In vivo efficiency of targeted norfloxacin against persistent, isoniazid-insensitive, Mycobacterium bovis BCG present in the physiologically hypoxic mouse liver

Persistence of Mycobacterium tuberculosis is a hypoxia-inducible state in which the bacteria are phenotypically insensitive to currently available antituberculous drugs. In humans, persistent M. tuberculosis is found in granulomatous lesions, either inside macrophages or in necrotic tissue, where the partial oxygen pressure (pO(2)) is very low. Persistent bacteria can remain silent for decades before overt tuberculosis develops. Due to insensitivity to classical drugs, M. tuberculosis persistence prevents rapid and definitive clearance of bacteria. Consequently, therapeutic molecules are required that are both active against persistent bacilli and able to reach their intramacrophagic location. In contrast to its native form, norfloxacin is active in vivo against Mycobacterium bovis BCG present in the lungs when temporarily linked to a macromolecular carrier targeted to macrophages. To study the efficiency of this macromolecular prodrug targeted to persistent mycobacteria confined inside macrophages, we established a short-term in vivo model based on the physiological pO(2) differences between lungs, spleen and liver. Whereas lungs and spleen are well oxygenated, the liver has a low pO(2) due to its portal irrigation. Therefore, studying mycobacteria in the liver yields information about in vivo persistent bacilli exposed to low pO(2). To our knowledge, no similar short-term in vivo model has been published to date. Using this model, we demonstrated the insensitivity to isoniazid of M. bovis BCG present in hypoxic sites, and showed that norfloxacin given as a mannosylated macrophage-targeted prodrug was able to kill these isoniazid-insensitive mycobacteria. This demonstrates that intracellular persistent mycobacteria are amenable to antibiotic treatment.     

A novel mycolic acid cyclopropane synthetase is required for cording, persistence, and virulence of Mycobacterium tuberculosis

Colonial morphology of pathogenic bacteria is often associated with virulence. For M. tuberculosis, the causative agent of tuberculosis (TB), virulence is correlated with the formation of serpentine cords, a morphology that was first noted by Koch. We identified a mycobacterial gene, pcaA, that we show is required for cording and mycolic acid cyclopropane ring synthesis in the cell wall of both BCG and M. tuberculosis. Furthermore, we show that mutants of pcaA fail to persist within and kill infected mice despite normal initial replication. These results indicate that a novel member of a family of cyclopropane synthetases is necessary for lethal chronic persistent M. tuberculosis infection and define a role for cyclopropanated lipids in bacterial pathogenesis.     

Inhibition of the sole type I signal peptidase of Mycobacterium tuberculosis is bactericidal under replicating and nonreplicating conditions

Proteins secreted by bacteria perform functions vital for cell survival and play a role in virulence in Mycobacterium tuberculosis. M. tuberculosis lepB (Rv2903c) encodes the sole homolog of the type I signal peptidase (SPase). The lepB gene is essential in M. tuberculosis, since we could delete the chromosomal copy only when a second functional copy was provided elsewhere. By placing expression under the control of an anhydrotetracycline-inducible promoter, we confirmed that reduced lepB expression was detrimental to growth. Furthermore, we demonstrated that a serine-lysine catalytic dyad, characteristic for SPase function, is required for LepB function. We confirmed the involvement of LepB in the secretion of a reporter protein fused to an M. tuberculosis signal peptide. An inhibitor of LepB (MD3; a beta-aminoketone) was active against M. tuberculosis, exhibiting growth inhibition and bactericidal activity. Overexpression of lepB reduced the susceptibility of M. tuberculosis to MD3, and downregulation resulted in increased susceptibility, suggesting that LepB is the true target of MD3. MD3 lead to a rapid loss of viability and cell lysis. Interestingly, the compound had increased potency in nonreplicating cells, causing a reduction in viable cell numbers below the detection limit after 24 h. These data suggest that protein secretion is required to maintain viability under starvation conditions and that secreted proteins play a critical role in generating and surviving the persistent state. We conclude that LepB is a promising novel target for drug discovery in M. tuberculosis, since its inhibition results in rapid killing of persistent and replicating organisms.     

