A Mycobacterium marinum mel2 Mutant Is Defective for Growth in ...

INFECTION AND IMMUNITY, Jan. 2007, p. 127–134 0019-9567/07/$08.00⫹0 doi:10.1128/IAI.01000-06 Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Vol. 75, No. 1

A Mycobacterium marinum mel2 Mutant Is Defective for Growth in Macrophages That Produce Reactive Oxygen and Reactive Nitrogen Species䌤 Selvakumar Subbian,1 Parmod K. Mehta,1 Suat L. G. Cirillo,1 Luiz E. Bermudez,2 and Jeffrey D. Cirillo1* Department of Microbial and Molecular Pathogenesis, Texas A&M University Health Sciences Center, 471 Reynolds Medical Building, College Station, Texas 77843,1 and Department of Biomedical Sciences, College of Veterinary Medicine, and Department of Microbiology, College of Sciences, Oregon State University, Corvallis, Oregon 973312 Received 23 June 2006/Returned for modification 12 September 2006/Accepted 29 September 2006

Macrophages produce reactive oxygen species (ROS) and reactive nitrogen species (RNS) in response to bacterial infections. Mycobacteria are relatively resistant to ROS, but RNS inhibit growth of, and possibly even kill, mycobacteria in activated macrophages. We recently constructed a Mycobacterium marinum mel2 locus mutant, which is known to affect macrophage infection. We found previously that the mel2 locus confers resistance to ROS and RNS in laboratory medium, suggesting that this locus might play a similar role during growth in macrophages. Since J774A.1 murine macrophages produce high levels of ROS and RNS upon activation with gamma interferon (IFN-␥), we examined the effects of IFN-␥ on ROS and RNS production by these cells as well as the effects on growth of M. marinum in these cells. We found that an M. marinum mutant with mutation of the first gene in the mel2 locus, melF, is defective for growth in IFN-␥-plus-lipopolysaccharidetreated J774A.1 cells and that this defect is abrogated by the presence of either inhibitors of nitric oxide synthase or ROS scavengers. Furthermore, the M. marinum melF mutant displays a defect at late stages in the mouse footpad model of infection. These phenotypic characteristics could be complemented fully by the entire mel2 locus but only partially by the presence of melF alone, supporting data suggesting that this insertion mutation has polar effects on downstream genes in the mel2 locus. These observations demonstrate that the M. marinum mel2 locus plays a role in resistance to ROS and RNS produced by activated macrophages. More than 150 Mycobacterium marinum infections are observed in humans each year (16), usually associated with aquatic sports, fishing, or aquarium maintenance. Mycobacterium marinum causes primarily skin lesions on the extremities in humans (24, 27). This is most likely due to its low temperature for optimal growth of between 25 and 35°C (9), since human skin temperatures are thought to be in the range of 33 to 35°C (43), which is similar to the case for another mycobacterial skin pathogen, Mycobacterium leprae (11). During skin infections in humans (12) and mice (9, 13), M. marinum replicates within macrophages, and disease is characterized by granuloma formation, with some similarities to those in human tuberculosis (12, 22). Macrophages from numerous species (4, 21, 33, 40) and epithelial cell lines (44, 45) can be efficiently infected by and allow growth of M. marinum. Thus, M. marinum, similar to other pathogenic mycobacteria, can resist or avoid the bactericidal mechanisms of macrophages. Mycobacteria are relatively resistant to the reactive oxygen species (ROS) produced by macrophages (8), but their intracellular growth is inhibited by activation with gamma interferon (IFN-␥) through the production of reactive nitrogen species (RNS) by nitric oxide synthase (7, 18, 31). Further-

