Department of Entomology, University of
Minnesota, St. Paul, Minnesota 55108,1 and
Max von Pettenkofer-Institut für Hygiene und Medizinische
Mikrobiologie, Ludwig-Maximilians-Universität München,
D-80336 Munich, Germany2
Received 21 August 1998/Returned for modification 28 September
1998/Accepted 23 March 1999
 |
INTRODUCTION |
The Lyme disease spirochete
Borrelia burgdorferi sensu stricto cycles in nature
between small mammals and Ixodes ticks. This ability to
invade and infect two physiologically quite divergent hosts involves
alterations in the outer surface protein (Osp) composition of B. burgdorferi, most notably, outer surface protein A (OspA)
and outer surface protein (OspC). These changes occur while
spirochetes are being transmitted from the tick to the mammalian host.
Factors such as temperature (7, 29-31) and blood meal (23, 29) have been demonstrated to influence synthesis of several B. burgdorferi proteins. One of the most notable
effects is an increase in OspC production and a decline in OspA
production in spirochetes during nymphal attachment and feeding
(6, 8-10, 25, 29). This differential expression of OspA and
OspC during tick feeding coincides with an increase in the infectivity
of spirochetes for the mammalian host (23, 24). OspA has
been shown to be expressed in significant amounts in most B. burgdorferi strains in culture (2, 4, 10, 17,
32). An increase in temperature enhanced the expression of
OspC in spirochetes cultured in BSK medium, whereas no effect on
OspA was noted (7).
The aim of this research was to compare OspA and OspC expression in
B. burgdorferi sensu stricto cocultivated with tick
(Ixodes scapularis) cells with those cultivated
axenically in BSK-H medium. We also examined how temperature
affects the expression of OspA and OspC in B. burgdorferi in the two culture systems. Here we report that
temperature modulates the expression of both OspA and OspC in B. burgdorferi cocultivated with I. scapularis cells.
(This work is part of the Ph.D. dissertation of Marygorret Obonyo.)
| |
MATERIALS AND METHODS |
Spirochete strains and cultivation.
Strains JMNT (14,
22) and N40 (3) of B. burgdorferi
sensu stricto (passage 3), previously maintained at 34°C, were stored
frozen in liquid nitrogen. Before each experiment, frozen spirochetes
were thawed at 37°C, transferred to complete BSK-H (Sigma, St. Louis,
Mo.) medium supplemented with 6.3% rabbit serum (Gibco, Grand Island,
N.Y.) and 1.4% gelatin (Difco, Detroit, Mich.), and incubated at
34°C for 4 days prior to cocultivation with tick cells or axenic
cultivation in BSK-H medium.
I. scapularis tick cell line ISE6 (18) was
used. The line was maintained in L15B (19) medium containing
5% heat-inactivated fetal bovine serum (Sigma), 10% tryptose
phosphate broth (Difco), and 1% lipoprotein cholesterol concentrate
(ICN Pharmaceuticals, Inc., Costa Mesa, Calif.). Cells, seeded into
24-well tissue culture plates (1 ml/well) or 25-cm2 tissue
culture flasks (5 ml/flask), were grown at 31°C until the cell
density reached 2.5 × 106 cells/ml (usually by 7 days). Spirochetes, previously grown in BSK-H medium, were counted with
a Petroff-Hausser bacterial counting chamber (Hausser Scientific,
Horsham, Pa.) and were diluted in L15BS medium (13, 19)
(1.25 × 107 borreliae/ml). The spent tick cell
culture medium (L15B) was replaced with an equivalent volume of
spirochetes in L15BS medium (13, 19), and cultures were
incubated at 31, 34, or 37°C. For comparison, spirochetes cultured
axenically were diluted to 105 spirochetes/ml in fresh
complete BSK-H medium and were incubated at 31, 34, or 37°C.
Microscopic evaluation.
