Protection ofMycobacterium tuberculosisfrom Reactive Oxygen Species Conferred by themel2Locus Impacts Persistence and Dissemination

Abstract

Persistence of Mycobacterium tuberculosis in humans represents a major roadblock to elimination of tuberculosis. We describe identification of a locus in M. tuberculosis, mel2, that displays similarity to bacterial bioluminescent loci and plays an important role during persistence in mice. We constructed a deletion of the mel2 locus and found that the mutant displays increased susceptibility to reactive oxygen species (ROS). Upon infection of mice by aerosol the mutant grows normally until the persistent stage, where it does not persist as well as wild type. Histopathological analyses show that infection with the mel2 mutant results in reduced pathology and both CFU and histopathology indicate that dissemination of the mel2 mutant to the spleen is delayed. These data along with growth in activated macrophages and infection of Phox ؊/؊ and iNOS ؊/؊ mice and bone marrow-derived macrophages suggest that the primary mechanism by which mel2 affects pathogenesis is through its ability to confer resistance to ROS. These studies provide the first insight into the mechanism of action for this novel class of genes that are related to bioluminescence genes. The role of mel2 in resistance to ROS is important for persistence and dissemination of M. tuberculosis and suggests that homologues in other bacterial species are likely to play a role in pathogenesis.Despite extensive efforts to eradicate tuberculosis, caused by Mycobacterium tuberculosis, worldwide and prevent the spread of antibiotic-resistant strains, tuberculosis remains one of the most frequent causes of death in humans. Currently, one-third of the world's population is thought to be persistently infected with tuberculosis (6,19,20). A better understanding of the mechanisms that lead to persistence in humans is needed before it will be possible to develop rational strategies to prevent establishment of latency and block reactivation from it. Although the mouse model, even when infected by the natural low-dose aerosol route, does not replicate all aspects of pathogenesis by tuberculosis, its cost-effectiveness and the presence of numerous reagents make it an important tool for examination of the acute and persistent stages of infection (26,50,51). As a result, much of our knowledge regarding the role of the host in controlling infections as well as the bacterial factors involved has been obtained using the mouse model (26,31,34).Initially, tubercle bacilli encounter naïve alveolar macrophages in the mouse lung that produce low levels of reactive oxygen species (ROS) and undergo an oxidative burst in response to infection (8, 52). In the absence of ROS production, there is a modest transient advantage for tuberculosis in the lung, suggesting that ROS initially assist in the control of bacterial growth (16). This innate immune response does little to prevent the growth of the bacteria in mouse lungs, since for the first month after infection, bacterial numbers increase to a million or more. This time point corresponds to the peaks in the numbers of CD4 and CD8 T cel...

Imaging tuberculosis with endogenous β-lactamase reporter enzyme fluorescence in live mice

“…1 where multiple strains were used, Mtb strain Erdman was used for all experiments. All strains were grown as described previously (49). Bacterial numbers were confirmed in all experiments by plating dilutions for colony-forming units.…”

Random inducible controlled expression (RICE) for identification of mycobacterial virulence genes

“…Bioluminescence genes have been suggested to play a role in defense against reactive oxygen species 15–17 . Furthermore, it is recognized that M. tuberculosis undergoes oxidative damage during latency 18 and we have shown that mel2 facilitates persistence of M. tuberculosis in mice by defending against reactive oxygen species 12 .…”

“…The second component of this model, the NADPH-dependent FMN oxidoreductase (SsuE), has similarity to MelG, encoded by the second ORF in the mel2 locus, which consists of multiple domains with flavin reductase-like motifs. The resulting working model, summarized in Figure 2, could explain the observed role of the mel2 locus in resistance to oxidative damage and its similarity to the lux locus found in Vibrio fisheri 12–14 . We expect to gain insight into the validity of this model through analysis of the biochemical and physiological roles of these proteins.…”

“…Several independent pools of M. smegmatis and individual clones of M. marinum cosmid libraries that represented the entire M. marinum genome were used to identify clones that conferred efficient adherence and entry into macrophages. As a result of these screens we have identified several new mycobacterial genes that are involved in host cell interactions and many of these are present in M. tuberculosis 4, 5 and have similar functions in M. tuberculosis 12–14 . One of the potential limitations of RICE is that over expression in this manner might lead to toxicity for the bacteria, but this problem can be easily circumvented by conditional expression using an inducible promoter.…”

Common Killing Mechanism for Bactericidal Antibacterial Compounds

“…8 In addition, Mtb can also detoxify ROS with proteins expressed from the mel2 locus. 9 These and other means of detoxification of ROS would prevent the generation of 8-OdG after treatment with the aforementioned biocidal antibiotics. The exact role that 8-OdG degradation and detoxification may play in antimicrobial resistance will need to be confirmed through further in vitro and in vivo experimentation.…”