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Influence of culture medium on the resistance and response of Mycobacterium bovis BCG to reactive nitrogen intermediates
Microbes and Infection 8 (2006) 434–441 www.elsevier.com/locate/micinf
Original article
Influence of culture medium on the resistance and response of Mycobacterium bovis BCG to reactive nitrogen intermediates Walter Florio *, Giovanna Batoni, Semih Esin, Daria Bottai, Giuseppantonio Maisetta, Flavia Favilli, Franca L. Brancatisano, Mario Campa Dipartimento di Patologia Sperimentale, Biotecnologie Mediche, Infettivologia ed Epidemiologia, Università di Pisa, Via S. Zeno 35-39, 56127 Pisa, Italy Received 9 March 2005; accepted 15 July 2005 Available online 16 September 2005
Abstract The aim of the present work was to evaluate the influence of the culture medium on the resistance and response of Mycobacterium bovis BCG to reactive nitrogen intermediates, in vitro. BCG was grown in Sauton, Dubos or Middlebrook 7H9 medium and exposed to sodium nitroprusside (SNP) for up to 7 days. The percentage of bacilli that survived was significantly lower in Middlebrook 7H9 than in Sauton or Dubos medium. Addition of SNP to Middlebrook 7H9 caused an increase in the RedOx potential in either the absence or the presence of BCG, while addition of the compound to Sauton medium gave rise to an increase in the RedOx potential only in the absence of bacteria, whereas a decrease in the RedOx potential was observed in the presence of BCG. The resistance of BCG to SNP in the different media did not correlate with the concentration of peroxynitrite in culture supernatants. BCG grown in different media showed a differential protein expression pattern, as assessed by two-dimensional gel electrophoresis. Exposure of BCG to sub-lethal concentrations of SNP in Middlebrook 7H9, but not in Sauton medium, revealed a differential expression of at least 38 protein species. Altogether these results demonstrate that the growth medium may have a remarkable influence on the resistance and the response of BCG to SNP and suggest that the different resistance of BCG in the two media is unlikely to be due to a differential antioxidant effect of the medium itself. © 2005 Elsevier SAS. All rights reserved. Keywords: Mycobacterium bovis; BCG; Nitroprusside; Peroxynitrite; Two-dimensional gel electrophoresis
1. Introduction Nitric oxide (NO) and related reactive nitrogen intermediates (RNI) are highly reactive compounds produced by macrophages after activation by TNF-a and/or IFN-c [1], stimulation with LPS [2] or infection with various microorganisms, including Mycobacterium tuberculosis [3]. Production of NO by macrophages is catalyzed by the inducible isoform of NO synthase (NOS2), and represents a powerful effector mechanism against a number of pathogenic bacteria [4]. The protective role of NOS2 against M. tuberculosis has been established in several mouse models of experimental infection
Abbreviations: BCG, bacillus Calmette-Guérin; DCDHF, 2,7dichlorodihydrofluorescein; 2-DE, two-dimensional gel electrophoresis; NO, nitric oxide; NOS2, inducible nitric oxide synthase; RNI, reactive nitrogen intermediates; SNP, sodium nitroprusside. * Corresponding author. Tel.: +39 050 2213 691; fax: +39 050 2213 711. E-mail address: [email protected] (W. Florio). 1286-4579/$ - see front matter © 2005 Elsevier SAS. All rights reserved. doi:10.1016/j.micinf.2005.07.013
[5–8], and it has been shown that NOS2 may be expressed at high levels by alveolar macrophages from patients with untreated, culture-positive tuberculosis [9], indicating that RNI may play an important role in the response to tuberculosis infection also in humans. Despite the evidence of NOS2 expression in vivo, production of NOS2 by human monocytes/macrophages in cell culture systems has long been variable and controversial [10–14]. Therefore, the relevance of RNI production as a mycobactericidal activity of human macrophages remains a matter of considerable debate. The potential importance of RNI in the control of tuberculous infection has stimulated the research on the mechanisms by which M. tuberculosis resists the toxic activity of these compounds. Recent studies have shown that exposure of M. tuberculosis to RNI, in vitro, may induce the differential expression of at least 53 genes, most of which correspond to hypoxia-induced genes [15,16]. Few studies have been reported on the response of tubercle bacillus to RNI at the protein level, and a few proteins have been shown to be
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expressed differentially after exposure to various RNI generators [17–19]. Using metabolic labeling and twodimensional gel electrophoresis (2-DE), Garbe et al. [17] detected a number of polypeptides differentially expressed by M. tuberculosis after exposure to RNI in vitro. The a-crystallin homologue heat shock protein was identified as one of the most abundant proteins of M. tuberculosis after incubation with several chemically diverse NO donors, whereas incubation of the bacillus with sodium nitroprusside (SNP) or S-nitroso-N-acetyl-D,L-penicillamine, but not with other NO donors, resulted in induction of a 19 kDa protein, named Nox19, which contains amino-acidic regions homologous to sequences found in a subclass of nonheme bacterial ferritins [18]. Since iron metabolism is linked to the mycobacterial response to oxidative stress by shared regulatory components [20,21], induction of Nox19 might be part of a complex regulatory mechanism. In general, the resistance and the response of mycobacteria to RNI may be influenced by several experimental variables, such as the type of RNI generator [18] and its concentration [16], the growth phase of the culture when the stress is applied [19], the duration of the stress [15], and possibly others. In the present study, we evaluated the influence of the culture medium on the resistance of Mycobacterium bovis BCG to the RNI generator SNP, and investigated the response of the microorganism after exposure to sub-lethal concentrations of this toxic compound by utilizing a functional proteomics approach.
