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Journal of Bacteriology, April 2008, p. 2981-2986, Vol. 190, No. 8
0021-9193/08/$08.00+0     doi:10.1128/JB.01857-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Nitrate Enhances the Survival of Mycobacterium tuberculosis during Inhibition of Respiration{triangledown}

Charles D. Sohaskey*

Department of Veterans Affairs Medical Center, Long Beach, California 90822,1 Department of Microbiology and Molecular Genetics, University of California, Irvine, California 927172

Received 26 November 2007/ Accepted 8 February 2008


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ABSTRACT
 
When oxygen is slowly depleted from growing cultures of Mycobacterium tuberculosis, they enter a state of nonreplicating persistence that resembles the dormant state seen with latent tuberculosis. In this hypoxic state, nitrate reductase activity is strongly induced. Nitrate in the medium had no effect on long-term persistence during gradual oxygen depletion (Wayne model) for up to 46 days, but significantly enhanced survival during sudden anaerobiosis. This enhancement required a functional nitrate reductase. Thioridazine is a member of the class of phenothiazines that act, in part, by inhibiting respiration. Thioridazine was toxic to both actively growing and nonreplicating cultures of M. tuberculosis. At a sublethal concentration of thioridazine, nitrate in the medium improved the growth. At lethal concentrations of thioridazine, nitrate increased survival during aerobic incubation as well as in microaerobic cultures that had just entered nonreplicating persistence (NRP-1). In contrast, the survival of anaerobic persistent (NRP-2) cultures exposed to thioridazine was not increased by the addition of nitrate. Nitrate reduction is proposed to play a role during the sudden interruption of aerobic respiration due to causes such as hypoxia, thioridazine, or nitric oxide.


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INTRODUCTION
 
It has been estimated that over 2 billion people are infected with Mycobacterium tuberculosis, the causative agent of tuberculosis (9). This is a staggering one-third of the world's population. Most of these infections are latent, showing no obvious symptoms. However, reactivation does occur and can progress to active disease. During latency, tubercle bacilli show increased resistance to many of the antibiotics that are used to treat active disease. There is a clear necessity to identify drugs that can target M. tuberculosis in this latent state. Understanding latency is important for the eventual control and eradication of tuberculosis.

In the human host, tubercle bacilli reside in granulomas, structures that limit the availability of oxygen. This condition is reproduced in vitro by the Wayne model (27). Growing cultures of M. tuberculosis in sealed tubes gradually deplete the available oxygen and enter a microaerobic state called nonreplicating persistent stage 1 (NRP-1). This stage is characterized by the cessation of bacterial replication, a strong induction of respiratory nitrate reductase activity, and a change in energy metabolism (20, 22, 28). With further incubation, the oxygen levels continue to decrease until cultures enter the anaerobic stage, NRP-2. At this point, most RNA and protein synthesis has ceased and the bacteria are resistant to many antibiotics.

One of the activities induced in M. tuberculosis during oxygen deprivation is the respiratory reduction of nitrate to nitrite. This activity provides energy for the bacteria entering the NRP state (28, 29). In culture, the induction of nitrate reductase activity is not due to an increase in the levels of the nitrate reductase enzyme encoded by narGHJI (22). Instead, hypoxia results in an induction of the nitrate transporter encoded by narK2. There is an added level of regulation, as NarK2 is active only in the absence of oxygen or when aerobic respiration is inhibited (21). In a similar manner, narK2, but not narGHJI, is induced during chronic infection of mice (20) or after exposure to nitric oxide (25).

Nitric oxide is proposed to play an important role in controlling the replication of M. tuberculosis in humans, inducing dormancy and preventing reactivation (8, 16). Nitric oxide and hypoxia both interfere with oxygen utilization in M. tuberculosis and produce a nonreplicating state (25). Both also induce nitrate respiration. Nitric oxide is unstable and quickly breaks down, with nitrate being one of the stable end products. Therefore, at the time that M. tuberculosis enters a nonreplicating state, there is an increase in available nitrate.

During the inhibition of aerobic respiration, nitrate reduction may play a key role in the survival of M. tuberculosis. The role of nitrate reduction during inhibition was analyzed in growing and NRP cultures. Because of the short-lived nature of nitric oxide, the phenothiazine thioridazine was used to inhibit respiration.


