Bakh Institute of Biochemistry, Moscow, Russia1
Institute of Biological Sciences, University of Wales, Aberystwyth, UK2
Author for correspondence: Arseny Kaprelyants. Tel: +7 95 954 40 47. Fax: +7 95 954 27 32. e-mail: arseny{at}inbi.ras.ru
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ABSTRACT |
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Keywords: dormancy, Nocardiaceae, VBNC, Rpf, Micrococcus luteus
Abbreviations: ADC, albumen, glucose and NaCl; CTC, 5-cyano-2,3-ditolyl tetrazolium chloride; MPN, most probable number; Rpf, resuscitation-promoting factor; SN, supernatant; VBNC, viable but non-culturable
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INTRODUCTION |
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Micrococcus luteus is a member of the Actinomycetales and is therefore related to Mycobacterium tuberculosis. This organism can persist in a dormant state, following growth to stationary phase in batch culture and starvation for several months in the spent growth medium (Kaprelyants et al., 1993 ; Kaprelyants & Kell, 1993a
). Dormant Micrococcus luteus cells have lost the ability to grow on agar plates; however, the addition of supernatant (SN) from growing Micrococcus luteus cultures to dormant cells has been shown to restore the ability to divide freely, thereby resuscitating the cells to normal, colony-forming bacteria (Kaprelyants & Kell, 1996
; Kaprelyants et al., 1994
). Micrococcus luteus cells secrete a resuscitation-promoting factor (Rpf), which promotes the resuscitation of dormant cells (Kaprelyants et al., 1996
). Rpf has subsequently been shown to be a small protein that is active at picomolar concentrations (Kaprelyants et al., 1999
; Mukamolova et al., 1998
). Genes encoding Rpf-like proteins are widely distributed throughout the Actinomycetales; database searches have revealed that similar genes are present in mycobacteria, corynebacteria and streptomycetes (Kell & Young, 2000
). If, as seems likely, the Rpf-like proteins of these organisms have similar functional roles to that of Micrococcus luteus Rpf, we may be able to predict the existence of a non-culturable, dormant state in other members of the Actinomycetales.
The aim of the present study was to find conditions for the in vitro transition of two organisms belonging to the Corynebacterineae (fast-growing R. rhodochrous and slow-growing Mycobacterium tuberculosis) to a non-culturable (possibly dormant) state. We also tested the hypothesis that secreted bacterial proteins may stimulate resuscitation of these two organisms.
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METHODS |
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Scanning electron microscopy.
Cells were fixed for 30 min in 3% glutaraldehyde, followed by treatment with 0·1 M sodium cacodylate buffer (pH 7·2). Samples were air-dried, sputter-coated with 20 nm Au particles and examined using a GSM-840A (Japan) scanning electron microscope.
Respiratory activity, membrane energization and the permeability barrier.
The respiratory activity of the bacterial cells was determined by using the fluorescent redox dye CTC (5-cyano-2,3-ditolyl tetrazolium chloride; Polysciences), as described by Kaprelyants & Kell (1993b ). Briefly, the bacteria were incubated in the presence of 4 mM CTC (freshly prepared in 10 mM sodium phosphate buffer, pH 7·0) for 30 min at 37 °C. Fluorescence was monitored with a Nikon fluorescence microscope with excitation at 530550 nm. Cells were stained with propidium iodide (4 µM in phosphate buffer), to assess the state of the membrane permeability barrier. Rhodamine 123 was used to monitor membrane energization, as previously described (Kaprelyants & Kell, 1992
). Rhodamine 123 accumulation was sensitive to the uncoupler carbonylcyanide-3-chlorophenyl-hydrazone (CCCP). Cells were studied under the fluorescence microscope (excitation at 510560 nm and emission at 590 nm for propidium iodide; excitation at 450490 nm and emission at 520 nm for rhodamine 123).
Estimation of the redox potential.
The changes in the oxidation/reduction potential of the growth medium during R. rhodochrous growth were measured in undiluted samples (3 ml) taken from the bacterial culture. Measurement of the redox potential was done with a platinum electrode (and an AgCl reference electrode; normal potential at 20 °C was 201±2 mV) by using an I-130.2M ionometer (Soyusanalitpribor) under aerobic conditions at 20 °C, with intensive stirring until a constant value was reached.