2. Materials and methods 2.1. Bacterial cultures and stress challenge experiments M. bovis BCG, strain Pasteur (Pasteur Merieux), was grown in roller bottles in modified Sauton medium containing 0.05% Tween 80 (v/v) [22]. For treatment with SNP, an aliquot of a mid-log phase culture was harvested by centrifugation (4600 rpm, 10 min, 25 °C), and the bacterial pellet was inoculated at a cell density of 3 × 106 c.f.u. per ml of modified Sauton medium, without Tween 80, Middlebrook 7H9 medium supplemented with 0.5% bovine serum albumin, 0.2% glucose and 0.085% NaCl or Dubos medium added of 0.1% bovine serum albumin. Cultures were grown in roller bottles at 37 °C for 5 days, then SNP (Sigma) was added from a freshly prepared 1 M solution to a final concentration of 25, 5, 1, or 0 mM (non-stressed control cultures). Inoculum size and culture conditions were optimized so that BCG cultures in the different media were all in the same (midlogarithmic) phase when SNP was added. Cultures were incubated in the presence of SNP for 24 h, 3 or 7 days. For evaluation of c.f.u., a 5-ml sample of the culture was taken at different times after addition of SNP, and bacteria were finely suspended by vortex with 1 mm diameter glass beads. Tenfold serial dilutions of each bacterial suspension were plated onto Middlebrook 7H11 agar supplemented with 10% OADC
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enrichments (Difco). The plates were sealed in plastic envelopes and incubated at 37 °C for 3 weeks before counting the number of colonies. For preparation of BCG lysates, SNP was added to midlog phase cultures to a final concentration of 1 mM; after 2 h incubation with the compound, cultures were harvested by centrifugation, the bacterial pellet was washed once with sterile MilliQ H2O, then frozen at –80 °C or immediately processed for subsequent 2-DE analysis (see below). 2.2. Measurement of RedOx potential The RedOx potential of culture media and bacterial culture supernatants, before or after addition of SNP, was measured by using a Hamilton electrochemical sensor (Hamilton Bonaduz AG). The electrode was dipped in the sample for at least 30 s before reading the RedOx potential, and it was washed extensively with MilliQ H2O between two successive measurements. 2.3. Measurement of peroxynitrite concentration The concentration of peroxynitrite generated by SNP in the different culture media was measured by using the method described by Crow [23], with some modifications. A 14.5 mM stock solution of 2,7-dichlorodihydrofluorescein (DCDHF) (Cayman Chemical) was prepared by dissolving DCDHF diacetate in 100 mM NaOH, and stored in aliquots at –20 °C. DCDHF was added to samples to a final concentration of 100 µM, and oxidation reactions were carried out in polypropylene tubes for 30 min at room temperature. Then, the reaction was stopped by adding L-ascorbic acid (Carlo Erba) from a 100 mM stock solution to a final concentration of 6 mM. Addition of L-ascorbic acid effectively prevented further slow oxidation of DCDHF without affecting the absorbance spectrum of samples in the full wavelength range of visible light (data not shown). A Hewlett–Packard Agilent 8453 spectrophotometer was used to measure the A500. Preliminary experiments were performed to ascertain that DCDHF was not reacting with bacterial products in culture supernatants. The OD500 of samples from cultures without SNP was < 10−2 for all media and at all times tested. Nevertheless, the supernatants of control cultures (no SNP added) were used as blanks in the assay for supernatants of cultures with SNP in corresponding media. The concentration of peroxynitrite reached a peak at 4–6 h after addition of SNP, then it began to slowly decrease (data not shown). 2.4. 2-DE For preparation of BCG lysates, bacteria were suspended in 9 M urea, 10 mg/ml DTT, 1% CHAPS (Sigma). Glass Beads (BioSpec Products, Inc.), 0.1 mm diameter, were added to the bacterial suspension, 1 g per milliliter of suspension, and bacteria were disrupted by vigorous shaking with a Mini Bead-Beater (BioSpec Products), six cycles of 20 s each at
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2500 rpm, keeping the bacterial suspension in ice between two successive cycles. The lysate was centrifuged for 5 min at 12,000 × g at room temperature, and IPG buffer was added to the supernatant (Amersham Biosciences) to a final concentration of 2% (v/v). A 280 µl aliquot of the resulting sample, containing 300–600 µg protein, was incorporated into immobiline dry strips, pH 4–7 (linear pH gradient) or pH 3–5.6 NL (non linear pH gradient) (Amersham Biosciences), by overnight rehydration at room temperature. Isoelectrofocusing was performed with a Multiphor II apparatus (Amersham Pharmacia Biotech) setting 300 V, 1 mA, 5 W for 10 min, followed by 3500 V, 1 mA, 5 W for 7 h at 10 °C. After isoelectrofocusing, the strips were usually frozen at –80 °C. Second dimension separation was carried out in 12.5% Laemmli polyacrylamide gels. Before loading on the gel, the strips were equilibrated for 5 min in 50 mM Tris–HCl, pH 6.8, 6 M urea, 30% glycerol (v/v), 1% SDS, 5 mg/ml DTT, then for 10 min in 50 mM Tris–HCl, pH 6.8, 10% glycerol (v/v), 2% SDS, 5 mg/ml DTT, containing bromophenol blue as a dye. Prestained proteins (Cell Signaling Technology) were used as molecular weight markers. The gels were stained with silver nitrate or Coomassie blue R-250 as previously described [24]. Stained gels were matched and analyzed with the ImageMaster 2D software (ImageMaster 2D, v. 4.01, Amersham Biosciences). For comparison of the relative protein amount in matched spots of different gels, protein spot volumes were normalized for the total volume of all the spots in the gel. Three independent samples were prepared for each biological condition, and individual samples were run in duplicate to evaluate the intrinsic variability of the technique. Individual gels from the same experiment were comparatively analyzed, and only protein spots showing at least a threefold difference in the normalized volume in a reproducible manner were considered differentially expressed.