Cells were suspended with a Pasteur
pipette, centrifuged onto glass microscope slides (Shandon Southern
Instruments, Pittsburgh, Pa.), air dried, and fixed in methanol. Some
slides were stained with 4% Giemsa (Gibco) solution and others were
used for indirect fluorescent-antibody (IFA) microscopy. For IFA
microscopy, cells were incubated with monoclonal antibody (MAb) H5332
(2) (provided by Tom Schwan, Rocky Mountain Laboratories,
National Institutes of Health) at 37°C for 30 min. Thereafter, slides
were washed once in phosphate-buffered saline (PBS) containing 5 mM
MgCl2 and were then reacted for 30 min at 37°C with
rhodamine-conjugated anti-mouse immunoglobulin G (Pierce, Rockford,
Ill.). The slides were washed once in PBS containing MgCl2
(5 mM) and were examined by fluorescence microscopy.
SDS-PAGE and immunoblotting.
Spirochetes grown axenically in
BSK-H medium at 31°C were harvested after 7 days, and those grown at
34 or 37°C were passaged into fresh medium when they reached the
mid-logarithmic phase of growth (4 days) and were harvested 3 days
later. Spirochetes were counted with a bacterial counting chamber and
were harvested by centrifugation (3,000 × g) for 30 min at 4°C. The resulting pellet was resuspended in Hank's balanced
salt solution (HBSS) and was recentrifuged (3,000 × g
for 30 min at 4°C). After a second wash with HBSS and a second round
of centrifugation, pelleted spirochetes were resuspended in 2× sodium
dodecyl sulfate (SDS) reducing buffer and were lysed by boiling for 5 min. Spirochetes grown with ISE6 cells in 25-cm2 tissue
culture flasks were suspended by pipetting and were separated from ISE6
cells by centrifugation (170 × g) at ambient
temperature for 5 min; those spirochetes remaining in suspension were
counted and were harvested as stated above for spirochetes cultured
axenically in BSK-H medium.
Whole-cell lysates (2 × 106 to 4 × 106 spirochetes/lane) were subjected to a discontinuous
SDS-polyacrylamide gel electrophoresis (PAGE) (15) with a
12% acrylamide separating gel. Electrophoresis was done at a constant
voltage of 190 V with the Mini-PROTEAN II system (Bio-Rad, Hercules,
Calif.). After electrophoresis the proteins were visualized by staining
the gels with Rapid Coomassie blue stain (Diversified Biotech, Boston,
Mass.).
For immunoblot analyses, gel-separated proteins were transferred to
polyvinylidene difluoride membranes (Immobilon-P; Millipore Corporation, Bedford, Mass.) with the Mini-PROTEAN II system (Bio-Rad) at 100 V for 1 h. Following transfer, the membranes were blocked with 5% nonfat dry milk-0.2% Tween 20 in PBS for 2 h, washed
three times in PBS, and reacted overnight at 4°C with a mixture of
mouse MAbs, MAbs H5332 (2), H4610 (26), and H9724
(1) (all provided by Tom Schwan, Rocky Mountain
Laboratories, National Institutes of Health), which are specific for
OspA, OspB, and flagellin, respectively, and MAb L22 2B8, which is
specific for OspC (32). After incubation with primary
antibodies, the blots were washed three times in PBS and were incubated
for 1 h with the secondary antibody, goat anti-mouse antibody
conjugated to horseradish peroxidase (Pierce). After three 10-min
washes of the blots in PBS, bound antibody was visualized with
3,3',5,5'-tetramethylbenzidine (Kirkegaard & Perry Laboratories, Inc.,
Gaithersburg, Md.) by standard methods.
Gels and immunoblots were scanned and the OspA and OspC band
intensities were determined with Kodak Digital Science Image Analysis
Software (Eastman Kodak Co., Rochester, N.Y.). These digitized images
were used to acquire molecular mass data and determine the staining
intensity for each protein (OspA, OspC, or flagellin). The amount of
flagellin was used as our standard to compare the differential
expression of OspA and OspC, since flagellin expression was not
influenced by temperature. Digitized images were used to estimate the
relative amounts of OspA and OspC to the amount of flagellin. For ease
of comparison, band intensities for flagellin were normalized to a
value of 1.0.
| |
RESULTS |
The availability of I. scapularis cell lines
(21) made it possible for us to experimentally analyze the
effect of temperature on Osp expression of B. burgdorferi cocultivated with tick cells. We had previously
demonstrated that spirochetes would not grow in L15BS without tick
cells (14) and that tick cells did not survive in BSK for
more than 2 days (13). When we initiated these experiments,
we considered that the changes in Osp expression were not rapid heat
shock responses which occur within a few hours (31).
