Department of Biochemistry, Microbiology, and Molecular Genetics1, and Electron Microscope Facility2, University of Rhode Island, Kingston, RI 02881, USA
Author for correspondence: David R. Nelson. Tel: +1 401 874 5902. Fax: +1 401 874 2202. e-mail: dnelson{at}uri.edu
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ABSTRACT |
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Keywords: Borrelia burgdorferi, spirochaete, serum starvation, cysts, Lyme disease
Abbreviations: CSF, cerebrospinal fluid; MPN, most probable number; SSP, serum starvation protein
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INTRODUCTION |
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B. burgdorferi cells alternate between tick vectors (members of the Ixodes ricinus complex) and mammals. Therefore, these spirochaetes are adapted to survive in a number of different environments. Further, B. burgdorferi cells exhibit substantial changes in protein synthesis and gene expression and antigenicity during different stages of their zoonotic life cycle (Akins et al., 1998 ; Das et al., 1997
; Fikrig et al., 1998
; Schwan et al., 1995
; de Silva et al., 1996
; de Silva & Fikrig, 1997
). For example, two major outer-membrane proteins (OspA and OspC) are differentially synthesized within the tick host. OspA is abundant on the surface of spirochaetes within the midguts of unfed ticks (de Silva et al., 1996
) whereas OspC is more readily detected on the surface of spirochaetes in fed ticks (Schwan et al., 1995
). Additionally, several B. burgdorferi genes appear to be expressed only within infected mammals (de Silva & Fikrig, 1997
; Akins et al., 1998
; Suk et al., 1995
).
Brorson & Brorson (1997) demonstrated that cells of B. burgdorferi undergo a morphological transformation from motile spirochaetes into non-motile, spherical cyst-forms when incubated for ~4 weeks in BarbourStoennerKelly (BSKII) medium without the addition of rabbit serum, a routinely added media supplement thought to provide cells with a source of fatty acids (Barbour & Hayes, 1986
). Cyst-forms have also been observed in Lyme disease patient tissues (Hulinska et al., 1994
; Aberer et al., 1997
). It has been reported that these forms of B. burgdorferi are viable and capable of transforming back into motile spirochaetes, although survival was not accurately quantified (Brorson & Brorson, 1997
). The significance of these forms remains to be seen, but it has been suggested that cysts may represent a different stage in the life cycle of the spirochaetes or possibly play a role in human disease (Brorson & Brorson, 1997
, 1998a
, b
).
In the work described here, we studied cyst formation under defined conditions to determine: (1) the kinetics of cyst formation, (2) the viability of cyst-forms, and (3) whether cysts form as the result of a starvation-induced programme involving differential protein synthesis. Our study suggests that B. burgdorferi cells possess a complex starvation response that involves loss of motility, induction of protein synthesis, and morphological changes.
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METHODS |
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Serum starvation and recovery experiments.
Exponential-phase cells (50 ml) grown in BSKII medium were centrifuged (9000 g, 10 min) at 4 °C. Supernatants were removed and cells were resuspended in 80 ml RPMI-1640 Select-Amine (RPMI) (Life Technologies). RPMI is a defined medium containing glucose, vitamins, and all 20 of the protein amino acids. Cultures were incubated at 33 °C in 100 ml screw-cap culture bottles (Fisher) at a concentration of 1·5x107 cells ml-1. Cell density was determined by phase-contrast microscopy using a haemocytometer (Reichart).
For the recovery experiments, exponential-phase cells were prepared and incubated in RPMI as described above. Viability of cells at various time points was determined by a three-tube most-probable-number (MPN) technique (American Public Health Association, 1975 ). Briefly, serum-starved cells (1 ml) were removed from RPMI by centrifugation (14000 g, 1 min), resuspended in BSKII (1 ml), and serially diluted (serial tenfold dilutions) into BSKII. Each dilution was done in triplicate. Tubes (1 ml) were incubated at 33 oC for up to 2 weeks or until growth was observed, whichever was first. The percentage of recovery was estimated by a standard method (American Public Health Association, 1975
).
Electron microscopy.
Vegetative cells and cysts were fixed by the addition of EM-grade formaldehyde (1% final concentration) to the culture medium. Fixed samples were centrifuged at 9000 g for 5 min and resuspended in 0·5 ml 3% glutaraldehyde in 0·1 M sodium cacodylate buffer (pH 7·2). Cells were mounted on 200-mesh carbon-coated grids and negatively stained with 1% phosphotungstic acid or 0·5% ammonium molybdate. The negatively stained preparations were examined and photographed using a JEOL 1200EX transmission electron microscope.
