RexAB Is Essential for the Mutagenic Repair of Staphylococcus aureus DNA Damage Caused by Co-trimoxazole

ABSTRACT Co-trimoxazole (SXT) is a combination therapeutic that consists of sulfamethoxazole and trimethoprim that is increasingly used to treat skin and soft tissue infections caused by methicillin-resistant Staphylococcus aureus (MRSA). However, the use of SXT is limited to the treatment of low-burden, superficial S. aureus infections and its therapeutic value is compromised by the frequent emergence of resistance. As a first step toward the identification of approaches to enhance the efficacy of SXT, we examined the role of bacterial DNA repair in antibiotic susceptibility and mutagenesis. We found that mutants lacking the DNA repair complex RexAB had a modest SXT MIC that was 2-fold lower than that seen with wild-type strains but were killed 50-fold to 5,000-fold more efficiently by the combination antibiotic at the breakpoint concentration. SXT-mediated DNA damage occurred via both thymidine limitation and the generation of reactive oxygen species and triggered induction of the SOS response in a RexAB-dependent manner. SOS induction was associated with a 50% increase in the mutation rate, which may contribute to emergence of resistant strains during SXT therapy. In summary, this work determined that SXT caused DNA damage in S. aureus via both thymidine limitation and oxidative stress and that the damage was repaired by the RexAB complex, leading to induction of the mutagenic SOS response. Small-molecule inhibitors of RexAB could therefore have therapeutic value by increasing the efficacy of SXT and decreasing the emergence of drug resistance during treatment of infections caused by S. aureus.

Download Full-text

RexAB is essential for the mutagenic repair ofStaphylococcus aureusDNA damage caused by co-trimoxazole

10.1101/568196 ◽

2019 ◽

Cited By ~ 1

Author(s):

Rebecca S. Clarke ◽

Maya S. Bruderer ◽

Kam Pou Ha ◽

Andrew M. Edwards

Keyword(s):

Dna Damage ◽

Dna Repair ◽

Sos Response ◽

Dna Double Strand Breaks ◽

Bacterial Dna ◽

Strand Breaks ◽

Therapeutic Value ◽

Resistant Strains ◽

Emergence Of Resistance

AbstractCo-trimoxazole (SXT) is a combination therapeutic that consists of sulfamethoxazole and trimethoprim that is increasingly used to treat skin and soft-tissue infections caused by methicillin-resistantStaphylococcus aureus(MRSA). However, the use of SXT is limited to the treatment of low-burden, superficialS. aureusinfections and its therapeutic value is compromised by the frequent emergence of resistance. As a first step towards the identification of approaches to enhance the efficacy of SXT, we examined the role of bacterial DNA repair in antibiotic susceptibility and mutagenesis. This revealed that SXT caused DNA damage inS. aureusvia both thymidine limitation and the generation of reactive oxygen species. Then, using mutants defective for DNA repair, it was found that repair of this damage required the RexAB nuclease/helicase complex, indicating that SXT causes DNA double-strand breaks. Furthermore, RexAB-mediated DNA repair led to induction of the SOS response, which resulted in an increased mutation rate and may explain the frequent emergence of resistant strains during SXT therapy. In summary, this work determined that SXT causes DNA damage inS. aureusvia both thymidine limitation and oxidative stress, which is repaired by the RexAB complex, leading to induction of the mutagenic SOS response. Small molecule inhibitors of RexAB could therefore have therapeutic value by increasing the efficacy of SXT and decreasing the emergence of drug-resistance during treatment of infections caused byS. aureus.

