Molecular and Physiological Effects of Mycobacterial oxyR Inactivation
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《细菌学杂志》
Department of Microbiology, University of Michigan Medical School, Ann Arbor, Michigan 48105,Department of Molecular Genetics and Microbiology,School of Pharmacy,Department of Cell Biology and Physiology, University of New Mexico Health Sciences Center, Albuquerque, New Mexico 87131,Department of Microbiology and Immunology, University of Maryland School of Medicine, Baltimore, Maryland 21201,Center for Veterinary Medicine, Food and Drug Administration, Laurel, Maryland 20857,Department of Medicine, University of Massachusetts Medical School, Worcester, Massachusetts 01605
ABSTRACT
The majority of slow-growing mycobacteria have a functional oxyR, the central regulator of the bacterial oxidative stress response. In contrast, this gene has been inactivated during the evolution of Mycobacterium tuberculosis. Here we inactivated the oxyR gene in Mycobacterium marinum, an organism used to model M. tuberculosis pathogenesis. Inactivation of oxyR abrogated induction of ahpC, a gene encoding alkylhydroperoxide reductase, normally activated upon peroxide challenge. The absence of oxyR also resulted in increased sensitivity to the front-line antituberculosis drug isoniazid. Inactivation of oxyR in M. marinum did not affect either virulence in a fish infection model or survival in human macrophages. Our findings demonstrate, at the genetic and molecular levels, a direct role for OxyR in ahpC regulation in response to oxidative stress. Our study also indicates that oxyR is not critical for virulence in M. marinum. However, oxyR inactivation confers increased sensitivity to isonicotinic acid hydrazide, suggesting that the natural loss of oxyR in the tubercle bacillus contributes to the unusually high sensitivity of M. tuberculosis to isoniazid.
INTRODUCTION
Tuberculosis is the number one cause of death from a single infectious agent in the world, with 8 million new cases of active disease each year, 2 million fatalities annually, and over a billion people latently infected (WHO fact sheet; http://www.who.int/mediacentre/factsheets/fs104/en/). The members of the Mycobacterium tuberculosis complex (16, 21), the causative agent of tuberculosis, and several other pathogenic mycobacteria, including M. marinum (7, 15, 32), have the ability to infect macrophages and survive within phagocytic cells (2, 3, 17). Despite several advances in the field in the wake of the availability of M. tuberculosis genomic information, much remains to be learned about the biology and pathogenesis of the tubercle bacillus. Evolutionarily, M. marinum is closely related to M. tuberculosis (38), causing tuberculosis like-disease in poikilothermic hosts, such as fish (6, 26). In humans, M. marinum can cause localized disease that is restricted to the extremities (39). Due to its close evolutionary link to M. tuberculosis and apparent parallels between the diseases caused by the two microbes, M. marinum has been used effectively to study mycobacterial pathogenesis (1, 14, 24, 25, 32, 34).
Recent studies of the oxidative stress response genes in mycobacteria have revealed that the two most significant mycobacterial pathogens, M. tuberculosis and M. leprae, paradoxically lack parts of their oxidative stress response defenses (11, 24). In M. tuberculosis, oxyR, the central regulator of bacterial oxidative and nitrosative stress response (18, 33), is surprisingly inactive (10, 11, 28); it is represented in the M. tuberculosis genome as an unannotated pseudogene (see Fig. 1A), located between the Rv2427c and Rv2428 (AhpC) open reading frames. M. leprae, on the other hand, has an intact oxyR but lacks a functional furA, the negative regulator of KatG (43). Despite the loss of oxyR in M. tuberculosis, several of the oxidative stress response genes in M. tuberculosis are preserved. One of the genes suspected to be under the control of OxyR is ahpC, encoding alkylhydroperoxide reductase (AhpC), which appears to be expressed only under static growth conditions in M. tuberculosis (31). AhpC has been suggested to protect mycobacteria from the deleterious effects of the combinatorial products of reactive oxygen and nitrogen intermediates and has been implicated in isoniazid (INH) resistance (9, 13, 22, 27, 31). The oxyR gene is highly conserved and is functional in most mycobacteria (other than the M. tuberculosis complex) with slow to moderate growth rates, including M. marinum (24). The counterintuitive loss of oxyR in M. tuberculosis appears paradoxical, since it would be expected that the elimination of oxyR might reduce mycobacterial fitness in the face of oxidative defense mechanisms of the host phagocytic cells. However, the exact effects of this M. tuberculosis evolutionary signature are not known.
Here we demonstrate, at the genetic and molecular levels, that OxyR regulates ahpC in response to peroxide-induced stress. oxyR inactivation showed neutral effects on M. marinum virulence. Our results also indicate that the M. marinum oxyR knockout mutant is more sensitive to INH than the wild-type parental strain.
MATERIALS AND METHODS
Bacterial strains, plasmids, and culture conditions. M. marinum ATCC 15069 and ATCC 927 were obtained from the American Type Culture Collection. M. marinum mutant strains 27.1 and 91.4 were derived from M. marinum strain ATCC 927 by insertion of an IS1096 element into cysD and a PE family gene using the pYUB285 transposon delivery plasmid. Mycobacteria were grown in Middlebrook 7H9 medium or 7H10 plates (Becton Dickinson, Sparks, MD) supplemented with albumin-dextrose-catalase (ADC) (Becton Dickinson, Sparks, MD) enrichment and 0.05% Tween 80. Medium was supplemented with 50 μg/ml kanamycin (Sigma, St. Louis, MO) when necessary. Escherichia coli was grown in LB broth or plates supplemented with 100 μg/ml of ampicillin and 5-bromo-4-chloro-3-indolyl--D-galactopyranoside (X-Gal) (both from Sigma, St. Louis, MO) when required. All incubations were carried out at 30°C unless otherwise noted.