Assessment of cell viability.
Bacterial suspensions were serially diluted in growth medium and duplicate 100 µl samples were plated in triplicate onto agar-solidified nutrient broth E (for R. rhodochrous) or onto agar-solidified, ADC-supplemented Sautons medium (for Mycobacterium tuberculosis). Plates were incubated at 37 °C. After 5 days of growth for R. rhodochrous and after 2 months of growth for Mycobacterium tuberculosis, c.f.u. values were enumerated. The detection limit was 5x100 c.f.u. ml-1. The same serially diluted samples of Mycobacterium tuberculosis were also employed for resuscitation and most probable number (MPN) assays. Using a fresh pipette tip each time, 100 µl samples were taken from each dilution. Ten of these samples were added to 10 replicate 10 ml Pyrex screw-cap tubes, each containing 2 ml Sautons medium supplemented with ADC. Rpf was added to five of the tubes to give a final concentration of 100 pM. Five additional samples were added to five replicate 10 ml Pyrex screw-cap tubes containing 2 ml filter-sterilized SN taken from cultures of Mycobacterium tuberculosis in the late-exponential phase of growth (3040 days post-inoculation). The 15 tubes for each dilution were incubated at 37 °C without shaking for 2 months.
Resuscitation and MPN assays for R. rhodochrous were performed in 48-well plastic plates (Corning) containing either 0·5 ml modified Sautons medium (see above) or 0·5 ml filter-sterilized SN taken from R. rhodochrous cultures. Some wells contained 125 pM Rpf. All wells were supplemented with 0·5% yeast extract (LabM). Appropriate serial dilutions of R. rhodochrous cells (50 µl) were added to each well. Plates were incubated at 37 °C, with agitation at 150 r.p.m. for 5 days. Tubes or wells with visible bacterial growth were counted as positive, and MPN values were calculated using standard statistical methods (de Man, 1975 ).
Spent medium preparation.
SNs were obtained from R. rhodochrous and Mycobacterium tuberculosis cultures harvested at the times indicated in Results. The R. rhodochrous SN was subjected to centrifugation (12000 g, 20 min). The SNs were sterilized by passage through a 0·22 µm filter (Whatman) before use. Proteinase treatment of the SNs was performed at 37 °C by adding trypsin (200 µg ml-1) or proteinase K immobilized on acrylic beads (0·04 U ml-1; Sigma), followed by the addition of trypsin inhibitor (400 µg ml-1) to samples containing trypsin. The time of incubation was 30 min for trypsin-treated SNs and 3 h for proteinase-K-treated SNs.
Bacterial counts.
The total number of Mycobacterium tuberculosis cells was determined microscopically using a Helbers chamber. A minimum of 100 bacteria was counted and the SD of the total counts was not in excess of 20%.
Preparation of recombinant Micrococcus luteus Rpf.
The Rpf protein of Micrococcus luteus (histidine-tagged recombinant form) was obtained as described by Mukamolova et al. (1998) . Mono Q ion-exchange purification was omitted in some experiments. The purified protein was stored in 10 mM Tris/HCl (pH 7·4) containing 50% (v/v) glycerol at -20 °C for up to 2 weeks, and the protein concentration was determined spectrophotometrically. Before use, all preparations were screened for growth-promoting activity using a small inoculum of Micrococcus luteus, as described by Mukamolova et al. (1998)
. Some preparations had poor activity; only those with substantial activity were employed for these experiments.
Preparation of antibody columns.
Rabbits were immunized three times at 3-week intervals by subcutaneous injection with 1 ml of a 50% (v/v) mixture of Rpf (1 mg ml-1 in water) and incomplete Freunds adjuvant (Sigma). Serum was collected 10 days after the last immunization, and the immunoglobulin fraction was purified by a standard protocol using polyethylene glycol. Similar methods were used by Micropharm (Newcastle Emlyn, Carmarthenshire, UK) to raise and purify antibodies to a truncated version of Rpf (residues A39L115) in sheep. Immunoglobulins were conjugated to CNBr-activated Sepharose 4B (Sigma) (Osterman, 1985 ).
