Deficiency of Double-Strand DNA Break Repair Does Not Impair Mycobacterium tuberculosis Virulence in Multiple Animal Models of Infection

ABSTRACTMycobacterium tuberculosispersistence within its human host requires mechanisms to resist the effector molecules of host immunity, which exert their bactericidal effects through damaging pathogen proteins, membranes, and DNA. Substantial evidence indicates that bacterial pathogens, includingM. tuberculosis, require DNA repair systems to repair the DNA damage inflicted by the host during infection, but the role of double-strand DNA break (DSB) repair systems is unclear. Double-strand DNA breaks are the most cytotoxic form of DNA damage and must be repaired for chromosome replication to proceed.M. tuberculosiselaborates three genetically distinct DSB repair systems: homologous recombination (HR), nonhomologous end joining (NHEJ), and single-strand annealing (SSA). NHEJ, which repairs DSBs in quiescent cells, may be particularly relevant toM. tuberculosislatency. However, very little information is available about the phenotype of DSB repair-deficientM. tuberculosisin animal models of infection. Here we testedM. tuberculosisstrains lacking NHEJ (a ΔkuΔligDstrain), HR (a ΔrecAstrain), or both (a ΔrecAΔkustrain) in C57BL/6J mice, C3HeB/FeJ mice, guinea pigs, and a mouse hollow-fiber model of infection. We found no difference in bacterial load, histopathology, or host mortality between wild-type and DSB repair mutant strains in any model of infection. These results suggest that the animal models tested do not inflict DSBs on the mycobacterial chromosome, that other repair pathways can compensate for the loss of NHEJ and HR, or that DSB repair is not required forM. tuberculosispathogenesis.

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Monkey Models of Tuberculosis: Lessons Learned

Infection and Immunity ◽

10.1128/iai.02850-14 ◽

2014 ◽

Vol 83 (3) ◽

pp. 852-862 ◽

Cited By ~ 45

Author(s):

Juliet C. Peña ◽

Wen-Zhe Ho

Keyword(s):

Mycobacterium Tuberculosis ◽

Animal Models ◽

Nonhuman Primates ◽

Lessons Learned ◽

Preclinical Testing ◽

Content Type ◽

Cynomolgus Macaques ◽

Drug Regimens

The use of animal models has been invaluable for studying the pathogenesis ofMycobacterium tuberculosisinfection, as well as for testing the efficacy of vaccines and drug regimens for tuberculosis. Among the applied animal models, nonhuman primates, particularly macaques, share the greatest anatomical and physiological similarities with humans. As such, macaque models have been used for investigating tuberculosis pathogenesis and preclinical testing of drugs and vaccines. This review focuses on published major studies which illustrate how the rhesus and cynomolgus macaques have enriched and may continue to advance the field of global tuberculosis research.

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Roles of Caenorhabditis elegans WRN Helicase in DNA Damage Responses, and a Comparison with Its Mammalian Homolog: A Mini-Review

Gerontology ◽

10.1159/000439200 ◽

2015 ◽

Vol 62 (3) ◽

pp. 296-303 ◽

Cited By ~ 6

Author(s):

Jin-Sun Ryu ◽

Hyeon-Sook Koo

Keyword(s):

Dna Damage ◽

Caenorhabditis Elegans ◽

Werner Syndrome ◽

Model Organisms ◽

Current Status ◽

Helicase Domain ◽

Replication Forks ◽

Dna Breaks ◽

Double Strand Dna Breaks ◽

Double Strand Dna

Werner syndrome protein (WRN) is unusual among RecQ family DNA helicases in having an additional exonuclease activity. WRN is involved in the repair of double-strand DNA breaks via the homologous recombination and nonhomologous end joining pathways, and also in the base excision repair pathway. In addition, the protein promotes the recovery of stalled replication forks. The helicase activity is thought to unwind DNA duplexes, thereby moving replication forks or Holliday junctions. The targets of the exonuclease could be the nascent DNA strands at a replication fork or the ends of double-strand DNA breaks. However, it is not clear which enzyme activities are essential for repairing different types of DNA damage. Model organisms such as mice, flies, and worms deficient in WRN homologs have been investigated to understand the physiological results of defects in WRN activity. Premature aging, the most remarkable characteristic of Werner syndrome, is also seen in the mutant mice and worms, and hypersensitivity to DNA damage has been observed in WRN mutants of all three model organisms, pointing to conservation of the functions of WRN. In the nematode Caenorhabditis elegans, the WRN homolog contains a helicase domain but no exonuclease domain, so that this animal is very useful for studying the in vivo functions of the helicase without interference from the activity of the exonuclease. Here, we review the current status of investigations of C. elegans WRN-1 and discuss its functional differences from the mammalian homologs.