Construction of plasmid vectors. To generate the plasmid used for inactivation of oxyR on the M. marinum chromosome via homologous recombination, a 1,672-bp fragment containing the complete ahpC-oxyR coding sequences was amplified by PCR from genomic DNA using oligonucleotides 5'-TATAGGCGCCCTACGCCGAAG-3' and 5'-ATAAGGCGCCCAGGCTCAGTTG-3' and then cloned into the PCR2.1 vector (Invitrogen, Carlsbad, CA). The resulting plasmid was restricted with EcoRI, and the excised fragment was subcloned into the EcoRI site of pUC19. An NdeI-SphI fragment was excised and inserted into the NdeI-SphI site of pSM243, a mycobacterial suicide vector carrying the sacB gene, to generate pEPM1. A 1.2-kb NheI-SpeI Kmr cassette from pMV206 was inserted into the NheI site of the M. marinum oxyR gene in pEPM1, followed by the insertion of a 2.0-kb XbaI-NheI xylE cassette from pHSX-1 (8) into the NheI site of the oxyR::Kmr construct, resulting in pEPM2 with a 4.9-kb fragment carrying the oxyR::xylE+ Kmr construct.
Allelic replacement. Plasmid pEPM2 (oxyR::xylE+ Kmr) was introduced into M. marinum ATCC 15069 by electroporation. Potential recombinants were selected in two steps. First, merodiploids were selected by plating cells on 7H10 medium supplemented with 0.2% pyruvate and kanamycin at 10 μg/ml. The presence of the fragment was tested for by screening for xylE activity (generation of yellow colonies upon spraying with catechol [Sigma, St. Louis, MO]) to distinguish real transformants from spontaneous Kmr mutants. Merodiploids were screened for legitimate recombination by PCR using primer 5'-GGTCCTCAGTCTCTGACCA-3', an oligonucleotide upstream of oxyR (sequence not carried in pEPM2), and primer 5'-TGCAGTTTCATTTGATGCTCGATGAGT-3', an oligonucleotide specific for the Kmr cassette. To select for the second recombination event that would integrate the oxyR::xylE+ Kmr construct into the chromosome, merodiploids were plated onto 7H10 plates containing 10% sucrose and kanamycin at 10 μg/ml. Colonies that were resistant to sucrose were screened by PCR using primers 5'-GGTCCTCAGTCTCTGACCA-3' and 5'-GTCCTCCATAGGCCGAAC-3', which hybridize upstream and downstream of the DNA fragment used for the disruption. These colonies were further screened by Southern blot hybridization. For complementation, a functional copy of oxyR was inserted into the M. marinum chromosome using an integrative plasmid encoding Hygr as previously described (42).
DNA and protein purification, Southern blot hybridization, and gel shift assays. Mycobacterial DNA was isolated as previously described (42). Two micrograms of DNA from M. marinum ATCC 15069 and M. marinum a21868 (oxyR::xylE+ Kmr) was digested with SacII, separated in a 1% agarose gel, and transferred to a Duralon membrane (Perkin-Elmer Life Sciences, Boston, MA). An oxyR-specific hybridization probe was generated by PCR using primers 5'-ATCCGGTTCGGCATCATCCCC-3' and 5'-GCAACTCGGACAGTGCCG-3' and was labeled with [-32P]dCTP (3,000 Ci/mmol; NEN DuPont) by random priming (Invitrogen, Carlsbad, CA). OxyR was overexpressed and purified according to procedures reported previously (12). A DNA-protein binding electrophoretic mobility shift assay was conducted as previously described (24).
Determination of sensitivity to INH and peroxides. Disk inhibition assays were used to determine differences in sensitivities of M. marinum ATCC 15069 and M. marinum a21868 to INH, hydrogen peroxide (H2O2), and cumene hydroperoxide (CHP). The strains were grown to mid-exponential phase (optical density at 600 nm [OD600], 0.5) in 7H9 medium, and single-cell suspensions were obtained by vortexing with 3-mm glass beads (Fisher Scientific, Pittsburgh, PA) and filtration through a 5-μm-pore-size filter (Osmonics, Inc., Minnetonka, MN). Two-hundred-microliter aliquots were mixed with soft 7H10 agar and poured onto 7H10 plates to grow a bacterial lawn. BBL paper disks (6 mm; Becton Dickinson, Sparks, MD) were impregnated with 10 μl of INH (2 mg/ml), H2O2 (300 mM), or CHP (30 mM) (all from Sigma, St. Louis, MO) or mixes of INH and peroxides (at the same concentrations) and were placed on top of the solidified soft agar. Zones of growth inhibition were measured after 3 to 4 days of incubation at 30°C. Six plates were done for each condition for both strains.
Determination of isoniazid MIC. MICs were determined using Etest strips (AB Biodisk) according to the manufacturer's recommendations and represent the mean and standard deviation of quadruplicate determinations.
Exposure of mycobacteria to hydrogen peroxide and immunoblot analysis. M. marinum ATCC 15069 and M. marinum a21868 were grown in 7H9 medium to an OD600 of 0.5, aliquoted into 50-ml portions, and treated with 0.02, 0.2, 2, and 10 mM H2O2 (Sigma) or 0.02, 0.1, 0.2, and 0.6 mM CHP (Sigma). Cells were incubated in a 37°C shaker for 2 h. Crude protein extracts were obtained by homogenization in a Mini Bead-beater (Biospec Products Inc., Bartlesville, OK) for 2 min using zirconium beads (diameter, 0.1 to 0.15 mm). The beads and cell debris were removed by centrifugation, and the resulting supernatants were used for immunoblot analysis. Twenty micrograms of total protein was run on sodium dodecyl sulfate (SDS)-polyacrylamide gels (11%), and proteins were transferred to Immobilon-P membranes (Millipore, Billerica, MA) by electroblotting. Western blot (immunoblot) analysis was performed using a rabbit antiserum to M. tuberculosis AhpC raised against purified AhpC (13) as the primary antibody and goat anti-rabbit immunoglobulin G conjugated to peroxidase (Kirkegaard & Perry Laboratories Inc., Gaithersburg, MD) as the secondary antibody. The anti-KatG antibody was from C. Barry. Bound antibody was visualized by formation of diaminobenzidine precipitate.