Therefore, borreliae were exposed to cells and temperature changes for
5 to 7 days to allow induction of the OspA-OspC response. Variability
in OspC expression with time of cultivation has been reported, even at
those temperatures that strongly induce OspC expression
(29). We reduced this variability by using borrelial inocula
that had an identical thermal prehistory (34°C) to initiate our
experimental cultures.
Phase-contrast microscopy revealed that B. burgdorferi
markedly disrupted ISE6 cell layers by day 5 in cultures grown at 34 or
37°C. By day 7, when spirochetes were harvested for SDS-PAGE and
immunoblot analyses, none of the tick cells were adherent in cultures
maintained at 34 or 37°C, while approximately 30% of the tick cells
were adherent in cultures maintained at 31°C. Control cells cultured
axenically in L15BS remained normal and adherent. Clusters of
spirochetes were attached to the tick cells and were found in the
spaces between the cells (Fig. 1). The
influence of temperature on OspA expression of borreliae cocultivated
with tick cells was also examined by IFA microscopy. Spirochetes
cocultivated with tick cells at 31 or 37°C for 48 h reacted
strongly with the OspA-specific MAb (Fig. 2A and
B). By day 5 the OspA-specific MAb
reactivity of borreliae cultured at 31°C was strong (Fig. 2C), while
in those spirochetes cultured at 37°C the amount of MAb binding had
declined noticeably (Fig. 2D). Moreover, at 31°C, spirochetes reacted
uniformly with the MAb (Fig. 2E), while at 37°C OspA expression was
patchy (Fig. 2F). Spirochetes cocultivated with the I. scapularis cells at 34°C did not show a noticeable change
in OspA expression within the 7 days.
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FIG. 1.
Giemsa-stained B. burgdorferi JMNT
(arrowhead) attached to tick cells (ISE6 cells). Bar, 10 µm.
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FIG. 2.
Epifluorescent image of rhodamine-labeled B. burgdorferi JMNT attached to tick cells (ISE6 cells). The cells
were harvested at selected times and centrifuged onto microscope
slides, and the spirochetes reacted with MAb to OspA. Panels A to D are
all at the same magnification (bar in panel D, 100 µm). (A) Cells
grown at 31°C after 48 h. (B) Cells grown at 37°C and
harvested after 48 h. (C) Cells grown at 31°C and harvested
after 5 days. (D) Cells grown at 37°C and harvested after 5 days.
Panels E and F are at the same magnification (bar, in panel F, 20 µm). (E) Cells grown at 31°C and harvested after 5. (F) Cells grown
at 37°C and harvested after 5 days.
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To quantify relative differences in the protein expression of
spirochetes cultured at 31, 34, or 37°C at the time of maximal tick
cell layer disruption, we harvested spirochetes for SDS-PAGE and
immunoblot analysis. Because SDS-PAGE profiles showed no major differences in borreliae harvested on day 5 or day 7 (data not shown),
spirochetes cocultivated for 7 days with ISE6 cells were harvested for
all subsequent analyses.
