scholarly journals CFP-10 from Mycobacterium tuberculosis Selectively Activates Human Neutrophils through a Pertussis Toxin-Sensitive Chemotactic Receptor

2014 ◽  
Vol 83 (1) ◽  
pp. 205-213 ◽  
Author(s):  
Amanda Welin ◽  
Halla Björnsdottir ◽  
Malene Winther ◽  
Karin Christenson ◽  
Tudor Oprea ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Pertussis Toxin ◽  
Human Neutrophils ◽  
Content Type ◽  
Chaperone Function ◽  
Type Vii Secretion ◽  
Chaperone Protein ◽  
Type Vii Secretion System ◽  
G Protein Coupled

Upon infection withMycobacterium tuberculosis, neutrophils are massively recruited to the lungs, but the role of these cells in combating the infection is poorly understood. Through a type VII secretion system,M. tuberculosisreleases a heterodimeric protein complex, containing a 6-kDa early secreted antigenic target (ESAT-6) and a 10-kDa culture filtrate protein (CFP-10), that is essential for virulence. Whereas the ESAT-6 component possesses multiple virulence-related activities, no direct biological activity of CFP-10 has been shown, and CFP-10 has been described as a chaperone protein for ESAT-6. We here show that the ESAT-6:CFP-10 complex induces a transient release of Ca2+from intracellular stores in human neutrophils. Surprisingly, CFP-10 rather than ESAT-6 was responsible for triggering the Ca2+response, in a pertussis toxin-sensitive manner, suggesting the involvement of a G-protein-coupled receptor. In line with this, the response was accompanied by neutrophil chemotaxis and activation of the superoxide-producing NADPH-oxidase. Neutrophils were unique among leukocytes in responding to CFP-10, as monocytes and lymphocytes failed to produce a Ca2+signal upon stimulation with theM. tuberculosisprotein. Hence, CFP-10 may contribute specifically to neutrophil recruitment and activation duringM. tuberculosisinfection, representing a novel biological role for CFP-10 in the ESAT-6:CFP-10 complex, beyond the previously described chaperone function.

scholarly journals A Duplicated ESAT-6 Region of ESX-5 Is Involved in Protein Export and Virulence of Mycobacteria

2015 ◽  
Vol 83 (11) ◽  
pp. 4349-4361 ◽  
Author(s):  
Swati Shah ◽  
Joe R. Cannon ◽  
Catherine Fenselau ◽  
Volker Briken
Keyword(s):  
Secretion System ◽  
Bacterial Virulence ◽  
Inflammasome Activation ◽  
Zebrafish Model ◽  
Content Type ◽  
Type Vii Secretion ◽  
Specific Subset ◽  
Host Cell Death ◽  
Type Vii Secretion System

ABSTRACTThe ESX-5 secretion system ofMycobacterium tuberculosisis important for bacterial virulence and for the secretion of the large PE/PPE protein family, whose genes constitute 10% of theM. tuberculosisgenome. A four-gene region of the ESX-5 system is duplicated three times in theM. tuberculosisgenome, but the functions of these duplicates are unknown. Here we investigated one of these duplicates: the region carrying theesxI,esxJ,ppe15, andpe8genes (ESX-5a). An ESX-5a deletion mutant in the model systemM. marinumbackground was deficient in the secretion of some members of the PE/PPE family of proteins. Surprisingly, we also identified other proteins that are not members of this family, thus expanding the range of ESX-5 secretion substrates. In addition, we demonstrated that ESX-5a is important for the virulence ofM. marinumin the zebrafish model. Furthermore, we showed the role of theM. tuberculosisESX-5a region in inflammasome activation but not host cell death induction, which is different from the case for theM. tuberculosisESX-5 system. In conclusion, the ESX-5a region is nonredundant with its ESX-5 paralog and is necessary for secretion of a specific subset of proteins inM. tuberculosisandM. marinumthat are important for bacterial virulence ofM. marinum. Our findings point to a role for the three ESX-5 duplicate regions in the selection of substrates for secretion via ESX-5, and hence, they provide the basis for a refined model of the molecular mechanism of this type VII secretion system.