Download Full-text

Opposing Roles for Two Molecular Forms of Replication Protein A in Rad51-Rad54-Mediated DNA Recombination in Plasmodium falciparum

mBio ◽

10.1128/mbio.00252-13 ◽

2013 ◽

Vol 4 (3) ◽

Cited By ~ 23

Author(s):

Anusha M. Gopalakrishnan ◽

Nirbhay Kumar

Keyword(s):

Dna Damage ◽

Dna Repair ◽

Antigenic Variation ◽

Protein A ◽

Dna Recombination ◽

Strand Exchange ◽

Content Type ◽

Dna Strand Exchange ◽

Dna Strand

ABSTRACT The bacterial RecA protein and its eukaryotic homologue Rad51 play a central role in the homologous DNA strand exchange reaction during recombination and DNA repair. Previously, our lab has shown that PfRad51, the Plasmodium falciparum homologue of Rad51, exhibited ATPase activity and promoted DNA strand exchange in vitro. In this study, we evaluated the catalytic functions of PfRad51 in the presence of putative interacting partners, especially P. falciparum homologues of Rad54 and replication protein A. PfRad54 accelerated PfRad51-mediated pairing between single-stranded DNA (ssDNA) and its homologous linear double-stranded DNA (dsDNA) in the presence of 0.5 mM CaCl2. We also present evidence that recombinant PfRPA1L protein serves the function of the bacterial homologue single-stranded binding protein (SSB) in initiating homologous pairing and strand exchange activity. More importantly, the function of PfRPA1L was negatively regulated in a dose-dependent manner by PfRPA1S, another RPA homologue in P. falciparum. Finally, we present in vivo evidence through comet assays for methyl methane sulfonate-induced DNA damage in malaria parasites and accompanying upregulation of PfRad51, PfRad54, PfRPA1L, and PfRPA1S at the level of transcript and protein needed to repair DNA damage. This study provides new insights into the role of putative Rad51-interacting proteins involved in homologous recombination and emphasizes the physiological role of DNA damage repair during the growth of parasites. IMPORTANCE Homologous recombination plays a major role in chromosomal rearrangement, and Rad51 protein, aided by several other proteins, plays a central role in DNA strand exchange reaction during recombination and DNA repair. This study reports on the characterization of the role of P. falciparum Rad51 in homologous strand exchange and DNA repair and evaluates the functional contribution of PfRad54 and PfRPA1 proteins. Data presented here provide mechanistic insights into DNA recombination and DNA damage repair mechanisms in this parasite. The importance of these research findings in future work will be to investigate if Rad51-dependent mechanisms are involved in chromosomal rearrangements during antigenic variation in P. falciparum. A prominent determinant of antigenic variation, the extraordinary ability of the parasite to rapidly change its surface molecules, is associated with var genes, and antigenic variation presents a major challenge to vaccine development.

Download Full-text

Genetic and Biochemical Analysis of CodY-Mediated Cell Aggregation in Staphylococcus aureus Reveals an Interaction between Extracellular DNA and Polysaccharide in the Extracellular Matrix

Journal of Bacteriology ◽

10.1128/jb.00593-19 ◽

2020 ◽

Vol 202 (8) ◽

Cited By ~ 2

Author(s):

Kevin D. Mlynek ◽

Logan L. Bulock ◽

Carl J. Stone ◽

Luke J. Curran ◽

Marat R. Sadykov ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Cell Surface ◽

