scholarly journals Oxidation of dCTP contributes to antibiotic lethality in stationary-phase mycobacteria

2018 ◽  
Vol 115 (9) ◽  
pp. 2210-2215 ◽  
Author(s):  
Xiao-Yong Fan ◽  
Bi-Kui Tang ◽  
Yuan-Yuan Xu ◽  
Ang-Xuan Han ◽  
Kun-Xiong Shi ◽  
...  
Keyword(s):  
Stationary Phase ◽  
Double Strand Breaks ◽  
Oxygen Species ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Metabolic Perturbation ◽  
Dead End ◽  
Induced Mutagenesis ◽  
Antibiotic Efficacy ◽  
Mechanistic View

Growing evidence shows that generation of reactive oxygen species (ROS) derived from antibiotic-induced metabolic perturbation contribute to antibiotic lethality. However, our knowledge of the mechanisms by which antibiotic-induced oxidative stress actually kills cells remains elusive. Here, we show that oxidation of dCTP underlies ROS-mediated antibiotic lethality via induction of DNA double-strand breaks (DSBs). Deletion of mazG-encoded 5-OH-dCTP–specific pyrophosphohydrolase potentiates antibiotic killing of stationary-phase mycobacteria, but did not affect antibiotic efficacy in exponentially growing cultures. Critically, the effect of mazG deletion on potentiating antibiotic killing is associated with antibiotic-induced ROS and accumulation of 5-OH-dCTP. Independent lines of evidence presented here indicate that the increased level of DSBs observed in the ΔmazG mutant is a dead-end event accounting for enhanced antibiotic killing. Moreover, we provided genetic evidence that 5-OH-dCTP is incorporated into genomic DNA via error-prone DNA polymerase DnaE2 and repair of 5-OH-dC lesions via the endonuclease Nth leads to the generation of lethal DSBs. This work provides a mechanistic view of ROS-mediated antibiotic lethality in stationary phase and may have broad implications not only with respect to antibiotic lethality but also to the mechanism of stress-induced mutagenesis in bacteria.

scholarly journals Staphylococcal DNA Repair Is Required for Infection

mBio ◽  
2020 ◽  
Vol 11 (6) ◽  
Author(s):  
Kam Pou Ha ◽  
Rebecca S. Clarke ◽  
Gyu-Lee Kim ◽  
Jane L. Brittan ◽  
Jessica E. Rowley ◽  
...  
Keyword(s):  
Reactive Oxygen Species ◽  
Staphylococcus Aureus ◽  
Human Blood ◽  
Immune Cells ◽  
Double Strand Breaks ◽  
Oxygen Species ◽  
Dna Double Strand Breaks ◽  
Gram Positive ◽  
Content Type ◽  
Strand Breaks

ABSTRACT To cause infection, Staphylococcus aureus must withstand damage caused by host immune defenses. However, the mechanisms by which staphylococcal DNA is damaged and repaired during infection are poorly understood. Using a panel of transposon mutants, we identified the rexBA operon as being important for the survival of Staphylococcus aureus in whole human blood. Mutants lacking rexB were also attenuated for virulence in murine models of both systemic and skin infections. We then demonstrated that RexAB is a member of the AddAB family of helicase/nuclease complexes responsible for initiating the repair of DNA double-strand breaks. Using a fluorescent reporter system, we were able to show that neutrophils cause staphylococcal DNA double-strand breaks through reactive oxygen species (ROS) generated by the respiratory burst, which are repaired by RexAB, leading to the induction of the mutagenic SOS response. We found that RexAB homologues in Enterococcus faecalis and Streptococcus gordonii also promoted the survival of these pathogens in human blood, suggesting that DNA double-strand break repair is required for Gram-positive bacteria to survive in host tissues. Together, these data demonstrate that DNA is a target of host immune cells, leading to double-strand breaks, and that the repair of this damage by an AddAB-family enzyme enables the survival of Gram-positive pathogens during infection. IMPORTANCE To cause infection, bacteria must survive attack by the host immune system. For many bacteria, including the major human pathogen Staphylococcus aureus, the greatest threat is posed by neutrophils. These immune cells ingest the invading organisms and try to kill them with a cocktail of chemicals that includes reactive oxygen species (ROS). The ability of S. aureus to survive this attack is crucial for the progression of infection. However, it was not clear how the ROS damaged S. aureus and how the bacterium repaired this damage. In this work, we show that ROS cause breaks in the staphylococcal DNA, which must be repaired by a two-protein complex known as RexAB; otherwise, the bacterium is killed, and it cannot sustain infection. This provides information on the type of damage that neutrophils cause S. aureus and the mechanism by which this damage is repaired, enabling infection.

