scholarly journals Individual Mycobacterium tuberculosis Resuscitation-Promoting Factor Homologues Are Dispensable for Growth In Vitro and In Vivo

2004 ◽  
Vol 72 (1) ◽  
pp. 515-526 ◽  
Author(s):  
JoAnn M. Tufariello ◽  
William R. Jacobs, ◽  
John Chan
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Stationary Phase ◽  
Essential Gene ◽  
Micrococcus Luteus ◽  
Expression Patterns ◽  
Active Growth ◽  
Prolonged Incubation ◽  
Aerosol Infection

ABSTRACT Mycobacterium tuberculosis possesses five genes with significant homology to the resuscitation-promoting factor (Rpf) of Micrococcus luteus. The M. luteus Rpf is a secreted ∼16-kDa protein which restores active growth to cultures of M. luteus rendered dormant by prolonged incubation in stationary phase. More recently, the Rpf-like proteins of M. tuberculosis have been shown to stimulate the growth of extended-stationary-phase cultures of Mycobacterium bovis BCG. These data suggest that the Rpf proteins can influence the growth of mycobacteria; however, the studies do not demonstrate specific functions for the various members of this protein family, nor do they assess the function of M. tuberculosis Rpf homologues in vivo. To address these questions, we have disrupted each of the five rpf-like genes in M. tuberculosis Erdman, and analyzed the mutants for their growth in vitro and in vivo. In contrast to M. luteus, for which rpf is an essential gene, we find that all of the M. tuberculosis rpf deletion mutant strains are viable; in addition, all show growth kinetics similar to Erdman wild type both in vitro and in mouse organs following aerosol infection. Analysis of rpf expression in M. tuberculosis cultures from early log phase through late stationary phase indicates that expression of the rpf-like genes is growth phase-dependent, and that the expression patterns of the five M. tuberculosis rpf genes, while overlapping to various degrees, are not uniform. We also provide evidence that mycobacterial rpf genes are expressed in vivo in the lungs of mice acutely infected with virulent M. tuberculosis.

scholarly journals Mutants of Mycobacterium tuberculosis Lacking Three of the Five rpf-Like Genes Are Defective for Growth In Vivo and for Resuscitation In Vitro

2005 ◽  
Vol 73 (5) ◽  
pp. 3038-3043 ◽  
Author(s):  
Katrina J. Downing ◽  
Vladimir V. Mischenko ◽  
Margarita O. Shleeva ◽  
Danielle I. Young ◽  
Michael Young ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Micrococcus Luteus ◽  
Most Probable Number ◽  
Infection Model ◽  
Term Survival ◽  
Probable Number ◽  
Mouse Infection Model ◽  
Nonculturable State

ABSTRACT Mycobacterium tuberculosis contains five genes, rpfA through rpfE, that bear significant homology to the resuscitation-promoting factor (rpf) gene of Micrococcus luteus, whose product is required to resuscitate the growth of dormant cultures of M. luteus and is essential for the growth of this organism. Previous studies have shown that deletion of any one of the five rpf-like genes did not affect the growth or survival of M. tuberculosis in vitro. In conjunction with the results of whole-genome expression profiling, this finding was indicative of their functional redundancy. In this study, we demonstrate that the single deletion mutants are phenotypically similar to wild-type M. tuberculosis H37Rv in vivo. The deletion of individual rpf-like genes had no discernible effect on the growth or long-term survival of M. tuberculosis in liquid culture, and the ability to resuscitate spontaneously from a nonculturable state in a most probable number assay was also unaffected for the three strains tested (the ΔrpfB, ΔrpfD, and ΔrpfE strains). In contrast, two multiple strains, KDT8 (ΔrpfA-mutation ΔrpfC ΔrpfB) and KDT9 (ΔrpfA ΔrpfC ΔrpfD), which lack three of the five rpf-like genes, were significantly yet differentially attenuated in a mouse infection model. These mutants were also unable to resuscitate spontaneously in vitro, demonstrating the importance of the Rpf-like proteins of M. tuberculosis in resuscitation from the nonculturable state. These results strongly suggest that the biological functions of the five rpf-like genes of M. tuberculosis are not wholly redundant and underscore the potential utility of these proteins as targets for therapeutic intervention.

