scholarly journals Generation of Attenuated Mycobacterium bovis Strains by Signature-Tagged Mutagenesis for Discovery of Novel Vaccine Candidates

2005 ◽  
Vol 73 (4) ◽  
pp. 2379-2386 ◽  
Author(s):  
Desmond M. Collins ◽  
Bronwyn Skou ◽  
Stefan White ◽  
Shalome Bassett ◽  
Lauren Collins ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Mycobacterium Bovis ◽  
Guinea Pigs ◽  
Essential Role ◽  
Immunoblot Analysis ◽  
Infection Model ◽  
Vaccine Candidates ◽  
Mouse Infection Model ◽  
Potential Vaccine

ABSTRACT Mycobacterium bovis, a member of the Mycobacterium tuberculosis complex, has a particularly wide host range and causes tuberculosis in most mammals, including humans. A signature tag mutagenesis approach, which employed illegitimate recombination and infection of guinea pigs, was applied to M. bovis to discover genes important for virulence and to find potential vaccine candidates. Fifteen attenuated mutants were identified, four of which produced no lesions when inoculated separately into guinea pigs. One of these four mutants had nine deleted genes including mmpL4 and sigK and, in guinea pigs with aerosol challenge, provided protection against tuberculosis at least equal to that of M. bovis BCG. Seven mutants had mutations near the esxA (esat-6) locus, and immunoblot analysis of these confirmed the essential role of other genes at this locus in the secretion of EsxA (ESAT-6) and EsxB (CFP10). Mutations in the eight other attenuated mutants were widely spread through the chromosome and included pks1, which is naturally inactivated in clinical strains of M. tuberculosis. Many genes identified were different from those found by signature tag mutagenesis of M. tuberculosis by use of a mouse infection model and illustrate how the use of different approaches enables identification of a wider range of attenuating mutants.

Role of prophylactic azithromycin to reduce airway inflammation and mortality in a RSV mouse infection model

2018 ◽  
Vol 53 (5) ◽  
pp. 567-574 ◽  
Author(s):  
Ricardo A. Mosquera ◽  
Wilfredo De Jesus-Rojas ◽  
James M. Stark ◽  
Aravind Yadav ◽  
Cindy K. Jon ◽  
...  
Keyword(s):  
Airway Inflammation ◽  
Infection Model ◽  
Mouse Infection Model ◽  
Mouse Infection

scholarly journals Proteomic and transcriptomic experiments reveal an essential role of RNA degradosome complexes in shaping the transcriptome of Mycobacterium tuberculosis

2019 ◽  
Vol 47 (11) ◽  
pp. 5892-5905 ◽  
Author(s):  
Przemysław Płociński ◽  
Maria Macios ◽  
Joanna Houghton ◽  
Emilia Niemiec ◽  
Renata Płocińska ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Essential Role ◽  
Rna Degradosome

scholarly journals MTSA-10, the Product of the Rv3874 Gene of Mycobacterium tuberculosis, Elicits Tuberculosis-Specific, Delayed-Type Hypersensitivity in Guinea Pigs

2000 ◽  
Vol 68 (2) ◽  
pp. 990-993 ◽  
Author(s):  
Roberto Colangeli ◽  
John S. Spencer ◽  
Pablo Bifani ◽  
Alan Williams ◽  
Konstantin Lyashchenko ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Skin Test ◽  
Mycobacterium Bovis ◽  
Mycobacterium Avium ◽  
Guinea Pigs ◽  
Nontuberculous Mycobacteria ◽  
Specific Antigen ◽  
Delayed Type Hypersensitivity ◽  
New Skin

ABSTRACT In a search for new skin test reagents specific for tuberculosis, we found that the antigen encoded by gene Rv3874 of Mycobacterium tuberculosis elicited delayed-type hypersensitivity in M. tuberculosis-infected guinea pigs but not in control animals immunized with Mycobacterium bovis bacillus Calmette-Guérin (BCG) or Mycobacterium avium. The antigen, which was named MTSA-10 (for M. tuberculosis-specific antigen 10), is a prime candidate for a component of a new tuberculin that will allow discrimination by a skin test of latent M. tuberculosis infection from vaccination with BCG or from sensitization with environmental, nontuberculous mycobacteria.

scholarly journals Requirement of the mymA Operon for Appropriate Cell Wall Ultrastructure and Persistence of Mycobacterium tuberculosis in the Spleens of Guinea Pigs

2005 ◽  
Vol 187 (12) ◽  
pp. 4173-4186 ◽  
Author(s):  
Amit Singh ◽  
Radhika Gupta ◽  
R. A. Vishwakarma ◽  
P. R. Narayanan ◽  
C. N. Paramasivan ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Parental Strain ◽  
Guinea Pigs ◽  
Cell Envelope ◽  
Sodium Dodecyl ◽  
Phenotypic Traits ◽  
Antitubercular Drugs ◽  
Mycolic Acids ◽  
Increased Sensitivity