scholarly journals DNA double-strand breaks in the Toxoplasma gondii-infected cells by the action of reactive oxygen species

2020 ◽  
Vol 13 (1) ◽  
Author(s):  
Haohan Zhuang ◽  
Chaoqun Yao ◽  
Xianfeng Zhao ◽  
Xueqiu Chen ◽  
Yimin Yang ◽  
...  
Keyword(s):  
Dna Damage ◽  
Toxoplasma Gondii ◽  
Double Strand Breaks ◽  
Host Cells ◽  
Oxygen Species ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Damage Response ◽  
Infected Cells

Abstract Background Toxoplasma gondii is an obligate parasite of all warm-blooded animals around the globe. Once infecting a cell, it manipulates the host’s DNA damage response that is yet to be elucidated. The objectives of the present study were three-fold: (i) to assess DNA damages in T. gondii-infected cells in vitro; (ii) to ascertain causes of DNA damage in T. gondii-infected cells; and (iii) to investigate activation of DNA damage responses during T. gondii infection. Methods HeLa, Vero and HEK293 cells were infected with T. gondii at a multiplicity of infection (MOI) of 10:1. Infected cells were analyzed for a biomarker of DNA double-strand breaks (DSBs) γH2AX at 10 h, 20 h or 30 h post-infection using both western blot and immunofluorescence assay. Reactive oxygen species (ROS) levels were measured using 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA), and ROS-induced DNA damage was inhibited by a ROS inhibitor N-acetylcysteine (NAC). Lastly, DNA damage responses were evaluated by detecting the active form of ataxia telangiectasia mutated/checkpoint kinase 2 (ATM/CHK2) by western blot. Results γH2AX levels in the infected HeLa cells were significantly increased over time during T. gondii infection compared to uninfected cells. NAC treatment greatly reduced ROS and concomitantly diminished γH2AX in host cells. The phosphorylated ATM/CHK2 were elevated in T. gondii-infected cells. Conclusions Toxoplasma gondii infection triggered DNA DSBs with ROS as a major player in host cells in vitro. It also activated DNA damage response pathway ATM/CHK2. Toxoplasma gondii manages to keep a balance between survival and apoptosis of its host cells for the benefit of its own survival.

BCR/ABL Oncogenic Kinase Promotes Unfaithful Repair of the Reactive Oxygen Species - Dependent DNA Double-Strand Breaks.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 712-712 ◽  
Author(s):  
Tomasz Skorski ◽  
Michal O. Nowicki ◽  
Rafal Falinski ◽  
Mateusz Koptyra ◽  
Artur Slupianek ◽  
...  
Keyword(s):  
Reactive Oxygen Species ◽  
Cell Cycle ◽  
Dna Damage ◽  
Oxidative Dna Damage ◽  
Double Strand Breaks ◽  
Transformed Cells ◽  
Oxygen Species ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Abl Kinase

