Differential release of tumor necrosis factor-α from murine peritoneal macrophages stimulated with virulent and avirulent species of mycobacteria

Abstract The ability of Mycobacterium tuberculosis H37Rv and H37Ra, M. bovis BCG and M. smegmatis to induce the secretion of tumor necrosis factor-α (TNF-α) by cultured murine peritoneal macrophages is inversely related to their virulence. The avirulent species of mycobacteria which were unable to persist in macrophages were capable of inducing significant levels of TNF-α compared to that formed in cultures infected with the virulent M. tuberculosis H37Rv. This difference was also associated with an inherent toxicity by live H37Rv for macrophage cultures. Heat-killed H37Rv was non-toxic and induced significant levels of TNF-α; in contrast, live and heat-killed suspensions of avirulent mycobacteria had an equivalent ability to trigger TNF-α secretion. The TNF-α response was dose-dependent, related directly to the percentage of infected cells, and peaked 6–12 h post-infection. An early and vigorous TNF-α response appears to be a marker of macrophage resistance, while the downregulation of this response seems associated with macrophage toxicity and unrestricted mycobacterial growth.     

Pathogenic Mycobacterium bovis strains differ in their ability to modulate the proinflammatory activation phenotype of macrophages

ABSTRACT: BACKGROUND: Tuberculosis, caused by Mycobacterium tuberculosis or Mycobacterium bovis, remains one of the leading infectious diseases worldwide. The ability of mycobacteria to rapidly grow in host macrophages is a factor contributing to enhanced virulence of the bacteria and disease progression. Bactericidal functions of phagocytes are strictly dependent on activation status of these cells, regulated by the infecting agent and cytokines. Pathogenic mycobacteria can survive the hostile environment of the phagosome through interference with activation of bactericidal responses. To study the mechanisms employed by highly virulent mycobacteria to promote their intracellular survival, we investigated modulating effects of two pathogenic M. bovis isolates and a reference M. tuberculosis H37Rv strain, differing in their ability to multiply in macrophages, on activation phenotypes of the cells primed with major cytokines regulating proinflammatory macrophage activity. RESULTS: Bone marrow- derived macrophages obtained from C57BL/6 mice were infected by mycobacteria after a period of cell incubation with or without treatment with IFN-gamma, inducing proinflammatory type-1 macrophages (M1), or IL-10, inducing anti-inflammatory type-2 cells (M2). Phenotypic profiling of M1 and M2 was then evaluated. The M. bovis strain MP287/03 was able to grow more efficiently in the untreated macrophages, compared with the strains B2 or H37Rv. This strain induced weaker secretion of proinflammatory cytokines, coinciding with higher expression of M2 cell markers, mannose receptor (MR) and arginase-1 (Arg-1). Treatment of macrophages with IFN-gamma and infection by the strains B2 and H37Rv synergistically induced M1 polarization, leading to high levels of inducible nitric oxide synthase (iNOS) expression, and reduced expression of the Arg-1. In contrast, the cells infected with the strain MP287/03 expressed high levels of Arg-1 which competed with iNOS for the common substrate arginine, leading to lower levels of NO production. CONCLUSIONS: The data obtained demonstrated that the strain, characterized by increased growth in macrophages, down- modulated classical macrophage activation, through induction of an atypical mixed M1/M2 phenotype.     

Aptamer inhibits <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis> (H37Rv) invasion of macrophage

