scholarly journals Mycobacterium bovis Requires P27 (LprG) To Arrest Phagosome Maturation and Replicate within Bovine Macrophages

2016 ◽  
Vol 85 (3) ◽  
Author(s):  
Cristina Lourdes Vázquez ◽  
María Verónica Bianco ◽  
Federico Carlos Blanco ◽  
Marina Andrea Forrellad ◽  
Maximiliano Gabriel Gutierrez ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Host Cell ◽  
Mutant Strain ◽  
Mycobacterium Bovis ◽  
Phagosome Maturation ◽  
Wild Type ◽  
Content Type ◽  
Bovine Macrophage ◽  
Wild Type Phenotype ◽  
First Time

ABSTRACT Mycobacterium bovis causes tuberculosis in a wide variety of mammals, with strong tropism for cattle and eventually humans. P27, also called LprG, is among the proteins involved in the mechanisms of the virulence and persistence of M. bovis and Mycobacterium tuberculosis. Here, we describe a novel function of P27 in the interaction of M. bovis with its natural host cell, the bovine macrophage. We found that a deletion in the p27-p55 operon impairs the replication of M. bovis in bovine macrophages. Importantly, we show for the first time that M. bovis arrests phagosome maturation in a process that depends on P27. This effect is P27 specific since complementation with wild-type p27 but not p55 fully restored the wild-type phenotype of the mutant strain; this indicates that P55 plays no important role during the early events of M. bovis infection. In addition, we also showed that the presence of P27 from M. smegmatis decreases the association of LAMP-3 with bead phagosomes, indicating that P27 itself blocks phagosome-lysosome fusion by modulating the traffic machinery in the cell host.

scholarly journals Activation of 2,4-Diaminoquinazoline in Mycobacterium tuberculosis by Rv3161c, a Putative Dioxygenase

2018 ◽  
Vol 63 (1) ◽  
Author(s):  
Eduard Melief ◽  
Shilah A. Bonnett ◽  
Edison S. Zuniga ◽  
Tanya Parish
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Mutant Strain ◽  
Type Strain ◽  
Wild Type Strain ◽  
Type A ◽  
Wild Type ◽  
Metabolite Analysis ◽  
Content Type ◽  
Data Support ◽  
In The Wild

ABSTRACT The diaminoquinazoline series has good potency against Mycobacterium tuberculosis. Resistant isolates have mutations in Rv3161c, a putative dioxygenase. We carried out metabolite analysis on a wild-type strain and an Rv3161c mutant strain after exposure to a diaminoquinazoline. The parental compound was found in intracellular extracts from the mutant but not the wild type. A metabolite consistent with a monohydroxylated form was identified in the wild type. These data support the hypothesis that Rv3161c metabolizes diaminoquinazolines in M. tuberculosis.

scholarly journals Identification of the Missing trans-Acting Enoyl Reductase Required for Phthiocerol Dimycocerosate and Phenolglycolipid Biosynthesis in Mycobacterium tuberculosis

2007 ◽  
Vol 189 (13) ◽  
pp. 4597-4602 ◽  
Author(s):  
Roxane Siméone ◽  
Patricia Constant ◽  
Christophe Guilhot ◽  
Mamadou Daffé ◽  
Christian Chalut
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Mutant Strain ◽  
Mycobacterium Bovis ◽  
Reductase Activity ◽  
Polyketide Synthases ◽  
Type I ◽  
Wild Type ◽  
Enoyl Reductase ◽  
Phthiocerol Dimycocerosates ◽  
Activity Domain

ABSTRACT Phthiocerol dimycocerosates (DIM) and phenolglycolipids (PGL) are functionally important surface-exposed lipids of Mycobacterium tuberculosis. Their biosynthesis involves the products of several genes clustered in a 70-kb region of the M. tuberculosis chromosome. Among these products is PpsD, one of the modular type I polyketide synthases responsible for the synthesis of the lipid core common to DIM and PGL. Bioinformatic analyses have suggested that this protein lacks a functional enoyl reductase activity domain required for the synthesis of these lipids. We have identified a gene, Rv2953, that putatively encodes an enoyl reductase. Mutation in Rv2953 prevents conventional DIM formation and leads to the accumulation of a novel DIM-like product. This product is unsaturated between C-4 and C-5 of phthiocerol. Consistently, complementation of the mutant with a functional pks15/1 gene from Mycobacterium bovis BCG resulted in the accumulation of an unsaturated PGL-like substance. When an intact Rv2953 gene was reintroduced into the mutant strain, the phenotype reverted to the wild type. These findings indicate that Rv2953 encodes a trans-acting enoyl reductase that acts with PpsD in phthiocerol and phenolphthiocerol biosynthesis.