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HPF1 dynamically controls the PARP1/2 balance between initiating and elongating ADP-ribose modifications

Nature Communications ◽

10.1038/s41467-021-27043-8 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Marie-France Langelier ◽

Ramya Billur ◽

Aleksandr Sverzhinsky ◽

Ben E. Black ◽

John M. Pascal

Keyword(s):

Dna Damage ◽

Dna Damage Response ◽

Chain Elongation ◽

Dna Breaks ◽

Data Support ◽

Damage Response ◽

Hit And Run ◽

Dna Break ◽

Structural Insights ◽

In Cells

AbstractPARP1 and PARP2 produce poly(ADP-ribose) in response to DNA breaks. HPF1 regulates PARP1/2 catalytic output, most notably permitting serine modification with ADP-ribose. However, PARP1 is substantially more abundant in cells than HPF1, challenging whether HPF1 can pervasively modulate PARP1. Here, we show biochemically that HPF1 efficiently regulates PARP1/2 catalytic output at sub-stoichiometric ratios matching their relative cellular abundances. HPF1 rapidly associates/dissociates from multiple PARP1 molecules, initiating serine modification before modification initiates on glutamate/aspartate, and accelerating initiation to be more comparable to elongation reactions forming poly(ADP-ribose). This “hit and run” mechanism ensures HPF1 contributions to PARP1/2 during initiation do not persist and interfere with PAR chain elongation. We provide structural insights into HPF1/PARP1 assembled on a DNA break, and assess HPF1 impact on PARP1 retention on DNA. Our data support the prevalence of serine-ADP-ribose modification in cells and the efficiency of serine-ADP-ribose modification required for an acute DNA damage response.

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Monitoring Tuberculosis Drug Activity in Live Animals by Using Near-Infrared Fluorescence Imaging

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01280-19 ◽

2019 ◽

Vol 63 (12) ◽

Author(s):

Raphael Sommer ◽

Stewart T. Cole

Keyword(s):

Mycobacterium Tuberculosis ◽

Near Infrared ◽

Imaging System ◽

Limit Of Detection ◽

Bacterial Load ◽

Genetically Engineered ◽

Fluorescent Imaging ◽

Effective Vaccine ◽

Content Type ◽

Immunodeficient Mice

ABSTRACT Worldwide, tuberculosis (TB) is the leading cause of death due to infection with a single pathogenic agent, Mycobacterium tuberculosis. In the absence of an effective vaccine, new, more powerful antibiotics are required to halt the growing spread of multidrug-resistant strains and to shorten the duration of TB treatment. However, assessing drug efficacy at the preclinical stage remains a long and fastidious procedure that delays the progression of drugs down the pipeline and towards the clinic. In this investigation, we report the construction, optimization, and characterization of genetically engineered near-infrared (NIR) fluorescent reporter strains of the pathogens Mycobacterium marinum and Mycobacterium tuberculosis that enable the direct visualization of bacteria in infected zebrafish and mice, respectively. Fluorescence could be measured precisely in infected immunodeficient mice, while its intensity appeared to be below the limit of detection in immunocompetent mice, probably because of the lower bacterial load obtained in these animals. Furthermore, we show that the fluorescence level accurately reflects the bacterial load, as determined by CFU enumeration, thus enabling the efficacy of antibiotic treatment to be assessed in live animals in real time. The NIR fluorescent imaging system disclosed here is a valuable resource for TB research and can serve to accelerate drug development.

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Efficient 5-OP-RU-Induced Enrichment of Mucosa-Associated Invariant T Cells in the Murine Lung Does Not Enhance Control of Aerosol Mycobacterium tuberculosis Infection

Infection and Immunity ◽

10.1128/iai.00524-20 ◽

2020 ◽

Vol 89 (1) ◽

pp. e00524-20 ◽

Cited By ~ 3

Author(s):

Charles Kyriakos Vorkas ◽

Olivier Levy ◽

Miroslav Skular ◽

Kelin Li ◽

Jeffrey Aubé ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Bacterial Infections ◽

