Background:The role of the MmpL11 transporter in mycobacteria is not understood. Results: Mycobacterium smegmatis mmpL11 mutants accumulate mycolic acid precursors and fail to transport monomeromycolyl diacylglycerol and mycolate ester wax to the bacterial surface. Conclusion: MmpL11 contributes to mycobacterial cell wall biosynthesis. Significance: MmpL11 plays a conserved role in mycobacterial cell wall biogenesis that is important for M. tuberculosis virulence.

The mycobactericidal properties of macrophages include the delivery of bacteria to a hydrolytic lysosome enriched in bactericidal Ubiquitin-derived peptides (Ub-peptides). To better understand interactions of ubiquitin-derived peptides with mycobacteria, we further characterized the structure and function of the bactericidal Ub-peptide Ub2. We found that Ub2 adopts a β-sheet conformation in the context of sodium dodecyl sulfate (SDS) micelles and phospholipid (POPC:POPG, 1:1) vesicles that was dependent upon the primary sequence of the peptide. Point mutations in Ub2 that reduced the net charge of the peptide decreased Ub2 bactericidal activity. We investigated Ub-peptide function in the context of model membranes and intact bacteria. Differential scanning calorimetry analysis demonstrated that Ub2 inserts into and perturbs model phospholipid vesicles. In addition, we demonstrate that Ub2 disrupts the integrity of the mycobacterial membrane, equilibrates the transmembrane potential and localizes within both the mycobacterial membrane and cytoplasm of treated bacteria. Finally, we identified additional bactericidal Ub-peptides and characterized their activity and structure. This study provides new insight into the mycobactericidal mechanisms of Ub-peptides.

The mycobactericidal properties of macrophages include the generation of reactive oxygen intermediates and the delivery of bacteria to a hydrolytic lysosome enriched in bactericidal ubiquitin-derived peptides (Ubpeptides). To better understand the interactions of ubiquitin-derived peptides with mycobacteria and identify putative mycobacterial intrinsic resistance mechanisms, we screened for transposon mutants with increased susceptibility to the bactericidal Ub-peptide Ub2. We isolated 27 Mycobacterium smegmatis mutants that were hypersusceptible to Ub2. Two mutants were isolated that possessed mutations in the msmeg_0166 gene, which encodes a transcriptional regulator. The msmeg_0166 mutants were also hypersusceptible to other host antimicrobial peptides and oxidative stress. In characterizing msmeg_0166, we found that it encodes a repressor of oxyS, and therefore we have renamed the gene roxY. We demonstrate that RoxY and OxyS contribute to M. smegmatis resistance to oxidative stress. An ahpD transposon mutant was also isolated in our screen for Ub-peptide hypersusceptibility. Overexpression of oxyS in M. smegmatis reduced transcription of the ahpCD genes, which encode a peroxide detoxification system. Our data indicate that RoxY, OxyS, and AhpD play a role in the mycobacterial oxidative stress response and are important for resistance to host antimicrobial peptides.Despite the availability of antibiotics to combat tuberculosis, it is one of the leading causes of death due to infectious disease. The World Health Organization estimates that Mycobacterium tuberculosis infects one-third of the world's population, and 8 million new cases of tuberculosis are reported annually (http://www.who.int/mediacentre/factsheets/fs104/en/). M. tuberculosis infections persist for decades despite the host immune response that exposes the bacterium to a number of stressful environments during the course of infection. The nature of these host defenses and the repertoire of M. tuberculosis resistance mechanisms are still being elucidated. The ability of M. tuberculosis to survive within the host macrophage is key to its pathogenesis. While the pathogen is well-equipped to establish a niche in resting macrophages, activation of the host macrophage shifts the balance toward mycobacterial clearance (26,38). The mycobactericidal activity of activated macrophages includes both oxidative and nonoxidative mechanisms. Studies with mice indicate that reactive oxygen intermediates (ROI) and reactive nitrogen intermediates (RNI) function in control of tuberculosis infection: M. tuberculosis infection of NADPH phagosome oxidase (NOX2)-deficient mice indicates a role for ROI in the lung, and Nos2 Ϫ/Ϫ mice are quickly killed upon infection with M. tuberculosis (1,5,17). Enhanced fusion of the bacterium-containing vacuole with the macrophage lysosome also contributes to the mycobactericidal activity of activated macrophages. The induction of autophagy in Mycobacterium bovis BCG-and M. tuberculosis-infected macrophages by serum starvation, rapamy...

Non-human primates, notably rhesus macaques (Macaca mulatta, RM), provide a robust experimental model to investigate the immune response to and effective control of Mycobacterium tuberculosis infections. Changes in the function of immune cells and immunosenescence may contribute to the increased susceptibility of the elderly to tuberculosis. The goal of this study was to examine the impact of age on M. tuberculosis host-pathogen interactions following infection of primary alveolar macrophages derived from young and aged rhesus macaques. Of specific interest to us was whether the mycobactericidal capacity of autophagic macrophages was reduced in older animals since decreased autophagosome formation and autophagolysosomal fusion has been observed in other cells types of aged animals. Our data demonstrate that alveolar macrophages from old RM are as competent as those from young animals for autophagic clearance of M. tuberculosis infection and controlling mycobacterial replication. While our data do not reveal significant differences between alveolar macrophage responses to M. tuberculosis by young and old animals, these studies are the first to functionally characterize autophagic clearance of M. tuberculosis by alveolar macrophages from RM.