Intracellular Survival Of Mycobacteria Research Articles

Environment dependent expression of mycobacterium hormone sensitive lipases: expression pattern under ex-vivo and individual in-vitro stress conditions in M. tuberculosis H37Ra.

Hormone-sensitive lipase (HSL) is a neutral lipase capable of hydrolysing various kinds of lipids. In comparison to single human Hormone Sensitive Lipase (hHSL), that is induced under nutritional stress, twelve serine hydrolases are annotated as HSL in Mycobacterium tuberculosis (mHSL). Mycobacterium is exposed to multiple stresses inside the host. Therefore, the present study was carried out to investigate if mHSL are also expressed under stress condition and if there is any correlation between various stress conditions and expression pattern of mHSL. The expression pattern of mHSL under different environmental conditions (in-vitro and ex-vivo) were studied using qRT-PCR in M. tuberculosis H37Ra strain with 16S rRNA as internal control. Out of 12, only two genes (lipU and lipY) were expressed at very low level in mid log phase culture under aerobic conditions, while 9 genes were expressed at stationary phase of growth. Ten mHSLs were expressed post-infection under ex-vivo conditions in time dependent manner. LipH and lipQ did not express at any time point under ex-vivo condition. The relative expression of most of the genes under individual stress was much higher than observed in ex-vivo conditions. The expression pattern of genes varied with change in stress condition. Different sets of mHSL genes were expressed under different individual stress conditions pointing towards the requirement of different mHSL to combat different stress conditions. Overall, most of the mHSLs have demonstrated stress dependent expression pointing towards their role in intracellular survival of mycobacteria.

ESAT-6 impedes IL-18 mediated phagosome lysosome fusion via microRNA-30a upon Calcimycin treatment in mycobacteria infected macrophages

The weaponry possessed by Mycobacterium tuberculosis (M. tb) in the form of immunodominant antigens hijack the host immune system to give a survival advantage to this intracellular fiend, but the mechanism of this control is not entirely known. Since we have previously reported the mechanism of autophagy inhibition by early secreted antigenic target 6 kDa (ESAT-6) through microRNA (miR)-30a-3p in Calcimycin treated differentiated THP-1 (dTHP-1) cells, the present study was undertaken to deduce the effect of miR-30a on the immunomodulatory profile of ESAT-6 treated cells and the mechanism involved thereof, if any. Initially, the effect of recombinant ESAT-6 (rESAT-6) on the immunomodulatory profile in Calcimycin-treated phorbol 12-myristate 13-acetate (PMA) dTHP-1 cells was checked. Later, transfection studies using miR-30a-3p inhibitor or -5p mimic highlighted the contrary roles of different arms of the same miRNA in regulating IL-18 response by ESAT-6 in dTHP-1 cells after Calcimycin treatment. By using either IL-18 neutralizing antibody or inhibitors of phosphoinositide 3-kinase (PI3K)/NF-κB/phagosome-lysosome fusion in the miRNA-30a transfected background, IL-18 mediated signaling and intracellular killing of mycobacteria was reversed in the presence of ESAT-6. Overall, the results of this study conclusively prove the contrary roles of miR-30a-3p and miR-30a-5p in regulating IL-18 signaling by ESAT-6 in dTHP-1 cells upon Calcimycin treatment that affected phagosome-lysosome fusion and intracellular survival of mycobacteria.

MicroRNA-325-3p Facilitates Immune Escape of Mycobacterium tuberculosis through Targeting LNX1 via NEK6 Accumulation to Promote Anti-Apoptotic STAT3 Signaling.

