Incorporation of cysteine by Borrelia burgdorferi and Borrelia hermsii



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Institute of Microbiology, University of Bologna, S. Orsola Hospital, via Massarenti 9, 40138 Bologna, Italy Received August 15, 1991 Revision received March 3, 1992 Accepted March 9, 1992 SAMBRI, V., and CEVENINI, R. 1992. Incorporation of cysteine by Borrelia burgdorferi and Borrelia hermsii. Can. J . Microbiol. 38: 1016-1021. The growth rate of Borrelia burgdorferi and Borrelia hermsii in BSK I1 medium prepared with cysteine-free or cysteinecontaining (0.185-5.92 mM) CMRL 1066 medium was studied. In media with cysteine-free CMRL 1066, growth of borreliae was detectable, although it was reduced by approximately 80%. Bacterial growth was maximal when the concentration of cysteine in CMRL 1066 reached 1.48 mM, which represents the standard cysteine concentrations of the medium; higher concentrations inhibited the growth of borreliae. Cysteine incorporation, measured by the uptake of radiolabeled cysteine, showed that cysteine enters B. burgdorferi and B. hermsii cells by passive diffusion. Labeling studies of borreliae with [35~]cysteine indicated that B. burgdorferi has several cysteine-containing proteins, including ones at 22, 30 (OspA), and 34 kDa (OspB), whereas B. hermsii showed only two [35~]cysteine-incorporating proteins, at 22 and 24 kDa, which were exposed onto the outer cell surface. In addition, most of the c steine-incorporating proY teins could be biosynthetically radiolabeled when bacterial cells were grown in vitro with [ Hlpalmitate, and the differences in cysteine incorporation observed between B. burgdorferi and B. hermsii were found to be correlated with differences in lipoproteins. Key words: Borrelia burgdorferi, Borrelia hermsii, cysteine, cysteine uptake, lipoproteins, outer surface proteins. SAMBRI, V., and CEVENINI, R. 1992. Incorporation of cysteine by Borrelia burgdorferi and Borrelia hermsii. Can. J . Microbiol. 38: 1016-1021. On a ktudie le taux de croissance de Borrelia burgdorferi et de Borrelia hermsii dans le milieu BSK I1 prepark avec du milieu CMRL 1066 dkpourvu de cystkine ou avec cysteine (0,185 a 5,92 mM). Dans le milieu CMRL 1066 sans cystkine, Borrelia peut se dkvelopper mais sa croissance est rkduite d'environ 80%. La croissance bactkrienne est maximale lorsque le milieu CMRL 1066 contient 1,48 mM de cystkine, ce qui correspond a la concentration de cystkine standard du milieu. Des concentrations de cystkine plus elevkes inhibent la croissance. L'incorporation de la cystkine a kt6 mesurke d'aprks la captation de cystkine marquke avec une substance radioactive. I1 semble que la cystkine pknktre dans les cellules de B. burgdorferi et de B. hermsii par diffusion passive. Ces ktudes par marquage chez Borrelia avec la [35~]cystkine indiquent que chez B. burgdorferi on retrouve de la cystkine incorporke dans quelques protkines de 22, 30 (OspA) et de 34 kDa (OspB) alors que chez B. hermsii la [35~]cystkine est incorporke dans seulement deux protkines de 22 et de 24 kDa qui sont localisees sur la surface externe. La majoritk des protkines qui incorporent de la cystkine peuvent 2tre radiomarqukes lors de la biosynthkse si les cellules bactkriennes sont cultivkes in vitro avec du [3~:]palmitate. Les diffkrences observees entre B. burgdorferi et B. hermsii dans l'incorporation de la cystkine sont en corrklation avec des diffkrences dans les lipoprotkines. Mots clks : Borrelia burgdorferi, Borrelia hermsii, cystkine, captation de la cystkine, lipoprotkines, protkines de la surface externe. [Traduit par la rkdaction]

