scholarly journals Rv0180c contributes to Mycobacterium tuberculosis cell shape and to infectivity in mice and macrophages

2021 ◽  
Vol 17 (11) ◽  
pp. e1010020
Author(s):  
Delphine Payros ◽  
Henar Alonso ◽  
Wladimir Malaga ◽  
Arnaud Volle ◽  
Serge Mazères ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Cell Shape ◽  
Cell Envelope ◽  
Membrane Receptors ◽  
Host Cells ◽  
Wild Type ◽  
Genes Encoding ◽  
The Difference ◽  
Complement Receptor 3 ◽  
New Perspective

Mycobacterium tuberculosis, the main causative agent of human tuberculosis, is transmitted from person to person via small droplets containing very few bacteria. Optimizing the chance to seed in the lungs is therefore a major adaptation to favor survival and dissemination in the human population. Here we used TnSeq to identify genes important for the early events leading to bacterial seeding in the lungs. Beside several genes encoding known virulence factors, we found three new candidates not previously described: rv0180c, rv1779c and rv1592c. We focused on the gene, rv0180c, of unknown function. First, we found that deletion of rv0180c in M. tuberculosis substantially reduced the initiation of infection in the lungs of mice. Next, we established that Rv0180c enhances entry into macrophages through the use of complement-receptor 3 (CR3), a major phagocytic receptor for M. tuberculosis. Silencing CR3 or blocking the CR3 lectin site abolished the difference in entry between the wild-type parental strain and the Δrv0180c::km mutant. However, we detected no difference in the production of both CR3-known carbohydrate ligands (glucan, arabinomannan, mannan), CR3-modulating lipids (phthiocerol dimycocerosate), or proteins in the capsule of the Δrv0180c::km mutant in comparison to the wild-type or complemented strains. By contrast, we established that Rv0180c contributes to the functionality of the bacterial cell envelope regarding resistance to toxic molecule attack and cell shape. This alteration of bacterial shape could impair the engagement of membrane receptors that M. tuberculosis uses to invade host cells, and open a new perspective on the modulation of bacterial infectivity.

scholarly journals Identification of Genes Encoding Exported Mycobacterium tuberculosis Proteins Using a Tn552′phoA In Vitro Transposition System

2000 ◽  
Vol 182 (10) ◽  
pp. 2732-2740 ◽  
Author(s):  
Miriam Braunstein ◽  
Thomas J. Griffin ◽  
Jordan I. Kriakov ◽  
Sarah T. Friedman ◽  
Nigel D. F. Grindley ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Protective Immunity ◽  
Cell Envelope ◽  
Open Reading Frames ◽  
Signal Peptides ◽  
Genomic Libraries ◽  
Target Dna ◽  
Genes Encoding ◽  
Associated Proteins

ABSTRACT Secreted and cell envelope-associated proteins are important to both Mycobacterium tuberculosis pathogenesis and the generation of protective immunity to M. tuberculosis. We used an in vitro Tn552′phoA transposition system to identify exported proteins of M. tuberculosis. The system is simple and efficient, and the transposon inserts randomly into target DNA. M. tuberculosis genomic libraries were targeted with Tn552′phoA transposons, and these libraries were screened in M. smegmatis for active PhoA translational fusions. Thirty-two different M. tuberculosis open reading frames were identified; eight contain standard signal peptides, six contain lipoprotein signal peptides, and seventeen contain one or more transmembrane domains. Four of these proteins had not yet been assigned as exported proteins in the M. tuberculosisdatabases. This collection of exported proteins includes factors that are known to participate in the immune response of M. tuberculosis and proteins with homologies, suggesting a role in pathogenesis. Nine of the proteins appear to be unique to mycobacteria and represent promising candidates for factors that participate in protective immunity and virulence. This technology of creating comprehensive fusion libraries should be applicable to other organisms.

scholarly journals Structural Basis and Functional Consequence of Helicobacter pylori CagA Multimerization in Cells

2006 ◽  
Vol 281 (43) ◽  
pp. 32344-32352 ◽  
Author(s):  
Shumei Ren ◽  
Hideaki Higashi ◽  
Huaisheng Lu ◽  
Takeshi Azuma ◽  
Masanori Hatakeyama
Keyword(s):  
Helicobacter Pylori ◽  
Tyrosine Phosphorylation ◽  
Cell Shape ◽  
Host Cells ◽  
Structural Basis ◽  
Sequence Motif ◽  
Wild Type ◽  
Src Homology 2 ◽  
Src Homology ◽  
In Cells