Biofilm Formation ◽

Biochemical Analysis ◽

Cell Aggregation ◽

Extracellular Dna ◽

Dependent Manner ◽

Content Type ◽

Mutant Cells

ABSTRACT The global regulator CodY links nutrient availability to the regulation of virulence factor gene expression in Staphylococcus aureus, including many genes whose products affect biofilm formation. Antithetical phenotypes of both biofilm deficiency and accumulation have been reported for codY-null mutants; thus, the role of CodY in biofilm development remains unclear. codY mutant cells of a strain producing a robust biofilm elaborate proaggregation surface-associated features not present on codY mutant cells that do not produce a robust biofilm. Biochemical analysis of the clinical isolate SA564, which aggregates when deficient for CodY, revealed that these features are sensitive to nuclease treatment and are resistant to protease exposure. Genetic analyses revealed that disrupting lgt (the diacylglycerol transferase gene) in codY mutant cells severely weakened aggregation, indicating a role for lipoproteins in the attachment of the biofilm matrix to the cell surface. An additional and critical role of IcaB in producing functional poly-N-acetylglucosamine (PIA) polysaccharide in extracellular DNA (eDNA)-dependent biofilm formation was shown. Moreover, overproducing PIA is sufficient to promote aggregation in a DNA-dependent manner regardless of source of nucleic acids. Taken together, our results point to PIA synthesis as the primary determinant of biofilm formation when CodY activity is reduced and suggest a modified electrostatic net model for matrix attachment whereby PIA associates with eDNA, which interacts with the cell surface via covalently attached membrane lipoproteins. This work counters the prevailing view that polysaccharide- and eDNA/protein-based biofilms are mutually exclusive. Rather, we demonstrate that eDNA and PIA can work synergistically to form a biofilm. IMPORTANCE Staphylococcus aureus remains a global health concern and exemplifies the ability of an opportunistic pathogen to adapt and persist within multiple environments, including host tissue. Not only does biofilm contribute to persistence and immune evasion in the host environment, it also may aid in the transition to invasive disease. Thus, understanding how biofilms form is critical for developing strategies for dispersing biofilms and improving biofilm disease-related outcomes. Using biochemical, genetic, and cell biology approaches, we reveal a synergistic interaction between PIA and eDNA that promotes cell aggregation and biofilm formation in a CodY-dependent manner in S. aureus. We also reveal that envelope-associated lipoproteins mediate attachment of the biofilm matrix to the cell surface.

Download Full-text

Apoptosis-Like Death, an Extreme SOS Response in Escherichia coli

mBio ◽

10.1128/mbio.01426-14 ◽

2014 ◽

Vol 5 (4) ◽

Cited By ~ 55

Author(s):

Ariel Erental ◽

Ziva Kalderon ◽

Ann Saada ◽

Yoav Smith ◽

Hanna Engelberg-Kulka

Keyword(s):

Escherichia Coli ◽

Dna Damage ◽

Cell Death ◽

Dna Repair ◽

Sos Response ◽

Mitochondrial Apoptosis ◽

Apoptotic Pathway ◽

Content Type ◽

Cycle Arrest ◽

Transport Chain

ABSTRACT In bacteria, SOS is a global response to DNA damage, mediated by the recA-lexA genes, resulting in cell cycle arrest, DNA repair, and mutagenesis. Previously, we reported that Escherichia coli responds to DNA damage via another recA-lexA-mediated pathway resulting in programmed cell death (PCD). We called it apoptosis-like death (ALD) because it is characterized by membrane depolarization and DNA fragmentation, which are hallmarks of eukaryotic mitochondrial apoptosis. Here, we show that ALD is an extreme SOS response that occurs only under conditions of severe DNA damage. Furthermore, we found that ALD is characterized by additional hallmarks of eukaryotic mitochondrial apoptosis, including (i) rRNA degradation by the endoribonuclease YbeY, (ii) upregulation of a unique set of genes that we called extensive-damage-i nduced (Edin) genes, (iii) a decrease in the activities of complexes I and II of the electron transport chain, and (iv) the formation of high levels of OH˙ through the Fenton reaction, eventually resulting in cell death. Our genetic and molecular studies on ALD provide additional insight for the evolution of mitochondria and the apoptotic pathway in eukaryotes. IMPORTANCE The SOS response is the first described and the most studied bacterial response to DNA damage. It is mediated by a set of two genes, recA-lexA, and it results in DNA repair and thereby in the survival of the bacterial culture. We have shown that Escherichia coli responds to DNA damage by an additional recA-lexA-mediated pathway resulting in an apoptosis-like death (ALD). Apoptosis is a mode of cell death that has previously been reported only in eukaryotes. We found that E. coli ALD is characterized by several hallmarks of eukaryotic mitochondrial apoptosis. Altogether, our results revealed that recA-lexA is a DNA damage response coordinator that permits two opposite responses: life, mediated by the SOS, and death, mediated by the ALD. The choice seems to be a function of the degree of DNA damage in the cell.