3. Results 3.1. Resistance of BCG to SNP in different culture media In order to evaluate the influence of the culture medium on the resistance of BCG to RNI, the resistance of the bacillus to the RNI generator SNP was tested in three different culture media for mycobacteria: modified Sauton medium, a synthetic medium that is commonly used for preparation of mycobacterial culture filtrates as it does not contain exogenous proteins; Middlebrook 7H9 medium, utilized in several studies on the response of mycobacteria to RNI [17,19]; Dubos medium, another synthetic medium, included in this study as it does not contain citric acid or other organic acids that might have an antioxidant effect. BCG was inoculated in the different culture media at a cell density of 3 × 106 c.f.u. per ml and, after 5 days of growth (mid-log phase), SNP was added to cultures at different final concentrations: 1, 5, 25 or 0 mM (control cultures). The percentage of survival of BCG after 24 h, 3 and 7 days incubation with SNP was determined
by plating serial dilutions of the cultures onto Middlebrook 7H11/OADC agar plates, and counting the number of colonies after a 3 weeks’ incubation at 37 °C. Exposure of BCG to different concentrations of SNP caused a dose- and timedependent decrease in the percentage of survival of the microorganism, in all three media. Nevertheless, BCG cultured in Sauton or Dubos medium was significantly more resistant to the bactericidal effect of SNP than bacilli grown in Middlebrook 7H9 (Fig. 1). At the lowest concentration of SNP, a significant difference in the percentage of survival was observed only after 7 days incubation with the compound (Fig. 1a), whereas at the higher concentrations of SNP the difference between Middlebrook 7H9 and Sauton or Dubos became significant after 24 h or 3 days, respectively (Fig. 1b and c). The Sauton and Middlebrook 7H9 media, where the resistance of BCG was the highest and the lowest, respectively, were selected for further study. 3.2. Evaluation of the antioxidant effect of the medium RNI are highly reactive compounds with a short half-life in aqueous solutions, as they rapidly react with molecules that are susceptible to oxidation [25,26]. The chemical composition of the medium, and hence its antioxidant effect, might affect the effective concentration of RNI to which the bacterium is exposed. In order to establish whether the different susceptibility of BCG to SNP observed in Sauton compared to Middlebrook 7H9 medium was due to a differential antioxidant effect exerted by the two different media, the variation of the RedOx potential was measured after addition of SNP, both in the presence and in the absence of bacteria. As shown in Fig. 2, in the absence of bacteria, addition of SNP caused a comparable increase in the RedOx potential in the two different media. Similarly, addition of SNP to cultures in Middlebrook 7H9 determined an increase in the RedOx potential, in relation to the concentration of the RNI generator (Fig. 3). Surprisingly, addition of SNP to cultures in Sauton medium caused a decrease in the RedOx potential, at all the tested concentrations of the compound. 3.3. Generation of peroxynitrite by SNP In order to assess whether SNP was able to generate peroxynitrite in the culture systems used to test the resistance of BCG, a colorimetric assay, based on the specific oxidation of DCDHF by peroxynitrite [23], was adapted to measure the concentration of the compound after addition of SNP to the Sauton and Middlebrook 7H9 media, and to the corresponding BCG cultures. The results show that peroxynitrite was indeed generated after addition of SNP to the culture media, and that the concentration of peroxynitrite in the two different media was comparable at all the SNP concentrations tested (Fig. 4). In general, the concentration of peroxynitrite was also comparable in culture supernatants of BCG grown in Sauton or Middlebrook 7H9, with a single exception: after a 7 days incubation with 25 mM SNP, the concentration of per-
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Fig. 1. Percentage of survival of BCG grown in Sauton (circles), Dubos (triangles) or Middlebrook 7H9 medium (squares), and exposed to 1 mM (a), 5 mM (b) or 25 mM SNP (c). The data reported represent the mean ± S.E.M. of three independent experiments. *: P < 0.05; **: P < 0.01; ***: P < 0.001, Sauton vs. Middlebrook 7H9; ♦: P < 0.05; ♦♦: P < 0.01; ♦♦♦: P < 0.001, Dubos vs. Middlebrook 7H9.
Fig. 2. Variation of the RedOx potential (DORP) after addition of 1 mM (a), 5 mM (b) or 25 mM SNP (c) to the Sauton (light bars) or Middlebrook 7H9 medium (dark bars), without bacteria. The data reported represent the mean ± S.E.M. of three independent experiments.
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Fig. 3. Variation of the RedOx potential (DORP) after addition of 1 mM (a), 5 mM (b) or 25 mM SNP (c) to BCG cultures in Sauton (light bars) or Middlebrook 7H9 medium (dark bars). The data reported represent the mean ± S.E.M. of three independent experiments.
Fig. 4. Peroxynitrite concentration, 4 h after addition of different concentrations of SNP to the Sauton (circles) or Middlebrook 7H9 medium (triangles). The experiment was repeated three times with the same overall results.
oxynitrite was even higher in culture supernatants of BCG cultivated in Sauton than in Middlebrook 7H9 (Fig. 5). Therefore, the peroxynitrite concentration does not seem to correlate with the different resistance of BCG grown in the different media. 3.4. Influence of the medium and of exposure to SNP on protein expression by BCG To investigate the possibility that the culture medium might influence the expression of phenotypic characters by BCG, the protein expression profile of the bacillus cultivated in the two different media was comparatively analyzed by 2-DE, at the mid-log phase of growth. Interestingly, several proteins
were differentially expressed by the microorganisms grown in the two media. In particular, thirteen protein species were more abundant in lysates of BCG grown in Middlebrook 7H9, whereas another two polypeptides, with acidic pI, were present in higher amounts in lysates of the bacillus cultivated in Sauton medium (Fig. 6). The differentially expressed protein species were confined to a pI range between 4.2 and 5.6, and all but one had an apparent molecular mass lower than 30 kDa (Fig. 6). To investigate the effect of exposure to sub-lethal concentrations of SNP on protein expression by BCG, mid-log phase cultures of the bacillus grown in the different media were added 1 mM SNP; after two hours incubation with or without the compound, cultures were harvested and bacilli were processed for 2-DE analysis. As shown in Fig. 7, exposure of BCG cultures in Middlebrook 7H9 to SNP resulted in the differential expression of 38 protein species by the microorganism. In particular, the expression of 16 proteins was increased after incubation with SNP, whereas another 22 polypeptides were present in lower amounts in the lysates of BCG exposed to the compound than in non-exposed, control cultures. The differentially expressed proteins were distributed over a wide range of molecular mass but confined to the acidic pI range 3–5.6 (Fig. 7). No additional differentially expressed protein spots were observed after 2-DE of BCG lysates in the pI range 4–7 (data not shown). When BCG cultivated in Sauton medium was exposed to SNP, the differences in the protein expression pattern between exposed and
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Fig. 5. Peroxynitrite concentration after addition of 1 mM (a), 5 mM (b) or 25 mM SNP (c) to BCG cultures in Sauton (circles) or Middlebrook 7H9 medium (triangles). The data reported represent the mean ± S.E.M. of three independent experiments.