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MATERIALS AND METHODS
 
Culture conditions. M. tuberculosis H37Rv from the culture collection of this laboratory was used. Cultures were grown in Dubos Tween-albumin broth (DTA) (Difco, Detroit, MI) at a starting density of 2.5 x 106 cells/ml. Aerobic cultures were incubated at 37°C on an Innova model 4000 rotary shaker-incubator at a speed of 275 rpm (New Brunswick Scientific Co., Inc., Edison, NJ). Growth was monitored at 580 nm. Plating was on Dubos oleic albumin agar. Nitrate, when used, was added at a final concentration of 5 mM.

MIC. M. tuberculosis in mid-log phase was diluted in 10 ml DTA to a cell density of 2.5 x 106 cells/ml. Thioridazine or ethanol (as the control) was added, and cultures in triplicate were incubated aerobically at 37°C. The MIC was defined as the lowest concentration that resulted in at least 90% inhibition, as measured at the time a control culture reached an optical density of 580 nm (OD580) of 1.0. The 50% inhibitory concentration (IC50) was defined as the concentration that produces a 50% inhibition under the same conditions as above. Thioridazine hydrochloride (Sigma, St. Louis, MO) was dissolved in ethanol.

Measurement of oxygen utilization. Mid-log-phase cultures containing approximately 2.5 x 108 cells/ml (OD580 of ~0.4) were aliquoted to 8-ml screw-cap tubes. Methylene blue to 0.0003% was added. Thioridazine ranging from 100 µg/ml to 0.8 µg/ml was added. Tubes were sealed with Parafilm and incubated at 37°C. Decolorization of methylene blue, indicating the utilization of oxygen, was monitored at 665 nm for 8 h.

Sudden anaerobiosis. Cultures were grown to mid-log phase in DTA with no nitrate. These were pooled, and the OD580 adjusted to 0.100. When indicated, nitrate was added at a final concentration of 5 mM. Then 8 ml of cultures was aliquoted in completely filled screw-cap tubes. Oxyrase was added to remove oxygen (Oxyrase, Inc. Mansfield, OH). These tubes were tightly capped and incubated at 37°C. Control tubes with methylene blue as an indicator of the oxygen concentration were completely decolorized within 2 h. At intervals, a tube was opened for plating and then discarded. Each curve was repeated twice.

Nitrite assays. Aerobic or NRP-1 cultures, containing approximately 108 CFU/ml, were opened and dispensed into either loosely capped tubes (aerobic incubation) or tightly capped tubes (anaerobic incubation). Oxyrase was used to remove oxygen. At intervals, samples were removed to determine nitrite concentrations by the Griess reaction (22). The cell-free nitrate reductase assay was as published previously (22).

Survival with thioridazine. Aerobically growing cultures were diluted to approximately 108 CFU/ml in DTA. Thioridazine or ethanol was added, and aerobic incubation continued.

For NRP cultures (Wayne model), conditions were as described previously by using a headspace ratio of 0.5 (27). Culture tubes were sealed with septum caps and wrapped with Parafilm. At the time of shiftdown into NRP-1, approximately 67 h after inoculation, thioridazine or ethanol was carefully injected to avoid the introduction of air bubbles. Each curve was repeated twice. For NRP-2 cultures, thioridazine was injected at 208 h after the OD580 had leveled off. After 200 h, oxygen levels are less than 0.06% saturation (27). At intervals, a tube was opened for plating and then discarded.

Statistics. Growth and survival plots were compared using Student's two-tailed t test. P values of ≤0.01 were considered significant.


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RESULTS
 
Effect of nitrate on anaerobic survival. To determine the effect of nitrate on M. tuberculosis during anaerobiosis, two approaches were used. In the first, cultures with or without nitrate were incubated in the Wayne model to allow time for them to adapt to a slowly decreasing oxygen concentration. The survival levels of wild-type H37Rv and the nitrate reductase mutant RVW1 were analyzed. RVW1 is an M. tuberculosis narGHJI knockout mutant with no detectable nitrate reductase activity (22). To monitor survival, samples were plated starting at the time of the initial deflection of the growth curve into NRP-1 and then at intervals for over 40 days (Fig. 1A). There was little change in viability over the course of the experiment, with less than a 1-log decrease from the initial time point to day 47. This result suggests that nitrate reduction is not essential for survival during shiftdown to nonreplicating persistence in the Wayne model.


