Contribution of theMycobacterium tuberculosisMmpL Protein Family to Virulence and Drug Resistance

Abstract

The genome sequence of Mycobacterium tuberculosis revealed the presence of 12 membrane proteins proposed to have a function in the transport of lipids. Insertional inactivation of 11 of these has revealed that only 1 (MmpL3) is apparently essential for viability. Five of these proteins are conserved within the genome of Mycobacterium leprae. The drug susceptibilities of these 11 mutants to a broad spectrum of agents are unaltered, suggesting that unlike their function in other organisms, these proteins do not play a significant role in intrinsic drug resistance. Each of these mutants was assessed for growth kinetics and lethality in a murine low-dose aerosol model of tuberculosis, and four were found to be impaired in one or both measures of virulence. Two of these, with mutations of MmpL4 and the previously characterized MmpL7, which transports phthiocerol dimycocerosate, were found to have both impaired growth kinetics and impaired lethality. Mutants with inactivation of MmpL8, which transports a precursor of the sulfatides, or MmpL11, which transports an unknown substrate, were found to establish infection normally but to be significantly attenuated for lethality in time-to-death studies. These studies support the concept that MmpL-mediated lipid secretion both contributes to the innate ability of the pathogen to survive intracellularly and also contributes directly to the host-pathogen dialogue that determines the ultimate outcome of infection.

Discovery and characterization of a unique mycobacterial heme acquisition system

“…5 C and D). These results imply extracellular to intracellular heme transport and offer concrete support of the previous proposal that several MmpL proteins may be involved in mycobacterial metabolite import (32). The high sequence homology and close genomic proximity of MmpL3 and MmpL11 (and also the hemophore Rv0203), and the similar heme binding properties of their soluble domains, support roles of both MmpL3 and MmpL11 in heme uptake.…”

“…Thus, we postulate that MmpL11 and MmpL3 are both heme transporters. Previously, it was shown that mmpL3 is likely to be essential (32), and our attempts to delete the potential heme uptake region (Rv0201c-Rv0207c) or mmpL3 alone from MtbΔmbtB were also unsuccessful. To evaluate the involvement of MmpL11 in heme uptake, a double mutant with deleted Rv0202c (mmpL11) was constructed by replacing most of the mmpL11 coding region with an apramycin resistance cassette and its promoter in-frame (Fig.…”

Rapid and spontaneous loss of phthiocerol dimycocerosate (PDIM) from Mycobacterium tuberculosis grown in vitro: implications for virulence studies

“…This cell-wall-associated lipid is composed of a long-chain b-diol (phthiocerol) esterified to two multi-methyl branched fatty acids (mycocerosic acids). At least three independent studies have confirmed that M. tuberculosis mutants deficient in PDIM synthesis or translocation are severely attenuated in vivo (Camacho et al, 1999;Cox et al, 1999;Domenech et al, 2005). None of these studies have reported an in vitro defect associated with the loss of PDIM.…”

“…In each of these two studies, the level of in vivo attenuation afforded through the loss of PDIM was approximately 50-fold in mouse lungs by just 3 weeks post-infection. We have subsequently described the same level of attenuation for an MmpL7 mutant generated in H37Rv (Domenech et al, 2005). MmpL7 is the transmembrane transport protein necessary for PDIM export.…”

“…), during our previous work on polyketide synthase (PKS) biosynthetic pathways and putative lipid-transport proteins in M. tuberculosis (Domenech et al, 2004(Domenech et al, , 2005Reed et al, 2004) we observed that a disproportionately high number of gene-replacement mutants that we had generated in the H37Rv background were completely devoid of PDIM. This was despite the fact that we were targeting genes in pathways that were not predicted to have an impact on PDIM biosynthesis.…”