more, mice that are respiratory burst deficient as a result of a defect in NADPH oxidase, similar to chronic granulomatous disease patients, whose macrophages do not produce significant levels of ROS, are relatively resistant to mycobacterial infection (1). RNS can inhibit growth of mycobacteria and are thought to be important for protective immunity (7, 8, 18, 23, 31), making the mechanisms of mycobacterial resistance to RNS an active area of study. Interestingly, Mycobacterium tuberculosis is relatively resistant to peroxynitrite anion compared to less pathogenic mycobacteria (51). Mycobacteria have several mechanisms of resistance to ROS and RNS (34), but the specific role of each of these pathways during pathogenesis is not well understood. We recently identified the mycobacterial mel2 locus, which is important for the ability of M. marinum to infect human, mouse, and fish macrophages (20). In silico analysis of the genes present in the mel2 locus demonstrated that these genes have striking similarity to bioluminescence or lux genes from other organisms (20). It is particularly intriguing that the melF gene within this locus displays high similarity to luxA, a monooxygenase gene that has been implicated in resistance to ROS in bioluminescent bacteria (30, 41, 49). Interestingly, we found that an M. marinum mutant with mutation in the mel2 locus has increased susceptibility to ROS and RNS in vitro (S. Subbian, P. K. Mehta, S. L. G. Cirillo, and J. D. Cirillo, submitted for publication). As a result of these observations, we asked in the current study whether the role of the M. marinum mel2 locus in resistance to ROS and RNS is responsible for its effects on macrophage infection. We found that the M. mari-

* Corresponding author. Mailing address: Department of Microbial and Molecular Pathogenesis, Texas A&M University Health Sciences Center, 471 Reynolds Medical Building, College Station, TX 77843. Phone: (979) 458-0778. Fax: (979) 845-3479. E-mail: jdcirillo@medicine .tamhsc.edu. 䌤 Published ahead of print on 9 October 2006. 127