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 Role of Metal-Dependent Regulation of ESX-3 Secretion in Intracellular Survival of Mycobacterium tuberculosis

2016 ◽  
Vol 84 (8) ◽  
pp. 2255-2263 ◽  
Author(s):  
Emir Tinaztepe ◽  
Jun-Rong Wei ◽  
Jenelle Raynowska ◽  
Cynthia Portal-Celhay ◽  
Victor Thompson ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Cell Envelope ◽  
Bacterial Pathogen ◽  
Transcriptional Response ◽  
Secretion Systems ◽  
Content Type ◽  
Type Vii Secretion ◽  
Significant Difference ◽  
Iron And Zinc

More people die every year fromMycobacterium tuberculosisinfection than from infection by any other bacterial pathogen. Type VII secretion systems (T7SS) are used by both environmental and pathogenic mycobacteria to secrete proteins across their complex cell envelope. In the nonpathogenMycobacterium smegmatis, the ESX-1 T7SS plays a role in conjugation, and the ESX-3 T7SS is involved in metal homeostasis. InM. tuberculosis, these secretion systems have taken on roles in virulence, and they also are targets of the host immune response. ESX-3 secretes a heterodimer composed of EsxG (TB9.8) and EsxH (TB10.4), which impairs phagosome maturation in macrophages and is essential for virulence in mice. Given the importance of EsxG and EsxH during infection, we examined their regulation. WithM. tuberculosis, the secretion of EsxG and EsxH was regulated in response to iron and zinc, in accordance with the previously described transcriptional response of theesx-3locus to these metals. While iron regulated theesx-3expression in bothM. tuberculosisandM. smegmatis, there is a significant difference in the dynamics of this regulation. InM. smegmatis, theesx-3locus behaved like other iron-regulated genes such asmbtB. InM. tuberculosis, both iron and zinc modestly repressedesx-3expression. Diminished secretion of EsxG and EsxH in response to these metals altered the interaction ofM. tuberculosiswith macrophages, leading to impaired intracellularM. tuberculosissurvival. Our findings detail the regulatory differences ofesx-3inM. tuberculosisandM. smegmatisand demonstrate the importance of metal-dependent regulation of ESX-3 for virulence inM. tuberculosis.

scholarly journals Genomics of Atlantic Forest Mycobacteriaceae strains unravels a mobilome diversity with a novel integrative conjugative element and plasmids harbouring T7SS

2020 ◽  
Vol 6 (7) ◽  
Author(s):  
Sergio Mascarenhas Morgado ◽  
Ana Carolina Paulo Vicente
Keyword(s):  
Atlantic Forest ◽  
Mobile Genetic Elements ◽  
Content Type ◽  
Genetic Elements ◽  
Link Type ◽  
Type Vii Secretion ◽  
Integrative Conjugative Element ◽  
Type Vii Secretion System ◽  
Integrative Conjugative Elements

Mobile genetic elements (MGEs) are agents of bacterial evolution and adaptation. Genome sequencing provides an unbiased approach that has revealed an abundance of MGEs in prokaryotes, mainly plasmids and integrative conjugative elements. Nevertheless, many mobilomes, particularly those from environmental bacteria, remain underexplored despite their representing a reservoir of genes that can later emerge in the clinic. Here, we explored the mobilome of the Mycobacteriaceae family, focusing on strains from Brazilian Atlantic Forest soil. Novel Mycolicibacterium and Mycobacteroides strains were identified, with the former ones harbouring linear and circular plasmids encoding the specialized type-VII secretion system (T7SS) and mobility-associated genes. In addition, we also identified a T4SS-mediated integrative conjugative element (ICEMyc226) encoding two T7SSs and a number of xenobiotic degrading genes. Our study uncovers the diversity of the Mycobacteriaceae mobilome, providing the evidence of an ICE in this bacterial family. Moreover, the presence of T7SS genes in an ICE, as well as plasmids, highlights the role of these mobile genetic elements in the dispersion of T7SS.

