A Dose-Finding Study to Guide Use of Verapamil as an Adjunctive Therapy in Tuberculosis.

Induction of mycobacterial efflux pumps is a cause of Mycobacterium tuberculosis (Mtb) drug tolerance, a barrier to shortening antitubercular treatment. Verapamil inhibits Mtb efflux pumps that mediate tolerance to rifampin, a cornerstone of tuberculosis (TB) treatment. Verapamil's mycobacterial efflux pump inhibition also limits Mtb growth in macrophages in the absence of antibiotic treatment. These findings suggest that verapamil could be used as an adjunctive therapy for TB treatment shortening. However, verapamil is rapidly and substantially metabolized when co-administered with rifampin. We determined in a dose-escalation clinical trial of persons with pulmonary TB that rifampin-induced clearance of verapamil can be countered without toxicity by the administration of larger than usual doses of verapamil. An oral dosage of 360 mg sustained-release (SR) verapamil given every 12 hours concomitantly with rifampin achieved median verapamil exposures of 903.1 ng.h/mL (area under the curve (AUC)0-12 h ) in the 18 participants receiving this highest studied verapamil dose; these AUC findings are similar to those in persons receiving daily doses of 240 mg verapamil SR but not rifampin. Moreover, norverapamil:verapamil, R:S verapamil, and R:S norverapamil AUC ratios were all significantly greater than those of historical controls receiving SR verapamil in the absence of rifampin. Thus, rifampin administration favors the less-cardioactive verapamil metabolites and enantiomers that retain similar Mtb efflux inhibitory activity to verapamil, increasing overall benefit. Finally, rifampin exposures were 50% greater after verapamil administration, which may also be advantageous. Our findings suggest that a higher dosage of verapamil can be safely used as adjunctive treatment in rifampin-containing treatment regimens.

A dose-finding study to guide use of verapamil as an adjunctive therapy in tuberculosis.

Induction of mycobacterial efflux pumps is a cause of Mycobacterium tuberculosis (Mtb) drug tolerance, a barrier to shortening antitubercular treatment. Verapamil inhibits Mtb efflux pumps that mediate tolerance to rifampin, a cornerstone of tuberculosis treatment. Verapamil's mycobacterial efflux pump inhibition also limits Mtb growth in macrophages in the absence of antibiotic treatment. These findings suggest that verapamil could be used as an adjunctive therapy for TB treatment shortening. However, verapamil is rapidly and substantially metabolized when co-administered with rifampin. We determined in a dose-escalation clinical trial that rifampin-induced clearance of verapamil can be countered without toxicity by the administration of larger than usual doses of verapamil. An oral dosage of 360 mg sustained-release (SR) verapamil given every 12 hours concomitantly with rifampin achieved median verapamil exposures of 903.1 ng.h/ml (AUC 0-12h), similar to those in persons receiving daily doses of 240 mg verapamil SR but not rifampin. Norverapamil:verapamil, R:S verapamil and R:S norverapamil AUC ratios were all significantly greater than those of historical controls receiving SR verapamil in the absence of rifampin, suggesting that rifampin administration favors the less-cardioactive verapamil metabolites and enantiomers. Finally, rifampin exposures were significantly greater after verapamil administration. Our findings suggest that a higher dosage of verapamil can be safely used as adjunctive treatment in rifampin-containing treatment regimens.

Redefining tuberculosis: an interview with Lalita Ramakrishnan.

Professor Lalita Ramakrishnan is at the forefront of modern tuberculosis (TB) research. She has developed vital tools, most notably a robust zebrafish model, to study this disease, leading to seminal discoveries uncovering bacterial and host interactions throughout infection. Her group has harnessed this knowledge to develop new treatments for TB and shape clinical research. By unveiling these complex interactions, they have also improved our understanding of fundamental biology of macrophages and other infectious diseases, such as leprosy.

Gaucher disease protects against tuberculosis.

