S-Nitrosoglutathione cytotoxicity to Mycobacterium smegmatis and its use to isolate stationary phase survival mutants

FEMS Microbiology Letters 239 (2004) 221–228 www.fems-microbiology.org

S-Nitrosoglutathione cytotoxicity to Mycobacterium smegmatis and its use to isolate stationary phase survival mutants Marjan J. Smeulders, Jacquie Keer 1, Kathryn M. Gray, Huw D. Williams

*

Department of Biological Sciences, Imperial College London, Sir Alexander Fleming Building, South Kensington Campus, London SW7 2AZ, UK Received 7 April 2004; received in revised form 16 August 2004; accepted 23 August 2004 First published online 11 September 2004 Edited by R.S. Buxton

Abstract We report that stationary phase Mycobacterium smegmatis is more sensitive than exponential phase cells to the nitric oxide donor S-Nitrosoglutathione (GSNO). This finding was used to select for both spontaneous and transposon mutants of M. smegmatis with increased resistance to GSNO in stationary phase. Some of these mutants were also defective in stationary phase survival, demonstrating a link between sensitivity to GSNO and stationary phase survival. Transduction of the disrupted region from seven selected mutants indicated that the transposon insertion was linked to the GSNO-resistance and stationary phase survival phenotypes. For five mutants, the disrupted genes were identified. Three were homologous to genes with possible roles in nutrient scavenging, including: (i) a putative amino acid efflux pump, (ii) a putative thioesterase and (iii) an enoyl-CoA-hydratase. One mutant was disrupted in the atpD gene, encoding the b chain of F1 F0 ATP synthase. We independently isolated a stationary phase survival mutant disrupted in the atpA gene (encoding the a chain) of the F1 F0 ATP synthase of the same operon, suggesting an important role for efficient ATP synthesis in stationary phase survival.
2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. Keywords: Starvation; Nitric oxide; Tuberculosis; ATP synthase; LysE; Fatty acid oxidation

1. Background One of the keys to the success of Mycobacterium tuberculosis as a pathogen, lies in the fact that it can survive within the human host for long periods without showing clinical signs of disease even after the primary infection has been successfully treated with antibiotics (reviewed in [1]). Whether M. tuberculosis cells in the persistent phase of infection reside in a state of dormancy or in a state of continuous but * Corresponding author. Tel.: +44 020 75945383; fax: +44 020 75842056. E-mail address: [email protected] (H.D. Williams). 1 Present address: LGC, Queens Road, Middlesex TW11 0LY, UK.

suppressed cell turnover, kept in check by the hostÕs immune system, is not known [2,3]. There is some evidence to suggest that the bacteria persist in a state of non-growth or stationary phase [4–6]. Therefore, we have been studying the mechanisms used by mycobacteria to survive for prolonged periods in stationary phase, in particular the response of the fast growing, non-pathogenic M. smegmatis to nutrient limited stationary phase [7–9]. We have previously isolated mutants with defects in stationary phase survival by screening a large number of individual transposon mutants for loss of culturability after 1 month of incubation in stationary phase [7]. These mutants had defects in stationary phase survival under oxygen-sufficient conditions in rich, lemco medium and under

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2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.femsle.2004.08.030