Fig. 6. 2-DE of lysates of BCG grown in Sauton (a) or Middlebrook 7H9 medium (b). Squares: proteins expressed in higher amounts in Sauton medium; ellipses: proteins expressed in higher amounts in Middlebrook 7H9 medium.
non-exposed bacilli were minimal and comparable to those due to the intrinsic variability of the employed method, even with SNP concentrations up to 25 mM and/or after incubation periods up to 24 h (data not shown). 4. Discussion In the present study, the influence of the growth medium on the resistance of BCG to the RNI generator SNP was evaluated. A significant difference was observed in the percentage of survival of BCG exposed to SNP in different culture media, indicating that the growth medium and, in general, the milieu may exert a remarkable influence on the resistance of the
microorganism to the toxic activity of RNI. In the light of this observation, the bactericidal activity of physiological concentrations of RNI produced by activated macrophages against phagocytosed bacilli cannot be easily inferred from that observed in an arbitrary in vitro system. For instance, the low pH and the presence of other toxic radicals, metals and/or oxidizable groups inside of the acidified phagolysosome might have a dramatic influence on the effective concentration, halflife and toxicity of NO and other RNI generated by the activity of NOS2, in vivo. The permeability to RNI and the scavenging properties of the cell envelope of intracellular bacilli represent another set of variables that might exert a predominant influence on the vulnerability of tubercle bacilli to the toxic activity of these compounds.
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Fig. 7. 2-DE of lysates of BCG after two hours exposure to 1 mM SNP (a) or no SNP (b) in Middlebrook 7H9. Squares: proteins expressed in higher amounts after exposure of BCG to SNP; ellipses: proteins expressed in lower amounts after exposure of BCG to SNP.
To evaluate whether a possible differential antioxidant effect exerted by the two different media might influence the sensitivity of BCG to SNP, the variation of the RedOx potential of the medium was measured after addition of SNP, either in the presence or absence of bacteria. Addition of SNP determined an increase in the RedOx potential in both the two media, without bacteria, and in culture supernatants of BCG grown in Middlebrook 7H9. On the contrary, addition of SNP to cultures in Sauton medium was followed by a decrease in the RedOx potential. It is possible that RNI and/or other oxidizing compounds generated by SNP reacted with some bacterial component and/or product, expressed/produced by BCG when the bacillus is grown in Sauton but not in Middlebrook 7H9, giving rise to products with strong reducing power (DORP < 0). Actually, it has been shown that the culture medium may influence the composition and the thickness of the cell wall of tubercle bacillus and other mycobacterial species [27,28], and that some mycobacterial cell wall constituents, namely type I phenolic glycolipids, lipophosphoglycans and lipoarabinomannan, may exert a detoxifying effect on toxic radicals [29–31]. Therefore, the different resistance to SNP might be due to differentially expressed bacterial components, rather than to an antioxidant effect of the medium. SNP has been widely used as a NO generator, but there is evidence that it might exert its biological effects also by generating peroxynitrite, a strong oxidant of biological systems [32,33], and/or other radicals [34–36]. To assess whether peroxynitrite was actually generated by SNP, and to evaluate a possible correlation between the concentration of this strong oxidizing agent and the resistance of BCG to SNP in the two different media used, we measured the concentration of peroxynitrite in culture supernatants of BCG exposed to SNP. The results of these experiments show that peroxynitrite was indeed generated at µM concentrations by SNP, but the concentration of peroxynitrite did not correlate with the different sensitivity of BCG to SNP in the different culture media. This observation suggests that the bactericidal effect of SNP was
not mainly due to peroxynitrite. This hypothesis is consistent with the observation of Yu et al. [37] who demonstrated that tubercle bacillus is relatively resistant to peroxynitrite, whereas it is effectively killed by exposure to µM concentrations of NO2 and, to a lesser extent, by NO. Alternatively, it is possible that BCG cultivated in different media expressed differential phenotypic characters that might exert an influence on the resistance of the microorganism to the bactericidal effect of peroxynitrite. To investigate the effect of culture medium and exposure to SNP on protein expression by BCG, we comparatively analyzed the 2-DE protein expression pattern of the microorganism grown in the two different media and exposed or nonexposed to SNP. The comparative analysis of somatic proteins of the bacillus grown in the different media showed that the protein expression profile of BCG might vary in relation to the culture medium. An inducible response to RNI was observed when the microorganism was exposed to the nitrosative stress in Middlebrook 7H9, but not in Sauton medium, indicating that the culture medium may also influence the responsiveness of BCG to RNI. It is interesting to note that when BCG was exposed to SNP in Sauton medium, no relevant differences in the protein expression pattern between exposed and non-exposed bacilli were observed (data not shown). It is tempting to speculate that the acidic proteins expressed in higher amounts by BCG grown in Sauton medium, compared to BCG cultivated in Middlebrook 7H9, were those involved in the higher resistance to RNI of the bacillus cultivated in that medium. Identification of such proteins will help to elucidate their possible role in the resistance and adaptive response of the bacillus to RNI. Acknowledgments The present work was supported by ″Progetti P.R.I.N.″, protocols No. 2002067349-001 and No. 2004067822-001, Roma, Italy.
