Abstract
Mycobacterium tuberculosis H37Rv possesses five resuscitation-promoting factors, RpfA–E, which are required for the resuscitation of dormancy in mycobacteria induced by prolonged incubation of the culture in stationary phase. This study explores the transcriptional profile of all the rpf-like genes of M. tuberculosis H37Rv in the exponential phase, stationary phase, non-culturable phase and Rpf-mediated resuscitation phase. The relative expression was also monitored under acid stress, nutrient starvation and low-oxygen (hypoxia) conditions by real-time quantitative PCR. We show differential relative expression of the rpf genes during different stages of growth and under stress. During early resuscitation, all rpf genes were expressed with maximal expression ratios for rpfA and rpfD. rpfC was consistently expressed during all stages of growth and nutrient starvation. Acid stress induced higher relative expression of rpfD and rpfE and hypoxia of rpfC and rpfE. These results therefore provide further evidence that the rpf genes perform distinct roles during cell growth and cell survival under different physiological stresses, and are consistent with the rpf-like genes being differentially regulated.
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A set of supplementary methods, describing the protocol for cDNA synthesis, a supplementary figure, showing amplification efficiency for each primer set, and a supplementary table, showing the data used to calculate the normalized ratios, are available with the online version of this paper.
Edited by: J. Cavet
INTRODUCTION
Persistence and reactivation play significant roles in the pathogenesis of Mycobacterium tuberculosis. Only a proportion of persons infected with M. tuberculosis develop active tuberculosis, while the majority of them harbour the bacilli for an extended period of time in the form of latent infection without producing clinical symptoms. Approximately one-third of the world's population is infected with latent M. tuberculosis, and run the risk of reactivation, causing active disease (WHO, 2010; Gedde-Dahl, 1952). Hence, an understanding of mycobacterial persistence and reactivation is crucial for effective control of the disease.
The translation from replicating to a dormant state and then to reactivation remains an enigma. In recent years, a class of protein has been identified that act like growth factors for dormant mycobacteria. The protein known as resuscitation-promoting factor was originally identified in Micrococcus luteus as a ∼16–17 kDa protein that is secreted by actively growing bacteria. The protein in picomolar concentrations is able to resuscitate dormant Micrococcus luteus or mycobacteria generated by prolonged incubation in spent culture medium (Mukamolova et al., 1998a, b; Shleeva et al., 2002). M. tuberculosis contains five Rpf homologues which share sequence as well as functional homology with Rpf of Micrococcus luteus in their capacity to resuscitate dormant Mycobacterium bovis BCG or M. tuberculosis in vitro (Mukamolova et al., 2002). The five rpf-like genes of M. tuberculosis H37Rv are distributed throughout the chromosome and are designated rpfA (Rv0867c), rpfB (Rv1009), rpfC (Rv1884c), rpfD (Rv2389c) and rpfE (Rv2450c) (). The presence of five rpf-like genes in M. tuberculosis is intriguing, as all five genes are expressed in vitro and in vivo in the lungs of mice acutely infected with M. tuberculosis (Tufariello et al., 2004; Mukamolova et al., 2002; Downing et al., 2004). Transcripts for all the rpf-like genes are detectable at the earliest time points, although relative expression has been shown to differ at later time points during growth in culture (Tufariello et al., 2004; Mukamolova et al., 2002). Strains harbouring deletions of individual rpf-like genes from M. tuberculosis do not show significant alteration of growth in vitro and in vivo (Tufariello et al., 2004; Downing et al., 2004). Earlier studies have confirmed that rpfA–E are collectively dispensable for growth of M. tuberculosis in vitro and in vivo and suggest a functional hierarchy within this multi-gene family under different conditions (Kana et al., 2008). Deletion of individual rpfB–E genes has been shown to be accompanied by upregulation of some or all of the remaining rpf-like genes (Downing et al., 2004), although later studies with multiple rpf mutants have clearly shown that rpf-like genes do not compensate for the loss of other genes by transcriptional upregulation; instead they suggest that rpf-like genes are likely to be regulated by other distinct mechanisms (Downing et al., 2005; Kana et al., 2008). In this respect, it is worth noting that rpfA has been shown to be regulated by cAMP receptor protein (Rickman et al., 2005), while rpfC is positively regulated by the sigma factor SigD (Raman et al., 2004). RpfB and RpfE have been shown to interact with mycobacterial endopeptidase RipA (Rpf-interacting protein), resulting in peptidoglycan hydrolysis. RipA does not interact with RpfA, C and D, adding to the complexity of Rpf function and regulation (Hett et al., 2007).
