Testing Chemical and Genetic Modulators in Mycobacterium tuberculosis Infected Cells Using Phenotypic Assays

2015 ◽  
pp. 387-411 ◽  
Author(s):  
Vincent Delorme ◽  
Ok-Ryul Song ◽  
Alain Baulard ◽  
Priscille Brodin
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Infected Cells ◽  
Genetic Modulators

scholarly journals Plasma membrane damage causes NLRP3 activation and pyroptosis during Mycobacterium tuberculosis infection

2019 ◽  
Author(s):  
Kai S. Beckwith ◽  
Marianne S. Beckwith ◽  
Sindre Ullmann ◽  
Ragnhild Sætra ◽  
Haelin Kim ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Plasma Membrane ◽  
Membrane Damage ◽  
Common Mechanism ◽  
Mycobacterium Tuberculosis Infection ◽  
Plasma Membrane Damage ◽  
Cytosolic Side ◽  
Infected Cells ◽  
Mediated Contact ◽  
Spread Of Infection

AbstractMycobacterium tuberculosis (Mtb) is a major global health problem and causes extensive cytotoxicity in patient cells and tissues. Here we define an NLRP3, caspase-1 and gasdermin D-mediated pathway to pyroptosis in human monocytes following exposure to Mtb. We demonstrate an ESX-1 mediated, contact-induced plasma membrane (PM) damage response that occurs during phagocytosis or from the cytosolic side of the PM after phagosomal rupture in Mtb infected cells. This PM injury in turn causes K+ efflux and activation of NLRP3 dependent IL-1β release and pyroptosis, facilitating the spread of Mtb to neighbouring cells. Further we reveal a dynamic interplay of pyroptosis with ESCRT-mediated PM repair. Collectively, these findings reveal a novel mechanism for pyroptosis and spread of infection acting through dual PM disturbances both during and after phagocytosis. We also highlight dual PM damage as a common mechanism utilized by other NLRP3 activators that have previously been shown to act through lysosomal damage.Graphical abstract

scholarly journals Cutting Edge: IL-1R1 Mediates Host Resistance to Mycobacterium tuberculosis by Trans-Protection of Infected Cells

2018 ◽  
Vol 201 (6) ◽  
pp. 1645-1650 ◽  
Author(s):  
Andrea C. Bohrer ◽  
Claire Tocheny ◽  
Maike Assmann ◽  
Vitaly V. Ganusov ◽  
Katrin D. Mayer–Barber
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Host Resistance ◽  
Cutting Edge ◽  
Infected Cells

scholarly journals Mycobacterium tuberculosis and Mycobacterium bovis derived proteins induce caspase-independent apoptosis in bovine macrophages

2019 ◽  
Vol 6 (1) ◽  
Author(s):  
Anaid Maciel-Rivera ◽  
Susana Flores-Villalva ◽  
Itzel Jímenez-Vázquez ◽  
Omar Catalán-Barcenas ◽  
Clara Inés Espitia-Pinzón ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Mycobacterium Bovis ◽  
Crude Protein ◽  
Tuberculosis Complex ◽  
Mycobacterium Infection ◽  
Independent Mechanism ◽  
Infected Cells ◽  
Genus Mycobacterium ◽  
Different Response ◽  
Dependent Pathway

Species of the genus Mycobacterium are capable of inducing cell apoptosis. Infected cells with M. tuberculosis undergo apoptosis through a caspase-dependent pathway. We have previously shown that whole Mycobacterium bovis cells and derived crude protein extracts were able to trigger apoptosis through a caspase independent mechanism. However, the identity of the protein or protein fractions capable of inducing apoptosis has not been determined. In this study, bovine macrophages were incubated in the presence of Mycobacterium tuberculosis recombinant proteins and Mycobacterium bovis protein extracts to identify proteins capable of inducing apoptosis. A subgroup of proteins and protein extracts from strains of the Mycobacterium tuberculosis complex were shown to induce DNA fragmentation in bovine macrophages through a caspase independent pathway. Our findings suggest that bovine macrophages may elicit a different response to mycobacterium infection than that displayed by murine or human cells.

Proteomic Analysis of Differentially Expressed Proteins in Mycobacterium Tuberculosis- Infected Macrophages

2019 ◽  
Vol 17 ◽  
Author(s):  
Shuang Tian ◽  
Dongjun Yang ◽  
Qian Long ◽  
Min Ling
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Differentially Expressed ◽  
Tandem Mass ◽  
Differentially Expressed Proteins ◽  
Cellular Processes ◽  
Tandem Mass Tag ◽  
Proliferation And Apoptosis ◽  
Liquid Chromatography Tandem Mass ◽  
Infected Cells ◽  
Cellular Components