We compared the differential expression of OspA and OspC by
B. burgdorferi JMNT and N40 cocultivated with tick
cells or cultured axenically. Because expression of flagellin is not
influenced by temperature (29, 31), the amount of OspA or
OspC was related to the amount of flagellin as the standard in each
lane. Coomassie blue staining indicated that strain JMNT cocultivated
with tick cells expressed high levels of OspC at 34 and 37°C but not
at 31°C (Fig. 3A). OspC production in
spirochetes cocultivated with tick cells at 34 or 37°C was greater
than that in spirochetes cultured in BSK-H medium. OspA production was
greater when spirochetes were grown axenically in BSK-H medium than
when they were cocultivated with tick cells. Furthermore, at 37°C
there was a concomitant decline in OspA production. As expected, the
amount of flagellin was not affected by temperature or cocultivation
with tick cells. To confirm our SDS-PAGE observations we did an
immunoblot analysis using a mixture containing MAbs to OspA, OspB,
OspC, and flagellin. Reactivity with MAb L22 2B8 showed that OspC was
present at high levels in JMNT cocultivated with tick cells at 37°C
as well as in spirochetes cultured axenically at 37°C (Fig. 3B). At
34°C the spirochetes cocultivated with ISE6 cells also expressed high levels of OspC, while much less OspC was present on spirochetes cultured axenically at 34°C. Borreliae grown at 31°C in either system also produced OspC, but the signal was weaker. The results obtained with MAbs to OspA and OspB indicated that spirochetes cocultivated with tick cells at 37°C expressed low levels of OspA and
OspB, whereas spirochetes cocultivated with tick cells at 31°C
expressed more OspA and OspB. Borreliae cultured axenically produced high levels of OspA and OspB at all three temperatures. Similar SDS-PAGE and immunoblotting data were obtained with strain N40.
OspC production by strain N40 cocultivated with tick cells at 34 or
37°C was also greater than that by spirochetes cultured in BSK-H
medium. Again, OspA production was highest for spirochetes grown
axenically in BSK-H medium. At 37°C there was a decline in OspA
production by N40 cocultivated with tick cells. Our immunoblot analysis
with the MAb mixture confirmed our SDS-PAGE observations.
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FIG. 3.
Analysis of B. burgdorferi JMNT by
SDS-PAGE with Rapid Coomassie blue staining (A) or immunoblotting (B)
with MAbs to OspA, OspB, OspC, and flagellin as probes. Spirochetes
were cocultivated with a vector tick cell line (ISE6 cells) or were
grown axenically in BSK medium at 37, 34, or 31°C. Molecular mass
markers (in kilodaltons) are shown on the left.
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To confirm the differential expressions of OspA and OspC, we analyzed
our gels and immunoblots using image analysis software. This data
analysis confirmed that OspC production increased with temperature in
both strains (strains JMNT and N40) cocultivated with tick cells (Fig.
4). For both strains the level of OspA
production declined with an increase in temperature and the amount of
OspA relative to the amount of flagellin was lowest in borreliae
cocultivated with tick cells at 37°C (Fig.
5).
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FIG. 4.
Amount of OspC relative to amount of flagellin in
B. burgdorferi JMNT (A) and N40 (B). Spirochetes were
cocultivated with tick cells (open circles) or were grown axenically in
BSK-H medium (closed circles) at 31, 34, or 37°C. The gels were
scanned to determine OspA, OspC, and flagellin band intensities by
using Kodak Image Analysis Software. Flagellin was used as a
reference protein, and the ratio of the amount of a protein band to the
amount of flagellin was used to generate these graphs.
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FIG. 5.
Amount of OspA relative to amount of flagellin in
B. burgdorferi JMNT (A) and N40 (B). Spirochetes were
cocultivated with tick cells (open circles) or were grown axenically in
BSK-H medium (closed circles) at 31, 34, or 37°C. Gels were scanned
to determine OspA, OspC, and flagellin band intensities by using Kodak
Image Analysis Software. Flagellin was used as a reference protein, and
the ratio of the amount of a protein band to the amount of flagellin
was used to generate these graphs.
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DISCUSSION |
Successful transmission of the Lyme disease spirochete
(B. burgdorferi) from ticks to mammalian hosts, and
vice versa, presumably requires the induction of major physiological
changes in the spirochete. Temporal changes in the protein composition
of the spirochete's outer surface, mainly in OspA and OspC, during
this transition has led to the hypothesis that these Osps are pivotal
to infectivity for the mammalian host and survival in the vector
(27). Spirochetes within the gut of unfed and questing ticks
express mainly OspA but little or no OspC (29) and are
confined mainly to the luminal surfaces of gut cells (5,
33). Spirochetes in homogenates prepared from unfed ticks are not
infectious for mammals (23, 24). When an infected tick feeds
on a mammalian host several physiological changes occur and the tick's
body temperature increases from ambient temperature to more than 34°C
(20). During the blood meal the spirochetes start to migrate
from the gut to invade the salivary glands and ultimately the mammalian
host via the saliva (9, 33). The infectivity of spirochetes
for the mammalian host increases during the tick's blood meal
(23). Several studies have demonstrated a downregulation of
spirochetal OspA and upregulation of OspC during the early stages of
the tick's blood meal (6, 8, 10, 25, 29). Downregulation of
OspA expression occurs within 72 h of feeding, and it is estimated
that only one-third of the borreliae in the gut and salivary glands are
OspA positive at that time (10).