Introduction Borrelia burgdorferi and Borrelia hermsii, which belong to the genus Borrelia in the bacterial order of Spirochetales (Holt 1978; Kelly 1978), are helical-shaped bacteria transmitted to humans by tick bite. Borrelia burgdorferi is the causative agent of Lyme disease (Burgdorfer et al. 1982; Steere et al. 1983; Steere 1989); B. hermsii causes the disease known as American tick-borne relapsing fever (Burgdorfer 1985). The nutritional needs for ,these host-associated parasites are complex. In the past, continuous propagation of borreliae mainly involved the use of animal sera and protein-rich fluids, until Kelly formulated a fully defined medium (Kelly 1971). Stoenner enriched Kelly's basic formulation by using Yeastolate (Difco, U.S.A.) and CMRL 1066 (Parker et al. 1957), a tissue culture medium containing amino acids, vitamins, and other growth factors (Stoenner 1974). Among the ingredients of CMRL 1066 is the amino acid L-cysteineHCl at 1.48 mM. ' ~ u t h o rto whom all correspondence should be addressed. Printed in Canada / Imprime au Canada

A medium enriched in L-cysteine is used for the growth of organisms belonging to the genus Legionella (Feeley et al. 1978), and deprivation of cysteine has been shown to interfere with the transformation of chlamydiae from reproductive to infective form (Stirling et al. 1983; Newhall 1987). Labeling studies with ["~]c~steineindicated that cysteine-containing proteins are present on the outer membrane of chlamydiae (Newhall and Jones 1983; Hatch et al. 1984) and legionellae (Butler et al. 1985). Because spirochetes of the genus Borrelia require CMRL 1066, which contains the amino acid L-cysteine in a considerable amount, as a basic component for the unique medium able to allow optimal growth conditions (Barbour 1984), the aim of this study was to evaluate the effect of varying cysteine concentrations on the growth of borreliae, and the uptake and incorporation of this amino acid in Borrelia cells.

Materials and methods Bacterial strains and culture Borrelia burgdorferi strain IRS (ATCC 3521 1) (Barbour et al. 1983; Sambri et al. 1991a) and B. hermsii strain HS-1 (ATCC


- B. burgdorferi

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B. herrnsii


~ ~ y s t e i nconcentration, e mM

FIG. 1. Effect of L-cysteine on growth of B. burgdorferi and B. hermsii. Borreliae were grown for 7 days in BSK I1 medium prepared with CMRL 1066 lacking cystine and methionine, and containing increasing concentrations of cysteine.

35209) (Stoenner et al. 1982) were used. Borrelia burgdorferi and B. hermsii were passed in vitro several times before being used for these experiments. Borreliae were grown in modified Barbour-Stoenner-Kelly medium (BSK 11), prepared using CMRL 1066 (Biochrom KG, Berlin, Germany) (Barbour 1984) as previously described (Sambri and Lovett 1990). Briefly, an inoculum of lo6 spirochetes, determined with the Petroff-Hausser counting chamber (Johnson et al. 1987), was added to each 250-mL plastic bottle, containing 150 mL of BSK 11, and incubated at 34°C for 5 days.

37"C, p H 7.4, for 1 h. Experiments were stopped at fixed times (5, 15, 30, 45, and 60 min) throughout the study. Some experiments were carried out either at 4°C or in the presence of 50 mM sodium arsenate (Sigma, U.S.A.). After the desired incubation time intervals, transport was terminated by centrifugation at 15 000 x g for 20 min at room temperature. One hundred microlitres of supernatant and the pelleted bacteria were washed twice in phosphate-buffered saline (PBS) (6000 x g for 60 min at 4"C), applied directly to 0.45-pm cellulose acetate membrane filters, and counted for radioactivity after addition of Ready-Solv E P (Beckman, U.S.A.), using a Packard 2000 CA liquid scintillation analyzer.