Figure 1
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  		FIG. 1. Effect of nitrate on survival during hypoxia. Cultures were supplemented with (broken lines and open symbols), or without (solid lines and filled symbols) 5 mM NO3–. Circles, wild type; squares, RVW1 ({Delta}narG). The standard deviations are indicated by error bars. (A) Gradual hypoxia in the Wayne model. (B) Sudden anaerobiosis. Oxyrase was added to remove oxygen. For all time points except the initial one, the P value was <0.01, which we determined by Student's t test, comparing the CFU/ml of WT cultures with nitrate to that of cultures without nitrate.

In the second approach, the effect of nitrate on survival during sudden anaerobiosis was determined. Sudden anaerobiosis was defined as the creation of anaerobic conditions in a time less than the 18-h doubling time of M. tuberculosis. Aerobically growing cultures of M. tuberculosis with and without nitrate were sealed, and the oxygen was removed with Oxyrase. Methylene blue, used as an indicator of the oxygen level, faded within 2 h. Samples were plated at intervals to determine viability (Fig. 1B). In contrast to the gradual onset of hypoxia in the Wayne model, sudden anaerobiosis was quickly lethal to all cultures. The wild type showed a 3-log decrease in 26 days, with an overall half-life of 59 h. Cultures with nitrate showed increased survival and a significantly higher half-life of 86 h. To determine whether the increased survival was due to nitrate reduction, the survival of RVW1 during sudden anaerobiosis was measured. The half-life of RVW1 was 49 h both with and without nitrate, indicating that nitrate reduction was responsible for the enhanced survival of the wild type.

MIC. Nitrate enhanced survival during sudden interruption of aerobic respiration. Both nitric oxide and thioridazine also interfere with aerobic respiration, although they do so by different mechanisms (7, 25, 30). Nitric oxide has a short half-life, breaking down into several compounds, including nitrate and nitrite. Both of these interfere with attempts to measure nitrate reductase activity. Therefore, in place of nitric oxide, thioridazine was used to determine the effect of nitrate during the inhibition of respiration.

The MIC and IC50 values of thioridazine on actively growing cultures of M. tuberculosis were determined. The MIC of 7.5 µg/ml was similar to values in previously published reports (7, 17), while the IC50 was 2.5 µg/ml. At the MIC, there was greater than 90% inhibition of growth (Fig. 2A). Cultures with 5 mM nitrate were also inhibited by this level of thioridazine, but to a lesser extent (significance after 223 h of growth was a P value of 0.01). The MIC and IC50 values of the narG mutant RVW1 were identical to those of the wild type, but nitrate had no effect on growth with thioridazine (Fig. 2B).


Figure 2
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  		FIG. 2. Minimum inhibitory level of thioridazine. OD580 growth curves of M. tuberculosis with thioridazine. The circles represent growth in DTA, while the triangles represent growth in DTA containing 7.5 µg/ml of thioridazine. Solid symbols and lines are cultures without nitrate, while those with empty symbols and dotted lines contain 5 mM NaNO3. (A) Wild type. (B) RVW1 ({Delta}narG). The standard deviations are indicated by error bars.

Thioridazine is reported to have multiple targets. To determine the frequency of spontaneous resistance, ~109 CFU was plated on 15 µg/ml and 30 µg/ml thioridazine. No resistant mutants were isolated, indicating a frequency of spontaneous resistance of less than 10–9.

The effect of thioridazine on oxygen utilization by M. tuberculosis was determined with the redox indicator methylene blue. Complete inhibition of decolorization was seen at 60 µg/ml, indicating strong inhibition of respiration. Fifty percent inhibition was seen at fourfold the MIC (30 µg/ml).