Radioactive labelling and protein precipitation.
Exponential-phase cells (50 ml) grown in BSKII were centrifuged (9000 g, 4 °C) and resuspended as described above except that methionine was excluded in the formulation of RPMI. After the culture had been allowed to incubate at 33 °C for 5 min, 10 µCi ml-1 (370 kBq ml-1) of Tran35S-Label (specific activity 43·48 TBq mmol-1; ICN Pharmaceuticals) was added. Cells were labelled for either 2 h or up to 17 h in some cyst preparations where the low rate of incorporation required longer periods of labelling. Radioactive labelling was stopped by adding excess methionine (10 mM). Labelled cells were then centrifuged (9000 g, 10 min, 4 °C) and washed twice in 10 mM HEPES buffer (pH 7·6, 4 °C). Cells were disrupted by sonication and protein was TCA-precipitated in order to concentrate the 35S-labelled protein as described previously (Girouard et al., 1993 ; Scorpio et al., 1994
). TCA-precipitated proteins were solubilized in solubilization-reduction mixture (Laemmli, 1970
). Radioactive incorporation was determined by liquid scintillation counting of the solubilized protein.
Electrophoresis and Western blotting.
Serum-starved (48 h) or exponential-phase cells grown in BSKII were centrifuged (9000 g, 10 min) and washed three times in 10 mM HEPES buffer (pH 7·6, 4 °C). Cells were disrupted by sonication and protein concentrations in unlabelled cell extracts were estimated by the method of Bradford (1976) with a Bio-Rad protein assay kit.
One-dimensional SDS-PAGE was performed in 1·5 mm thick slab gels (5% stacking, 1013% separating) as described by Laemmli (1970) , using an SE 600 vertical electrophoresis unit. Equal protein (20 µg) was loaded in each gel lane. The separated proteins were either stained with Coomassie blue or electroblotted to nitrocellulose by a modification of the procedure described by Towbin et al. (1979)
using a Bio-Rad Transblot Cell (400 mA, 60 min) (Girouard et al., 1993
). The nitrocellulose membranes were briefly stained with 0·1% Ponceau S (Sigma) to mark the positions of molecular mass standards and then blocked in Tris-buffered saline (TBS: 50 mM Tris, 150 mM NaCl; pH 7·4) plus 2% Tween 20 (Sigma) for 20 min. The membranes were washed twice for 5 min in TBS+0·05% Tween 20. The transferred proteins were reacted overnight at 4 °C with sera from B. burgdorferi-infected rhesus monkeys (L913 or K205) (Philipp et al., 1993
; Roberts et al., 1998
), from a chronically infected Lyme disease patient (J1), or with monoclonal antibodies at the appropriate dilution (see figure legends) in TBS plus 0·05% Tween 20. Monkey sera and patient sera were generous gifts of Mario Philipp (Tulane Regional Primate Research Center, Covington, LA, USA) and Thomas Mather (University of Rhode Island), respectively. Mouse monoclonal antibodies H9724, H5332, 4B8F4 and 149, specific for flagellin, OspA, OspC and GroEL, respectively, were kindly provided by Barbara Johnson (Division of Vector-Borne Diseases, Ft Collins, CO, USA).
Proteins were separated by isoelectric focusing (IEF) using the Multiphor II system (Pharmacia) with Immobiline DryStrips (pH 310) according to the manufacturers protocol. Equal radioactive counts of 35S-labelled proteins (solubilized after TCA precipitation) were loaded onto IEF gels and gels were run to equilibrium at 20 °C. IEF gels were then incubated for 10 min in equilibrium buffer (50 mM Tris pH 8·8, 6 M urea, 30%, v/v, glycerol, 2% SDS) before being loaded onto a vertical SDS-PAGE gel. Vertical electrophoresis (the second dimension) was performed using the SE 600 apparatus as described above. Separated proteins were visualized by fluorography as previously described (Carreiro et al., 1990 ). Dried gels were exposed to Fuji RX film (Fisher) at -70 °C for 37 d. Fluorograms were analysed by laser densitometry using a Molecular Dynamics Personal Densitometer (Molecular Dynamics) and Image QuaNT v.5.0 software, as previously described (Garcia et al., 1997
).
MALDI-MS protein identification.