Cysteine loading and starvation Borrelia burgdorferi and B. hermsii were grown in BSK I1 medium prepared with CMRL 1066 lacking cysteine, cystine, and methionine and containing 6 % normal rabbit serum "trace hemolyzed" (Pel-Freez, Rogers, Ark., U .S.A.) (Barbour 1984). To determine the minimum concentration of cysteine allowing maximal growth of borreliae, glass tubes (three for each concentration of amino acid) containing BSK 11, made with CMRL 1066 with increasing millimolar concentrations (0.0-5.92 mM) of cysteine (L-cysteine.HC1, Merck, Germany) and lacking cystine and methionine, were inoculated with 1 x 10' live microorganisms. After 7 days incubation the number of viable bacteria was scored for each tube, as described above. Analogous experiments were performed by adding cystine (0.0-0.08 mM) or methionine (0.00.1 mM) alone or in combination (keeping one of the two substance at the usual concentration contained in CMRL 1066 and raising the other one) to BSK I1 made with CMRL 1066 lacking the above amino acids.

Expression of cysteine uptake data Results were expressed as micromoles cysteine uptake per minute per microgram ( x l o p 3 ) bacterial protein. The experiments were performed in triplicate and the results from different experiments pooled together by plotting the bound fractions versus time. From the known specific activity of the substrate and disintegrations per minute of either the extracellular fluid (unbound) or bacteria (bound cysteine), uptake of cysteine per microgram protein was calculated for each time point; the slope of the linear regression of uptake versus time was uptake velocity in micromol per minute per microgram protein for each concentration of cysteine. Uptake velocities were plotted against dose, to get a full dose-response study. Because the uptake was linearly related to dose over the whole range of concentrations observed, kinetics parameters for uptake fitting a classical saturation kinetics, i.e., Michaelis-Menton kinetics (active transport), were not considered.

Cysteine incorporation measurements We considered cysteine uptake as the binding of cysteine to bacterial cell membranes, irrespective of such following intracellular events as incorporation into proteins, which is discussed below. Uptake of radiolabeled cysteine was measured by an incubation technique (Hardison et al. 1988). Incubations were initiated by the addition of 2 x 10' bacteria to 1 mL of BSK I1 medium prepared with cysteine-free CMRL 1066 medium, to which had been added 15 pCi (1 Ci = 37 GBq) of ~ - [ ~ ' ~ ] c ~ s t (>HI0 e i n e Ci/rnmol; NEN Du Pont, U.S.A.) together with from 0.185 to 1.48 pmol unlabeled L-cysteine (Merck, Germany). The incubation was carried out at

SDS-PA GE Sodium dodecyl sulphate - polyacrylamide gel electrophoresis (SDS-PAGE) was performed following the method of Laemmli (1970) as previously described (Cevenini et al. 1987). Briefly, a 12.5% (w/v) concentrated acrylamide (Bio-Rad Laboratories; U.S.A.) separating gel was loaded with the samples dissolved in solubilization buffer (0.1 % bromophenol blue (Merck, Germany), 2% SDS (BDH, U.K.), 10% glycerol (Carlo Erba, Italy), 0.1 M Tris-HC1 (Sigma, U.S.A.). In some experiments, 10% (v/v) 2-mercaptoethanol (ME) (Merck, Germany) was added t o the solubilization buffer. Electrophoresis was run at 20 mA, constant

CAN. J. MICROBIOL. VOL. 38, 1992

- B. burgdorferi

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- - - 0. hermsii

L-cysteine concentration, mM FIG. 2. Uptake of L-cysteineby B. burgdorferi and B. hermsii. Bacteria were incubated in the presence of increasing concentrations of the amino acid. Results are expressed as micromoles of cysteine uptake per minute per microgram ( x l o p 3 )of bacterial protein.