Effect of thioridazine on nitrate reduction. Since thioridazine interferes with aerobic respiration, its effect on nitrate respiration was determined also. The change in the concentration of nitrite was used as an indication of nitrate reductase activity in aerobic cultures of M. tuberculosis in which both nitrate and thioridazine were added (Table 1). The addition of thioridazine resulted in a fivefold increase in the rate of nitrite production during a 12-h time frame. The experiment was repeated with hypoxic NRP cultures. Due to the production of the nitrate transporter NarK2, the control NRP cultures showed a 54-fold increase in activity relative to that of the aerobic cultures (22). The same NRP cultures treated with thioridazine had activities similar to those of the control, indicating no effect on nitrate reductase activity (Table 1).


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  		TABLE 1. Rate of nitrite production during exposure to thioridazinea

The NarK2 transporter is inactive in aerobic conditions unless respiration is inhibited (21). Since thioridazine inhibits respiration, its effect on NarK2 in the presence of oxygen was determined. An NRP culture was opened to introduce oxygen and incubated aerobically. It showed low-nitrite production (Table 1). With the addition of thioridazine, there was an eightfold induction of nitrite production. This activity was similar to the level produced by an aerobic culture incubated with thioridazine, indicating no activation of NarK2.

A cell-free assay was used to determine whether thioridazine had an effect on nitrate reductase enzyme. Methyl viologen was used as the electron donor. The control reaction had 39 ± 2 U of activity. This was similar to a reaction with 30 µg/ml thioridazine that produced 38 ± 2 U of activity.

Next, the effect of thioridazine on nitrate reduction during aerobic growth was determined. Cultures were grown aerobically, and both nitrate (5 mM) and thioridazine (30 µg/ml) were added. Nitrite concentrations were determined daily for 8 days (Fig. 3). Replication ceased with the addition of thioridazine, while the control culture continued to grow (data not shown). As seen previously, there was a short-term increase in nitrite production, but after 12 h of exposure to thioridazine, nitrite production ceased abruptly.


Figure 3
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  		FIG. 3. Effect of thioridazine on nitrite synthesis. Cultures with nitrate were grown aerobically (•) and with thioridazine ({circ}), and nitrite concentrations were determined. The standard deviations are indicated by error bars.

Effect of thioridazine on actively growing cultures. The effect of inhibition of respiration by thioridazine on the survival of aerobically growing M. tuberculosis with and without nitrate was determined. Cultures were treated with thioridazine, and viability was determined by plating for 4 days (Fig. 4). The untreated controls continue to grow until they entered stationary phase on day 3. The cultures with thioridazine showed a decline in the CFU, resulting in a decrease of more than 99% by day 4. The cultures with both thioridazine and nitrate also showed a decline in CFU, however, at a significantly reduced rate. After day 2, there was a log difference in viability between thioridazine-treated cultures with nitrate and those without nitrate. The half-life of cultures with thioridazine was 13 h, but it was 38 h for those containing both thioridazine and nitrate.


Figure 4
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  		FIG. 4. Effect of nitrate on aerobic growth during thioridazine treatment. Aerobic cultures without (solid lines and filled symbols) or with 5 mM NO3– (dotted lines and empty symbols) were treated with thioridazine. Circles, control cultures with ethanol; triangles, cultures with 30 µg/ml thioridazine. The standard deviations are indicated by error bars. For all time points except the initial one, the P value was <0.01, as we determined by Student's t test, comparing the CFU/ml of thioridazine-containing cultures with nitrate to that of cultures without nitrate.

Effect of thioridazine on NRP cultures. There are no reports of the effect of thioridazine on nonreplicating persistent cultures. To determine the effect on microaerobic cultures, thioridazine was injected right after shiftdown to NRP-1 and viability followed by plating (Fig. 5A). Cultures entering NRP-1 were sensitive to thioridazine, and they showed a 4-log decrease in CFU by day 8. Thioridazine was significantly less toxic in the presence of nitrate, with a decrease in CFU of only 2 logs.


Figure 5
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  		FIG. 5. Effect of nitrate on the survival of NRP cultures treated with thioridazine. Cultures were grown in the Wayne model without (solid lines and filled symbols) or with 5 mM NO3– (dotted lines and empty symbols). At early NRP-1 (A) or NRP-2 (B), thioridazine (30 µg/ml) (triangles) or ethanol (circles) was injected. The standard deviations are indicated by error bars.