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num mel2 mutant is defective for growth in activated J774A.1 macrophages and displays a moderate decrease in growth in the mouse footpad model of infection. The M. marinum mel2 mutant does not display any growth defect in macrophages in the presence of ROS scavengers or nitric oxide synthase inhibitors, demonstrating that this growth defect is dependent on the production of both ROS and RNS. These observations provide further insight into the molecular mechanisms used by M. marinum to overcome host defense mechanisms and cause disease. MATERIALS AND METHODS Strains and growth conditions. M. marinum strain M, a clinical isolate obtained from the skin of a patient (40), was used in these studies. M. marinum strains were grown at 33°C in 7H9 broth (Difco, Detroit, MI) supplemented with 0.5% glycerol, 10% albumin-dextrose complex (ADC), and 0.25% Tween 80 (M-ADC-TW) for 5 days. The M. marinum mel2 mutant carries a mini-Mu transposon insertion in the melF gene as described previously (20). The M. marinum melF::pJDC79 strain is a melF mutant that carries the plasmid pMV262 (48), carrying the melF gene from M. tuberculosis, which has previously been shown to complement the macrophage infection defect of the M. marinum mel2 mutant (20). The M. marinum melF::pJDC75 strain is a melF mutant that carries the single-copy integrating plasmid pYUB178 (37), with the entire M. tuberculosis mel2 locus cloned into its single NheI site. The number of viable bacteria was determined for each assay by using the LIVE/DEAD assay (Molecular Probes, Eugene, OR) and by plating dilutions for CFU on 7H9 (M-ADC) agar (Difco, Detroit, MI). All inocula used were ⬎99% viable. Where appropriate, kanamycin was added at a concentration of 25 ␮g/ml (Escherichia coli) or 10 ␮g/ml (M. marinum). Cell culture. The murine macrophage cell line J774A.1 (ATCC TIB67) was maintained at 37°C and 5% CO2 in high-glucose Dulbecco’s modified Eagle medium (Gibco, Bethesda, MD) supplemented with 10% heat-inactivated fetal bovine serum (Gibco) and 2 mM L-glutamine. Cells were equilibrated at 33°C for 1 hour prior to assays involving M. marinum. Cell viability was determined by exclusion of trypan blue. Aliquots of the cells were incubated at room temperature for 3 min in 0.2% trypan blue (wt/vol) in phosphate-buffered saline (PBS) and washed five times with PBS prior to quantitation of viable cells in a hemocytometer. Cell viability in all assays was ⬎90%, and none of the cellular treatments significantly affected cellular viability. In addition, the viabilities of the cells remained comparable throughout all time points used in these studies. Mouse footpad model of infection. C57BL6/J mice were infected with approximately 1 ⫻ 104 CFU intradermally in the right footpad, as described previously, for evaluation of M. marinum virulence in mammals (13). There were five mice in each experimental group for each time point. Total footpad CFU were determined for the right foot by plating dilutions of homogenized tissue on M-ADC agar. Antimycobacterial activity of macrophages. The ability of the different mycobacterial strains to survive and replicate in macrophages was evaluated using the J774A.1 cell line, essentially as described previously (20). Macrophages were seeded in 24-well tissue culture dishes (Costar) at 1 ⫻ 106 cells per well and treated with 100 U murine IFN-␥ (Boehringer Mannheim) plus 1 ␮g/ml lipopolysaccharide (E. coli LPS; Difco) for 24 h at 37°C. The medium was replaced before use, cells were equilibrated to 33°C for 1 hour, and the cells were infected at a multiplicity of infection of 0.1 bacterium per cell for 30 min at 33°C. The cells were then washed twice with PBS, and the medium was replaced with medium containing 30 ␮g/ml amikacin to prevent extracellular bacterial replication, as described previously (20). The medium was then removed from triplicate sets of wells at various time points, and the cells were lysed using 1 ml of 0.1% Triton X-100 (Sigma) for 10 min. Dilutions were plated on M-ADC agar to determine intracellular CFU. Survival is expressed as the increase in CFU compared to the CFU at time zero (30 min), i.e., x-fold increase ⫽ CFU Tx/CFU T0. We evaluated the role of ROS and RNS in the mycobactericidal activity of macrophages by scavenging ROS or inhibiting RNS production in macrophages with various compounds. We treated macrophages with diazabicyclooctane (DABCO; 0.1 and 1 mM), mannitol (1 and 10 mM), or superoxide dismutase (SOD; 150 and 1,500 U) after infection with mycobacteria to scavenge ROS throughout the incubation period. The role of RNS was examined in the same manner, using L-arginine (10 mM), NG-monomethyl-L-arginine monoacetate (NMMA; 250 and 500 ␮M), or NG-nitro-L-arginine methyl ester hydrochloride (NAME; 1 and 2 mM).

INFECT. IMMUN. Measurement of ROS and RNS levels. Macrophages were examined for production of ROS and RNS in the presence of lipopolysaccharide (1 ␮g/ml; Difco), murine gamma interferon (100 U/ml; Boehringer Mannheim), phorbol 12myristate 13-acetate (PMA [100 ng/ml]; Sigma), M. marinum, or combinations of these reagents for 24 h at 37°C. ROS levels were evaluated by measuring H2O2 production with horseradish peroxidase-dependent oxidation of phenol red (38) and comparison to a standard curve developed with known concentrations of H2O2. Basically, the cells were stimulated with the reagents and/or bacteria for 24 h, the medium was removed, and 100 ␮l of assay solution (28 mM phenol red, 100 U/ml horseradish peroxidase in Hanks balanced salt solution) containing the same stimulants was added. The plates were incubated at 37°C for 1 h, and the reaction was stopped by adding 10 ␮l of 1 N NaOH. The plates were allowed to equilibrate for 3 min and read at 630 nm. Levels of RNS were determined by measuring the NO2⫺ content through reaction with the Griess reagent as described previously (25). Basically, the medium was removed from the wells, and an equal volume was mixed with Griess reagent and incubated at room temperature for 10 min. The optical density at 540 nm was then determined and compared against a standard curve constructed using NaNO3 to determine the actual concentration of NO2⫺. Statistical analyses. All experiments were carried out in triplicate and repeated at least three times, unless otherwise noted. The significance of the results was determined using the Student t test or analysis of variance. P values of ⬍0.05 were considered significant.

