ABSTRACT
Treatment of tuberculosis, a complex granulomatous disease, requires long-term multidrug therapy to overcome tolerance, an epigenetic drug resistance that is widely attributed to nonreplicating bacterial subpopulations. Here, we deploy Mycobacterium marinum-infected zebrafish larvae for in vivo characterization of antitubercular drug activity and tolerance. We describe the existence of multidrug-tolerant organisms that arise within days of infection, are enriched in the replicating intracellular population, and are amplified and disseminated by the tuberculous granuloma. Bacterial efflux pumps that are required for intracellular growth mediate this macrophage-induced tolerance. This tolerant population also develops when Mycobacterium tuberculosis infects cultured macrophages, suggesting that it contributes to the burden of drug tolerance in human tuberculosis. Efflux pump inhibitors like verapamil reduce this tolerance. Thus, the addition of this currently approved drug or more specific efflux pump inhibitors to standard antitubercular therapy should shorten the duration of curative treatment.
Subject(s)
Drug Tolerance , Macrophages/microbiology , Mycobacterium marinum/physiology , Mycobacterium tuberculosis/physiology , Tuberculosis/microbiology , Animals , Antitubercular Agents/therapeutic use , Bacterial Proteins/antagonists & inhibitors , Bacterial Proteins/metabolism , Disease Models, Animal , Granuloma/physiopathology , Humans , Larva/microbiology , Membrane Transport Modulators/pharmacology , Membrane Transport Proteins/metabolism , Mycobacterium Infections, Nontuberculous/drug therapy , Mycobacterium Infections, Nontuberculous/pathology , Mycobacterium Infections, Nontuberculous/physiopathology , Mycobacterium marinum/drug effects , Tuberculosis/drug therapy , Tuberculosis/pathology , Tuberculosis/physiopathology , Verapamil/pharmacology , Zebrafish/microbiologyABSTRACT
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.
Subject(s)
Mycobacterium tuberculosis , Tuberculosis , Humans , Rifampin/pharmacology , Proton Pump Inhibitors/pharmacology , Antitubercular Agents/pharmacology , Verapamil/pharmacology , Macrophages , Tuberculosis/drug therapy , Drug Tolerance , Bacterial Proteins , Microbial Sensitivity TestsABSTRACT
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.
Subject(s)
Macrophages/physiology , Mycobacterium tuberculosis/genetics , Rifampin/pharmacology , ATP-Binding Cassette Transporters/genetics , Antitubercular Agents/pharmacology , Bacterial Proteins/genetics , Drug Resistance, Multiple, Bacterial/genetics , Humans , Isoniazid/pharmacology , Loss of Function Mutation , Microbial Sensitivity Tests , Mycobacterium tuberculosis/isolation & purification , THP-1 Cells , Tuberculosis, Multidrug-Resistant/genetics , Tuberculosis, Multidrug-Resistant/microbiologyABSTRACT
Drug tolerance likely represents an important barrier to tuberculosis treatment shortening. We previously implicated the Mycobacterium tuberculosis efflux pump Rv1258c as mediating macrophage-induced tolerance to rifampicin and intracellular growth. In this study, we infected the human macrophage-like cell line THP-1 with drug-sensitive and drug-resistant M. tuberculosis strains and found that tolerance developed to most antituberculosis drugs, including the newer agents moxifloxacin, PA-824, linezolid, and bedaquiline. Multiple efflux pump inhibitors in clinical use for other indications reversed tolerance to isoniazid and rifampicin and slowed intracellular growth. Moreover, verapamil reduced tolerance to bedaquiline and moxifloxacin. Verapamil's R isomer and its metabolite norverapamil have substantially less calcium channel blocking activity yet were similarly active as verapamil at inhibiting macrophage-induced drug tolerance. Our finding that verapamil inhibits intracellular M. tuberculosis growth and tolerance suggests its potential for treatment shortening. Norverapamil, R-verapamil, and potentially other derivatives present attractive alternatives that may have improved tolerability.
Subject(s)
Antitubercular Agents/pharmacology , Drug Resistance, Bacterial/drug effects , Macrophages/physiology , Mycobacterium tuberculosis/drug effects , Verapamil/analogs & derivatives , Verapamil/pharmacology , Bacterial Proteins/antagonists & inhibitors , Calcium Channel Blockers/pharmacology , Carrier Proteins/antagonists & inhibitors , Cell Line , Humans , Microbial Sensitivity TestsABSTRACT
The need for lengthy treatment to cure tuberculosis stems from phenotypic drug resistance, also known as drug tolerance, which has been previously attributed to slowed bacterial growth in vivo. We discuss recent findings that challenge this model and instead implicate macrophage-induced mycobacterial efflux pumps in antimicrobial tolerance. Although mycobacterial efflux pumps may have originally served to protect against environmental toxins, in the pathogenic mycobacteria, they appear to have been repurposed for intracellular growth. In this light, we discuss the potential of efflux pump inhibitors such as verapamil to shorten tuberculosis treatment by their dual inhibition of tolerance and growth.
