Division of labor among Mycobacterium smegmatis RNase H enzymes: RNase H1 activity of RnhA or RnhC is essential for growth whereas RnhB and RnhA guard against killing by hydrogen peroxide in stationary phase.

RNase H enzymes sense the presence of ribonucleotides in the genome and initiate their removal by incising the ribonucleotide-containing strand of an RNA:DNA hybrid. Mycobacterium smegmatis encodes four RNase H enzymes: RnhA, RnhB, RnhC and RnhD. Here, we interrogate the biochemical activity and nucleic acid substrate specificity of RnhA. We report that RnhA (like RnhC characterized previously) is an RNase H1-type magnesium-dependent endonuclease with stringent specificity for RNA:DNA hybrid duplexes. Whereas RnhA does not incise an embedded mono-ribonucleotide, it can efficiently cleave within tracts of four or more ribonucleotides in duplex DNA. We gained genetic insights to the division of labor among mycobacterial RNases H by deleting the rnhA, rnhB, rnhC and rnhD genes, individually and in various combinations. The salient conclusions are that: (i) RNase H1 activity is essential for mycobacterial growth and can be provided by either RnhC or RnhA; (ii) the RNase H2 enzymes RnhB and RnhD are dispensable for growth and (iii) RnhB and RnhA collaborate to protect M. smegmatis against oxidative damage in stationary phase. Our findings highlight RnhC, the sole RNase H1 in pathogenic mycobacteria, as a candidate drug discovery target for tuberculosis and leprosy.

Protective effect of 4,4'-diaminodiphenylsulphone against oxidative stress but not to apoptotic stress in human diploid fibroblasts.

The antibiotic drug 4,4'-diaminodiphenylsulphone (DDS) is used to treat several dermatologic diseases, including Hansen's disease. This study confirmed the antioxidant nature of DDS in hydrogen peroxide (H(2)O(2))-induced oxidative stress and assessed its role in other apoptotic stresses in human diploid fibroblasts (HDFs). Oxidative stress was effectively reduced by DDS in a dose-dependent manner. Moreover, the oxidative stress-induced increases in the levels of the p53 and p21 proteins were inhibited by pre-treatment with DDS. In addition, H(2)O(2) and DDS increased the level of cytochrome P450 (CYP450) IIE1 in HDFs, implicating a role for DDS in H(2)O(2) scavenging via the activation of CYP450. DDS treatment increased the activity of catalase (CAT), glutathione peroxidase (GPx) and glutathione reductase (GR), as well as the GSH/GSSG ratio, indicating activation of the glutathione system against oxidative stress. However, DDS showed no protective effects on HDFs against other apoptotic stimuli, such as thapsigargin and staurosporine, suggesting that DDS would act only against oxidative stress. Therefore, in addition to its antibiotic function, DDS is a potent antioxidant against H(2)O(2)-induced oxidative stress in HDFs.

Effect of reactive oxygen intermediaries on the viability and infectivity of Mycobacterium lepraemurium.

Murine leprosy is a natural disease of the mouse, the most popular model animal used in biomedical research; the disease is caused by Mycobacterium lepraemurium (MLM), a successful parasite of macrophages. The aim of the study was to test the hypothesis that MLM survives within macrophages because it highly resists the toxic effects of the reactive oxygen intermediaries produced by these cells in response to infection by the microorganism. MLM cells were incubated in the presence of horseradish peroxidase (HRPO)-H(2)O(2)-halide for several periods of time. The peroxidative effect of this system was investigated by assessing the changes occurred in (a) lipid composition; (b) viability; and (c) infectivity of the microorganism. Changes in the lipid composition of peroxidated- vs. intact-MLM were analysed by thin layer chromatography. The effect of the peroxidative system on the viability and infectivity of MLM was measured by the alamar blue reduction assay and by its ability to produce an infection in the mouse, respectively. Peroxidation of MLM produced drastic changes in the lipid envelope of the microorganism, killed the bacteria and abolished their ability to produce an in vivo infection in the mouse. In vitro, MLM is highly susceptible to the noxious effects of the HRPO-H(2)O(2)-halide system. Although the lipid envelope of MLM might protect the microorganism from the peroxidative substances produced at 'physiological' concentrations in vivo, the success of MLM as a parasite of macrophages might rather obey for other reasons. The ability of MLM to enter macrophages without triggering these cells' oxidative response and the lack of granular MPO in mature macrophages might better explain its success as an intracellular parasite of these cells.

In vitro susceptibility of Mycobacterium leprae to oxygen-mediated damage.