Tuberculosis (TB) is an infectious disease caused by Mycobacterium tuberculosis that poses threats to the public. M. tuberculosis survives in macrophages by escaping from immune surveillance and clearance, which exacerbates the bacterial proliferation. However, the molecular mechanisms of this immune escape have not yet been fully understood. Using multiple cell and mouse models, we found that microRNA-325-3p (miR-325-3p) is upregulated after M. tuberculosis infection and Mir325-deficient mice show resistance to M. tuberculosis We demonstrated that miR-325-3p directly targets LNX1, an E3 ubiquitin ligase of NEK6, and that this hampers the proteasomal degradation of NEK6 in macrophages. The abnormal accumulation of NEK6 leads to the activation of STAT3 signaling, thus inhibiting the process of apoptosis and promoting the intracellular survival of M. tuberculosis Our findings not only reveal a new immune escape pathway of M. tuberculosis but also may provide new insights into the development of therapeutic approaches for drug-resistant TB.IMPORTANCE Intracellular survival of Mycobacterium tuberculosis results in bacterial proliferation and the spread of infection in lungs, consequently deteriorating the conditions of tuberculosis (TB) patients. This research discovers a new immune escape pathway of M. tuberculosis by modulating host miR-325-3p expression, thus leading to the intracellular survival of M. tuberculosis These findings make a contribution to the understanding of the immune escape of M. tuberculosis, and they provide a theoretical basis for the development of therapeutic approaches for drug-resistant TB.

Rv2037c, a stress induced conserved hypothetical protein of Mycobacterium tuberculosis, is a phospholipase: Role in cell wall modulation and intracellular survival

The genome of Mycobacterium tuberculosis encode for several hypothetical proteins that needed to be characterized. Rv2037c, a hypothetical protein, was 25 and 4 folds upregulated under acidic and nutritive stress, respectively in M. tuberculosis H37Ra. The protein demonstrated lipolytic activity with pNP-decanoate with optimum pH 8.0 and temperature 40 °C. In addition, the protein demonstrated phospholipase activity. To understand the effect of rv2037c on mycobacterium physiology, the gene was cloned and expressed in M. smegmatis. The protein was found in membrane and extracellular fraction. The expression of rv2037c in M. smegmatis (MS_Rv2037c) altered colony morphology and cell surface features like enhanced biofilm and pellicle formation. MS_Rv2037c decreased cell-wall permeability, enhanced TDM content, resistance against various stresses and antibiotics. MS_Rv2037c demonstrated better infection and intracellular survival capability in infected THP-1 macrophage. Macrophages treated with Rv2037c demonstrated irregular cell membrane. Mice infected with MS_Rv2037c had higher bacterial load in lung, liver and spleen compared to control. Rv2037c induced the production of pro-inflammatory cytokines TNFα and IL12, suggesting its role in immune-modulation. Recombinant protein also generated humoral response in EPTB and MDR-TB patients. The results pointed towards the crucial role of this enzyme in cell-wall modulation, infection and intracellular survival of mycobacterium.

M1 macrophage dependent-p53 regulates the intracellular survival of mycobacteria

Tumor suppressor p53 is not only affects immune responses but also contributes to antibacterial activity. However, its bactericidal function during mycobacterial infection remains unclear. In this study, we found that the p53-deficient macrophages failed to control Mycobacterium tuberculosis (Mtb), manifested as a lower apoptotic cell death rate and enhanced intracellular survival. The expression levels of p53 during Mtb infection were stronger in M1 macrophages than in M2 macrophages. The TLR2/JNK signaling pathway plays an essential role in the modulation of M1 macrophage polarization upon Mtb infection. It facilitates p53-mediated apoptosis through the production of reactive oxygen species, nitric oxide and inflammatory cytokines in Mtb-infected M1 macrophages. In addition, nutlin-3 effectively abrogated the intracellular survival of mycobacteria in both TB patients and healthy controls after H37Ra infection for 24 h, indicating that the enhancement of p53 production effectively suppressed the intracellular survival of Mtb in hosts. These results suggest that p53 can be a new therapeutic target for TB therapy.

Mitofusin 2-Deficiency Suppresses Mycobacterium tuberculosis Survival in Macrophages.

