Reversible gene silencing through frameshift indels and frameshift scars provide adaptive plasticity for Mycobacterium tuberculosis

AbstractMycobacterium tuberculosis can adapt to changing environments by non-heritable mechanisms. Frame-shifting insertions and deletions (indels) may also participate in adaptation through gene disruption, which could be reversed by secondary introduction of a frame-restoring indel. We present ScarTrek, a program that scans genomic data for indels, including those that together disrupt and restore a gene’s reading frame, producing “frame-shift scars” suggestive of reversible gene inactivation. We use ScarTrek to analyze 5977 clinical M. tuberculosis isolates. We show that indel frequency inversely correlates with genomic linguistic complexity and varies with gene-position and gene-essentiality. Using ScarTrek, we detect 74 unique frame-shift scars in 48 genes, with a 3.74% population-level incidence of unique scar events. We find multiple scars in the ESX-1 gene cluster. Six scars show evidence of convergent evolution while the rest shared a common ancestor. Our results suggest that sequential indels are a mechanism for reversible gene silencing and adaptation in M. tuberculosis.

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Mucosal Influenza Vector Vaccine Carrying TB10.4 and HspX Antigens Provides Protection against Mycobacterium tuberculosis in Mice and Guinea Pigs

Vaccines ◽

10.3390/vaccines9040394 ◽

2021 ◽

Vol 9 (4) ◽

pp. 394

Author(s):

Mariia Sergeeva ◽

Ekaterina Romanovskaya-Romanko ◽

Natalia Zabolotnyh ◽

Anastasia Pulkina ◽

Kirill Vasilyev ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Vaccine Candidate ◽

Cell Immune Response ◽

Lung Pathology ◽

Ns1 Protein ◽

Reading Frame ◽

Intranasal Immunization ◽

Vector Vaccine ◽

Boost Immunization ◽

New Strategies

New strategies providing protection against tuberculosis (TB) are still pending. The airborne nature of Mycobacterium tuberculosis (M.tb) infection assumes that the mucosal delivery of the TB vaccine could be a more promising strategy than the systemic route of immunization. We developed a mucosal TB vaccine candidate based on recombinant attenuated influenza vector (Flu/THSP) co-expressing truncated NS1 protein NS1(1–124) and a full-length TB10.4 and HspX proteins of M.tb within an NS1 protein open reading frame. The Flu/THSP vector was safe and stimulated a systemic TB-specific CD4+ and CD8+ T-cell immune response after intranasal immunization in mice. Double intranasal immunization with the Flu/THSP vector induced protection against two virulent M.tb strains equal to the effect of BCG subcutaneous injection in mice. In a guinea pig TB model, one intranasal immunization with Flu/THSP improved protection against M.tb when tested as a vaccine candidate for boosting BCG-primed immunity. Importantly, enhanced protection provided by a heterologous BCG-prime → Flu/THSP vector boost immunization scheme was associated with a significantly reduced lung and spleen bacterial burden (mean decrease of 0.77 lg CFU and 0.72 lg CFU, respectively) and improved lung pathology 8.5 weeks post-infection with virulent M.tb strain H37Rv.

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Introducing COCOS: codon consequence scanner for annotating reading frame changes induced by stop-lost and frame shift variants

Bioinformatics ◽

10.1093/bioinformatics/btw820 ◽

2017 ◽

pp. btw820

Author(s):

Mariusz Butkiewicz ◽

Jonathan L. Haines ◽

William S. Bush

Keyword(s):

Reading Frame ◽

Frame Shift

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Recombinant Expression and Characterization of the Major β-Lactamase of Mycobacterium tuberculosis

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.42.6.1375 ◽

1998 ◽

Vol 42 (6) ◽

pp. 1375-1381 ◽

Cited By ~ 39

Author(s):

Rama Kishan R. Voladri ◽

David L. Lakey ◽

Steven H. Hennigan ◽

Barbara E. Menzies ◽

Kathryn M. Edwards ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Recombinant Expression ◽