In order to evaluate factors responsible for the failure of Mycobacterium leprae to multiply in cell-free cultures in vitro studies were undertaken to determine the possible poisoning of the organism by hydroxide and superoxide radicals produced in the growth medium. The superoxide dismutase activity was very low, 10% of the levels found in armadillo cells, while measured activity of catalase and glutathione peroxidase was negligible. Susceptibility of M. leprae to hydrogen peroxide was enhanced by potassium iodide but not by lactoperoxidase. The addition of high amounts of catalase completely prevented hydrogen peroxide-mediated killing of M. leprae. Superoxide generated by the action of xanthine oxidase on xanthine was lethal to M. leprae, but superoxide dismutase added to the reaction mixture gave significant protection. Thus superoxide radicals may be a major cause for the sudden termination of growth of M. leprae in primary cultures and also for failure of subcultures.

Identification of a gene involved in the biosynthesis of cyclopropanated mycolic acids in Mycobacterium tuberculosis.

Mycolic acids represent a major constituent of the mycobacterial cell wall complex, which provides the first line of defense against potentially lethal environmental conditions. Slow-growing pathogenic mycobacteria such as Mycobacterium tuberculosis modify their mycolic acids by cyclopropanation, whereas fast-growing saprophytic species such as Mycobacterium smegmatis do not, suggesting that this modification may be associated with an increase in oxidative stress experienced by the slow-growing species. We have demonstrated the transformation of the distal cis double bond in the major mycolic acid of M. smegmatis to a cis-cyclopropane ring upon introduction of cosmid DNA from M. tuberculosis. This activity was localized to a single gene (cma1) encoding a protein that was 34% identical to the cyclopropane fatty acid synthase from Escherichia coli. Adjacent regions of the DNA sequence encode open reading frames that display homology to other fatty acid biosynthetic enzymes, indicating that some of the genes required for mycolic acid biosynthesis may be clustered in this region. M. smegmatis overexpressing the cma1 gene product significantly resist killing by hydrogen peroxide, suggesting that this modification may be an important adaptation of slow-growing mycobacteria to oxidative stress.

Cytokines in mycobacterial infections: in vitro and ex vivo studies.

Different species of mycobacteria differ in their capacity to induce the production of tumor necrosis factor-alpha (TNF-alpha) by human monocytes in vitro. Whereas M. tuberculosis is a potent inducer of TNF-alpha, M. leprae is much less potent. TNF-alpha production is found to be associated with the availability of H2O2 generated by activated monocytes, as superoxide enhancing H2O2 concentration increases and catalase degrading H2O2 decreases TNF-alpha production. Furthermore, M. kansasii with high intrinsic catalase induce less TNF-alpha than mycobacteria with low intrinsic catalase. In vitro infection of monocytes with M. tuberculosis leads to an impairment of the antigen-presenting capacity, as determined by a reduction of antigen-induced T cell proliferation and interferon gamma (IFN-gamma) production. Of crucial importance is this impairment is the M. tuberculosis-induced down-modulation of MHC class II antigens. The role of TNF-alpha in vivo is reflected in patients with various forms of leprosy. In skin lesions of lepromatous leprosy patients TNF-alpha, interleukin 1 beta (IL-1 beta), and INF-gamma production are found to be rare, whereas these cytokines are well expressed in skin lesions of patients with tuberculoid leprosy. After multidrug chemotherapy an increase of local cytokine production is found. Taken together, these findings suggest that components of mycobacteria may interfere with local cell-mediated immune reactions in vivo. The molecular mechanisms involved in these local responses need to be defined.

Susceptibilities of Mycobacterium leprae and M. avium complex to the H2O2-Fe-mediated halogenation system supplemented with antimicrobial agents.

The susceptibilities of Mycobacterium leprae and M. avium complex (MAC) to the H2-O2-Fe-mediated halogenation system supplemented with antimicrobial agents were evaluated by fluorescein diacetate-ethidium bromide (FDA/EB) staining. In the case of M. leprae, the number of greenstained bacteria (intact cells) was reduced in the presence of the H2O2-Fe-mediated halogenation system supplemented with agents possessing antileprosy activity, such as rifampin, 4,4'-diaminodiphenylsulfone (dapsone), clofazimine, and ofloxacin. In the case of the MAC strain, although viable units of the organisms were reduced by the halogenation system alone, the number of greenstained cells in the FDA/EB stain was not reduced, even when the halogenation system was used in combination with ofloxacin. Because stainability of the cells is related to structural and functional intactness of the membrane, differences between M. leprae and the MAC strain imply possible differences in the rigidity of the cell membrane.

Susceptibility of Mycobacterium leprae to the bactericidal activity of mouse peritoneal macrophages and to hydrogen peroxide.

Macrophages from athymic nude mice were infected in vitro with Mycobacterium leprae to study the intracellular fate of this organism. Using the proportional bactericidal test, we have shown that the viability of M. leprae declines rapidly within these macrophages, although results of clearance experiments demonstrate that live and killed organisms are cleared at comparable rates. We have also shown that M. leprae is susceptible to the bactericidal effects of hydrogen peroxide and we suggest that hydrogen peroxide generated by macrophages is responsible for the killing of intracellular M. leprae.