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RESULTS |
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To avoid problems associated with heterogeneity, we also determined whether similar non-culturable cells could be formed in the more homogeneous cultures that are produced when Mycobacterium tuberculosis is grown in the presence of a suitable detergent. To this end, cultures were grown in flasks containing ADC-supplemented Sautons medium with 0·05% Tween 80, which were sealed with a rubber cap and agitated at 200 r.p.m. Sampling was done using a syringe needle to avoid introducing oxygen from the laboratory atmosphere. Under these conditions, we also found a decrease in the c.f.u. count in the total cell population, with kinetics similar to those shown in Fig. 5(b) for filtered cells. Moreover, the culture contained many of the typical ovoid and coccoid cells after 4 months of cultivation (not shown). When such cells were plated onto agar medium c.f.u. counts of zero were obtained, even after the plates were incubated for a 2 month period (Fig. 6a
). However, in contrast to cells grown in static culture without Tween 80, this state of non-culturability was maintained for the remainder of the experimental period (48 months post-inoculation).
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Mycobacterium tuberculosis cells grown in the presence of Tween 80 were also able to resuscitate in liquid medium. Table 2 shows that 5-month-old and 6-month-old cells, which had completely lost the ability to form colonies on solid medium, were resuscitated in liquid medium. An additional 12 log increase in resuscitation was obtained in the presence of SN. These results are very similar to those observed with cells obtained by filtration of heterogeneous cultures.
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DISCUSSION |
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As we found for Micrococcus luteus, rather strict culture conditions must be satisfied to observe the transition of the majority of the cell population to a non-culturable state (Mukamolova et al., 1998 ). For R. rhodochrous, inoculum age (Fig. 1b
), medium composition, speed of flask agitation and culture volume in relation to the flask capacity significantly influenced the formation of non-culturable bacteria. However, in contrast to Micrococcus luteus, non-culturability was a transient phenomenon for R. rhodochrous cells. For each culture, there was a window of several hours during which about 99·9% of the bacteria in the culture had lost culturability. Some previously published results also show a transient decrease in the c.f.u. count when a fast-growing organism, Mycobacterium smegmatis, is held in stationary phase (Dick et al., 1998
; Smeulders et al., 1999
). The authors did not draw this specific conclusion from their data and considered this as a death phase. Recently, Keer et al. (2000)
have found that several mutants of Mycobacterium smegmatis, defective in stationary phase survival under aerobic or anaerobic conditions, revealed a transient decrease in viability followed by an increase in culturability. The authors suggest at least two possible explanations for this effect: (a) re-growth (cryptic growth) of viable cells, and (b) formation of non-culturable (and possibly dormant) bacteria in stationary phase and their subsequent resuscitation (Keer et al., 2000
, 2001
). Since the authors did not measure culturability by MPN counts, direct evidence was neither sought nor obtained for resuscitation of non-culturable cells.
In our study, we applied MPN assays to measure viability. This method permits numerical estimation of the number of resuscitatable cells in liquid medium at high dilutions, effectively circumventing the problem of re-growth of residual viable cells during resuscitation (Kaprelyants et al., 1994 ; Kell et al., 1998
). This assay revealed the presence of non-culturable cells of R. rhodochrous during a rather short time interval in stationary phase. The transient character of the loss of cell viability during stationary phase suggests that there are sequential processes occurring. First, there is a transition to a non-culturable state. This then seems to be followed by the (cryptic?) growth of residual viable bacteria, possibly accompanied by the growth of resuscitated bacteria after 8696 h of incubation. The individual contributions of (a) residual viable cells and (b) resuscitated cells to the observed growth could vary, depending on the particular set of culture conditions (inoculum age, aeration conditions, growth medium, etc.). Non-culturable cells may be formed under many different conditions, but we have found conditions where resuscitatable cells make up the majority of the bacterial population (and are therefore experimentally accessible). Several questions remain. For example, why do R. rhodochrous cultures start to lose their culturability? Why do the viable bacteria fail to grow during the period of transition to the non-culturable state, and why do they start to grow subsequently? During the transition to non-culturability, we found evidence for the presence of an inhibitory component(s) in the SN, which is no longer present once the c.f.u. counts start to increase again. Further studies are needed to elucidate the possible role of accumulation/destruction of this inhibitor in culture behaviour.