Download Full-text

Regulation of Cell Division in Bacteria by Monitoring Genome Integrity and DNA Replication Status

Journal of Bacteriology ◽

10.1128/jb.00408-19 ◽

2019 ◽

Vol 202 (2) ◽

Cited By ~ 5

Author(s):

Peter E. Burby ◽

Lyle A. Simmons

Keyword(s):

Cell Cycle ◽

Dna Damage ◽

Cell Division ◽

Dna Replication ◽

Cell Cycle Progression ◽

Genome Instability ◽

Sos Response ◽

Bacterial Cells ◽

Cycle Progression ◽

Content Type

ABSTRACT All organisms regulate cell cycle progression by coordinating cell division with DNA replication status. In eukaryotes, DNA damage or problems with replication fork progression induce the DNA damage response (DDR), causing cyclin-dependent kinases to remain active, preventing further cell cycle progression until replication and repair are complete. In bacteria, cell division is coordinated with chromosome segregation, preventing cell division ring formation over the nucleoid in a process termed nucleoid occlusion. In addition to nucleoid occlusion, bacteria induce the SOS response after replication forks encounter DNA damage or impediments that slow or block their progression. During SOS induction, Escherichia coli expresses a cytoplasmic protein, SulA, that inhibits cell division by directly binding FtsZ. After the SOS response is turned off, SulA is degraded by Lon protease, allowing for cell division to resume. Recently, it has become clear that SulA is restricted to bacteria closely related to E. coli and that most bacteria enforce the DNA damage checkpoint by expressing a small integral membrane protein. Resumption of cell division is then mediated by membrane-bound proteases that cleave the cell division inhibitor. Further, many bacterial cells have mechanisms to inhibit cell division that are regulated independently from the canonical LexA-mediated SOS response. In this review, we discuss several pathways used by bacteria to prevent cell division from occurring when genome instability is detected or before the chromosome has been fully replicated and segregated.

Download Full-text

A Role for Histone H2B During Repair of UV-Induced DNA Damage in Saccharomyces cerevisiae

Genetics ◽

10.1093/genetics/160.4.1375 ◽

2002 ◽

Vol 160 (4) ◽

pp. 1375-1387

Author(s):

Emmanuelle M D Martini ◽

Scott Keeney ◽

Mary Ann Osley

Keyword(s):

Dna Damage ◽

Dna Repair ◽

Chromatin Structure ◽

Specific Effect ◽

Cell Killing ◽

Cyclobutane Pyrimidine Dimers ◽

Histone H2b ◽

Uv Sensitivity ◽

Pyrimidine Dimers

Abstract To investigate the role of the nucleosome during repair of DNA damage in yeast, we screened for histone H2B mutants that were sensitive to UV irradiation. We have isolated a new mutant, htb1-3, that shows preferential sensitivity to UV-C. There is no detectable difference in bulk chromatin structure or in the number of UV-induced cis-syn cyclobutane pyrimidine dimers (CPD) between HTB1 and htb1-3 strains. These results suggest a specific effect of this histone H2B mutation in UV-induced DNA repair processes rather than a global effect on chromatin structure. We analyzed the UV sensitivity of double mutants that contained the htb1-3 mutation and mutations in genes from each of the three epistasis groups of RAD genes. The htb1-3 mutation enhanced UV-induced cell killing in rad1Δ and rad52Δ mutants but not in rad6Δ or rad18Δ mutants, which are defective in postreplicational DNA repair (PRR). When combined with other mutations that affect PRR, the histone mutation increased the UV sensitivity of strains with defects in either the error-prone (rev1Δ) or error-free (rad30Δ) branches of PRR, but did not enhance the UV sensitivity of a strain with a rad5Δ mutation. When combined with a ubc13Δ mutation, which is also epistatic with rad5Δ, the htb1-3 mutation enhanced UV-induced cell killing. These results suggest that histone H2B acts in a novel RAD5-dependent branch of PRR.