Cell Activation ◽

Bacterial Load ◽

Cell Subset ◽

Mycobacterium Tuberculosis Infection ◽

Class I Mhc ◽

Content Type ◽

Mait Cells ◽

Mait Cell

ABSTRACTMucosa-associated invariant T (MAIT) cells are an innate-like T cell subset in mammals that recognize microbial vitamin B metabolites presented by the evolutionarily conserved major histocompatibility complex class I (MHC I)-related molecule, MR1. Emerging data suggest that MAIT cells may be an attractive target for vaccine-induced protection against bacterial infections because of their rapid cytotoxic responses at mucosal services to a widely conserved bacterial ligand. In this study, we tested whether a MAIT cell priming strategy could protect against aerosol Mycobacterium tuberculosis infection in mice. Intranasal costimulation with the lipopeptide Toll-like receptor (TLR)2/6 agonist, Pam2Cys (P2C), and the synthetic MR1 ligand, 5-OP-RU, resulted in robust expansion of MAIT cells in the lung. Although MAIT cell priming significantly enhanced MAIT cell activation and expansion early after M. tuberculosis challenge, these MAIT cells did not restrict M. tuberculosis bacterial load. MAIT cells were depleted by the onset of the adaptive immune response, with decreased detection of granzyme B+ and gamma interferon (IFN-γ)+ MAIT cells relative to that in uninfected P2C/5-OP-RU-treated mice. Decreasing the infectious inoculum, varying the time between priming and aerosol infection, and testing MAIT cell priming in nitric oxide synthase 2 (NOS2)-deficient mice all failed to reveal an effect of P2C/5-OP-RU-induced MAIT cells on M. tuberculosis control. We conclude that intranasal MAIT cell priming in mice induces early MAIT cell activation and expansion after M. tuberculosis exposure, without attenuating M. tuberculosis growth, suggesting that MAIT cell enrichment in the lung is not sufficient to control M. tuberculosis infection.

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Phase I study of sequential sapacitabine and seliciclib in patients with advanced solid tumors.

Journal of Clinical Oncology ◽

10.1200/jco.2012.30.15_suppl.3053 ◽

2012 ◽

Vol 30 (15_suppl) ◽

pp. 3053-3053

Author(s):

Geoffrey Shapiro ◽

Eunice Lee Kwak ◽

James M. Cleary ◽

Sara Tolaney ◽

Leena Gandhi ◽

...

Keyword(s):

Dna Damage ◽

Phase I ◽

Mutation Carrier ◽

Phase I Study ◽

Brca Mutation ◽

Fold Increase ◽

Brca Mutation Carrier ◽

Dna Breaks ◽

Dsb Repair ◽

Skin Biopsies

3053 Background: Sapacitabine is an orally administered nucleoside analogue; the active metabolite CNDAC generates ss DNA breaks that are converted to ds DNA breaks (DSB) during subsequent replication, resulting in cell death if unrepaired. CNDAC-induced DSB repair is dependent on homologous recombination (HR). Seliciclib is a potent orally bioavailable inhibitor of CDK2, 7 and 9, and sensitizes cells to CNDAC by decreasing DSB repair via compromise of HR protein activation and transcriptional inhibition of HR components. This phase I study evaluates sequential sapacitabine and seliciclib. Methods: Dose escalation was conducted in patients with incurable solid tumors and adequate organ function with sapacitabine b.i.d. x 7 consecutive days (d1-7), seliciclib b.i.d. x 3 consecutive days (d8-10) followed by 11 days of rest. At least 3 patients were evaluated per dose level. MTD was the highest dose level at which less than one-third of at least 6 patients experienced cycle 1 DLT. Skin biopsies were obtained to assess DNA damage following sapacitabine (d8 vs pre-treatment) and further augmentation of DNA damage after seliciclib (d11 vs d8). Results: 27 patients were treated. The MTD and RP2D is sapacitabine 50 mg b.i.d./seliciclib 1200 mg b.i.d. DLTs were reversible transaminase elevations and neutropenia. The most frequent adverse events (all cycles, regardless of causality) included anorexia, fatigue, abdominal pain, dizziness, nausea, anemia, neutropenia, creatinine elevation, hyperglycemia, hyperbilirubinemia, hypophosphatemia, hypokalemia and hypomagnesemia, the majority mild to moderate in intensity. Skin biopsies showed a 2.3-fold increase in H2AX staining post-sapacitabine (n=16; p=0.007) and a further 0.58-fold increase post-seliciclib (n=12; p=0.069). Two confirmed PRs occurred in patients with pancreatic and breast cancer, both BRCA mutation carriers. SD as best response >/= 12 weeks was observed in 6 additional patients, including one BRCA mutation carrier with ovarian cancer (ongoing at 24 weeks). Conclusions: Sequential sapacitabine and seliciclib is safe with preliminary antitumor activity. BRCA mutation carrier status may be a potential biomarker for response across multiple tumor types.

