Microbial Pathogenesis 1991 ; 10 : 137-148
Characterization of Borrelia burgdorferi invasion of cultured endothelial cells Laurie E . Comstock and D . Denee Thomas* 'Department of Microbiology and Immunology, Bowman Gray School of Medicine, Wake Forest University Medical Center, Winston-Salem, North Carolina 27103, U .S.A . (Received August 16, 1990; accepted in revised form October 31, 1990)
Comstock, L . E . (Dept of Microbiology and Immunology, Bowman Gray School of Medicine, Wake Forest University Medical Center, Winston-Salem, North Carolina 27103, U .S .A .) and D . D . Thomas . Characterization of Borrelia burgdorferi invasion of cultured endothelial cells . Microbial Pathogenesis 1991 ; 10 : 137-148 . Borrelia burgdorferi can adhere to cultured endothelial cells and penetrate through cell monolayers by passing through intercellular tight junctions and through the host cell cytoplasm . Borrelia burgdorferi strains which were isolated from different sources and areas of the U .S . all demonstrated similar invasive capabilities . Bacterial penetration from the apical to the basal surface of the monolayer was 20 times more efficient than from the basal to the apical surface . Borreliae which were non-viable as a result of either heat treatment or ultraviolet (UV) irradiation showed reduced association with the endothelial cell monolayer and loss of invasive capabilities . Borreliae were able to invade when protein synthesis was inhibited with streptomycin or chloramphenicol . When assays were conducted at 4'C, bacterial penetration of the monolayer was completely inhibited . Treatment of borreliae with proteases affecting outer surface proteins greatly reduced cell association and bacterial invasion . Key words : bacterial attachment ; bacterial invasion ; Borrelia burgdorferi ; endothelial cells .
Introduction Lyme borreliosis is a multi-system disorder which can affect the myocardium, central nervous system and joints . Increasing evidence suggests that the existence of bacteria at these sites is necessary for the ensuing symptoms .' -' Since infection begins in the bloodstream and lymphatics, penetration across the endothelial cell lining of blood vessels and subsequent dissemination from the bloodstream to tissues may be important prerequisites for secondary and tertiary stages of disease . Previously, we have shown that
burgdorferi can attach to the apical surface of
cultured human umbilical vein endothelial (HUVE) cells, gain entry into the cells, s .9 translocate across the cytoplasm, and emerge from the basal surface of the monolayer . These bacteria are also able to penetrate by passing through the intercellular tight junctions of the monolayer . 10 In this study, we characterized the invasion process . Our findings have revealed that the borreliae which invaded endothelial cells did not maintain an intracellular residence,
`Author to whom correspondence and requests for reprints should be addressed at : Department of Microbiology and Immunology, Bowman Gray School of Medicine, Wake Forest University Medical Center, 300 S . Hawthorne Rd, Winston-Salem, N .C . 27103, U .S .A. 0882-4010/91/020137+12 $03 .00/0
1991 Academic Press Limited
L . E . Comstock and D . D . Thomas
Table 1 Penetration of endothelial cell monolayers by B . burgdorferi strains' Strainb HB19 p .6 ECM-NY p .10 Sh-2-82 p .9 N-40 p .4 CA-2-87 p .9
% in lower chamber' 8 .1+0 .3 8 .0±0 .4 8 .2±0 .3 7 .8+0 .2 7 .5+0 .4
'Suspensions containing 5X10 8 bacteria were added to HUVE cell monolayers . 'B . burgdorferi isolates with corresponding number of passages in culture . `Reflects the percentage of the borreliae added to the monolayer that had penetrated to the media below after 4 h . Values represent average ± SD for samples performed in triplicate in at least three separate experiments .