Following two-dimensional PAGE separation of labelled proteins and autoradiography, the protein corresponding to serum starvation protein s (SSP s) was cut from the gel and subjected to in-gel trypsin digestion and MALDI-MS protein identification as described by Williams et al. (1996) .
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RESULTS |
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Determination of viability during serum starvation
When rabbit serum or BSK was added to RPMI containing 48 h serum-starved cells, the cysts opened within 10 s to yield intact, but non-motile spirochaete cells (Fig. 2). A newly emerged spirochaete with an empty cyst is shown in Fig. 2C
. Cysts did not open when 20% sucrose was added to the culture. Cells began to regain motility 1215 h after emerging from the cysts. Since RPMI+S is not a growth medium for B. burgdorferi, viability experiments were performed by MPN determinations in BSKII as described in Methods. The data presented in Table 1
show that B. burgdorferi cells could be grown from cultures starved for serum for up to 8 d. Cells starved for 2 weeks were not recoverable even after >30 d incubation in BSKII. In contrast, no viable cells could be recovered by 5 d from cultures incubated in RPMI+S, nor could viable cells be recovered from cultures starved in HEPES buffer after 1 d (Table 1
). It is important to note that spirochaetes incubated in HEPES buffer or distilled water formed round cells that clumped together. These structures resembled spheroplasts and were repeatedly demonstrated to be nonviable by MPN determinations.
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DISCUSSION |
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It has previously been reported that B. burgdorferi spirochaetes develop into cysts in vitro when they are incubated either in BSKII-S for several weeks (Brorson & Brorson, 1997 ) or in CSF (37 °C) for 24 h (Brorson & Brorson, 1998a
). We began our study by observing cyst formation in BSKII-S. We estimate that only 3050% of the cells transform into cysts after 4 weeks. We hypothesize that cells form cysts in response to starvation for serum or the abundant fatty acids and lipids in serum. Slow formation of cysts in BSKII-S is probably due to the presence of some fatty acids or lipids in the large amount of BSA (50 g l-1) in the medium. We thought that elimination of all fatty acids and lipids from the medium would increase both the rate of cyst formation and the percentage of cells which undergo the transformation. Using RPMI without the addition of serum, cells become non-motile within a few hours and 90% of vegetative spirochaetes form cysts within 48 h. A cyst appears to contain an intact, coiled non-motile spirochaete within a membrane (Fig. 1
). It is unclear whether the membrane is part of the original outer membrane of the vegetative cell or whether it is constructed during cyst formation.
Cyst formation is an active cellular response to serum starvation. The addition of tetracycline inhibits cyst formation, demonstrating that cyst formation requires protein synthesis and that cysts are not merely degenerative forms. Further, the temporal pattern of protein synthesis during cyst formation strongly suggests that encystment is a global response to a specific starvation (see Fig. 3 and Table 2
). Additionally, cysts are able to survive longer than B. burgdorferi cells incubated in either similar media plus serum or buffer alone (Table 1
). In contrast to a recent report that describes rapid cyst formation in distilled water (Brorson & Brorson, 1998b
), we did not observe the formation of viable cysts in HEPES or distilled water. We did observe that B. burgdorferi incubated for more than 1 h in distilled water or HEPES buffer formed rounded, clumping, spheroplast-like cells, but these structures were consistently shown to be nonviable by quantitative methods. When serum or BSK is added to these types of cell structures they appear to rupture, releasing cytoplasmic contents, unlike the emergence of intact spirochaete-shaped cells that we observed (Fig. 2B
, C
).
Morphological changes by B. burgdorferi cells in response to adverse environmental conditions have been described by others (Barbour & Hayes, 1986 ; Brorson & Brorson, 1997
, 1998a
; Burgdorfer & Hayes, 1989
; Kersten et al., 1995
; Preac Mursic et al., 1989
). For example, the formation of vesicles or blebs and gemmae has been shown to occur when cells are exposed to physiological stress such as changes in pH, depletion of metabolites, and ageing (Burgdorfer & Hayes, 1989
) or exposure to antibiotics (Kersten et al., 1995
; Preac Mursic et al., 1989
). Little is known about the physiological role of these forms, although blebs and gemmae have been shown to contain DNA (Garon et al., 1989
) and may be involved in the exchange of genetic information. Unlike cyst-forms, blebs and gemmae have not been shown to be viable, capable of transforming back into motile, vegetative cells. We have also observed blebs and gemmae in our cultures, particularly when cells are exposed to antibiotics and extreme pH stress, but it is clear that these vesicles are not cysts.
