ROS - Reactive Oxygen Species and the bacterial response to lethal stress

Introduction

This work analises the importance of ROS on stress responses in bacteria. ROS or Reactive Oxygen Species are naturally occuring yet extremely reactive molecules that cause cellular damage and death. 3 ROS species in particular are the subject of study for the purpose of this presentation – superoxide, hydrogen peroxide and hydroxil radical. The stress factors that cause primary damage in the cell activate ROS and in turn ROS will activate other mechanisms to regulate positively or negatively causing secondary.

What is ROS?

ROS, or Reactive Oxygen Species are: -Molecules like hydrogen peroxide (H2O2) -Radicals like the hydroxyl radical (OH) The superoxide anion, both an ion and a radical (O2-).

ROS accumulation in the cells if on a moderate level, can be beneficial to the cells and an inducer of protective functions. However if ROS accumulation is uncontrolled it will kill the cell.

Stress Factors (Antibiotics)

There are various stress factors, for this presentation we’ll be talking about antibiotics, which provoke cellular damage that can lead to cell death. It is important to distinguish cellular death from growth inhibition.

The current opinion is that the stress response goes through the ROS cascade and that it can be programmed cell response as “live-or-die”. The stress factors cause the primary cell damage, of which can be of various types. e

Quinolones cause inhibition of topoisomerases, in Gram+ they inhibit topoisomerase II (DNA girase) as opposed in Gram- where they inhibit topoisomerase IV. Aminoglycosides are bactericides which inhibit protein synthesis, blocking the 30s subunit of the bacterial ribosome, impending the correct translation and its corresponding protein synthesis. Beta-Lactam are antibiotics that inhibit the synthesis of peptidoglycan.

Vancomicine only operates in Gram+ . It inhibits the cell wall synthesis, blocking peptidoglycan’s incorporation through active competition with its binding sites. Poliximines break the cell membrane structure by interacting with its phospholipids.

They selectevely toxic for Gram- bacterias and are produced in Gram+ bacterias.

They mostly affect the outer membranes due to the specificity by lipidsaccarides molecules.

The antibiotics were tested to verify their effect on ROS. It is known that many on them promote the activation of the SoxRS regulon.

The antibiotics were tested in normal growth conditions and their effect on ROS production was measured through various probes. Alternative growth methods were later made, one of them in anaerobic conditions, another one in chemical inhibition of ROS accumulation, and a third with a deficiency of a ROS scavenger enzyme. In normal growth conditions all showed accumulation of ROS.

Anaerobiose blocks or reduces ROS such as chemical inhibition.

The enzyme deficiency increases ROS accumulation, in some cases, the overexpression decreases ROS. The chemical inhibition is done with dypiridyl, na iron chelator which suppresses Fenton’s reaction, thiourea, a ROS scavenger, glutatione and vitamin C, ROS neutralizing antioxidants, nitric acid and hydrogen sulfide, ROS scavenger stimulant gases – superoxide dismutase and catalase/peroxidase.

The obtained data supports the following hypothesis: The antibiotics activate the ROS cascade.

The anaerobiose inhibits the functoning of ROS due to lack of O2 reagent. The chemical inhibition blocks ROS neutralizing the ROS cascade, and the ROS regulator enzyme deficiencies inhibit the regulation of ROS levels (this regulation tends to keep a low value). As such, the overexpression of this enzyme decreases the quantity of ROS.

Connection of ROS and stress-mediated killing

The article tells us that an increase in hydroxyl radical (detected by a fluorescent dye,HPF) accompanied killing by ampicillin, norfloxacin and kanamycin while no surge in HPF fluorescence occured with five different bacteriostatic agents or with bactericidal compounds at sub-lethal concentrations. Not only that, hydroxyl radical accumulation and cell death were reduced by treatment with an ROS scavenger and dipyridyl, an iron chelator that suppresses the aforementioned Fenton reaction. These findings indicate that an accumulation of ROS could amplify effects of antibiotical stress-mediated lesions.