but either emerged or were subsequently degraded by the endothelial cells . Borrelial penetration did not appear to cause damage to the monolayer . Invasion was shown to be more efficient from the apical rather than the basal surface . Inactivation of borreliae with heat and UV light caused a significant inhibition of invasion . Cleavage of outer surface proteins with proteases greatly inhibited invasion into cells and penetration of the endothelial cell monolayer . Results Invasive capabilities of various B . burgdorferi strains Previously, we have shown that infectious B . burgdorferi strain HB19, which was isolated from the blood of a patient displaying primary Lyme disease symptoms,' can invade cultured endothelial cells and penetrate through a confluent monolayer . $ Other virulent B . burgdorferi strains isolated from different sources were tested for invasive capabilities . Strain ECM-87, which was isolated from a patient's skin lesion, displayed a degree of penetration of the endothelial cell monolayer similar to the human blood isolate (Table 1) . Three tick isolates, Sh-2-82 and N-40 (isolated from Ixodes dammini ticks in Eastern United States) and CA-2-87 (isolated from an Ixodes pacificus tick in California) demonstrated penetrative capabilities similar to the clinical isolates (Table 1) . Transmission electron microscopy revealed that all strains were able to enter into and translocate through the endothelial cell cytoplasm . Since the degree of invasion was similar for all the strains that were tested, HB19 was used for all further studies . Long term interaction of borreliae with HUVE cells Results from initial penetration studies indicated that borreliae enter endothelial cells grown in a monolayer and are able to emerge from the basal surface .' These studies did not address, however, whether all bacteria which enter subsequently exit the cell, whether some are degraded inside the endothelial cell, or whether some are able to survive intracellularly for a prolonged period of time . To determine whether borreliae are able to persist inside endothelial cells, bacteria were added to the apical surfaces of monolayers and allowed to invade for 4 h, by which time many bacteria were intracellular and many had penetrated through the monolayer . Borreliae which were free in the upper and lower chambers were washed away so that only intracellular
B . burgdorferi interaction with endothelial cells
and attached bacteria remained, and fresh medium was added . The monolayers were incubated at 37°C for an additional 24 h, at which time the cells were washed, fixed, and prepared for electron microscopy . Borreliae were observed in the media below the monolayer by dark-field microscopy, indicating that bacteria had emerged from the monolayer . Compared with approximately four intracellular bacteria per HUVE cell in cross-section following 4 h of incubation [Fig . 1(A)], electron microscopy revealed less than one intracellular bacterium per five HUVE cells 24 h after removal of the unassociated bacteria [Fig . 1 (B)] . Borreliae were occasionally seen, however, attached to the apical surface and in junctions . The few borreliae that did remain inside the host cells appeared unhealthy, as if in various stages of degradation [Fig . 1(C)] . The endothelial cell monolayers appeared structurally normal upon visual inspection using the electron microscope, were at least 95% viable by trypan blue exclusion conducted before and after invasion, and retained intercellular tight junctions [Fig . 1 (B)] .
Penetration of the basal surface
HUVE cell monolayers have polarity" like other epithelial cell types . The apical membrane surface composition differs from the basal surface" and this difference is maintained by the tight junctions formed between cells . To investigate whether B . burgdorferi were able to invade the H UVE cell from the basal side, 5 x 10 $ bacteria were added to the media below the filter/monolayer, and equal numbers were added above the monolayer in duplicate wells. Following incubation, penetration from the basal to the apical surface was quantitated by counting bacteria in the upper chamber and by visually inspecting the interior of the HUVE cells by transmission electron microscopy . Two separate experiments performed in triplicate revealed that the bacteria which had invaded through the monolayer from the basal to apical surface after 3 h of incubation accounted for a mean ± SD of 0 .2±0 .1% of the added bacteria, a 20-fold difference when compared with 4 .0±0 .4% for apical to basal penetration . Since the volume of the lower chamber is greater than the volume above the monolayer, this assay was also performed by applying equal concentrations of bacteria (2 .5 x 109 /ml) above or below the monolayer . Even though five times more bacteria were applied to the basal surface than to the apical surface, penetration from apical to basal surface was still 8 .8 times more efficient than from the basal to apical surface . In monolayers where borreliae were added to the basal surface, no bacteria were observed attached to the basal membrane by electron microscopy . Also, electron microscopy revealed less than one intracellular bacterium per four HOVE cells compared with approximately seven per cell in samples where bacteria were added to the apical surface . To determine whether gravity was affecting the system, bacteria were applied above or below the filter in the absence of endothelial cells . The number of bacteria which crossed the filter to each side was the same when equal concentrations were added . Therefore, gravity does not contribute to the differences in invasion from each side . Invasion at 4° C
Invasion of eukaryotic cells by several bacterial species has been shown to be inhibited when the incubation is conducted at 4°C . Borreliae were added to HUVE cell monolayers/filters which had been preincubated for 30 min at 4°C and were maintained at 4°C for the duration of the assay . Borreliae were able to attach under these conditions. However, when 50 HUVE cells were observed by electron microscopy, no bacteria were observed inside cells . In addition, no borreliae were detected below the monolayers after 4 h (Table 2) . Dark-field microscopy revealed that the borreliae were still vigorously motile after 4 h at 4°C and were able to replicate when placed in BSK
L . E . Comstock and D . D . Thomas
Fig . 1 . Transmission electron micrographs of borreliae-infected HUVE cell monolayers . Suspensions containing 5x108 borreliae were incubated on monolayers for 4 h prior to washing and fixation . (A) Monolayer fixed immediately following a 4 h incubation with borreliae . (B,C) following 4 h incubation with borreliae, the monolayer was washed, fresh media was added, and the monolayer was incubated an additional 24 h prior to fixation .