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carbon-starvation conditions [7]. Other mutants including a purF mutant and response regulator mutant, devR, were specifically affected in stationary phase survival under oxygen-starvation conditions [8], a model developed by Wayne and co-workers [10] to study mycobacterial persistence. An increasing number of mycobacterial genes and proteins have been identified that are induced upon oxygen starvation [11–15] and one of these is the 16 kDa a-crystallin small heat shock protein (Acr or HspX), encoded by the acr/hspX genes [16]. HspX is also a M. tuberculosis antigen and is required for growth of M. tuberculosis in macrophages [17] and is highly upregulated during exposure of M. tuberculosis to reactive nitrogen intermediates (RNI) [18]. Furthermore, it has recently been shown that there is overlap between induction of gene expression by oxygen-starvation and RNI, many of which genes are part of the DevR regulon [19,20]. RNI are an important host mechanism in the control of tuberculosis (reviewed in [21]). Mycobacteria exhibit a RNI response and several other genes have been identified that are involved in the protection against nitrosative stress [22,23,18,24]. De Groote et al. [25] reported that Salmonella typhimurium is more sensitive in stationary phase than in exponential phase to the nitric oxide donor S-nitrosoglutathione (GSNO). These authors found that a spontaneous GSNO-resistant mutant of S. typhimurium was defective in rpoS, the gene encoding the stationary phase r factor, suggesting that the general stationary phase response is involved in GSNO sensitivity in S. typhimurium [25]. Therefore, we were interested to determine whether mycobacterial resistance to GSNO is growth phase dependent and whether this was a tool that we could exploit to study the stationary phase physiology of mycobacteria.

2. Materials and methods 2.1. Strains and media M. smegmatis mc2 155 was grown into carbonstarved stationary phase as described previously [9]. Culture viability was assessed by viable plate counting on lab-lemco medium (Oxoid).

the molarity of GSH. PBS was composed of 8 g NaCl, 0.2 g KCl, 1.44 g Na2 HPO4 and 0.24 g KHPO4 per litre dH2O. GSNO sensitivity assays were based on those described by De Groote et al. [25]. (i) Disc diffusion assay. M. smegmatis cultures were grown to midexponetial phase or a specific time into stationary phase, diluted to 5 · 107 cfu ml 1 and 200 ll of each culture was spread onto a lab-lemco plate. Paper discs (13 mm diameter) were placed on the lawns and 70 ll of 1 M GSNO was added to these. Because GSNO degrades within 20 h, fresh GSNO was added to the discs twice a day, until the bacteria had grown enough to record the zone of inhibition (3 days). (ii) Microbroth dilution assay. This was used for measuring MBC (minimal bactericidal concentration) and MIC (minimal inhibitory concentration). Assays were done in 96-well microtitre plates: cultures were diluted to 5 · 107 cfu ml 1 and 50 ll volumes were added to wells containing 50 ll fresh carbon-limited minimal medium with GSNO concentrations ranging from 0.1 to 100 mM. Culture optical densities were read at 630 nm, using a Vmax Kinetic microplate reader (Molecular Devices) immediately after inoculation and after 20– 24 h of shaking incubation at 37 C. The MIC was recorded as the lowest concentration of GSNO giving maximum inhibition of growth (defined as no increase in OD630 after 24 h incubation). Five ll of culture from each well was then spotted onto lab-lemco plates and growth was recorded over the following 3 days to give the MBC, defined as the lowest concentration of GSNO resulting in no growth. 2.3. Isolation of spontaneous GSNO-resistant mutants Cultures at 9, 30 and 45 days into carbon-starved stationary phase were exposed to GSNO at concentrations of 2–4 mM for 24 h at 37 C, and then plated onto lab-lemco medium. Colonies were picked as soon as they became visible, streaked onto lab-lemco plates for glycerol stock preparation, and inoculated into 1 ml of fresh carbon-limited minimal medium. When these cultures were 5–6 days in stationary phase, their GSNO-resistance was tested by exposing samples of the culture to 0, 5, 10 and 25 mM GSNO as described above. Those mutants that survived a 24 h exposure to 25 mM GSNO were then tested for their starvation survival phenotype.

2.2. GSNO sensitivity assays GSNO was made by adding NaNO2 in 1 M HCl (made up in carbon-limited minimal medium or in PBS) to solid reduced glutathione (GSH) and neutralising to pH 7 with 1 M NaOH (made up in carbon-limited minimal medium or PBS) [25]. To ensure complete conversion of GSH to GSNO, NaNO2 was used in double

2.4. Isolation of GSNO-resistant mutants following transposon mutagenesis M. smegmatis Tn611 transposon mutants were made using the transposon delivery plasmid pCG79 [26] and a modified version of the method described previously [7]. To select for transposon mutants, 32 ll aliquots of the