Apoptosis is an important host defense mechanism against mycobacterial infection. However, the molecular mechanisms regulating apoptosis during mycobacterial infection are not well known. Recent reports suggest that bacterial infection regulates mitochondrial fusion and fission in various ways. Here, we investigated the role of mitochondria in Mycobacterium tuberculosis (Mtb)-infected macrophages. Mtb H37Rv (Rv) infection induced mitofusin 2 (MFN2) degradation, leading to mitochondrial fission. Interestingly, Mtb H37Ra (Ra) infection induced significantly greater mitochondrial fragmentation than Rv infection. Mtb-mediated Parkin, an E3 ubiquitin ligase, contributed to the degradation of MFN2. To evaluate the role of endoplasmic reticulum stress in the production of Parkin during Mtb infection, we analyzed Parkin production in 4-phenylbutyric acid (4-PBA)-pretreated macrophages. Pretreatment with 4-PBA reduced Parkin production in Mtb-infected macrophages. In contrast, the level of MFN2 production recovered to a level similar to that of the unstimulated control. In addition, Ra-infected macrophages had reduced mitochondrial membrane potential (MMP) compared to those infected with Rv. Interestingly, intracellular survival of mycobacteria was decreased in siMFN2-transfected macrophages; in contrast, overexpression of MFN2 in macrophages increased Mtb growth compared with the control.

Rv1273c, an ABC transporter of Mycobacterium tuberculosis promotes mycobacterial intracellular survival within macrophages via modulating the host cell immune response

Mycobacterium proteins, especially cell wall associated proteins, interact with host macrophage to regulate the functions and cytokine production. So, identification and characterization of such proteins is essential for understanding tuberculosis pathogenesis. The role of the ABC transporter proteins in the pathophysiology and virulence of Mycobacterium tuberculosis is not clearly understood. In the present study, Rv1273c, an ABC transporter, has been expressed in a non-pathogenic and fast growing Mycobacterium smegmatis strain to explore its role in host pathogen interactions. Over expression of Rv1273c resulted in enhanced intracellular survival in macrophage as well as modified cell wall architecture. We found altered colony morphology and cell surface properties that might be linked with remodelling of bacterial cell wall which may help in the intracellular survival of mycobacterium. However, the enhanced intracellular survival was not found to be the consequence of an increased resistance to intracellular stresses. The activation of macrophage by Rv1273c was associated with perturbed cytokine production. Pharmacological inhibition experiment and western immunoblotting suggested that this altered cytokine profile was mediated possibly by NF-kB and p38 pathway in macrophage. Overall, the present findings indicated that Rv1273c enhanced mycobacterium persistence and mediated the evasion of immune responses during infection.

LpqT improves mycobacteria survival in macrophages by inhibiting TLR2 mediated inflammatory cytokine expression and cell apoptosis

Tuberculosis is a severe infectious disease caused by Mycobacterium tuberculosis (Mtb). LpqT is a lipoprotein of Mtb identified as a candidate virulence factor by a high-throughput screen searching for genes important for mycobacteria intracellular survival. To investigate its function, we constructed M. smegmatis strains deficient of LpqT or overexpressing LpqT. Wildtype or LpqT modified M. smegmatis strains were used to infect macrophages and mice, and intracellular survival of mycobacteria was measured. We found that LpqT can improve M. smegmatis survival in macrophage cell line, bone marrow derived macrophages (BMDMs), and murine lungs. This survival promoting effect is dependent on TLR2 and Myd88. Western blot analysis of M. smegmatis infected macrophages showed that LpqT suppressed M. smegmatis induced NF-κB and MAPK phosphorylation, indicating that LpqT hampered TLR2 signal activation. In consistent with this, LpqT inhibited M. smegmatis induced inflammatory cytokine expression and cell apoptosis in macrophages, thus supported mycobacteria intracellular survival.