Biallelic mutations in the glucocerebrosidase (GBA1) gene cause Gaucher disease, characterized by lysosomal accumulation of glucosylceramide and glucosylsphingosine in macrophages. Gaucher and other lysosomal diseases occur with high frequency in Ashkenazi Jews. It has been proposed that the underlying mutations confer a selective advantage, in particular conferring protection against tuberculosis. Here, using a zebrafish Gaucher disease model, we find that the mutation GBA1 N370S, predominant among Ashkenazi Jews, increases resistance to tuberculosis through the microbicidal activity of glucosylsphingosine in macrophage lysosomes. Consistent with lysosomal accumulation occurring only in homozygotes, heterozygotes remain susceptible to tuberculosis. Thus, our findings reveal a mechanistic basis for protection against tuberculosis by GBA1 N370S and provide biological plausibility for its selection if the relatively mild deleterious effects in homozygotes were offset by significant protection against tuberculosis, a rampant killer of the young in Europe through the Middle Ages into the 19th century.

The human proton pump inhibitors inhibit Mycobacterium tuberculosis rifampicin efflux and macrophage-induced rifampicin tolerance.

Tuberculosis treatment requires months-long combination chemotherapy with multiple drugs, with shorter treatments leading to relapses. A major impediment to shortening treatment is that Mycobacterium tuberculosis becomes tolerant to the administered drugs, starting early after infection and within days of infecting macrophages. Multiple lines of evidence suggest that macrophage-induced drug tolerance is mediated by mycobacterial drug efflux pumps. Here, using assays to directly measure drug efflux, we find that M. tuberculosis transports the first-line antitubercular drug rifampicin through a proton gradient-dependent mechanism. We show that verapamil, a known efflux pump inhibitor, which inhibits macrophage-induced rifampicin tolerance, also inhibits M.tuberculosis rifampicin efflux. As with macrophage-induced tolerance, the calcium channel-inhibiting property of verapamil is not required for its inhibition of rifampicin efflux. By testing verapamil analogs, we show that verapamil directly inhibits M. tuberculosis drug efflux pumps through its human P-glycoprotein (PGP)-like inhibitory activity. Screening commonly used drugs with incidental PGP inhibitory activity, we find many inhibit rifampicin efflux, including the proton pump inhibitors (PPIs) such as omeprazole. Like verapamil, the PPIs inhibit macrophage-induced rifampicin tolerance as well as intramacrophage growth, which has also been linked to mycobacterial efflux pump activity. Our assays provide a facile screening platform for M. tuberculosis efflux pump inhibitors that inhibit in vivo drug tolerance and growth.

mTOR-regulated mitochondrial metabolism limits mycobacterium-induced cytotoxicity.

Necrosis of macrophages in the granuloma, the hallmark immunological structure of tuberculosis, is a major pathogenic event that increases host susceptibility. Through a zebrafish forward genetic screen, we identified the mTOR kinase, a master regulator of metabolism, as an early host resistance factor in tuberculosis. We found that mTOR complex 1 protects macrophages from mycobacterium-induced death by enabling infection-induced increases in mitochondrial energy metabolism fueled by glycolysis. These metabolic adaptations are required to prevent mitochondrial damage and death caused by the secreted mycobacterial virulence determinant ESAT-6. Thus, the host can effectively counter this early critical mycobacterial virulence mechanism simply by regulating energy metabolism, thereby allowing pathogen-specific immune mechanisms time to develop. Our findings may explain why Mycobacterium tuberculosis, albeit humanity's most lethal pathogen, is successful in only a minority of infected individuals.

Tumor necrosis factor induces pathogenic mitochondrial ROS in tuberculosis through reverse electron transport.

Tumor necrosis factor (TNF) is a critical host resistance factor against tuberculosis. However, excess TNF produces susceptibility by increasing mitochondrial reactive oxygen species (mROS), which initiate a signaling cascade to cause pathogenic necrosis of mycobacterium-infected macrophages. In zebrafish, we identified the mechanism of TNF-induced mROS in tuberculosis. Excess TNF in mycobacterium-infected macrophages elevates mROS production by reverse electron transport (RET) through complex I. TNF-activated cellular glutamine uptake leads to an increased concentration of succinate, a Krebs cycle intermediate. Oxidation of this elevated succinate by complex II drives RET, thereby generating the mROS superoxide at complex I. The complex I inhibitor metformin, a widely used antidiabetic drug, prevents TNF-induced mROS and necrosis of Mycobacterium tuberculosis-infected zebrafish and human macrophages; metformin may therefore be useful in tuberculosis therapy.