Control of stress-induced, ROS-mediated bacterial cell death

-Control exerted by ROS-detoxifying enzymes (SodA, SodB, SodC, AhpCF, KatG, KatE) -Deletion of genes katG or ahpCF results in hyperlethality to various antibiotics. -Over-expression of KatG or ahpCF however will mitigate ROS-mediated damage.

1. Upon exposure to stress, the MazE antitoxin is degraded and in so doing the MazF toxin is liberated. This toxin then goes on to cleave many celular RNAs leaving them truncated. These 5’ mRNA can be translated into truncated misfolded peptides which then lodge in cell membranes, in turn activating the Cpx envelope protein stress system.

The Cpx two-component signal transduction pathway mediates adaptation to envelope protein misfolding. However, there is experimental evidence that at least 50 genes in 34 operons are part of the Cpx regulon and many have functions that are undefined or unrelated to envelope protein maintenance. No comprehensive analysis of the Cpx regulon has been presented to date.

2. Following the activation of Cpx, a protein kinase -YihE- is expressed. The gene this protein originates from, yihE is located in an operon that is positively regulated by CpxR, the response regulator of the Cpx envelope stress-response system. This protein negatively regulates the toxin MazF inhibiting the activation of Cpx, and in so doing stopping cell death in the process.

nduction of Cpx can also induce expression of genes including protein that refold/degrade misfolded peptides and suppress Cpx induction giving it a protective function. However its wild-type can also be destructive as evidenced by deleting CpxR, the response regulator, which in turn protects from the lethal action of quinolones, gentamycin, and ampicillin.

Arc or,anoxic redox control, is a two-component system (TCS) and a complex signal transduction system that plays an important role in regulating energy metabolism at the level of transcription in bacteria. This system comprises the ArcB protein, a hybrid membrane-associated sensor kinase, and the ArcA protein, a typical response regulator.

3.Under anaerobic conditions, the ArcB kinase transmembranar sensor self phosphorilizes, phosphorilating ArcA, a global cytosolic transcriptional regulator which controls the expression of numerous operons involved in the aerobic/anaerobic metabolism. The article tells us that Arc could then stimulate TCA and perturb electron transfer complexes such as cytochrome bd oxidase which would then result in ROS increase.

ROS Cascade

Superoxide and hydrogen peroxide arise when molecular oxygen sporadically oxidizes redox enzymes that normally transfer electrons to other substrates. The ROS cascade is a sequence in which the following transformations occur: The superoxide anion is formed into hydrogen peroxide which in turns into hydroxil radical through Fenton chemistry with hydrogen peroxide as a substrate.

Hydroxil accumulates in the cell causing secondary lesions such as nucleic acid cleaving, protein carbonilation and lipid peroxidation, and eventually cell death.

ROS Effects

ROS accumulation in the cells if on a moderate level, can be beneficial to the cells and an inducer of protective functions. However if ROS accumulation is uncontrolled it will kill the cell. Bacteria contain protective proteins that detoxify ROS(SodA, SodB, SodC, AhpCF, KatG, KatE)as well as counter its damage (SoxRS, OxyRS, and SOS regulons)However, when it comes to hydroxyl radical, there hasn’t been any protein-based mechanism identified that is able to detoxify it.

Control of stress-induced, ROS-mediated bacterial cell death If stress is mild and transient, ROS accumulate to a moderate level that is suficient for ROS to be beneficial mutagens and inducers of protective functionsHowever, if stress is severe and persistent, Cpx-mediated perturbation of arc may lead to high levels of ROS that overwhelm protective elements.Therefore the level of stress influences the outcome of the stress response, response of cell death based on threshold levels of ROS.

4. When such an event occurs, ROS will cause lesions to the cell causing its death. If left unchecked, this process will stimulate further ROS production and in turn, causing more death. Contolled, this process can be beneficial by removing damaged cells from the overall population.

