All ETDs from UAB

Advisory Committee Chair

Michael Niederweis

Advisory Committee Members

Kevin Dybvig

Guillermo Marques

Andries Steyn

Charles Turnbough

Document Type

Dissertation

Date of Award

2010

Degree Name by School

Doctor of Philosophy (PhD) Heersink School of Medicine

Abstract

Mycobacterium tuberculosis (Mtb) infects one third of the global population and causes approximately 2,000,000 Tuberculosis-related deaths annually. Mycobacteria are Gram positive organisms but contain a unique outer membrane (OM) which is functionally similar but structurally different from those of Gram negative bacteria. The mycobacterial OM presents an efficient permeability barrier towards hydrophilic solutes. Slow permeation kinetics of hydrophilic molecules through membranes and several discoveries of cell wall channel-forming proteins indicate that mycobacterial OMs are functionalized by proteins. MspA is the primary porin of M. smegmatis and mediates diffusion of small, hydrophilic nutrients and antibiotics across the OM. Unlike porins of Gram negative bacteria, MspA forms a large, octameric ß-barrel and represents the founding member of a new class of OM proteins. Mtb does not encode Msp homologs and functions of Mtb OM proteins are unknown. Therefore, MspA serves to model transport of hydrophilic compounds across mycobacterial OMs. Here, the periplasmic L6 loop and the constriction zone were identified as determinants of transport through the MspA channel. The L6 loop affected transport of ions, monosaccharides, voltage-gating, expression, and channel stability, but was dispensable for porin function. Conversely, the constriction zone mutant MspA D90L was highly impaired for channel activity. As the role of the hydrophilic pathway across the OM during Mtb infection was unknown, we expressed MspA wt and D90L in Mtb during mouse infections to determine whether increased permeability would be beneficial due to increased nutrient uptake or detrimental due to toxic solute influx. Surface exposure of functional pores drastically reduced virulence, suggesting that toxic solute influx outweighs the benefits of increased nutrient uptake during infection. Because genes that reduce fitness are selected against in vivo, stable gene integration is required. Therefore, we constructed a new series of phage-based plasmids capable of multiple, stable integrations in mycobacterial chromosomes. When used in tandem, these vectors provide increased global expression of terminator-protected gene cassettes. Thus, this work describes how porins functionalize the hydrophilic pathway across mycobacterial OMs and defines low OM permeability as required for Mtb virulence. Additionally, new genetic tools were generated to enhance in vivo examination of genes in mycobacteria.

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