Metabolic labeling of proteins. M. marinum wild-type and oxyR-deleted mutant strains were grown to an OD600 of 0.5 in 7H9 medium and aliquoted into 5-ml aliquots in polypropylene tubes. H2O2 or CHP was added to final concentrations of 0.125, 0.25, 0.5, and 2 mM, followed by the addition of 10 μCi of [35S]methionine and [35S]cysteine (Expre35S35S protein labeling mix; 1,000 Ci/mmol; Perkin-Elmer Life Sciences, Boston, MA). After 2 h at 37°C, the cultures were centrifuged and washed once with phosphate-buffered saline (PBS). The bacterial pellets were resuspended in 100 μl of PBS and homogenized by bead beating as described above. The resulting supernatants were run in 11% SDS-polyacrylamide gels. Gels were dried and exposed to X-ray film (Kodak Inc., Rochester, NY) for autoradiography.
Catalase activity assay. The M. marinum wild-type strain (ATCC 15069), the oxyR-deleted strain, and the oxyR-complemented strain were grown at 30°C in 7H9 medium with 10% OADC (oleic acid-ADC) (supplemented with 50 μg/ml kanamycin for mutants) to an OD600 of 1.6 to 1.8. Crude protein extracts were obtained by homogenization by bead beating as described above. The beads and cell debris were removed by centrifugation, and the resulting supernatants were used for catalase activity assays with the Amplex Red catalase assay kit (Molecular Probes) according to the manufacturer's recommendations.
Survival in macrophages. Human peripheral blood monocyte-derived macrophages were infected at a multiplicity of infection of 10:1 with the respective strains of M. marinum either in the presence or in the absence of human gamma interferon (200 U/ml) and lipopolysaccharide (50 ng/ml). Cells were incubated at 37°C for 3 days. Cell lysates for the day 0 and day 3 time points were serially diluted and plated for CFU. This time point showed optimal differences in murine macrophages.
Fish model of tuberculosis. The M. marinum wild-type strain (ATCC 15069), the oxyR-deleted strain, and the oxyR-complemented strain were grown in 7H9 medium with 10% OADC (supplemented with 50 μg/ml kanamycin for mutants) to an OD600 of 1.6 to 1.8. The cells were harvested by centrifugation, and the pellet was resuspended in sterile PBS and sonicated for 3 min at power level 3, while being cooled using a cup horn accessory attached to a cell disruptor (heat systems, model W-220F; Branson Ultrasonics Corp., Danbury, CT). The inoculum was frozen in 1-ml aliquots at –80°C. Twenty fish per group were infected intraperitoneally either with 3.5 x 108 CFU of the wild-type strain or with 1.5 x 108 CFU of the oxyR knockout mutant strain or the complemented strain and were monitored for 21 days.
Statistical analyses. Statistical analyses (Fisher's least-squares determination) were performed with ANOVA (version 1.11), Abacus Software.
RESULTS AND DISCUSSION
Inactivation of oxyR in M. marinum. Since all members of the M. tuberculosis complex lack a functional oxyR and have additional mutations in oxyR binding sites (9), we resorted to inactivating the oxyR gene in M. marinum as a model system for studying mycobacterial virulence (15, 32). The M. marinum oxyR-ahpC locus has a genetic organization identical to that of M. tuberculosis and other pathogenic mycobacteria (Fig. 1A) (24). The inactivation was done by constructing a mycobacterial suicide vector, pEPM2, carrying a copy of the entire M. marinum ahpC-oxyR coding region with the oxyR gene disrupted by a xylE+ Kmr cassette at the NheI site of the gene (Fig. 1B). The xylE gene was used as a second screening marker for plasmid insertion given the high background of spontaneous resistance of mycobacteria when plated on Km-supplemented media (8). The plasmid also carries the counterselectable marker sacB, for isolation of second crossover events on sucrose-containing media. After electroporation into M. marinum and screening for xylE+ colonies among Kmr outgrowths, a merodiploid isolate that contained a legitimate recombination integration event was obtained. This strain, upon growth in the presence of sucrose (to select for loss of the sacB marker), gave rise to an isolate containing merodiploid resolution due to a second crossover, resulting in the oxyR::xylE+ Kmr mutant strain M. marinum a21868. The gene replacement was confirmed by Southern blot analysis (Fig. 1C).
OxyR is a regulator of the oxidative stress response and ahpC. OxyR has been well characterized in enteric bacteria (5), where it is known to regulate several genes involved in the peroxide stress response, including ahpC, katG, gorA, fur, dps, and oxyS. In M. tuberculosis, various deletions and point mutations have rendered the oxyR gene inactive. This inactivation has been linked to the silencing of the ahpC gene under certain conditions (31). Mycobacterial OxyR (from M. leprae, where oxyR is intact) has been purified (12) and shown to bind to the intergenic region between oxyR and ahpC (12, 24) where the promoters for these genes have been mapped (44). To date, however, there is no direct in vivo evidence demonstrating that oxyR controls the expression of ahpC or any other gene in mycobacteria. To test whether oxyR regulates ahpC and katG, we analyzed AhpC and KatG levels in wild-type (oxyR+) and mutant (oxyR::xylE+ Kmr) strains. M. marinum ATCC 15069 (wild-type strain) and M. marinum a21868 (oxyR::xylE+ Kmr) were exposed to increasing concentrations of either H2O2 or an organic peroxide, CHP, and protein extracts were immunoblotted for AhpC and KatG using corresponding antibodies (Fig. 2A and B). The wild-type strain showed increased steady-state levels of AhpC with increasing concentrations of H2O2, with a peak induction at 0.2 mM H2O2 (180% relative to the untreated control) and 0.2 mM CHP (210% of the control). Inspection of KatG levels in the same extracts (thus doubling as a loading control) in the wild-type strain showed a slight increase or drop (144% for H2O2 and 46% for CHP) in KatG levels when AhpC levels were high. However, in the oxyR::xylE+ Kmr mutant strain, AhpC and KatG levels remained similar and were not induced by peroxides (88% of the control for H2O2 and 46% of the control for CHP). At high peroxide levels (i.e., 10 mM), some loss of either AhpC or KatG was detected, possibly due to increased degradation and/or inability to increase production due to excessive damage. It is noteworthy that in most samples, KatG levels showed inverse intensity to AhpC in the oxyR+ strain, thus providing an internal control for ahpC expression. These experiments demonstrate that OxyR is required for induction of ahpC expression in response to peroxide stress.