The C terminus of the mycobacterium ESX-1 secretion system substrate ESAT-6 is required for phagosomal membrane damage and virulence.

SignificanceTuberculosis (TB), an ancient disease of humanity, continues to be a major cause of worldwide death. The causative agent of TB, Mycobacterium tuberculosis, and its close pathogenic relative Mycobacterium marinum, initially infect, evade, and exploit macrophages, a major host defense against invading pathogens. Within macrophages, mycobacteria reside within host membrane-bound compartments called phagosomes. Mycobacterium-induced damage of the phagosomal membranes is integral to pathogenesis, and this activity has been attributed to the specialized mycobacterial secretion system ESX-1, and particularly to ESAT-6, its major secreted protein. Here, we show that the integrity of the unstructured ESAT-6 C terminus is required for macrophage phagosomal damage, granuloma formation, and virulence.

Elevated cerebrospinal fluid cytokine levels in tuberculous meningitis predict survival in response to dexamethasone.

Adjunctive treatment with antiinflammatory corticosteroids like dexamethasone increases survival in tuberculosis meningitis. Dexamethasone responsiveness associates with a C/T variant in Leukotriene A4 Hydrolase (LTA4H), which regulates expression of the proinflammatory mediator leukotriene B4 (LTB4). TT homozygotes, with increased expression of LTA4H, have the highest survival when treated with dexamethasone and the lowest survival without. While the T allele is present in only a minority of the world's population, corticosteroids confer modest survival benefit worldwide. Using Bayesian methods, we examined how pretreatment levels of cerebrospinal fluid proinflammatory cytokines affect survival in dexamethasone-treated tuberculous meningitis. LTA4H TT homozygosity was associated with global cytokine increases, including tumor necrosis factor. Association between higher cytokine levels and survival extended to non-TT patients, suggesting that other genetic variants may also induce dexamethasone-responsive pathological inflammation. These findings warrant studies that tailor dexamethasone therapy to pretreatment cerebrospinal fluid cytokine concentrations, while searching for additional genetic loci shaping the inflammatory milieu.

A Bayesian analysis of the association between Leukotriene A4 Hydrolase genotype and survival in tuberculous meningitis.

Tuberculous meningitis has high mortality, linked to excessive inflammation. However, adjunctive anti-inflammatory corticosteroids reduce mortality by only 30%, suggesting that inflammatory pathophysiology causes only a subset of deaths. In Vietnam, the survival benefit of anti-inflammatory corticosteroids was most pronounced in patients with a C/T promoter variant in the leukotriene A4 hydrolase (LTA4H) gene encoding an enzyme that regulates inflammatory eicosanoids. LTA4H TT patients with increased expression had increased survival, consistent with corticosteroids benefiting individuals with hyper-inflammatory responses. However, an Indonesia study did not find an LTA4H TT genotype survival benefit. Here using Bayesian methods to analyse both studies, we find that LTA4H TT genotype confers survival benefit that begins early and continues long-term in both populations. This benefit is nullified in the most severe cases with high early mortality. LTA4H genotyping together with disease severity assessment may target glucocorticoid therapy to patients most likely to benefit from it.