There is an urgent need to develop new anti-tuberculosis drugs due to the rising tendency in tuberculosis (TB) around the world. It is known that Mycobacterium tuberculosis (M. tuberculosis) generally infects mammalian host via aerosol route. The pathogenic process has been fully studied that it can initially invade alveolar macrophage, then established stable residence within those phagocytic cells, suggesting that one of the possible ways to prevent this pathogen is to inhibit its invasion and growth in the macrophage. Aptamers from SELEX (Systematic Evolution of Ligands by Exponential Enrichment) have been used to rival virulent M. tuberculosis (H37Rv) in our previous work, and the materials to which aptamers bound were proved to be some outer membrane proteins of H37Rv. In the present study, the interaction between M. tuberculosis and macrophage in the presence of aptamers was investigated in more details. The results suggested that the selective aptamers significantly inhibited H37Rv invasion of macrophage in vitro, and the effect correspond to the binding affinity of these aptamers to H37Rv. The values of equilibrium dissociation constant (Kd) was calculated by flow cytometry, all in the nanomolar range, showed much higher affinity to H37Rv than M. bovis Bacillus Guerin (BCG). Moreover, the aptamer-treated H37Rv can stimulate IFN-γ, IL-15 and IL-17 secretion of macrophages compared with H37Rv (no treated). In summary, our data indicated that the NK2 aptamer not only acted as anti-tuberculosis agent by inhibiting virulent M. tuberculosis (H37Rv) invasion of macrophage, but also might be used as molecular probe for exploring the interaction between the outer membrane of M. tuberculosis and macrophage.     

Differential expression of NF-kappaB in mycobacteria infected THP-1 affects apoptosis

The present study was conducted to see the role of NF-kappaB in virulent (Mycobacterium tuberculosis H37Rv) and avirulent (M. tuberculosis H37Ra) mycobacterial infection in THP-1 cells. To inactivate NF-kappaB, pCMV-IkappaBalphaM dn containing THP-1 cell line was generated which showed marked increase in apoptosis with M. tuberculosis H37Rv and M. tuberculosis H37Ra. Infected THP-1-IkappaBalphaM dn cells showed decrease in mitochondrial membrane potential, cytochrome c release, activation of caspase-3 and enhanced TNF-alpha production. Increase in apoptosis of infected THP-1-IkappaBalphaM dn cells resulted in inhibition of intracellular mycobacterial growth. Differential NF-kappaB activation potential was observed with M. tuberculosis H37Rv and M. tuberculosis H37Ra. Both the strains activated NF-kappaB after 4 h in THP-1 cells however after 48 h only M. tuberculosis H37Rv activated NF-kappaB which lead to up-regulation of bcl-2 family anti-apoptotic member, bfl-1/A1. Our results indicated that NF-kappaB activation may be a determinant factor for the success of virulent mycobacteria within macrophages.     

Inhibition of bfl-1/A1 by siRNA inhibits mycobacterial growth in THP-1 cells by enhancing phagosomal acidification

Virulent tubercle bacilli inhibit apoptosis to establish a safe environment within the host cells. Here, we report that NF-kappaB dependent antiapoptotic protein bfl-1/A1 plays an important role in this process. Both virulent and avirulent mycobacteria bearing THP-1 cells expressed considerable amount of bfl-1/A1 after 4 h of infection. However, after 48 h of infection, bfl-1/A1 expression was evident only in Mycobacterium tuberculosis H37Rv but not in M. tuberculosis H37Ra infected cells. When parallel experiments were performed with Human monocyte-derived macrophages (MDMs), differential expression of bfl-1/A1 mRNA was observed in case of M. tuberculosis H37Rv and M. tuberculosis H37Ra infection. siRNA mediated inhibition of bfl-1/A1 induced apoptosis in M. tuberculosis H37Rv infected THP-1 and MDMs. Reduction in intracellular mycobacterial growth was observed in bfl-1/A1 siRNA transfected, M. tuberculosis H37Rv infected THP-1 cells. Enhancement of phagosome-lysosome fusion was observed in bfl-1/A1 siRNA treated and M. tuberculosis H37Rv infected THP-1 cells. These results clearly indicated that differential expression of bfl-1/A1 in M. tuberculosis H37Rv and M. tuberculosis H37Ra infected THP-1 cells probably account for the difference in infection outcome.     