scholarly journals Sigma Factor F Does Not Prevent Rifampin Inhibition of RNA Polymerase or Cause Rifampin Tolerance in Mycobacterium tuberculosis

2010 ◽  
Vol 192 (20) ◽  
pp. 5472-5479 ◽  
Author(s):  
Ruben C. Hartkoorn ◽  
Claudia Sala ◽  
Sophie J. Magnet ◽  
Jeffrey M. Chen ◽  
Florence Pojer ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Rna Polymerase ◽  
Stationary Phase ◽  
Sigma Factor ◽  
Potent Inhibitor ◽  
Sigma Factors ◽  
Antituberculosis Drugs ◽  
Axenic Cultures

ABSTRACT The tolerance of Mycobacterium tuberculosis to antituberculosis drugs is a major reason for the lengthy therapy needed to treat a tuberculosis infection. Rifampin is a potent inhibitor of RNA polymerase (RNAP) in vivo but has been shown to be less effective against stationary-phase bacteria. Sigma factor F is associated with bacteria entering stationary phase and has been proposed to impact rifampin activity. Here we investigate whether RNAP containing SigF is more resistant to rifampin inhibition in vitro and whether overexpression of sigF renders M. tuberculosis more tolerant to rifampin. Real-time and radiometric in vitro transcription assays revealed that rifampin equally inhibits transcription by RNAP containing sigma factors SigA and SigF, therefore ruling out the hypothesis that SigF may be responsible for increased resistance of the enzyme to rifampin in vitro. In addition, overexpression or deletion of sigF did not alter rifampin susceptibility in axenic cultures of M. tuberculosis, indicating that SigF does not affect rifampin tolerance in vivo.

scholarly journals Examination of Mycobacterium tuberculosis sigma factor mutants using low-dose aerosol infection of guinea pigs suggests a role for SigC in pathogenesis

Microbiology ◽  
2006 ◽  
Vol 152 (6) ◽  
pp. 1591-1600 ◽  
Author(s):  
Russell K. Karls ◽  
Jeannette Guarner ◽  
David N. McMurray ◽  
Kristin A. Birkness ◽  
Frederick D. Quinn
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Sigma Factor ◽  
Guinea Pigs ◽  
Low Dose ◽  
Sigma Factors ◽  
Respiratory Pathogen ◽  
Growth Deficiency ◽  
Aerosol Infection

Secondary sigma factors in bacteria direct transcription of defence regulons in response to specific stresses. To identify which sigma factors in the human respiratory pathogen Mycobacterium tuberculosis are important for adaptive survival in vivo, defined null mutations were created in individual sigma factor genes. In this study, in vitro growth virulence and guinea pig pathology of M. tuberculosis mutants lacking functional sigma factors (SigC, SigF, or SigM) were compared to the parent strain, H37Rv. None of the mutant strains exhibited a growth deficiency in Middlebrook 7H9 broth, nor were any impaired for intracellular replication in the human monocytic macrophage cell-line THP-1. Following low-dose aerosol infection of guinea pigs, however, differences could be detected. While a SigM mutant resulted in lung and spleen granulomas of comparable composition to those found in H37Rv-infected animals, a SigF mutant was partially attenuated, exhibiting necrotic spleen granulomas and ill-defined lung granulomas. SigC mutants exhibited attenuation in the lung and spleen; notably, necrotic granulomas were absent. These data suggest that while SigF may be important for survival in the lung, SigC is likely a key regulator of pathogenesis and adaptive survival in the lung and spleen. Understanding how SigC mediates survival in the host should prove useful in the development of anti-tuberculosis therapies.

scholarly journals TLR9 regulates Th1 responses and cooperates with TLR2 in mediating optimal resistance to Mycobacterium tuberculosis

2005 ◽  
Vol 202 (12) ◽  
pp. 1715-1724 ◽  
Author(s):  
Andre Bafica ◽  
Charles A. Scanga ◽  
Carl G. Feng ◽  
Cynthia Leifer ◽  
Allen Cheever ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Ex Vivo ◽  
Double Knockout ◽  
Susceptibility To Infection ◽  
Pathogen Challenge ◽  
In Vitro Response ◽  
Ifn Γ ◽  
Aerosol Infection