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Activation of the DNA Damage Checkpoint in Yeast Lacking the Histone Chaperone Anti-Silencing Function 1

Molecular and Cellular Biology ◽

10.1128/mcb.24.23.10313-10327.2004 ◽

2004 ◽

Vol 24 (23) ◽

pp. 10313-10327 ◽

Cited By ~ 72

Author(s):

Christopher Josh Ramey ◽

Susan Howar ◽

Melissa Adkins ◽

Jeffrey Linger ◽

Judson Spicer ◽

...

Keyword(s):

Cell Cycle ◽

Dna Damage ◽

Dna Replication ◽

Chromatin Structure ◽

Fluorescent Protein ◽

Histone Chaperone ◽

Dna Damage Checkpoint ◽

Genomic Integrity ◽

Dna Breaks ◽

Double Strand Dna

ABSTRACT The packaging of the eukaryotic genome into chromatin is likely to be important for the maintenance of genomic integrity. Chromatin structures are assembled onto newly synthesized DNA by the action of chromatin assembly factors, including anti-silencing function 1 (ASF1). To investigate the role of chromatin structure in the maintenance of genomic integrity, we examined budding yeast lacking the histone chaperone Asf1p. We found that yeast lacking Asf1p accumulate in metaphase of the cell cycle due to activation of the DNA damage checkpoint. Furthermore, yeast lacking Asf1p are highly sensitive to mutations in DNA polymerase alpha and to DNA replicational stresses. Although yeast lacking Asf1p do complete DNA replication, they have greatly elevated rates of DNA damage occurring during DNA replication, as indicated by spontaneous Ddc2p-green fluorescent protein foci. The presence of elevated levels of spontaneous DNA damage in asf1 mutants is due to increased DNA damage, rather than the failure to repair double-strand DNA breaks, because asf1 mutants are fully functional for double-strand DNA repair. Our data indicate that the altered chromatin structure in asf1 mutants leads to elevated rates of spontaneous recombination, mutation, and DNA damage foci formation arising during DNA replication, which in turn activates cell cycle checkpoints that respond to DNA damage.

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Optimized CRISPR-Cas9 Genome Editing for Leishmania and Its Use To Target a Multigene Family, Induce Chromosomal Translocation, and Study DNA Break Repair Mechanisms

mSphere ◽

10.1128/msphere.00340-16 ◽

2017 ◽

Vol 2 (1) ◽

Cited By ~ 28

Author(s):

Wen-Wei Zhang ◽

Patrick Lypaczewski ◽

Greg Matlashewski

Keyword(s):

Genome Editing ◽

Chromosomal Translocations ◽

Content Type ◽

Crispr System ◽

Repair Mechanisms ◽

Human Leishmaniasis ◽

Dna Break ◽

Dna Break Repair ◽

Break Repair ◽

Double Strand Dna

ABSTRACT Leishmania parasites cause human leishmaniasis. To accelerate characterization of Leishmania genes for new drug and vaccine development, we optimized and simplified the CRISPR-Cas9 genome-editing tool for Leishmania. We show that co-CRISPR targeting of the miltefosine transporter gene and serial transfections of an oligonucleotide donor significantly eased isolation of edited mutants. This cotargeting strategy was efficiently used to delete all 11 members of the A2 virulence gene family. This technical advancement is valuable, since there are many gene clusters and supernumerary chromosomes in the various Leishmania species and isolates. We simplified this CRISPR system by developing a gRNA and Cas9 coexpression vector which could be used to delete genes in various Leishmania species. This CRISPR system could also be used to generate specific chromosomal translocations, which will help in the study of Leishmania gene expression and transcription control. This study also provides new information about double-strand DNA break repair mechanisms in Leishmania. CRISPR-Cas9-mediated genome editing has recently been adapted for Leishmania spp. parasites, the causative agents of human leishmaniasis. We have optimized this genome-editing tool by selecting for cells with CRISPR-Cas9 activity through cotargeting the miltefosine transporter gene; mutation of this gene leads to miltefosine resistance. This cotargeting strategy integrated into a triple guide RNA (gRNA) expression vector was used to delete all 11 copies of the A2 multigene family; this was not previously possible with the traditional gene-targeting method. We found that the Leishmania donovani rRNA promoter is more efficient than the U6 promoter in driving gRNA expression, and sequential transfections of the oligonucleotide donor significantly eased the isolation of edited mutants. A gRNA and Cas9 coexpression vector was developed that was functional in all tested Leishmania species, including L. donovani, L. major, and L. mexicana. By simultaneously targeting sites from two different chromosomes, all four types of targeted chromosomal translocations were generated, regardless of the polycistronic transcription direction from the parent chromosomes. It was possible to use this CRISPR system to create a single conserved amino acid substitution (A189G) mutation for both alleles of RAD51, a DNA recombinase involved in homology-directed repair. We found that RAD51 is essential for L. donovani survival based on direct observation of the death of mutants with both RAD51 alleles disrupted, further confirming that this CRISPR system can reveal gene essentiality. Evidence is also provided that microhomology-mediated end joining (MMEJ) plays a major role in double-strand DNA break repair in L. donovani. IMPORTANCE Leishmania parasites cause human leishmaniasis. To accelerate characterization of Leishmania genes for new drug and vaccine development, we optimized and simplified the CRISPR-Cas9 genome-editing tool for Leishmania. We show that co-CRISPR targeting of the miltefosine transporter gene and serial transfections of an oligonucleotide donor significantly eased isolation of edited mutants. This cotargeting strategy was efficiently used to delete all 11 members of the A2 virulence gene family. This technical advancement is valuable, since there are many gene clusters and supernumerary chromosomes in the various Leishmania species and isolates. We simplified this CRISPR system by developing a gRNA and Cas9 coexpression vector which could be used to delete genes in various Leishmania species. This CRISPR system could also be used to generate specific chromosomal translocations, which will help in the study of Leishmania gene expression and transcription control. This study also provides new information about double-strand DNA break repair mechanisms in Leishmania.

