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1.
PLoS Pathog ; 14(4): e1006974, 2018 04.
Article in English | MEDLINE | ID: mdl-29698476

ABSTRACT

Mycobacterium tuberculosis (Mtb) remains a grave threat to world health with emerging drug resistant strains. One prominent feature of Mtb infection is the extensive reprogramming of host tissue at the site of infection. Here we report that inhibition of matrix metalloproteinase (MMP) activity by a panel of small molecule inhibitors enhances the in vivo potency of the frontline TB drugs isoniazid (INH) and rifampicin (RIF). Inhibition of MMP activity leads to an increase in pericyte-covered blood vessel numbers and appears to stabilize the integrity of the infected lung tissue. In treated mice, we observe an increased delivery and/or retention of frontline TB drugs in the infected lungs, resulting in enhanced drug efficacy. These findings indicate that targeting Mtb-induced host tissue remodeling can increase therapeutic efficacy and could enhance the effectiveness of current drug regimens.


Subject(s)
Antitubercular Agents/pharmacology , Granuloma, Respiratory Tract/drug therapy , Lung/drug effects , Matrix Metalloproteinase Inhibitors/pharmacology , Mycobacterium tuberculosis/drug effects , Small Molecule Libraries/pharmacology , Tuberculosis/drug therapy , Animals , Granuloma, Respiratory Tract/enzymology , Granuloma, Respiratory Tract/microbiology , Isoniazid/pharmacology , Lung/enzymology , Lung/microbiology , Mice , Mice, Inbred C57BL , Mycobacterium tuberculosis/enzymology , Rifampin/pharmacology , Tuberculosis/enzymology , Tuberculosis/microbiology
2.
J Biol Chem ; 293(17): 6497-6516, 2018 04 27.
Article in English | MEDLINE | ID: mdl-29530985

ABSTRACT

The cell wall of Mycobacterium tuberculosis (Mtb) is a complex structure that protects the pathogen in hostile environments. Peptidoglycan (PG), which helps determine the morphology of the cell envelope, undergoes substantial remodeling under stress. This meshwork of linear chains of sugars, cross-linked through attached peptides, is generated through the sequential action of enzymes termed transglycosylases and transpeptidases. The Mtb genome encodes two classical transglycosylases and four transpeptidases, the functions of which are not fully elucidated. Here, we present work on the yet uncharacterized transpeptidase PbpA and a nonclassical transglycosylase RodA. We elucidate their roles in regulating in vitro growth and in vivo survival of pathogenic mycobacteria. We find that RodA and PbpA are required for regulating cell length, but do not affect mycobacterial growth. Biochemical analyses show PbpA to be a classical transpeptidase, whereas RodA is identified to be a member of an emerging class of noncanonical transglycosylases. Phosphorylation of RodA at Thr-463 modulates its biological function. In a guinea pig infection model, RodA and PbpA are found to be required for both bacterial survival and formation of granuloma structures, thus underscoring the importance of these proteins in mediating mycobacterial virulence in the host. Our results emphasize the fact that whereas redundant enzymes probably compensate for the absence of RodA or PbpA during in vitro growth, the two proteins play critical roles for the survival of the pathogen inside its host.


Subject(s)
Bacterial Proteins , Glycosyltransferases , Granuloma, Respiratory Tract , Microbial Viability , Mycobacterium tuberculosis , Peptidyl Transferases , Animals , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Female , Genome, Bacterial , Glycosyltransferases/genetics , Glycosyltransferases/metabolism , Granuloma, Respiratory Tract/enzymology , Granuloma, Respiratory Tract/genetics , Granuloma, Respiratory Tract/pathology , Guinea Pigs , Male , Mice , Mice, Inbred BALB C , Mycobacterium tuberculosis/enzymology , Mycobacterium tuberculosis/genetics , Mycobacterium tuberculosis/pathogenicity , Peptidyl Transferases/genetics , Peptidyl Transferases/metabolism , Tuberculosis/enzymology , Tuberculosis/genetics , Tuberculosis/pathology
3.
Neuroimmunomodulation ; 18(4): 212-25, 2011.
Article in English | MEDLINE | ID: mdl-21389736