B . burgdorferi interaction with endothelial cells
Table 2 Adherence and invasion capabilities of B . burgdorferi following various modifications
Pre-treatment Experiment 1 Control' 4°C' UV light-" Heat' Experiment 2' Control Trypsin Proteinase K Pronase
host cell association'
Intracellular bacteria per 50 HUVE cells'
8 .0+0 .3 2 .0+0 .8 4.1+0 .3 4 .7+1 .4
75 49 41
3 .2+0 .5 0 0 0
100 100 100
148 0 0 0
8 .2+3 .3 2 .9±0 .5 2 .5+0 .5 3 .0+0 .3
65 70 63
4 .0+1 .9 0 .4±0 .2 0 .2+0 .1 0 .5+0 .4
90 95 87
272 6 0 3
'Expressed as the percentage of added bacteria that associated with the monolayer following a 4 h coincubation . Represents the average ± SD from three experiments performed in triplicate . 'Represents percentage decrease in cell association compared to the control . `Represents percentage of bacteria added to the monolayer that were found in lower chamber after 4 h . For all assays, 6 .5 mm diameter filters were used . Quantitative results from earlier assays, therefore, cannot be compared with those in this table . Reported as average ± SD from three experiments performed in triplicate . 'Quantitated using electron microscopy following a 4 h invasion period . Represents the number of bacteria observed in cross-section in 50 cells located in at least three different areas of the filter . 'Results obtained for controls for all three modifications (4°C, UV light, heat) were identical . 'HUVE cell monolayers were incubated at 4°C for 30 min prior to addition of borreliae and for the duration of the assay. "Borreliae were exposed to 15 W of UV-irradiation for 25 min at a distance of 30 cm . 'Borreliae were incubated at 56°C for 15 min prior to exposure to monolayers . 'Borreliae were treated with trypsin (0 .1 mg/ml), proteinase K (0 .2 mg/ml), or pronase (0 .2 mg/ml) in PBS or PBS alone for 40 min at 34°C prior to addition to monolayers .
II medium . The viability of endothelial cells incubated at 4°C and 37 ° C did not differ by trypan blue exclusion . Additionally, electron microscopic observations revealed that monolayers incubated at 4°C for 4 h maintained tight junctions and were visually similar to monolayers incubated at 37°C . When the co-incubation was conducted at 37°C, many intracellular borreliae were observed surrounded by host cell cytoskeletal components not only beneath invading bacteria at the host cell membrane, but also throughout their transport across the host cell cytoplasm to the basal surface (Fig . 2) . These results collectively suggest that the endothelial cell cytoskeleton may be involved in the invasion process . However, when endothelial cell monolayers were treated with cytochalasin B or D (1, 2 .5, or 5 µg/ml) or colchicine (2 x 10 -3 M) for 1 h prior to and during the co-incubation, bacterial invasion into cells was not decreased as determined by electron microscopic observation . Effect of UV inactivation and heat treatment of B . burgdorferi on invasion of HUVE cells
In order to determine whether borrelial viability is essential for attachment and invasion, the cell association and invasion assays were performed using UV-irradiated or heattreated bacteria . The borreliae were exposed to heat or UV light just to the point where they lost viability (i .e . loss of motility and inability to replicate) . These treatments did not cause the bacteria to clump . Following a 4 h co-incubation of bacteria with HUVE cell monolayers, no UV-irradiated or heat-killed borreliae were present in the media below the monolayers (Table 2) . Electron microscopy revealed that many UV- and
L. E . Comstock and D . D . Thomas
Fig . 2 . Transmission electron microscopy of intracellular B . burgdorferi surrounded by endothelial cell cytoskeleton .