Additional confirmation that OxyR induces the expression of ahpC was achieved by [35S]methionine metabolic labeling of newly synthesized polypeptides in the M. marinum oxyR+ and oxyR::xylE Kmr strains exposed to H2O2 or CHP and analysis by SDS-polyacrylamide gel electrophoresis (PAGE) and autoradiography (Fig. 3A). Several polypeptides were induced in the wild-type strain in response to both H2O2 and CHP treatments, including AhpC (the band was identified as previously described [9]), and at least seven additional polypeptides (p17.4, p18.4, p31, p33, p39, p41, p45) (Fig. 3A). In addition to AhpC, induction of at least one other polypeptide, of 39 kDa (p39), was affected in the oxyR mutant strain (Fig. 3A). A direct regulation of ahpC by OxyR was confirmed by DNA binding of OxyR to the ahpC promoter region by using an electrophoretic mobility shift assay (Fig. 2C). No positive gel shift was observed with the katG promoter region (Fig. 2C, lanes 5 and 6), consistent with a lack of negative effects of oxyR inactivation on the KatG response to H2O2 or CHP stimulation (Fig. 2A and B). Moreover, no significant differences in catalase activity were observed between strains (Fig. 3B). The specificity of mycobacterial OxyR interaction within binding sites has been established previously (12). These observations constitute the first direct genetic and molecular evidence for the role of OxyR as a regulator of the oxidative stress response in mycobacteria. However, the observation that several peroxide-inducible polypeptides retained a capacity for increased de novo synthesis despite the absence of OxyR suggests the presence of oxyR-independent oxidative stress response systems in M. marinum.
Inactivation of oxyR causes increased sensitivity to peroxides and INH. To test whether inactivation of oxyR resulted in increased M. marinum susceptibility to peroxides, disk inhibition assays were used and sensitivities to CHP compared for wild-type oxyR+ and oxyR::xylE+ Kmr mutant strains (Table 1). A significant (P < 0.01) increase was observed in the growth inhibition zone for the oxyR::xylE+ Kmr mutant strain in the presence of hydrogen (not shown) or organic peroxides compared to that of the parent strain, M. marinum ATCC 15069 (Table 1). Since it has been shown that a range of reactive radicals are generated during INH activation by mycobacterial catalase-peroxidase (9, 30, 37), we also tested whether the susceptibility of the oxyR mutant to INH was affected. We tested this using the previously published disk inhibition zone assay (19, 40, 43, 44) (Table 1). These results for INH were also confirmed by conventional MIC determination, which showed a significant decrease, from 26.3 ± 1.5 to 14.9 ± 0.7 μg/ml (P < 0.001), upon oxyR mutation. Complementation of oxyR by integration of a functional oxyR gene into the chromosome of the oxyR::xylE+ Kmr insertional mutant reversed the increased susceptibility to INH and CHP (Table 1). Furthermore, complementation reversed the decrease in the MIC of INH, putting it back to 25.1 ± 1.2 μg/ml, a value not significantly different from that for the wild type (P = 0.18). These results demonstrate that elimination of a functional OxyR results in increased susceptibility to INH in M. marinum, a mycobacterium otherwise highly resistant to this antituberculosis drug. These findings demonstrate that oxyR participates in the protection of the mycobacterial cell against INH action. Furthermore, the results also suggest that the antimycobacterial action of INH includes an oxidative component and that the loss of oxyR in M. tuberculosis, which is naturally highly sensitive to INH, contributes to its susceptibility to isoniazid.
Effect of oxyR inactivation on M. marinum virulence. To assess the effects of oxyR inactivation in a natural host model, we compared survival for M. marinum wild-type and mutant strains using the goldfish Carassius auratus. Groups of 20 fish were inoculated with either 3.5 x 108 CFU of the wild-type strain, 1.5 x 108CFU of the oxyR mutant strain, or 1.5 x 108 CFU of the oxyR-complemented strain, and fish survival was monitored. All the strains were able to cause disease in the fish model. In all cases, 10% of the fish died within 5 days and only 40% of the fish survived at day 9 (Fig. 4). These findings indicate that the oxyR mutant is as virulent as the wild-type strain in the fish infection model and that the inactivation of M. marinum oxyR does not affect the organism's fitness in this model.
M. marinum survival experiments were also carried out in human peripheral blood monocyte-derived macrophages. The data (Fig. 5) indicate no differences in survival between the wild-type and oxyR mutant strains, irrespective of the activation state of the macrophages. We conclude that oxyR inactivation did not undermine the viability of M. marinum in this assay. Thus, our results argue for a neutral virulence phenotype of oxyR inactivation in M. marinum.
Conclusions. The results shown here strengthen the notion that oxyR is a regulator of the oxidative stress response in mycobacteria and that it controls the expression of ahpC. The role of katG has already been demonstrated by Ng et al. (23), using gp91phox knockout mice and katG mutant M. tuberculosis. As shown in our study, oxyR inactivation does not seem to negatively affect KatG expression; hence, any effects observed here should be ascribed to ahpC or other genes regulated by OxyR, such as the gene encoding p39. The results presented in this study indicate a link between the loss of oxyR and mycobacterial INH susceptibility. Since AhpC has been shown to protect against combinatorial products of reactive oxygen and nitrogen species (4), while INH activation generates damaging radicals (29, 30, 41) including nitric oxide (36, 37), it is likely that the inability to induce AhpC renders oxyR mutant mycobacteria increasingly sensitive to INH. The increased INH sensitivity in the M. marinum oxyR mutant is also in keeping with the previously reported observations using ahpC mutants of M. smegmatis (43, 44) and oxyR mutants of enteric bacteria.
Our virulence analyses in a relevant host (fish model) show a neutral phenotype for the oxyR mutant in M. marinum. In other bacteria, mutants in oxyR or its effectors have been reported either to display (20) or not to show (35) overt virulence phenotypes. In the case of mycobacterial pathogens, the natural loss of oxyR in M. tuberculosis and the results with the oxyR inactivation in M. marinum presented here argue for the absence of a major virulence phenotype associated with oxyR elimination. It is thus possible that the evolutionary loss of oxyR in M. tuberculosis has not put this pathogen at a survival disadvantage during infection but nevertheless has serendipitously made it more vulnerable to drugs such as isoniazid.