ABSTRACT We had recently reported that the mymA operon (Rv3083 to Rv3089) of Mycobacterium tuberculosis is regulated by AraC/XylS transcriptional regulator VirS (Rv3082c) and is important for the cell envelope of M. tuberculosis. In this study, we further show that a virS mutant (MtbΔvirS) and a mymA mutant (Mtbmym::hyg) of M. tuberculosis exhibit reduced contents and altered composition of mycolic acids along with the accumulation of saturated C24 and C26 fatty acids compared to the parental strain. These mutants were markedly more susceptible to major antitubercular drugs at acidic pH and also showed increased sensitivity to detergent (sodium dodecyl sulfate) and to acidic stress than the parental strain. We show that disruption of virS and mymA genes impairs the ability of M. tuberculosis to survive in activated macrophages, but not in resting macrophages, suggesting the importance of the mymA operon in protecting the bacterium against harsher conditions. Infection of guinea pigs with MtbΔvirS, Mtbmym::hyg, and the parental strain resulted in an ∼800-fold-reduced bacillary load of the mutant strains compared with the parental strain in spleens, but not in the lungs, of animals at 20 weeks postinfection. Phenotypic traits were fully complemented upon reintroduction of the virS gene into MtbΔvirS. These observations show the important role of the mymA operon in the pathogenesis of M. tuberculosis at later stages of the disease.

scholarly journals Association of ω with the C-Terminal Region of the β′ Subunit Is Essential for Assembly of RNA Polymerase in Mycobacterium tuberculosis

2018 ◽  
Vol 200 (12) ◽  
Author(s):  
Chunyou Mao ◽  
Yan Zhu ◽  
Pei Lu ◽  
Lipeng Feng ◽  
Shiyun Chen ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Mycobacterium Tuberculosis ◽  
Rna Polymerase ◽  
Essential Role ◽  
Β Subunit ◽  
Content Type ◽  
E Coli ◽  
Core Enzyme ◽  
Loop Region

ABSTRACT The ω subunit is the smallest subunit of bacterial RNA polymerase (RNAP). Although homologs of ω are essential in both eukaryotes and archaea, this subunit has been known to be dispensable for RNAP in Escherichia coli and in other bacteria. In this study, we characterized an indispensable role of the ω subunit in Mycobacterium tuberculosis . Unlike the well-studied E. coli RNAP, the M. tuberculosis RNAP core enzyme cannot be functionally assembled in the absence of the ω subunit. Importantly, substitution of M. tuberculosis ω with ω subunits from E. coli or Thermus thermophilus cannot restore the assembly of M. tuberculosis RNAP. Furthermore, by replacing different regions in M. tuberculosis ω with the corresponding regions from E. coli ω, we found a nonconserved loop region in M. tuberculosis ω essential for its function in RNAP assembly. From RNAP structures, we noticed that the location of the C-terminal region of the β′ subunit (β′CTD) in M. tuberculosis RNAP but not in E. coli or T. thermophilus RNAP is close to the ω loop region. Deletion of this β′CTD in M. tuberculosis RNAP destabilized the binding of M. tuberculosis ω on RNAP and compromised M. tuberculosis core assembly, suggesting that these two regions may function together to play a role in ω-dependent RNAP assembly in M. tuberculosis . Sequence alignment of the ω loop and the β′CTD regions suggests that the essential role of ω is probably restricted to mycobacteria. Together, our study characterized an essential role of M. tuberculosis ω and highlighted the importance of the ω loop region in M. tuberculosis RNAP assembly. IMPORTANCE DNA-dependent RNA polymerase (RNAP), which consists of a multisubunit core enzyme (α 2 ββ′ω) and a dissociable σ subunit, is the only enzyme in charge of transcription in bacteria. As the smallest subunit, the roles of ω remain the least well studied. In Escherichia coli and some other bacteria, the ω subunit is known to be nonessential for RNAP. In this study, we revealed an essential role of the ω subunit for RNAP assembly in the human pathogen Mycobacterium tuberculosis , and a mycobacterium-specific ω loop that plays a role in this function was also characterized. Our study provides fresh insights for further characterizing the roles of bacterial ω subunit.

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 Influence of Mycobacterium bovis BCG Vaccination on Cellular Immune Response of Guinea Pigs Challenged with Mycobacterium tuberculosis

2008 ◽  
Vol 15 (8) ◽  
pp. 1248-1258 ◽  
Author(s):  
Diane Ordway ◽  
Marcela Henao-Tamayo ◽  
Crystal Shanley ◽  
Erin E. Smith ◽  
Gopinath Palanisamy ◽  
...  
Keyword(s):  
T Cells ◽  
Mycobacterium Tuberculosis ◽  
Mycobacterium Bovis ◽  
Guinea Pigs ◽  
Lung Damage ◽  
Cell Populations ◽  
Lung Pathology ◽  
Bcg Vaccination ◽  
Macrophage Populations ◽  
Cellular Immune