In this study, we have explored by real-time PCR the relative expression of all five rpf-like genes of M. tuberculosis H37Rv during different growth stages, ranging from exponential phase to the non-culturable (NC) state and subsequent Rpf-mediated resuscitation. The relative expression profiling was further extended to three different physiological stress conditions; namely, nutrient starvation, hypoxia and acid stress.
METHODS
Bacterial strains and culture conditions.
M. tuberculosis H37Rv was grown in Sauton's medium containing albumin–dextrose–catalase (ADC; Becton Dickinson) and Tween 80 (0.05 %, v/v) without shaking at 37 °C. For the resuscitation of NC cells of M. tuberculosis, filter-sterilized supernatant of actively growing M. tuberculosis culture (OD600 0.5) was used. Methylene blue (1.5 μg ml−1) was used as a redox indicator in hypoxia experiments.
Formation of non-culturable cells (NCC) and their resuscitation.
The NC state was achieved as described by Shleeva et al. (2003). Briefly, NCC of M. tuberculosis were obtained by prolonged incubation of culture in Sauton's medium containing Tween 80 (0.05 %, v/v) and ADC for 6 months at 37 °C (Shleeva et al., 2003; Salina et al., 2006). Samples were taken at regular intervals and passed through a 23-gauge syringe needle before plating to make the culture homogeneous. Plating was done on MB7H10 agar medium containing oleic acid–albumin–dextrose–catalase (OADC; Becton Dickinson). After 6 months of incubation, M. tuberculosis became NC, as c.f.u. levels were less than 5 cells ml−1. Resuscitation of NCC was induced by cell-free culture supernatant of exponential phase (OD600 0.5) M. tuberculosis H37Rv culture, as described by Shleeva et al. (2003). The cells were removed by centrifugation at 6000 g for 10 min and the supernatant was filter-sterilized with a 0.22 μm pore-size filter (Whatman) at 4 °C and used within 20 min.
RNA isolation from different growth stages.
An exponential-phase starter culture of M. tuberculosis H37Rv was used to inoculate the working culture at an approximate density of 106 c.f.u. ml−1 (based on the estimate that OD600 1=5×108 c.f.u. ml−1) in Sauton's medium containing ADC and Tween 80 at 37 °C without shaking. RNA extraction was done at different growth stages such as early exponential phase (OD600 0.2, 5-day-old culture), exponential phase (OD600 0.6, 3-week-old culture), stationary phase (OD600 2.5, 3-month-old culture), NC state (OD600 4.5, 6–month-old culture) and resuscitation phase (OD600 0.2, 3 days after resuscitation). From the various growth phases described above, 30 ml culture was harvested in triplicate from three independent experiments. RNA isolation was done according to the kit protocol (RNeasy Mini kit, Qiagen) and quantified by Qubit fluorometer (Invitrogen).
Acid stress.
M. tuberculosis H37Rv culture was grown in 100 ml Sauton's medium (pH 7.2) containing Tween 80 (0.05 %, v/v) at 37 °C without shaking. When the OD600 reached 0.6, cells were divided into three 30 ml aliquots, centrifuged at 6000 g for 10 min at room temperature, resuspended in 50 ml Sauton's medium (pH 4.5, pH 5.5 or pH 7.2) containing Tween 80 (0.05 %, v/v) and kept at 37 °C without shaking. After 24 h of acid shock, 30 ml culture from each flask was pelleted. RNA isolated from the culture grown in Sauton's medium at pH 7.2 was used as a control or positive calibrator for relative expression analysis. RNA isolation was done according to the kit protocol (RNeasy Mini kit, Qiagen) and quantified by Qubit fluorometer (Invitrogen). RNA was isolated from three independent experiments.