Helicobacter pylori cagA-positive strains are associated with gastric adenocarcinoma. The cagA gene product CagA is delivered into gastric epithelial cells where it localizes to the plasma membrane and undergoes tyrosine phosphorylation at the EPIYA-repeat region, which contains the EPIYA-A segment, EPIYA-B segment, and Western CagA-specific EPIYA-C or East Asian CagA-specific EPIYA-D segment. In host cells, CagA specifically binds to and deregulates SHP-2 phosphatase via the tyrosine-phosphorylated EPIYA-C or EPIYA-D segment, thereby inducing an elongated cell shape known as the hummingbird phenotype. In this study, we found that CagA multimerizes in cells in a manner independent of its tyrosine phosphorylation. Using a series of CagA mutants, we identified a conserved amino acid sequence motif (FPLXRXXXVXDLSKVG), which mediates CagA multimerization, within the EPIYA-C segment as well as in a sequence that located immediately downstream of the EPIYA-C or EPIYA-D segment. We also found that a phosphorylation-resistant CagA, which multimerizes but cannot bind SHP-2, inhibits the wild-type CagA-SHP-2 complex formation and abolishes induction of the hummingbird phenotype. Thus, SHP-2 binds to a preformed and tyrosinephosphorylated CagA multimer via its two Src homology 2 domains. These results, in turn, indicate that CagA multimerization is a prerequisite for CagA-SHP-2 interaction and subsequent deregulation of SHP-2. The present work raises the possibility that inhibition of CagA multimerization abolishes pathophysiological activities of CagA that promote gastric carcinogenesis.

scholarly journals Lipoprotein Processing Is Essential for Resistance of Mycobacterium tuberculosis to Malachite Green

2009 ◽  
Vol 53 (9) ◽  
pp. 3799-3802 ◽  
Author(s):  
Niaz Banaei ◽  
Eleanor Z. Kincaid ◽  
S.-Y. Grace Lin ◽  
Edward Desmond ◽  
William R. Jacobs ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Malachite Green ◽  
Cell Envelope ◽  
Sodium Dodecyl ◽  
Signal Peptidase ◽  
Wild Type ◽  
First Line ◽  
Green Is ◽  
Antituberculosis Drugs ◽  
Forming Efficiency

ABSTRACT Malachite green, a synthetic antimicrobial dye, has been used for over 50 years in mycobacterial culture medium to inhibit the growth of contaminants. The molecular basis of mycobacterial resistance to malachite green is unknown, although the presence of malachite green-reducing enzymes in the cell envelope has been suggested. The objective of this study was to investigate the role of lipoproteins in resistance of Mycobacterium tuberculosis to malachite green. The replication of an M. tuberculosis lipoprotein signal peptidase II (lspA) mutant (ΔlspA::lspA mut) on Middlebrook agar with and without 1 mg/liter malachite green was investigated. The lspA mutant was also compared with wild-type M. tuberculosis in the decolorization rate of malachite green and sensitivity to sodium dodecyl sulfate (SDS) detergent and first-line antituberculosis drugs. The lspA mutant has a 104-fold reduction in CFU-forming efficiency on Middlebrook agar with malachite green. Malachite green is decolorized faster in the presence of the lspA mutant than wild-type bacteria. The lspA mutant is hypersensitive to SDS detergent and shows increased sensitivity to first-line antituberculosis drugs. In summary, lipoprotein processing by LspA is essential for resistance of M. tuberculosis to malachite green. A cell wall permeability defect is likely responsible for the hypersensitivity of lspA mutant to malachite green.

scholarly journals WhiB5, a Transcriptional Regulator That Contributes to Mycobacterium tuberculosis Virulence and Reactivation

2012 ◽  
Vol 80 (9) ◽  
pp. 3132-3144 ◽  
Author(s):  
Stefano Casonato ◽  
Axel Cervantes Sánchez ◽  
Hirohito Haruki ◽  
Monica Rengifo González ◽  
Roberta Provvedi ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Chronic Infection ◽  
Null Mutant ◽  
Wild Type ◽  
Secretion Systems ◽  
Content Type ◽  
Type Vii Secretion ◽  
Expression Of Genes ◽  
Genes Encoding

ABSTRACTThe proteins belonging to the WhiB superfamily are small global transcriptional regulators typical of actinomycetes. In this paper, we characterize the role of WhiB5, aMycobacterium tuberculosisprotein belonging to this superfamily. A null mutant was constructed inM. tuberculosisH37Rv and was shown to be attenuated during both progressive and chronic mouse infections. Mice infected with the mutant had smaller bacillary burdens in the lungs but a larger inflammatory response, suggesting a role of WhiB5 in immunomodulation. Most interestingly, thewhiB5mutant was not able to resume growth after reactivation from chronic infection, suggesting that WhiB5 controls the expression of genes involved in this process. The mutant was also more sensitive than the wild-type parental strain toS-nitrosoglutathione (GSNO) and was less metabolically active following prolonged starvation, underscoring the importance of GSNO and starvation in development and maintenance of chronic infection. DNA microarray analysis identified 58 genes whose expression is influenced by WhiB5, includingsigM, encoding an alternative sigma factor, and genes encoding the constituents of two type VII secretion systems, namely, ESX-2 and ESX-4.