Subject(s)
ATP-Binding Cassette Transporters/antagonists & inhibitors , Antitubercular Agents/pharmacology , Mycobacterium tuberculosis/drug effects , Tuberculosis, Pulmonary/drug therapy , Verapamil/pharmacology , Virulence Factors/antagonists & inhibitors , ATP-Binding Cassette Transporters/genetics , ATP-Binding Cassette Transporters/metabolism , Biological Transport , Calcium Channel Blockers/pharmacology , Drug Resistance, Multiple, Bacterial/drug effects , Drug Synergism , Evolution, Molecular , Gene Expression , Humans , Macrophages/drug effects , Macrophages/microbiology , Mycobacterium tuberculosis/genetics , Mycobacterium tuberculosis/metabolism , Time Factors , Tuberculosis, Pulmonary/microbiology , Virulence Factors/genetics , Virulence Factors/metabolismABSTRACT
Efforts are underway globally to develop effective vaccines and drugs against M. tuberculosis (Mtb) to reduce the morbidity and mortality of tuberculosis. Improving detection of slow-growing mycobacteria could simplify and accelerate efficacy studies of vaccines and drugs in animal models and human clinical trials. Here, a real-time reverse transcription PCR (RT-PCR) assay was developed to detect pre-ribosomal RNA (pre-rRNA) of Mycobacterium bovis bacille Calmette-Guérin (BCG) and Mtb. This pre-rRNA biomarker is indicative of bacterial viability. In two different mouse models, the presence of pre-rRNA from BCG and Mtb in ex vivo tissues showed excellent agreement with slower culture-based colony-forming unit assays. The addition of a brief nutritional stimulation prior to molecular viability testing further differentiated viable but dormant mycobacteria from dead mycobacteria. This research has set the stage to evaluate pre-rRNA as a BCG and/or Mtb infection biomarker in future drug and vaccine clinical studies.
Subject(s)
Mycobacterium bovis , Mycobacterium tuberculosis , Tuberculosis , Animals , Mice , Humans , Mycobacterium bovis/genetics , Mycobacterium tuberculosis/genetics , BCG Vaccine , RNA Precursors , Tuberculosis/diagnosis , Tuberculosis/prevention & control , Vaccine Development , BiomarkersABSTRACT
The genus Mycobacterium, which is a member of the high G+C group of Gram-positive bacteria, includes important pathogens, such as M. tuberculosis and M. leprae. A recent publication in PNAS reported that M. marinum and M. bovis bacillus Calmette-Guérin produce a type of spore known as an endospore, which had been observed only in the low G+C group of Gram-positive bacteria. Evidence was presented that the spores were similar to endospores in ultrastructure, in heat resistance and in the presence of dipicolinic acid. Here, we report that the genomes of Mycobacterium species and those of other high G+C Gram-positive bacteria lack orthologs of many, if not all, highly conserved genes diagnostic of endospore formation in the genomes of low G+C Gram-positive bacteria. We also failed to detect the presence of endospores by light microscopy or by testing for heat-resistant colony-forming units in aged cultures of M. marinum. Finally, we failed to recover heat-resistant colony-forming units from frogs chronically infected with M. marinum. We conclude that it is unlikely that Mycobacterium is capable of endospore formation.
Subject(s)
Mycobacterium tuberculosis/physiology , Mycobacterium/physiology , Spores, Bacterial/physiology , Bacillus subtilis/genetics , Base Sequence , Chromosomes, Bacterial/genetics , DNA, Bacterial/genetics , Genome, Bacterial , Humans , Mycobacterium/genetics , Mycobacterium marinum/genetics , Mycobacterium marinum/physiology , Mycobacterium tuberculosis/genetics , Operon , Streptomyces/genetics , Tuberculosis/genetics , Tuberculosis/immunologyABSTRACT
Tuberculosis, caused by Mycobacterium tuberculosis (Mtb) is an ancient disease that has remained a leading cause of infectious death. Mtb has evolved drug resistance to every antibiotic regimen ever introduced, greatly complicating treatment, lowering rates of cure and menacing TB control in parts of the world. As technology has advanced, our understanding of antimicrobial resistance has improved, and our models of the phenomenon have evolved. In this review, we focus on recent research progress that supports an updated model for the evolution of drug resistance in Mtb. We highlight the contribution of drug tolerance on the path to resistance, and the influence of heterogeneity on tolerance. Resistance is likely to remain an issue for as long as drugs are needed to treat TB. However, with technology driving new insights and careful management of newly developed resources, antimicrobial resistance need not continue to threaten global progress against TB, as it has done for decades.