RESULTS M. marinum induces an oxidative burst in macrophages. Our previous observations demonstrated that our M. marinum mel2 locus mutant displays a defect in resistance to ROS compared to wild-type M. marinum in vitro (Subbian et al., submitted for publication). However, it remains unclear whether the mel2 locus plays a role in resistance to ROS during interactions with macrophages. Since it has not previously been shown that M. marinum induces an oxidative burst in macrophages, we examined whether ROS are produced at significant levels during infection by M. marinum. We measured the production of ROS, as assayed by H2O2 levels, in J774A.1 macrophages after infection by M. marinum compared to those after treatment with LPS, IFN-␥, and PMA (Fig. 1). We found that the M. marinum wild type and the mel2 mutant induced an oxidative burst in J774A.1 cells that was comparable to that induced by LPS from E. coli. Production of H2O2 by J774A.1 cells was not significantly greater for cells treated with both M. marinum and IFN-␥ than for those treated with M. marinum alone, suggesting that further activation of these cells will not increase the oxidative burst generated. These observations demonstrate that M. marinum induces a strong oxidative burst in J774A.1 cells. M. marinum mel2 mutant is not defective for growth in resting macrophages. Since M. marinum induces an oxidative burst in macrophages, it is likely that ROS play a significant role in the defense against these bacteria during infections. We first examined whether resting J774A.1 cells are able to mount a sufficiently strong response to infection by the M. marinum wild type and the mel2 mutant to allow differences to be observed in survival and replication (Fig. 2). The mutant and wild-type M. marinum strains were compared to complemented mutant strains that carry melF alone (pJDC79) as well as the entire mel2 locus (pJDC75), since our previous observations suggest that this insertion mutation has polar effects on the downstream genes in the locus (Subbian et al., submitted for publication). No significant difference in the growth of the mel2 mutant compared to that of wild-type M. marinum was observed in J774A.1 cells under standard culture conditions.

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FIG. 2. Growth of the M. marinum wild type, a mel2 mutant that carries an insertion in melF (melF), the mutant carrying a plasmid expressing melF alone (melF::pJDC79), and the mutant carrying an integrated single-copy plasmid expressing the entire mel2 locus (melF::pJDC75) in J774A.1 murine macrophages. The graph shows the increase in cell number (CFU Tx/CFU T0) for each strain after 24 h of growth in macrophages with 30 ␮g/ml amikacin to prevent extracellular bacterial replication. Data are the means and standard deviations for assays done in triplicate and are representative of three experiments. FIG. 1. Evaluation of the oxidative burst produced by 5 ⫻ 105 J774A.1 murine macrophages treated for 24 h with different agents, including LPS, murine IFN-␥, PMA, 5 ⫻ 104 CFU of Mycobacterium marinum or the M. marinum mel2 mutant that carries an insertion in melF (melF), and combinations of these treatments. H2O2 concentrations were determined using a standard curve created with known concentrations of H2O2. Data are the means and standard deviations for assays done in triplicate and are representative of two experiments.

Since we observed a strong oxidative burst after 24 h of infection with M. marinum and only very low H2O2 production in untreated cells, these cells may not produce enough ROS initially to show the phenotypic effects of the mel2 mutant. We reasoned that priming or activating J774A.1 cells prior to infection would create more stringent growth conditions for M. marinum, reminiscent of macrophages in a host that are already mounting a strong immune response to infection. Activated J774A.1 cells produce high levels of ROS and RNS. We compared the production of ROS by J774A.1 cells after treatment with PMA, LPS, IFN-␥, PMA plus LPS, and IFN-␥ plus LPS (Fig. 3A). Each of these treatments resulted in the production of high levels of ROS compared to those in untreated cells, suggesting that infection of J774A.1 cells pretreated with these compounds would allow us to examine the effects of ROS on growth of the M. marinum mel2 mutant. All treatments resulted in similar levels of ROS production, except for PMA treatment of J774A.1 cells, which produced slightly lower levels (P ⬍ 0.04) than the strongest ROS-producing treatment, IFN-␥ plus LPS. The production of ROS was examined prior to all subsequent experiments and was always