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When screening mutants for starvation survival phenotypes, strains were inoculated into 3 ml carbon-limited minimal medium in 30 ml polystyrene tubes, or 1 ml medium in 7 ml polystyrene tubes. To reduce the chance of suppression or reversion of stationary phase survival phenotypes, cultures of both GSNO-resistant spontaneous and transposon mutants were inoculated directly from glycerol stocks, giving a final glycerol concentration of 0.08, 0.16 (both carbon-limited) or 0.25% v/v (carbon-sufficient), depending on the size of the inoculum. In some experiments, cultures were inoculated from plates that had been inoculated with a sample from the glycerol stock and incubated until growth was just visible. Transposon mutants were always grown at the non-permissive temperature (42 C), in the presence of kanamycin (20 lg ml 1) with or without streptomycin (20 lg ml 1), to reduce any possibility of Tn611 movement. This was necessary because the whole plasmid pCG79 transposes into the chromosome. Bacteriophage I3 [27] was used to transduce the transposon insertions to clean genetic backgrounds [8]. 2.6. Recombinant DNA methods

3.1. Growth phase dependence of GSNO-resistance in M. smegmatis We investigated the growth phase dependence of M. smegmatis sensitivity to S-nitrosoglutathione (GSNO) using a disc diffusion and a microbroth dilution assay. Both assays showed that carbon-starved, stationary phase M. smegmatis were more sensitive to GSNO than exponential phase cells (Fig. 1). In the disc diffusion assay, exponentially growing cells had a significantly smaller inhibition zone than cells inoculated into the assay from cultures 1, 6 and 36 days into stationary phase (Fig. 1(a)). The microbroth dilution assay established a MIC for GSNO of 8 mM for exponential phase cultures, which was reduced to 4 mM after 24 h in stationary phase and to 0.5 mM 36 days into stationary phase. GSNO was also clearly bactericidal for M. smegmatis at markedly lower concentrations in stationary phase than in exponential phase. An MBC of 25 mM was found for exponential phase cultures, which decreased

radius of inhibition zone (mm)

2.5. Screening starvation-survival phenotypes

3. Results and discussion

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mutant library were inoculated into 5 ml carbon-limited minimal medium without glycerol (final concentration of glycerol was 0.08% v/v, resulting from the glycerol in the inoculum), at 42 C. When the cultures had been 2–3 days in stationary phase, GSNO was added to a concentration of 14 mM, and the culture incubated for a further 24 h after which the cells were centrifuged and resuspended in 1 ml lab-lemco medium. The whole mixture was plated over several lab-lemco plates with kanamycin and incubated at 42 C. When colonies were visible, they were picked and (i) inoculated into 1 ml carbon-limited minimal medium in a 7-ml polystyrene tube and (ii) inoculated into 200 ll carbon-limited minimal medium, containing 12.5% glycerol in a well of a microtitre plate. The 1 ml cultures were used to test GSNOresistance at 3–10 days stationary phase, and to test starvation survival. The microtitre plates were frozen immediately at 80 C as glycerol stock cultures.

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The DNA flanking the transposon insertion in the Tn611 mutants was identified using Ligation Mediated PCR (LM-PCR) [28]. Cloned products were sequenced and resulting sequence was compared with the SPTR database at HGMP (http://menu.hgmp.mrc.ac.uk/cgibin/blast), the unfinished M. smegmatis sequence at TIGR (http://www.tigr.org/cgi-bin/BlastSearch/blast.cgi?) and with the M. tuberculosis H37Rv genome sequence at the Sanger centre using BLAST (http://www.sanger.ac.uk/ Projects/M_tuberculosis/blast_server.shtml).

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Fig. 1. GSNO-resistance of exponentially growing and carbonstarved, stationary phase M. smegmatis. (a) Disc diffusion assay. The mean (±SD) inhibition zone is the distance between the edge of the inhibition zone and the edge of the disc. (b) Microbroth dilution assay. Representative data from 1 of at least 3 repeat experiments are shown. In both (a) and (b), the x-axis indicates the growth phase of the cultures used, either exponential phase (exp) or stationary phase (sp), and the time the culture had been in stationary phase in hours (hr) or days (d).

