Apurinic/Apyrimidinic Endonucleases of Mycobacterium tuberculosis Protect against DNA Damage but Are Dispensable for the Growth of the Pathogen in Guinea Pigs

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.

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Definition of the Mycobacterial SOS Box and Use To Identify LexA-Regulated Genes in Mycobacterium tuberculosis

Journal of Bacteriology ◽

10.1128/jb.184.12.3287-3295.2002 ◽

2002 ◽

Vol 184 (12) ◽

pp. 3287-3295 ◽

Cited By ~ 69

Author(s):

Elaine O. Davis ◽

Edith M. Dullaghan ◽

Lucinda Rand

Keyword(s):

Dna Damage ◽

Mycobacterium Tuberculosis ◽

Dna Polymerase ◽

Expression Data ◽

Repeat Family ◽

New Genes ◽

Lexa Binding ◽

Definition Of ◽

Inducible Genes ◽

Single Mismatch

ABSTRACT The bases of the mycobacterial SOS box important for LexA binding were determined by replacing each base with every other and examining the effect on the induction of a reporter gene following DNA damage. This analysis revealed that the SOS box was longer than originally thought by 2 bp in each half of the palindromic site. A search of the Mycobacterium tuberculosis genome sequence with the new consensus, TCGAAC(N)4GTTCGA, identified 4 sites which were perfect matches and 12 sites with a single mismatch which were predicted to bind LexA. Genes which could potentially be regulated by these SOS boxes were ascertained from their positions relative to the sites. Examination of expression data for these genes following DNA damage identified 12 new genes which are most likely regulated by LexA as well as the known M. tuberculosis DNA damage-inducible genes recA, lexA, and ruvC. Of these 12 genes, only 2 have a predicted function: dnaE2, a component of DNA polymerase III, and linB, which is similar to 1,3,4,6-tetrachloro-1,4-cylcohexadiene hydrolase. Curiously, of the remaining 10 genes predicted to be LexA regulated, 7 are members of the M. tuberculosis 13E12 repeat family, which has some of the characteristics of mobile elements.

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Drug treatment combined with BCG vaccination reduces disease reactivation in guinea pigs infected with Mycobacterium tuberculosis

Vaccine ◽

10.1016/j.vaccine.2011.12.114 ◽

2012 ◽

Vol 30 (9) ◽

pp. 1572-1582 ◽

Cited By ~ 13

Author(s):

Shaobin Shang ◽

Crystal A. Shanley ◽

Megan L. Caraway ◽

Eileen A. Orme ◽

Marcela Henao-Tamayo ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Drug Treatment ◽

Guinea Pigs ◽

Bcg Vaccination ◽

Disease Reactivation

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Vaccination with Recombinant Mycobacterium tuberculosis PknD Attenuates Bacterial Dissemination to the Brain in Guinea Pigs

PLoS ONE ◽

10.1371/journal.pone.0066310 ◽

2013 ◽

Vol 8 (6) ◽

pp. e66310 ◽

Cited By ~ 12

Author(s):

Ciaran Skerry ◽

Supriya Pokkali ◽

Michael Pinn ◽

Nicholas A. Be ◽

Jamie Harper ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Guinea Pigs ◽

Bacterial Dissemination ◽

The Brain

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Characterization of the Two Mycobacterium tuberculosis recA Promoters

Journal of Bacteriology ◽

10.1128/jb.185.20.6005-6015.2003 ◽

2003 ◽

Vol 185 (20) ◽

pp. 6005-6015 ◽

Cited By ~ 21

Author(s):

Krishna K. Gopaul ◽

Patricia C. Brooks ◽

Jean-François Prost ◽

Elaine O. Davis

Keyword(s):

Escherichia Coli ◽

Dna Damage ◽

Mycobacterium Tuberculosis ◽

Promoter Activity ◽

The Other ◽

Expression Level ◽

Promoter Elements ◽

Kinetics Of

ABSTRACT The recA gene of Mycobacterium tuberculosis is unusual in that it is expressed from two promoters, one of which, P1, is DNA damage inducible independently of LexA and RecA, while the other, P2, is regulated by LexA in the classical way (E. O. Davis, B. Springer, K. K. Gopaul, K. G. Papavinasasundaram, P. Sander, and E. C. Böttger, Mol. Microbiol. 46:791-800, 2002). In this study we characterized these two promoters in more detail. Firstly, we localized the promoter elements for each of the promoters, and in so doing we identified a mutation in each promoter which eliminates promoter activity. Interestingly, a motif with similarity to Escherichia coli σ70 −35 elements but located much closer to the −10 element is important for optimal expression of P1, whereas the sequence at the −35 location is not. Secondly, we found that the sequences flanking the promoters can have a profound effect on the expression level directed by each of the promoters. Finally, we examined the contribution of each of the promoters to recA expression and compared their kinetics of induction following DNA damage.