scholarly journals The Glycan-Rich Outer Layer of the Cell Wall of Mycobacterium tuberculosis Acts as an Antiphagocytic Capsule Limiting the Association of the Bacterium with Macrophages

2004 ◽  
Vol 72 (10) ◽  
pp. 5676-5686 ◽  
Author(s):  
Richard W. Stokes ◽  
Raymond Norris-Jones ◽  
Donald E. Brooks ◽  
Terry J. Beveridge ◽  
Dan Doxsee ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Surface Hydrophobicity ◽  
Host Cells ◽  
Microbial Pathogens ◽  
Mammalian Host ◽  
Human Macrophage ◽  
Bacterial Surface ◽  
Important Virulence Factor ◽  
Complement Receptor 3 ◽  
Macrophage Populations

ABSTRACT Mycobacterium tuberculosis, the causative agent of tuberculosis, is a facultative intracellular pathogen that infects macrophages and other host cells. We show that sonication of M. tuberculosis results in the removal of material from the surface capsule-like layer of the bacteria, resulting in an enhanced propensity of the bacteria to bind to macrophages. This effect is observed with disparate murine and human macrophage populations though, interestingly, not with freshly explanted alveolar macrophages. Enhanced binding to macrophages following sonication is significantly greater within members of the M. tuberculosis family (pathogens) than within the Mycobacterium avium complex (opportunistic pathogens) or for Mycobacterium smegmatis (saprophyte). Sonication does not affect the viability or the surface hydrophobicity of M. tuberculosis but does result in changes in surface charge and in the binding of mannose-specific lectins to the bacterial surface. The increased binding of sonicated M. tuberculosis was not mediated through complement receptor 3. These results provide evidence that the surface capsule on members of the M. tuberculosis family may be an important virulence factor involved in the survival of M. tuberculosis in the mammalian host. They also question the view that M. tuberculosis is readily ingested by any macrophage it encounters and support the contention that M. tuberculosis, like many other microbial pathogens, has an antiphagocytic capsule that limits and controls the interaction of the bacterium with macrophages.

scholarly journals The cGAS/STING Pathway Is Important for Dendritic Cell Activation but Is Not Essential to Induce Protective Immunity against Mycobacterium tuberculosis Infection

2018 ◽  
Vol 10 (3) ◽  
pp. 239-252 ◽  
Author(s):  
Fabio V. Marinho ◽  
Sulayman Benmerzoug ◽  
Stephanie  Rose ◽  
Priscila C. Campos ◽  
João T. Marques ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Cell Activation ◽  
Body Weight Gain ◽  
Host Cells ◽  
Health Concern ◽  
Wild Type ◽  
Activation Markers ◽  
Sting Pathway

Mycobacterium tuberculosis (Mtb) infection remains a major public health concern. The STING (stimulator of interferon genes) pathway contributes to the cytosolic surveillance of host cells. Most studies on the role of STING activation in Mtb infection have focused on macrophages. Moreover, a detailed investigation of the role of STING during Mtb infection in vivo is required. Here, we deciphered the involvement of STING in the activation of dendritic cells (DCs) and the host response to Mtb infection in vivo. In DCs, this adaptor molecule was important for Ifn-β expression and IL-12 production as well as for the surface expression of the activation markers CD40 and CD86. We also documented that Mtb DNA induces STING activation in murine fibroblasts. In vivo Mtb aerogenic infection induced the upregulation of the STING and cGAS (cyclic GMP-AMP synthase) genes, and Ifn-β pulmonary expression was dependent on both sensors. However, mice deficient for STING or cGAS presented a similar outcome to wild-type controls, with no major alterations in body weight gain, bacterial burden, or survival. Lung inflammation, proinflammatory cytokine production, and inflammatory cell recruitment were similar in STING- and cGAS-deficient mice compared to wild-type controls. In summary, although the STING pathway seems to be crucial for DC activation during Mtb infection, it is dispensable for host protection in vivo.

scholarly journals Mycobacterium tuberculosis controls host innate immune activation through cyclopropane modification of a glycolipid effector molecule