Subject(s)
Mycobacterium tuberculosis , Tuberculosis, Lymph Node , Humans , Mycobacterium tuberculosis/genetics , Antitubercular Agents/pharmacology , Antitubercular Agents/therapeutic use , Drug Resistance , BiologyABSTRACT
CD4 T cell effector function is required for optimal containment of Mycobacterium tuberculosis (Mtb) infection. IFNÉ£ produced by CD4 T cells is a key cytokine that contributes to protection. However, lung-infiltrating CD4 T cells have a limited ability to produce IFNÉ£, and IFNÉ£ plays a lesser protective role within the lung than at sites of Mtb dissemination. In a murine infection model, we observed that IFNÉ£ production by Mtb-specific CD4 T cells is rapidly extinguished within the granuloma but not within unaffected lung regions, suggesting localized immunosuppression. We identified a signature of TGFß signaling within granuloma-infiltrating T cells in both mice and rhesus macaques. Selective blockade of TGFß signaling in T cells resulted in an accumulation of terminally differentiated effector CD4 T cells, improved IFNÉ£ production within granulomas, and reduced bacterial burdens. These findings uncover a spatially localized immunosuppressive mechanism associated with Mtb infection and provide potential targets for host-directed therapy.
Subject(s)
Granuloma/immunology , T-Lymphocytes/immunology , Transforming Growth Factor beta/metabolism , Tuberculosis/immunology , Adaptive Immunity , Animals , CD4-Positive T-Lymphocytes , Cell Death , Cytokines , Disease Models, Animal , Female , Granuloma/microbiology , Inflammation , Interferon-gamma , Lung/microbiology , Macaca mulatta , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Mycobacterium tuberculosis , Th1 CellsABSTRACT
Mycobacterium tuberculosis (Mtb) infection is initiated in the distal airways, but the bacteria ultimately disseminate to the lung interstitium. Although various cell types, including alveolar macrophages (AM), neutrophils, and permissive monocytes, are known to be infected with Mtb, the initially infected cells as well as those that mediate dissemination from the alveoli to the lung interstitium are unknown. In this study, using a murine infection model, we reveal that early, productive Mtb infection occurs almost exclusively within airway-resident AM. Thereafter Mtb-infected, but not uninfected, AM localize to the lung interstitium through mechanisms requiring an intact Mtb ESX-1 secretion system. Relocalization of infected AM precedes Mtb uptake by recruited monocyte-derived macrophages and neutrophils. This dissemination process is driven by non-hematopoietic host MyD88/interleukin-1 receptor inflammasome signaling. Thus, interleukin-1-mediated crosstalk between Mtb-infected AM and non-hematopoietic cells promotes pulmonary Mtb infection by enabling infected cells to disseminate from the alveoli to the lung interstitium.
Subject(s)
Macrophages, Alveolar/immunology , Mycobacterium tuberculosis/immunology , Pulmonary Alveoli/immunology , Pulmonary Alveoli/microbiology , Tuberculosis/immunology , Tuberculosis/microbiology , Animals , Bacterial Proteins/metabolism , Granuloma/microbiology , Granuloma/pathology , Immunity, Innate/immunology , Inflammation/immunology , Mice , Mice, Inbred C57BL , Mice, Knockout , Myeloid Differentiation Factor 88/metabolism , Receptors, Interleukin-1/metabolismABSTRACT
The blockade of phagolysosomal fusion is considered a critical mycobacterial strategy to survive in macrophages. However, viable mycobacteria have been observed in phagolysosomes during infection of cultured macrophages, and mycobacteria have the virulence determinant MarP, which confers acid resistance in vitro. Here we show in mice and zebrafish that innate macrophages overcome mycobacterial lysosomal avoidance strategies to rapidly deliver a substantial proportion of infecting bacteria to phagolysosomes. Exploiting the optical transparency of the zebrafish, we tracked the fates of individual mycobacteria delivered to phagosomes versus phagolysosomes and discovered that bacteria survive and grow in phagolysosomes, though growth is slower. MarP is required specifically for phagolysosomal survival, making it an important determinant for the establishment of mycobacterial infection in their hosts. Our work suggests that if pathogenic mycobacteria fail to prevent lysosomal trafficking, they tolerate the resulting acidic environment of the phagolysosome to establish infection.