ACKNOWLEDGMENTS
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ABSTRACT
The majority of slow-growing mycobacteria have a functional oxyR, the central regulator of the bacterial oxidative stress response. In contrast, this gene has been inactivated during the evolution of Mycobacterium tuberculosis. Here we inactivated the oxyR gene in Mycobacterium marinum, an organism used to model M. tuberculosis pathogenesis. Inactivation of oxyR abrogated induction of ahpC, a gene encoding alkylhydroperoxide reductase, normally activated upon peroxide challenge. The absence of oxyR also resulted in increased sensitivity to the front-line antituberculosis drug isoniazid. Inactivation of oxyR in M. marinum did not affect either virulence in a fish infection model or survival in human macrophages. Our findings demonstrate, at the genetic and molecular levels, a direct role for OxyR in ahpC regulation in response to oxidative stress. Our study also indicates that oxyR is not critical for virulence in M. marinum. However, oxyR inactivation confers increased sensitivity to isonicotinic acid hydrazide, suggesting that the natural loss of oxyR in the tubercle bacillus contributes to the unusually high sensitivity of M. tuberculosis to isoniazid.
INTRODUCTION
Tuberculosis is the number one cause of death from a single infectious agent in the world, with 8 million new cases of active disease each year, 2 million fatalities annually, and over a billion people latently infected (WHO fact sheet; http://www.who.int/mediacentre/factsheets/fs104/en/). The members of the Mycobacterium tuberculosis complex (16, 21), the causative agent of tuberculosis, and several other pathogenic mycobacteria, including M. marinum (7, 15, 32), have the ability to infect macrophages and survive within phagocytic cells (2, 3, 17). Despite several advances in the field in the wake of the availability of M. tuberculosis genomic information, much remains to be learned about the biology and pathogenesis of the tubercle bacillus. Evolutionarily, M. marinum is closely related to M. tuberculosis (38), causing tuberculosis like-disease in poikilothermic hosts, such as fish (6, 26). In humans, M. marinum can cause localized disease that is restricted to the extremities (39). Due to its close evolutionary link to M. tuberculosis and apparent parallels between the diseases caused by the two microbes, M. marinum has been used effectively to study mycobacterial pathogenesis (1, 14, 24, 25, 32, 34).
Recent studies of the oxidative stress response genes in mycobacteria have revealed that the two most significant mycobacterial pathogens, M. tuberculosis and M. leprae, paradoxically lack parts of their oxidative stress response defenses (11, 24). In M. tuberculosis, oxyR, the central regulator of bacterial oxidative and nitrosative stress response (18, 33), is surprisingly inactive (10, 11, 28); it is represented in the M. tuberculosis genome as an unannotated pseudogene (see Fig. 1A), located between the Rv2427c and Rv2428 (AhpC) open reading frames. M. leprae, on the other hand, has an intact oxyR but lacks a functional furA, the negative regulator of KatG (43). Despite the loss of oxyR in M. tuberculosis, several of the oxidative stress response genes in M. tuberculosis are preserved. One of the genes suspected to be under the control of OxyR is ahpC, encoding alkylhydroperoxide reductase (AhpC), which appears to be expressed only under static growth conditions in M. tuberculosis (31). AhpC has been suggested to protect mycobacteria from the deleterious effects of the combinatorial products of reactive oxygen and nitrogen intermediates and has been implicated in isoniazid (INH) resistance (9, 13, 22, 27, 31). The oxyR gene is highly conserved and is functional in most mycobacteria (other than the M. tuberculosis complex) with slow to moderate growth rates, including M. marinum (24). The counterintuitive loss of oxyR in M. tuberculosis appears paradoxical, since it would be expected that the elimination of oxyR might reduce mycobacterial fitness in the face of oxidative defense mechanisms of the host phagocytic cells. However, the exact effects of this M. tuberculosis evolutionary signature are not known.
Here we demonstrate, at the genetic and molecular levels, that OxyR regulates ahpC in response to peroxide-induced stress. oxyR inactivation showed neutral effects on M. marinum virulence. Our results also indicate that the M. marinum oxyR knockout mutant is more sensitive to INH than the wild-type parental strain.
MATERIALS AND METHODS
Bacterial strains, plasmids, and culture conditions. M. marinum ATCC 15069 and ATCC 927 were obtained from the American Type Culture Collection. M. marinum mutant strains 27.1 and 91.4 were derived from M. marinum strain ATCC 927 by insertion of an IS1096 element into cysD and a PE family gene using the pYUB285 transposon delivery plasmid. Mycobacteria were grown in Middlebrook 7H9 medium or 7H10 plates (Becton Dickinson, Sparks, MD) supplemented with albumin-dextrose-catalase (ADC) (Becton Dickinson, Sparks, MD) enrichment and 0.05% Tween 80. Medium was supplemented with 50 μg/ml kanamycin (Sigma, St. Louis, MO) when necessary. Escherichia coli was grown in LB broth or plates supplemented with 100 μg/ml of ampicillin and 5-bromo-4-chloro-3-indolyl--D-galactopyranoside (X-Gal) (both from Sigma, St. Louis, MO) when required. All incubations were carried out at 30°C unless otherwise noted.
Construction of plasmid vectors. To generate the plasmid used for inactivation of oxyR on the M. marinum chromosome via homologous recombination, a 1,672-bp fragment containing the complete ahpC-oxyR coding sequences was amplified by PCR from genomic DNA using oligonucleotides 5'-TATAGGCGCCCTACGCCGAAG-3' and 5'-ATAAGGCGCCCAGGCTCAGTTG-3' and then cloned into the PCR2.1 vector (Invitrogen, Carlsbad, CA). The resulting plasmid was restricted with EcoRI, and the excised fragment was subcloned into the EcoRI site of pUC19. An NdeI-SphI fragment was excised and inserted into the NdeI-SphI site of pSM243, a mycobacterial suicide vector carrying the sacB gene, to generate pEPM1. A 1.2-kb NheI-SpeI Kmr cassette from pMV206 was inserted into the NheI site of the M. marinum oxyR gene in pEPM1, followed by the insertion of a 2.0-kb XbaI-NheI xylE cassette from pHSX-1 (8) into the NheI site of the oxyR::Kmr construct, resulting in pEPM2 with a 4.9-kb fragment carrying the oxyR::xylE+ Kmr construct.