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Original article
Influence of culture medium on the resistance and response of Mycobacterium bovis BCG to reactive nitrogen intermediates Walter Florio *, Giovanna Batoni, Semih Esin, Daria Bottai, Giuseppantonio Maisetta, Flavia Favilli, Franca L. Brancatisano, Mario Campa Dipartimento di Patologia Sperimentale, Biotecnologie Mediche, Infettivologia ed Epidemiologia, Università di Pisa, Via S. Zeno 35-39, 56127 Pisa, Italy Received 9 March 2005; accepted 15 July 2005 Available online 16 September 2005
Abstract The aim of the present work was to evaluate the influence of the culture medium on the resistance and response of Mycobacterium bovis BCG to reactive nitrogen intermediates, in vitro. BCG was grown in Sauton, Dubos or Middlebrook 7H9 medium and exposed to sodium nitroprusside (SNP) for up to 7 days. The percentage of bacilli that survived was significantly lower in Middlebrook 7H9 than in Sauton or Dubos medium. Addition of SNP to Middlebrook 7H9 caused an increase in the RedOx potential in either the absence or the presence of BCG, while addition of the compound to Sauton medium gave rise to an increase in the RedOx potential only in the absence of bacteria, whereas a decrease in the RedOx potential was observed in the presence of BCG. The resistance of BCG to SNP in the different media did not correlate with the concentration of peroxynitrite in culture supernatants. BCG grown in different media showed a differential protein expression pattern, as assessed by two-dimensional gel electrophoresis. Exposure of BCG to sub-lethal concentrations of SNP in Middlebrook 7H9, but not in Sauton medium, revealed a differential expression of at least 38 protein species. Altogether these results demonstrate that the growth medium may have a remarkable influence on the resistance and the response of BCG to SNP and suggest that the different resistance of BCG in the two media is unlikely to be due to a differential antioxidant effect of the medium itself. © 2005 Elsevier SAS. All rights reserved. Keywords: Mycobacterium bovis; BCG; Nitroprusside; Peroxynitrite; Two-dimensional gel electrophoresis
1. Introduction Nitric oxide (NO) and related reactive nitrogen intermediates (RNI) are highly reactive compounds produced by macrophages after activation by TNF-a and/or IFN-c [1], stimulation with LPS [2] or infection with various microorganisms, including Mycobacterium tuberculosis [3]. Production of NO by macrophages is catalyzed by the inducible isoform of NO synthase (NOS2), and represents a powerful effector mechanism against a number of pathogenic bacteria [4]. The protective role of NOS2 against M. tuberculosis has been established in several mouse models of experimental infection
Abbreviations: BCG, bacillus Calmette-Guérin; DCDHF, 2,7dichlorodihydrofluorescein; 2-DE, two-dimensional gel electrophoresis; NO, nitric oxide; NOS2, inducible nitric oxide synthase; RNI, reactive nitrogen intermediates; SNP, sodium nitroprusside. * Corresponding author. Tel.: +39 050 2213 691; fax: +39 050 2213 711. E-mail address: [email protected] (W. Florio). 1286-4579/$ - see front matter © 2005 Elsevier SAS. All rights reserved. doi:10.1016/j.micinf.2005.07.013
[5–8], and it has been shown that NOS2 may be expressed at high levels by alveolar macrophages from patients with untreated, culture-positive tuberculosis [9], indicating that RNI may play an important role in the response to tuberculosis infection also in humans. Despite the evidence of NOS2 expression in vivo, production of NOS2 by human monocytes/macrophages in cell culture systems has long been variable and controversial [10–14]. Therefore, the relevance of RNI production as a mycobactericidal activity of human macrophages remains a matter of considerable debate. The potential importance of RNI in the control of tuberculous infection has stimulated the research on the mechanisms by which M. tuberculosis resists the toxic activity of these compounds. Recent studies have shown that exposure of M. tuberculosis to RNI, in vitro, may induce the differential expression of at least 53 genes, most of which correspond to hypoxia-induced genes [15,16]. Few studies have been reported on the response of tubercle bacillus to RNI at the protein level, and a few proteins have been shown to be
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expressed differentially after exposure to various RNI generators [17–19]. Using metabolic labeling and twodimensional gel electrophoresis (2-DE), Garbe et al. [17] detected a number of polypeptides differentially expressed by M. tuberculosis after exposure to RNI in vitro. The a-crystallin homologue heat shock protein was identified as one of the most abundant proteins of M. tuberculosis after incubation with several chemically diverse NO donors, whereas incubation of the bacillus with sodium nitroprusside (SNP) or S-nitroso-N-acetyl-D,L-penicillamine, but not with other NO donors, resulted in induction of a 19 kDa protein, named Nox19, which contains amino-acidic regions homologous to sequences found in a subclass of nonheme bacterial ferritins [18]. Since iron metabolism is linked to the mycobacterial response to oxidative stress by shared regulatory components [20,21], induction of Nox19 might be part of a complex regulatory mechanism. In general, the resistance and the response of mycobacteria to RNI may be influenced by several experimental variables, such as the type of RNI generator [18] and its concentration [16], the growth phase of the culture when the stress is applied [19], the duration of the stress [15], and possibly others. In the present study, we evaluated the influence of the culture medium on the resistance of Mycobacterium bovis BCG to the RNI generator SNP, and investigated the response of the microorganism after exposure to sub-lethal concentrations of this toxic compound by utilizing a functional proteomics approach.