Results In BSK II prepared with cysteine-free C M 1066 ~ the ~ final cell yield of borreliae (Fig. was greatly inhibited. In fact, dark-field microscopy showed 85.4 and 78.3% A utoradiography reduction of viable bacteria, respectively, for B. hermsii and Autoradiography was used to demonstrate either [35~]cysteine B. burgdorferi, when grown in BSK I1 medium prepared and 14c-labeled amino acid or [3~]palmitate incorporation into with cysteine-free CMRL 1066 compared with the same proteins of borreliae, as previously described (Cevenini et al. medium containing 1.48 mM cysteine. The addition of 1991a). Tritiated palmitate used to label borreliae was dried under cysteine at concentrations of 2.96 and 5.92 mM severely nitrogen and suspended in a small volume of 95% sterile ethanol. inhibited the growth of borreliae. A cysteine concentration Final ethanol concentration was not more than 0.5%. Briefly, higher than 5.92 mM also caused a notable decrease of the ~ - [ ~ ' ~ ] c ~ s t and e i n eI4c-labeled amino acid mixture ( > 50 mCi/ pH of BSK I1 medium. When cystine (from 0.0 to 0.08 mM) mmol; Amersham Co., U.K.) or [9,10-(n)-~~]~almitic acid (speand methionine (from 0.0 to 0.1 mM), alone or in combinacific activity 53 Ci/mmol, Amersham Co., U.K.) was added to an initial culture of spirochetes (0.25 x 10' bacteria) at a concentration, were substituted for cysteine in CMRL 1066, the numtion of 5, 1, and 250 pCi/mL, respectively. When the number of ber of viable organisms was comparable with that found in spirochetes in the culture reached 2 x 1 0 ' / m ~ , the bacterial cultures grown in cysteine-free CMRL 1066. suspension was spun down in a Sorvall (Du Pont Co., U.S.A.) Because the uptake rate of labeled cysteine into borreliae RC-5B centrifuge at 18 000 rpm, using a model SS-34 rotor for cells at cysteine concentrations less than 0.1 mM was too 20 min at 20°C; the resulting pellet was washed in 0.01 M sodium low to provide reliable results, incorporation experiments phosphate buffer, containing 0.15 M NaCl, pH 7.2 (PBS), and were performed with higher concentrations. At low concenstored frozen at - 70°C until use. trations growth yield cannot be correlated with uptake. In After electrophoresis, gels were prepared for autoradiography the range of concentrations ,that did not prove toxic to the by incubation in 150 mL of Enlightning (NEN - Du Pont, U.S.A.) cells (i.e., up to 5.92 mM), the uptake rate of cysteine was for 30 min before drying under vacuum at 80°C. Fluorograms were obtained by exposing for 72 h, using a Kodak X-Omat AR film linear with dose for both B. burgdorferi and B. hermsii (Eastman-Kodak Co., U .S.A.) with intensifying screens at - 70°C, (Fig. 2), indicating that cysteine is entering bacteria by and developed following standard procedure. passive diffusion. The falloff of uptake at cysteine concentrations higher than 5.92 mM was not considered in setting Immune-ascites production up the dose-response curve owing to the altered conditions The production of mouse immune ascites containing monoof the cells. The uptake rates were independent of both low specific polyclonal antibodies against the 22- and 24-kDa proteins of B. hermsii has been previously described (Cevenini et al. 1991b). temperature (4OC) and presence of 50 mM sodium arsenate (data not shown). The uptake rates were similar for the two Surface immunofluorescence assay (SIFA) species of bacteria, being the slope of the linear component Immunoglobulin binding to strain IRS and strain HS-1 cell sur(b = (pmol-min-' .pg x lo-' p r o t e i n - ' / m ~ ) did not faces was assessed by using living bacteria, mouse monospecific differ significantly between B. burgdorferi (b = 0.1 1) and polyclonal antibodies, and fluorescein-conjugated rabbit antimouse B. hermsii (b = 0.086). To determine whether the compoglobulins (Dako, Copenhagen, Denmark), as previously described (Sambri et al. 1991b). sition of the cellular protein reflected the uptake of cysteine, current, until the dye front had migrated 12 cm into the separating gel. ~ e l were s fixedin 7% acetic acid, 30% methanol (v/v)-aqueoui solution, then stained with Coomassie Brilliant Blue (0.025% (w/v) solution, Sigma, U.S.A.) or processed for autoradiography.