To investigate the role of Toll-like receptor (TLR)9 in the immune response to mycobacteria as well as its cooperation with TLR2, a receptor known to be triggered by several major mycobacterial ligands, we analyzed the resistance of TLR9−/− as well as TLR2/9 double knockout mice to aerosol infection with Mycobacterium tuberculosis. Infected TLR9−/− but not TLR2−/− mice displayed defective mycobacteria-induced interleukin (IL)-12p40 and interferon (IFN)-γ responses in vivo, but in common with TLR2−/− animals, the TLR9−/− mice exhibited only minor reductions in acute resistance to low dose pathogen challenge. When compared with either of the single TLR-deficient animals, TLR2/9−/− mice displayed markedly enhanced susceptibility to infection in association with combined defects in proinflammatory cytokine production in vitro, IFN-γ recall responses ex vivo, and altered pulmonary pathology. Cooperation between TLR9 and TLR2 was also evident at the level of the in vitro response to live M. tuberculosis, where dendritic cells and macrophages from TLR2/9−/− mice exhibited a greater defect in IL-12 response than the equivalent cell populations from single TLR9-deficient animals. These findings reveal a previously unappreciated role for TLR9 in the host response to M. tuberculosis and illustrate TLR collaboration in host resistance to a major human pathogen.

scholarly journals In Vitro and In Vivo Activities of the Riminophenazine TBI-166 against Mycobacterium tuberculosis

2019 ◽  
Vol 63 (5) ◽  
Author(s):  
Jian Xu ◽  
Bin Wang ◽  
Lei Fu ◽  
Hui Zhu ◽  
Shaochen Guo ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Skin Pigmentation ◽  
Multidrug Resistant ◽  
Content Type ◽  
Resistant Tuberculosis ◽  
Wild Strains ◽  
Skin Discoloration ◽  
Aerosol Infection

ABSTRACT The riminophenazine agent clofazimine (CFZ) is repurposed as an important component of the new short-course multidrug-resistant tuberculosis regimen and significantly shortens first-line regimen for drug-susceptible tuberculosis in mice. However, CFZ use is hampered by its unwelcome skin discoloration in patients. A new riminophenazine analog, TBI-166, was selected as a potential next-generation antituberculosis riminophenazine following an extensive medicinal chemistry effort. Here, we evaluated the activity of TBI-166 against Mycobacterium tuberculosis and its potential to accumulate and discolor skin. The in vitro activity of TBI-166 against both drug-sensitive and drug-resistant M. tuberculosis is more potent than that of CFZ. Spontaneous mutants resistant to TBI-166 were found at a frequency of 2.3 × 10−7 in wild strains of M. tuberculosis. TBI-166 demonstrates activity at least equivalent to that of CFZ against intracellular M. tuberculosis and in low-dose aerosol infection models of acute and chronic murine tuberculosis. Most importantly, TBI-166 causes less skin discoloration than does CFZ despite its higher tissue accumulation. The efficacy of TBI-166, along with its decreased skin pigmentation, warrants further study and potential clinical use.

scholarly journals Mycobacterium tuberculosis protein kinase K confers survival advantage during early infection in mice and regulates growth in culture and during persistent infection: implications for immune modulation

Microbiology ◽  
2010 ◽  
Vol 156 (9) ◽  
pp. 2829-2841 ◽  
Author(s):  
Vandana Malhotra ◽  
Lourdes T. Arteaga-Cortés ◽  
Gwendolyn Clay ◽  
Josephine E. Clark-Curtiss
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Stationary Phase ◽  
Persistent Infection ◽  
Immune Modulation ◽  
Acute Infection ◽  
Early Infection ◽  
Inhibition Of Growth ◽  
Significant Expression

Mycobacterium tuberculosis serine/threonine protein kinases (STPKs) are key regulators of growth and metabolism; however, evidence for their roles in virulence is limited. In a preliminary screen based on comparative expression between strains H37Rv and H37Ra, six STPK genes, pknD, pknG, pknH, pknJ, pknK and pknL, showed higher expression in H37Rv. In the second screen, STPK expression was analysed in H37Rv-infected human macrophages. Interestingly, significant expression of pknK was detected only at 18 h post-infection, suggesting its involvement in early infection events. We have investigated the roles of PknK in vitro and in vivo. PknK levels were induced under stationary phase and deletion of pknK resulted in increased resistance of the mutant to acidic pH, hypoxia, oxidative and stationary-phase stresses in vitro. These results, together with the increased survival of the ΔpknK strain during persistent infection in mice, reveal a role for PknK in adaptive mechanisms that slow the growth of mycobacteria. A novel finding of this study was the inhibition of growth of ΔpknK strain during acute infection in mice that correlated with the significant upregulation of tumour necrosis factor as well as the simultaneous downregulation of interleukin-12p40, interferon-γ and induced nitric oxide synthase transcripts. Finally, we provide evidence for the localization of PknK during infection and discuss its implications in pathogenesis.