2. Materials and methods 2.1. Bacterial cultures and stress challenge experiments M. bovis BCG, strain Pasteur (Pasteur Merieux), was grown in roller bottles in modified Sauton medium containing 0.05% Tween 80 (v/v) [22]. For treatment with SNP, an aliquot of a mid-log phase culture was harvested by centrifugation (4600 rpm, 10 min, 25 °C), and the bacterial pellet was inoculated at a cell density of 3 × 106 c.f.u. per ml of modified Sauton medium, without Tween 80, Middlebrook 7H9 medium supplemented with 0.5% bovine serum albumin, 0.2% glucose and 0.085% NaCl or Dubos medium added of 0.1% bovine serum albumin. Cultures were grown in roller bottles at 37 °C for 5 days, then SNP (Sigma) was added from a freshly prepared 1 M solution to a final concentration of 25, 5, 1, or 0 mM (non-stressed control cultures). Inoculum size and culture conditions were optimized so that BCG cultures in the different media were all in the same (midlogarithmic) phase when SNP was added. Cultures were incubated in the presence of SNP for 24 h, 3 or 7 days. For evaluation of c.f.u., a 5-ml sample of the culture was taken at different times after addition of SNP, and bacteria were finely suspended by vortex with 1 mm diameter glass beads. Tenfold serial dilutions of each bacterial suspension were plated onto Middlebrook 7H11 agar supplemented with 10% OADC
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enrichments (Difco). The plates were sealed in plastic envelopes and incubated at 37 °C for 3 weeks before counting the number of colonies. For preparation of BCG lysates, SNP was added to midlog phase cultures to a final concentration of 1 mM; after 2 h incubation with the compound, cultures were harvested by centrifugation, the bacterial pellet was washed once with sterile MilliQ H2O, then frozen at –80 °C or immediately processed for subsequent 2-DE analysis (see below). 2.2. Measurement of RedOx potential The RedOx potential of culture media and bacterial culture supernatants, before or after addition of SNP, was measured by using a Hamilton electrochemical sensor (Hamilton Bonaduz AG). The electrode was dipped in the sample for at least 30 s before reading the RedOx potential, and it was washed extensively with MilliQ H2O between two successive measurements. 2.3. Measurement of peroxynitrite concentration The concentration of peroxynitrite generated by SNP in the different culture media was measured by using the method described by Crow [23], with some modifications. A 14.5 mM stock solution of 2,7-dichlorodihydrofluorescein (DCDHF) (Cayman Chemical) was prepared by dissolving DCDHF diacetate in 100 mM NaOH, and stored in aliquots at –20 °C. DCDHF was added to samples to a final concentration of 100 µM, and oxidation reactions were carried out in polypropylene tubes for 30 min at room temperature. Then, the reaction was stopped by adding L-ascorbic acid (Carlo Erba) from a 100 mM stock solution to a final concentration of 6 mM. Addition of L-ascorbic acid effectively prevented further slow oxidation of DCDHF without affecting the absorbance spectrum of samples in the full wavelength range of visible light (data not shown). A Hewlett–Packard Agilent 8453 spectrophotometer was used to measure the A500. Preliminary experiments were performed to ascertain that DCDHF was not reacting with bacterial products in culture supernatants. The OD500 of samples from cultures without SNP was < 10−2 for all media and at all times tested. Nevertheless, the supernatants of control cultures (no SNP added) were used as blanks in the assay for supernatants of cultures with SNP in corresponding media. The concentration of peroxynitrite reached a peak at 4–6 h after addition of SNP, then it began to slowly decrease (data not shown). 2.4. 2-DE For preparation of BCG lysates, bacteria were suspended in 9 M urea, 10 mg/ml DTT, 1% CHAPS (Sigma). Glass Beads (BioSpec Products, Inc.), 0.1 mm diameter, were added to the bacterial suspension, 1 g per milliliter of suspension, and bacteria were disrupted by vigorous shaking with a Mini Bead-Beater (BioSpec Products), six cycles of 20 s each at
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2500 rpm, keeping the bacterial suspension in ice between two successive cycles. The lysate was centrifuged for 5 min at 12,000 × g at room temperature, and IPG buffer was added to the supernatant (Amersham Biosciences) to a final concentration of 2% (v/v). A 280 µl aliquot of the resulting sample, containing 300–600 µg protein, was incorporated into immobiline dry strips, pH 4–7 (linear pH gradient) or pH 3–5.6 NL (non linear pH gradient) (Amersham Biosciences), by overnight rehydration at room temperature. Isoelectrofocusing was performed with a Multiphor II apparatus (Amersham Pharmacia Biotech) setting 300 V, 1 mA, 5 W for 10 min, followed by 3500 V, 1 mA, 5 W for 7 h at 10 °C. After isoelectrofocusing, the strips were usually frozen at –80 °C. Second dimension separation was carried out in 12.5% Laemmli polyacrylamide gels. Before loading on the gel, the strips were equilibrated for 5 min in 50 mM Tris–HCl, pH 6.8, 6 M urea, 30% glycerol (v/v), 1% SDS, 5 mg/ml DTT, then for 10 min in 50 mM Tris–HCl, pH 6.8, 10% glycerol (v/v), 2% SDS, 5 mg/ml DTT, containing bromophenol blue as a dye. Prestained proteins (Cell Signaling Technology) were used as molecular weight markers. The gels were stained with silver nitrate or Coomassie blue R-250 as previously described [24]. Stained gels were matched and analyzed with the ImageMaster 2D software (ImageMaster 2D, v. 4.01, Amersham Biosciences). For comparison of the relative protein amount in matched spots of different gels, protein spot volumes were normalized for the total volume of all the spots in the gel. Three independent samples were prepared for each biological condition, and individual samples were run in duplicate to evaluate the intrinsic variability of the technique. Individual gels from the same experiment were comparatively analyzed, and only protein spots showing at least a threefold difference in the normalized volume in a reproducible manner were considered differentially expressed.