Intraphagosomal Mycobacterium tuberculosis acquires iron from both extracellular transferrin and intracellular iron pools. Impact of interferon-gamma and hemochromatosis

Mycobacterium tuberculosis multiplies within the macrophage phagosome and requires iron for growth. We examined the route(s) by which intracellular M. tuberculosis acquires iron. During intracellular growth of the virulent Erdman M. tuberculosis strain in human monocyte-derived macrophages (MDM), M. tuberculosis acquisition of (59)Fe from transferrin (TF) provided extracellularly (exogenous source) was compared with acquisition when MDM were loaded with (59)Fe from TF prior to M. tuberculosis infection (endogenous sources). M. tuberculosis (59)Fe acquisition required viable bacteria and was similar from exogenous and endogenous sources at 24 h and greater from exogenous iron at 48 h. Interferon-gamma treatment of MDM reduced (59)Fe uptake from TF 51% and TF receptor expression by 34%. Despite this, intraphagosomal M. tuberculosis iron acquisition in IFN-gamma-treated cells was decreased by only 30%. Macrophages from hereditary hemochromatosis patients have altered iron metabolism. Intracellular M. tuberculosis acquired markedly less iron in MDM from these individuals than in MDM from healthy donors, regardless of the iron source (exogenous and endogenous): 36 +/- 3.8% and 17 +/- 9.6% of control, respectively. Thus, intraphagosomal M. tuberculosis can acquire iron from both extracellular TF and endogenous macrophage sources. Acquisition of iron from macrophage cytoplasmic iron pools may be critical for the intracellular growth of M. tuberculosis. This acquisition is altered by IFN-gamma treatment to a small extent, but is markedly reduced in macrophages from hemochromatosis patients.     

Antitubercular activity of trifluoperazine, a calmodulin antagonist

Trifluoperazine, a calmodulin antagonist, completely inhibited the growth of mycobacteria. The minimum inhibitory concentrations in shake cultures in a synthetic medium containing 0.2% Tween 80 were 5 and 8 micrograms/ml, respectively, for the human pathogenic strain Mycobacterium tuberculosis H37Rv and M. tuberculosis resistant to isoniazid. When added to a growing culture of M. tuberculosis H37Rv on the 10th day (mid exponential phase), trifluoperazine 50 micrograms/ml further arrested growth of this organism. It is suggested that trifluoperazine or similar calmodulin antagonists might be useful as antitubercular drugs.     

Differential regulation of protein kinase C isoforms of macrophages by pathogenic and non-pathogenic mycobacteria

Given the fact that Mycobacterium tuberculosis (Mtb) may respond to the intracellular milieu of the macrophage with the induction of environmentally regulated genes required for survival and growth of the bacteria we assumed that the protein kinases may also be the factors in Mycobacterium-macrophage interaction. Since, protein kinases play a major role in various critical cellular processes including regulation of immune responses, we describe the fate of expression and phosphorylation of protein kinase C in macrophage cell lines exposed to Mtb H37Rv and raised the question whether the change in the events of expression and phosphorylation are the results of direct interaction of bacilli with macrophages and/or, are also indirectly mediated by specific cytokines that are induced in response to exposure. Our results show that only novel PKCs are phosphorylated during infection of macrophages by pathogenic and non-pathogenic mycobacteria and the alteration is a result of direct host-bacilli association which is independent of cytokines as mediators. Expression of PKC-alpha (conventional PKC isoform) was down regulated by Mtb H37Rv. In contrast the non-pathogenic fast grower Mycobacterium smegmatis (MS) increased the expression and phosphorylation of PKC-alpha. PKC-alpha was also increased in macrophages treated with serum of mice immunized with Mtb H37Rv. The study has shown that pathogenic and non-pathogenic mycobacteria categorically select the type of protein kinases C for activation/deactivation.     

Requirement of gene fadD33 for the growth of Mycobacterium tuberculosis in a hepatocyte cell line