scholarly journals The Mycobacterium tuberculosis SecA2 System Subverts Phagosome Maturation To Promote Growth in Macrophages

2012 ◽  
Vol 80 (3) ◽  
pp. 996-1006 ◽  
Author(s):  
Jonathan Tabb Sullivan ◽  
Ellen F. Young ◽  
Jessica R. McCann ◽  
Miriam Braunstein
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Protein Export ◽  
Growth Defect ◽  
Phagosome Maturation ◽  
Wild Type ◽  
Intracellular Growth ◽  
Maturation Arrest ◽  
Content Type ◽  
Late Endosomes ◽  
Export System

The ability ofMycobacterium tuberculosisto grow in macrophages is critical to the virulence of this important pathogen. One wayM. tuberculosisis thought to maintain a hospitable niche in macrophages is by arresting the normal process of phagosomes maturing into acidified phagolysosomes. The process of phagosome maturation arrest byM. tuberculosisis not fully understood, and there has remained a need to firmly establish a requirement for phagosome maturation arrest forM. tuberculosisgrowth in macrophages. Other intracellular pathogens that control the phagosomal environment use specialized protein export systems to deliver effectors of phagosome trafficking to the host cell. InM. tuberculosis, the accessory SecA2 system is a specialized protein export system that is required for intracellular growth in macrophages. In studying the importance of the SecA2 system in macrophages, we discovered that SecA2 is required for phagosome maturation arrest. Shortly after infection, phagosomes containing a ΔsecA2mutant ofM. tuberculosiswere more acidified and showed greater association with markers of late endosomes than phagosomes containing wild-typeM. tuberculosis. We further showed that inhibitors of phagosome acidification rescued the intracellular growth defect of the ΔsecA2mutant, which demonstrated that the phagosome maturation arrest defect of the ΔsecA2mutant is responsible for the intracellular growth defect. This study demonstrates the importance of phagosome maturation arrest forM. tuberculosisgrowth in macrophages, and it suggests there are effectors of phagosome maturation that are exported into the host environment by the accessory SecA2 system.

scholarly journals A Nonribosomal Peptide Synthase Gene Driving Virulence in Mycobacterium tuberculosis

mSphere ◽  
2018 ◽  
Vol 3 (5) ◽  
Author(s):  
Kiranmai Bhatt ◽  
Henrique Machado ◽  
Nuno S. Osório ◽  
Jeremy Sousa ◽  
Filipa Cardoso ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Mutant Strain ◽  
Infectious Agent ◽  
Initial Growth ◽  
Wild Type ◽  
Nonribosomal Peptide ◽  
Content Type ◽  
Ifn Γ ◽  
Slow Growing

ABSTRACT Nonribosomal peptide synthases produce short peptides in a manner that is distinct from classical mRNA-dependent ribosome-mediated translation. The Mycobacterium tuberculosis genome harbors a nonribosomal peptide synthase gene, nrp, which is part of a gene cluster proposed to be involved in the biosynthesis of isonitrile lipopeptides. Orthologous clusters are found in other slow-growing pathogenic mycobacteria and actinomycetes. To probe the role of the nrp gene in infection, we generated an nrp deletion mutant in M. tuberculosis H37Rv and tested its virulence in immunocompetent (C57BL/6) mice. The nrp mutant strain displayed lower initial growth rates in the lungs and a defective dissemination to the spleens of infected mice. Mice infected with the mutant strain also survived for twice as long as those infected with wild-type M. tuberculosis and, remarkably, showed subdued pathology, despite similar bacterial loads at later stages of infection. The differences in the course of infection between wild-type and nrp mutant strains were accompanied by distinct dynamics of the immune response. Most strikingly, the nrp mutant was highly attenuated in immunodeficient (SCID-, recombination activating 2 [RAG2]-, and gamma interferon [IFN-γ]-deficient) mice, suggesting that macrophages control the nrp mutant more efficiently than they control the wild-type strain. However, in the presence of IFN-γ, both strains were equally controlled. We propose that the nrp gene and its associated cluster are drivers of virulence during the early stages of infection. IMPORTANCE Over 10 million people developed tuberculosis (TB) in 2016, and over 1.8 million individuals succumbed to the disease. These numbers make TB the ninth leading cause of death worldwide and the leading cause from a single infectious agent. Therefore, finding novel therapeutic targets in Mycobacterium tuberculosis, the pathogen that causes most cases of human TB, is critical. In this study, we reveal a novel virulence factor in M. tuberculosis, the nrp gene. The lack of nrp highly attenuates the course of M. tuberculosis infection in the mouse model, which is particularly relevant in immune-deficient hosts. This is very relevant as TB is particularly incident in immune-suppressed individuals, such as HIV patients.