Nutrient starvation.
An exponentially growing M. tuberculosis H37Rv culture (OD600 0.6) in Sauton's medium with Tween 80 (0.05 %, v/v) was centrifuged at 6000 g for 10 min at room temperature. Cells were washed twice with PBS with Tween 80 (0.05 %, v/v) pH 7.2 and resuspended in 100 ml PBS-Tween (pH 7.2). The culture was incubated at 37 °C without shaking. Cells were harvested at 0, 24 and 96 h. Cells harvested at 0 h served as a control (Betts et al., 2002). RNA isolation was done by using the RNeasy Mini kit (Qiagen) and quantified by Qubit fluorometer (Invitrogen). The experiment was repeated three times with three independent experimental set-ups.
Hypoxia conditions.
An aerobic exponentially growing culture of M. tuberculosis H37Rv was diluted to OD600 0.005 in MB7H9 medium containing ADC, Tween 80 (0.05 %, v/v) and the redox dye methylene blue (1.5 μg ml−1). The culture was transferred to sealed rubber-capped tubes (50 % headspace ratio) and kept standing at 37 °C. Anaerobiosis was monitored by decolorization of the redox indicator methylene blue (Boon et al., 2001). Complete decolorization of the dye was obtained after 25 days incubation at 37 °C. For control samples, tubes were not sealed and were kept at 37 °C with periodic exposure to air by shaking for the same period of time. The cells were pelleted on the 26th day in triplicate, and RNA was isolated by using the RNeasy Mini kit (Qiagen) and quantified by Qubit fluorometer (Invitrogen).
cDNA synthesis.
Each RNA sample was treated with RNase-free DNase I (Fermentas) and heat-inactivated according to the manufacturer's instructions. RNA integrity was checked by formaldehyde agarose gel electrophoresis. After staining with ethidium bromide, three sharp bands of 23S, 16S and 5S rRNA were seen. DNA contamination was checked by (no reverse transcription) PCR for each RNA sample in a gradient cycler (Mastercycler gradient, Eppendorf) using a 16S rRNA primer. No amplification was seen after 30 cycles of amplification. PCR cycling parameters were: initial denaturation at 95 °C for 5 min, denaturation at 94 °C for 1 min, annealing at 60 °C for 30 s, extension at 72 °C for 30 s for 30 cycles, followed by a final extension of 10 min at 70 °C. PCR was performed in a 20 μl reaction mix containing 1.25 U DreamTaq DNA polymerase (Fermentas), 16S rRNA primers (0.2 μM each), RNA template (100 ng), MgCl2 (final concentration 2.0 mM) and dNTPs (final concentration 0.2 mM). PCR products were electrophoresed in a 2.5 % agarose gel and visualized by ethidium bromide staining.
A 300–400 ng RNA sample was used for cDNA synthesis using random hexamer primers with the RevertAid First Strand H Minus cDNA synthesis kit (Fermentas). A detailed protocol for cDNA synthesis is available with the online version of this paper. Real-time quantitative PCR was performed in a LightCycler 480II instrument (Roche) using a LightCycler 480 SYBR Green I Master kit (Roche). Primers were designed using dnastar primer design software and purchased from Sigma–Aldrich. All primer sets were optimized for annealing temperature and concentration to ensure that only a single product of the correct size would be amplified. The optimum annealing temperature and primer concentration were found to be 68 °C and 0.5 μM.
PCR efficiency and real-time PCR.
Amplification efficiency for each primer set was calculated by serially diluting exponential-phase cDNA template. Efficiency values were calculated by the software in LightCycler 480II and were in the optimum range. Efficiency values for each primer set are given in Table 1⇓ and Supplementary Fig. S1, and showed high linearity in the investigated range of 0.1–100 ng cDNA.