ABSTRACT

Granulomatous structures are highly dynamic during active mycobacterial infection, with accompanying responsive inflammation contributing to modulation of pathology throughout the course of disease. The heightened inflammatory response coinciding with initiation and maintenance of newly developing granulomatous structures must be limited to avoid excessive damage to bystander tissue. Modulating the cellular bioavailability of glucocorticoids by local regulation of 11ßHSD enzymes within responding tissue and parenchyma would allow controlled inflammatory response during infection. Mycobacterial glycolipid trehalose 6,6'-dimycolate was used to induce strong pulmonary granulomatous inflammation immunopathology. Pulmonary corticosterone was significantly increased at days 3 and 5 after administration. An inverse relationship of 11ßHSD1 and 11ßHSD2 message correlated with pathology development. Immunohistochemical analysis also demonstrated that 11ßHSD2 is expressed in proximity to granulomatous lesions. A role for pro-inflammatory IL-6 cytokine in regulation of converting enzymes to control the granulomatous response was confirmed using gene-disrupted IL-6-/- mice. A model is proposed linking IL-6 to endocrine-derived factors which allows modification of active corticosterone into inert 11-dehydrocorticosterone at the site of granuloma formation to limit excessive parenchymal damage.


Subject(s)
11-beta-Hydroxysteroid Dehydrogenase Type 2/metabolism , Granuloma, Respiratory Tract/enzymology , Granuloma, Respiratory Tract/pathology , Interleukin-6/metabolism , 11-beta-Hydroxysteroid Dehydrogenase Type 2/immunology , Animals , Cord Factors/toxicity , Corticosterone/analysis , Corticosterone/metabolism , Cytokines/biosynthesis , Cytokines/immunology , Disease Progression , Enzyme-Linked Immunosorbent Assay , Female , Gene Expression , Gene Expression Regulation/immunology , Granuloma, Respiratory Tract/immunology , Immunohistochemistry , Interleukin-6/immunology , Mice , Mice, Inbred C57BL , Mice, Knockout , RNA, Messenger/analysis , Radioimmunoassay , Reverse Transcriptase Polymerase Chain Reaction
4.
Respir Res ; 12: 13, 2011 Jan 20.
Article in English | MEDLINE | ID: mdl-21251246

ABSTRACT

BACKGROUND: Remodeling of lung tissues during the process of granuloma formation requires significant restructuring of the extra-cellular matrix and cathepsins K, L and S are among the strongest extra-cellular matrix degrading enzymes. Cathepsin K is highly expressed in various pathological granulomatous infiltrates and all three enzymes in their active form are detected in bronchoalveolar lavage fluids from patients with sarcoidosis. Granulomatous inflammation is driven by T-cell response and cathepsins S and L are actively involved in the regulation of antigen presentation and T-cell selection. Here, we show that the disruption of the activities of cathepsins K, L, or S affects the development of lung granulomas in a mouse model of sarcoidosis. METHODS: Apolipoprotein E-deficient mice lacking cathepsin K or L were fed Paigen diet for 16 weeks and lungs were analyzed and compared with their cathepsin-expressing littermates. The role of cathepsin S in the development of granulomas was evaluated using mice treated for 8 weeks with a potent and selective cathepsin S inhibitor. RESULTS: When compared to wild-type litters, more cathepsin K-deficient mice had lung granulomas, but individually affected mice developed smaller granulomas that were present in lower numbers. The absence of cathepsin K increased the number of multinucleated giant cells and the collagen content in granulomas. Cathepsin L deficiency resulted in decreased size and number of lung granulomas. Apoe-/- mice treated with a selective cathepsin S inhibitor did not develop lung granulomas and only individual epithelioid cells were observed. CONCLUSIONS: Cathepsin K deficiency affected mostly the occurrence and composition of lung granulomas, whereas cathepsin L deficiency significantly reduced their number and cathepsin S inhibition prevented the formation of granulomas.


Subject(s)
Cathepsin K/deficiency , Cathepsin L/deficiency , Cathepsins/antagonists & inhibitors , Granuloma, Respiratory Tract/prevention & control , Lung/drug effects , Protease Inhibitors/pharmacology , Sarcoidosis, Pulmonary/drug therapy , Animals , Apolipoproteins E/deficiency , Apolipoproteins E/genetics , CD4-Positive T-Lymphocytes/immunology , Cathepsin K/genetics , Cathepsin L/genetics , Cathepsins/metabolism , Collagen/metabolism , Disease Models, Animal , Granuloma, Respiratory Tract/enzymology , Granuloma, Respiratory Tract/genetics , Granuloma, Respiratory Tract/immunology , Granuloma, Respiratory Tract/pathology , Hypertrophy , Lung/enzymology , Lung/immunology , Lung/pathology , Mice , Mice, Knockout , Sarcoidosis, Pulmonary/enzymology , Sarcoidosis, Pulmonary/genetics , Sarcoidosis, Pulmonary/immunology , Sarcoidosis, Pulmonary/pathology , Thymus Gland/enzymology , Thymus Gland/pathology , Time Factors
5.
Neuroimmunomodulation ; 16(3): 147-54, 2009.
Article in English | MEDLINE | ID: mdl-19246936

