Structure-function analysis of MmpL7-mediated lipid transport in mycobacteria

Mycobacterial membrane protein Large (MmpL7) is a Resistance-Nodulation-Division (RND) family transporter required for the export of the virulence lipid, phthiocerol dimycocerosate (PDIM), in Mycobacterium tuberculosis. Using a null mutant of the related, vaccine strain Mycobacterium bovis BCG, we show that MmpL7 is also involved in the transport of the structurally related phenolic glycolipid (PGL), which is also produced by the hypervirulent M. tuberculosis strain HN878, but absent in M. tuberculosis H37Rv. Furthermore, we generated an in silico model of M. tuberculosis MmpL7 that revealed MmpL7 as a functional outlier within the MmpL-family, missing a canonical proton-relay signature sequence, suggesting that it employs a yet-unidentified mechanism for energy coupling for transport. In addition, our analysis demonstrates that the periplasmic porter domain 2 insert (PD2-insert), which doesn't share any recognisable homology, is highly alpha-helical in nature, suggesting an organisation similar to that seen in the hopanoid PD3/4 domains. Using the M. bovis BCG mmpL7 mutant for functional complementation with mutated alleles of mmpL7, we were able to identify residues present in the transmembrane domains TM4 and TM10, and the PD2 domain insert that play a crucial role in PDIM transport, and in certain cases, biosynthesis of PDIM

Molecular mechanisms of cell wall lipid transport in Mycobacterium tuberculosis

Presently, Tuberculosis (Tb), caused by Mycobacterium tuberculosis (Mtb), remains a global health threat, particularly with the emergence of drug-resistant strains and HIV and Tb co-infection that challenges the current Tb treatment. Preventative measures and several anti-Tb drugs target the lipids of the mycobacterial outer membrane consisting of mycolic acids amongst an array of glycolipids. In Mtb, there are thirteen outer membrane lipid transporters designated mycobacterial membrane protein large (MmpL) proteins. These proteins are also involved in heme acquisition and possibly drug resistance and nitrosative stress adaptation, contributing to mycobacterial survival and pathogenesis. MmpL proteins are structurally and phylogenetically classed as resistance-nodulation-division (RND) transporters that use protonmotive force (PMF) to mediate substrate translocation. Unlike their RND counterparts in Gram-negative bacteria the structure and mechanisms of these transporters in Mycobacteria are yet to be determined. This work aimed to investigate the mechanism of three transport systems: MmpL3, MmpL7 and DrrABC (encoded by mmpL3, mmpL7 and drrABC genes) using molecular modelling, genetics and lipid and protein biochemistry. mmpL3 is essential for trehalose monomycolate (TMM) transport. While mmpL7 is required for the export of two structurally related lipids, phthiocerol dimycocerosates (PDIM) and phenolic glycolipids (PGL). With the use of different membrane extraction agents, we were able to identify and propose improvements for the structural characterisation of MmpL3 and MmpL7. Molecular analysis and the MmpL7 homology model facilitated the identification of residues in the transmembrane and periplasmic domain that were verified by genetics and lipid analysis as critical for PDIM synthesis and transport. Even though MmpL7 is an RND transporter, there was no evidence of conserved PMF sites or a network forming proton translocation channel. Conveniently within the same genetic context is drrC, part of the drrABC cluster, that has a co-dependent relationship with mmpL7 in PDIM transport. Interestingly the overexpression of only drrAB genes function as a multi-drug ATP-binding cassette (ABC) pump. Structural modelling and molecular analysis revealed that drrABC encodes a heterodimeric ABC transporter where drrB and drrC encode the transmembrane spanning domains that create a pore or channel for substrate entry/exit, while drrA encodes the nucleotide binding domain for ATP binding and hydrolysis mediated transport suggesting a potential mechanism for PDIM/PGL transport. Genetics and biochemical analyses verified that all drrABC genes were required for PDIM transport across the membrane. Indeed, this work provides evidence that MmpL7 does not operate as an independent transporter but instead serves as a scaffold linking lipid synthesis and transport. The knowledge gained from such investigations related to MmpL proteins endeavours to aid in better understanding of anti-Tb drug development

Construction and phenotypic characterization of Mycobacterium smegmatis mutants deficient in DNA glycosylases

A dissertation submitted to the Faculty of Health science, University of the Witwatersrand, Johannesburg, in fulfilment of the requirements for the degree of Master of Science in Medicine. Johannesburg 2013The causative pathogen of tuberculosis, Mycobacterium tuberculosis (Mtb), is equipped with several DNA repair mechanisms for continued survival within the host. One such mechanism is Base Excision Repair (BER) that repairs DNA damage caused by reactive oxygen and nitrogen species (ROS/RNS) generated by the host immune cells during infection. BER is dependent on DNA glycosylases namely: formamidopyrimidine (Fpg/MutM/Fapy), endonucleaseVIII (Nei) and endonucleaseIII (Nth) with Nei being structurally similar to Fpg but functionally similar to Nth. Bioinformatics analysis of the genome sequences of Mtb and its non-pathogenic relative Mycobacterium smegmatis (Msm) identified a unique duplication of Fpg and Nei glycosylases and a single nth gene in the same chromosomal context in both organisms. Previously, it has been shown that the lack of Fpg/Nei glycosylases in Msm display no differences in growth and survival under normal and oxidative stress conditions with no increase in spontaneous mutation rates as compared to the parental strain, suggesting that nth maybe significant for mycobacterial genome maintenance. Hence, in this study the nth gene was site specifically inactivated by homologous recombination in the parental Msm strain and in selected combinatorial mutant strains deficient in the Fpg/Nei glycosylases. Loss of the nth allele in the panel of mutants was genotypically confirmed by PCR and southern blot analyses. Inactivation of the nth gene did not affect the in vitro growth of the mutant strains under normal culture conditions. Interestingly, UV induced DNA damage of the single nth mutant resulted in a dramatic increase in mutation frequency that was not observed in any of the mutants. The progressive loss of fpg, nei and nth genes showed exaggerated reduced survival under oxidative stress. The subsequent deletion of nth in mutants deficient in fpg/nei resulted in a dramatic increase in spontaneous mutation rates and frequencies, implying that nth is integral for the repair of both spontaneous and induced DNA damage. Undoubtedly, these results indicate that Msm nth encoding the Nth glycosylase is involved in DNA repair and has anti-mutator properties. Furthermore, nth together with fpg and nei is part of a robust DNA repair system that maintains the integrity of the mycobacterial genome