Issue Date:
2009
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 haemolyticum, a Gram-positive bacterium, is an under-reportedagent of disease, causing pharyngitis, wound infections and a variety of invasive diseases.This work characterized a known A. haemolyticum toxin, phospholipase D (PLD), anddetermined its possible role in bacterial virulence. In addition, a novel toxin, arcanolysin(ALN), was identified and characterized. A draft genome sequence was determined andseveral additional virulence factors that may aid in disease pathogenesis were identified.PLD was present in all strains of A. haemolyticum tested, and was expressedmaximally during logarithmic growth. Recombinant PLD caused lipid raftrearrangement on the surface of HeLa cells in a dose-dependent manner. Thisrearrangement allowed maximal bacterial adhesion to the host, with a pld knockoutadhering only 39.7% to HeLa cells as compared to wildtype. Loss of production of PLDdid not affect bacterial invasion. However, PLD expressed by intracellular bacteria wascytotoxic to host cells, as determined by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium/phenazine methosulfate(MTS/PMS) viability assays. PLD caused host cell death via necrosis as determined bytransmission electron microscopy. PLD did not induce apoptosis, as caspases 3/7 and 9were not elevated in HeLa cells infected with wildtype A. haemolyticum.A. haemolyticum also expresses a Cholesterol-Dependent Cytolysin (CDC), ALN.Like pld, aln was present in all strains tested. ALN displays a variant undecapeptide andan unusual N-terminal extension not found in most other CDCs. Recombinant ALN11shows significantly increased activity against cultured cells and erythrocytes of humanorigin, compared with intermediate activity on rabbit and hamster cells, and low to noactivity on bovine and ovine cells as measured by hemolysis, cytotoxicity and membranebinding assays. ALN was less inhibited by free cholesterol when compared with otherCDCs, indicating the possibility of alternative receptor binding.The A. haemolyticum genome was sequenced to >20X coverage, and assembled to50 contigs covering ~95% of the genome. The genome is ~1.95Mb with a mol %G+C of53.1% and contained no plasmids. pld and aln have a reduced mol %G+C of 47.2% and46.5%, respectively, indicating the possibility of gene acquisition by horizontal transfer.Initial bioinformatics analysis identified genes encoding a protease, an extracellularDNase, two neuraminidases and three fimbrial biosynthetic operons were also identifiedwithin the genome.
Type:
text; Electronic Dissertation
Keywords:
aln; arcanobacterium; haemolyticum; pld; virulence
Degree Name:
Ph.D.
Degree Level:
doctoral
Degree Program:
Microbiology; Graduate College
Degree Grantor:
University of Arizona
Advisor:
Jost, B. Helen
Committee Chair:
Jost, B. Helen

Full metadata record

DC FieldValue Language
dc.language.isoENen_US
dc.titleIdentification and Characterization of Arcanobacterium haemolyticum Virulence Factorsen_US
dc.creatorLucas, Erynn Ainsleeen_US
dc.contributor.authorLucas, Erynn Ainsleeen_US
dc.date.issued2009en_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 haemolyticum, a Gram-positive bacterium, is an under-reportedagent of disease, causing pharyngitis, wound infections and a variety of invasive diseases.This work characterized a known A. haemolyticum toxin, phospholipase D (PLD), anddetermined its possible role in bacterial virulence. In addition, a novel toxin, arcanolysin(ALN), was identified and characterized. A draft genome sequence was determined andseveral additional virulence factors that may aid in disease pathogenesis were identified.PLD was present in all strains of A. haemolyticum tested, and was expressedmaximally during logarithmic growth. Recombinant PLD caused lipid raftrearrangement on the surface of HeLa cells in a dose-dependent manner. Thisrearrangement allowed maximal bacterial adhesion to the host, with a pld knockoutadhering only 39.7% to HeLa cells as compared to wildtype. Loss of production of PLDdid not affect bacterial invasion. However, PLD expressed by intracellular bacteria wascytotoxic to host cells, as determined by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium/phenazine methosulfate(MTS/PMS) viability assays. PLD caused host cell death via necrosis as determined bytransmission electron microscopy. PLD did not induce apoptosis, as caspases 3/7 and 9were not elevated in HeLa cells infected with wildtype A. haemolyticum.A. haemolyticum also expresses a Cholesterol-Dependent Cytolysin (CDC), ALN.Like pld, aln was present in all strains tested. ALN displays a variant undecapeptide andan unusual N-terminal extension not found in most other CDCs. Recombinant ALN11shows significantly increased activity against cultured cells and erythrocytes of humanorigin, compared with intermediate activity on rabbit and hamster cells, and low to noactivity on bovine and ovine cells as measured by hemolysis, cytotoxicity and membranebinding assays. ALN was less inhibited by free cholesterol when compared with otherCDCs, indicating the possibility of alternative receptor binding.The A. haemolyticum genome was sequenced to >20X coverage, and assembled to50 contigs covering ~95% of the genome. The genome is ~1.95Mb with a mol %G+C of53.1% and contained no plasmids. pld and aln have a reduced mol %G+C of 47.2% and46.5%, respectively, indicating the possibility of gene acquisition by horizontal transfer.Initial bioinformatics analysis identified genes encoding a protease, an extracellularDNase, two neuraminidases and three fimbrial biosynthetic operons were also identifiedwithin the genome.en_US
dc.typetexten_US
dc.typeElectronic Dissertationen_US
dc.subjectalnen_US
dc.subjectarcanobacteriumen_US
dc.subjecthaemolyticumen_US
dc.subjectplden_US
dc.subjectvirulenceen_US
thesis.degree.namePh.D.en_US
thesis.degree.leveldoctoralen_US
thesis.degree.disciplineMicrobiologyen_US
thesis.degree.disciplineGraduate Collegeen_US
thesis.degree.grantorUniversity of Arizonaen_US
dc.contributor.advisorJost, B. Helenen_US
dc.contributor.chairJost, B. Helenen_US
dc.contributor.committeememberJost, B. Helenen_US
dc.contributor.committeememberBillington, Stephen J.en_US
dc.contributor.committeememberPierson, L. S.en_US
dc.identifier.proquest10521en_US
dc.identifier.oclc659752251en_US
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