Gel Filtration ◽

Multidrug Resistant ◽

Chromosomal Dna ◽

Reading Frame ◽

Major Mechanism ◽

Dna Library ◽

Class A ◽

Resistant Tuberculosis

ABSTRACT New antibiotic regimens are needed for the treatment of multidrug-resistant tuberculosis. Mycobacterium tuberculosis has a thick peptidoglycan layer, and the penicillin-binding proteins involved in its biosynthesis are inhibited by clinically relevant concentrations of β-lactam antibiotics. β-Lactamase production appears to be the major mechanism by whichM. tuberculosis expresses β-lactam resistance. β-Lactamases from the broth supernatant of 3- to 4-week-old cultures of M. tuberculosis H37Ra were partially purified by sequential gel filtration chromatography and chromatofocusing. Three peaks of β-lactamase activity with pI values of 5.1, 4.9, and 4.5, respectively, and which accounted for 10, 78, and 12% of the total postchromatofocusing β-lactamase activity, respectively, were identified. The β-lactamases with pI values of 5.1 and 4.9 were kinetically indistinguishable and exhibited predominant penicillinase activity. In contrast, the β-lactamase with a pI value of 4.5 showed relatively greater cephalosporinase activity. An open reading frame in cosmid Y49 of the DNA library of M. tuberculosis H37Rv with homology to known class A β-lactamases was amplified from chromosomal DNA of M. tuberculosis H37Ra by PCR and was overexpressed in Escherichia coli. The recombinant enzyme was kinetically similar to the pI 5.1 and 4.9 enzymes purified directly from M. tuberculosis. It exhibited predominant penicillinase activity and was especially active against azlocillin. It was inhibited by clavulanic acid andm-aminophenylboronic acid but not by EDTA. We conclude that the major β-lactamase of M. tuberculosis is a class A β-lactamase with predominant penicillinase activity. A second, minor β-lactamase with relatively greater cephalosporinase activity is also present.

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CHMP1 is a novel nuclear matrix protein affecting chromatin structure and cell-cycle progression

Journal of Cell Science ◽

10.1242/jcs.114.13.2383 ◽

2001 ◽

Vol 114 (13) ◽

pp. 2383-2393 ◽

Cited By ~ 4

Author(s):

Daniel R. Stauffer ◽

Tiffani L. Howard ◽

Thihan Nyun ◽

Stanley M. Hollenberg

Keyword(s):

Gene Silencing ◽

Nuclear Matrix ◽

Cell Cycle Progression ◽

Matrix Protein ◽

Multivesicular Body ◽

Polycomb Group ◽

Condensed Chromatin ◽

Stable Gene ◽

Body Protein ◽

Reading Frame

The Polycomb-group (PcG) is a diverse set of proteins required for maintenance of gene silencing during development. In a screen for conserved partners of the PcG protein Polycomblike (Pcl), we have identified a new protein, human CHMP1 (CHromatin Modifying Protein; CHarged Multivesicular body Protein), which is encoded by an alternative open reading frame in the PRSM1 gene and is conserved in both complex and simple eukaryotes. CHMP1 contains a predicted bipartite nuclear localization signal and distributes as distinct forms to the cytoplasm and the nuclear matrix in all cell lines tested. We have constructed a stable HEK293 cell line that inducibly overexpresses CHMP1 under ecdysone control. Overexpressed CHMP1 localizes to a punctate subnuclear pattern, encapsulating regions of nuclease-resistant, condensed chromatin. These novel structures are also frequently surrounded by increased histone H3 phosphorylation and acetylation. CHMP1 can recruit a PcG protein, BMI1, to these regions of condensed chromatin and can cooperate with co-expressed vertebrate Pcl in a Xenopus embryo PcG assay; this is consistent with a role in PcG function. In combination, these observations suggest that CHMP1 plays a role in stable gene silencing within the nucleus.

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Characterization of Guided Entry of Tail-Anchored Proteins 3 Homologues in Mycobacterium tuberculosis

Journal of Bacteriology ◽

10.1128/jb.00159-19 ◽

2019 ◽

Vol 201 (14) ◽

Author(s):

Kuan Hu ◽

Ashley T. Jordan ◽

Susan Zhang ◽

Avantika Dhabaria ◽

Amanda Kovach ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Structure Prediction ◽

