L.M.B.O Asad

Several pathways of hydrogen peroxide action that damage the E. coli genome

Hydrogen peroxide is an important reactive oxygen species (ROS) that arises either during the aerobic respiration process or as a by-product of water radiolysis after exposure to ionizing radiation. The reaction of hydrogen peroxide with transition metals imposes on cells an oxidative stress condition that can result in damage to cell components such as proteins, lipids and principally to DNA, leading to mutagenesis and cell death. Escherichia coli cells are able to deal with these adverse events via DNA repair mechanisms, which enable them to recover their genome integrity. These include base excision repair (BER), nucleotide excision repair (NER) and recombinational repair. Other important defense mechanisms present in Escherichia coli are OxyR and SosRS anti-oxidant inducible pathways, which are elicited by cells to avoid the introduction of oxidative lesions by hydrogen peroxide. This review summarizes the phenomena of lethal synergism between UV irradiation (254 nm) and H2O2, the cross-adaptive response between different classes of genotoxic agents and hydrogen peroxide, and the role of copper ions in the lethal response to H2O2 under low-iron conditions.

Hydrogen peroxide induces protection against lethal effects of cumene hydroperoxide in Escherichia coli cells: an Ahp dependent and OxyR independent system?

Pretreatment with 2.5 mM H2O2 protects bacterial cells against cumene hydroperoxide killing. This response is independent of the OxyR system, but possibly involves the participation of Ahp protein, since ahp mutants are not protected. Treatment of bacterial cells with high H2O2 concentrations caused an alteration on the electrophoretic profile of the smaller subunit (22-kDa) of Ahp. This alteration does not require novel gene products and is not dependent on the OxyR protein. In this way, we propose that the modification of the 22-kDa subunit of Ahp by high H2O2 concentration may be responsible for the protection against the lethal effects of cumene hydroperoxide.

Participation of stress-inducible systems and enzymes involved in BER and NER in the protection of Escherichia coli against cumene hydroperoxide

We studied the participation of the stress-inducible systems, as the OxyR, SoxRS and SOS regulons in the protection of Escherichia coli cells against lethal effects of cumene hydroperoxide (CHP). Moreover, we evaluated the participation of BER and NER in the repair of the DNA damage produced by CHP. Our results suggest that the hypersensitivity observed in the oxyR mutants to the lethal effect of CHP does not appear to be due to SOS inducing DNA lesions, but rather to cell membrane damage. On the other hand, DNA damage induced by CHP appears to be repaired by enzymes involved in BER and NER pathways. In this case, Fpg protein and UvrABC complex could be involved cooperatively in the elimination of a specific DNA lesion. Finally, we have detected the requirement for the uvrA gene function in SOS induction by CHP treatment.

Iron-limited condition modulates biofilm formation and interaction with human epithelial cells of enteroaggregative Escherichia coli (EAEC).

Aims:  The aim of this study was to investigate the influence of low iron availability on biofilm formation and adherence to HEp‐2 cells of enteroaggregative Escherichia coli (EAEC) strains isolated from diarrhoea cases.

Corynebacterium diphtheriae putative tellurite-resistance protein (CDCE8392_0813) contributes to the intracellular survival in human epithelial cells and lethality of Caenorhabditis elegans

Corynebacterium diphtheriae, the aetiologic agent of diphtheria, also represents a global medical challenge because of the existence of invasive strains as causative agents of systemic infections. Although tellurite (TeO32-) is toxic to most microorganisms, TeO32--resistant bacteria, including C. diphtheriae, exist in nature. The presence of TeO32--resistance (TeR) determinants in pathogenic bacteria might provide selective advantages in the natural environment. In the present study, we investigated the role of the putative TeR determinant (CDCE8392_813gene) in the virulence attributes of diphtheria bacilli. The disruption of CDCE8392_0813 gene expression in the LDCIC-L1 mutant increased susceptibility to TeO32- and reactive oxygen species (hydrogen peroxide), but not to other antimicrobial agents. The LDCIC-L1 mutant also showed a decrease in both the lethality of Caenorhabditis elegans and the survival inside of human epithelial cells compared to wild-type strain. Conversely, the haemagglutinating activity and adherence to and formation of biofilms on different abiotic surfaces were not regulated through the CDCE8392_0813 gene. In conclusion, the CDCE8392_813 gene contributes to the TeR and pathogenic potential of C. diphtheriae.

Mutagenicity induced by UVC in Escherichia coli cells: reactive oxygen species involvement.

