The Mycobacterium tuberculosis (Mtb) electron transport chain (ETC) has received significant attention as a drug target, however its vulnerability may be affected by its flexibility in response to disruption. Here we determine the effect of the ETC inhibitors bedaquiline, Q203 and clofazimine on the Mtb ETC, and the value of the ETC as a drug target, by measuring Mtb's respiration using extracellular flux technology. We find that Mtb's ETC rapidly reroutes around inhibition by these drugs and increases total respiration to maintain ATP levels. Rerouting is possible because Mtb rapidly switches between terminal oxidases, and, unlike eukaryotes, is not susceptible to back pressure. Increased ETC activity potentiates clofazimine's production of reactive oxygen species, causing rapid killing in vitro and in a macrophage model. Our results indicate that combination therapy targeting the ETC can be exploited to enhance killing of Mtb.

Hydrogen sulfide (H 2 S) is involved in numerous pathophysiological processes and shares overlapping functions with CO and •NO. However, the importance of host-derived H 2 S in microbial pathogenesis is unknown. Here we show that Mtb-infected mice deficient in the H 2 S-producing enzyme cystathionine β-synthase (CBS) survive longer with reduced organ burden, and that pharmacological inhibition of CBS reduces Mtb bacillary load in mice. Highresolution respirometry, transcriptomics and mass spectrometry establish that H 2 S stimulates Mtb respiration and bioenergetics predominantly via cytochrome bd oxidase, and that H 2 S reverses •NO-mediated inhibition of Mtb respiration. Further, exposure of Mtb to H 2 S regulates genes involved in sulfur and copper metabolism and the Dos regulon. Our results indicate that Mtb exploits host-derived H 2 S to promote growth and disease, and suggest that host-directed therapies targeting H 2 S production may be potentially useful for the management of tuberculosis and other microbial infections.

The ubiquitous gasotransmitter hydrogen sulfide (H2S) has been recognized to play a crucial role in human health. Using cystathionine γ-lyase (CSE)-deficient mice, we demonstrate an unexpected role of H2S inMycobacterium tuberculosis(Mtb) pathogenesis. We showed thatMtb-infected CSE−/−mice survive longer than WT mice, and support reduced pathology and lower bacterial burdens in the lung, spleen, and liver. Similarly, in vitroMtbinfection of macrophages resulted in reduced colony forming units in CSE−/−cells. Chemical complementation of infected WT and CSE−/−macrophages using the slow H2S releaser GYY3147 and the CSE inhibitor DL-propargylglycine demonstrated that H2S is the effector molecule regulatingMtbsurvival in macrophages. Furthermore, we demonstrate that CSE promotes an excessive innate immune response, suppresses the adaptive immune response, and reduces circulating IL-1β, IL-6, TNF-α, and IFN-γ levels in response toMtbinfection. Notably,Mtbinfected CSE−/−macrophages show increased flux through glycolysis and the pentose phosphate pathway, thereby establishing a critical link between H2S and central metabolism. Our data suggest that excessive H2S produced by the infected WT mice reduce HIF-1α levels, thereby suppressing glycolysis and production of IL-1β, IL-6, and IL-12, and increasing bacterial burden. Clinical relevance was demonstrated by the spatial distribution of H2S-producing enzymes in human necrotic, nonnecrotic, and cavitary pulmonary tuberculosis (TB) lesions. In summary, CSE exacerbates TB pathogenesis by altering immunometabolism in mice and inhibiting CSE or modulating glycolysis are potential targets for host-directed TB control.

Iron is an essential factor for the growth and virulence of Mycobacterium tuberculosis (Mtb). However, little is known about the mechanisms by which the host controls iron availability during infection. Since ferritin heavy chain (FtH) is a major intracellular source of reserve iron in the host, we hypothesized that the lack of FtH would cause dysregulated iron homeostasis to exacerbate TB disease. Therefore, we used knockout mice lacking FtH in myeloid-derived cell populations to study Mtb disease progression. We found that FtH plays a critical role in protecting mice against Mtb, as evidenced by increased organ burden, extrapulmonary dissemination, and decreased survival in Fth−/− mice. Flow cytometry analysis showed that reduced levels of FtH contribute to an excessive inflammatory response to exacerbate disease. Extracellular flux analysis showed that FtH is essential for maintaining bioenergetic homeostasis through oxidative phosphorylation. In support of these findings, RNAseq and mass spectrometry analyses demonstrated an essential role for FtH in mitochondrial function and maintenance of central intermediary metabolism in vivo. Further, we show that FtH deficiency leads to iron dysregulation through the hepcidin–ferroportin axis during infection. To assess the clinical significance of our animal studies, we performed a clinicopathological analysis of iron distribution within human TB lung tissue and showed that Mtb severely disrupts iron homeostasis in distinct microanatomic locations of the human lung. We identified hemorrhage as a major source of metabolically inert iron deposition. Importantly, we observed increased iron levels in human TB lung tissue compared to healthy tissue. Overall, these findings advance our understanding of the link between iron-dependent energy metabolism and immunity and provide new insight into iron distribution within the spectrum of human pulmonary TB. These metabolic mechanisms could serve as the foundation for novel host-directed strategies.