Download Full-text

DNA Damage Response in Multiple Myeloma: The Role of the Tumor Microenvironment

Cancers ◽

10.3390/cancers13030504 ◽

2021 ◽

Vol 13 (3) ◽

pp. 504

Author(s):

Takayuki Saitoh ◽

Tsukasa Oda

Keyword(s):

Multiple Myeloma ◽

Dna Damage ◽

Dna Repair ◽

Tumor Microenvironment ◽

Dna Damage Response ◽

Genetic Instability ◽

Dna Repair Mechanisms ◽

Damage Response ◽

Repair Mechanisms

Multiple myeloma (MM) is an incurable plasma cell malignancy characterized by genomic instability. MM cells present various forms of genetic instability, including chromosomal instability, microsatellite instability, and base-pair alterations, as well as changes in chromosome number. The tumor microenvironment and an abnormal DNA repair function affect genetic instability in this disease. In addition, states of the tumor microenvironment itself, such as inflammation and hypoxia, influence the DNA damage response, which includes DNA repair mechanisms, cell cycle checkpoints, and apoptotic pathways. Unrepaired DNA damage in tumor cells has been shown to exacerbate genomic instability and aberrant features that enable MM progression and drug resistance. This review provides an overview of the DNA repair pathways, with a special focus on their function in MM, and discusses the role of the tumor microenvironment in governing DNA repair mechanisms.

Download Full-text

The Staphylococcus aureus KdpDE Two-Component System Couples Extracellular K+Sensing and Agr Signaling to Infection Programming

Infection and Immunity ◽

10.1128/iai.01180-10 ◽

2011 ◽

Vol 79 (6) ◽

pp. 2154-2167 ◽

Cited By ~ 56

Author(s):

Ting Xue ◽

Yibo You ◽

De Hong ◽

Haipeng Sun ◽

Baolin Sun

Keyword(s):

Staphylococcus Aureus ◽

Uptake System ◽

Main Function ◽

Two Component System ◽

Component System ◽

Content Type ◽

Two Component ◽

Virulence Regulator ◽

External K

ABSTRACTThe Kdp system is widely distributed among bacteria. InEscherichia coli, the Kdp-ATPase is a high-affinity K+uptake system and its expression is activated by the KdpDE two-component system in response to K+limitation or salt stress. However, information about the role of this system in many bacteria still remains obscure. Here we demonstrate that KdpFABC inStaphylococcus aureusis not a major K+transporter and that the main function of KdpDE is not associated with K+transport but that instead it regulates transcription for a series of virulence factors through sensing external K+concentrations, indicating that this bacterium might modulate its infectious status through sensing specific external K+stimuli in different environments. Our results further reveal thatS. aureusKdpDE is upregulated by the Agr/RNAIII system, which suggests that KdpDE may be an important virulence regulator coordinating the external K+sensing and Agr signaling during pathogenesis in this bacterium.

Download Full-text

The Role of Posttranslational Modifications in DNA Repair

BioMed Research International ◽

10.1155/2020/7493902 ◽

2020 ◽

Vol 2020 ◽

pp. 1-13

Author(s):

Miaomiao Bai ◽

Dongdong Ti ◽

Qian Mei ◽

Jiejie Liu ◽

Xin Yan ◽

...