scholarly journals In Vitro and In Vivo Activities of Macrolide Derivatives against Mycobacterium tuberculosis

2005 ◽  
Vol 49 (4) ◽  
pp. 1447-1454 ◽  
Author(s):  
Kanakeshwari Falzari ◽  
Zhaohai Zhu ◽  
Dahua Pan ◽  
Huiwen Liu ◽  
Poonpilas Hongmanee ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Vero Cells ◽  
Infection Model ◽  
Gram Positive Bacteria ◽  
Fold Reduction ◽  
Dependent Inhibition ◽  
Low Cytotoxicity ◽  
Aerosol Infection

ABSTRACTExisting macrolides have never shown definitive clinical efficacy in tuberculosis. Recent reports suggest that ribosome methylation is involved in macrolide resistance inMycobacterium tuberculosis, a mechanism that newer macrolides have been designed to overcome in gram-positive bacteria. Therefore, selected macrolides and ketolides (descladinose) with substitutions at positions 9, 11,12, and 6 were assessed for activity againstM. tuberculosis, and those with MICs of ≤4 μM were evaluated for cytotoxicity to Vero cells and J774A.1 macrophages. Several compounds with 9-oxime substitutions or aryl substitutions at position 6 or on 11,12 carbamates or carbazates demonstrated submicromolar MICs. For the three macrolide-ketolide pairs, macrolides demonstrated superior activity. Four compounds with low MICs and low cytotoxicity also effected significant reductions in CFU in infected macrophages. Active compounds were assessed for tolerance and the ability to reduce CFU in the lungs of BALB/c mice in an aerosol infection model. A substituted 11,12 carbazate macrolide demonstrated significant dose-dependent inhibition ofM. tuberculosisgrowth in mice, with a 10- to 20-fold reduction of CFU in lung tissue. Structure-activity relationships, some of which are unique toM. tuberculosis, suggest several synthetic directions for further improvement of antituberculosis activity. This class appears promising for yielding a clinically useful agent for tuberculosis.

Tuberkulose - Screening

2011 ◽  
Vol 68 (7) ◽  
pp. 381-387
Author(s):  
Otto Schoch
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Interferon Gamma ◽  
Wird Eine ◽  
Interferon Gamma Release Assays

Das primäre Ziel der Aktivitäten zur bevölkerungsbezogenen Tuberkulosekontrolle ist die Identifizierung von Patienten mit sputummikroskopisch positiver Lungentuberkulose. Wenn diese Patienten umgehend therapiert werden, haben sie nicht nur eine optimale Heilungschance, sondern übertragen auch den Krankheitserreger nicht weiter auf andere Personen. Das Screening, die systematische Suche nach Tuberkulose, erfolgt in der Regel radiologisch bei der Suche nach Erkrankten, während immunologische Teste bei der Suche nach einer Infektion mit Mycobacterium tuberculosis zur Anwendung kommen. Diese Infektion, die ein erhöhtes Risiko für die Entwicklung einer Tuberkulose-Erkrankung mit sich bringt, wird im Rahmen der Umgebungsuntersuchungen oder bei Hochrisikogruppen gesucht. Neben dem traditionellen in vivo Mantoux Hauttest stehen heute die neueren in vitro Blutteste, die sogenannten Interferon Gamma Release Assays (IGRA) zur Verfügung, die unter anderem den Vorteil einer höheren Spezifität mit sich bringen, weil die verwendeten Antigene der Mykobakterien-Wand beim Impfstamm Bacille Calmitte Guerin (BCG) und bei den meisten atypischen Mykobakterien nicht vorhanden sind. Zudem kann bei Immunsupprimierten dank einer mitgeführten Positivkontrolle eine Aussage über die Wahrscheinlichkeit eines falsch negativen Testresultates gemacht werden. Bei neu diagnostizierter Infektion mit Mycobacterium tuberculosis wird eine präventive Chemotherapie mit Isoniazid während 9 Monaten durchgeführt.

