Identification and characterization of a Mycobacterium tuberculosis gene that enhances mycobacterial survival within macrophages

Persistent Link:
http://hdl.handle.net/10150/279898
Title:
Identification and characterization of a Mycobacterium tuberculosis gene that enhances mycobacterial survival within macrophages
Author:
Wei, Jun
Issue Date:
2001
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:
The virulence of Mycobacterium tuberculosis (Mtb) depends on its ability to multiply and survive within host macrophages. In screening for Mtb genes that play a role in the intracellular survival, a Mtb gene ( eis) was identified that enhanced survival of Mycobacterium smegmatis in both human monocytes and in the human macrophage-like cell line U-937 when introduced on the multi-copy plasmid pOLYG. When a single chromosomal copy of eis was introduced into M. smegmatis, using an integrative vector, the construct still exhibited increased intracellular survival in U-937 cells. The eis gene was found in the genomic DNA of various M. tuberculosis strains and of Mycobacterium bovis BCG but not in that of M. smegmatis or 10 other mycobacterial species. Western blot analysis showed that the eis gene could produce a 42-kD protein product in both M. smegmatis and M. tuberculosis. The role of the eis gene in M. tuberculosis intracellular survival and multiplication was investigated by inactivation of the eis gene via allelic exchange. A mutated eis allele ( eis::hyg) was delivered at the eis locus, using the suicide vector pMJ10, in both Mtb strains H37Rv and H37Ra. Complemented mutants were also constructed by reintroducing a wild-type eis into the chromosome attB site. Southern and Western blot analysis demonstrated that the eis gene was disrupted and no Eis protein was produced, and that complemented strains regained the ability to produce Eis. Wild-type M. tuberculosis, eis knockout mutant and complemented strain were then evaluated for their capacity to survive and multiply within U-937 cells. The eis mutant survived and multiplied, as its parental strain, in U-937 cells over a 7-day period. These findings suggest that eis may not be required for the short-term multiplication of M. tuberculosis in U-937 cells. Further in vivo study needs to be done to clarify the role of eis in the pathogenesis of tuberculosis.
Type:
text; Dissertation-Reproduction (electronic)
Keywords:
Biology, Microbiology.
Degree Name:
Ph.D.
Degree Level:
doctoral
Degree Program:
Graduate College; Microbiology and Immunology
Degree Grantor:
University of Arizona
Advisor:
Friedman, Richard L.

Full metadata record

DC FieldValue Language
dc.language.isoen_USen_US
dc.titleIdentification and characterization of a Mycobacterium tuberculosis gene that enhances mycobacterial survival within macrophagesen_US
dc.creatorWei, Junen_US
dc.contributor.authorWei, Junen_US
dc.date.issued2001en_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.abstractThe virulence of Mycobacterium tuberculosis (Mtb) depends on its ability to multiply and survive within host macrophages. In screening for Mtb genes that play a role in the intracellular survival, a Mtb gene ( eis) was identified that enhanced survival of Mycobacterium smegmatis in both human monocytes and in the human macrophage-like cell line U-937 when introduced on the multi-copy plasmid pOLYG. When a single chromosomal copy of eis was introduced into M. smegmatis, using an integrative vector, the construct still exhibited increased intracellular survival in U-937 cells. The eis gene was found in the genomic DNA of various M. tuberculosis strains and of Mycobacterium bovis BCG but not in that of M. smegmatis or 10 other mycobacterial species. Western blot analysis showed that the eis gene could produce a 42-kD protein product in both M. smegmatis and M. tuberculosis. The role of the eis gene in M. tuberculosis intracellular survival and multiplication was investigated by inactivation of the eis gene via allelic exchange. A mutated eis allele ( eis::hyg) was delivered at the eis locus, using the suicide vector pMJ10, in both Mtb strains H37Rv and H37Ra. Complemented mutants were also constructed by reintroducing a wild-type eis into the chromosome attB site. Southern and Western blot analysis demonstrated that the eis gene was disrupted and no Eis protein was produced, and that complemented strains regained the ability to produce Eis. Wild-type M. tuberculosis, eis knockout mutant and complemented strain were then evaluated for their capacity to survive and multiply within U-937 cells. The eis mutant survived and multiplied, as its parental strain, in U-937 cells over a 7-day period. These findings suggest that eis may not be required for the short-term multiplication of M. tuberculosis in U-937 cells. Further in vivo study needs to be done to clarify the role of eis in the pathogenesis of tuberculosis.en_US
dc.typetexten_US
dc.typeDissertation-Reproduction (electronic)en_US
dc.subjectBiology, Microbiology.en_US
thesis.degree.namePh.D.en_US
thesis.degree.leveldoctoralen_US
thesis.degree.disciplineGraduate Collegeen_US
thesis.degree.disciplineMicrobiology and Immunologyen_US
thesis.degree.grantorUniversity of Arizonaen_US
dc.contributor.advisorFriedman, Richard L.en_US
dc.identifier.proquest3002536en_US
dc.identifier.bibrecord.b41431960en_US
All Items in UA Campus Repository are protected by copyright, with all rights reserved, unless otherwise indicated.