Keyword(s):

Dna Damage ◽

Dna Repair ◽

Protein Interactions ◽

Posttranslational Modifications ◽

Genome Stability ◽

Protein Localization ◽

Complex Structure ◽

Protein Protein Interactions ◽

Regulate Protein

The human body is a complex structure of cells, which are exposed to many types of stress. Cells must utilize various mechanisms to protect their DNA from damage caused by metabolic and external sources to maintain genomic integrity and homeostasis and to prevent the development of cancer. DNA damage inevitably occurs regardless of physiological or abnormal conditions. In response to DNA damage, signaling pathways are activated to repair the damaged DNA or to induce cell apoptosis. During the process, posttranslational modifications (PTMs) can be used to modulate enzymatic activities and regulate protein stability, protein localization, and protein-protein interactions. Thus, PTMs in DNA repair should be studied. In this review, we will focus on the current understanding of the phosphorylation, poly(ADP-ribosyl)ation, ubiquitination, SUMOylation, acetylation, and methylation of six typical PTMs and summarize PTMs of the key proteins in DNA repair, providing important insight into the role of PTMs in the maintenance of genome stability and contributing to reveal new and selective therapeutic approaches to target cancers.

Download Full-text

Nucleotide Excision Repair Protein Rad23 Regulates Cell Virulence Independent of Rad4 in Candida albicans

mSphere ◽

10.1128/msphere.00062-20 ◽

2020 ◽

Vol 5 (1) ◽

Cited By ~ 1

Author(s):

Jia Feng ◽

Shuangyan Yao ◽

Yansong Dong ◽

Jing Hu ◽

Malcolm Whiteway ◽

...

Keyword(s):

Dna Damage ◽

Candida Albicans ◽

Nucleotide Excision Repair ◽

Dna Damage Response ◽

Excision Repair ◽

Content Type ◽

Virulence Regulation ◽

Nucleotide Excision ◽

Damage Response

ABSTRACT In the pathogenic yeast Candida albicans, the DNA damage response contributes to pathogenicity by regulating cell morphology transitions and maintaining survival in response to DNA damage induced by reactive oxygen species (ROS) in host cells. However, the function of nucleotide excision repair (NER) in C. albicans has not been extensively investigated. To better understand the DNA damage response and its role in virulence, we studied the function of the Rad23 nucleotide excision repair protein in detail. The RAD23 deletion strain and overexpression strain both exhibit UV sensitivity, confirming the critical role of RAD23 in the nucleotide excision repair pathway. Genetic interaction assays revealed that the role of RAD23 in the UV response relies on RAD4 but is independent of RAD53, MMS22, and RAD18. RAD4 and RAD23 have similar roles in regulating cell morphogenesis and biofilm formation; however, only RAD23, but not RAD4, plays a negative role in virulence regulation in a mouse model. We found that the RAD23 deletion strain showed decreased survival in a Candida-macrophage interaction assay. Transcriptome sequencing (RNA-seq) and quantitative real-time PCR (qRT-PCR) data further revealed that RAD23, but not RAD4, regulates the transcription of a virulence factor, SUN41, suggesting a unique role of RAD23 in virulence regulation. Taking these observations together, our work reveals that the RAD23-related nucleotide excision pathway plays a critical role in the UV response but may not play a direct role in virulence. The virulence-related role of RAD23 may rely on the regulation of several virulence factors, which may give us further understanding about the linkage between DNA damage repair and virulence regulation in C. albicans. IMPORTANCE Candida albicans remains a significant threat to the lives of immunocompromised people. An understanding of the virulence and infection ability of C. albicans cells in the mammalian host may help with clinical treatment and drug discovery. The DNA damage response pathway is closely related to morphology regulation and virulence, as well as the ability to survive in host cells. In this study, we checked the role of the nucleotide excision repair (NER) pathway, the key repair system that functions to remove a large variety of DNA lesions such as those caused by UV light, but whose function has not been well studied in C. albicans. We found that Rad23, but not Rad4, plays a role in virulence that appears independent of the function of the NER pathway. Our research revealed that the NER pathway represented by Rad4/Rad23 may not play a direct role in virulence but that Rad23 may play a unique role in regulating the transcription of virulence genes that may contribute to the virulence of C. albicans.

Download Full-text