Mycobacterial protein tyrosine kinase, PtkA phosphorylates PtpA at tyrosine residues and the mechanism is stalled by the novel series of inhibitors

Phosphorylation and dephosphorylation are the key mechanisms for mycobacterial physiology and play critical roles in mycobacterial survival and in its pathogenesis. Mycobacteria evade host immune mechanism by inhibiting phagosome – lysosome fusion in which mycobacterial protein tyrosine phosphatase A (PtpA;TP) plays an indispensable role. Tyrosine kinase (PtkA;TK) activated by autophosphorylation; phosphorylates TP, which subsequently leads to increase in its phosphatase activity. The phosphorylated TP is secreted in phagosome of macrophage. In the present study, we have shown that the phosphorylation at two sites of TP; Y128 and Y129 are critical for TK-mediated phosphatase activity. The disruption of this interaction between TK and TP inhibits activation of later which further leads to the decrease in intracellular survival of mycobacteria. Furthermore, the proof of concept has been established using benzylbenzofurans and benzofuranamides, which inhibit the growth and intracellular survival of mycobacteria, associate with the functional sites of TP and contend with the TK. This binding was further restated by looking at the anchorage of protein–protein and the protein–inhibitor complexes in the homology-based structure models and by surface plasmon resonance analysis.

Peroxiredoxin-1 of macrophage is critical for mycobacterial infection and is controlled by early secretory antigenic target protein through the activation of p38 MAPK

Early secretory antigenic target protein (ESAT-6) is an important virulent factor which plays a crucial role in Mycobacterium tuberculosis (MTB) pathogenesis. Here, we demonstrate the role of ESAT-6 in phagocytosis and intracellular survival of mycobacteria through a mechanism mediated by regulation of a host protein; Peroxiredoxin-1 (Prdx-1). Prdx-1 is an anti-apoptotic and stress response protein which protects cells from damage by ROS and H2O2. The J774 A.1 cells infected with MTB or over-expressing ESAT-6 through eukaryotic promoter vector showed elevated expression of Prdx-1. Further investigation revealed that the up-regulation of Prdx-1 is mediated through the activation of one of the MAP kinases, p38. The NRF-2, a transcriptional activator of Prdx-1 is translocated to the nucleus upon phosphorylation by p38 and subsequently, regulates expression of Prdx-1. Inhibition of the p38 MAPK by a specific inhibitor, SB203580, abrogates the ESAT-6 mediated induction of Prdx-1 expression as well as the phosphorylation of NRF-2 in a time-dependent manner. The inhibition of Prdx-1 expression by specific siRNA in J774 A.1 cells resulted in the reduced bacterial uptake and intracellular survival of the mycobacteria. This is the first report proclaiming that the ESAT-6 regulates Prdx-1 which is involved in the increase of mycobacterial uptake and survival. The intermediate mechanisms involve the increased Prdx-1 production in macrophages through the activation of p38 and NRF-2 dependent signaling.

Glucocorticoids Suppress Antimicrobial Autophagy and Nitric Oxide Production and Facilitate Mycobacterial Survival in Macrophages

Chronic administration of glucocorticoids has been shown to render individuals highly susceptible to mycobacterial infection and lead to reactivation of latent bacilli. However, the effect of glucocorticoids on innate anti-mycobacterial defense, especially in macrophages remains largely unknown. Here, we found that glucocorticoids inhibited the innate immune response, antimicrobial nitric oxide production and autophagy in mycobacteria-challenged macrophages. Meanwhile, maturation and acidification of mycobacterial phagosomes were attenuated in RAW264.7 cells after glucocorticoids treatment. Consequently, we observed a glucocorticoid-induced increase in the survival of intracellular mycobacteria in both primary macrophages and cell lines. Glucocorticoids treatment decreased the activation of TBK1 kinase, which promotes the maturation of autophagosomes. Inhibition of TBK1 also decreased the production of nitric oxide. Furthermore, several autophagy-related genes were down-regulated, while activation of the Akt/mTOR signaling pathway was increased after glucocorticoids treatment, which may account for autophagy inhibition during mycobacterial infection. Restoration of autophagy with the agonist rapamycin abolished glucocorticoid-mediated enhancement of mycobacterial survival, suggesting that glucocorticoids blocked anti-mycobacterial defense via autophagy inhibition. Collectively, this study demonstrates that glucocorticoids impair innate antimicrobial autophagy and promote mycobacterial survival in macrophages, which is a novel mechanism for glucocorticoid-mediated immunosuppression. Our findings may provide important clues for tuberculosis prevention.