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proteins of borreliae grown in the presence of [35~]cysteine were separated by SDS-PAGE and analyzed by autoradiography (Fig. 3). At least 10 proteins incorporating cysteine were found among B. burgdorferi polypeptides, the most prominent being proteins with molecular mass 53-58, 34 (OspB), 30 (OspA), and 22 kDa (Fig. 3, lane 4). Only two cysteine-containing proteins, with molecular mass 22 and 24 kDa, were found in B. hermsii (Fig. 3, lane 2). Comparison of the polypeptide profiles of whole cells of B. burgdorferi and B. hermsii intrinsically radiolabeled with 14c-labeled amino acids and [)'s] cysteine showed the cysteine-rich nature of the 22-kDa protein of B. burgdorferi (Fig. 3). The presence (10% v/v) or the absence of 2-mercaptoethanol in solubilization buffer and heating at 100°C of preparations did not modify the electrophoretic motility of the cysteinecontaining proteins (data not shown). The incorporation of [ 3 ~ ~ p a l m i t ainto t e B. burgdorferi and B. hermsii proteins was also performed. Radiolabeled proteins were separated by SDS-PAGE and analyzed by autoradiography. Whole B. burgdorferi and B. hermsii cells incorporated ~ ~ ~ j ~ a l r n i respectively, tate, into at least seven and nine proteins (Fig. 4), in particular into the 30- (OspA) and 34-kDa (OspB) proteins of B. burgdorferi and the 22- and 24-kDa of B. hermsii. Experiments by SIFA, with mouse monospecific polyclonal antibodies active with the 22- and 24-kDa proteins of B. hermsii HS-1 strain, showed that these proteins were exposed onto the surface of viable B. hermsii cells. Discussion Previous studies have demonstrated that BSK I1 medium is, as far as is known, the medium that allows the best conditions for the growth of both B. hermsii and B. burgdorferi (Barbour 1984). The results of the present study show that a 1.48 mM cysteine concentration in the CMRL 1066 medium, which is a component of BSK I1 medium, is both optimal and critical for Borrelia growth, a closely concentration dependent growth rate being observed: in cysteinefree media, Borrelia growth rate was actually inhibited; it was slightly promoted by increases in cysteine concentrations from 0.046 to 0.74 mM; it reached the maximum at concentrations of cysteine as high as 1.48 mM. Further increases in cysteine concentrations severely inhibited growth of the borreliae. It is known that cysteine may be toxic to or may inhibit the growth of bacteria (Carlsson et al. 1979; Himelbloom and Hassan 1986), fungi (Bhuvaneswaran et al. 1964), and yeasts (Allen and Hussey 1971). For borreliae, this amino acid seems to be both required and deleterious, and consequently is an important limiting factor for the bacterial growth. The rate of incorporation of cysteine by Borrelia cells was linear with the amino acid concentration in the growth medium, and was not modified by low temperature or sodium arsenate, which depletes ATP reserves and also reduces the proton-motive force. Therefore, the uptake of cysteine by borreliae, at least within an amino acid concentration ranging from 0.185 to 5.92 mM in CMRL 1066 medium, does not support the concept of an active carrier-mediated process (Kay and Gronlund 1969), at least at cysteine concentrations in the medium as high as those investigated in the present study. The occurrence of passive together with active uptake of cysteine has been reported

FIG. 3. SDS-PAGE protein profile of B. hermsii (strain HS-1) and B. burgdorferi (strain IRS) labeled with I4c-labeled amino acid mixture (lanes 1 and 3, respectively) and [35~]cysteine-labeled B. hermsii strain HS-1 (lane 2) and B. burgdorferi strain IRS (lane 4). The 22-kDa protein of B. burgdorferi was preferentially radiolabeled with [35~]cysteine compared with the I4c-labeled amino acid mixture, indicating that it is cysteine rich. Lane 5 contains molecular weight markers.