between 9 and 15 ␮M under our standard experimental conditions, i.e., treatment with IFN-␥ plus LPS. Since the M. marinum mel2 mutant displays greater susceptibility than the wild type to both ROS and RNS in vitro (Subbian et al., submitted for publication), we examined whether treating J774A.1 cells with these compounds resulted in the production of RNS as well as ROS. We found that treatment with PMA, LPS, and IFN-␥ individually did not increase basal levels of RNS produced by J774A.1 cells but that both PMA plus LPS and IFN-␥ plus LPS increased RNS production by these cells (Fig. 3B). Treatment of J774A.1 cells with IFN-␥ plus LPS increased RNS production a great deal more than did treatment with PMA plus LPS (P ⬍ 0.001). Based on these observations, we concluded that treatment of IFN-␥ plus LPS leads to maximal levels of production of both ROS and RNS in J774A.1 cells. Thus, we chose to use J774A.1 cells treated with IFN-␥ plus LPS for further studies because they appear to mimic cells that are mounting a strong immune response to infection and will provide stringent conditions to evaluate the fitness of the M. marinum mel2 mutant under oxidative stress. M. marinum mel2 mutant is defective for growth in activated macrophages. We examined the ability of the M. marinum mel2 mutant to survive and replicate in J774A.1 cells activated by treatment with IFN-␥ plus LPS compared to that of resting (untreated) cells (Fig. 4A). The mel2 mutant only displayed a significant (P ⬍ 0.01) defect for intracellular growth in J774A.1 cells when they were activated with IFN-␥ plus LPS, not when they were untreated. Complementation studies demonstrated

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FIG. 3. (A) Production of H2O2 by 5 ⫻ 105 J774A.1 murine macrophages treated for 24 h with different agents, including LPS, murine IFN-␥, PMA, and combinations of these compounds. H2O2 concentrations were determined using a standard curve created with known concentrations of H2O2. (B) Production of nitrite (NO2⫺) by 5 ⫻ 105 J774A.1 murine macrophages treated for 24 h with different agents, including LPS, murine IFN-␥, and PMA. Nitrite concentrations were determined using a standard curve created with NaNO2. Data are the means and standard deviations for assays done in triplicate and are representative of two experiments.

that the mel2 mutant displays a defect in growth within activated J774A.1 cells (P ⬍ 0.01) that can be complemented partially with the melF gene and completely with the entire mel2 locus (Fig. 4B). Partial complementation was not expected with melF alone because our earlier studies suggested that the insertion mutation in melF has polar effects on downstream genes in the mel2 locus. These observations are consistent with the susceptibility of this mutant to ROS and RNS in vitro and suggest that the primary defect of the mel2 mutant

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FIG. 4. (A) Growth of M. marinum wild type and the mel2 mutant that carries an insertion in melF (melF) in resting (untreated) and activated (IFN-␥ ⫹ LPS) J774A.1 cells. The murine IFN-␥ concentration used was 100 U/ml, and the LPS concentration was 1 ␮g/ml. (B) Growth of M. marinum wild type, the mel2 mutant that carries an insertion in melF (melF), the mutant carrying a plasmid expressing melF alone (melF::pJDC79), and the mutant carrying an integrated single-copy plasmid expressing the entire mel2 locus (melF::pJDC75) in IFN-␥- and LPS-treated J774A.1 murine macrophages. The graphs show the increase in cell number (CFU Tx/CFU T0) for each strain after 24 h of growth in macrophages with 30 ␮g/ml amikacin to prevent extracellular bacterial replication. Data are the means and standard deviations for assays done in triplicate and are representative of three experiments.

during growth in macrophages is a result of its greater susceptibility to ROS and RNS produced by macrophages. ROS scavengers rescue the growth defect of the mel2 mutant. If growth of the mel2 mutant in macrophages is inhibited by the presence of ROS produced by these cells, the addition of ROS scavengers should rescue this defect and allow wildtype growth. We treated activated macrophages with three ROS scavengers, namely, mannitol, DABCO, and SOD, during infection by wild-type M. marinum and the mel2 mutant.

