Characterisation of the Mycobacterium tuberculosis DRRABC ATP-binding cassette transporter

Nash, Angus A. (2003) Characterisation of the Mycobacterium tuberculosis DRRABC ATP-binding cassette transporter. PhD thesis, University of Glasgow.

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Mycobacterium tuberculosis, the aetiological agent of pulmonary tuberculosis (TB), is a re-emerging threat to global public health. TB continues to account for in excess of 2 million deaths annually with mortality and morbidity set to rise over the next decade as a consequence of the global HTV/AIDS pandemic. Many of the problems associated with treating this ancient disease are related to the intrinsic drug resistance of the causal organism, a factor that is compounded by the relatively small number of effective chemotherapeutic agents available. The current situation is further exacerbated by the emergence of multi-drug resistant strains of M. tuberculosis (MDR-TB) in certain parts of south-east Asia and eastern Europe. The global spread of such strains, fuelled by the increased availability of air travel, represents a serious threat to even the rich, industrialised nations of the West since successful treatment of MDR-TB requires the use of expensive and highly toxic drugs. These various factors miderline the urgent need for new and effective TB drug treatments. The development of such agents will undoubtedly require a much-improved understanding of M tuberculosis at the most basic biochemical and genetic levels. To this end the completion of the TB genome sequencing project in 1998 represents a major breakthrough. The genome provides not only a detailed blueprint of every potential drug target that might be used in treatment of the disease, but also every conceivable drug resistance mechanism employed by the organism to evade our current treatments. Active efflux is one of the key strategies used by microorganisms to avoid the deleterious effects of xenotoxic compounds, such transport processes being catalysed by an array of membrane-associated proteins. The ATP-binding cassette (ABC) transporters have been identified as one of the largest and most widely distributed families of such transmembrane transport systems. Virtually ubiquitous in nature, ABC transporters have been found in the genomes of every organism from the simplest archaea through to man. Whilst the vast majority of ABC transporters mediate standard cellular processes such as nutrient acquisition, a significant number have been shown to exhibit a broad substrate range and the capability to transport structurally diverse molecules. It is the expression of these broad-substrate transporters that is most commonly linked with drug resistance phenotypes. Little is currently known about the contribution of ABC transporter systems to the intrinsic drug resistance of M tuberculosis. In fact, despite the presence of a large number of ABC transporters in the genome, very little is known about the biological role of any M. tuberculosis ABC transporter. Against this background it was the remit of this thesis to investigate the structure and function of just one such system. The M tuberculosis drrABC operon was identified during the genome sequencing project as encoding a potential antibiotic resistance mechanism. The basis of this assumption was a significant level of sequence homology between the drrABC genes and those encoding a known antibiotic efflux ABC transporter from the evolutionarily related organism Streptomyces peucetius. The overall experimental strategy adopted was to clone and over-express the three genes of the drrABC operon in a non-pathogenic heterologous host (E. coli). It was hoped that this approach might generate large enough quantities of the individual proteins to investigate not only the biological function of the DrrABC transporter but also the structure of isolated components. Expression of the mycobacterial genes in E. coli proved to be a challenging undertaking, differences in the genetics of the host and donor organisms making co-expression of all three genes impossible. Expression of the individual proteins in isolation was somewhat more successful, with the ATP-binding sub-unit of the transporter, the DrrA protein, over-expressed and purified as a catalytically active fusion protein. The membrane- associated DrrB protein was successfully expressed in E. coli yet proved highly unstable and resistant to extraction from the membrane. The same problems applied to the second membrane-bound component of the transporter, DrrC. Issues of low expression and protein instability are not uncommon when working with membrane proteins and it appears that the M tuberculosis DrrB and C proteins are no different in this respect. The over-expression and purification of DrrA allowed a partial characterisation of the catalytic activity of the protein using traditional biochemical methods. As a cation- dependent ATPase DrrA was shown to exhibit biological activity broadly consistent with a role as the catalytic sub-unit of an ABC transporter. As such DrrA is only the second M tuberculosis protein for which such activity has been demonstrated. The exact biological function of the DrrABC transporter remains a matter of some doubt. Research by one group of scientists suggest that the transporter is indeed a functional antibiotic efflux mechanism, whilst another group argue that its primary role is the transmembrane transport of an important mycobacterial cell wall component. Whilst these two roles are not mutually exclusive, they do suggest that DrrABC may yet prove to be an attractive target for drug intervention. Unambiguous demonstration of the roles of this transporter, and the many other M tuberculosis transmembrane transport systems, will require a great deal of further research and the development of improved expression systems.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Additional Information: Adviser: Prof. Adrian R. Walmsley.
Keywords: Microbiology.
Colleges/Schools: College of Medical Veterinary and Life Sciences
Supervisor's Name: Supervisor, not known
Date of Award: 2003
Depositing User: Enlighten Team
Unique ID: glathesis:2003-71421
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 10 May 2019 10:49
Last Modified: 07 Jun 2021 09:24

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