heat-killed bacteria were attached to the apical membrane surface of the HUVE cells, but none were inside the 50 HUVE cells that were observed in cross-section . The cell association results support the observation that these treatments did not prevent the borreliae from adhering to the host cell monolayers . The cell association assay does not distinguish between attached and intracellular bacteria . Therefore, the 49% and 41% inhibition of bacterial association with the monolayer following UV-irradiation and heat treatment respectively may be attributable to a lack of invasion rather than a decrease in adherence . Effect of bacterial treatment with protein synthesis inhibitors on adherence to and invasion of HUVE cells The preceding results showed that borrelial viability is important for invasion into HUVE cells . To determine whether de novo borrelial protein synthesis is necessary for entry into HOVE cells, bacteria were treated with chloramphenicol or streptomycin prior to and during the course of the assays . The concentrations and conditions used significantly reduced protein synthesis of borreliae treated with streptomycin and completely reduced protein synthesis in samples treated with chloramphenicol (Fig . 3) . These drug concentrations, however, did not reduce borrelial motility during the 4 h course of the assays. Neither cell association nor invasion was significantly affected by treating the bacteria with these protein synthesis inhibitors . Results from three separate experiments revealed that 8 .8±0 .3% and 8 .2±0 .4% of streptomycin and chloramphenicol treated borreliae associated with the monolayer compared to 8 .5±0 .3% association for untreated controls . Similarly, 2 .4±0 .4% and 2 .2±0 .5% of streptomycin and chloramphenicol treated borreliae invaded through the monolayers after 4 h compared to 2 .9±0 .9% invading when untreated bacteria were added . Electron microscopy revealed that, as with untreated bacteria, borreliae treated with streptomycin and chloramphenicol were able to penetrate by passing through the endothelial cells .
B . burgdorferi
interaction with endothelial cells
Fig . 3 . Inhibition of B. burgdorferi protein synthesis following antibiotic treatment . Samples containing 108 borreliae in 200µl M199-FCS were incubated with (A) no addition, (B) 25 µg/ml chloramphenicol, or (C) 375 pg/ml streptomycin . All samples were incubated for 1 h at 34°C . Next, 10 µCi of [ 35 S]-methionine was added to each sample, and incubation was continued for an additional 4 h at 37'C . Bacteria were washed three times and subjected to SDS-PAGE and autoradiography. 5 x
Inhibition of invasion following treatment of borreliae with proteases It has been shown that when intact B . burgdorferi are treated with trypsin, an outer surface protein of 34 kilodaltons (kDa) is cleaved, and when treated with proteinase K, this same protein and an additional outer surface protein of 31 kDa are cleaved . 14 We have previously implicated these molecules as contributing to borrelial association with HUVE cells .' Therefore, we sought to determine the ability of borreliae to adhere to and invade into HUVE cells when these molecules were cleaved from the bacterial surface . During the 40 min course of the protease treatments in PBS, borreliae lost motility . However, when proteases were removed and the bacteria were resuspended in M199-FCS, they regained motility and were viable, as determined by their ability to replicate when placed in BSK ll . Protease-treated bacteria were added to HUVE cell monolayers in media containing 25 yg/ml chloramphenicol to prevent de novo synthesis of surface proteins . Figure 4 shows that the 31 and 34 kDa proteins were not resynthesized when the bacteria were incubated with the monolayer for 4 h in the presence of chloramphenicol . Results showed a large inhibition in both cell association and invasion (Table 2) . Bacteria which were treated with either trypsin or pronase, both of which cleaved the 34 kDa protein (Fig . 4), were inhibited approximately 64% in cell association and approximately 88% in penetration of the monolayer (Table 2) . When borreliae were treated with proteinase K, which cleaved both the 31 and 34 kDa proteins, there was a slightly greater inhibition of both cell association (70%) and bacterial penetration (95%) . Additionally, when monolayers were observed in crosssection using the electron microscope, only six and three trypsin-and-pronase-treated borreliae, respectively, were observed inside 50 HUVE cells following 4 h of incubation .
L . E . Conistock and D D . Thomas
Fig . 4 . Coomassie blue-stained profile of B . burgdorferi proteins following treatment with proteases . Borreliae were suspended in PBS with no addition (lane 1 ), trypsin (0 .1 mg/ml ; lane 2), proteinase K (0 .2 mg/ml ; lane 3), or pronase (0 .2 mg/ml ; lane 4) and incubated for 40 min at 34°C . Reactions were stopped by washing organisms and resuspending in M199-FCS containing 25 pg/ml chloramphenicol and added to monolayers . Unassociated bacteria were collected after 4 h, washed, and subjected to SDS-PAGE . Molecular mass standards are shown on the left .