The extent of resuscitation often exceeded the proportion of active cells in the population (about 10% in all experiments, according to Rhodamine-123 and CTC staining) and in some experiments approached 100%. This fact indicates that inactive cells, with a morphology characteristic of dormancy (small coccoidal cells with enhanced phase-contrast; Mukamolova et al., 1995 ), can resuscitate. Therefore, we suggest that incubation of R. rhodochrous cells under the conditions described resulted in the formation of a population of dormant bacteria, with a characteristic non-culturable phenotype.
Mycobacterium tuberculosis held in stationary phase in capped tubes also revealed the transient formation of non-culturable cells (on a different time scale). However, because of the marked non-homogeneity of Mycobacterium tuberculosis cultures grown without detergent, filtration was required to isolate the fraction of non-culturable cells. The filtered fraction containing non-culturable bacteria may not include all such bacteria in the population; aggregates, unable to pass through the filter, may also contain such bacteria. The increase in the c.f.u. count after 4 months of incubation in stationary phase (Fig. 5b) could be due to the growth of initially viable cells, to the resuscitation of non-culturable cells, or to a mixture of both of these factors in the highly heterogeneous population. The substantially homogeneous populations grown in the presence of Tween 80 were unable to form colonies for a long period of time (Fig. 6a
). We suspect that it may also be possible to establish stable populations of non-culturable cells for R. rhodochrous in stationary phase, but the appropriate conditions for this were not identified here.
There is a substantial difference between the non-culturable cells of the two species studied, in respect of their ability to resuscitate. For R. rhodochrous, SN was needed for resuscitation (Fig. 4a), whereas significant resuscitation (up to 5 logs) of Mycobacterium tuberculosis cells occurred in liquid Sautons medium without any additions (Fig. 6b
). SN or Rpf further increased resuscitation of Mycobacterium tuberculosis cells by up to 2 logs (Fig. 6b
). These data clearly demonstrate the fact that the term non-culturability has an operational meaning only (Barer, 1997
; Kell et al., 1998
); Mycobacterium tuberculosis cells that were unable to form colonies on plates were able to grow in liquid medium, as was reported by Biketov et al. (2000)
.
As previously seen with Micrococcus luteus (Kaprelyants et al., 1994 ), SN taken from exponentially growing cells of both species studied possessed a resuscitation-promoting activity. In R. rhodochrous SN, activity was associated with a small protein, which probably belongs to the Rpf family as SN passed through an affinity column containing anti-Rpf antibodies had significantly reduced activity. Indeed, immunoblotting of this SN after SDS-PAGE revealed several protein bands that interacted with specific anti-Rpf antibodies (M.O. Shleeva, unpublished results). Moreover, Southern hybridization of BamHI-, PstI-, PvuII- or XhoI-digested R. rhodochrous DNA using a rpf-specific probe revealed the presence of four hybridizing bands (data not shown). Recombinant Micrococcus luteus Rpf stimulated resuscitation of R. rhodochrous, but it was less active than SN.
A similar conclusion could be made with respect to the active compound in Mycobacterium tuberculosis SN, as Rpf also showed stimulatory activity (although it was less active than SN; Fig. 6b). Moreover, the Rpf-like proteins of Mycobacterium tuberculosis do indeed have growth factor activity and are able to stimulate the growth of non-culturable cells of Mycobacterium bovis BCG held in prolonged stationary phase (G.V. Mukamolova, unpublished results). In addition, non-culturable cells have also been found after persistence of Mycobacterium tuberculosis in murine macrophages ex vivo. The viability of these cells was restored by incubation in the presence of Rpf in liquid medium (Biketov et al., 2000
).