scholarly journals Mycobacterium tuberculosis Toxin CpnT Is an ESX-5 Substrate and Requires Three Type VII Secretion Systems for Intracellular Secretion

mBio ◽  
2021 ◽  
Vol 12 (2) ◽  
Author(s):  
B. Izquierdo Lafuente ◽  
R. Ummels ◽  
C. Kuijl ◽  
W. Bitter ◽  
A. Speer
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Host Cells ◽  
Secretion Systems ◽  
Macrophage Infection ◽  
Content Type ◽  
Secretion Pathway ◽  
Terminal Domain ◽  
Type Vii Secretion ◽  
Type Vii Secretion System ◽  
Axenic Conditions

ABSTRACT CpnT, a NAD+ glycohydrolase, is the only known toxin that is secreted by Mycobacterium tuberculosis. CpnT is composed of two domains; the C-terminal domain is the toxin, whereas the N-terminal domain is required for secretion. CpnT shows characteristics of type VII secretion (T7S) substrates, including a predicted helix-turn-helix domain followed by a secretion motif (YxxxE). Disruption of this motif indeed abolished CpnT secretion. By analyzing different mutants, we established that CpnT is specifically secreted by the ESX-5 system in Mycobacterium marinum under axenic conditions and during macrophage infection. Surprisingly, intracellular secretion of CpnT was also dependent on both ESX-1 and ESX-4. These secretion defects could be partially rescued by coinfection with wild-type bacteria, indicating that secreted effectors are involved in this process. In summary, our data reveal that three different type VII secretion systems have to be functional in order to observe intracellular secretion of the toxin CpnT. IMPORTANCE For decades, it was believed that the intracellular pathogen M. tuberculosis does not possess toxins. Only fairly recently it was discovered that CpnT is a potent secreted toxin of M. tuberculosis, causing necrotic cell death in host cells. However, until now the secretion pathway remained unknown. In our study, we were able to identify CpnT as a substrate of the mycobacterial type VII secretion system. Pathogenic mycobacteria have up to five different type VII secretion systems, called ESX-1 to ESX-5, which play distinct roles for the pathogen during growth or infection. We were able to elucidate that CpnT is exclusively secreted by the ESX-5 system in bacterial culture. However, to our surprise we discovered that, during infection studies, CpnT secretion relies on intact ESX-1, ESX-4, and ESX-5 systems. We elucidate for the first time the intertwined interplay of three different and independent secretion systems to secrete one substrate during infection.

scholarly journals ESX-1-Independent Horizontal Gene Transfer by Mycobacterium tuberculosis Complex Strains

mBio ◽  
2021 ◽  
Vol 12 (3) ◽  
Author(s):  
Jan Madacki ◽  
Mickael Orgeur ◽  
Guillem Mas Fiol ◽  
Wafa Frigui ◽  
Laurence Ma ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Mycobacterium Smegmatis ◽  
Secretion System ◽  
Dna Transfer ◽  
Chromosomal Dna ◽  
Content Type ◽  
Type Vii Secretion ◽  
Wide Range ◽  
Population Structures ◽  
Type Vii Secretion System