Gene fadD33 of Mycobacterium tuberculosis, one of the 36 homologues of gene fadD of Escherichia coli identified in the M. tuberculosis genome, predictively encodes an acyl-CoA synthase, an enzyme involved in fatty acids metabolism. The gene is underexpressed in the attenuated strain M. tuberculosis H37Ra relative to virulent H37Rv and plays a role in M. tuberculosis virulence in BALB/c mice by supporting mycobacterial replication in the liver. In the present paper, we investigated the role of fadD33 expression in bacterial growth within the hepatocyte cell line HepG2, as well as in human monocyte-derived THP-1 cells and peripheral blood mononuclear cells. M. tuberculosis H37Rv proved able to grow within HepG2 cells, while the intracellular replication of M. tuberculosis H37Ra was markedly impaired; complementation of strain H37Ra with gene fadD33 restored its replication to the levels of H37Rv. Moreover, disruption of gene fadD33 by allelic exchange mutagenesis reduced the intracellular growth of M. tuberculosis H37Rv, and complementation of the fadD33-disrupted mutant with gene fadD33 restored bacterial replication. Conversely, fadD33 expression proved unable to influence M. tuberculosis growth in human phagocytes, as fadD33-disrupted M. tuberculosis H37Rv mutant, as well as fadD33-complemented M. tuberculosis H37Ra, grew within THP-1 cells and peripheral monocytes basically at the same rates as parent H37Rv and H37Ra strains. The results of these experiments indicate that gene fadD33 expression confers growth advantage to M. tuberculosis in immortalized hepatocytes, but not in macrophages, thus emphasizing the importance of fadD33 in liver-specific replication of M. tuberculosis.     

Overexpression of Mycobacterium tuberculosis manB, a phosphomannomutase that increases phosphatidylinositol mannoside biosynthesis in Mycobacterium smegmatis and mycobacterial association with human macrophages

Mycobacterium tuberculosis (M. tb) pathogenesis involves the interaction between the mycobacterial cell envelope and host macrophage, a process mediated, in part, by binding of the mannose caps of M. tb lipoarabinomannan (ManLAM) to the macrophage mannose receptor (MR). A presumed critical step in the biosynthesis of ManLAM, and other mannose-containing glycoconjugates, is the conversion of mannose-6-phosphate to mannose-1-phosphate, by a phosphomannomutase (PMM), to produce GDP-mannose, the primary mannose-donor in mycobacteria. We have identified four M. tb H37Rv genes with similarity to known PMMs. Using in vivo complementation of PMM and phosphoglucomutase (PGM) deficient strains of Pseudomonas aeruginosa, and an in vitro enzyme assay, we have identified both PMM and PGM activity from one of these genes, Rv3257c (MtmanB). MtmanB overexpression in M. smegmatis produced increased levels of LAM, lipomannan, and phosphatidylinositol mannosides (PIMs) compared with control strains and led to a 13.3 +/- 3.9-fold greater association of mycobacteria with human macrophages, in a mannan-inhibitable fashion. This increased association was mediated by the overproduction of higher order PIMs that possess mannose cap structures. We conclude that MtmanB encodes a functional PMM involved in the biosynthesis of mannosylated lipoglycans that participate in the association of mycobacteria with macrophage phagocytic receptors.     

Construction and evaluation of luciferase reporter phages for the detection of active and non-replicating tubercle bacilli

The luciferase reporter phages (LRP) show great promise for diagnostic mycobacteriology. Though conventional constructs developed from lytic phages such as D29 and TM4 are highly specific, they lack sensitivity. We have isolated and characterized Che12, the first true temperate phage infecting M. tuberculosis. Since the tuberculosis (TB) cases among HIV infected population result from the reactivation of latent bacilli, it would be useful to develop LRP that can detect dormant bacteria. During dormancy, pathogenic mycobacteria switch their metabolism involving divergent genes than during normal, active growth phase. Since the promoters of these genes can potentially function during dormancy, they were exploited for the construction of novel mycobacterial luciferase reporter phages. The promoters of hsp60, isocitrate lyase (icl), and alpha crystallin (acr) genes from M. tuberculosis were used for expressing firefly luciferase gene (FFlux) in both Che12 and TM4 phages and their efficiency was evaluated in detecting dormant bacteria from clinical isolates of M. tuberculosis. These LRP constructs exhibited detectable luciferase activity in dormant as well as in actively growing M. tuberculosis. The TM4 ts mutant based constructs showed about one log increase in light output in three of the five tested clinical isolates and in M. tuberculosis H37Rv compared to conventional lytic reporter phage, phAE129. By refining the LRP assay format further, an ideal rapid assay can be designed not only to diagnose active and dormant TB but also to differentiate the species and to find their drug susceptibility pattern.     