Allelic replacement. Plasmid pEPM2 (oxyR::xylE+ Kmr) was introduced into M. marinum ATCC 15069 by electroporation. Potential recombinants were selected in two steps. First, merodiploids were selected by plating cells on 7H10 medium supplemented with 0.2% pyruvate and kanamycin at 10 μg/ml. The presence of the fragment was tested for by screening for xylE activity (generation of yellow colonies upon spraying with catechol [Sigma, St. Louis, MO]) to distinguish real transformants from spontaneous Kmr mutants. Merodiploids were screened for legitimate recombination by PCR using primer 5'-GGTCCTCAGTCTCTGACCA-3', an oligonucleotide upstream of oxyR (sequence not carried in pEPM2), and primer 5'-TGCAGTTTCATTTGATGCTCGATGAGT-3', an oligonucleotide specific for the Kmr cassette. To select for the second recombination event that would integrate the oxyR::xylE+ Kmr construct into the chromosome, merodiploids were plated onto 7H10 plates containing 10% sucrose and kanamycin at 10 μg/ml. Colonies that were resistant to sucrose were screened by PCR using primers 5'-GGTCCTCAGTCTCTGACCA-3' and 5'-GTCCTCCATAGGCCGAAC-3', which hybridize upstream and downstream of the DNA fragment used for the disruption. These colonies were further screened by Southern blot hybridization. For complementation, a functional copy of oxyR was inserted into the M. marinum chromosome using an integrative plasmid encoding Hygr as previously described (42).
DNA and protein purification, Southern blot hybridization, and gel shift assays. Mycobacterial DNA was isolated as previously described (42). Two micrograms of DNA from M. marinum ATCC 15069 and M. marinum a21868 (oxyR::xylE+ Kmr) was digested with SacII, separated in a 1% agarose gel, and transferred to a Duralon membrane (Perkin-Elmer Life Sciences, Boston, MA). An oxyR-specific hybridization probe was generated by PCR using primers 5'-ATCCGGTTCGGCATCATCCCC-3' and 5'-GCAACTCGGACAGTGCCG-3' and was labeled with [-32P]dCTP (3,000 Ci/mmol; NEN DuPont) by random priming (Invitrogen, Carlsbad, CA). OxyR was overexpressed and purified according to procedures reported previously (12). A DNA-protein binding electrophoretic mobility shift assay was conducted as previously described (24).
Determination of sensitivity to INH and peroxides. Disk inhibition assays were used to determine differences in sensitivities of M. marinum ATCC 15069 and M. marinum a21868 to INH, hydrogen peroxide (H2O2), and cumene hydroperoxide (CHP). The strains were grown to mid-exponential phase (optical density at 600 nm [OD600], 0.5) in 7H9 medium, and single-cell suspensions were obtained by vortexing with 3-mm glass beads (Fisher Scientific, Pittsburgh, PA) and filtration through a 5-μm-pore-size filter (Osmonics, Inc., Minnetonka, MN). Two-hundred-microliter aliquots were mixed with soft 7H10 agar and poured onto 7H10 plates to grow a bacterial lawn. BBL paper disks (6 mm; Becton Dickinson, Sparks, MD) were impregnated with 10 μl of INH (2 mg/ml), H2O2 (300 mM), or CHP (30 mM) (all from Sigma, St. Louis, MO) or mixes of INH and peroxides (at the same concentrations) and were placed on top of the solidified soft agar. Zones of growth inhibition were measured after 3 to 4 days of incubation at 30°C. Six plates were done for each condition for both strains.
Determination of isoniazid MIC. MICs were determined using Etest strips (AB Biodisk) according to the manufacturer's recommendations and represent the mean and standard deviation of quadruplicate determinations.
Exposure of mycobacteria to hydrogen peroxide and immunoblot analysis. M. marinum ATCC 15069 and M. marinum a21868 were grown in 7H9 medium to an OD600 of 0.5, aliquoted into 50-ml portions, and treated with 0.02, 0.2, 2, and 10 mM H2O2 (Sigma) or 0.02, 0.1, 0.2, and 0.6 mM CHP (Sigma). Cells were incubated in a 37°C shaker for 2 h. Crude protein extracts were obtained by homogenization in a Mini Bead-beater (Biospec Products Inc., Bartlesville, OK) for 2 min using zirconium beads (diameter, 0.1 to 0.15 mm). The beads and cell debris were removed by centrifugation, and the resulting supernatants were used for immunoblot analysis. Twenty micrograms of total protein was run on sodium dodecyl sulfate (SDS)-polyacrylamide gels (11%), and proteins were transferred to Immobilon-P membranes (Millipore, Billerica, MA) by electroblotting. Western blot (immunoblot) analysis was performed using a rabbit antiserum to M. tuberculosis AhpC raised against purified AhpC (13) as the primary antibody and goat anti-rabbit immunoglobulin G conjugated to peroxidase (Kirkegaard & Perry Laboratories Inc., Gaithersburg, MD) as the secondary antibody. The anti-KatG antibody was from C. Barry. Bound antibody was visualized by formation of diaminobenzidine precipitate.
Metabolic labeling of proteins. M. marinum wild-type and oxyR-deleted mutant strains were grown to an OD600 of 0.5 in 7H9 medium and aliquoted into 5-ml aliquots in polypropylene tubes. H2O2 or CHP was added to final concentrations of 0.125, 0.25, 0.5, and 2 mM, followed by the addition of 10 μCi of [35S]methionine and [35S]cysteine (Expre35S35S protein labeling mix; 1,000 Ci/mmol; Perkin-Elmer Life Sciences, Boston, MA). After 2 h at 37°C, the cultures were centrifuged and washed once with phosphate-buffered saline (PBS). The bacterial pellets were resuspended in 100 μl of PBS and homogenized by bead beating as described above. The resulting supernatants were run in 11% SDS-polyacrylamide gels. Gels were dried and exposed to X-ray film (Kodak Inc., Rochester, NY) for autoradiography.