3. Results 3.1. Resistance of BCG to SNP in different culture media In order to evaluate the influence of the culture medium on the resistance of BCG to RNI, the resistance of the bacillus to the RNI generator SNP was tested in three different culture media for mycobacteria: modified Sauton medium, a synthetic medium that is commonly used for preparation of mycobacterial culture filtrates as it does not contain exogenous proteins; Middlebrook 7H9 medium, utilized in several studies on the response of mycobacteria to RNI [17,19]; Dubos medium, another synthetic medium, included in this study as it does not contain citric acid or other organic acids that might have an antioxidant effect. BCG was inoculated in the different culture media at a cell density of 3 × 106 c.f.u. per ml and, after 5 days of growth (mid-log phase), SNP was added to cultures at different final concentrations: 1, 5, 25 or 0 mM (control cultures). The percentage of survival of BCG after 24 h, 3 and 7 days incubation with SNP was determined
by plating serial dilutions of the cultures onto Middlebrook 7H11/OADC agar plates, and counting the number of colonies after a 3 weeks’ incubation at 37 °C. Exposure of BCG to different concentrations of SNP caused a dose- and timedependent decrease in the percentage of survival of the microorganism, in all three media. Nevertheless, BCG cultured in Sauton or Dubos medium was significantly more resistant to the bactericidal effect of SNP than bacilli grown in Middlebrook 7H9 (Fig. 1). At the lowest concentration of SNP, a significant difference in the percentage of survival was observed only after 7 days incubation with the compound (Fig. 1a), whereas at the higher concentrations of SNP the difference between Middlebrook 7H9 and Sauton or Dubos became significant after 24 h or 3 days, respectively (Fig. 1b and c). The Sauton and Middlebrook 7H9 media, where the resistance of BCG was the highest and the lowest, respectively, were selected for further study. 3.2. Evaluation of the antioxidant effect of the medium RNI are highly reactive compounds with a short half-life in aqueous solutions, as they rapidly react with molecules that are susceptible to oxidation [25,26]. The chemical composition of the medium, and hence its antioxidant effect, might affect the effective concentration of RNI to which the bacterium is exposed. In order to establish whether the different susceptibility of BCG to SNP observed in Sauton compared to Middlebrook 7H9 medium was due to a differential antioxidant effect exerted by the two different media, the variation of the RedOx potential was measured after addition of SNP, both in the presence and in the absence of bacteria. As shown in Fig. 2, in the absence of bacteria, addition of SNP caused a comparable increase in the RedOx potential in the two different media. Similarly, addition of SNP to cultures in Middlebrook 7H9 determined an increase in the RedOx potential, in relation to the concentration of the RNI generator (Fig. 3). Surprisingly, addition of SNP to cultures in Sauton medium caused a decrease in the RedOx potential, at all the tested concentrations of the compound. 3.3. Generation of peroxynitrite by SNP In order to assess whether SNP was able to generate peroxynitrite in the culture systems used to test the resistance of BCG, a colorimetric assay, based on the specific oxidation of DCDHF by peroxynitrite [23], was adapted to measure the concentration of the compound after addition of SNP to the Sauton and Middlebrook 7H9 media, and to the corresponding BCG cultures. The results show that peroxynitrite was indeed generated after addition of SNP to the culture media, and that the concentration of peroxynitrite in the two different media was comparable at all the SNP concentrations tested (Fig. 4). In general, the concentration of peroxynitrite was also comparable in culture supernatants of BCG grown in Sauton or Middlebrook 7H9, with a single exception: after a 7 days incubation with 25 mM SNP, the concentration of per-
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Fig. 1. Percentage of survival of BCG grown in Sauton (circles), Dubos (triangles) or Middlebrook 7H9 medium (squares), and exposed to 1 mM (a), 5 mM (b) or 25 mM SNP (c). The data reported represent the mean ± S.E.M. of three independent experiments. *: P < 0.05; **: P < 0.01; ***: P < 0.001, Sauton vs. Middlebrook 7H9; ♦: P < 0.05; ♦♦: P < 0.01; ♦♦♦: P < 0.001, Dubos vs. Middlebrook 7H9.
Fig. 2. Variation of the RedOx potential (DORP) after addition of 1 mM (a), 5 mM (b) or 25 mM SNP (c) to the Sauton (light bars) or Middlebrook 7H9 medium (dark bars), without bacteria. The data reported represent the mean ± S.E.M. of three independent experiments.
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Fig. 3. Variation of the RedOx potential (DORP) after addition of 1 mM (a), 5 mM (b) or 25 mM SNP (c) to BCG cultures in Sauton (light bars) or Middlebrook 7H9 medium (dark bars). The data reported represent the mean ± S.E.M. of three independent experiments.
Fig. 4. Peroxynitrite concentration, 4 h after addition of different concentrations of SNP to the Sauton (circles) or Middlebrook 7H9 medium (triangles). The experiment was repeated three times with the same overall results.
oxynitrite was even higher in culture supernatants of BCG cultivated in Sauton than in Middlebrook 7H9 (Fig. 5). Therefore, the peroxynitrite concentration does not seem to correlate with the different resistance of BCG grown in the different media. 3.4. Influence of the medium and of exposure to SNP on protein expression by BCG To investigate the possibility that the culture medium might influence the expression of phenotypic characters by BCG, the protein expression profile of the bacillus cultivated in the two different media was comparatively analyzed by 2-DE, at the mid-log phase of growth. Interestingly, several proteins
were differentially expressed by the microorganisms grown in the two media. In particular, thirteen protein species were more abundant in lysates of BCG grown in Middlebrook 7H9, whereas another two polypeptides, with acidic pI, were present in higher amounts in lysates of the bacillus cultivated in Sauton medium (Fig. 6). The differentially expressed protein species were confined to a pI range between 4.2 and 5.6, and all but one had an apparent molecular mass lower than 30 kDa (Fig. 6). To investigate the effect of exposure to sub-lethal concentrations of SNP on protein expression by BCG, mid-log phase cultures of the bacillus grown in the different media were added 1 mM SNP; after two hours incubation with or without the compound, cultures were harvested and bacilli were processed for 2-DE analysis. As shown in Fig. 7, exposure of BCG cultures in Middlebrook 7H9 to SNP resulted in the differential expression of 38 protein species by the microorganism. In particular, the expression of 16 proteins was increased after incubation with SNP, whereas another 22 polypeptides were present in lower amounts in the lysates of BCG exposed to the compound than in non-exposed, control cultures. The differentially expressed proteins were distributed over a wide range of molecular mass but confined to the acidic pI range 3–5.6 (Fig. 7). No additional differentially expressed protein spots were observed after 2-DE of BCG lysates in the pI range 4–7 (data not shown). When BCG cultivated in Sauton medium was exposed to SNP, the differences in the protein expression pattern between exposed and
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Fig. 5. Peroxynitrite concentration after addition of 1 mM (a), 5 mM (b) or 25 mM SNP (c) to BCG cultures in Sauton (circles) or Middlebrook 7H9 medium (triangles). The data reported represent the mean ± S.E.M. of three independent experiments.