Abstract The oncogenic BCR/ABL tyrosine kinase induces constitutive DNA damage in Philadelphia chromosome (Ph1)-positive leukemia cells. We find that BCR/ABL kinase - induced reactive oxygen species (ROS) cause chronic oxidative DNA damage as indicated by an enzymatic assay detecting oxidized bases. These DNA lesions result in DNA double-strand breaks (DSBs) detected by comet assay, immunofluorescent gamma-H2AX nuclear foci and linker-ligation PCR (LL-PCR). Combined analysis of the length of LL-PCR products and the sequences of two reference genes DR-GFP and Na+/K+ ATPase revealed that ROS dependent DSBs occurred in the regions containing multiple, 5–9bp long stretches of G/C, in concordance with the notion that oxidative DNA damage is predominantly detected in G/C-rich sequences. Elevated numbers of DSBs were detected in BCR/ABL cell lines, murine bone marrow cells transformed with BCR/ABL and in CML patient samples, in comparison to normal counterparts. Inhibition of the BCR/ABL kinase by STI571 and diminishion of ROS activity by the ROS scavenger PDTC reduced DSBs formation. Cell cycle analysis revealed that most of these DSBs occur during S and G2/M phases, and are probably associated with the stalled replication forks. Homologous recombination repair (HRR) and non-homologous end-joining (NHEJ) represent two major mechanisms of DSBs repair in S and G2/M cell cycle phase. Using the in vivo recombination assay consisting of the DSB-dependent reconstitution of the green fluorescent protein (GFP) gene we found that HRR is stimulated in BCR/ABL-positive cells. In addition, in vitro assay measuring the activity of NHEJ revealed that this repair process is also activated by the BCR/ABL kinase. RAD51 and Ku70 play a key role in HRR and NHEJ, respectively. The reaction sites of HRR and NHEJ in the nuclei could be visualized by double-immunofluorescence detecting co-localization of gamma-H2AX foci (DSBs sites) with RAD51 (HRR sites) or Ku70 (NHEJ sites). Equal co-localization frequency of gamma-H2AX foci with RAD51 and Ku70 was detected, suggesting that both HRR and NHEJ play an important role in reparation of ROS-dependent DSBs in BCR/ABL-transformed cells. Analysis of the DSBs repair products in the reporter DR-GFP gene in BCR/ABL cells identified ~40% of HRR and ~60% of NHEJ events. Sequencing revealed point-mutations in HRR products and large deletions in NHEJ products in BCR/ABL-positive cells, but not in non-transformed cells. We propose that the following series of events may contribute to genomic instability of Ph1-positive leukemias: BCR/ABL → ROS → oxidative DNA damage → DSBs in proliferating cells → unfaithful HRR and NHEJ repair. Since BCR/ABL share many similarities with other members of the fusion tyrosine kinases (FTKs) family, these events may contribute to genomic instability of hematological malignancies caused by FTKs.

scholarly journals Through Its Nonstructural Protein NS1, Parvovirus H-1 Induces Apoptosis via Accumulation of Reactive Oxygen Species

2010 ◽  
Vol 84 (12) ◽  
pp. 5909-5922 ◽  
Author(s):  
Georgi Hristov ◽  
Melanie Krämer ◽  
Junwei Li ◽  
Nazim El-Andaloussi ◽  
Rodrigo Mora ◽  
...  
Keyword(s):  
Reactive Oxygen Species ◽  
Nonstructural Protein ◽  
Reactive Oxygen ◽  
Double Strand Breaks ◽  
Oxygen Species ◽  
Ns1 Protein ◽  
Dna Double Strand Breaks ◽  
Caspase 9 ◽  
Strand Breaks ◽  
Cycle Arrest

ABSTRACT The rat parvovirus H-1 (H-1PV) attracts high attention as an anticancer agent, because it is not pathogenic for humans and has oncotropic and oncosuppressive properties. The viral nonstructural NS1 protein is thought to mediate H-1PV cytotoxicity, but its exact contribution to this process remains undefined. In this study, we analyzed the effects of the H-1PV NS1 protein on human cell proliferation and cell viability. We show that NS1 expression is sufficient to induce the accumulation of cells in G2 phase, apoptosis via caspase 9 and 3 activation, and cell lysis. Similarly, cells infected with wild-type H-1PV arrest in G2 phase and undergo apoptosis. Furthermore, we also show that both expression of NS1 and H-1PV infection lead to higher levels of intracellular reactive oxygen species (ROS), associated with DNA double-strand breaks. Antioxidant treatment reduces ROS levels and strongly decreases NS1- and virus-induced DNA damage, cell cycle arrest, and apoptosis, indicating that NS1-induced ROS are important mediators of H-1PV cytotoxicity.