Catalase activity assay. The M. marinum wild-type strain (ATCC 15069), the oxyR-deleted strain, and the oxyR-complemented strain were grown at 30°C in 7H9 medium with 10% OADC (oleic acid-ADC) (supplemented with 50 μg/ml kanamycin for mutants) to an OD600 of 1.6 to 1.8. Crude protein extracts were obtained by homogenization by bead beating as described above. The beads and cell debris were removed by centrifugation, and the resulting supernatants were used for catalase activity assays with the Amplex Red catalase assay kit (Molecular Probes) according to the manufacturer's recommendations.
Survival in macrophages. Human peripheral blood monocyte-derived macrophages were infected at a multiplicity of infection of 10:1 with the respective strains of M. marinum either in the presence or in the absence of human gamma interferon (200 U/ml) and lipopolysaccharide (50 ng/ml). Cells were incubated at 37°C for 3 days. Cell lysates for the day 0 and day 3 time points were serially diluted and plated for CFU. This time point showed optimal differences in murine macrophages.
Fish model of tuberculosis. The M. marinum wild-type strain (ATCC 15069), the oxyR-deleted strain, and the oxyR-complemented strain were grown in 7H9 medium with 10% OADC (supplemented with 50 μg/ml kanamycin for mutants) to an OD600 of 1.6 to 1.8. The cells were harvested by centrifugation, and the pellet was resuspended in sterile PBS and sonicated for 3 min at power level 3, while being cooled using a cup horn accessory attached to a cell disruptor (heat systems, model W-220F; Branson Ultrasonics Corp., Danbury, CT). The inoculum was frozen in 1-ml aliquots at –80°C. Twenty fish per group were infected intraperitoneally either with 3.5 x 108 CFU of the wild-type strain or with 1.5 x 108 CFU of the oxyR knockout mutant strain or the complemented strain and were monitored for 21 days.
Statistical analyses. Statistical analyses (Fisher's least-squares determination) were performed with ANOVA (version 1.11), Abacus Software.
RESULTS AND DISCUSSION
Inactivation of oxyR in M. marinum. Since all members of the M. tuberculosis complex lack a functional oxyR and have additional mutations in oxyR binding sites (9), we resorted to inactivating the oxyR gene in M. marinum as a model system for studying mycobacterial virulence (15, 32). The M. marinum oxyR-ahpC locus has a genetic organization identical to that of M. tuberculosis and other pathogenic mycobacteria (Fig. 1A) (24). The inactivation was done by constructing a mycobacterial suicide vector, pEPM2, carrying a copy of the entire M. marinum ahpC-oxyR coding region with the oxyR gene disrupted by a xylE+ Kmr cassette at the NheI site of the gene (Fig. 1B). The xylE gene was used as a second screening marker for plasmid insertion given the high background of spontaneous resistance of mycobacteria when plated on Km-supplemented media (8). The plasmid also carries the counterselectable marker sacB, for isolation of second crossover events on sucrose-containing media. After electroporation into M. marinum and screening for xylE+ colonies among Kmr outgrowths, a merodiploid isolate that contained a legitimate recombination integration event was obtained. This strain, upon growth in the presence of sucrose (to select for loss of the sacB marker), gave rise to an isolate containing merodiploid resolution due to a second crossover, resulting in the oxyR::xylE+ Kmr mutant strain M. marinum a21868. The gene replacement was confirmed by Southern blot analysis (Fig. 1C).
OxyR is a regulator of the oxidative stress response and ahpC. OxyR has been well characterized in enteric bacteria (5), where it is known to regulate several genes involved in the peroxide stress response, including ahpC, katG, gorA, fur, dps, and oxyS. In M. tuberculosis, various deletions and point mutations have rendered the oxyR gene inactive. This inactivation has been linked to the silencing of the ahpC gene under certain conditions (31). Mycobacterial OxyR (from M. leprae, where oxyR is intact) has been purified (12) and shown to bind to the intergenic region between oxyR and ahpC (12, 24) where the promoters for these genes have been mapped (44). To date, however, there is no direct in vivo evidence demonstrating that oxyR controls the expression of ahpC or any other gene in mycobacteria. To test whether oxyR regulates ahpC and katG, we analyzed AhpC and KatG levels in wild-type (oxyR+) and mutant (oxyR::xylE+ Kmr) strains. M. marinum ATCC 15069 (wild-type strain) and M. marinum a21868 (oxyR::xylE+ Kmr) were exposed to increasing concentrations of either H2O2 or an organic peroxide, CHP, and protein extracts were immunoblotted for AhpC and KatG using corresponding antibodies (Fig. 2A and B). The wild-type strain showed increased steady-state levels of AhpC with increasing concentrations of H2O2, with a peak induction at 0.2 mM H2O2 (180% relative to the untreated control) and 0.2 mM CHP (210% of the control). Inspection of KatG levels in the same extracts (thus doubling as a loading control) in the wild-type strain showed a slight increase or drop (144% for H2O2 and 46% for CHP) in KatG levels when AhpC levels were high. However, in the oxyR::xylE+ Kmr mutant strain, AhpC and KatG levels remained similar and were not induced by peroxides (88% of the control for H2O2 and 46% of the control for CHP). At high peroxide levels (i.e., 10 mM), some loss of either AhpC or KatG was detected, possibly due to increased degradation and/or inability to increase production due to excessive damage. It is noteworthy that in most samples, KatG levels showed inverse intensity to AhpC in the oxyR+ strain, thus providing an internal control for ahpC expression. These experiments demonstrate that OxyR is required for induction of ahpC expression in response to peroxide stress.