Tuberculosis: a problem with persistence

Mycobacterium tuberculosis is one of most successful pathogens of mankind, infecting one-third of the global population and claiming two million lives every year. The ability of the bacteria to persist in the form of a long-term asymptomatic infection, referred to as latent tuberculosis, is central to the biology of the disease. The persistence of bacteria in superficially normal tissue was recognized soon after the discovery of the tubercle bacillus, and much of our knowledge about persistent populations of M. tuberculosis dates back to the first half of the last century. Recent advances in microbial genetics and host immunity provide an opportunity for renewed investigation of this persistent threat to human health.     

The peptidoglycan of stationary-phase Mycobacterium tuberculosis predominantly contains cross-links generated by L,D-transpeptidation

Our understanding of the mechanisms used by Mycobacterium tuberculosis to persist in a "dormant" state is essential to the development of therapies effective in sterilizing tissues. Gene expression profiling in model systems has revealed a complex adaptive response thought to endow M. tuberculosis with the capacity to survive several months of combinatorial antibiotic treatment. We show here that this adaptive response may involve remodeling of the peptidoglycan network by substitution of 4-->3 cross-links generated by the D,D-transpeptidase activity of penicillin-binding proteins by 3-->3 cross-links generated by a transpeptidase of L,D specificity. A candidate gene, previously shown to be upregulated upon nutrient starvation, was found to encode an L,D-transpeptidase active in the formation of 3-->3 cross-links. The enzyme, Ldt(Mt1), was inactivated by carbapenems, a class of beta-lactam antibiotics that are poorly hydrolyzed by the M. tuberculosis beta-lactamases. Ldt(Mt1) and carbapenems may therefore represent a target and a drug family relevant to the eradication of persistent M. tuberculosis.     

Enhanced susceptibility of multidrug resistant strains of Mycobacterium tuberculosis to granulysin peptides correlates with a reduced fitness phenotype

Previously it was shown that the antimicrobial protein granulysin possesses potent membranolytic activity against Mycobacterium tuberculosis. Here we demonstrate that granF2 and G13, which are two short synthetic peptides derived from granulysin, inhibited the in vitro growth of clinical isolates of both multidrug resistant and drug susceptible strains of M. tuberculosis. Importantly, a particularly high activity against multidrug resistant M. tuberculosis correlated with a reduced growth rate compared to drug susceptible strains. A synergistic antibacterial effect of granF2 was further observed in combination with ethambutol, a compound with a documented effect on cell wall permeability. This finding suggests that granF2 and ethambutol exert their functions at different levels of the mycobacterial surface. Upon infection of macrophages in vitro, granF2 but not G13 efficiently reduced the intracellular growth of multidrug resistant M. tuberculosis in the presence of the pore-forming protein streptolysin O. The apoptotic function of granF2 apparently promoted destruction of host cells whereby the peptide gained access to and killed intracellular bacteria. Thus, a cost of resistance and a subsequent reduced fitness, measured as decreased growth among multidrug resistant strains of M. tuberculosis, could be associated with increased susceptibility to natural immune defense mechanisms, such as antimicrobial peptides of granulysin. However, a robust cell wall and the membrane of cells still provide physical shelter for the bacteria that may spare sensitive M. tuberculosis stains from being killed.     

Nucleoside diphosphate kinase of Mycobacterium tuberculosis acts as GTPase-activating protein for Rho-GTPases

Several bacterial pathogens secrete proteins into the host cells that act as GTPase-activating proteins (GAPs) for Rho-GTPases and convert GTP-bound active form to GDP-bound inactive form. However, no such effector molecule has been identified in Mycobacterium tuberculosis. In this study, we show that culture supernatant of M. tuberculosis H(37)Rv harbors a protein that stimulates the conversion of GTP-bound Rho-GTPases to the GDP-bound form. Nucleoside diphosphate kinase (Ndk) was identified as this culture supernatant protein that stimulated in vitro GTP hydrolysis by members of Rho-GTPases. The histidine-117 mutant of Ndk, which is impaired for autophosphorylation and nucleotide-binding activities, shows GAP activity. These results suggest that Ndk of M. tuberculosis functions as a Rho-GAP to downregulate Rho-GTPases, and this activity may aid in pathogenesis of the bacteria.     