scholarly journals Regulation of Reactive Oxygen Species byAtmIs Essential for Proper Response to DNA Double-Strand Breaks in Lymphocytes

2006 ◽  
Vol 178 (1) ◽  
pp. 103-110 ◽  
Author(s):  
Keisuke Ito ◽  
Keiyo Takubo ◽  
Fumio Arai ◽  
Hitoshi Satoh ◽  
Sahoko Matsuoka ◽  
...  
Keyword(s):  
Reactive Oxygen Species ◽  
Reactive Oxygen ◽  
Double Strand Breaks ◽  
Oxygen Species ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Proper Response

scholarly journals Identification of a signature of evolutionarily conserved stress-induced mutagenesis in cancer

2021 ◽  
Author(s):  
Luis Humberto Cisneros ◽  
Kimberly J Bussey ◽  
Charles Vasque
Keyword(s):  
Double Strand Breaks ◽  
Whole Genome Sequencing Data ◽  
Dna Double Strand Breaks ◽  
Sequencing Data ◽  
Single Nucleotide ◽  
Strand Breaks ◽  
Evolutionarily Conserved ◽  
Induced Mutagenesis ◽  
Genomic Locations ◽  
Inherited Mutations

The clustering of mutations observed in cancer cells is reminiscent of the stress-induced mutagenesis (SIM) response in bacteria. SIM employs error-prone polymerases resulting in mutations concentrated around DNA double-strand breaks with an abundance that decays with genomic distance. We performed a quantitative study on single nucleotide variant calls for whole-genome sequencing data from 1950 tumors and non-inherited mutations from 129 normal samples. We introduce statistical methods to identify mutational clusters and quantify their distribution pattern. Our results show that mutations in both normal and cancer samples are indeed clustered and have shapes indicative of SIM. We found the genomic locations of groups of close mutations are more likely to be prevalent across normal samples than in cancer suggesting loss of regulation over the mutational process during carcinogenesis.

scholarly journals RexAB promotes the survival of Staphylococcus aureus exposed to multiple classes of antibiotics

2021 ◽  
Author(s):  
Rebecca S. Clarke ◽  
Kam Pou Ha ◽  
Andrew M. Edwards
Keyword(s):  
Reactive Oxygen Species ◽  
Staphylococcus Aureus ◽  
Dna Damage ◽  
Sos Response ◽  
Double Strand Breaks ◽  
Oxygen Species ◽  
Bacterial Survival ◽  
Wild Type ◽  
Dna Double Strand Breaks ◽  
Strand Breaks

Antibiotics inhibit essential bacterial processes, resulting in arrest of growth and in some cases cell death. Many antibiotics are also reported to trigger endogenous production of reactive oxygen species (ROS), which damage DNA, leading to induction of the mutagenic SOS response associated with the emergence of drug resistance. However, the type of DNA damage that arises and how this triggers the SOS response is largely unclear. We found that several different classes of antibiotic triggered dose-dependent induction of the SOS response in Staphylococcus aureus , indicative of DNA damage, including some bacteriostatic drugs. The SOS response was heterogenous and varied in magnitude between strains and antibiotics. However, in many cases, full induction of the SOS response was dependent upon the RexAB helicase/nuclease complex, which processes DNA double strand breaks to produce single-stranded DNA and facilitate RecA nucleoprotein filament formation. The importance of RexAB in repair of DNA was confirmed by measuring bacterial survival during antibiotic exposure, with most drugs having significantly greater bactericidal activity against rexB mutants relative to wild type strains. For some, but not all antibiotics there was no difference in bactericidal activity between wild type and rexB mutant under anaerobic conditions, indicative of a role for reactive oxygen species in mediating DNA damage. Taken together, this work confirms previous observations that several classes of antibiotics cause DNA damage in S. aureus and extends them by showing that processing of DNA double strand breaks by RexAB is a major trigger of the mutagenic SOS response and promotes bacterial survival.

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