BackgroundMycobacterium tuberculosis is a virulent bacillus causing tuberculosis, a disease responsible for million deaths each year worldwide. In order to understand its mechanism of pathogenesis in humans and to help control tuberculosis, functions of numerous Mycobacterium tuberculosis genes are being characterized. In this study we report the dual functionality of tlyA gene product of Mycobacterium tuberculosis annotated as Rv1694, a 268 amino acid long basic protein.ResultsThe recombinant purified Rv1694 protein was found to exhibit hemolytic activity in vitro. It showed concentration and time-dependent hemolysis of rabbit and human erythrocytes. Multiple oligomeric forms (dimers to heptamers) of this protein were seen on the membranes of the lysed erythrocytes. Like the oligomers of conventional, well-known, pore-forming toxins, the oligomers of Rv1694 were found to be resistant to heat and SDS, but were susceptible to reducing agents like β-mercaptoethanol as it had abolished the hemolytic activity of Rv1694 indicating the role of disulfide bond(s). The Rv1694 generated de novo by in vitro transcription and translation also exhibited unambiguous hemolysis confirming the self assembly and oligomerization properties of this protein. Limited proteolytic digestion of this protein has revealed that the amino terminus is susceptible while in solution but is protected in presence of membrane. Striking feature of Rv1694 is its presence on the cell wall of E. coli as visualized by confocal microscopy. The surface expression is consistent with the contact dependent haemolytic ability of E. coli expressing this protein. Also, immune serum specific to this protein inhibits the contact dependent hemolysis. Moreover, Rv1694 protein binds to and forms stable oligomers on the macrophage phagosomal membranes. In addition to all these properties, E. coli expressing Rv1694 was found to be susceptible to the antibiotic capreomycin as its growth was significantly slower than mock vector transformed E. coli. The S30 extract of E. coli expressing the Rv1694 had poor translational activity in presence of capreomycin, further confirming its methylation activity. Finally, incorporation of methyl group of [3H]-S-adenosylmethionine in isolated ribosomes also confirmed its methylation activity.ConclusionsThe Rv1694 has an unusual dual activity. It appears to contain two diverse functions such as haemolytic activity and ribosomal RNA methylation activity. It is possible that the haemolytic activity might be relevant to intra-cellular compartments such as phagosomes rather than cell lysis of erythrocytes and the self-assembly trait may have a potential role after successful entry into macrophages by Mycobacterium tuberculosis.

SummaryHeme oxygenase-1 (HO-1) is a cytoprotective enzyme that controls inflammatory responses and redox homeostasis; however, its role during pulmonary tuberculosis (TB) remains unclear. Using freshly resected human TB lung tissue, we examined the role of HO-1 within the cellular and pathological spectrum of TB. Flow cytometry and histopathological analysis of human TB lung tissues showed that HO-1 is expressed primarily in myeloid cells and that HO-1 levels in these cells were directly proportional to cytoprotection. HO-1 mitigates TB pathophysiology by diminishing myeloid cell-mediated oxidative damage caused by reactive oxygen and/or nitrogen intermediates, which control granulocytic karyorrhexis to generate a zonal HO-1 response. Using whole-body or myeloid-specific HO-1-deficient mice, we demonstrate that HO-1 is required to control myeloid cell infiltration and inflammation to protect against TB progression. Overall, this study reveals that zonation of HO-1 in myeloid cells modulates free-radical-mediated stress, which regulates human TB immunopathology.

Mycobacterium tuberculosis (M.tb) has evolved mechanisms to evade its destruction in phagolysosomes, where it successfully survives and replicates within phagocytes. Recent studies have shown that virulent strains of M.tb can translocate from the phagosome into the cytosol of dendritic cells (DC). The molecular mechanisms by which virulent M.tb strains can escape the phagosome remain unknown. Here we show that the virulent M.tb strain H37Rv, but not the vaccine strain Bacille Calmette-Guérin (BCG), escapes from the phagolysosome and enters the cytosol by interfering with the TLR-2-MyD88 signaling pathway. Using H37Rv mutants, we further demonstrate that the region of difference-1 (RD-1) locus and ESAT-6, a gene within the RD-1 locus, play an important role in the capacity of M.tb to migrate from the phagosome to the cytosol of macrophages. H37Rv, BCG, H37RvΔRD1, and H37RvΔESAT6 were able to translocate to the cytosol in macrophages derived from TLR-2- and MyD88-deficient animals, whereas only virulent H37Rv was able to enter the cytosol in macrophages from wild type mice. Therefore, signaling through the TLR-2–MyD88 pathway in macrophages plays an important role in confining M.tb within phagolysomes. Virulent strains of M.tb have evolved mechanisms to subvert this pathway, thus facilitating their translocation to the cytosol and to escape the toxic microenvironment of the phagosome or phagolysosome.

Signals modulating the production of Mycobacterium tuberculosis (Mtb) virulence factors essential for establishing long-term persistent infection are unknown. The WhiB3 redox regulator is known to regulate the production of Mtb virulence factors, however the mechanisms of this modulation are unknown. To advance our understanding of the mechanisms involved in WhiB3 regulation, we performed Mtb in vitro, intraphagosomal and infected host expression analyses. Our Mtb expression analyses in conjunction with extracellular flux analyses demonstrated that WhiB3 maintains bioenergetic homeostasis in response to available carbon sources found in vivo to establish Mtb infection. Our infected host expression analysis indicated that WhiB3 is involved in regulation of the host cell cycle. Detailed cell-cycle analysis revealed that Mtb infection inhibited the macrophage G1/S transition, and polyketides under WhiB3 control arrested the macrophages in the G0-G1 phase. Notably, infection with the Mtb whiB3 mutant or polyketide mutants had little effect on the macrophage cell cycle and emulated the uninfected cells. This suggests that polyketides regulated by Mtb WhiB3 are responsible for the cell cycle arrest observed in macrophages infected with the wild type Mtb. Thus, our findings demonstrate that Mtb WhiB3 maintains bioenergetic homeostasis to produce polyketide and lipid cyclomodulins that target the host cell cycle. This is a new mechanism whereby Mtb modulates the immune system by altering the host cell cycle to promote long-term persistence. This new knowledge could serve as the foundation for new host-directed therapeutic discovery efforts that target the host cell cycle.