ABSTRACT

OBJECTIVE: Tuberculosis has a staggering influence on world health, resulting in nearly 2 million deaths per year. The influence of glucocorticoids during Mycobacterium tuberculosis infection has been under investigation for decades; however, the identity of mycobacterial factors and the mechanism by which glucocorticoids are tissue specifically regulated to influence immune function during acute granuloma formation are unknown. METHODS: One factor implicated in initiating immunopathology during M. tuberculosis infection is trehalose-6,6'-dimycolate (TDM), a glycolipid component of the mycobacterial cell wall. Intravenous administration of TDM causes inflammatory responses in lungs of mice similar to M. tuberculosis infection and has been used as a successful model to examine proinflammatory regulation and early events involved in the manifestation of pathology. RESULTS AND CONCLUSION: IL-6, IL-1alpha and TNF-alpha mRNA and protein peaked during the initiation of granuloma formation. Pulmonary corticosterone levels were elevated when the proinflammatory response was greatest, dropping to half of that upon the establishment of granuloma pathology on day 7. It is hypothesized that once corticosterone reaches the site of inflammation, the enzymes 11beta-hydroxysteroid dehydrogenases (11betaHSDs) can influence bioavailability by interconverting corticosterone and the inert metabolite 11-dehydrocorticosterone. RT-PCR demonstrated that pulmonary 11betaHSD type 1 mRNA decreased 4-fold and 11betaHSD type 2 (11betaHSD2) mRNA expression increased 2.5-fold on day 3 after injection, suggesting that corticosterone regulation in the lung, specifically the reduction of active corticosterone by 11betaHSD2, may influence the progression of granuloma formation in response to the mycobacterial glycolipid.


Subject(s)
11-beta-Hydroxysteroid Dehydrogenase Type 2/metabolism , Cord Factors/metabolism , Granuloma, Respiratory Tract/enzymology , Granuloma, Respiratory Tract/microbiology , Tuberculosis, Pulmonary/enzymology , Tuberculosis, Pulmonary/microbiology , 11-beta-Hydroxysteroid Dehydrogenase Type 1/genetics , 11-beta-Hydroxysteroid Dehydrogenase Type 1/metabolism , 11-beta-Hydroxysteroid Dehydrogenase Type 2/genetics , Animals , Corticosterone/analogs & derivatives , Corticosterone/metabolism , Cytokines/genetics , Cytokines/metabolism , Disease Models, Animal , Down-Regulation/physiology , Female , Granuloma, Respiratory Tract/physiopathology , Immune Tolerance/physiology , Lung/enzymology , Lung/microbiology , Lung/physiopathology , Mice , Mice, Inbred BALB C , Mycobacterium tuberculosis/metabolism , RNA, Messenger/metabolism , Tuberculosis, Pulmonary/physiopathology , Up-Regulation/physiology
6.
Eur Respir J ; 33(1): 134-41, 2009 Jan.
Article in English | MEDLINE | ID: mdl-18715875

ABSTRACT

Tuberculosis (TB) pleural disease is complicated by extensive tissue destruction. Matrix metalloproteinase (MMP)-1 and -9 are implicated in immunopathology of pulmonary and central nervous system TB. There are few data on MMP activity in TB pleurisy. The present study investigated MMP-1, -2 and -9 and their specific inhibitors (tissue inhibitor of metalloproteinase (TIMP)-1 and -2) in tuberculous effusions, and correlated these with clinical and histopathological features. Clinical data, routine blood tests, and pleural fluid/biopsy material were obtained from 89 patients presenting with pleural effusions in a TB-endemic area. MMP-1, -2 and -9 were measured by zymography or western blot, and TIMP-1 and -2 by ELISA. Pleural biopsies were examined microscopically, cultured for acid-alcohol fast bacilli and immunostained for MMP-9. Tuberculous pleural effusions contained the highest concentrations of MMP-9 compared with malignant effusions or heart failure transudates. MMP-9 concentrations were highest in effusions from patients with granulomatous biopsies: median (interquartile range) 108 (61-218) pg x mL(-1) versus 43 (12-83) pg x mL(-1) in those with nongranulomatous pleural biopsies. MMP-1 and -2 were not upregulated in tuberculous pleural fluid. The ratio of MMP-9:TIMP-1 was significantly higher in TB effusions. Tuberculous pleurisy is characterised by a specific pattern of matrix metalloproteinase-9 upregulation, correlating with the presence of granulomas and suggesting a specific role for matrix metalloproteinase-9 in inflammatory responses in tuberculous pleural disease.