: Mycobacterium tuberculosis (MTB) and Mycobacterium avium (MA) belong to the intracellular parasitic bacteria. To better understand how MTB survives in macrophages and the different pathogenic mechanisms of MTB and MA, the tandem mass tag (TMT) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) were used for analysis of the differentially expressed proteins in MTB-infected macrophages and MA-infected macrophages. A total of 682 proteins were found to be differentially expressed in MTB-infected cells in comparison with MA-infected cells. Gene Ontology annotation revealed the involvement of 682 differentially expressed proteins in cellular components, biological processes and molecular functions including binding, catalytic activity, metabolic processes, cellular processes, cell part, cell proliferation and apoptosis, etc. Among these, 10 proteins (O60812, P06576, O43660-2, E9PL10, O00442, M0R050, Q9H8H0, Q9BSJ8, P41240 and Q8TD57-3) were down-regulated in MTB-infected cells. We found that M0R050, O00442, Q9H8H0, O60812 and O43660 are interactive proteins which participate in a multitude of cellular RNA processing, suggesting that these five down-regulated proteins might repress the synthesis of some resistant proteins in MTB-infected cells to promote MTB survival in macrophages.

scholarly journals Identification of Autophagy-Inhibiting Factors of Mycobacterium tuberculosis by High-Throughput Loss-of-Function Screening

2020 ◽  
Vol 88 (12) ◽  
Author(s):  
Emily J. Strong ◽  
Kristen L. Jurcic Smith ◽  
Neeraj K. Saini ◽  
Tony W. Ng ◽  
Steven A. Porcelli ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Mammalian Target Of Rapamycin ◽  
Survival Rates ◽  
Host Cells ◽  
Loss Of Function ◽  
Necrosis Factor Alpha ◽  
Content Type ◽  
Targeted Deletion ◽  
Infected Cells ◽  
Factor Alpha

ABSTRACT The interaction of host cells with mycobacteria is complex and can lead to multiple outcomes ranging from bacterial clearance to progressive or latent infection. Autophagy is recognized as one component of host cell responses that has an essential role in innate and adaptive immunity to intracellular bacteria. Many microbes, including Mycobacterium tuberculosis, have evolved to evade or exploit autophagy, but the precise mechanisms and virulence factors are mostly unknown. Through a loss-of-function screening of an M. tuberculosis transposon mutant library, we identified 16 genes that contribute to autophagy inhibition, six of which encoded the PE/PPE protein family. Their expression in Mycobacterium smegmatis confirmed that these PE/PPE proteins inhibit autophagy and increase intracellular bacterial persistence or replication in infected cells. These effects were associated with increased mammalian target of rapamycin (mTOR) activity and also with decreased production of tumor necrosis factor alpha (TNF-α) and interleukin-1β (IL-1β). We also confirmed that the targeted deletion of the pe/ppe genes in M. tuberculosis resulted in enhanced autophagy and improved intracellular survival rates compared to those of wild-type bacteria in the infected macrophages. Differential expression of these PE/PPE proteins was observed in response to various stress conditions, suggesting that they may confer advantages to M. tuberculosis by modulating its interactions with host cells under various conditions. Our findings demonstrated that multiple M. tuberculosis PE/PPE proteins are involved in inhibiting autophagy during infection of host phagocytes and may provide strategic targets in developing therapeutics or vaccines against tuberculosis.

scholarly journals The Down-Regulation of Cathepsin G in THP-1 Monocytes after Infection with Mycobacterium tuberculosis Is Associated with Increased Intracellular Survival of Bacilli

2004 ◽  
Vol 72 (10) ◽  
pp. 5712-5721 ◽  
Author(s):  
Carlos A. Rivera-Marrero ◽  
Julie Stewart ◽  
William M. Shafer ◽  
Jesse Roman
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Gene Array ◽  
Antimicrobial Properties ◽  
Cathepsin G ◽  
Array Analysis ◽  
Down Regulation ◽  
Monocytic Cells ◽  
Reverse Transcription Pcr ◽  
Gene Array Analysis ◽  
Infected Cells

ABSTRACT Cathepsin G (CatG) is a serine protease found in the azurophilic granules of monocytes that is known to have antimicrobial properties, but its role in Mycobacterium tuberculosis infection is unknown. We found that M. tuberculosis infection of human THP-1 monocytic cells induced the down-regulation of CatG mRNA expression, as demonstrated by gene array analysis and reverse transcription-PCR. This was associated with a concomitant decrease in CatG protein and enzymatic activity. In contrast, the expression of lysosomal cathepsins B and D was up-regulated in infected cells. This effect was also observed when THP-1 cells were induced to differentiate into adherent macrophages by exposure to bacterial lipopolysaccharide (LPS). In agreement with this, CatG expression was null in adherent macrophages isolated from bronchoalveolar lavages and normal blood. We wanted to determine if the down-regulation of CatG would be relevant to M. tuberculosis infection. First, we found that addition of CatG to THP-1 cells prior to infection resulted in decreased bacillary viability, presumably due to extracellular killing of bacilli. However, pretreatment of cells with LPS, which decreases intracellular CatG expression, resulted in increased bacillary viability. Second, we found that CatG cationic peptides killed M. tuberculosis bacilli and were five- to sevenfold more bactericidal than full-length CatG. These observations suggest that M. tuberculosis infection of human monocytic cells results in a “cathepsin switch” with down-regulation of CatG rendering M. tuberculosis bacilli more viable. Therefore, the down-regulation of CatG in macrophages is advantageous to M. tuberculosis bacilli and possibly is an important mechanism by which M. tuberculosis is able to evade the host immune defenses.