结核分枝杆菌毒素-抗毒素系统与生物膜

结核分枝杆菌(Mycobacterium tuberculosis)是引起结核病的病原菌。其处于持续生存的休眠状态时,可导致长期无症状感染,称为结核潜伏感染。研究显示,结核分枝杆菌染色体中存在大量 “毒素-抗毒素系统”(toxin-antitoxin system,TAS),某些TAS在潜伏感染中发挥作用,可调节细菌生长和诱导细菌进入休眠状态;某些TAS参与生物膜形成和应激反应,但其影响生物膜形成的机制尚未阐明。生物膜中的结核分枝杆菌对多种抗结核药物耐药,且能抵抗宿主免疫系统防御;休眠状态的结核分枝杆菌对抗结核药物通常也是耐受的,给结核病治疗带来了巨大挑战。本文就近年来结核分枝杆菌TAS与生物膜的研究及抗结核药物对生物膜形成的影响进行综述。     

Hypoxia: a window into Mycobacterium tuberculosis latency

Tuberculosis is a massive public health problem on a global scale and the success of Mycobacterium tuberculosis is linked to its ability to persist within humans for long periods without causing any overt disease symptoms. Hypoxia is predicted to be a key host-induced stress limiting growth of the pathogen in vivo . However, multiple studies in vitro and in vivo indicate that M. tuberculosis adapts to oxygen limitation by entering into a metabolically altered state, while awaiting the opportunity to reactivate. Molecular signatures of bacteria adapted to hypoxia in vitro are accumulating, although correlations to human disease are only now being established. Similarly, defining the mechanisms that control this adaptation is an active area of research. In this review we discuss the historical precedents linking hypoxia and latency, and the gathering knowledge of M. tuberculosis hypoxic responses. We also examine the role of these responses in tuberculosis latency, and identify promising avenues for future studies.     

Selective killing of nonreplicating mycobacteria

Antibiotics are typically more effective against replicating rather than nonreplicating bacteria. However, a major need in global health is to eradicate persistent or nonreplicating subpopulations of bacteria such as Mycobacterium tuberculosis (Mtb). Hence, identifying chemical inhibitors that selectively kill bacteria that are not replicating is of practical importance. To address this, we screened for inhibitors of dihydrolipoamide acyltransferase (DlaT), an enzyme required by Mtb to cause tuberculosis in guinea pigs and used by the bacterium to resist nitric oxide-derived reactive nitrogen intermediates, a stress encountered in the host. Chemical screening for inhibitors of Mtb DlaT identified select rhodanines as compounds that almost exclusively kill nonreplicating mycobacteria in synergy with products of host immunity, such as nitric oxide and hypoxia, and are effective on bacteria within macrophages, a cellular reservoir for latent Mtb. Compounds that kill nonreplicating pathogens in cooperation with host immunity could complement the conventional chemotherapy of infectious disease.     

A family of autocrine growth factors in Mycobacterium tuberculosis

Mycobacterium tuberculosis and its close relative, Mycobacterium bovis (BCG) contain five genes whose predicted products resemble Rpf from Micrococcus luteus. Rpf is a secreted growth factor, active at picomolar concentrations, which is required for the growth of vegetative cells in minimal media at very low inoculum densities, as well as the resuscitation of dormant cells. We show here that the five cognate proteins from M. tuberculosis have very similar characteristics and properties to those of Rpf. They too stimulate bacterial growth at picomolar (and in some cases, subpicomolar) concentrations. Several lines of evidence indicate that they exert their activity from an extra-cytoplasmic location, suggesting that they are also involved in intercellular signalling. The five M. tuberculosis proteins show cross-species activity against M. luteus, Mycobacterium smegmatis and M. bovis (BCG). Actively growing cells of M. bovis (BCG) do not respond to these proteins, whereas bacteria exposed to a prolonged stationary phase do. Affinity-purified antibodies inhibit bacterial growth in vitro, suggesting that sequestration of these proteins at the cell surface might provide a means to limit or even prevent bacterial multiplication in vivo. The Rpf family of bacterial growth factors may therefore provide novel opportunities for preventing and controlling mycobacterial infections.     