Roles of endoplasmic reticulum stress-mediated apoptosis in M1-polarized macrophages during mycobacterial infections.

Alteration of macrophage function has an important regulatory impact on the survival of intracellular mycobacteria. We found that macrophages infected with attenuated Mycobacterium tuberculosis (Mtb) strain H37Ra had elevated expression of M1-related molecules, whereas the M2 phenotype was dominant in macrophages infected with virulent Mtb H37Rv. Further, the TLR signalling pathway played an important role in modulating macrophage polarization against Mtb infection. Interestingly, endoplasmic reticulum (ER) stress was significantly increased in M1 polarized macrophages and these macrophages effectively removed intracellular Mtb, indicating that ER stress may be an important component of the host immune response to Mtb in M1 macrophages. This improved understanding of the mechanisms that regulate macrophage polarization could provide new therapeutic strategies for tuberculosis.

Targeting Drug-Sensitive and -Resistant Strains of Mycobacterium tuberculosis by Inhibition of Src Family Kinases Lowers Disease Burden and Pathology.

In view of emerging drug resistance among bacterial pathogens, including Mycobacterium tuberculosis, the development of novel therapeutic strategies is increasingly being sought. A recent paradigm in antituberculosis (anti-TB) drug development is to target the host molecules that are crucial for intracellular survival of the pathogen. We previously showed the importance of Src tyrosine kinases in mycobacterial pathogenesis. Here, we report that inhibition of Src significantly reduced survival of H37Rv as well as multidrug-resistant (MDR) and extremely drug-resistant (XDR) strains of M. tuberculosis in THP-1 macrophages. Src inhibition was also effective in controlling M. tuberculosis infection in guinea pigs. In guinea pigs, reduced M. tuberculosis burden due to Src inhibition also led to a marked decline in the disease pathology. In agreement with the theoretical framework of host-directed approaches against the pathogen, Src inhibition was equally effective against an XDR strain in controlling infection in guinea pigs. We propose that Src inhibitors could be developed into effective host-directed anti-TB drugs, which could be indiscriminately used against both drug-sensitive and drug-resistant strains of M. tuberculosis. IMPORTANCE The existing treatment regimen for tuberculosis (TB) suffers from deficiencies like high doses of antibiotics, long treatment duration, and inability to kill persistent populations in an efficient manner. Together, these contribute to the emergence of drug-resistant tuberculosis. Recently, several host factors were identified which help intracellular survival of Mycobacterium tuberculosis within the macrophage. These factors serve as attractive targets for developing alternate therapeutic strategies against M. tuberculosis. This strategy promises to be effective against drug-resistant strains. The approach also has potential to considerably lower the risk of emergence of new drug-resistant strains. We explored tyrosine kinase Src as a host factor exploited by virulent M. tuberculosis for intracellular survival. We show that Src inhibition can effectively control tuberculosis in infected guinea pigs. Moreover, Src inhibition ameliorated TB-associated pathology in guinea pigs. Thus, Src inhibitors have strong potential to be developed as possible anti-TB drugs.

RD-1 encoded EspJ protein gets phosphorylated prior to affect the growth and intracellular survival of mycobacteria.

Mycobacterium tuberculosis (MTB) synchronizes a number of processes and controls a series of events to subvert host defense mechanisms for the sake of residing inside macrophages. Besides these, MTB also possesses a wide range of signal enzyme systems, including eleven serine threonine protein kinases (STPKs). The present study describes STPK modulated modification in one of the hypothetical proteins of the RD1 region; EspJ (ESX-1 secretion associated protein), which is predicted to be involved in virulence of MTB. We have employed knock-out MTB, and M. bovis BCG as a surrogate strain to elaborate the consequence of the phosphorylation of EspJ. The molecular and mass spectrometric analyses in this study, confirmed EspJ as one of the substrates of STPKs. The ectopic expression of phosphoablative mutants of espJ in M. bovis BCG also articulated the effect of phosphorylation on the growth and in survival of mycobacteria. Importantly, the level of phosphorylation of EspJ also differed between pathogenic H37 Rv (Rv) and non pathogenic H37 Ra (Ra) strains of MTB. This further suggested that to a certain extent, the STPKs mediated phosphorylation may be accountable, in determining the growth and in intra-cellular survival of mycobacteria.