Subject(s)
Granuloma, Respiratory Tract/etiology , Matrix Metalloproteinase 9/metabolism , Tuberculosis, Pleural/enzymology , Tuberculosis, Pleural/pathology , Adult , Aged , Case-Control Studies , Cohort Studies , Female , Granuloma, Respiratory Tract/enzymology , Granuloma, Respiratory Tract/pathology , Humans , Male , Matrix Metalloproteinase 1/metabolism , Middle Aged , Pleural Effusion/enzymology , Pleural Effusion/etiology , Pleural Effusion/pathology , Tissue Inhibitor of Metalloproteinase-1/metabolism , Tissue Inhibitor of Metalloproteinase-2/metabolism , Tuberculosis, Pleural/complications
8.
J Immunol ; 170(10): 5210-8, 2003 May 15.
Article in English | MEDLINE | ID: mdl-12734369

ABSTRACT

Containment of intracellularly viable microorganisms requires an intricate cooperation between macrophages and T cells, the most potent mediators known to date being IFN-gamma and TNF. To identify novel mechanisms involved in combating intracellular infections, experiments were performed in mice with selective defects in the lymphotoxin (LT)/LT beta R pathway. When mice deficient in LT alpha or LT beta were challenged intranasally with Mycobacterium tuberculosis, they showed a significant increase in bacterial loads in lungs and livers compared with wild-type mice, suggesting a role for LT alpha beta heterotrimers in resistance to infection. Indeed, mice deficient in the receptor for LT alpha(1)beta(2) heterotrimers (LT beta R-knockout (KO) mice) also had significantly higher numbers of M. tuberculosis in infected lungs and exhibited widespread pulmonary necrosis already by day 35 after intranasal infection. Furthermore, LT beta R-KO mice were dramatically more susceptible than wild-type mice to i.p. infection with Listeria monocytogenes. Compared with wild-type mice, LT beta R-KO mice had similar transcript levels of TNF and IFN-gamma and recruited similar numbers of CD3(+) T cells inside granulomatous lesions in M. tuberculosis-infected lungs. Flow cytometry revealed that the LT beta R is expressed on pulmonary macrophages obtained after digestion of M. tuberculosis-infected lungs. LT beta R-KO mice showed delayed expression of inducible NO synthase protein in granuloma macrophages, implicating deficient macrophage activation as the most likely cause for enhanced susceptibility of these mice to intracellular infections. Since LIGHT-KO mice proved to be equally resistant to M. tuberculosis infection as wild-type mice, these data demonstrate that signaling of LT alpha(1)beta(2) heterotrimers via the LT beta R is an essential prerequisite for containment of intracellular pathogens.


Subject(s)
Intracellular Fluid/immunology , Listeria monocytogenes/immunology , Listeriosis/immunology , Lymphotoxin-alpha/metabolism , Mycobacterium tuberculosis/immunology , Receptors, Tumor Necrosis Factor/physiology , Tuberculosis, Pulmonary/immunology , Administration, Intranasal , Animals , Bone Marrow Transplantation/immunology , Genetic Predisposition to Disease , Granuloma, Respiratory Tract/enzymology , Granuloma, Respiratory Tract/genetics , Granuloma, Respiratory Tract/immunology , Granuloma, Respiratory Tract/microbiology , Interferon-gamma/biosynthesis , Interferon-gamma/deficiency , Intracellular Fluid/microbiology , Listeriosis/genetics , Listeriosis/microbiology , Lung/enzymology , Lung/immunology , Lung/microbiology , Lung/pathology , Lymphotoxin beta Receptor , Lymphotoxin-alpha/deficiency , Lymphotoxin-alpha/genetics , Macrophage Activation/immunology , Macrophages/enzymology , Macrophages/immunology , Macrophages/microbiology , Macrophages/pathology , Membrane Proteins/deficiency , Membrane Proteins/genetics , Mice , Mice, Inbred C57BL , Mice, Knockout , Necrosis , Nitric Oxide Synthase/biosynthesis , Nitric Oxide Synthase Type II , Radiation Chimera , Receptors, Tumor Necrosis Factor/biosynthesis , Receptors, Tumor Necrosis Factor/deficiency , Receptors, Tumor Necrosis Factor/genetics , Tuberculosis, Pulmonary/genetics , Tuberculosis, Pulmonary/microbiology , Tuberculosis, Pulmonary/prevention & control , Tumor Necrosis Factor Ligand Superfamily Member 14 , Tumor Necrosis Factor-alpha/biosynthesis , Tumor Necrosis Factor-alpha/deficiency , Tumor Necrosis Factor-alpha/genetics
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