ABSTRACT Mycobacterium bovis bacillus Calmette-Guérin (BCG) currently remains the only licensed vaccine for the prevention of tuberculosis. In this study, we used a newly described flow cytometric technique to monitor changes in cell populations accumulating in the lungs and lymph nodes of naïve and vaccinated guinea pigs challenged by low-dose aerosol infection with virulent Mycobacterium tuberculosis. As anticipated, vaccinated guinea pigs controlled the growth of the challenge infection more efficiently than controls did. This early phase of bacterial control in immune animals was associated with increased accumulation of CD4 and CD8 T cells, including cells expressing the activation marker CD45, as well as macrophages expressing class II major histocompatibility complex molecules. As the infection continued, the numbers of T cells in the lungs of vaccinated animals waned, whereas the numbers of these cells expressing CD45 increased. Whereas BCG vaccination reduced the influx of heterophils (neutrophils) into the lungs, an early B-cell influx was observed in these vaccinated animals. Overall, vaccine protection was associated with reduced pathology and lung damage in the vaccinated animals. These data provide the first direct evidence that BCG vaccination accelerates the influx of protective T-cell and macrophage populations into the infected lungs, diminishes the accumulation of nonprotective cell populations, and reduces the severity of lung pathology.

scholarly journals Immunogenicity of Mycobacterium tuberculosis Antigens in Mycobacterium bovis BCG-Vaccinated and M. bovis-Infected Cattle

2006 ◽  
Vol 74 (8) ◽  
pp. 4566-4572 ◽  
Author(s):  
A. S. Mustafa ◽  
Y. A. Skeiky ◽  
R. Al-Attiyah ◽  
M. R. Alderson ◽  
R. G. Hewinson ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Mycobacterium Bovis ◽  
Infected Cattle ◽  
Subunit Vaccines ◽  
Vaccine Candidates ◽  
Future Evaluation ◽  
Research Challenge ◽  
Interferon Responses ◽  
Recombinant Bcg

ABSTRACT The development of novel vaccine strategies supplementing Mycobacterium bovis BCG (BCG) constitutes an urgent research challenge. To identify potential subunit vaccine candidates, we have tested a series of eight recently identified Mycobacterium tuberculosis antigens in M. bovis-infected and BCG-vaccinated cattle. These antigens were characterized on the basis of their ability to induce in vitro gamma interferon responses in infected or BCG-vaccinated calves. We were able to establish a hierarchy of these antigens based on how frequently they were recognized in both groups of animals. In particular, we were able to prioritize frequently recognized proteins like Rv0287, Rv1174, and Rv1196 for future evaluation as subunit vaccines to be used in BCG-protein heterologous prime-boost vaccination scenarios. In addition, the antigen most dominantly recognized in M. bovis-infected cattle in this study, Rv3616c, was significantly less frequently recognized by BCG vaccinees and could be a target to improve BCG, for example, by increasing its secretion, in a recombinant BCG vaccine.

Mycobacterium bovis BCG vaccination modulates TNF-α production after pulmonary challenge with virulent Mycobacterium tuberculosis in guinea pigs

Tuberculosis ◽  
2007 ◽  
Vol 87 (2) ◽  
pp. 155-165 ◽  
Author(s):  
Toshiko Yamamoto ◽  
Todd M. Lasco ◽  
Kazuyuki Uchida ◽  
Yoshitaka Goto ◽  
Amminikutty Jeevan ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Mycobacterium Bovis ◽  
Guinea Pigs ◽  
Mycobacterium Bovis Bcg ◽  
Bcg Vaccination ◽  
Tnf Α

scholarly journals Skin Test Performed with Highly Purified Mycobacterium tuberculosis Recombinant Protein Triggers Tuberculin Shock in Infected Guinea Pigs

2005 ◽  
Vol 73 (6) ◽  
pp. 3301-3306 ◽  
Author(s):  
Stephen T. Reece ◽  
Nicole Stride ◽  
Pamela Ovendale ◽  
Steven G. Reed ◽  
Antonio Campos-Neto
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Recombinant Protein ◽  
Skin Test ◽  
Guinea Pigs ◽  
Lung Parenchyma ◽  
Necrosis Factor Alpha ◽  
Vaccine Candidates ◽  
Factor Alpha ◽  
Necrosis Factor ◽  
Present Experimental Evidence

ABSTRACT Tuberculin shock due to inoculation of Mycobacterium tuberculosis antigens in patients with tuberculosis is a serious syndrome originally described over 100 years ago by Robert Koch. Here, we present experimental evidence that a single M. tuberculosis recombinant protein, CFP-10, triggers this syndrome. Intradermal inoculation of CFP-10 elicits in M. tuberculosis-infected mice high levels of serum tumor necrosis factor alpha and causes tuberculin shock in infected guinea pigs characterized by hypothermia and death within 6 to 48 h after the antigen inoculation. Autopsies of these animals revealed intense polycythemia and hemorrhagic patches in the lung parenchyma, a pathological observation consistent with tuberculin shock. These results point to the possible occurrence of tuberculin shock in sensitive individuals inoculated with highly purified M. tuberculosis recombinant proteins as vaccine candidates or skin test reagents.

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