Synthesis of isonicotinic acid N'-arylidene-N-[2-oxo-2-(4-aryl-piperazin-1-yl)-ethyl]-hydrazides as antituberculosis agents

A new series of antituberculosis agents 6-9 was designed, synthesized and evaluated for antituberculosis activity against Mycobacterium tuberculosis H37Rv and clinical isolates in an agar dilution method. Compound 9h showed comparable in vitro activity (MIC) to isoniazid against M. tuberculosis H37Rv and clinical isolates (sensitive strains) and superior activity against resistant strains of M. tuberculosis.     

Dehalogenation of haloalkanes by Mycobacterium tuberculosis H37Rv and other mycobacteria

Haloalkane dehalogenases convert haloalkanes to their corresponding alcohols by a hydrolytic mechanism. To date, various haloalkane dehalogenases have been isolated from bacteria colonizing environments that are contaminated with halogenated compounds. A search of current databases with the sequences of these known haloalkane dehalogenases revealed the presence of three different genes encoding putative haloalkane dehalogenases in the genome of the human parasite Mycobacterium tuberculosis H37Rv. The ability of M. tuberculosis and several other mycobacterial strains to dehalogenate haloaliphatic compounds was therefore studied. Intact cells of M. tuberculosis H37Rv were found to dehalogenate 1-chlorobutane, 1-chlorodecane, 1-bromobutane, and 1,2-dibromoethane. Nine isolates of mycobacteria from clinical material and four strains from a collection of microorganisms were found to be capable of dehalogenating 1,2-dibromoethane. Crude extracts prepared from two of these strains, Mycobacterium avium MU1 and Mycobacterium smegmatis CCM 4622, showed broad substrate specificity toward a number of halogenated substrates. Dehalogenase activity in the absence of oxygen and the identification of primary alcohols as the products of the reaction suggest a hydrolytic dehalogenation mechanism. The presence of dehalogenases in bacterial isolates from clinical material, including the species colonizing both animal tissues and free environment, indicates a possible role of parasitic microorganisms in the distribution of degradation genes in the environment.     

The polyketide Pks1 contributes to biofilm formation in Mycobacterium tuberculosis

Infections caused by biofilms are abundant and highly persistent, displaying phenotypic resistance to high concentrations of antimicrobials and modulating host immune systems. Tuberculosis (TB), caused by Mycobacterium tuberculosis, shares these qualities with biofilm infections. To identify genetic determinants of biofilm formation in M. tuberculosis, we performed a small-scale transposon screen using an in vitro pellicle biofilm assay. We identified five M. tuberculosis mutants that were reproducibly attenuated for biofilm production relative to that of the parent strain H37Rv. One of the most attenuated mutants is interrupted in pks1, a polyketide synthase gene. When fused with pks15, as in some M. tuberculosis isolates, pks1 contributes to synthesis of the immunomodulatory phenolic glycolipids (PGLs). However, in strains such as H37Rv with split pks15 and pks1 loci, PGL is not produced and pks1 has no previously defined role. We showed that pks1 complementation restores biofilm production independently of the known role of pks1 in PGL synthesis. We also assessed the relationship among biofilm formation, the pks15/1 genotype, and M. tuberculosis phylogeography. A global survey of M. tuberculosis clinical isolates revealed surprising sequence variability in the pks15/1 locus and substantial variation in biofilm phenotypes. Our studies identify novel M. tuberculosis genes that contribute to biofilm production, including pks1. In addition, we find that the ability to make pellicle biofilms is common among M. tuberculosis isolates from throughout the world, suggesting that this trait is relevant to TB propagation or persistence.     