2005 ◽  
Vol 201 (4) ◽  
pp. 535-543 ◽  
Author(s):  
Vivek Rao ◽  
Nagatoshi Fujiwara ◽  
Steven A. Porcelli ◽  
Michael S. Glickman
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Fine Structure ◽  
Immune Activation ◽  
Cell Envelope ◽  
Granulomatous Inflammation ◽  
Host Cells ◽  
Specific Cell ◽  
Health Crisis ◽  
Trehalose Dimycolate ◽  
Necessary And Sufficient

Mycobacterium tuberculosis (Mtb) infection remains a global health crisis. Recent genetic evidence implicates specific cell envelope lipids in Mtb pathogenesis, but it is unclear whether these cell envelope compounds affect pathogenesis through a structural role in the cell wall or as pathogenesis effectors that interact directly with host cells. Here we show that cyclopropane modification of the Mtb cell envelope glycolipid trehalose dimycolate (TDM) is critical for Mtb growth during the first week of infection in mice. In addition, TDM modification by the cyclopropane synthase pcaA was both necessary and sufficient for proinflammatory activation of macrophages during early infection. Purified TDM isolated from a cyclopropane-deficient pcaA mutant was hypoinflammatory for macrophages and induced less severe granulomatous inflammation in mice, demonstrating that the fine structure of this glycolipid was critical to its proinflammatory activity. These results established the fine structure of lipids contained in the Mtb cell envelope as direct effectors of pathogenesis and identified temporal control of host immune activation through cyclopropane modification of TDM as a critical pathogenic strategy of Mtb.

scholarly journals Potential Plasticity of the Mannoprotein Repertoire Associated to Mycobacterium tuberculosis Virulence Unveiled by Mass Spectrometry-Based Glycoproteomics

Molecules ◽  
2020 ◽  
Vol 25 (10) ◽  
pp. 2348
Author(s):  
Laure Tonini ◽  
Bashir Sadet ◽  
Alexandre Stella ◽  
David Bouyssié ◽  
Jérôme Nigou ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Mycobacterium Tuberculosis ◽  
Large Scale ◽  
Therapeutic Targets ◽  
Multidrug Resistant ◽  
Host Cells ◽  
Bacterial Protein ◽  
Wild Type ◽  
In The Wild ◽  
Resistant Strains

To date, Mycobacterium tuberculosis (Mtb) remains the world’s greatest infectious killer. The rise of multidrug-resistant strains stresses the need to identify new therapeutic targets to fight the epidemic. We previously demonstrated that bacterial protein-O-mannosylation is crucial for Mtb infectiousness, renewing the interest of the bacterial-secreted mannoproteins as potential drug-targetable virulence factors. The difficulty of inventorying the mannoprotein repertoire expressed by Mtb led us to design a stringent multi-step workflow for the reliable identification of glycosylated peptides by large-scale mass spectrometry-based proteomics. Applied to the differential analyses of glycoproteins secreted by the wild-type Mtb strain—and by its derived mutant invalidated for the protein-O-mannosylating enzyme PMTub—this approach led to the identification of not only most already known mannoproteins, but also of yet-unknown mannosylated proteins. In addition, analysis of the glycoproteome expressed by the isogenic recombinant Mtb strain overexpressing the PMTub gene revealed an unexpected mannosylation of proteins, with predicted or demonstrated functions in Mtb growth and interaction with the host cell. Since in parallel, a transient increased expression of the PMTub gene has been observed in the wild-type bacilli when infecting macrophages, our results strongly suggest that the Mtb mannoproteome may undergo adaptive regulation during infection of the host cells. Overall, our results provide deeper insights into the complexity of the repertoire of mannosylated proteins expressed by Mtb, and open the way to novel opportunities to search for still-unexploited potential therapeutic targets.

scholarly journals Deviant Expression of Rab5 on Phagosomes Containing the Intracellular Pathogens Mycobacterium tuberculosis andLegionella pneumophila Is Associated with Altered Phagosomal Fate

2000 ◽  
Vol 68 (5) ◽  
pp. 2671-2684 ◽  
Author(s):  
Daniel L. Clemens ◽  
Bai-Yu Lee ◽  
Marcus A. Horwitz
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Hela Cells ◽  
Legionella Pneumophila ◽  
Critical Role ◽  
Small Gtpase ◽  
Biologically Active ◽  
Host Cells ◽  
Molecular Characteristics ◽  
Wild Type ◽  
Immunogold Staining