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FIG. 6. Growth of M. marinum wild type and the mel2 mutant that carries an insertion in melF (melF) in IFN-␥- and LPS-treated J774A.1 murine macrophages in the presence of L-arginine (L-arg) and the nitric oxide synthase inhibitors NMMA (A) and NAME (B). The graphs show the increase in cell number (CFU Tx/CFU T0) for each strain after 24 h of growth in macrophages with 30 ␮g/ml amikacin to prevent extracellular bacterial replication. Data are the means and standard deviations for assays done in triplicate and are representative of three experiments.

FIG. 5. Growth of M. marinum wild type and the mel2 mutant that carries an insertion in melF (melF) in IFN-␥- and LPS-treated J774A.1 murine macrophages in the presence of ROS scavengers mannitol (A), DABCO (B), and SOD (C). The graphs show the increase in cell number (CFU Tx/CFU T0) for each strain after 24 h of growth in macrophages with 30 ␮g/ml amikacin to prevent extracellular bacterial replication. Data are the means and standard deviations for assays done in triplicate and are representative of three experiments.

All three ROS scavengers reduced ROS production to basal levels, allowed recovery of wild-type levels of growth in macrophages (Fig. 5), and improved growth of the wild-type bacteria. None of these scavengers affected growth in resting macrophages (data not shown), since they most likely did not produce sufficient ROS prior to infection with mycobacteria to inhibit bacterial growth. These observations suggest that ROS reduce the ability of M. marinum to survive and/or replicate in mammalian macrophages. In addition, the production of ROS

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TABLE 1. Growth of the M. marinum melF mutant in mouse footpads CFU/g of footpad

Experimental groupa M. M. M. M.

marinum marinum::pMV262 marinum melF::pMV262 marinum melF::pJDC79

1 day

3 wk

8.3 ⫻ 104 ⫾ 0.9 ⫻ 104 7.9 ⫻ 104 ⫾ 0.4 ⫻ 104 8.0 ⫻ 104 ⫾ 0.5 ⫻ 104 7.9 ⫻ 104 ⫾ 0.7 ⫻ 104

3.5 ⫻ 104 ⫾ 0.7 ⫻ 105 3.7 ⫻ 104 ⫾ 0.5 ⫻ 105 2.1 ⫻ 104 ⫾ 0.6 ⫻ 105b 3.1 ⫻ 104 ⫾ 0.4 ⫻ 105

a An inoculum of 104 bacteria was used for all experimental groups. Data represent the means for duplicate platings of tissues from 10 mice per group. b Significantly different (P ⬍ 0.05) from the result obtained with wild-type M. marinum at the same time point.