Next the effect of thioridazine in anaerobic NRP-2 cultures was determined. Cultures were grown for 200 h in the Wayne model, at which point oxygen levels had dropped below 0.06% saturation (27). Thioridazine was injected, and survival was measured by plating (Fig. 5B). Cultures exposed to thioridazine showed decreased survival, with a 4-log decrease in CFU after 6 days.


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DISCUSSION
 
Anaerobiosis. There is growing evidence that in the human host, the availability of oxygen is limiting for Mycobacterium tuberculosis during some stages of infection (6, 29). Direct measurement of the gaseous content of tubercles showed decreased oxygen levels (13). Also in activated macrophage, an oxygen gradient was detected between the phagosome and the extracellular space (14). M. tuberculosis is an obligate aerobe requiring oxygen for replication. A fundamental aspect of the Wayne model is that the depletion of oxygen is gradual, providing time for cultures to adapt. This allows persistence of M. tuberculosis for at least 46 days under hypoxic conditions with little decrease in viability (Fig. 1A). Without this period of adaptation, viability decreases with a half-life of 59 h (Fig. 1B).

In response to gradual hypoxia, M. tuberculosis stops replicating and there is a decrease in protein and RNA synthesis. These responses produce a lower energy demand on the cell and were thought to be major adaptations for anaerobic survival (29). During the microaerobic transition stage (NRP-1), more than 100 genes are induced, and many of these are regulated by the DosR/DevR system (19, 25). Interestingly, although DosR/DevR mutants show normal growth cessation, they had decreased survival in the Wayne model (5, 25). This result suggests that the decreased metabolism by itself is not sufficient for anaerobic survival and, instead, that genes regulated directly or indirectly by DosR/DevR may be responsible for the long-term persistence during anaerobiosis.

M. tuberculosis can use nitrate as a final electron acceptor, although this does not permit growth in the absence of oxygen (22). Nitrate had no effect on survival in the Wayne model, but had a significant effect during sudden anaerobiosis. The narG nitrate reductase mutant was unaffected by the presence of nitrate, indicating a role for nitrate respiration in the increased survival.

The nitrate reductase system has been identified as an important virulence factor for M. tuberculosis. It was suggested that the elevated levels of nitrate reductase activity of some lineages of M. tuberculosis may have increased their virulence, and thus, their evolutionary success (12). Transcripts of narG were detected in granulomas from the lungs of tuberculosis patients showing expression in vivo (10, 18).

A narG knockout mutant of the closely related Mycobacterium bovis BCG showed reduced virulence and lung damage in immunocompetent and SCID mice (11). In comparison to infections with wild-type M. bovis BCG, SCID mice infected with the narG mutant had reduced numbers of granulomas and fewer bacteria in both the liver and lungs. The narG mutant still produced tissue damage in the lungs of immunocompetent mice, but was cleared from many organs, unlike the wild-type strain.

The expression of narG and narK2 by M. tuberculosis in the Wayne model is similar to that seen both in mice and in vitro after exposure to nitric oxide (20, 25). In mice, narG was constitutively expressed, while narK2 expression was upregulated at the time that M. tuberculosis shifted from active growth to chronic infection (20).

Inhibition of respiration. Thioridazine belongs to a class of antipsychotic drugs called phenothiazines. They have been shown to inhibit the growth of M. tuberculosis (1, 4) by targeting the type II NADH dehydrogenase (30, 31), succinate dehydrogenase (7), and the binding of calcium to proteins (2). Nitrate reductase was not a direct target, as there was no inhibition in a cell-free assay. The inhibition of respiration by thioridazine would be abrupt, producing an effect similar to sudden anaerobiosis or exposure to nitric oxide.

Thioridazine briefly increased the rate of nitrate reduction in aerobic cultures (Table 1). This was not due to increased transport of nitrate by narK2, since the transcription of this gene was not induced by thioridazine (7), but it may be due to the shunting of electrons from cytochrome oxidase to nitrate reductase. The increase in nitrate reductase activity lasted for approximately 12 h, at which time the production of nitrite ceased (Fig. 3). Nitrate reduction resulted in an enhanced ability to resist the inhibitory effects of low concentrations of thioridazine (Fig. 2). At higher lethal concentrations of thioridazine, there was increased survival in aerobic cultures due to the presence of nitrate (Fig. 4). This increase in growth rate at MIC levels or the increased survival at higher concentrations was not seen with the narG mutant.