Bacterial Species ◽

Normal Growth ◽

Anion Transport ◽

Content Type ◽

Reading Frame ◽

Anion Transporters ◽

Anion Transporter

ABSTRACT We characterized an operon in Mycobacterium tuberculosis, Rv3679-Rv3680, in which each open reading frame is annotated to encode “anion transporter ATPase” homologues. Using structure prediction modeling, we found that Rv3679 and Rv3680 more closely resemble the guided entry of tail-anchored proteins 3 (Get3) chaperone in eukaryotes. Get3 delivers proteins into the membranes of the endoplasmic reticulum and is essential for the normal growth and physiology of some eukaryotes. We sought to characterize the structures of Rv3679 and Rv3680 and test if they have a role in M. tuberculosis pathogenesis. We solved crystal structures of the nucleotide-bound Rv3679-Rv3680 complex at 2.5 to 3.2 Å and show that while it has some similarities to Get3 and ArsA, there are notable differences, including that these proteins are unlikely to be involved in anion transport. Deletion of both genes did not reveal any conspicuous growth defects in vitro or in mice. Collectively, we identified a new class of proteins in bacteria with similarity to Get3 complexes, the functions of which remain to be determined. IMPORTANCE Numerous bacterial species encode proteins predicted to have similarity with Get3- and ArsA-type anion transporters. Our studies provide evidence that these proteins, which we named BagA and BagB, are unlikely to be involved in anion transport. In addition, BagA and BagB are conserved in all mycobacterial species, including the causative agent of leprosy, which has a highly decayed genome. This conservation suggests that BagAB constitutes a part of the core mycobacterial genome and is needed for some yet-to-be-determined part of the life cycle of these organisms.

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The Largest Open Reading Frame (pks12) in the Mycobacterium tuberculosis Genome Is Involved in Pathogenesis and Dimycocerosyl Phthiocerol Synthesis

Infection and Immunity ◽

10.1128/iai.71.7.3794-3801.2003 ◽

2003 ◽

Vol 71 (7) ◽

pp. 3794-3801 ◽

Cited By ~ 49

Author(s):

Tatiana D. Sirakova ◽

Vinod S. Dubey ◽

Hwa-Jung Kim ◽

Michael H. Cynamon ◽

Pappachan E. Kolattukudy

Keyword(s):

Fatty Acids ◽

Mycobacterium Tuberculosis ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Immunoblot Analysis ◽

Open Reading Frame ◽

Pcr Analysis ◽

Reading Frame ◽

Sds Page

ABSTRACT The cell wall lipids in Mycobacterium tuberculosis are probably involved in pathogenesis. The largest open reading frame in the genome of M. tuberculosis H37Rv, pks12, is unique in that it encodes two sets of domains needed to produce fatty acids. A pks12-disrupted mutant was produced, and disruption was confirmed by both PCR analysis and Southern blotting. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis showed that a 430-kDa protein band present in the wild type was missing in the mutant. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MS) and liquid chromatography (LC)-MS analysis of tryptic peptides showed that 54 peptides distributed throughout this protein matched the pks12-encoded sequence. Biochemical analysis using [1-14C]propionate as the radiotracer showed that the pks12 mutant was deficient in the synthesis of dimycocerosyl phthiocerol (DIM). SDS-PAGE, immunoblot analysis of proteins, and analysis of fatty acids showed that the mutant can produce mycocerosic acids. Thus, the pks12 gene is probably involved in the synthesis of phthiocerol, the diol required for DIM synthesis. Growth of the pks12 mutant was attenuated in mouse alveolar macrophage cell line MH-S, and the virulence of the mutant in vivo was highly attenuated in a murine model. Thus, pks12 probably participates in DIM production and its expression is involved in pathogenesis.

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PPE Protein (Rv3873) from DNA Segment RD1 of Mycobacterium tuberculosis: Strong Recognition of Both Specific T-Cell Epitopes and Epitopes Conserved within the PPE Family

Infection and Immunity ◽

10.1128/iai.71.11.6116-6123.2003 ◽

2003 ◽

Vol 71 (11) ◽

pp. 6116-6123 ◽

Cited By ~ 84

Author(s):

Limei Meng Okkels ◽

Inger Brock ◽

Frank Follmann ◽

Else Marie Agger ◽

Sandra M. Arend ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

T Cell ◽

Mononuclear Cells ◽

Vaccine Development ◽

Significant Proportion ◽

Putative Open Reading Frame ◽

T Cell Epitopes ◽

Peripheral Blood Mononuclear ◽

Reading Frame ◽

T Cell Antigens

ABSTRACT Proteins encoded by DNA segment RD1 of Mycobacterium tuberculosis have recently been demonstrated to play important roles in bacterial virulence, vaccine development, and diagnostic reagent design. Previously, we characterized two immunodominant T-cell antigens, the early secreted antigen target (ESAT-6) and the 10-kDa culture filtrate protein (CFP10), which are encoded by the esx-lhp operon in this region. In the present study we characterized a third putative open reading frame in this region, rv3873, which encodes a PPE protein. We found that the rv3873 gene is expressed in M. tuberculosis H37Rv and that the native protein, Rv3873, is predominantly associated with the mycobacterial cell or wall. When tested as a His-tagged recombinant protein, Rv3873 stimulated high levels of gamma interferon secretion in peripheral blood mononuclear cells isolated from tuberculosis (TB) patients, as well as from healthy tuberculin purified protein derivative-positive donors. In contrast to other RD1-encoded antigens, Rv3873 was also found to be recognized by a significant proportion of Mycobacterium bovis BCG-vaccinated donors. Epitope mapping performed with overlapping peptides revealed a broad pattern of T-cell recognition comprising both TB-specific epitopes and epitopes also recognized by BCG-vaccinated donors. The immunodominant epitope (residues 118 to 135) for both TB patients and BCG-vaccinated individuals was found to be highly conserved among a large number of PPE family members.