scholarly journals Mutations in fbiD (Rv2983) as a Novel Determinant of Resistance to Pretomanid and Delamanid in Mycobacterium tuberculosis

2020 ◽  
Vol 65 (1) ◽  
pp. e01948-20
Author(s):  
Dalin Rifat ◽  
Si-Yang Li ◽  
Thomas Ioerger ◽  
Keshav Shah ◽  
Jean-Philippe Lanoix ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Clinical Evidence ◽  
Clinical Samples ◽  
Loss Of Function ◽  
Wild Type ◽  
Content Type ◽  
Solid Media ◽  
High Level ◽  
Level Resistance

ABSTRACTThe nitroimidazole prodrugs delamanid and pretomanid comprise one of only two new antimicrobial classes approved to treat tuberculosis (TB) in 50 years. Prior in vitro studies suggest a relatively low barrier to nitroimidazole resistance in Mycobacterium tuberculosis, but clinical evidence is limited to date. We selected pretomanid-resistant M. tuberculosis mutants in two mouse models of TB using a range of pretomanid doses. The frequency of spontaneous resistance was approximately 10−5 CFU. Whole-genome sequencing of 161 resistant isolates from 47 mice revealed 99 unique mutations, of which 91% occurred in 1 of 5 genes previously associated with nitroimidazole activation and resistance, namely, fbiC (56%), fbiA (15%), ddn (12%), fgd (4%), and fbiB (4%). Nearly all mutations were unique to a single mouse and not previously identified. The remaining 9% of resistant mutants harbored mutations in Rv2983 (fbiD), a gene not previously associated with nitroimidazole resistance but recently shown to be a guanylyltransferase necessary for cofactor F420 synthesis. Most mutants exhibited high-level resistance to pretomanid and delamanid, although Rv2983 and fbiB mutants exhibited high-level pretomanid resistance but relatively small changes in delamanid susceptibility. Complementing an Rv2983 mutant with wild-type Rv2983 restored susceptibility to pretomanid and delamanid. By quantifying intracellular F420 and its precursor Fo in overexpressing and loss-of-function mutants, we provide further evidence that Rv2983 is necessary for F420 biosynthesis. Finally, Rv2983 mutants and other F420H2-deficient mutants displayed hypersusceptibility to some antibiotics and to concentrations of malachite green found in solid media used to isolate and propagate mycobacteria from clinical samples.

scholarly journals Establishing Virulence Associated Polyphosphate Kinase 2 as a drug target for Mycobacterium tuberculosis

2016 ◽  
Vol 6 (1) ◽  
Author(s):  
Mamta Singh ◽  
Prabhakar Tiwari ◽  
Garima Arora ◽  
Sakshi Agarwal ◽  
Saqib Kidwai ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Mutant Strain ◽  
Guinea Pigs ◽  
High Throughput Screening ◽  
Drug Target ◽  
Wild Type Strain ◽  
Wild Type ◽  
Inorganic Polyphosphate ◽  
Phosphate Donor ◽  
Microbial Stress