ABSTRACT The intracellular human pathogens Legionella pneumophila and Mycobacterium tuberculosis reside in altered phagosomes that do not fuse with lysosomes and are only mildly acidified. The L. pneumophila phagosome exists completely outside the endolysosomal pathway, and the M. tuberculosisphagosome displays a maturational arrest at an early endosomal stage along this pathway. Rab5 plays a critical role in regulating membrane trafficking involving endosomes and phagosomes. To determine whether an alteration in the function or delivery of Rab5 could play a role in the aberrant development of L. pneumophila and M. tuberculosis phagosomes, we have examined the distribution of the small GTPase, Rab5c, in infected HeLa cells overexpressing Rab5c. Both pathogens formed phagosomes in HeLa cells with molecular characteristics similar to their phagosomes in human macrophages and multiplied in these host cells. Phagosomes containing virulent wild-type L. pneumophila never acquired immunogold staining for Rab5c, whereas phagosomes containing an avirulent mutant L. pneumophila (which ultimately fused with lysosomes) transiently acquired staining for Rab5c after phagocytosis. In contrast, M. tuberculosis phagosomes exhibited abundant staining for Rab5c throughout its life cycle. To verify that the overexpressed, recombinant Rab5c observed on the bacterial phagosomes was biologically active, we examined the phagosomes in HeLa cells expressing Rab5c Q79L, a fusion-promoting mutant. Such HeLa cells formed giant vacuoles, and after incubation with various particles, the giant vacuoles acquired large numbers of latex beads, M. tuberculosis, and avirulent L. pneumophila but not wild-type L. pneumophila, which consistently remained in tight phagosomes that did not fuse with the giant vacuoles. These results indicate that whereas Rab5 is absent from wild-type L. pneumophilaphagosomes, functional Rab5 persists on M. tuberculosisphagosomes. The absence of Rab5 on the L. pneumophilaphagosome may underlie its lack of interaction with endocytic compartments. The persistence of functional Rab5 on the M. tuberculosis phagosomes may enable the phagosome to retard its own maturation at an early endosomal stage.

scholarly journals IRG1 controls immunometabolic host response and restricts intracellular Mycobacterium tuberculosis infection

2019 ◽  
Author(s):  
Eik Hoffmann ◽  
Arnaud Machelart ◽  
Imène Belhaouane ◽  
Nathalie Deboosere ◽  
Anne-Marie Pauwels ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Host Response ◽  
Immune Cell ◽  
Isocitrate Lyase ◽  
Metabolic Reprogramming ◽  
Host Cells ◽  
Wild Type ◽  
Term Survival ◽  
Major Player ◽  
Deficient Mice

AbstractMycobacterium tuberculosis (Mtb), the pathogen causing human tuberculosis, has evolved multiple strategies to successfully prevent clearance by immune cells and to establish dissemination and long-term survival in the host. The modulation of host immunity to maximize pathogen elimination while minimizing inflammation-mediated tissue damage may provide another tool to fight drug-resistant Mtb strains. Metabolic reprogramming of immune cell populations can dramatically influence the outcome of immune responses and modulate antimicrobial properties of infected host cells, nicely demonstrating that metabolites are tightly linked to immune cell effector functions. One important endogenous metabolite of the Krebs cycle is itaconate, which has potent bactericidal activity by inhibiting isocitrate lyase and the glyoxylate shunt within prokaryotes including mycobacteria. Recent findings show that itaconate and the catalytic enzyme responsible for its generation in mammalian cells, i.e. IRG1 (immune-responsive gene 1), also modify inflammatory signaling of infected cells enhancing host defense pathways.Here, we demonstrate that IRG1 is recruited to Mtb-containing phagosomes and that it influences the host response controlling Mtb infection. While IRG1 deficiency does not affect uptake of Mtb by macrophages and dendritic cells (DCs) in vitro, it increases the intracellular replication of Mtb. Concomitantly, in comparison to wild type cells, IRG1-deficient macrophages and DCs have increased levels of lipid droplets, a correlate of inflammation. These intracellular organelles store triacylglycerol and phospholipids that are hijacked by Mtb as reservoir of host nutrients. Exposure of IRG1-deficient mice to M. bovis BCG via the intranasal route induced neither lethality nor severe lung immunopathology, while IRG1-deficient mice were highly susceptible to Mtb infection resulting in animal death three weeks post-infection linked to exacerbated inflammation and high mycobacterial burden. The lungs of infected IRG1-deficient mice displayed large areas of necrotizing granulomatous inflammation and neutrophil infiltration, accompanied by reduced levels of B and T lymphocytes and increased levels of alveolar and interstitial macrophage populations, compared to their wild type counterparts. Therefore, our findings demonstrate that IRG1 is a major player in controlling the acute phase of Mtb infection with a specific effect on pathogenic mycobacteria.

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