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Environmental and social factors impacting on epidemic and endemic tuberculosis: a modelling analysis

Royal Society Open Science ◽

10.1098/rsos.170726 ◽

2018 ◽

Vol 5 (1) ◽

pp. 170726 ◽

Cited By ~ 5

Author(s):

Chacha M. Issarow ◽

Nicola Mulder ◽

Robin Wood

Keyword(s):

Mycobacterium Tuberculosis ◽

Social Factors ◽

Environmental Conditions ◽

Population Level ◽

Transmission Probability ◽

Infectious Dose ◽

Air Volume ◽

Effective Contact ◽

Modelling Analysis ◽

The Mean

Tuberculosis (TB) transmission results from the interaction between infective sources and susceptible individuals within enabling socio-environmental conditions. As TB is an airborne pathogen, the transmission probability is determined by the volume of air inhaled from an infected source and the concentration of Mycobacterium tuberculosis containing respirable particles (doses) per volume of air. In this study, we model the contributions of infectious dose production, prevalence of infectious cases and daily rebreathed air volume (RAV) for defining the boundary conditions necessary to sustain endemic TB transmission at the population level. Results suggest that in areas with high RAV (range 300–1000 l d −1 ), such as prisons, TB transmission is contributed by both super-spreaders (exhaling ≥10 infectious doses hr −1 ) and lower infectivity individuals (exhaling less than 10 infectious doses hr −1 ). In settings with a low quantity of RAV (less than 100 l d −1 ), TB transmission occurs only from super-spreaders. Point-source epidemics occur in low rebreathed environments when super-spreaders infect a number of susceptibles but subsequent transmission is limited by the mean infectivity of secondary cases. By contrast, endemic TB occurs in poor socio-environmental conditions where mean infectivity cases are able to maintain a sufficiently high effective contact number.

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Enhancement of the Local CD8+ T-Cellular Immune Response to Mycobacterium tuberculosis in BCG-Primed Mice after Intranasal Administration of Influenza Vector Vaccine Carrying TB10.4 and HspX Antigens

Vaccines ◽

10.3390/vaccines9111273 ◽

2021 ◽

Vol 9 (11) ◽

pp. 1273

Author(s):

Kirill Vasilyev ◽

Anna-Polina Shurygina ◽

Natalia Zabolotnykh ◽

Mariia Sergeeva ◽

Ekaterina Romanovskaya-Romanko ◽

...

Keyword(s):

Immune Response ◽

Mycobacterium Tuberculosis ◽

Cellular Immune Response ◽

Vaccine Candidate ◽

Protective Efficacy ◽

Adaptive Immune Response ◽

Reading Frame ◽

Vector Vaccine ◽

Specific Effector ◽

Cellular Immune

BCG is the only licensed vaccine against Mycobacterium tuberculosis (M.tb) infection. Due to its intramuscular administration route, BCG is unable to induce a local protective immune response in the respiratory system. Moreover, BCG has a diminished ability to induce long-lived memory T-cells which are indispensable for antituberculosis protection. Recently we described the protective efficacy of new mucosal TB vaccine candidate based on recombinant attenuated influenza vector (Flu/THSP) co-expressing TB10.4 and HspX proteins of M.tb within an NS1 influenza protein open reading frame. In the present work, the innate and adaptive immune response to immunization with the Flu/THSP and the immunological properties of vaccine candidate in the BCG-prime → Flu/THSP vector boost vaccination scheme are studied in mice. It was shown that the mucosal administration of Flu/THSP induces the incoming of interstitial macrophages in the lung tissue and stimulates the expression of co-stimulatory CD86 and CD83 molecules on antigen-presenting cells. The T-cellular immune response to Flu/THSP vector was mediated predominantly by the IFNγ-producing CD8+ lymphocytes. BCG-prime → Flu/THSP vector boost immunization scheme was shown to protect mice from severe lung injury caused by M.tb infection due to the enhanced T-cellular immune response, mediated by antigen-specific effector and central memory CD4+ and CD8+ T-lymphocytes.

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