FIG. 4. Comparison of proteins from B. hermsii (lane 1) and B. burgdorferi (lane 2: + , OspA; , OspB) biosynthetically labeled with [3~]palmitate.Proteins were separated by SDSPAGE and visualized by fluorography. Lane 3 contains the molecular weight markers. in Lactobacillus sake (Shay et al. 1988). In the range of cysteine concentrations used in the present investigation, passive diffusion is the driving force for cysteine incorporation into cells. However, it is possible that at lower cysteine concentrations, an active incorporation helps passive dif-

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fusion; at increasing cysteine concentrations, the former being saturated, the latter becomes by far the predominant. The dramatic decrease in Borrelia growth rate observed beyond the optimal 1.48 mM cysteine concentration in the medium agrees well with the concept of passive diffusion, since Borrelia cells seem unable to regulate the intracellular cysteine concentration. The membrane permeability of the two Borrelia species, moreover, seems quite similar, as inferred by the similar incorporation rates of cysteine in both B. burgdorferi and B. hermsii. In contrast, there was an interesting difference in the pattern of cysteine incorporation into polypeptides between the two species: B. burgdorferi possessed several cysteineincorporating proteins, whereas B. hermsii showed only two, at 22 and 24 kDa. Previous studies (Burgdorfer 1985) have reported a difference in polypeptide composition between B. burgdorferi and B. hermsii, in particular demonstrating the presence of outer surface proteins with different molecular masses. It is noteworthy that in our study the most prominent cysteine-containing proteins were the 30- (OspA) and 34-kDa (OspB) polypeptides for B. burgdorferi and the 22- and 24-kDa polypeptides for B. hermsii. These latter also proved to be exposed onto the surface of bacterial cells. Brandt et al. (1990) demonstrated that the OspA and OspB proteins of B. burgdorferi are lipoproteins. We showed that the two major cysteine-incorporating proteins of B. hermsii, which are respectively 22- and 24-kDa proteins, could be radiolabeled during in vitro incubation . we did not of the organisms with [ ' ~ l ~ a l m i t a t eAlthough perform biochemical analysis of these proteins, it is reasonable that these molecules contain covalently attached fatty acids since they remained radiolabeled after boiling in SDS and electrophoresis in SDS-polyacrylamide gels. These results indicate that the differences in cysteine incorporation observed between B. burgdorferi and B. hermsii are correlated with differences in lipoproteins and that the cysteine site of the proteins of B. burgdorferi and very likely of B. hermsii are lipidation sites. Acknowledgements This work was supported in part by a grant (4243/ 1991) from Regione Emilia-Romagna. The bacterial strains were a gift from Michael A. Lovett, M.D., Ph.D., UCLA, Los Angeles, U.S.A. The authors thank Mr. Enzo Della Bella and Miss Francesca Massaria for their technical work. Allen, E.H., and Hussey, G.G. 1971. Inhibition of the growth of Helminthosporium carbonum by L-cysteine. Can. J. Microbiol. 17: 101-103. Barbour, A.G. 1984. Isolation and cultivation of Lyme disease spirochetes. Yale J. Biol. Med. 57: 521-525. Barbour, A.G., Burgdorfer, W., Hayes, S.F., et al. 1983. Isolation of a cultivable spirochete from Ixodes ricinus ticks of Switzerland. Curr. Microbiol. 8: 123-126. Bhuvaneswaran, C.A., Sreenivasan, C.A., and Rege, D.V. 1964. Effect of cysteine on respiration of catalase synthesis by Saccharomyces cerevisiae. Biochem. J. 92: 21 9-243. Brandt, M.E., Riley, B.S., Radolf, J.D., and Norgard, M.V. 1990. Immunogenic integral membrane poteins of Borrelia burgdorferi are lipoproteins. Infect. Immun. 58: 983-991. Burgdorfer , W. 1985. Borrelia. In Manual of clinical microbiology. 4th ed. Edited by H.E. Lennette, A. Balows, W.J. Hausler, Jr., and H.J. Shadomy. American Society for Microbiology, Washington, D.C. pp. 479-484.

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Incorporation of cysteine by Borrelia burgdorferi and Borrelia hermsii.

The growth rate of Borrelia burgdorferi and Borrelia hermsii in BSK II medium prepared with cysteine-free or cysteine-containing (0.185-5.92 mM) CMRL ...
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