Uptake of unnatural trehalose analogs as a reporter for Mycobacterium tuberculosis

The detection of tuberculosis currently relies upon insensitive and unspecific techniques; newer diagnostics would ideally co-opt specific bacterial processes to provide real-time readouts. The trehalose mycolyltransesterase enzymes (antigens 85A, 85B and 85C (Ag85A, Ag85B, Ag85C)) serve as essential mediators of cell envelope function and biogenesis in Mycobacterium tuberculosis. Through the construction of a systematically varied sugar library, we show here that Ag85 enzymes have exceptionally broad substrate specificity. This allowed exogenously added synthetic probes to be specifically incorporated into M. tuberculosis growing in vitro and within macrophages. Even bulky substituents, such as a fluorescein-containing trehalose probe (FITC-trehalose), were incorporated by growing bacilli, thereby producing fluorescent bacteria; microscopy revealed selective labeling of poles and membrane. Addition of FITC-trehalose to M. tuberculosis-infected macrophages allowed selective, sensitive detection of M. tuberculosis within infected mammalian macrophages. These studies suggest that analogs of trehalose may prove useful as probes of function and for other imaging modalities.     

Anaerobic nitrate reductase (narGHJI) activity of Mycobacterium bovis BCG in vitro and its contribution to virulence in immunodeficient mice

Mycobacterium tuberculosis and Mycobacterium bovis cause tuberculosis, which is responsible for the deaths of more people each year than any other bacterial infectious disease. Disseminated disease with Mycobacterium bovis BCG, the only currently available vaccine against tuberculosis, occurs in immunocompetent and immunodeficient individuals. Although mycobacteria are obligate aerobes, they are thought to face an anaerobic environment during infection, notably inside abscesses and granulomas. The purpose of this study was to define a metabolic pathway that could allow mycobacteria to exist under these conditions. Recently, the complete genome of M. tuberculosis has been sequenced, and genes homologous to an anaerobic nitrate reductase (narGHJI), an enzyme allowing nitrate respiration when oxygen is absent, were found. Here, we show that the narGHJI cluster of M. tuberculosis is functional as it conferred anaerobic nitrate reductase activity to Mycobacterium smegmatis. A narG mutant of M. bovis BCG was generated by targeted gene deletion. The mutant lacked the ability to reduce nitrate under anaerobic conditions. Both mutant and M. bovis BCG wild type grew equally well under aerobic conditions in vitro. Histology of immunodeficient mice (SCID) infected with M. bovis BCG wild type revealed large granulomas teeming with acid-fast bacilli; all mice showed signs of clinical disease after 50 days and succumbed after 80 days. In contrast, mice infected with the mutant had smaller granulomas containing fewer bacteria; these mice showed no signs of clinical disease after more than 200 days. Thus, it seems that nitrate respiration contributes significantly to virulence of M. bovis BCG in immunodeficient SCID mice.     

The Ser/Thr protein kinase PknB is essential for sustaining mycobacterial growth

The receptor-like protein kinase PknB from Mycobacterium tuberculosis is encoded by the distal gene in a highly conserved operon, present in all actinobacteria, that may control cell shape and cell division. Genes coding for a PknB-like protein kinase are also found in many more distantly related gram-positive bacteria. Here, we report that the pknB gene can be disrupted by allelic replacement in M. tuberculosis and the saprophyte Mycobacterium smegmatis only in the presence of a second functional copy of the gene. We also demonstrate that eukaryotic Ser/Thr protein kinase inhibitors, which inactivate PknB in vitro with a 50% inhibitory concentration in the submicromolar range, are able to kill M. tuberculosis H37Rv, M. smegmatis mc(2)155, and Mycobacterium aurum A+ with MICs in the micromolar range. Furthermore, significantly higher concentrations of these compounds are required to inhibit growth of M. smegmatis strains overexpressing PknB, suggesting that this protein kinase is the molecular target. These findings demonstrate that the Ser/Thr protein kinase PknB is essential for sustaining mycobacterial growth and support the development of protein kinase inhibitors as new potential antituberculosis drugs.     