Kinase Targets for Mycolic Acid Biosynthesis in Mycobacterium tuberculosis

2019 ◽  
Vol 12 (1) ◽  
pp. 27-49 ◽  
Author(s):  
Shahinda S.R. Alsayed ◽  
Chau C. Beh ◽  
Neil R. Foster ◽  
Alan D. Payne ◽  
Yu Yu ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Enzymatic Activity ◽  
Bacterial Pathogen ◽  
Building Blocks ◽  
Mycolic Acid ◽  
Adaptive Responses ◽  
Mycolic Acids ◽  
Hydroxylated Fatty Acids

Background:Mycolic acids (MAs) are the characteristic, integral building blocks for the mycomembrane belonging to the insidious bacterial pathogen Mycobacterium tuberculosis (M.tb). These C60-C90 long α-alkyl-β-hydroxylated fatty acids provide protection to the tubercle bacilli against the outside threats, thus allowing its survival, virulence and resistance to the current antibacterial agents. In the post-genomic era, progress has been made towards understanding the crucial enzymatic machineries involved in the biosynthesis of MAs in M.tb. However, gaps still remain in the exact role of the phosphorylation and dephosphorylation of regulatory mechanisms within these systems. To date, a total of 11 serine-threonine protein kinases (STPKs) are found in M.tb. Most enzymes implicated in the MAs synthesis were found to be phosphorylated in vitro and/or in vivo. For instance, phosphorylation of KasA, KasB, mtFabH, InhA, MabA, and FadD32 downregulated their enzymatic activity, while phosphorylation of VirS increased its enzymatic activity. These observations suggest that the kinases and phosphatases system could play a role in M.tb adaptive responses and survival mechanisms in the human host. As the mycobacterial STPKs do not share a high sequence homology to the human’s, there have been some early drug discovery efforts towards developing potent and selective inhibitors.Objective:Recent updates to the kinases and phosphatases involved in the regulation of MAs biosynthesis will be presented in this mini-review, including their known small molecule inhibitors.Conclusion:Mycobacterial kinases and phosphatases involved in the MAs regulation may serve as a useful avenue for antitubercular therapy.

scholarly journals Overexpression of the Oral Mucosa-Specific microRNA-31 Promotes Skin Wound Closure

2019 ◽  
Vol 20 (15) ◽  
pp. 3679 ◽  
Author(s):  
Lin Chen ◽  
Alyne Simões ◽  
Zujian Chen ◽  
Yan Zhao ◽  
Xinming Wu ◽  
...  
Keyword(s):  
Wound Healing ◽  
Oral Mucosa ◽  
Wound Closure ◽  
Expression Patterns ◽  
Skin Wound ◽  
Skin Wound Healing ◽  
Specific Expression ◽  
Keratinocyte Proliferation

Wounds within the oral mucosa are known to heal more rapidly than skin wounds. Recent studies suggest that differences in the microRNAome profiles may underlie the exceptional healing that occurs in oral mucosa. Here, we test whether skin wound-healing can be accelerating by increasing the levels of oral mucosa-specific microRNAs. A panel of 57 differentially expressed high expresser microRNAs were identified based on our previously published miR-seq dataset of paired skin and oral mucosal wound-healing [Sci. Rep. (2019) 9:7160]. These microRNAs were further grouped into 5 clusters based on their expression patterns, and their differential expression was confirmed by TaqMan-based quantification of LCM-captured epithelial cells from the wound edges. Of these 5 clusters, Cluster IV (consisting of 8 microRNAs, including miR-31) is most intriguing due to its tissue-specific expression pattern and temporal changes during wound-healing. The in vitro functional assays show that ectopic transfection of miR-31 consistently enhanced keratinocyte proliferation and migration. In vivo, miR-31 mimic treatment led to a statistically significant acceleration of wound closure. Our results demonstrate that wound-healing can be enhanced in skin through the overexpression of microRNAs that are highly expressed in the privileged healing response of the oral mucosa.

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