scholarly journals Diacylated Sulfoglycolipids Are Novel Mycobacterial Antigens Stimulating CD1-restricted T Cells during Infection with Mycobacterium tuberculosis

2004 ◽  
Vol 199 (5) ◽  
pp. 649-659 ◽  
Author(s):  
Martine Gilleron ◽  
Steffen Stenger ◽  
Zaima Mazorra ◽  
Frederick Wittke ◽  
Sabrina Mariotti ◽  
...  
Keyword(s):  
T Cells ◽  
Mycobacterium Tuberculosis ◽  
T Cell ◽  
Interferon Γ ◽  
Cell Clones ◽  
Previous Contact ◽  
Infected Cells ◽  
Lipid Antigen ◽  
Mycobacterial Antigens

Mycobacterial lipids comprise a heterogeneous group of molecules capable of inducing T cell responses in humans. To identify novel antigenic lipids and increase our understanding of lipid-mediated immune responses, we established a panel of T cell clones with different lipid specificities. Using this approach we characterized a novel lipid antigen belonging to the group of diacylated sulfoglycolipids purified from Mycobacterium tuberculosis. The structure of this sulfoglycolipid was identified as 2-palmitoyl or 2-stearoyl-3-hydroxyphthioceranoyl-2′-sulfate-α-α′-d-trehalose (Ac2SGL). Its immunogenicity is dependent on the presence of the sulfate group and of the two fatty acids. Ac2SGL is mainly presented by CD1b molecules after internalization in a cellular compartment with low pH. Ac2SGL-specific T cells release interferon γ, efficiently recognize M. tuberculosis–infected cells, and kill intracellular bacteria. The presence of Ac2SGL-responsive T cells in vivo is strictly dependent on previous contact with M. tuberculosis, but independent from the development of clinically overt disease. These properties identify Ac2SGL as a promising candidate to be tested in novel vaccines against tuberculosis.

Mycobacterium tuberculosis–specific CD8+T Cells Preferentially Recognize Heavily Infected Cells

2003 ◽  
Vol 168 (11) ◽  
pp. 1346-1352 ◽  
Author(s):  
Deborah A. Lewinsohn ◽  
Amy S. Heinzel ◽  
James M. Gardner ◽  
Liqing Zhu ◽  
Mark R. Alderson ◽  
...  
Keyword(s):  
T Cells ◽  
Mycobacterium Tuberculosis ◽  
Cd8 T Cells ◽  
Infected Cells

scholarly journals Interleukin-8 Is Differentially Expressed by Human-Derived Monocytic Cell Line U937 Infected with Mycobacterium tuberculosis H37Rv and Mycobacterium marinum

2003 ◽  
Vol 71 (10) ◽  
pp. 5480-5487 ◽  
Author(s):  
Chang-Hwa Song ◽  
Ji-Sook Lee ◽  
Hwa-Jung Kim ◽  
Jeong-Kyu Park ◽  
Tae-Hyun Paik ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Cell Line ◽  
Mycobacterium Marinum ◽  
Interleukin 8 ◽  
Differentially Expressed ◽  
Northern Hybridization ◽  
U937 Cells ◽  
Monocytic Cell ◽  
Monocytic Cell Line ◽  
Infected Cells

ABSTRACT Although Mycobacterium marinum is closely related to Mycobacterium tuberculosis H37Rv genomically, the clinical outcome in humans is quite different for M. marinum and M. tuberculosis infections. We investigated possible factors in the host macrophages for determining differential pathological responses to M. tuberculosis and M. marinum using an in vitro model of mycobacterial infection. Using suppression-subtractive hybridization, we identified 12 differentially expressed genes in the human monocytic cell line U937 infected with M. tuberculosis and M. marinum. Of those genes, the most frequently recovered transcript encoded interleukin-8 (IL-8). Northern hybridization revealed that IL-8 mRNA was highly upregulated in M. tuberculosis-infected U937 cells compared with M. marinum-infected cells. In addition, enzyme-linked immunosorbent assay showed that IL-8 protein secretion was significantly elevated in M. tuberculosis-infected U937 cells, human primary monocytes, and monocyte-derived macrophages compared with that in M. marinum-infected cells. The depressed IL-8 expression was unique in M. marinum-infected cells compared with cells infected with other strains of mycobacteria, including M. tuberculosis H37Ra, Mycobacterium bovis BCG, or Mycobacterium smegmatis. When the expression of NF-κB was assessed in mycobacterium-infected U937 cells, IκBα proteins were significantly degraded in M. tuberculosis-infected cells compared with M. marinum-infected cells. Collectively, these results suggest that differential IL-8 expression in human macrophages infected with M. tuberculosis and M. marinum may be critically associated with distinct host responses in tuberculosis. Additionally, our data indicate that differential signal transduction pathways may underlie the distinct patterns of IL-8 secretion in cells infected by the two mycobacteria.

Sign in / Sign up

Export Citation Format

Share Document