Development of an in vitro model system to study the interactions between Mycobacterium marinum and teleost neutrophils

The lack of a reliable mammalian neutrophil in vitro culture system has restricted our ability to examine their precise roles in mycobacterial infections. Previously, we developed the procedures for the isolation and culture of primary kidney-derived neutrophil-like cells from goldfish that are functionally and morphologically similar to mammalian neutrophils. The cultured primary goldfish neutrophils exhibited prolonged viability and functional effector responses. In this study, we demonstrate that when exposed to live or heat-killed Mycobacterium marinum, goldfish neutrophils increased their mRNA levels for several pro-inflammatory cytokines (il-1β1, il-1β2, tnfα-1, tnfα-2) and the cytokine receptors (ifngr1-1, tnfr1, tnfr2). These neutrophils also exhibited chemotaxis toward live mycobacteria, internalized the bacilli, and produced reactive oxygen intermediates (ROI) in response to pathogen exposure. The survival of intracellular mycobacteria was significantly reduced in activated neutrophils, indicating a robust killing response by these teleost granulocytes. We suggest that this goldfish primary neutrophil in vitro model system will provide important information regarding neutrophil-mediated host defense mechanisms against mycobacteria in teleosts as well as in higher vertebrates.

Quantitative Proteomics Analysis of PknG Functions in <italic>Mycobacterium</italic>

The eukaryotic like serine/threonine protein kinase G (PknG) from pathogenic mycobacteria plays an important role in inhibiting phagosome-lysosome fusion and mediating intracellular survival of mycobacteria in macrophages. Furthermore, PknG has broad functions in regulation of bacterial metabolism, survival, virulence, and drug-resistance in mycobacteria. In the present work, we construct the PknG - overexpression Mycobacterium smegmatis strain PknG-mc2155, and show that overexpression of PknG arrests bacterial growth and decreases the cellular glutamate and glutamine levels. Using quantitative proteomic technology, we systematically analyze the differentially expressed proteins between PknG-mc2155 and mc2155 strains. We have identified 176 differentially expressed proteins, in which 152 proteins are upregulated and 24 are downregulated in the PknG-mc2155 strain. Our data suggest that the decrease of bacterial growth rate is associated with the down-regulation of metabolic enzymes such as GlpK, ALD, and DesA in PknG-mc2155 cells, and the up-regulation of Ag85A, Ag85C, and SecA2 contributes to enhanced bacterial virulence. From our proteomic data, we identify a new phosphorylation site on Thr-329 residue of PknG. Analysis also shows that gltA and glmM are phosphorylated in PknG-mc2155 cells, indicating that gltA and glmM are potential substrates of PknG. Based on the proteomic data, the working mechanisms of PknG are proposed that provide new evidence for understanding functions of PknG in mycobacteria. Our data also demonstrates that quantitative proteomic analysis is an important tool for understanding functions and working mechanisms of bacteria proteins.

Phosphorylation of pyruvate kinase A by protein kinase J leads to the altered growth and differential rate of intracellular survival of mycobacteria.

PknJ (Rv2088) is a serine/threonine protein kinase of mycobacteria which is present in Mycobacterium tuberculosis (MTB), but its gene is absent in Mycobacterium smegmatis (MS); a fast grower and nonpathogenic species of mycobacteria. The heterologous expression of MTB-specific PknJ in MS altered the growth of recombinant mycobacteria highlighting one of the characteristics of this protein. This nature of the protein was further confirmed when Mycobacterium bovis BCG (BCG) containing antisense copy of pknJ resulted in the increased growth of BCG. The real-time RNA quantification analysis pointed out toward increased expression of this protein during infection of THP-1 macrophage cells which further emphasized that the protein is essential for the intracellular survival of mycobacteria. The differential in gel electrophoresis (DIGE) data followed by mass spectroscopy suggested that PknJ is involved in regulation of pyruvate kinase A (Rv1617). Since pyruvate kinase (PK) A is one of the key enzymes which controls glycolytic cycle in mycobacteria, we looked for its interaction with PknJ during extracellular and intracellular growth of mycobacteria. In order to identify the specific residue(s) involved in post-translational modification, the phospho-null mutants of PK were generated, and their substrate specificities in response to PknJ were assessed through kinase assay. The findings thus underlined that the PK activity is predominantly dependent on the threonine residue at the 94(th) position and further suggested that this site may be plausible in intracellular survival of mycobacteria upon phosphorylation with PknJ.