This result contrasts with 272 intracellular bacteria in 50 HOVE cells when the borreliae were untreated . Trypsin- and pronase-treated bacteria were, however, occasionally observed attached to the apical surface of the monolayer . Electron microscopy revealed that borreliae treated with proteinase K were not only inhibited in their ability to enter into HUVE cells (0 in 50 cells), but also had a greater inhibition of attachment, since none were observed attached to the apical surface of 50 HUVE cells . Also a smaller percentage of the proteinase K-treated borreliae associated with the monolayer, compared to bacteria treated with trypsin or pronase .
Discussion and conclusions Previously, we demonstrated the ability of a B . burgdorferi strain isolated from human blood to invade, translocate through, and emerge from cultured endothelial cells .' In this study, not only was this process further examined, but additional strains were analysed to determine whether invasiveness was characteristic of B . burgdorferi strains isolated from different sources . For each strain examined, the percentage of borreliae that invaded through the HUVE cell monolayer was approximately equivalent . The adherence of B. burgdorferi to endothelial cells has been shown to be specific .' However, the host cell receptor(s) to which borreliae attach has not been identified . Because borreliae penetrated from the apical to basal surface of the endothelial cell
B . burgdorferi interaction with endothelial cells
monolayer much more efficiently than from the basal to apical surface, these receptors may be selectively located in the apical membrane . It may be argued that the position of the filter at the basal surface presents an unfair advantage for bacteria invading from the apical membrane . However, the pore size of the filter is approximately 10 times the helical width of a bacterium, thereby allowing easy access of the borreliae to the basal surface . Also, because of their small diameter, the borreliae have ample space to migrate between the basal surface of the monolayer and the filter such that most of the basal surface is accessible to the bacteria . Lastly, the filter is also a barrier for the bacteria penetrating from the apical to basal surface because they must also move between the basal membrane and the filter to a pore before they become present in the media in the lower chamber . Bacteria penetrated from the apical to the basal surface approximately nine times more efficiently than from the basal to the apical surface . This difference occurred even when five times more bacteria were added to the basal surface . We believe this more than compensates for the barrier posed by the filter, The discrepancy in invasion of the two surfaces probably reflects the different compositions of the basal and apical surfaces, whether it be because of protein content, abundance of microvilli on the apical surface, or differential distribution of cytoskeletal components near the two surfaces . The location of B . burgdorferi in the body of an infected human during asymptomatic periods has not been determined . Our results demonstrate that borreliae do not appear to maintain a prolonged intracellular residence in cultured endothelial cells . Borreliae that entered these cells either escaped unharmed or were, presumably, degraded within the cytoplasm . This process appeared to cause no damage to the endothelial cells . The results from this in vitro study do not, however, rule out the possibility of prolonged borrelial residence in endothelial or other cell types in vivo . Some bacteria, such as Salmonella and Shigella, must be viable to trigger invasion of host cells,' 1,16 while even non-viable Chlamydia and Yersinia are able to enter into host cells ." , " We have demonstrated that non-viable borreliae were able to attach to HUVE cells but were unable to invade and penetrate through the monolayer . It is possible that the UV-irradiation and heat-treatment denatured an important invasionmediating molecule . However, the treatments were mild and were conducted just to the point where the borreliae lost viability . We, therefore, believe that borrelial viability is extremely important for invasion . Salmonella invasion of epithelial cells has been shown to be dependent on the growth phase of the bacterial culture ." Bacterial protein synthesis is required for invasion by Salmonella, 15 therefore, stationary phase cultures lose their invasiveness . Since inhibition of bacterial protein synthesis did not inhibit borrelial invasion, and since borreliae enter rapidly upon contact with host cells,' invasion was not dependent upon the synthesis of new invasion-mediating proteins . Removal of outer surface proteins with various proteases significantly inhibited attachment to and invasion of HUVE cells by B . burgdorferi. Coomassie staining of SDS-PAGE gels revealed that trypsin and pronase cleaved the 34 kDa protein, also known as Osp B, from the bacteria . Proteinase K cleaved Osp B and an abundant outer surface protein of 31 kDa (Osp A) . It has also been shown that a protein of M, 66 kDa is sensitive to both trypsin and proteinase K treatment . 