ABSTRACT Current models of horizontal gene transfer (HGT) in mycobacteria are based on “distributive conjugal transfer” (DCT), an HGT type described in the fast-growing, saprophytic model organism Mycobacterium smegmatis, which creates genome mosaicism in resulting strains and depends on an ESX-1 type VII secretion system. In contrast, only few data on interstrain DNA transfer are available for tuberculosis-causing mycobacteria, for which chromosomal DNA transfer between two Mycobacterium canettii strains was reported, a process which, however, was not observed for Mycobacterium tuberculosis strains. Here, we have studied a wide range of human- and animal-adapted members of the Mycobacterium tuberculosis complex (MTBC) using an optimized filter-based mating assay together with three selected strains of M. canettii that acted as DNA recipients. Unlike in previous approaches, we obtained a high yield of thousands of recombinants containing transferred chromosomal DNA fragments from various MTBC donor strains, as confirmed by whole-genome sequence analysis of 38 randomly selected clones. While the genome organizations of the obtained recombinants showed mosaicisms of donor DNA fragments randomly integrated into a recipient genome backbone, reminiscent of those described as being the result of ESX-1-mediated DCT in M. smegmatis, we observed similar transfer efficiencies when ESX-1-deficient donor and/or recipient mutants were used, arguing that in tubercle bacilli, HGT is an ESX-1-independent process. These findings provide new insights into the genetic events driving the pathoevolution of M. tuberculosis and radically change our perception of HGT in mycobacteria, particularly for those species that show recombinogenic population structures despite the natural absence of ESX-1 secretion systems. IMPORTANCE Data on the bacterial sex-mediated impact on mycobacterial evolution are limited. Hence, our results presented here are of importance as they clearly demonstrate the capacity of a wide range of human- and animal-adapted Mycobacterium tuberculosis complex (MTBC) strains to transfer chromosomal DNA to selected strains of Mycobacterium canettii. Most interestingly, we found that interstrain DNA transfer among tubercle bacilli was not dependent on a functional ESX-1 type VII secretion system, as ESX-1 deletion mutants of potential donor and/or recipient strains yielded numbers of recombinants similar to those of their respective parental strains. These results argue that HGT in tubercle bacilli is organized in a way different from that of the most widely studied Mycobacterium smegmatis model, a finding that is also relevant beyond tubercle bacilli, given that many mycobacteria, like, for example, Mycobacterium avium or Mycobacterium abscessus, are naturally devoid of an ESX-1 secretion system but show recombinogenic, mosaic-like genomic population structures.

scholarly journals Mutation ofRv2887, amarR-Like Gene, Confers Mycobacterium tuberculosis Resistance to an Imidazopyridine-Based Agent

2015 ◽  
Vol 59 (11) ◽  
pp. 6873-6881 ◽  
Author(s):  
Kathryn Winglee ◽  
Shichun Lun ◽  
Marco Pieroni ◽  
Alan Kozikowski ◽  
William Bishai
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Drug Targets ◽  
Efflux Pump ◽  
Transcriptional Regulator ◽  
Cross Resistance ◽  
Loss Of Function ◽  
Strong Activity ◽  
Content Type ◽  
Novel Drug

ABSTRACTDrug resistance is a major problem inMycobacterium tuberculosiscontrol, and it is critical to identify novel drug targets and new antimycobacterial compounds. We have previously identified an imidazo[1,2-a]pyridine-4-carbonitrile-based agent, MP-III-71, with strong activity againstM. tuberculosis. In this study, we evaluated mechanisms of resistance to MP-III-71. We derived three independentM. tuberculosismutants resistant to MP-III-71 and conducted whole-genome sequencing of these mutants. Loss-of-function mutations inRv2887were common to all three MP-III-71-resistant mutants, and we confirmed the role ofRv2887as a gene required for MP-III-71 susceptibility using complementation. The Rv2887 protein was previously unannotated, but domain and homology analyses suggested it to be a transcriptional regulator in the MarR (multiple antibiotic resistance repressor) family, a group of proteins first identified inEscherichia colito negatively regulate efflux pumps and other mechanisms of multidrug resistance. We found that two efflux pump inhibitors, verapamil and chlorpromazine, potentiate the action of MP-III-71 and that mutation ofRv2887abrogates their activity. We also used transcriptome sequencing (RNA-seq) to identify genes which are differentially expressed in the presence and absence of a functional Rv2887 protein. We found that genes involved in benzoquinone and menaquinone biosynthesis were repressed by functional Rv2887. Thus, inactivating mutations ofRv2887, encoding a putative MarR-like transcriptional regulator, confer resistance to MP-III-71, an effective antimycobacterial compound that shows no cross-resistance to existing antituberculosis drugs. The mechanism of resistance ofM. tuberculosisRv2887mutants may involve efflux pump upregulation and also drug methylation.