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determine whether acquisition of a GSNO-resistance mutation affected stationary phase survival, 19 strains were grown into carbon-limited stationary phase and their viability monitored for 1 month. Three out of these 19 cultures (those of strains A1, A5 and A6) lost all viability over a month of carbon starvation (Fig. 2(a)). Therefore, while it is clear that the GSNO-resistance can be acquired in a number of ways, there is a link between sensitivity to GSNO and the ability to survive in stationary phase in a number of these mutants. When the starvation survival experiment of the three stationary phase survival mutants was repeated after a number of subcultures of these strains, all three had lost the severe starvation survival phenotype (Fig. 2(b)). The difference with wild type was reduced to <10-fold for A1 and A6, and A5 did not show reduced survival compared with wild type (Fig. 2(b)). Also, all three mutants had lost the GSNO-resistance (results not shown), suggesting that there was high selective pressure to lose the mutation by reversion, or to acquire a suppressing mutation, both during growth and during starvation. Similar suppression of stationary phase survival phenotypes has already been reported for M. smegmatis [7].

to 10 mM for 8 h stationary phase cultures and progressively reduced to 0.5 mM in cultures that had been in stationary phase for 36 days (Fig. 1(b)). These data clearly demonstrate that stationary phase cultures of mycobacteria are more sensitive to GSNO than exponential phase cultures. Perhaps nutrient limited stationary phase M. smegmatis cells induce nutrient scavenging systems to be able to scavenge any type of nutrient they may encounter in stationary phase, including peptides such as GSNO. M. tuberculosis has two peptide permease operons, dppDCBA and oppADCB [29]. In the incomplete genome sequence of M. smegmatis (http:// www.tigr.org/cgi-bin/BlastSearch/blast.cgi?), an oppADCB operon is present and highly homologous to the M. tuberculosis operon. There are also homologues of the genes from the M. tuberculosis dppDCBA operon in M. smegmatis. It is possible that one of these permeases can transport GSNO, or part of the tripeptide, into the cell where the nitric oxide radical can dissociate and damage intracellular components. Intriguingly, a peptide permease mutant of M. bovis BCG is resistant to GSNO [30]. 3.2. Spontaneous GSNO-resistant cultures are defective in stationary phase survival

3.3. Isolation of GSNO-resistant mutants by transposon mutagenesis

We next investigated whether there was a link between GSNO-sensitivity and stationary phase survival. To address this we isolated a total of 38 putative, spontaneous GSNO-resistant mutants from stationary phase cultures exposed to GSNO. Out of the 38 strains tested, 12 survived exposure to 25 mM GSNO for 24 h, 10 had an MBC between 10 and 25 mM, 6 between 5 and 10 mM and 10 below 5 mM. Cultures of a number of these mutants showed visible clearing in stationary phase. To

The spontaneous GSNO-resistant M. smegmatis mutants showed that we could isolate stationary phase survival mutants following a positive selection for GSNO-resistance. Therefore, we decided to attempt to select GSNO-resistant mutants from a transposon mutant library. Although this approach would not eliminate the suppression problem, by maintaining kanamycin selection the risk of transposon loss and therefore reversion

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Fig. 2. Stationary phase survival of wild type (wt) M. smegmatis and four mutant strains. Strains A1, A3, A5 and A6 were survivors from a 9 day stationary phase culture and were resistant to 25 mM GSNO. Strain A3 is an example of strains that did not lose viability compared with wild type. (a) starvation survival of strains inoculated from plates immediately after isolation. (b) starvation survival of strains after multiple subcultures and inoculated from glycerol stocks after 3 months at 80 C. Strains were grown in 2 ml Hartmans-de Bont minimal medium in 30 ml polystyrene tubes (Sterilin) and the culture viability monitored by plating onto lab-lemco medium. The detection limit was 10 cfu/ml.