Primer sequences with amplicon size and melting temperature
Amplification efficiency for each primer set was calculated using a LightCycler 480II instrument by serially diluting exponential-phase cDNA template in the investigated range of 0.1–100 ng cDNA with high linearity.
For LightCycler reactions, a master mix of the following components was prepared: 7.0 μl PCR-grade water, 1.0 μl (0.5 μM) forward primer, 1.0 μl (0.5 μM) reverse primer, 10 μl 2× Master Mix, 1.0 μl cDNA (50–100 ng). A multiwell plate was sealed with sealing foil, centrifuged at 1500 g for 1 min and loaded into the LightCycler 480II instrument. Amplification was performed in triplicate wells for each sample analysed; a control reaction with no template (water) was run with all reactions. In each set of reactions, 16S rRNA was used as a reference gene for normalization of cDNA amount. Real-time PCR analysis was performed using the following optimized assay conditions: denaturation program (95 °C for 10 min); amplification and quantification program repeated for 45 cycles (95 °C for 10 s, 68 °C for 20 s, 72 °C for 30 s with a single fluorescence measurement); melting curve program (95 °C for 10 s, 70 °C for 1 min with continuous fluorescence measurement at 97 °C); and finally a cooling step at 40 °C for 30 s.
Relative quantification analysis was done using an efficiency-calibrated model (Pfaffl., 2001), and the normalized ratio was calculated as:
Melting curve analysis was performed after each run in a LightCycler 480II instrument to confirm the specificity of the primers.
Statistical analysis.
CP values for each run were used to calculate mean CP with sd and se. The normalized ratio was calculated using the efficiency-calibrated model (see Supplementary Table S1). The relative expression ratio was calculated from triplicate normalized ratios for each gene with sd (Table 2⇓). Student's t test was performed to determine the significance of the relative expression ratios. P<0.05 was considered significant (*), P<0.01 was considered highly significant (**) and P>0.05 was considered not significant.
Relative expression of all five rpf-like genes of M. tuberculosis H37Rv during different growth stages and under physiological stress conditions
The mean expression ratio and sd were calculated from the normalized ratio of three independent experiments. A.S., acid stress; N.S., nutrient starvation.
RESULTS
rpf-like genes are differentially expressed during growth of the cell
The relative expression of all five rpf genes (rpfA–E) was followed in exponential, stationary, NC and resuscitation phases. All five rpfs (rpfA–E) were expressed in the early exponential phase (Tufariello et al., 2004; this study), and hence RNA from this stage was taken as a positive calibrator for quantification of relative expression in different growth stages (Fig. 1⇓). rpfB, rpfC and rpfE had a higher expression ratio in exponential phase; the relative expression of rpfB and rpfE was reduced in stationary phase. The expression ratio of rpfD was lower in exponential phase but higher in the stationary and NC phases. However, only rpfC was found to be upregulated consistently in the exponential to stationary and NC phases. The relative expression of rpfA remained lower in exponential and NC phase but higher in resuscitation phase. Thus, from the data it appears that rpfC and rpfD have a role in stationary phase adaptation and persistence, while rpfB and rpfE may be required in exponential phase. Interestingly, in resuscitation phase, the relative expression of all five rpf genes was enhanced, with the maximal expression ratio of rpfA and rpfD on the third day of resuscitation (Fig. 1⇓, Table 2⇑). On the 10th day of resuscitation, the relative expression of all five rpf genes was almost identical to that observed in exponential phase (data not shown).
Relative expression analysis of all five rpf genes (rpfA–E) was followed in exponential, stationary, NC and resuscitation phases by real-time PCR. M. tuberculosis H37Rv was grown in Sauton's medium containing ADC and Tween 80 at 37 °C without shaking. RNA extraction was done at different growth stages as described in Methods. Early exponential-phase RNA was used as a positive calibrator and 16S rRNA was used as a reference gene for normalization. Data represent mean relative expression values of three independent experiments with sd values (Table 2). P<0.05 was considered significant (*), P<0.01 was considered highly significant (**) and P>0.05 was considered not significant.