Our results indicate that a tick cell culture system is a good model
system for reproducing in vitro those events that occur while
spirochetes are in the tick. Expression of spirochetal Osps in feeding
ticks differs from what is observed in BSK medium, as suggested by the
host immune response, which is directed against OspA and OspB after
needle inoculation (25, 28). The differential expression of
OspA and OspC that we observed for B. burgdorferi cocultivated with I. scapularis cells was similar to
that observed for borreliae resident within feeding I. scapularis ticks. We observed a decline in OspA production and an
increase in OspC production which was more pronounced in spirochetes
cocultivated with tick cells at 37°C than in those cultured
axenically in BSK-H medium at the same temperature. Our IFA microscopy,
SDS-PAGE, and immunoblotting results all revealed this decline in OspA
production. OspC production was enhanced at 37°C in both strains
(strains JMNT and N40) cocultivated with tick cells. These results
agree with those from previous studies that have shown the upregulation of OspC production in B. burgdorferi cultured at higher
temperatures and its decline at lower temperatures (7, 29,
31). However, a temperature-induced decline in OspA production
was not reported in those studies. Our results suggest that, along with
temperature, tick cell contact or other culture parameters, e.g.,
osmolality, may be involved in the modulation of spirochetal expression
of OspA. Manipulation of the tick cell system therefore should be the
next step in the search for answers about the regulation of Osp
expression. Answers to such questions could provide clues about
transmission mechanisms in ticks during attachment and feeding. Such
studies could provide information on the molecular mechanisms controlling the Osp phenotypes of the spirochetes that we observe in
nature and assist us in understanding and predicting the efficacy of
Lyme disease vaccines based on recombinant Osps. The role of OspA in
the adhesion of spirochetes to tick cells remains to be determined. MAb H5332, which is directed against OspA, did not interfere with the binding of B. burgdorferi to
I. scapularis cells (14). Also, mutants
of B. burgdorferi sensu stricto that lack OspA
have been found to adhere well to cultured I. scapularis cells (12). This would suggest that, at
least in vitro, OspA does not solely mediate binding of spirochetes to
tick cell membranes. Furthermore, our results indicate that while
spirochetes had downregulated OspA at 37°C they still had detectable
levels of OspA by immunoblotting. These results were not unexpected
because Osps, once they are present, will persist on the spirochetal
surface for several divisions (31).
Our interpretation of the differential expression of OspA and OspC is
based on investigations with North American strains of B. burgdorferi sensu stricto. In Europe, at least three different genospecies of B. burgdorferi sensu lato that are
pathogenic for humans exist, and the three genospecies display
different patterns of Osp expression in culture and in the vector tick.
Borrelia afzelii, for example, has been shown to express
both OspA and OspC in unfed nymphs of Ixodes ricinus
(11, 16). However, as reported for B. burgdorferi sensu stricto in I. scapularis, B. afzelii also downregulates OspA, while it continues
to express OspC in feeding I. ricinus (16).
Further investigations with strains from different genospecies are
needed to clarify the function and dynamics of OspA and OspC expression
in both the tick and the mammalian host.
We thank Russell Johnson for support and helpful suggestions. We
thank Tom Schwan (Rocky Mountain Laboratories, National Institute of
Health) for providing us with MAbs H5332, H4610, and H9724 against
OspA, OspB, and flagellin, respectively. We thank Stephen Barthold
(Center for Comparative Medicine, University of California, Davis) for
providing strain N40.
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Abstract
Introduction