Additional confirmation that OxyR induces the expression of ahpC was achieved by [35S]methionine metabolic labeling of newly synthesized polypeptides in the M. marinum oxyR+ and oxyR::xylE Kmr strains exposed to H2O2 or CHP and analysis by SDS-polyacrylamide gel electrophoresis (PAGE) and autoradiography (Fig. 3A). Several polypeptides were induced in the wild-type strain in response to both H2O2 and CHP treatments, including AhpC (the band was identified as previously described [9]), and at least seven additional polypeptides (p17.4, p18.4, p31, p33, p39, p41, p45) (Fig. 3A). In addition to AhpC, induction of at least one other polypeptide, of 39 kDa (p39), was affected in the oxyR mutant strain (Fig. 3A). A direct regulation of ahpC by OxyR was confirmed by DNA binding of OxyR to the ahpC promoter region by using an electrophoretic mobility shift assay (Fig. 2C). No positive gel shift was observed with the katG promoter region (Fig. 2C, lanes 5 and 6), consistent with a lack of negative effects of oxyR inactivation on the KatG response to H2O2 or CHP stimulation (Fig. 2A and B). Moreover, no significant differences in catalase activity were observed between strains (Fig. 3B). The specificity of mycobacterial OxyR interaction within binding sites has been established previously (12). These observations constitute the first direct genetic and molecular evidence for the role of OxyR as a regulator of the oxidative stress response in mycobacteria. However, the observation that several peroxide-inducible polypeptides retained a capacity for increased de novo synthesis despite the absence of OxyR suggests the presence of oxyR-independent oxidative stress response systems in M. marinum.
Inactivation of oxyR causes increased sensitivity to peroxides and INH. To test whether inactivation of oxyR resulted in increased M. marinum susceptibility to peroxides, disk inhibition assays were used and sensitivities to CHP compared for wild-type oxyR+ and oxyR::xylE+ Kmr mutant strains (Table 1). A significant (P < 0.01) increase was observed in the growth inhibition zone for the oxyR::xylE+ Kmr mutant strain in the presence of hydrogen (not shown) or organic peroxides compared to that of the parent strain, M. marinum ATCC 15069 (Table 1). Since it has been shown that a range of reactive radicals are generated during INH activation by mycobacterial catalase-peroxidase (9, 30, 37), we also tested whether the susceptibility of the oxyR mutant to INH was affected. We tested this using the previously published disk inhibition zone assay (19, 40, 43, 44) (Table 1). These results for INH were also confirmed by conventional MIC determination, which showed a significant decrease, from 26.3 ± 1.5 to 14.9 ± 0.7 μg/ml (P < 0.001), upon oxyR mutation. Complementation of oxyR by integration of a functional oxyR gene into the chromosome of the oxyR::xylE+ Kmr insertional mutant reversed the increased susceptibility to INH and CHP (Table 1). Furthermore, complementation reversed the decrease in the MIC of INH, putting it back to 25.1 ± 1.2 μg/ml, a value not significantly different from that for the wild type (P = 0.18). These results demonstrate that elimination of a functional OxyR results in increased susceptibility to INH in M. marinum, a mycobacterium otherwise highly resistant to this antituberculosis drug. These findings demonstrate that oxyR participates in the protection of the mycobacterial cell against INH action. Furthermore, the results also suggest that the antimycobacterial action of INH includes an oxidative component and that the loss of oxyR in M. tuberculosis, which is naturally highly sensitive to INH, contributes to its susceptibility to isoniazid.
Effect of oxyR inactivation on M. marinum virulence. To assess the effects of oxyR inactivation in a natural host model, we compared survival for M. marinum wild-type and mutant strains using the goldfish Carassius auratus. Groups of 20 fish were inoculated with either 3.5 x 108 CFU of the wild-type strain, 1.5 x 108CFU of the oxyR mutant strain, or 1.5 x 108 CFU of the oxyR-complemented strain, and fish survival was monitored. All the strains were able to cause disease in the fish model. In all cases, 10% of the fish died within 5 days and only 40% of the fish survived at day 9 (Fig. 4). These findings indicate that the oxyR mutant is as virulent as the wild-type strain in the fish infection model and that the inactivation of M. marinum oxyR does not affect the organism's fitness in this model.
M. marinum survival experiments were also carried out in human peripheral blood monocyte-derived macrophages. The data (Fig. 5) indicate no differences in survival between the wild-type and oxyR mutant strains, irrespective of the activation state of the macrophages. We conclude that oxyR inactivation did not undermine the viability of M. marinum in this assay. Thus, our results argue for a neutral virulence phenotype of oxyR inactivation in M. marinum.
Conclusions. The results shown here strengthen the notion that oxyR is a regulator of the oxidative stress response in mycobacteria and that it controls the expression of ahpC. The role of katG has already been demonstrated by Ng et al. (23), using gp91phox knockout mice and katG mutant M. tuberculosis. As shown in our study, oxyR inactivation does not seem to negatively affect KatG expression; hence, any effects observed here should be ascribed to ahpC or other genes regulated by OxyR, such as the gene encoding p39. The results presented in this study indicate a link between the loss of oxyR and mycobacterial INH susceptibility. Since AhpC has been shown to protect against combinatorial products of reactive oxygen and nitrogen species (4), while INH activation generates damaging radicals (29, 30, 41) including nitric oxide (36, 37), it is likely that the inability to induce AhpC renders oxyR mutant mycobacteria increasingly sensitive to INH. The increased INH sensitivity in the M. marinum oxyR mutant is also in keeping with the previously reported observations using ahpC mutants of M. smegmatis (43, 44) and oxyR mutants of enteric bacteria.
Our virulence analyses in a relevant host (fish model) show a neutral phenotype for the oxyR mutant in M. marinum. In other bacteria, mutants in oxyR or its effectors have been reported either to display (20) or not to show (35) overt virulence phenotypes. In the case of mycobacterial pathogens, the natural loss of oxyR in M. tuberculosis and the results with the oxyR inactivation in M. marinum presented here argue for the absence of a major virulence phenotype associated with oxyR elimination. It is thus possible that the evolutionary loss of oxyR in M. tuberculosis has not put this pathogen at a survival disadvantage during infection but nevertheless has serendipitously made it more vulnerable to drugs such as isoniazid.
ACKNOWLEDGMENTS
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