Relative expression of rpfs under stress conditions
The expression of all five rpf-like genes of M. tuberculosis H37Rv was analysed in mildly acidic [acid stress (A.S.) pH 5.5] and highly acidic (A.S. pH 4.5) conditions compared with the control grown at normal pH, as described in Methods. RNA of the control culture was used as a positive calibrator for real-time quantitative PCR (qRT-PCR). In mildly acidic (pH 5.5) and highly acidic conditions (pH 4.5), the relative expression of rpfE and rpfD was increased but the expression ratios of rpfA, rpfB and rpfC were decreased. In highly acidic conditions, rpfE had the maximum expression ratio among all rpfs (Fig. 2⇓, Table 2⇑).
Relative expression of all five rpf-like genes of M. tuberculosis H37Rv was analysed under mildly acidic (pH 5.5, A.S. 5.5) and highly acidic (pH 4.5, A.S. 4.5) conditions. Acid stress was generated as described in Methods. RNA isolated from the culture grown in Sauton's medium, pH 7.2, was used as a control or positive calibrator for relative expression analysis. 16S rRNA was used as a reference gene for normalization. Data represent mean relative expression values of three independent experiments with sd values (Table 2). P<0.05 was considered significant (*), P<0.01 was considered highly significant (**) and P>0.05 was considered not significant.
The relative expression of all five rpf genes was observed under hypoxia conditions generated as described in Methods. A control culture was used as a positive calibrator for relative expression analysis. After 25 days of culture incubation, complete decolorization of the redox dye methylene blue was observed. At this time point, the rpfE and rpfC expression ratios were increased, with a maximum expression ratio of rpfE. The relative expression of rpfA, rpfB and rpfD was lower compared with the control (Fig. 3⇓, Table 2⇑). We analysed the relative expression of all five rpf-like genes during 24 and 96 h of nutrient starvation (N.S.), using 0 h RNA as a positive calibrator and control. It was observed that during 24 h of nutrient stress, the relative expression of all five rpf genes increased with respect to the control. During prolonged incubation of the culture for 96 h, rpfC and rpfD had increased expression ratios but the relative expression of rpfA, rpfB and rpfE was decreased (Fig. 4⇓, Table 2⇑).
Relative expression analysis of all five rpf-like genes of M. tuberculosis H37Rv under hypoxia. Hypoxia was generated as described in Methods. RNA isolated from the culture grown aerobically in MB7H9 medium containing ADC and Tween 80 (0.05 %. v/v) was used as a control or positive calibrator for relative expression analysis. 16S rRNA was used as a reference gene for normalization. Data represent mean relative expression values of triplicate experiments with sd values (Table 2). P<0.05 was considered significant (*), P<0.01 was considered highly significant (**) and P>0.05 was considered not significant.
Relative expression of all five rpf-like genes during 24 and 96 h of nutrient starvation (N.S.) analysed by real-time PCR. Nutrient stress was generated as described in Methods. Cells harvested at 0 h served as control or positive calibrator for relative expression analysis. 16S rRNA was used as a reference gene for normalization. Data represent mean relative expression values of three independent experiments with sd values (Table 2). P<0.05 was considered significant (*), P<0.01 was considered highly significant (**) and P>0.05 was considered not significant.
DISCUSSION
In this paper, we have analysed the expression of the five rpf-like genes of M. tuberculosis H37Rv under different growth and physiological stress conditions. It was found that all rpf genes were expressed in early resuscitation phase with maximum relative expression for rpfA and rpfD. Only rpfC was observed to show consistently increased relative expression during all stages of growth and nutrient starvation. It was also observed that acid stress induced higher relative expression of rpfD and rpfE and hypoxia of rpfC and rpfE. The earlier results on expression of the five rpf (A–E) genes in axenic culture from early exponential to stationary phase by semiquantitative RT-PCR revealed the expression of all five genes at early time points but differences at later stages of growth (Mukamolova et al., 2002; Downing et al., 2004; Tufariello et al., 2004). The studies showed that rpfE was expressed maximally in the early exponential phase of growth, with expression falling markedly and remaining low throughout (Tufariello et al., 2004). Three of the other rpf-like genes, rpfA rpfB and rpfD, had higher expression in exponential phase but reduced expression in stationary phase. Only rpfC (rv1884c) was expressed at an undiminished level throughout when compared with early exponential phase (Tufariello et al., 2004). In extended stationary phase (4-month-old culture) of M. tuberculosis, expression of all five rpf-like genes can be detected (Tufariello et al., 2004), while no expression is observed in stationary phase and 5-month-old cultures of M. bovis BCG (Mukamolova et al., 2002). Differential expression of the five rpf-like genes in M. tuberculosis appears to be related to differential regulation and this is supported by functional and mutational analysis of rpf genes.