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 Lipid Virulence Factors of Mycobacterium tuberculosis Exert Multilayered Control over Autophagy-Related Pathways in Infected Human Macrophages

Cells ◽  
2020 ◽  
Vol 9 (3) ◽  
pp. 666 ◽  
Author(s):  
Aïcha Bah ◽  
Merlin Sanicas ◽  
Jérôme Nigou ◽  
Christophe Guilhot ◽  
Catherine Astarie-Dequeker ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Virulence Factors ◽  
Defense Mechanism ◽  
Immune Defense ◽  
Knock Out ◽  
Human Macrophages ◽  
Type Vii Secretion ◽  
Type Vii Secretion System ◽  
Phagosomal Membrane ◽  
Phthiocerol Dimycocerosates

Autophagy is an important innate immune defense mechanism that controls Mycobacterium tuberculosis (Mtb) growth inside macrophages. Autophagy machinery targets Mtb-containing phagosomes via xenophagy after damage to the phagosomal membrane due to the Type VII secretion system Esx-1 or via LC3-associated phagocytosis without phagosomal damage. Conversely, Mtb restricts autophagy-related pathways via the production of various bacterial protein factors. Although bacterial lipids are known to play strategic functions in Mtb pathogenesis, their role in autophagy manipulation remains largely unexplored. Here, we report that the lipid virulence factors sulfoglycolipids (SLs) and phthiocerol dimycocerosates (DIMs) control autophagy-related pathways through distinct mechanisms in human macrophages. Using knock-out and knock-in mutants of Mtb and Mycobacterium bovis BCG (Bacille Calmette Guerin) and purified lipids, we found that (i) Mtb mutants with DIM and SL deficiencies promoted functional autophagy via an MyD88-dependent and phagosomal damage-independent pathway in human macrophages; (ii) SLs limited this pathway by acting as TLR2 antagonists; (iii) DIMs prevented phagosomal damage-independent autophagy while promoting Esx-1-dependent xenophagy; (iv) and DIMs, but not SLs, limited the acidification of LC3-positive Mtb compartments. In total, our study reveals an unexpected and intricate role for Mtb lipid virulence factors in controlling autophagy-related pathways in human macrophages, thus providing further insight into the autophagy manipulation tactics deployed by intracellular bacterial pathogens.

scholarly journals Metabolic Switching of Mycobacterium tuberculosis during Hypoxia Is Controlled by the Virulence Regulator PhoP

2020 ◽  
Vol 202 (7) ◽  
Author(s):  
Prabhat Ranjan Singh ◽  
Anil Kumar Vijjamarri ◽  
Dibyendu Sarkar
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Nitrogen Metabolism ◽  
Electron Acceptor ◽  
Latent Infection ◽  
Critical Role ◽  
Hypoxic Stress ◽  
Content Type ◽  
Metabolic Switching ◽  
Virulence Regulator

ABSTRACT Mycobacterium tuberculosis retains the ability to establish an asymptomatic latent infection. A fundamental question in mycobacterial physiology is to understand the mechanisms involved in hypoxic stress, a critical player in persistence. Here, we show that the virulence regulator PhoP responds to hypoxia, the dormancy signal, and effectively integrates hypoxia with nitrogen metabolism. We also provide evidence to demonstrate that both under nitrogen limiting conditions and during hypoxia, phoP locus controls key genes involved in nitrogen metabolism. Consistently, under hypoxia a ΔphoP strain shows growth attenuation even with surplus nitrogen, the alternate electron acceptor, and complementation of the mutant restores bacterial growth. Together, our observations provide new biological insights into the role of PhoP in integrating nitrogen metabolism with hypoxia by the assistance of the hypoxia regulator DosR. The results have significant implications on the mechanism of intracellular survival and growth of the tubercle bacilli under a hypoxic environment within the phagosome. IMPORTANCE M. tuberculosis retains the unique ability to establish an asymptomatic latent infection. To understand the mechanisms involved in hypoxic stress which play a critical role in persistence, we show that the virulence regulator PhoP is linked to hypoxia, the dormancy signal. In keeping with this, phoP was shown to play a major role in M. tuberculosis growth under hypoxia even in the presence of surplus nitrogen, the alternate electron acceptor. Our results showing regulation of hypoxia-responsive genes provide new biological insights into role of the virulence regulator in metabolic switching by sensing hypoxia and integrating nitrogen metabolism with hypoxia by the assistance of the hypoxia regulator DosR.

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