Zhang and co-workers have recently reported that non-culturable cells accumulate during prolonged incubation of Mycobacterium tuberculosis H37Ra cells in stationary phase in the presence of Tween 80 (Sun & Zhang, 1999 ; Zhang et al., 2001
). In contrast to our study, their cell populations contained a significant number of colony-forming units. Slight differences in culture conditions may account for this (e.g. we maintained bacteria in capped tubes without oxygen input). They showed that SN from growing Mycobacterium tuberculosis cultures contained phospholipids, which increased the number of colony-forming units in aged cultures when added to plates and permitted growth from low inocula in liquid medium. Another active compound isolated from the SN was an 8 kDa protein, Rv1174c. Chemically synthesized peptides corresponding to three different segments of Rv1174c were added at micromolar concentrations (i.e. much higher than those at which Rpf is active) to liquid medium inoculated with old cells. After 510 days incubation, the number of recoverable colony-forming units was enhanced. We suggest that the phospholipids and micromolar concentrations of peptides employed by the authors may have promoted repair of injured cells (this may be especially true for the dried cells with zero c.f.u. counts used in this study) (Ray & Speck, 1973
). Since the authors did not measure viability by MPN, re-growth of a residual portion of viable cells cannot be excluded in some experiments and the extent of the proposed resuscitation activity cannot be quantified.
The transition of mycobacterial cells to dormancy in the Wayne model in vitro is based on the formation of non-replicating bacteria during adaptation to anaerobic conditions (Wayne, 1994 ; Wayne & Hayes, 1996
; Wayne & Sohaskey, 2001
). Protein synthesis in this state is generally reduced (Hu et al., 1998
), although certain genes, such as acr, are up-regulated (Desjardin et al., 2001
; Hu et al., 1999
; Yuan et al., 1996
). The model has been established in several different species, viz. Mycobacterium tuberculosis, Mycobacterium smegmatis and Mycobacterium bovis BCG (Dick et al., 1998
; Lim et al., 1999
; Wayne, 1994
). However, in contrast to our study, mycobacterial cells in Waynes model retain high viability and develop sensitivity to metronidazole as they become anaerobic (Wayne & Sramek, 1994
), indicating that they remain metabolically active. Therefore, they may not be considered truly dormant, but rather in a state of (O2) starvationsurvival (Kaprelyants et al., 1993
). We suggest that the difference in culturability may reflect the different times of persistence of Mycobacterium tuberculosis cells in a non-growing state (days in Waynes model and months in our study). Restoration of culturability requires resuscitation in liquid medium, which may either occur spontaneously or require the provision of compounds (e.g. growth factors) present in the SN of growing cells. The proportion of cells able to resuscitate spontaneously and those requiring growth factors may vary from one experiment to another, and resuscitation may depend on the particular conditions employed for the establishment of non-culturability.
Prolonged incubation of Mycobacterium tuberculosis cells in stationary phase may reflect more accurately the situation in vivo. In this connection we may stress that the essence of the Cornell model of dormant Mycobacterium tuberculosis is the creation of a period of sterility in vivo, induced by the administration of antibiotics after infection, when Mycobacterium tuberculosis cannot be detected by c.f.u. counts (Wayne, 1994 ) (in contrast to PCR; de Wit et al., 1995
). As shown in Fig. 7
, Waynes model may possibly represent a step towards the establishment of true dormancy in Mycobacterium tuberculosis, which is characterized by the emergence of non-culturable cells that need to be resuscitated before they can resume active growth. The necessity for anaerobic conditions to produce non-culturable cells of Mycobacterium tuberculosis in our experiments may reflect a mechanistic link between Waynes model and the model employed here. More work is required to establish whether the proposed sequence of events does indeed take place during the transition to, and reactivation from, the latent state in vivo. The observed activity of SN or Rpf with non-culturable bacterial populations suggests that one or more of the cognate Mycobacterium tuberculosis proteins could be involved in mechanisms of latency and reactivation of tuberculosis in vivo.
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ACKNOWLEDGEMENTS |
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Received 24 October 2001;
revised 10 December 2001;
accepted 8 January 2002.