RpfB has been shown to interact with a putative mycobacterial endopeptidase, designated Rpf-interacting protein A (RipA) (Hett et al., 2007). The two proteins colocalize in the septa of actively dividing cells, suggesting a role for the RipA–RpfB complex in peptidoglycan hydrolysis during cell division. RipA also interacts with RpfE but not with RpfA, RpfC and RpfD, suggesting that these Rpfs may act via a distinct mechanism and/or on a different substrate or pathway (Hett et al., 2007, 2008). Similarly, colony formation is delayed in M. tuberculosis with mutated rpfB, and colonies are much smaller than those of the wild type (Tufariello et al., 2004). The higher expression of rpfB and rpfE observed in exponential phase in our study may thus be related to cell division and growth. RpfC is by far the most abundant resuscitation-promoting factor in M. tuberculosis (Mukamolova et al., 2002; Tufariello et al., 2004). We also found a consistently higher expression ratio of rpfC from exponential to stationary to NC phase. rpfC appears to be directly regulated by the sigma factor SigD, and in vitro transcription of rpfC has been found to be completely dependent on SigD (Raman et al., 2004). The expression of SigD has been linked to the stringent response, with an increased expression in response to starvation that is partly Rel-dependent (Raman et al., 2004). We also observed enhanced rpfC expression during nutrient starvation (Fig. 3⇑). Hence, rpfC may have a role in stationary-phase adaptation and persistence.
Expression of the rpf-like genes during resuscitation of NCC to active growth by M. tuberculosis culture supernatant was also examined. Samples were taken on days 3 and 10 of resuscitation. Day 3 was chosen as changes in the composition of the cell population have been reported to occur as early as 3 days after inoculation of NCC in resuscitation medium (Shleeva et al., 2003). The progression of the culture from the NC phase to the resuscitation phase on day 3 resulted in an increase in expression of all five rpf genes. The expression of all the rpfs in early resuscitation phase is anticipated, as it is a recovery phase from the NC state to active growth. The higher expression ratio of all the rpf genes in early resuscitation phase correlates with mutational studies, which clearly suggest that deletion of three or more genes results in impairment of resuscitation from an NC state (Kana et al., 2008; Downing et al., 2005). Deletion of any one of the five rpf-like genes does not affect the growth and ability to resuscitate spontaneously from an NC state in vitro (Downing et al., 2005). Similarly single-deletion mutants have been found to be phenotypically similar to wild-type M. tuberculosis H37Rv in vivo (Downing et al., 2005). However, the multiple mutant strains KDT8 (ΔrpfA ΔrpfB ΔrpfC) and KDT9 (ΔrpfA ΔrpfC ΔrpfD), which lack three of the five rpf-like genes, are significantly but differentially attenuated in a mouse infection model. These mutants are also unable to resuscitate spontaneously in vitro (Downing et al., 2005). In another study, quadruple mutants (ΔrpfACBD and ΔrpfACBE) of M. tuberculosis were found to be significantly attenuated in a murine infection model, being severely impaired in growth and persistence (Kana et al., 2008). We observed higher relative expression of rpfA and rpfD during early resuscitation. rpfA has been shown to be subject to regulation by the cAMP receptor protein (Rickman et al., 2005), and is reported to be the most potent resuscitation-promoting factor that exhibits activity at femtomolar concentrations (Mukamolova et al., 2002). Among all five Rpf-like proteins of M. tuberculosis, RpfD is considered the most efficient in terms of resuscitation-promoting activity (Cohen-Gonsaud et al., 2005).