is at least partially responsible for the growth defect observed with the mel2 mutant. NOS inhibitors rescue the growth defect of the mel2 mutant. We also examined the role of RNS during growth of the mel2 mutant in activated macrophages. The addition of the nitric oxide synthase (NOS) inhibitor NMMA or NAME to activated macrophages during infection reduced RNS production approximately 10-fold, resulting in recovery of wild-type levels of mel2 mutant growth in these cells (Fig. 6). Similar to the addition of ROS scavengers, the addition of NOS inhibitors enhanced the growth of wild-type M. marinum within these cells. We demonstrated the specificity of the NOS inhibitors by the addition of L-arginine, which competitively blocks the inhibitory activity of these compounds on NOS. In all cases, the presence of L-arginine caused the mel2 mutant to display a defect in macrophages similar to that in untreated cells (Fig. 6). These inhibitors did not affect growth in resting macrophages (data not shown), most likely because they did not produce significant RNS. These data suggest that the mel2 locus plays a role in the resistance of M. marinum to both ROS and RNS during growth in activated macrophages. The mel2 mutant affects growth of M. marinum in mouse footpads. ROS and RNS are important components of the mammalian immune response to bacterial pathogens. In particular, RNS have been shown to be a key component of protection against mycobacterial infections (7, 8, 18, 31). Since M. marinum infection presents almost exclusively as granulomatous skin lesions on the extremities in humans (3, 10, 12, 16), we wished to utilize an animal model that closely mimics the disease in humans to examine the role of the mel2 mutant in virulence. Mouse footpads can be infected by M. marinum and produce granulomas similar to the skin lesions observed during M. marinum infections in humans. This model was previously shown to allow evaluation of virulence in different M. marinum strains (13). We utilized this model to examine the effects of the mel2 locus in the ability of M. marinum to grow in mouse footpads (Table 1). The melF mutant displayed a defect in the ability to replicate in mouse footpads during the first 3 weeks after infection (P ⬍ 0.05). The presence of the vector backbone alone did not affect growth of either the wild-type bacteria or the mel2 mutant. In contrast, the melF gene on a plasmid allowed complementation of this defect. The modest growth defect observed in the mouse footpad model of infection suggests that this model is not as sensitive to defects that affect susceptibility to the host immune response as our in vitro activated macrophage model. Possibly, this is

due to a reduced immune response in the extremities. However, these data do suggest that the mel2 locus plays a role in the virulence of M. marinum in mammals. DISCUSSION In the current study, we found that the M. marinum mel2 locus plays an important role in the ability of M. marinum to survive and replicate in macrophages producing ROS and RNS. Pathogenic mycobacteria must withstand toxic oxygen and nitrogen species produced by macrophages and within granulomatous lesions created during an immune response in order to cause disease. We showed that M. marinum induces a strong oxidative burst in J774A.1 cells, which is consistent with the ability of these bacteria to induce Toll-like receptor 1 and 2 genes in zebrafish (32). ROS and RNS represent key components of innate immunity and are also produced at high levels as part of an acquired immune response. Chronic granulomatous disease patients are defective in the production of ROS, but this deficiency does not significantly affect their susceptibility to mycobacterial infections (6). In contrast, the importance of RNS in defense against mycobacterial infections is well established (7, 23, 31). Despite their susceptibility to other RNS, M. tuberculosis strains are relatively resistant to peroxynitrite anion (51), an RNS that kills most other bacterial species (5, 52). The molecular mechanisms of mycobacterial resistance to ROS and RNS have been an area of intense investigation and suggest that there are multiple pathways involved in resistance (34). Pathways that have been described include superoxide dismutase (39), catalase (35), alkyl hydroperoxide reductase (50), nucleotide excision repair (15), the proteasome (14), the noxR systems (19, 42), methionine sulfoxide reductase (17, 47), and dihydrolipoamide acyltransferase (46). Whether these pathways represent redundancy due to the importance of defense against reactive species in general or whether each pathway is primarily responsible for resistance to specific reactive species remains to be determined. The growth defect observed in macrophages infected with the mel2 mutant was observed only in the presence of both ROS and RNS. These data fit well with our previous observation that the mel2 mutant is much more susceptible to the presence of both ROS and RNS in vitro. We utilized several ROS scavengers in these studies to demonstrate the specific involvement of ROS in macrophages. We also demonstrated the role of RNS in preventing growth of the mel2 mutant in macrophages by using two different NOS inhibitors. Furthermore, we demonstrated the specificity of the NOS inhibitors by the addition of L-arginine, the normal substrate for NOS (26), which alleviates the inhibitory activity of these compounds (2, 8). The fact that the mel2 mutant displays a greater defect when exposed to a combination of both ROS and RNS implies that this locus is important for growth in environments where both of these reactive species are present, such as during infection of mammals. In the current studies, we observed a significant difference between the mel2 mutant and the wild type 3 weeks after infection of mouse footpads. Possibly, this was due to the presence of other pathways that confer resistance to either ROS or RNS by itself at earlier time points. It is possible that oxyR, an important regulator of the oxidative stress response in other bacteria, or genes that it regulates