14 It is also probable that less abundant, protease-sensitive, outer surface proteins, not visible by Coomassie staining, are also cleaved from the bacteria by these proteases . Therefore, using this method alone, it is not possible to implicate a specific molecule in mediating attachment or invasion . Based on our data, however, it appears that trypsin and pronase remove invasion-mediating protein(s), and that proteinase K treatment removes proteins involved in mediating adherence and invasion . This premise is based on the observation
L . E . Comstock and D . D . Thomas
that borrelia treated with trypsin and pronase appear to be inhibited in their ability to invade, yet many bacteria were visualized attached to the apical surface . Bacteria treated with proteinase K were inhibited in attachment and invasion, as observed by electron microscopy . Osp A and B are likely candidates for mediating these functions, however, conclusive proof awaits further studies . A proposed scheme for B . burgdorferi invasion of HOVE cells based on the accumulated data is as follows . The bacteria attach to HUVE cells by interaction of bacterial protein(s) with the endothelial cell membrane . We believe this adhesinreceptor interaction is not sufficient to trigger internalization but, rather, may allow for separate invasion-mediating molecules to react with the endothelial cell surface . The fact that several treatments inhibited invasion, but not attachment, supports the hypothesis that two distinct sets of molecules or domains may be responsible for attachment and invasion . Borreliaf viability is necessary for invasion . Our data suggest that borrelial motility, rather than de novo synthesis of bacterial proteins, may be essential because any treatment that rendered the borreliae non-motile significantly inhibited invasion . The motility of attached bacteria may allow for less surface-exposed or hidden invasion-mediating molecules or epitopes to interact with the endothelial cell . This study demonstrates that B . burgdorferi play an active role in their invasion of non-professional phagocytes . Further studies will be directed at determining which outer surface proteins and domains mediate adherence and invasion and determining the importance of invasion to the pathogenesis of disease .
Materials and methods Bacteria . Borrelia burgdorferi HB19, 5 was provided by Dr Alan Barbour, University of Texas Health Science Center at San Antonio . Strains Sh-2-82, ECM-87, and CA-2-87 were provided by Dr Tom Schwan, Rocky Mountain Laboratories, Hamilton, Montana, and their isolations were described previously . 20 Strain N-40 was provided by Dr Allen Steere, Tufts University Medical Center, Boston, Massachusetts . These strains were used at passage 10 or lower . Borreliae were maintained in BSK II medium 21 at 34°C, and were used at log phase of growth (7 x 10' -10 x 10' organisms/ml) . Cultured eukaryotic cells. Human umbilical vein endothelial cells were regularly isolated from freshly delivered human umbilical cords by the method of Jaffe . 22 Cells were maintained in 5% CO 2 at 37°C in Medium 199 (M199 ; GIBCO, Grand Island, New York) supplemented with 20% fetal calf serum (FCS ; GIBCO), 100 µg/ml heparin (Sigma Chemical Co, St Louis, Missouri) and 50 ng/ml endothelial cell growth supplement (GIBCO) . Endothelial character of the cells was assured by demonstrating that cells at various passages stained positively with FITClabeled rabbit antihuman Factor VIII (von Willebrand factor) . Experiments were conducted using cells from passage 15 or fewer . Cell association assay. The ability of B . burgdorferi to adhere to HUVE cells was assessed as previously described .' Briefly, borreliae were intrinsically radiolabeled with [ 35 S]-methionine, 9 washed with phosphate- buffered saline (PBS), and resuspended to a density of 10' bacteria/ml in M199 containing 15% FCS (M199-FCS) . Aliquots (0 .5 ml) were added to confluent HUVE cell monolayers grown in 24-well plates . Following incubation at 37°C, monolayers with associated organisms were washed, solubilized, mixed with scintillation cocktail, and counted by scintillation . Invasion assay. Penetration assays were performed essentially as described previously . 23 For initial experiments, 5x104 HUVE cells were seeded onto sterile polycarbonate filters (3µm, 13mm diameter; Nuclepore, Pleasanton, California) which were mounted on plastic chemotaxis chambers (PC-2, ADAPS, Dedham, Massachusetts) . For experiments involving basal invasion and modifications of borreliae, 2 .5x104 HUVE cells were seeded onto sterile polycarbonate