In addition to loss of motility and cyst formation, B. burgdorferi cells responded to serum starvation by inducing the synthesis of a number of proteins (Table 2). Using monoclonal antibodies to well-characterized antigens, we identified one of the starvation-induced proteins (SSP o; see Table 2
). It appears that SSP o (OspA) synthesis increases about twofold when compared to cells incubated in RPMI+S. The upregulation of OspA during serum starvation is not surprising since OspA is the major outer-membrane protein detected in unfed ticks. Additionally, it has been suggested that OspA and OspC are inversely regulated by nutrient availability (Das et al., 1997
; Schwan et al., 1995
). However, an increased accumulation of OspA was not detected by Western blot analysis (Fig. 4
). This may be due to either increased turnover of OspA during cyst formation or the loss of OspA-containing membrane during cyst formation (Fig. 2
). We successfully identified another SSP (SSP s) by MALDI-MS as a homologue of VlsE, a protein shown to be involved in antigenic variation of B. burgdorferi cells (Zhang et al., 1997
). VlsE and its role in starvation-induced forms warrant further study. It is tempting to speculate that some of the proteins upregulated during serum starvation regulate this response.
Bacteria have evolved elaborate strategies that enable them to survive periods of starvation (Siegele & Kolter, 1992 ). Examples of morphological changes that occur in response to starvation include the formation of endospores (Losick & Youngman, 1984
), ultramicrocells (Kjelleberg et al., 1987
) and fruiting bodies (Kaiser, 1984
). Bacterial cells have also been shown to alter fatty acid composition, surface properties and cell walls in response to starvation (Kjelleberg et al., 1987
; Nyström & Kjelleberg, 1989
). Further, the carbon-starvation response of Vibrio sp. strain S14 has been shown to be dependent upon a spoT homologue (Ostling et al., 1996
). As in B. burgdorferi cyst formation, the Vibrio S14 carbon-starvation response (or ultramicrocell formation) involves: (1) loss of motility, (2) change in morphology to a spherical form, and (3) expression of a number of genes. Burgdorfer & Hayes (1989)
suggested that cyst formation of B. burgdorferi cells may be part of a complex developmental cycle. An adaptive response by B. burgdorferi to periods of starvation would seem to be essential for an organism that lacks critical biosynthetic pathways and depends largely on its environment for metabolites.
The response of bacteria to fatty acid starvation is not well known, as most bacteria studied are capable of fatty acid synthesis. Escherichia coli responds to fatty acid starvation by accumulating spoT-dependent guanosine tetraphosphate (ppGpp) and inhibiting stable rRNA synthesis (Seyfzadeh et al., 1993 ). ppGpp is an effector molecule involved in the physiological response of some bacteria to nutritional stress (Cashel & Rudd, 1987
; Nyström, 1993
). The SpoT protein is a bifunctional enzyme involved in the degradation and synthesis of ppGpp in bacteria (Cashel & Rudd, 1987
). Interestingly, the genome of B. burgdorferi contains a putative spoT gene (Fraser et al., 1997
). We are currently investigating the possibility that the putative spoT is involved in the regulation of the serum-starvation-induced proteins described in this study.
Cysts have been observed in vitro in human CSF (Brorson & Brorson, 1998a ) and in vivo in the tissues of Lyme-disease-infected humans (Aberer et al., 1997
; Hulinska et al., 1994
). It is not known whether the cysts observed in those studies are the same as the cysts described in this report. However, if viable cysts form in the body, they may represent a strategy that facilitates the survival of B. burgdorferi cells during nutritionally adverse conditions in host tissues. By forming cysts, it is also conceivable that B. burgdorferi cells evade detection by the immune system. We have not yet investigated the surface properties of cysts; thus, the clinical significance of these forms is not clear. However, Hulinska et al. (1994)
demonstrated that the surface of cyst-forms found in the erythema chronicum migrans of infected-human tissue was non-reactive to antibodies against OspA, whereas the content of the cysts did react with OspA. It would be interesting to determine whether the surfaces of cysts described in this study are antigenically different from the surface of vegetative cells.
This study presents data describing the physiological response of B. burgdorferi to a specific starvation. Our data suggest that B. burgdorferi cells, although possessing a small genome and extremely limited biosynthetic capabilities, rapidly respond to conditions of serum starvation by inducing a programme that involves loss of motility, change in morphology, and rapid induction of proteins.
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ACKNOWLEDGEMENTS |
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Received 22 June 1999;
revised 13 September 1999;
accepted 28 September 1999.