Subject(s)
Anti-Bacterial Agents/metabolism , Carboxylic Acids/metabolism , Lysosomes/microbiology , Macrophages/microbiology , Microbial Viability/drug effects , Mycobacterium marinum/physiology , Stress, Physiological , Animals , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Cells, Cultured , Disease Models, Animal , Host-Pathogen Interactions , Mice, Inbred C57BL , Mycobacterium Infections, Nontuberculous/microbiology , Mycobacterium marinum/drug effects , Mycobacterium marinum/growth & development , Virulence Factors/genetics , Virulence Factors/metabolism , ZebrafishABSTRACT
Three structural features of lipid A (addition of palmitate [C16 fatty acid], addition of aminoarabinose [positively charged amino sugar residue], and retention of 3-hydroxydecanoate [3-OH C10 fatty acid]) were determined for Pseudomonas aeruginosa isolates from patients with cystic fibrosis (CF; n=86), from the environment (n=13), and from patients with other conditions (n=14). Among P. aeruginosa CF isolates, 100% had lipid A with palmitate, 24.6% with aminoarabinose, and 33.3% retained 3-hydroxydecanoate. None of the isolates from the environment or from patients with other conditions displayed these modifications. These results indicate that unique lipid A modifications occur in clinical P. aeruginosa CF isolates.
Subject(s)
Cystic Fibrosis/microbiology , Lipid A/chemistry , Lung Diseases/microbiology , Pseudomonas Infections/microbiology , Pseudomonas aeruginosa/chemistry , Pseudomonas aeruginosa/classification , Arabinose/analogs & derivatives , Arabinose/analysis , Child , Child, Preschool , Chronic Disease , Cystic Fibrosis/epidemiology , Decanoic Acids/analysis , Humans , Infant , Lung Diseases/epidemiology , Palmitates/analysis , Prevalence , Pseudomonas Infections/epidemiology , Pseudomonas aeruginosa/isolation & purification , Pseudomonas aeruginosa/pathogenicityABSTRACT
Yersinia pestis is an important human pathogen that is maintained in flea-rodent enzootic cycles in many parts of the world. During its life cycle, Y. pestis senses host-specific environmental cues such as temperature and regulates gene expression appropriately to adapt to the insect or mammalian host. For example, Y. pestis synthesizes different forms of lipid A when grown at temperatures corresponding to the in vivo environments of the mammalian host and the flea vector. At 37 degrees C, tetra-acylated lipid A is the major form; but at 26 degrees C or below, hexa-acylated lipid A predominates. In this study, we show that the Y. pestis msbB (lpxM) and lpxP homologs encode the acyltransferases that add C12 and C(16:1) groups, respectively, to lipid IV(A) to generate the hexa-acylated form, and that their expression is upregulated at 21 degrees C in vitro and in the flea midgut. A Y. pestis deltamsbB deltalpxP double mutant that did not produce hexa-acylated lipid A was more sensitive to cecropin A, but not to polymyxin B. This mutant was able to infect and block fleas as well as the parental wild-type strain, indicating that the low-temperature-dependent change to hexa-acylated lipid A synthesis is not required for survival in the flea gut.
Subject(s)
Acyltransferases/metabolism , Gene Expression Regulation, Bacterial , Lipid A/metabolism , Temperature , Up-Regulation , Yersinia pestis/genetics , Yersinia pestis/metabolism , Acyltransferases/genetics , Animals , Anti-Bacterial Agents/pharmacology , Anti-Infective Agents/pharmacology , Antimicrobial Cationic Peptides/pharmacology , Female , Gastrointestinal Tract/microbiology , Genes, Bacterial/genetics , Lipid A/chemistry , Male , Microbial Sensitivity Tests , Mutation , Polymyxin B/pharmacology , Siphonaptera/microbiology , Yersinia pestis/drug effects , Yersinia pestis/growth & developmentABSTRACT
Lipopolysaccharide (LPS) is the major surface component of gram-negative bacteria, and a component of LPS, lipid A, is recognized by the innate immune system through the Toll-like receptor 4/MD-2 complex. Pseudomonas aeruginosa, an environmental gram-negative bacterium that opportunistically infects the respiratory tracts of patients with cystic fibrosis (CF), can synthesize various structures of lipid A. Lipid A from P. aeruginosa strains isolated from infants with CF has a specific structure that includes the removal of the 3 position 3-OH C10 fatty acid. Here we demonstrate increased expression of the P. aeruginosa lipid A 3-O-deacylase (PagL) in isolates from CF infants compared to that in environmental isolates. PagL activity was increased in environmental isolates by growth in medium limited for magnesium and decreased by growth at low temperature in laboratory-adapted strains of P. aeruginosa. P. aeruginosa PagL was shown to be an outer membrane protein by isopycnic density gradient centrifugation. Heterologous expression of P. aeruginosa pagL in Salmonella enterica serovar Typhimurium and Escherichia coli resulted in removal of the 3-OH C14 fatty acid from lipid A, indicating that P. aeruginosa PagL recognizes either 3-OH C10 or 3-OH C14. Finally, deacylated lipid A species were not observed in some clinical P. aeruginosa isolates from patients with severe pulmonary disease, suggesting that loss of PagL function can occur during long-term adaptation to the CF airway.