5. Elevated levels of ROS(superoxide) can induce the SoxRS-MarAB-AcrAB efflux pump system to try and export toxic stressors thereby suppressing the primary lesion formation. AcrAB expression can be induced by MarA and SoxS regulators, seeing as these act as a transcriptional AcrAB activator and it’s produced as a response to antibiotic stress.

6. ROS accumulation also induces the SoxRS-OxyRS regulons that counter the damage caused by ROS. SoxR contains an iron-sulfur cluster that becomes activated by oxidation, which is triggered by superoxide(The transcriptional regulator OxyR however is triggered by Hydrogen Peroxide).

SoxRS

The SoxR protein is an transcription activator that when oxidated, activates the trancription of the SoxRS operon, increasing SoxS’s expression. The process starts with the increase of superoxide, when that happens, genes like SodA, SodB and SodC that code specific superoxide dismutase enzymes and these are responsible for the conversion of superoxide into (O2-) into hydrogen peroxide (H2O2) and molecular oxygen (O2).

Oxidized SoxR then activates transcription of SoxS, which in turn activates the Sox regulon detoxifying the cell (Members of the Sox regulon include the genes sodA, nfsA and nfo.), effectively stopping the ROS mediated-cell death process until ROS quantity is too high for these countermeasures to handle.

OxyRS OxyR is a transcription activator sensitive to peroxides and is activated with the oxidation of cistein residue, through the intramolecular dissulfide bridge formation between the cistein residue, which results in a conformational change that permits the interaction between OxyR and Rna polymerase. The genes involved in the protection against peroxides are AhpC and KatG. AhpC has the ability of reducing peroxide due to an hydrophobic framework in the active site and the KatG gene that when codified forms the catalase-peroxidase enzyme which converts peroxide into water and oxygen.

Byfunctional Elements

Acording to this scenario, the level of stress influences the outcome of the stress response, such an idea suggests the existence of byfuctional elements and threshold-based cell death responses. (superoxide O2-, kills bacteria at high concentrations triggered by plumbagin, moderate concentrations induce a variety of protective genes , largely through the SoxRS regulon).

Superoxide

Lethal in high concentrations, promoted by plumbagin, a metabolic generator of superoxide, plumbagin is toxic, genotoxic and mutagenic .Moderate concentrations induce several gene protectors mainly through SoxRS regulon.In low amounts, the induction of drug effluxion systems----Superoxide combines with nitric oxide to form peroxinitrite (ONOO-) which is an oxidant capable of to damage DNA and proteins.In moderate amounts, reduce the lethality mediated by antibiotics.

MazF Promotes stress mediated programmed cell death, helps stressed cells enter a stress tolerant metabolic dormancy. Cpx

Induction of Cpx can also induce expression of genes including protein that refold/degrade misfolded peptides and suppress Cpx induction giving it a protective function.However its wild-type can also be destructive as evidenced by deleting CpxR, the response regulator, which in turn protects from the lethal action ofquinolones, gentamycin, and ampicillin.

Deletion of CpxR, the response regulator protects from quinolone glutamycin and ampicilin. Perhaps the destructive activity of wild type Cpx is due to the redox-sensing Arc system. Arc could then stimulate TCA and perturb electron transfer complexes such as cytochrome bd oxidase which would then result in ROS increase. Conclusions

Even though the “live or die” stress response has a potential of explaining the many complexities associated with ROS, there is still much left to know. For example it’s unknown how information from the initial stress induced lesion is transduced to the MazF-Cpx-ROS pathway as well as if non-oxygen reactive oxidants play a role similar to ROS when bacteria experience stress during anaerobic growth. In addition, if ways are found to boost intracellular ROS production, new strategies for increasing antimicrobial lethality could be found. It’s also important to consider how clinically important the relation between ROS and antimicrobial lethality can be, certain factors that interfere with antimicrobial lethality can compromise efficacy and promote antimicrobial resistance. One such factors is the comsuption of antioxidant dietary supplements.

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