Phagosomes containing mycobacteria begin to acidify rapidly after phagocytosis and the pH drops rapidly to 5.5 or lower. The acidification signal is used by mycobacteria to induce the expression of genes needed to alter phagosomal maturation (Hackam et al., 1998; Oh & Straubinger, 1996; Sturgill-Koszycki et al., 1994). The induction of rpfE under mildly acidic conditions has been reported previously (Fisher et al., 2002). The loss of rpfE from the triple mutant ΔrpfACB has been shown to have pronounced effect on persistence of tubercle bacilli in the lungs of mice (Kana et al., 2008). We observed that in highly acidic conditions the expression ratios of rpfE and rpfD were highest among all rpfs, which suggests a role for both rpfE and rpfD in acid-induced stress conditions.
M. tuberculosis is generally regarded as a strictly aerobic bacillus, although it can survive for a long period of time under microaerophilic conditions (Wayne & Hayes, 1996). An in vitro model to study this persistence state is the Wayne dormancy model, in which the bacteria downregulate their metabolism due to reduced access to oxygen (Wayne & Hayes, 1996). rpfE has been shown to have a role in persistence, and rpfC is regulated by the transcriptional regulator SigD required for stationary phase adaptation and persistence (Raman et al., 2004). Our results suggest that both rpfC and rpfE have a role in cell survival during hypoxia-induced persistence, and that rpfD and rpfE have a role in adaptation of M. tuberculosis in acid-induced stress conditions, indicating that rpfE is important for the survival of M. tuberculosis H37Rv under stress conditions induced by low oxygen and pH.
M. tuberculosis H37Rv arrests growth, decreases its respiration and becomes resistant to front-line drugs such as isoniazid, rifampicin and metronidazole under nutrient-starved conditions (Betts et al., 2002). It has been shown that the respiration rate of a nutrient-starved culture declines rapidly over the first 96 h of starvation (Betts et al., 2002). We have observed that during 96 h of nutrient stress rpfC and rpfD had higher relative expression, and the expression ratio of rpfA, rpfB and rpfE was decreased. As discussed above, rpfC is under the control of the putative sigma factor SigD, which shows enhanced expression during nutrient starvation (Betts et al., 2002). Starvation-induced expression of sigD is blunted in a relA mutant strain (Dahl et al., 2003), suggesting that sigD is a part of the RelA regulon required for the stringent response for survival of M. tuberculosis during nutrient starvation. It has been reported that expression of relA was induced under nutrient starvation (Betts et al., 2002), suggesting the increased expression of sigD together with increased expression of rpfC in nutrient starvation.
Conclusion
These studies are consistent with rpfA and rpfD playing important roles in reducing the lag phase during resuscitation, as they have maximum expression ratios in the initial phase of resuscitation (3 days after resuscitation). Only rpfC has a higher expression ratio from exponential phase to the NC state compared with early exponential phase, suggesting that rpfC has a consistently increased relative expression among all rpfs in M. tuberculosis. The results suggest that acid stress induces expression of rpfD and rpfE and that hypoxia induces expression of rpfC and rpfE. Nutrient starvation induces all the rpf genes in the early phase (24 h), but later after 96 h, rpfC and rpfD have higher relative expression. The five rpf genes are differentially expressed under different growth stages and stress conditions, suggesting that they have distinct roles during cell growth and cell survival under stress conditions.
Acknowledgments
We thank the Director for facilities and support. R. K. G. is Senior Research Fellow, Council of Scientific and Industrial Research (CSIR). Statistical analysis was done with the help of Mr Mukesh Srivastava, of the Biometry Division of the Institute. We are grateful to Mr Brijesh Singh (Roche Diagnostics, India) and Dr Vikas Srivastava for statistical analysis. Support from CSIR and the Department of Biotechnology of the Ministry of Science and Technology is acknowledged. This is Central Drug Research Institute (CDRI) communication number 7914.