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Polyphosphate Deficiency in Mycobacterium tuberculosis Is Associated with Enhanced Drug Susceptibility and Impaired Growth in Guinea Pigs

Journal of Bacteriology ◽

10.1128/jb.00038-13 ◽

2013 ◽

Vol 195 (12) ◽

pp. 2839-2851 ◽

Cited By ~ 42

Author(s):

R. Singh ◽

M. Singh ◽

G. Arora ◽

S. Kumar ◽

P. Tiwari ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Guinea Pigs ◽

Drug Susceptibility

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Slow induction of RecA by DNA damage in Mycobacterium tuberculosis

Microbiology ◽

10.1099/00221287-147-12-3271 ◽

2001 ◽

Vol 147 (12) ◽

pp. 3271-3279 ◽

Cited By ~ 41

Author(s):

K. G. Papavinasasundaram ◽

Nicola A. Thomas ◽

Patricia C. Brooks ◽

Farahnaz Movahedzadeh ◽

Peter J. Jenner ◽

...

Keyword(s):

Dna Damage ◽

Mycobacterium Tuberculosis

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Arginine-deprivation–induced oxidative damage sterilizes Mycobacterium tuberculosis

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1808874115 ◽

2018 ◽

Vol 115 (39) ◽

pp. 9779-9784 ◽

Cited By ~ 22

Author(s):

Sangeeta Tiwari ◽

Andries J. van Tonder ◽

Catherine Vilchèze ◽

Vitor Mendes ◽

Sherine E. Thomas ◽

...

Keyword(s):

Oxidative Stress ◽

Dna Damage ◽

Mycobacterium Tuberculosis ◽

De Novo ◽

Target Space ◽

Biosynthesis Pathway ◽

Arginine Biosynthesis ◽

Arginine Deprivation

Reactive oxygen species (ROS)-mediated oxidative stress and DNA damage have recently been recognized as contributing to the efficacy of most bactericidal antibiotics, irrespective of their primary macromolecular targets. Inhibitors of targets involved in both combating oxidative stress as well as being required for in vivo survival may exhibit powerful synergistic action. This study demonstrates that the de novo arginine biosynthetic pathway in Mycobacterium tuberculosis (Mtb) is up-regulated in the early response to the oxidative stress-elevating agent isoniazid or vitamin C. Arginine deprivation rapidly sterilizes the Mtb de novo arginine biosynthesis pathway mutants ΔargB and ΔargF without the emergence of suppressor mutants in vitro as well as in vivo. Transcriptomic and flow cytometry studies of arginine-deprived Mtb have indicated accumulation of ROS and extensive DNA damage. Metabolomics studies following arginine deprivation have revealed that these cells experienced depletion of antioxidant thiols and accumulation of the upstream metabolite substrate of ArgB or ArgF enzymes. ΔargB and ΔargF were unable to scavenge host arginine and were quickly cleared from both immunocompetent and immunocompromised mice. In summary, our investigation revealed in vivo essentiality of the de novo arginine biosynthesis pathway for Mtb and a promising drug target space for combating tuberculosis.

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972. A Mycobacterium tuberculosis Secreted Lipid Triggers Cough Through a Neuronal Cough Receptor

Open Forum Infectious Diseases ◽

10.1093/ofid/ofz359.074 ◽

2019 ◽

Vol 6 (Supplement_2) ◽

pp. S33-S33

Author(s):

Paxton Cruz ◽

Cody Ruhl ◽

Michael Shiloh

Keyword(s):

Mycobacterium Tuberculosis ◽

Disease Transmission ◽

Guinea Pigs ◽

Cough Reflex ◽

Nociceptive Neurons ◽

Active Pulmonary Tuberculosis ◽

Neuronal Receptor ◽

Musculoskeletal Systems ◽

Cough Receptor

Abstract Background A hallmark symptom of active pulmonary tuberculosis vital for disease transmission is cough. The current paradigm for tuberculosis-related cough is that it results from airway damage or irritation. However, there is limited experimental data to support this theory, and whether Mycobacterium tuberculosis (Mtb) induces cough to facilitate its own transmission has not been explored. The cough reflex is a complex and coordinated event involving both the nervous and musculoskeletal systems initiated by particulate or chemical molecules activating nociceptive neurons, which sense pain or irritation. This activation induces a signaling cascade ultimately resulting in a cough. Respiratory nociceptive neurons innervate the airway of humans and most mammals and thus are poised to respond to noxious molecules to help protect the lung from damage. Because Mtb is a lung pathogen, cough is a primary mechanism of Mtb transmission, and respiratory nociceptive neurons activate cough, we hypothesized that Mtb produces molecules that stimulate cough thereby facilitating its spread from infected to uninfected individuals. We previously identified a cough molecule produced by Mtb, and in this work characterize its neuronal receptor using genetics, biochemistry, and pharmacology. Methods We used an in vitro neuronal activation bioassay to study Mtb cough-inducing molecules. We also used a biochemical assay to identify the cough receptor. Finally, we used gene silencing, biochemistry, and pharmacologic inhibition to validate and characterize the activity of the newly discovered cough receptor. Results We isolated a complex lipid produced by Mtb that activates nociceptive neurons. Both an organic Mtb extract and the purified molecule alone were sufficient to induce cough in a conscious guinea pig cough model and guinea pigs infected with wild-type Mtb cough much more frequently than guinea pigs infected with Mtb strains unable to produce nociceptive molecules. Using genetics, biochemistry, and pharmacology techniques, we identified and validated a cough receptor for the Mtb lipid expressed on nociceptive neurons. Conclusion We conclude that Mtb produces a molecule that activates nociceptive neurons and induces cough through a specific neuronal receptor. These findings have significant implications for our understanding of Mtb transmission. Disclosures All Authors: No reported Disclosures.

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