Deducing the mechanism of action of compounds identified in phenotypic screens by integrating their multiparametric profiles with a reference genetic screen

Cell-based high-content screens are increasingly used to discover bioactive small molecules. However, identifying the mechanism of action of the selected compounds is a major bottleneck. Here we describe a protocol consisting of experimental and computational steps to identify the cellular pathways modulated by chemicals, and their mechanism of action. The multiparametric profiles from a 'query' chemical screen are used as constraints to select genes with similar profiles from a 'reference' genetic screen. In our case, the query screen is the intracellular survival of mycobacteria and the reference is a genome-wide RNAi screen of endocytosis. The two disparate screens are bridged by an 'intermediate' chemical screen of endocytosis, so that the similarity in the multiparametric profiles between the chemical and the genetic perturbations can generate a testable hypothesis of the cellular pathways modulated by the chemicals. This approach is not assay specific, but it can be broadly applied to various quantitative, multiparametric data sets. Generation of the query system takes 3-6 weeks, and data analysis and integration with the reference data set takes an 3 additional weeks.

Differential macrophage response to slow- and fast-growing pathogenic mycobacteria.

Nontuberculous mycobacteria (NTM) have recently been recognized as important species that cause disease even in immunocompetent individuals. The mechanisms that these species use to infect and persist inside macrophages are not well characterised. To gain insight concerning this process we used THP-1 macrophages infected with M. abscessus, M. fortuitum, M. celatum, and M. tuberculosis. Our results showed that slow-growing mycobacteria gained entrance into these cells with more efficiency than fast-growing mycobacteria. We have also demonstrated that viable slow-growing M. celatum persisted inside macrophages without causing cell damage and without inducing reactive oxygen species (ROS), as M. tuberculosis caused. In contrast, fast-growing mycobacteria destroyed the cells and induced high levels of ROS. Additionally, the macrophage cytokine pattern induced by M. celatum was different from the one induced by either M. tuberculosis or fast-growing mycobacteria. Our results also suggest that, in some cases, the intracellular survival of mycobacteria and the immune response that they induce in macrophages could be related to their growth rate. In addition, the modulation of macrophage cytokine production, caused by M. celatum, might be a novel immune-evasion strategy used to survive inside macrophages that is different from the one reported for M. tuberculosis.

Imatinib-Sensitive Tyrosine Kinases Regulate Mycobacterial Pathogenesis and Represent Therapeutic Targets against Tuberculosis

The lengthy course of treatment with currently used antimycobacterial drugs and the resulting emergence of drug-resistant strains have intensified the need for alternative therapies against Mycobacterium tuberculosis (Mtb), the etiologic agent of tuberculosis. We show that Mtb and Mycobacterium marinum use ABL and related tyrosine kinases for entry and intracellular survival in macrophages. In mice, the ABL family tyrosine kinase inhibitor, imatinib (Gleevec), when administered prophylactically or therapeutically, reduced both the number of granulomatous lesions and bacterial load in infected organs and was also effective against a rifampicin-resistant strain. Further, when coadministered with current first-line drugs, rifampicin or rifabutin, imatinib acted synergistically. These data implicate host tyrosine kinases in entry and intracellular survival of mycobacteria and suggest that imatinib may have therapeutic efficacy against Mtb. Because imatinib targets host, it is less likely to engender resistance compared to conventional antibiotics and may decrease the development of resistance against coadministered drugs.