All ETDs from UAB

Advisory Committee Chair

Michael Niederweis

Advisory Committee Members

David M Bedwell

William H Benjamin

Kevin F Dybvig

Andries J Steyn

Charles L Turnbough

Document Type

Dissertation

Date of Award

2009

Degree Name by School

Doctor of Philosophy (PhD) Heersink School of Medicine

Abstract

The extraordinary capacity of Mycobacterium tuberculosis (Mtb) to adapt to environmental changes during infection contributes to its success as a pathogen. While the unique outer membrane (OM) of mycobacteria functions as a permeability barrier for toxic molecules, uptake of nutrients is required to sustain viability of Mtb. Whereas hydrophobic molecules can diffuse through membranes, uptake of small hydrophilic compounds is mediated by water-filled channels, porins. For example, Msp-like porins of M. smegmatis (Ms) were shown to be required for uptake of hydrophilic beta-lactam antibiotics, which are also known to diffuse through porins in gram-negative bacteria. Because the structure of the OM of Ms and Mtb is similar, we hypothesized that disruption of an unknown porin in Mtb would increase resistance to beta-lactams. A transposon library of M. bovis BCG (BCG) was screened for mutants with increased resistance to ampicillin. Mutations were found in genes required for the biosynthesis of the OM as well as two previously uncharacterized genes, rv0194 and rv3903c. Overexpression of rv0194 in BCG and Ms showed that Rv0194 functions as an ATP-binding cassette efflux pump that confers resistance to beta-lactams, suggesting that Mtb has tripartite drug efflux pumps that span the entire cell envelope similar to that of E. coli. A mutation in rv3903c largely increased resistance to hydrophilic drugs and conferred resistance to nitric oxide both in vitro and in vivo. Uptake of glycerol was minimal in the rv3903c mutant resulting in decreased growth rate, which can be restored when grown on medium with the hydrophobic nutrient, oleic acid, as the sole carbon source. The recombinant N-terminal domain of Rv3903c purified from Ms and E. coli had a channel activity with a primary single conductance of 4.0±0.2 nS. Furthermore, it was able to complement the growth defect of the rv3903c mutant and an msp porin mutant of Ms. We suggest that Rv3903c is the first identified porin of Mtb in a new class of proteins with an unusual signal sequence and unique domain organization. Taken together, this work significantly advances our understanding of the transport mechanisms for nutrients and toxic compounds in mycobacteria.

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