Reactivation of M. tuberculosis infection in trans-membrane tumour necrosis factor mice

Of those individuals who are infected with M. tuberculosis, 90% do not develop active disease and represents a large reservoir of M. tuberculosis with the potential for reactivation of infection. Sustained TNF expression is required for containment of persistent infection and TNF neutralization leads to tuberculosis reactivation. In this study, we investigated the contribution of soluble TNF (solTNF) and transmembrane TNF (Tm-TNF) in immune responses generated against reactivating tuberculosis. In a chemotherapy induced tuberculosis reactivation model, mice were challenged by aerosol inhalation infection with low dose M. tuberculosis for three weeks to establish infection followed chemotherapeutic treatment for six weeks, after which therapy was terminated and tuberculosis reactivation investigated. We demonstrate that complete absence of TNF results in host susceptibility to M. tuberculosis reactivation in the presence of established mycobacteria-specific adaptive immunity with mice displaying unrestricted bacilli growth and diffused granuloma structures compared to WT control mice. Interestingly, bacterial re-emergence is contained in Tm-TNF mice during the initial phases of tuberculosis reactivation, indicating that Tm-TNF sustains immune pressure as in WT mice. However, Tm-TNF mice show susceptibility to long term M. tuberculosis reactivation associated with uncontrolled influx of leukocytes in the lungs and reduced IL-12p70, IFNγ and IL-10, enlarged granuloma structures, and failure to contain mycobacterial replication relative to WT mice. In conclusion, we demonstrate that both solTNF and Tm-TNF are required for maintaining immune pressure to contain reactivating M. tuberculosis bacilli even after mycobacteria-specific immunity has been established.     

Studies on neutral exopolysaccharides produced by the ectomycorrhiza Thelephora terrestris

We developed an in vitro tissue-culture model to analyze the process involved in mycobacterial spread through lung epithelial cell monolayers. A549 cells were infected with low numbers of viable Mycobacterium tuberculosis bacilli expressing the gfp gene. Subsequent addition of a soft agarose overlay prevented the dispersal of the bacilli from the initial points of attachment. By fluorescence microscopy the bacteria were observed to infect and grow within the primary target cells; this was followed by lysis of the infected cells and subsequent infection of adjacent cells. This process repeated itself until an area of clearing (plaque formation) was observed. The addition of amikacin after initial infection did not prevent intracellular growth; however, subsequent plaque formation was not observed. Plaque formation was also observed after infection with Mycobacterium bovis BCG bacilli, but the plaques were smaller than those formed after infection with M. tuberculosis. These observations reinforce the possibility that cell-to-cell spreading of M. tuberculosis bacilli, particularly early in the course of infection within lung macrophages, pneumocytes, and other cells, may be an important component in the infectious process.     

Codominance of TLR2-dependent and TLR2-independent modulation of MHC class II in Mycobacterium tuberculosis infection in vivo

Mycobacterium tuberculosis is an exceptionally successful human pathogen. A major component of this success is the ability of the bacteria to infect immunocompetent individuals and to evade eradication by an adaptive immune response that includes production of the macrophage-activating cytokine, IFN-gamma. Although IFN-gamma is essential for arrest of progressive tuberculosis, it is insufficient for efficacious macrophage killing of the bacteria, which may be due to the ability of M. tuberculosis to inhibit selected macrophage responses to IFN-gamma. In vitro studies have determined that mycobacterial lipoproteins and other components of the M. tuberculosis cell envelope, acting as agonists for TLR2, inhibit IFN-gamma induction of MHC class II. In addition, M. tuberculosis peptidoglycan and IL-6 secreted by infected macrophages inhibit IFN-gamma induction of MHC class II in a TLR2-independent manner. To determine whether TLR2-dependent inhibition of macrophage responses to IFN-gamma is quantitatively dominant over the TLR2-independent mechanisms in vivo, we prepared mixed bone marrow chimeric mice in which the hemopoietic compartment was reconstituted with a mixture of TLR(+/+) and TLR2(-/-) cells. When the chimeric mice were infected with M. tuberculosis, the expression of MHC class II on TLR2(+/+) and TLR2(-/-) macrophages from the lungs of individual infected chimeric mice was indistinguishable. These results indicate that TLR2-dependent and -independent mechanisms of inhibition of responses to IFN-gamma are equivalent in vivo, and that M. tuberculosis uses multiple pathways to abrogate the action of an important effector of adaptive immunity. This work was supported by National Institutes of Health Grants AI 065357-AI 020010.