Nonreplicating cultures of M. tuberculosis are more resistant to antibiotics, with cultures in anaerobic NRP-2 generally more resistant than those in microaerobic NRP-1 (27). NRP-1 cultures were sensitive to thioridazine injected at the time of shiftdown (Fig. 5A). Survival increased when nitrate was present in the medium. This result implies that aerobic respiration was a main target of thioridazine under microaerobic conditions. NRP-2 cultures were also sensitive to thioridazine, although there was no effect of nitrate (Fig. 5B). NRP-2 cultures are anaerobic, resulting in greatly reduced metabolism. Therefore, in NRP-2 cultures the main target of thioridazine is probably not respiration.

These results suggest that one role of nitrate reductase in M. tuberculosis is to allow the electron transport chain to remain active when aerobic respiration is partially or completely inhibited. This inhibition could be due to sudden anaerobiosis or to the addition of a respiratory poison, such as thioridazine or nitric oxide. The reason for the loss of viability during sudden anaerobiosis is not clear. It may be due to an imbalance in the NADH/NAD+ ratio and/or the decrease in ATP levels due to the collapse of the proton gradient. Gradual hypoxia produced little change in either ATP or cell viability, while more rapid hypoxia produced a decrease in ATP levels that paralleled a decrease in viability (27). A role of nitrate reductase during the sudden interruption in aerobic respiration may be to maintain the proton gradient for ATP production or to control the redox balance of the cell by dissipating excess reducing equivalents.

The nitrate reductase system is important for M. tuberculosis survival during exposure to compounds besides thioridazine. Pyrazinamide, one of the frontline antibiotics in the treatment of tuberculosis, is proposed to act by collapsing the proton gradient (32). Survival was increased in the presence of nitrate as an alternate electron acceptor under anaerobic conditions (26). Since nitrate present in the medium can change drug susceptibility, it should be included when testing compounds that affect respiration.

Potential use of thioridazine for treatment. There has been increasing interest in the possible use of phenothiazines for the treatment of tuberculosis (2, 23, 30). This is due in part to the increase in multidrug-resistant and extensively drug-resistant tuberculosis and to the development of novel thioridazine analogs (15, 30). The current first-line antibiotics are effective in the treatment of active tuberculosis. This is not true for multidrug-resistant or extensively drug-resistant strains of M. tuberculosis, which can be difficult to treat, even with alternate antibiotics. Thioridazine was active against multidrug-resistant M. tuberculosis (1, 4, 17) and acted synergistically with rifampin and streptomycin (24). Importantly, thioridazine was lethal to dormant M. tuberculosis in both NRP-1 and NRP-2 cultures (Fig. 5). While the MIC in vitro is 7.5 µg/ml, a concentration of approximately 0.5 µg/ml is the maximum achievable concentration in humans (4). M. tuberculosis strains residing in macrophages are susceptible to thioridazine even at 0.1 µg/ml, because macrophages concentrate phenothiazines into the vacuoles where M. tuberculosis reside (3, 17). In active tuberculosis, most tubercle bacilli are extracellular; they are where the concentration of thioridazine would not be inhibitory. The necessity for M. tuberculosis to be intracellular to allow for the concentration of thioridazine would limit its use to early stage or latent disease. Nitrate present from the breakdown of nitric oxide would be available to M. tuberculosis, further reducing the toxicity of thioridazine. For these reasons, thioridazine is not a promising candidate for the treatment of tuberculosis.


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ACKNOWLEDGMENTS
 
This study was supported by the Medical Research Services of the U.S. Department of Veterans Affairs.

I thank Sylors Chem and Michelle Thissen for comments on the manuscript.


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FOOTNOTES
 
* Mailing address: Tuberculosis Research Laboratory (151), Department of Veterans Affairs Medical Center, 5901 East Seventh Street, Long Beach, CA 90822. Phone: (562) 826-8000, ext. 4912. Fax: (562) 826-5675. E-mail: chuck{at}sohaskey.com Back

{triangledown} Published ahead of print on 22 February 2008. Back


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Journal of Bacteriology, April 2008, p. 2981-2986, Vol. 190, No. 8
0021-9193/08/$08.00+0     doi:10.1128/JB.01857-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.



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