Abstract Inorganic polyphosphate (PolyP) plays an essential role in microbial stress adaptation, virulence and drug tolerance. The genome of Mycobacterium tuberculosis encodes for two polyphosphate kinases (PPK-1, Rv2984 and PPK-2, Rv3232c) and polyphosphatases (ppx-1, Rv0496 and ppx-2, Rv1026) for maintenance of intracellular PolyP levels. Microbial polyphosphate kinases constitute a molecular mechanism, whereby microorganisms utilize PolyP as phosphate donor for synthesis of ATP. In the present study we have constructed ppk-2 mutant strain of M. tuberculosis and demonstrate that PPK-2 enzyme contributes to its ability to cause disease in guinea pigs. We observed that ppk-2 mutant strain infected guinea pigs had significantly reduced bacterial loads and tissue pathology in comparison to wild type infected guinea pigs at later stages of infection. We also report that in comparison to the wild type strain, ppk-2 mutant strain was more tolerant to isoniazid and impaired for survival in THP-1 macrophages. In the present study we have standardized a luciferase based assay system to identify chemical scaffolds that are non-cytotoxic and inhibit M. tuberculosis PPK-2 enzyme. To the best of our knowledge this is the first study demonstrating feasibility of high throughput screening to obtain small molecule PPK-2 inhibitors.

scholarly journals Extending the Definition of the GyrB Quinolone Resistance-Determining Region in Mycobacterium tuberculosis DNA Gyrase for Assessing Fluoroquinolone Resistance in M. tuberculosis

2012 ◽  
Vol 56 (4) ◽  
pp. 1990-1996 ◽  
Author(s):  
Alix Pantel ◽  
Stéphanie Petrella ◽  
Nicolas Veziris ◽  
Florence Brossier ◽  
Sylvaine Bastian ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Hot Spot ◽  
Dna Gyrase ◽  
Quinolone Resistance ◽  
Fluoroquinolone Resistance ◽  
Wild Type ◽  
Content Type ◽  
Highly Active ◽  
Definition Of

ABSTRACTFluoroquinolone (FQ) resistance is emerging inMycobacterium tuberculosis. The main mechanism of FQ resistance is amino acid substitution within the quinolone resistance-determining region (QRDR) of the GyrA subunit of DNA gyrase, the sole FQ target inM. tuberculosis. However, substitutions in GyrB whose implication in FQ resistance is unknown are increasingly being reported. The present study clarified the role of four GyrB substitutions identified inM. tuberculosisclinical strains, two located in the QRDR (D500A and N538T) and two outside the QRDR (T539P and E540V), in FQ resistance. We measured FQ MICs and also DNA gyrase inhibition by FQs in order to unequivocally clarify the role of these mutations in FQ resistance. Wild-type GyrA, wild-type GyrB, and mutant GyrB subunits produced from engineeredgyrBalleles by mutagenesis were overexpressed inEscherichia coli, purified to homogeneity, and used to reconstitute highly active gyrase complexes. MICs and DNA gyrase inhibition were determined for moxifloxacin, gatifloxacin, ofloxacin, levofloxacin, and enoxacin. All these substitutions are clearly implicated in FQ resistance, underlining the presence of a hot spot region housing most of the GyrB substitutions implicated in FQ resistance (residues NTE, 538 to 540). These findings help us to refine the definition of GyrB QRDR, which is extended to positions 500 to 540.

scholarly journals The Helicobacter pylori Autotransporter ImaA Tempers the Bacterium's Interaction with α5β1 Integrin

2016 ◽  
Vol 85 (1) ◽  
Author(s):  
William E. Sause ◽  
Daniela Keilberg ◽  
Soufiane Aboulhouda ◽  
Karen M. Ottemann
Keyword(s):  
Helicobacter Pylori ◽  
Host Cell ◽  
Type Iv Secretion ◽  
Host Cells ◽  
Wild Type ◽  
Content Type ◽  
Type Iv ◽  
H Pylori ◽  
Α5β1 Integrin ◽  
Cell Interface

ABSTRACT The human pathogen Helicobacter pylori uses the host receptor α5β1 integrin to trigger inflammation in host cells via its cag pathogenicity island (cag PAI) type IV secretion system (T4SS). Here, we report that the H. pylori ImaA protein (HP0289) decreases the action of the cag PAI T4SS via tempering the bacterium's interaction with α5β1 integrin. Previously, imaA-null mutants were found to induce an elevated inflammatory response that was dependent on the cag PAI T4SS; here we extend those findings to show that the elevated response is independent of the CagA effector protein. To understand how ImaA could be affecting cag PAI T4SS activity at the host cell interface, we utilized the Phyre structural threading program and found that ImaA has a region with remote homology to bacterial integrin-binding proteins. This region was required for ImaA function. Unexpectedly, we observed that imaA mutants bound higher levels of α5β1 integrin than wild-type H. pylori, an outcome that required the predicted integrin-binding homology region of ImaA. Lastly, we report that ImaA directly affected the amount of host cell β1 integrin but not other cellular integrins. Our results thus suggest a model in which H. pylori employs ImaA to regulate interactions between integrin and the T4SS and thus alter the host inflammatory strength.

