Aerobic organisms use molecular oxygen (O2) for respiration or oxidation of nutrients to obtain energy. Phagocytic cells that use NADPH dehydrogenases are able to generate reactive by-products of oxygen, such as superoxide anion radicals (O2”), hydrogen peroxide (H2O2), and higher hydroxyl radicals ("OH). The biological targets for these highly reactive oxygen species are DNA, RNA, proteins and lipids. Much of the damage is caused by hydroxyl radicals generated from H2O2 via the Fenton reaction, which requires iron (or another metal) and a source of reducing equivalents to regenerate the metal (Meneghini, 1997). The polyunsaturated fatty acids in membranes are damaged by free radicals, which is followed by a decrease in membrane fluidity as a result of the lipid peroxidation. After that, the events are amplified and more radicals are formed, including aldehydes that are very reactive and can damage proteins (Humphries & Szweda, 1998). DNA is also a main target of reactive oxygen intermediates, which contribute to base and sugar modifications. Several types of DNA damage are documented, including oxidative
C hapter 1: Introduction
adduction in the base radical (Meneghini, 1997). All these modifications are deleterious to the cell, since they lead to a loss of function of membranes and proteins, and block DNA replication or cause mutations. At the cellular level, when proteins are exposed to reactive oxygen molecules, modifications of amino acid side chains occur and the protein structure is altered. A study with E. coli has suggested that disulfide bonds are generated in the proteins of the cytosol when exposed to hydrogen peroxide, a situation referred to as “disulfide stress” (Aslund et ah, 1999). Irreversible oxidation of amino acid residues in a protein can be induced by metal ion-catalysed oxidation mechanisms or the Fenton reaction, which leads to the peptide bond cleavage (Cabiscol et al., 2000). All modifications will lead to functional changes that will disturb cellular metabolism. A Western blot technique was employed to identify the most susceptible proteins involved in oxidative damage. The proteins identified as potential targets were involved in different processes such as glucose catabolism (enolase), chaperone function (DNA K), protein synthesis (EF-G), outer membrane function (OmpA), and the P-subunit of ATPase (Tamarit et a l, 1998).
E. coli cells posses a specific defence against peroxides, mediated by the transcriptional activator OxyR, and another against superoxide, controlled by the two-stage SoxRS system (Storz & Imlay, 1999). The E. coli OxyR transcriptional factor activates the expression of several antioxidant defensive genes in response to hydrogen peroxide, including katG (hydroperoxidase), ahpCF (alkyl hydroperoxide reductase), oxyS (a regulatory RNA), dps (a non-specific DNA-binding protein), fu r (ferric uptake regulation), trx2 (thioredoxin) and grxA (glutaredoxin) (Storz & Zheng, 2000). E. coli
oxyR-deletion mutants are hypersensitive to hydrogen peroxide and fail to activate the expression of OxyR-regulated genes in response to hydrogen peroxide (Zheng & Storz,
2000; Zheng et ah, 2001). The SoxR transcriptional factor activates the expression of a single gene, soxS, in response to exposure to superoxide-generating agents and to nitric oxide. The elevated levels of the SoxS protein then lead to increased expression of several genes, including sodA (superoxide dismutase), zw f (glucos-6-phosphate dehydrogenase), jp r (NAPDH:flavodoxin oxidoreductase), fldA (flavodoxin 1), fumC
(fumarase C), acnA (aconitase), nfo (endonuclease IV), and micF (a regulatory RNA) (Storz and Zheng, 2000).
In comparison with E. coli, mycobacteria exhibit differences in their ability to mount an oxidative stress response. The M. tuberculosis equivalent of oxyR, the central regulator of the peroxide response, has been shown to be a pseudogene, inactivated via multiple lesions (Deretic et ah, 1995). Despite the loss of oxyR, other oxidative stress response genes are preserved in the tubercle bacilli. The gene ahpC which codes for a non- hemoprotein alkyl hydroperoxidase forms a part of the antioxidant defence system of M. tuberculosis (Hillas et ah, 2000). âhpC is upregulated in isoniazid-resistant M.
tuberculosis as a mechanism used by the organism to compensate for the loss of the KatG antioxidant activity (Deretic et ah, 1995; Sherman et ah, 1999). However, in a recent study with M tuberculosis, the importance of ahpC in virulence could not be demonstrated (Springer et ah, 2001). In this study the inactivation of the ahpC gene of
M. tuberculosis had no effect on growth during acute infection in mice and also did not affect the in vitro sensitivity to H2O2. Contrary to enteric bacteria where OxyR regulates both katG and ahpC (Zhang et ah, 1998), in M. tuberculosis and M smegmatis furA
(encoding a homologue of ferric uptake regulator Fur) is located immediately upstream of the katG gene. Zarht and colleagues (2001) demonstrated that FurA acts as a negative regulator of katG in M. smegmatis. Fur or Fur homologues regulate genes induced in
C hapter I: Introduction
response to oxidative stress, including sodA and sodB, encoding manganese and iron superoxide dismutases (SOD), the 8-hydroxyguanine endonuclease gene, catalase and peroxidase genes, alkyl hydroperoxidase genes, the soxRS genes and the oxyR gene (Hantke, 2001).
Iron deficiency can also lead to oxidative stress, presumably by decreasing the activity of heme-containing enzymes that are involved in the protection against reactive oxygen intermediates (Hantke, 2001), such as the M. tuberculosis catalase/peroxidase KatG. Excess of iron might lead to oxidative stress and DNA, protein and lipid damage via the Fenton reaction, in which iron catalyses the synthesis of highly reactive hydroxyl radicals from hydrogen peroxides. It has been suggested that IdeR, the repressor of the mycobacterial iron regulon, is necessary for an effective oxidative-stress response in M. smegmatis. Moreover, the same study showed that M. smegmatis ideR mutants have lower levels of katG and sodA mRNA and protein than the wild type strain (Dussurget and Smith, 1998). Gold et at. (2001) have shown that M. tuberculosis IdeR is also involved in iron metabolism, the oxidative stress response and consequently in the survival in macrophages.
Extracytoplasmic function (ECF) sigma factors in E. coli and Pseudomonas aeruginosa
have been shown to play a role in the regulation of gene expression required for survival following to exposure to stress (Fernandes et al., 1999). Based on these observations Raman et al. (2001) investigated the role of sigH during oxidative stress. They demonstrated that sigH mutants of M. tuberculosis are impaired in survival under different types of oxidative stress. They also demonstrated that in response to oxidative stress, SigH strongly induces the transcription of genes that have been shown to be
involved in the response to the stress, including thioredoxin reductase, the heat shock proteins DnaK and ClpB, and the stress-responsive sigma factors SigE and SigB.