Tuberculous meningitis is a serious infection of the lining of the brain, which affects over 100,000 people a year. Without treatment, it is always fatal: even with proper antibiotics, about a quarter of patients do not survive and many will have permanent brain damage. Overactive inflammation is thought to contribute to this process. Corticosteroid drugs, which dampen the inflammatory process, are therefore often used during treatment. However, they merely reduce mortality by 30%, suggesting that only some people benefit from them. Two recent studies have linked the genetic makeup of individuals who have tuberculous meningitis to how they respond to corticosteroids. There were, in particular, differences in the LTA4H gene that codes for an inflammation-causing protein. According to these results, only individuals carrying high-inflammation versions of the LTA4H gene would benefit from the treatment. Yet a third study did not find any effect of the genetic background of patients. All three papers used frequentist statistics to draw their conclusions, only examining the percentage of people who survived in each group. Yet, this type of analysis can miss important details. It also does not work well when the number of patients is small, or when the effectiveness of a drug varies during the course of an illness. Another method, called Bayesian statistics, can perform better under these limitations. In particular, it takes into account the probability of an event based on prior knowledge ­ for instance, that the risk of dying varies smoothly with time. Here, Whitworth et al. used Bayesian statistics to reanalyse the data from these studies, demonstrating that death rates were correlated with the type of LTA4H gene carried by patients. In particular, corticosteroid treatment worked best for people with the high inflammation versions of the gene. However, regardless of genetic background, corticosteroids were not effective if patients were extremely sick before being treated. The work by Whitworth et al. demonstrates the importance of using Bayesian statistics to examine the effectiveness of medical treatments. It could help to design better protocols for tuberculous meningitis treatment, tailored to the genetic makeup of patients.

Tumor Necrosis Factor and Schistosoma mansoni egg antigen omega-1 shape distinct aspects of the early egg-induced granulomatous response.

Infections by schistosomes result in granulomatous lesions around parasite eggs entrapped within the host tissues. The host and parasite determinants of the Schistosoma mansoni egg-induced granulomatous response are areas of active investigation. Some studies in mice implicate Tumor Necrosis Factor (TNF) produced in response to the infection whereas others fail to find a role for it. In addition, in the mouse model, the S. mansoni secreted egg antigen omega-1 is found to induce granulomas but the underlying mechanism remains unknown. We have recently developed the zebrafish larva as a model to study macrophage recruitment and granuloma formation in response to Schistosoma mansoni eggs. Here we use this model to investigate the mechanisms by which TNF and omega-1 shape the early granulomatous response. We find that TNF, specifically signaling through TNF receptor 1, is not required for macrophage recruitment to the egg and granuloma initiation but does mediate granuloma enlargement. In contrast, omega-1 mediates initial macrophage recruitment, with this chemotactic activity being dependent on its RNase activity. Our findings further the understanding of the role of these host- and parasite-derived factors and show that they impact distinct facets of the granulomatous response to the schistosome egg.

Schistosoma mansoni Eggs Modulate the Timing of Granuloma Formation to Promote Transmission.

Schistosome eggs provoke the formation of granulomas, organized immune aggregates, around them. For the host, the granulomatous response can be both protective and pathological. Granulomas are also postulated to facilitate egg extrusion through the gut lumen, a necessary step for parasite transmission. We used zebrafish larvae to visualize the granulomatous response to Schistosomamansoni eggs and inert egg-sized beads. Mature eggs rapidly recruit macrophages, which form granulomas within days. Beads also induce granulomas rapidly, through a foreign body response. Strikingly, immature eggs do not recruit macrophages, revealing that the eggshell is immunologically inert. Our findings suggest that the eggshell inhibits foreign body granuloma formation long enough for the miracidium to mature. Then parasite antigens secreted through the eggshell trigger granulomas that facilitate egg extrusion into the environment. In support of this model, we find that only mature S. mansoni eggs are shed into the feces of mice and humans.

Mycobacterium marinum phthiocerol dimycocerosates enhance macrophage phagosomal permeabilization and membrane damage.