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could be reduced. Also, having a drug resistance marker linked to the mutation would enable it to be transduced into clean genetic backgrounds (free of suppressor mutations). A Tn611 mutant library pool was grown into stationary phase for a few days, GSNO was added to a final concentration of 14 mM, and after 24 h exposure to GSNO the cultures were plated to recover surviving cells. Putative GSNO-resistant mutants were tested for GSNO resistance in stationary phase. Seven out of 22 cultures from 11 independent mutagenesis experiments yielded colonies after GSNO treatment. 420 colonies were picked in total, of which 58 survived exposure to 15–20 mM GSNO to varying degrees in a microbroth dilution assay. 3.4. Identification of GSNO-resistant transposon mutants defective in stationary phase survival We tested the stationary phase survival of 43 transposon mutants that were resistant to 20–25 mM GSNO when tested after initial isolation. Between 37–55 days into carbon starved stationary phase, 15 of the mutants showed a 10–105-fold reduction in stationary phase survival compared with wild type M. smegmatis. Unfortunately, many of the most promising mutants would not recover from glycerol stocks. This suggested that the mutation that reduced the cellÕs ability to survive or recover from stationary phase (our survival assays do not distinguish between recovery and survival phenotypes) might also have caused decreased resistance to freezing or recovery from cold shock. Seven mutants were recovered for further study. To demonstrate linkage between the Tn611 insertion and the GSNO-resistance/stationary phase survival phenotypes, as well as to assess the presence of possible accumulated suppressor mutations, phage I3 [27] was used to transduce the transposon mutation from the seven mutants into wild type M. smegmatis [7,8]. For 1 mutant, 3C3, no transductant was obtained. Southern hybridisation confirmed that parent and transductant strains contained identical transposon insertion regions (data not shown). All the transductants were more resistant to GSNO than wild type and similar to their parent strains, suggesting that in these mutants the transposon insertion was linked to the GSNO-resistance (data not shown). They had similar generation times to the wild type in exponential phase. Stationary phase survival of the mutants and their transductants was investigated (Fig. 3). After approx. 50 days in carbon-starved stationary phase, wild type M. smegmatis viability was reduced 100–1000fold. This is greater than the loss of viability at 37  C [7]. After 10 days in stationary phase, all mutants and transductants had 10–104-fold fewer viable cells than wild type M. smegmatis and in some mutants and transductants there was a further loss of viability to below detectable levels. Initially mutants and their transductants showed similar rates of loss of survival. However, the tim-

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ing and speed of reversion of the survival phenotype to wild type levels, which occurred in most cultures at some stage during the survival experiment, varied between mutants and their transductants. After reversion to wild type levels, there was often further loss of survival and recovery (Fig. 3). This is almost certainly a reflection of the dynamic nature of stationary phase cultures of M. smegmatis [7]. New variants can arise in M. smegmatis stationary phase in as similar way to the Growth Advantage in Stationary Phase (GASP) mutants that take over stationary phase populations of E. coli [31]. The dynamic nature of the phenotypes of the GSNO-resistant, stationary phase survival mutants may result from successive takeovers of the population by GASP mutants, which in some way confer transient survival advantages on the GSNO-resistance genotype. 3.5. Identification of the disrupted genes For each of the seven mutants selected for further study, the DNA flanking the transposon insertion in the Tn611 mutants was identified and sequenced. For mutant 3B3, no PCR products were obtained. For mutant 3E3, the two flanking fragments gave different gene homologies. We attempted without success to clone the transposon insertion of these mutants with two other methods. For the remaining five mutants, the DNA fragments up and downstream of the transposon insertion were identified by LM-PCR as the same gene and these can therefore be unambiguously identified. Mutant 3C3 had the transposon insertion in a gene with homology to M. tuberculosis genes Rv1986 and Rv0488 and a Streptomyces coelicolor putative membrane transporter [29,32]. These are all homologues of the lysE gene from Corynebacterium glutamicum, which encodes an exporter of lysine and arginine. LysE is required for growth on media rich in these amino acids or peptides containing them, preventing metabolic imbalance by removing excess lysine and arginine from the cell [33,34]. Mutant 3C8 was disrupted in a gene with homology to Rv0466, a M. tuberculosis gene annotated as a putative thioesterase [29] with homology to some plant thioesterase [35,36]. Mutant 3C11 had a gene disruption in a homologue of Rv0876c, an unknown membrane protein that in M. tuberculosis is neighboured on either side by other unknown proteins [29] In M. smegmatis, the arrangement is similar with approx. 140 bp upstream of the Rv0876c homologue a divergently transcribed Rv0877 homologue (154/235 (65%) amino acid identity), and immediately downstream in the same operon a Rv0875c homologue (91/148 (61%) amino acid identity). Mutant 8H1 was disrupted in a gene most homologous to an enoyl-CoA hydratase homologue from Bordetella pertussis [37], and its closest mycobacterial homologue (E value 2 · 10 50) was echA13 (Rv1935c),