Fig. 6. 2-DE of lysates of BCG grown in Sauton (a) or Middlebrook 7H9 medium (b). Squares: proteins expressed in higher amounts in Sauton medium; ellipses: proteins expressed in higher amounts in Middlebrook 7H9 medium.
non-exposed bacilli were minimal and comparable to those due to the intrinsic variability of the employed method, even with SNP concentrations up to 25 mM and/or after incubation periods up to 24 h (data not shown). 4. Discussion In the present study, the influence of the growth medium on the resistance of BCG to the RNI generator SNP was evaluated. A significant difference was observed in the percentage of survival of BCG exposed to SNP in different culture media, indicating that the growth medium and, in general, the milieu may exert a remarkable influence on the resistance of the
microorganism to the toxic activity of RNI. In the light of this observation, the bactericidal activity of physiological concentrations of RNI produced by activated macrophages against phagocytosed bacilli cannot be easily inferred from that observed in an arbitrary in vitro system. For instance, the low pH and the presence of other toxic radicals, metals and/or oxidizable groups inside of the acidified phagolysosome might have a dramatic influence on the effective concentration, halflife and toxicity of NO and other RNI generated by the activity of NOS2, in vivo. The permeability to RNI and the scavenging properties of the cell envelope of intracellular bacilli represent another set of variables that might exert a predominant influence on the vulnerability of tubercle bacilli to the toxic activity of these compounds.
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Fig. 7. 2-DE of lysates of BCG after two hours exposure to 1 mM SNP (a) or no SNP (b) in Middlebrook 7H9. Squares: proteins expressed in higher amounts after exposure of BCG to SNP; ellipses: proteins expressed in lower amounts after exposure of BCG to SNP.
To evaluate whether a possible differential antioxidant effect exerted by the two different media might influence the sensitivity of BCG to SNP, the variation of the RedOx potential of the medium was measured after addition of SNP, either in the presence or absence of bacteria. Addition of SNP determined an increase in the RedOx potential in both the two media, without bacteria, and in culture supernatants of BCG grown in Middlebrook 7H9. On the contrary, addition of SNP to cultures in Sauton medium was followed by a decrease in the RedOx potential. It is possible that RNI and/or other oxidizing compounds generated by SNP reacted with some bacterial component and/or product, expressed/produced by BCG when the bacillus is grown in Sauton but not in Middlebrook 7H9, giving rise to products with strong reducing power (DORP < 0). Actually, it has been shown that the culture medium may influence the composition and the thickness of the cell wall of tubercle bacillus and other mycobacterial species [27,28], and that some mycobacterial cell wall constituents, namely type I phenolic glycolipids, lipophosphoglycans and lipoarabinomannan, may exert a detoxifying effect on toxic radicals [29–31]. Therefore, the different resistance to SNP might be due to differentially expressed bacterial components, rather than to an antioxidant effect of the medium. SNP has been widely used as a NO generator, but there is evidence that it might exert its biological effects also by generating peroxynitrite, a strong oxidant of biological systems [32,33], and/or other radicals [34–36]. To assess whether peroxynitrite was actually generated by SNP, and to evaluate a possible correlation between the concentration of this strong oxidizing agent and the resistance of BCG to SNP in the two different media used, we measured the concentration of peroxynitrite in culture supernatants of BCG exposed to SNP. The results of these experiments show that peroxynitrite was indeed generated at µM concentrations by SNP, but the concentration of peroxynitrite did not correlate with the different sensitivity of BCG to SNP in the different culture media. This observation suggests that the bactericidal effect of SNP was
not mainly due to peroxynitrite. This hypothesis is consistent with the observation of Yu et al. [37] who demonstrated that tubercle bacillus is relatively resistant to peroxynitrite, whereas it is effectively killed by exposure to µM concentrations of NO2 and, to a lesser extent, by NO. Alternatively, it is possible that BCG cultivated in different media expressed differential phenotypic characters that might exert an influence on the resistance of the microorganism to the bactericidal effect of peroxynitrite. To investigate the effect of culture medium and exposure to SNP on protein expression by BCG, we comparatively analyzed the 2-DE protein expression pattern of the microorganism grown in the two different media and exposed or nonexposed to SNP. The comparative analysis of somatic proteins of the bacillus grown in the different media showed that the protein expression profile of BCG might vary in relation to the culture medium. An inducible response to RNI was observed when the microorganism was exposed to the nitrosative stress in Middlebrook 7H9, but not in Sauton medium, indicating that the culture medium may also influence the responsiveness of BCG to RNI. It is interesting to note that when BCG was exposed to SNP in Sauton medium, no relevant differences in the protein expression pattern between exposed and non-exposed bacilli were observed (data not shown). It is tempting to speculate that the acidic proteins expressed in higher amounts by BCG grown in Sauton medium, compared to BCG cultivated in Middlebrook 7H9, were those involved in the higher resistance to RNI of the bacillus cultivated in that medium. Identification of such proteins will help to elucidate their possible role in the resistance and adaptive response of the bacillus to RNI. Acknowledgments The present work was supported by ″Progetti P.R.I.N.″, protocols No. 2002067349-001 and No. 2004067822-001, Roma, Italy.
W. Florio et al. / Microbes and Infection 8 (2006) 434–441
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