Abstract We previously demonstrated that reactive oxygen species (ROS) could be involved in the DNA damage induced by ultraviolet-C (UVC). In this study, we evaluated singlet oxygen (1O2) involvement in UVC-induced mutagenesis in Escherichia coli cells. First, we found that treatment with sodium azide, an 1O2 chelator, protected cells against UVC-induced lethality. The survival assay showed that the fpg mutant was more resistant to UVC lethality than the wild-type strain. The rifampicin mutagenesis assay showed that UVC mutagenesis was inhibited five times more in cells treated with sodium azide, and stimulated 20% more fpg mutant. These results suggest that 1O2 plays a predominant role in UVC-induced mutagenesis. 1O2 generates a specific mutagenic lesion, 8-oxoG, which is repaired by Fpg protein. This lesion was measured by GC–TA reversion in the CC104 strain, its fpg mutant (BH540), and both CC104 and BH540 transformed with the plasmid pFPG (overexpression of Fpg protein). This assay showed that mutagenesis was induced 2.5-fold in the GC–TA strain and 7-fold in the fpg mutant, while the fpg mutant transformed with pFPG was similar to GC–TA strain. This suggests that UVC can also cause ROS-mediated mutagenesis and that the Fpg protein may be involved in this repair.

H2O2-induced cross-protection against UV-C killing in Escherichia coli is blocked in a lexA (Def) background

Pretreatment with 2.5 mM H2O2 protects E. coli cells against UV-C killing, a phenomenon independent of LexA cleavage. In this paper, we observe that this cross-protection response is neither dependent on the dinY gene product nor on the system that controls dinY, since H2O2 is able to induce cross-protection but not to induce the dinY gene in a lexA-noninducible strain [lexA (Ind-)]. Moreover, this response is not induced in a lexA (Def) background, suggesting that the expression of the SOS regulon may inhibit this cross-protection response.

The role of DNA base excision repair in filamentation in Escherichia coli K-12 adhered to epithelial HEp-2 cells

Base excision repair (BER) is dedicated to the repair of oxidative DNA damage caused by reactive oxygen species generated by chemical and physical agents or by metabolism which can react with DNA and cause a variety of mutations. Epithelial cells are typically the first type of host cell to come into contact with potential microbial invaders. In this work, we have evaluated whether the adherence to human epithelial cells causes DNA damage and associated filamentation. Experiments concerning adherence to HEp-2 cells were carried out with mutants deficient in BER that were derived from Escherichia coli K-12. Since the removal of mannose during bacterial interaction with HEp-2 cells allows adhesion through mannose-sensitive adhesins, the experiments were also performed in the presence and the absence of mannose. Our results showed enhanced filamentation for the single xth (BW9091) and triple xth nfo nth (BW535) mutants in adherence assays with HEp-2 cells performed without d-mannose. The increased filamentation growth was inhibited by complementation of BER mutants with a wild type xth gene. Moreover, we measured SOS induction of bacteria adhered to HEp-2 cells in the presence and absence of d-mannose through of SOS-chromotest assay and we observed a higher β-galactosidase expression in the absence of mannose. In this context, data showed evidence that bacterial attachment to HEp-2 epithelial surfaces can generate DNA lesions and SOS induction.

Effects of low iron conditions on the repair of DNA lesions induced by Cumene hydroperoxide in Escherichia coli cells

In the present study, we evaluated the sensitivity of different Escherichia coli strains to Cumene hydroperoxide (CHP) treatment under distinct conditions of Fe2+ availability. Our results showed that the pretreatment with an iron chelator (dipyridyl) protects all the tested strains against CHP toxic effects, but it was not sufficient to abolish the CHP induced mutagenesis. On the other hand, simultaneous pretreatment with both dipyridyl and neocuproine (copper chelator) leads to a complete protection against CHP mutagenic effects. Our data suggest the participation of copper ion in the CHP mutagenesis induced in E. coli.

The role of Fpg protein in UVC-induced DNA lesions

Abstract We previously demonstrated that reactive oxygen species (ROS) could be involved in ultraviolet-C (UVC)-induced DNA damage in Escherichia coli cells. In the present study, we evaluated the involvement of the GO system proteins in the repair of the 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxoG, GO) lesion, which is ROS-induced oxidative damage. We first found that the mutant strain Δfur, which produces an accumulation of iron, and the cells treated with 2,2′-dipyridyl, a iron chelator, were both as resistant to UVC-induced lethality as the wild strain. The 8-oxoG could be mediated by singlet oxygen (1O2). The Fpg protein repaired this lesion when it was linked to C (cytosine), whereas the MutY protein repaired 8-oxoG when it was linked to A (adenine). The survival assay showed that the Fpg protein, but not the MutY protein, was important to UVC-induced lethality and interacted with the UvrA protein, a nucleotide excision repair (NER) protein involved in UVC repair. The GC–TA reversion assay in the mutant strains from the ‘8-oxoG-repair’ GO system showed that UVC-induced mutagenesis in the fpg mutants, but not in the MutY strain. The transformation assay demonstrated that the Fpg protein is important in UVC repair. These results suggest that UVC could also cause indirect ROS-mediated DNA damage and the Fpg protein plays a predominant role in repairing this indirect damage.