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Fig. 3. Stationary phase survival of wild type M. smegmatis (d) and 7 Tn611 mutants (n), and their transductants (open symbols). (a) 3B3 and 1 transductant. (b) 3C3. (c) 3C8 and 1 transductant. (d and e) 3C11 and 5 transductants. (f) 3E3 and 2 transductants. (g) 3F3 and 1 transductant. (h) transductants of mutant 8H1. Experiments were carried out as described in the legend to Fig. 2, but cultures grown at 42 C.

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one of the 21 enoyl-CoA-hydratase homologues present on the genome [29]. Enoyl-CoA-hydratases classically function in the b-oxidation pathway for fatty acid catabolism. Host cell lipids are an important source of nutrients for M. tuberculosis in vivo [38,39]. 3F3 had the transposon inserted in a gene with homology to the M. tuberculosis atpD gene (Rv1310), which is part of the atpBEFHAGDC operon and encodes the b chain of the ATP synthase complex [40]. Interestingly, we had previously isolated a stationary phase survival mutant, 3911H, in the screen described by Keer et al [7]. This mutant displayed a 1000-fold lower survival than wild type M. smegmatis at 40 days stationary phase in lab-lemco medium (data not shown). The disrupted gene was also identified by LM-PCR and found to be homologous to M. tuberculosis Rv1308, which codes for the a chain of the ATP synthase operon. In E. coli, mutants in the atpIBEFHAGDC operon can still grow by generating energy from fermentable substrates but they fail to grow on oxidisable substrates such as succinate [40]. In our experiments, the sole carbon source present in the culture medium was glycerol. Glycerol enters the Embden Meyerhof pathway (EMP), after being first converted to L -glycerol-3-phosphate (requiring 1 ATP), and then to the EMP intermediate dihydroxyacetone phosphate (requiring 1 NAD(P)+). The further metabolism via the EMP leads to a net yield of 1 ATP per glycerol molecule, apparently sufficient for the growth of the atpD and atpA mutants. However, perhaps under energy/carbon starved conditions there is a premium on efficient energy generation to ensure prolonged survival. These results indicate that the sensitivity of stationary phase M. smegmatis to GSNO can be successfully used to isolate both spontaneous and Tn611 mutants that are resistant to GSNO in stationary phase and that are also defective in stationary phase survival. At present, we have no clear explanation how the stationary phase survival phenotype relates to increased GSNO-resistance in stationary phase. We have identified three genes required for successful stationary phase survival, which have a potential role in scavenging of nutrients released by dead and dying cells. The lysE homologue might be important in retaining metabolic balance when peptides and amino acids are used, whereas the putative thioesterase and the echA13 homologue may be required for lipid degradation. This supports our previous findings that stationary phase cultures of M. smegmatis are dynamic with cryptic growth playing an important role in overall population survival [9]. Acknowledgement This work was funded by the Wellcome Trust, via a Wellcome Prize Fellowship to M.J.S.

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