B . burgdorferi interaction with endothelial cells
membrane culture plate inserts (3µm, 6 .5mm diameter ; Nuclepore) . In both cases, chambers were placed in 24-well plates containing 1 ml M199-FCS/well and incubated for 48 h, at which time the monolayers were confluent and possessed high transendothelial electrical resistance . 25 Bacteria were quantitated by darkfield microscopy, centrifuged for 15 min at 17 000 xg, and resuspended in M199-FCS . For each assay, 0.2 ml samples containing 5 x 108 bacteria were added to the upper portions of the chambers (above the monolayers) . Following a 4 h incubation at 37°C in 5% CO 2 in air, aliquots from beneath the filters were removed and spirochetes were quantitated by darkfield microscopy . SDS-PAGE and autoradiography . Sodium dodecyl sulphate polyacrylamide gel electrophoresis was conducted using the discontinuous buffer system described by Laemmli . 24 Samples were boiled for 10 min in electrophoresis sample buffer containing 60 mm Tris, 2% SDS, 10% glycerol and 50 mm dithiothreitol . Stacking gels contained 5% acrylamide and separating gels 10% acrylamide . Following electrophoresis, gels were stained with Coomassie Brilliant Blue, treated with Amplify fluorographic reagent (Amersham Corp, Arlington Heights, Illinois), dried, and exposed to Kodak XAR film (Eastman Kodak, Rochester, New York) for 4 days at -70°C . UV-irradiation and heat treatment of borreliae . Borrelia (108 organisms/ml in PBS) were irradiated with UV light by exposure to a 15 W germicidal lamp at a distance of 17 cm for 10 min . The bacteria were then centrifuged, resuspended to the appropriate concentration in M199-FCS, and used in the cell association or invasion assay . Heat treatment consisted of resuspending borreliae to a concentration of 10 8 /ml in M199-FCS and placing the tube in a waterbath at 56°C for 15 min . For subsequent cell association assays, bacteria were added directly to monolayers . For invasion assays, bacteria were centrifuged, resuspended to the appropriate concentration in M199-FCS, and added to monolayers . Controls were resuspended to the same concentrations in PBS or M199-FCS and kept at room temperature for the duration of the treatments . Bacterial treatment with protein synthesis inhibitors . For some assays, bacteria were preincubated with 25 pg/ml chloramphenicol (Sigma) or 375 pg/ml streptomycin (Sigma) in M199-FCS for one hr at 34°C prior to addition to the monolayer . Protein synthesis inhibitors were also present at the same concentrations for the duration of the assays . The control was incubated in M199-FCS without antibiotics for the same period prior to addition to the monolayer . Protease treatments of borreliae . Borreliae were centrifuged, washed once with PBS and resuspended to 10 9/ml in PBS containing either 0 .1 mg/mI trypsin (GIBCO), 0 .2 mg/ml proteinase K (Boehringer Mannheim, Indianapolis, Indiana), 0 .2 mg/ml pronase (Boehringer Mannheim), or PBS alone . These bacteria were incubated for 40 min at 34°C, washed, and resuspended to the appropriate concentration in M199-FCS containing 25 pg/ml chloramphenicol . Suspensions were then added to wells or chambers for cell association or invasion assays as described above . Electron microscopy . Transmission electron microscopy was performed as described previously .' After bacteria were incubated with HUVE cell monolayers, the filters were removed from the chambers, rinsed in PBS, fixed in glutaraldehyde, stained, dehydrated and embedded in epoxy resin . Transverse thin sections were placed on copper mesh grids and stained with lead citrate and uranyl acetate prior to observation in a Philips TEM 400 microscope . Intracellular bacteria were quantitated by counting the number of borreliae present in 50 cross-sectioned HUVE cells following 4 h of co-incubation . Owing to the length and spiral shape of the borreliae, we cannot be certain that a single bacterium was counted only once . However, all samples were counted in a consistent manner and determinations can therefore be compared to each other . We thank Linda Higbie for excellent technical assistance, and Jon Lewis and Ken Grant for advice regarding electron microscopy . Alan Barbour, Tom Schwan, and Allen Steere kindly provided B. burgdorferi strains . Our grateful thanks are extended to each of these persons . This work was supported by Public Health Service grant Al-26804 from the National Institute of Allergy and Infectious Diseases .
L . E . Comstock and D . D . Thomas
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