All ETDs from UAB

Advisory Committee Chair

Michael E Niederweis

Advisory Committee Members

David M Bedwell

Paul A Goepfert

John L Hartman Iv

Andries J Steyn

Document Type

Dissertation

Date of Award

2012

Degree Name by School

Doctor of Philosophy (PhD) Heersink School of Medicine

Abstract

Mycobacterium tuberculosis is a dangerous human pathogen that has the capacity to adapt to and survive within the harsh phagosomal environment of macrophages. One obstacle that must be overcome by pathogens is to acquire iron, an essential component and cofactor of proteins required for vital cellular processes. M. tuberculosis meets its iron demands in the face of an array of sequestration strategies of the host by synthesizing and secreting two siderophores called mycobactin and carboxymycobactin. Siderophores are low molecular weight high affinity iron binding compounds that steal iron away from host stores and have been shown to be important for virulence of M. tuberculosis. Much remains unknown regarding the secretion and uptake of siderophores. Genes encoding the predicted outer membrane proteins, MmpS4 and MmpS5, are upregulated under low iron conditions. They are also located in operons with proteins of the MmpL family which have been shown to be transporters of complex lipids of M. tuberculosis. Because of this, we hypothesized that MmpS4 and MmpS5 may secrete a complex lipid that is required for adaptation of M. tuberculosis growth under low iron conditions. In order to test this hypothesis mmpS4 and mmpS5 single deletion mutants as well as an mmpS4/S5 double deletion mutant were constructed in M. tuberculosis. While the single gene deletion mutants grew similarly to the parent wt strain under low iron conditions, the mmpS4/S5 double deletion mutant exhibited an extreme growth defect under these conditions leading to the conclusion that MmpS4 and MmpS5 are required for growth in vitro of M. tuberculosis in low iron conditions. Iron sensing and uptake of siderophores are not affected in the mmpS4/S5 double deletion mutant. TLC analysis of the culture filtrate showed that this strain is unable to secrete siderophores, even though it retains the ability to synthesize them. Sub-cellular fractionation experiments confirmed that MmpS4 and MmpS5 are membrane-associated proteins. This is the first evidence of membrane-associated components of a novel siderophore secretion system in mycobacteria demonstrating that the siderophore secretion system is required for virulence of M. tuberculosis in mice. The mmpS4/S5 double deletion strain failed to proliferate and survive in mice. This work significantly advances our knowledge of siderophore-dependent iron utilization in mycobacteria and is the first study to show a role of MmpS proteins in siderophore secretion by M. tuberculosis.

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