Bfl-1/A1 acts as a negative regulator of autophagy in mycobacteria infected macrophages

Expression of Bcl-2 family protein, Bfl-1/A1 has been found to differ considerably amongst macrophages infected with virulent Mycobacterium tuberculosis H37Rv or with avirulent M. tuberculosis H37Ra. Present work was undertaken to deduce the significance of differential expression of Bfl-1/A1 in the outcome of mycobacterial infection. We have studied the role of Bfl-1/A1 particularly in autophagy formation in tubercle bacilli infected cells since autophagy has been recognized as a component of innate immunity against pathogenic mycobacteria. First, we have confirmed that upon infection virulent strain H37Rv retain Bfl-1/A1 for longer period and impose autophagosome maturation block within infected cells as evident from confocal microscopy. Moreover, down regulation of Bfl-1/A1 by siRNA induced autophagy formation and reduced bacterial growth. Furthermore, even the avirulent strain H37Ra resist autophagosome maturation and survive if the cellular level of Bfl-1 is maintained in THP-1 cells by stable transfection (Bfl-1 overexpressing cells). No noteworthy difference in mTOR expression was observed between normal THP-1 and Bfl-1 overexpressing THP-1 cells infected with either strain of mycobacteria. Interestingly, we found that not only mTOR but also Bfl-1/A1 is involved in rapamycin induced autophagy in mycobacteria infected macrophages. We have found that Bfl-1 physically interacts with Beclin 1 in Bfl-1 overexpressing THP-1 as well as in H37Rv infected THP-1 cells as they co-precipitated. Taken together, our results clearly demonstrated that Bfl-1/A1 negatively regulates autophagy and expression of Bfl-1/A1 in H37Rv infected macrophages provides the bacteria a survival strategy to overcome host defense.     

Identification of cyclic AMP-regulated genes in Mycobacterium tuberculosis complex bacteria under low-oxygen conditions

Mycobacterium tuberculosis is the etiological agent of tuberculosis (TB), which kills approximately 2 million people a year despite current treatment options. A greater understanding of the biology of this bacterium is needed to better combat TB disease. The M. tuberculosis genome encodes as many as 15 adenylate cyclases, suggesting that cyclic AMP (cAMP) has an important, yet overlooked, role in mycobacteria. This study examined the effect of exogenous cAMP on protein expression in Mycobacterium bovis BCG grown under hypoxic versus ambient conditions. Both shaking and shallow standing cultures were examined for each atmospheric condition. Different cAMP-dependent changes in protein expression were observed in each condition by two-dimensional gel electrophoresis. Shaking low-oxygen cultures produced the most changes (12), while standing ambient conditions showed the fewest (2). Five upregulated proteins, Rv1265, Rv2971, GroEL2, PE_PGRS6a, and malate dehydrogenase, were identified from BCG by mass spectrometry and were shown to also be regulated by cAMP at the mRNA level in both M. tuberculosis H37Rv and BCG. To our knowledge, these data provide the first direct evidence for cAMP-mediated gene regulation in TB complex mycobacteria.     

NK1.1+ cells and IL-22 regulate vaccine-induced protective immunity against challenge with Mycobacterium tuberculosis

We previously found that human NK cells lyse Mycobacterium tuberculosis-infected monocytes and alveolar macrophages and upregulate CD8(+) T cell responses. We also found that human NK cells produce IL-22, which inhibits intracellular growth of M. tuberculosis, and that NK cells lyse M. tuberculosis-expanded CD4(+)CD25(+)FOXP3(+) T regulatory cells (Tregs). To determine the role of NK cells during the protective immune response to vaccination in vivo, we studied the NK cell and T cell responses in a mouse model of vaccination with bacillus Calmette-Guérin (BCG), followed by challenge with virulent M. tuberculosis H37Rv. BCG vaccination enhanced the number of IFN-γ-producing and IL-22-producing NK cells. Depletion of NK1.1(+) cells at the time of BCG vaccination increased the number of immunosuppressive Tregs (CD4(+)CD25(hi), 95% Foxp3(+)) after challenge with M. tuberculosis H37Rv, and NK1.1(+) cells lysed expanded but not natural Tregs in BCG-vaccinated mice. Depletion of NK1.1(+) cells at the time of BCG vaccination also increased the bacillary burden and reduced T cell responses after challenge with M. tuberculosis H37Rv. IL-22 at the time of vaccination reversed these effects and enhanced Ag-specific CD4(+) cell responses in BCG-vaccinated mice after challenge with M. tuberculosis H37Rv. Our study provides evidence that NK1.1(+) cells and IL-22 contribute to the efficacy of vaccination against microbial challenge.