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Mode of Action of Clofazimine and Combination Therapy with Benzothiazinones against Mycobacterium tuberculosis

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00395-15 ◽

2015 ◽

Vol 59 (8) ◽

pp. 4457-4463 ◽

Cited By ~ 57

Author(s):

Benoit Lechartier ◽

Stewart T. Cole

Keyword(s):

Mycobacterium Tuberculosis ◽

Bacterial Load ◽

Multidrug Resistant ◽

Content Type ◽

Resistant Tuberculosis ◽

Multidrug Resistant Tuberculosis ◽

Drug Resistant Strains ◽

Transfer Chain

ABSTRACTClofazimine (CZM) is an antileprosy drug that was recently repurposed for treatment of multidrug-resistant tuberculosis. InMycobacterium tuberculosis, CZM appears to act as a prodrug, which is reduced by NADH dehydrogenase (NDH-2), to release reactive oxygen species upon reoxidation by O2. CZM presumably competes with menaquinone (MK-4), a key cofactor in the mycobacterial electron transfer chain, for its reduction by NDH-2. We studied the effect of MK-4 supplementation on the activity of CZM againstM. tuberculosisand found direct competition between CZM and MK-4 for the cidal effect of CZM, against nonreplicating and actively growing bacteria, as MK-4 supplementation blocked the drug's activity against nonreplicating bacteria. We demonstrated that CZM, like bedaquiline, is synergisticin vitrowith benzothiazinones such as 2-piperazino-benzothiazinone 169 (PBTZ169), and this synergy also occurs against nonreplicating bacteria. The synergy between CZM and PBTZ169 was lost in an MK-4-rich medium, indicating that MK-4 is the probable link between their activities. The efficacy of the dual combination of CZM and PBTZ169 was testedin vivo, where a great reduction in bacterial load was obtained in a murine model of chronic tuberculosis. Taken together, these data confirm the potential of CZM in association with PBTZ169 as the basis for a new regimen against drug-resistant strains ofM. tuberculosis.

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High Mycobacterium tuberculosis Bacillary Loads Detected by Tuberculosis Molecular Bacterial Load Assay in Patient Stool: a Potential Alternative for Nonsputum Diagnosis and Treatment Response Monitoring of Tuberculosis

Microbiology Spectrum ◽

10.1128/spectrum.02100-21 ◽

2022 ◽

Author(s):

Emmanuel Musisi ◽

Abdul Sessolo ◽

Sylvia Kaswabuli ◽

Josephine Zawedde ◽

Patrick Byanyima ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Treatment Response ◽

Bacterial Load ◽

Diagnosis And Treatment ◽

Response Monitoring ◽

Sample Type ◽

Content Type ◽

Potential Alternative

This paper highlights the value of stool as a sample type for diagnosis of tuberculosis. While other studies have used DNA-based assays like the Xpert MTB/RIF and culture to detect Mycobacterium tuberculosis in stool, this is the first study that has applied TB-MBLA, an RNA-based assay, to quantify TB bacteria in stool.

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