Phthiocerol dimycocerosates (PDIMs) are a class of mycobacterial lipids that promote virulence in Mycobacterium tuberculosis and Mycobacterium marinum. It has recently been shown that PDIMs work in concert with the M. tuberculosis Type VII secretion system ESX-1 to permeabilize the phagosomal membranes of infected macrophages. As the zebrafish-M. marinum model of infection has revealed the critical role of PDIM at the host-pathogen interface, we set to determine if PDIMs contributed to phagosomal permeabilization in M. marinum. Using an ΔmmpL7 mutant defective in PDIM transport, we find the PDIM-ESX-1 interaction to be conserved in an M. marinum macrophage infection model. However, we find PDIM and ESX-1 mutants differ in their degree of defect, with the PDIM mutant retaining more membrane damaging activity. Using an in vitro hemolysis assay-a common surrogate for cytolytic activity, we find that PDIM and ESX-1 differ in their contributions: the ESX-1 mutant loses hemolytic activity while PDIM retains it. Our observations confirm the involvement of PDIMs in phagosomal permeabilization in M. marinum infection and suggest that PDIM enhances the membrane disrupting activity of pathogenic mycobacteria and indicates that the role they play in damaging phagosomal and red blood cell membranes may differ.

SLeuthing Tuberculous Cough.

Cough, a hallmark of tuberculosis, transmits the disease. Ruhl et al. find that a Mycobacterium tuberculosis (Mtb)-specific lipid, SL-1, stimulates human nociceptive neurons and makes guinea pigs cough. Mtb extract, but not SL-1, also stimulates non-nociceptive neurons that participate in the cough reflex, suggesting additional cough-inducing mechanisms.

Mycobacterium tuberculosis pathogenicity viewed through the lens of molecular Koch's postulates.

Thirty years ago Stanley Falkow formulated molecular Koch's postulates as a framework to help dissect the contribution of microbial genes to their pathogenicity (Box 1). Three years later, his advice led me to develop Mycobacterium marinum, a close genetic relative of Mycobacterium tuberculosis, as a model for tuberculosis pathogenesis. Here, I discuss insights into M. tuberculosis pathogenicity from studying M. marinum in the zebrafish, and frame them in terms of molecular Koch's postulates. The highly orchestrated life cycle of M. tuberculosis is achieved in substantial measure not by "traditional" pathogen-exclusive virulence genes acquired along its evolutionary history, but rather by genes that are shared with its environmental ancestors. Together, these genes support its tactics of subterfuge and exploitation to overcome host immunity so as to produce the transmissible disease that ensures the evolutionary survival of this obligate human pathogen.

TNF Induces Pathogenic Programmed Macrophage Necrosis in Tuberculosis through a Mitochondrial-Lysosomal-Endoplasmic Reticulum Circuit.

Necrosis of infected macrophages constitutes a critical pathogenetic event in tuberculosis by releasing mycobacteria into the growth-permissive extracellular environment. In zebrafish infected with Mycobacterium marinum or Mycobacterium tuberculosis, excess tumor necrosis factor triggers programmed necrosis of infected macrophages through the production of mitochondrial reactive oxygen species (ROS) and the participation of cyclophilin D, a component of the mitochondrial permeability transition pore. Here, we show that this necrosis pathway is not mitochondrion-intrinsic but results from an inter-organellar circuit initiating and culminating in the mitochondrion. Mitochondrial ROS induce production of lysosomal ceramide that ultimately activates the cytosolic protein BAX. BAX promotes calcium flow from the endoplasmic reticulum into the mitochondrion through ryanodine receptors, and the resultant mitochondrial calcium overload triggers cyclophilin-D-mediated necrosis. We identify ryanodine receptors and plasma membrane L-type calcium channels as druggable targets to intercept mitochondrial calcium overload and necrosis of mycobacterium-infected zebrafish and human macrophages.

Diverse Clinical Isolates of Mycobacterium tuberculosis Develop Macrophage-Induced Rifampin Tolerance.

The Mycobacterium tuberculosis lineage 4 strains CDC1551 and H37Rv develop tolerance to multiple antibiotics upon macrophage residence. To determine whether macrophage-induced tolerance is a general feature of clinical M. tuberculosis isolates, we assessed macrophage-induced drug tolerance in strains from lineages 1-3, representing the other predominant M. tuberculosis strains responsible for tuberculosis globally. All 3 lineages developed isoniazid tolerance. While lineage 1, 3, and 4 strains developed rifampin tolerance, lineage 2 Beijing strains did not. Their failure to develop tolerance may be explained by their harboring of a loss-of-function mutation in the Rv1258c efflux pump that is linked to macrophage-induced rifampicin tolerance.