scholarly journals Nicotinamide Limits Replication of Mycobacterium tuberculosis and Bacille Calmette-Guérin Within Macrophages

2019 ◽  
Vol 221 (6) ◽  
pp. 989-999
Author(s):  
Jason D Simmons ◽  
Glenna J Peterson ◽  
Monica Campo ◽  
Jenny Lohmiller ◽  
Shawn J Skerrett ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Nicotinic Acid ◽  
Mycobacterium Bovis ◽  
Related Compound ◽  
Antimicrobial Properties ◽  
Broth Culture ◽  
Wild Type ◽  
Bacille Calmette Guerin ◽  
Macrophage Function ◽  
Modest Activity

Abstract Novel antimicrobials for treatment of Mycobacterium tuberculosis are needed. We hypothesized that nicotinamide (NAM) and nicotinic acid (NA) modulate macrophage function to restrict M. tuberculosis replication in addition to their direct antimicrobial properties. Both compounds had modest activity in 7H9 broth, but only NAM inhibited replication in macrophages. Surprisingly, in macrophages NAM and the related compound pyrazinamide restricted growth of bacille Calmette-Guérin but not wild-type Mycobacterium bovis, which both lack a functional nicotinamidase/pyrazinamidase (PncA) rendering each strain resistant to these drugs in broth culture. Interestingly, NAM was not active in macrophages infected with a virulent M. tuberculosis mutant encoding a deletion in pncA. We conclude that the differential activity of NAM and nicotinic acid on infected macrophages suggests host-specific NAM targets rather than PncA-dependent direct antimicrobial properties. These activities are sufficient to restrict attenuated BCG, but not virulent wild-type M. bovis or M. tuberculosis.

scholarly journals Fluorescence-Reported Allelic Exchange Mutagenesis-Mediated Gene Deletion Indicates a Requirement for Chlamydia trachomatis Tarp during In Vivo Infectivity and Reveals a Specific Role for the C Terminus during Cellular Invasion

2020 ◽  
Vol 88 (5) ◽  
Author(s):  
Susmita Ghosh ◽  
Elizabeth A. Ruelke ◽  
Joshua C. Ferrell ◽  
Maria D. Bodero ◽  
Kenneth A. Fields ◽  
...  
Keyword(s):  
Chlamydia Trachomatis ◽  
Host Cell ◽  
Host Cells ◽  
Actin Binding ◽  
Wild Type ◽  
Content Type ◽  
Allelic Exchange ◽  
Binding Domains

ABSTRACT The translocated actin recruiting phosphoprotein (Tarp) is a multidomain type III secreted effector used by Chlamydia trachomatis. In aggregate, existing data suggest a role of this effector in initiating new infections. As new genetic tools began to emerge to study chlamydial genes in vivo, we speculated as to what degree Tarp function contributes to Chlamydia’s ability to parasitize mammalian host cells. To address this question, we generated a complete tarP deletion mutant using the fluorescence-reported allelic exchange mutagenesis (FRAEM) technique and complemented the mutant in trans with wild-type tarP or mutant tarP alleles engineered to harbor in-frame domain deletions. We provide evidence for the significant role of Tarp in C. trachomatis invasion of host cells. Complementation studies indicate that the C-terminal filamentous actin (F-actin)-binding domains are responsible for Tarp-mediated invasion efficiency. Wild-type C. trachomatis entry into HeLa cells resulted in host cell shape changes, whereas the tarP mutant did not. Finally, using a novel cis complementation approach, C. trachomatis lacking tarP demonstrated significant attenuation in a murine genital tract infection model. Together, these data provide definitive genetic evidence for the critical role of the Tarp F-actin-binding domains in host cell invasion and for the Tarp effector as a bona fide C. trachomatis virulence factor.

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