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would be likely candidates for a role in resistance in vivo, but no obvious virulence defect is observed for an M. marinum oxyR mutant in fish (36). At later time points, the mel2 locus may allow mycobacteria to persist in the presence of an immune response that results in exposure of the bacteria to higher levels of ROS and RNS simultaneously. Studies using later time points after infection and animals that have been vaccinated previously with attenuated mycobacteria should help to test this hypothesis. The involvement of both ROS and RNS in the mel2 mutant phenotype may implicate a specific role for this locus in resistance to peroxynitrite, which is produced by the reaction of superoxide with nitric oxide (28, 29, 34). This concept would fit well with a role for this locus in later stages of infection, where the bacteria are faced with activated macrophages producing high levels of both ROS and RNS. In addition, this locus affects the growth of M. marinum in mouse footpads at later time points, further supporting its role during pathogenesis, particularly in persistence in the face of an immune response. It is intriguing that the mel2 locus displays similarity to bioluminescence systems in other bacterial species (20; Subbian et al., submitted for publication). These observations suggest that mel2 represents a previously unrecognized pathway for resistance of bacterial pathogens to ROS and RNS and support the concept that bioluminescence systems may have evolved from oxidative stress defense mechanisms, as previously suggested (41). Further studies are needed to determine how widespread this pathway for resistance to ROS and RNS is in bacterial pathogens and to dissect the biochemical mechanisms involved. At present, these data provide new insight into the mechanisms of resistance of M. marinum to ROS and RNS produced by macrophages. The role of the M. marinum mel2 locus in persistence in the face of a strong immune response suggests that these genes may be useful for the development of novel treatment and prevention strategies. ACKNOWLEDGMENTS This work was supported by grant AI47866 from the National Institutes of Health. We thank David McMurray and James Samuel for critical reviews of the manuscript. REFERENCES 1. Adams, L. B., M. C. Dinauer, D. E. Morgenstern, and J. L. Krahenbuhl. 1997. Comparison of the roles of reactive oxygen and nitrogen intermediates in the host response to Mycobacterium tuberculosis using transgenic mice. Tuber. Lung Dis. 78:237–246. 2. Adams, L. B., S. G. Franzblau, Z. Vavrin, J. B. Hibbs, Jr., and J. L. Krahenbuhl. 1991. L-Arginine-dependent macrophage effector functions inhibit metabolic activity of Mycobacterium leprae. J. Immunol. 147:1642–1646. 3. Bailey, J. P., Jr., S. J. Stevens, W. M. Bell, H. G. Mealing, Jr., D. H. Loebl, and E. H. Cook. 1982. Mycobacterium marinum infection. A fishy story. JAMA 247:1314. 4. Barker, L. P., K. M. George, S. Falkow, and P. L. C. Small. 1997. Differential trafficking of live and dead Mycobacterium marinum organisms in macrophages. Infect. Immun. 65:1497–1504. 5. Brunelli, L., J. P. Crow, and J. S. Beckman. 1995. The comparative toxicity of nitric oxide and peroxynitrite to Escherichia coli. Arch. Biochem. Biophys. 316:327–334. 6. Casanova, J. L., and L. Abel. 2002. Genetic dissection of immunity to mycobacteria: the human model. Annu. Rev. Immunol. 20:581–620. 7. Chan, J., K. Tanaka, D. Carroll, J. Flynn, and B. R. Bloom. 1995. Effects of nitric oxide synthase inhibitors on murine infection with Mycobacterium tuberculosis. Infect. Immun. 63:736–740. 8. Chan, J., Y. Xing, R. S. Magliozzo, and B. R. Bloom. 1992. Killing of virulent Mycobacterium tuberculosis by reactive nitrogen intermediates produced by activated murine macrophages. J. Exp. Med. 175:1111–1122.

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