Entry of Spiroplasma citri into Circulifer haematoceps Cells Involves Interaction between Spiroplasma Phosphoglycerate Kinase and Leafhopper Actin

Spiroplasma strain, leafhoppers, and cell line culture. S. citri GII3, originally isolated from its leafhopper vector C. haematoceps captured in Morocco (52), was cultivated in SP4 medium (50) at 32°C.

Healthy C. haematoceps leafhoppers were reared in an insect-proof cage on stock plants (Mattiola incana) at 30°C. Microinjection of S. citri GII3 into C. haematoceps has been described previously (21). Injected leafhoppers were maintained for 2 weeks on stock plants before salivary glands were removed.

The nonphagocyte cell line Ciha-1 (Circulifer haematoceps 1) (18) was cultured at 32°C in Schneider's Drosophila medium (Invitrogen) supplemented with 1% histidine buffer (0.057 M histidine monohydrate, pH 6.2), 10% Grace's insect cell culture medium, 0.5% G-5 supplement, and 10% (vol/vol) heat-inactivated fetal bovine serum.

Far Western blotting experiments.Leafhopper salivary glands were dissected in phosphate-buffered saline (PBS; 2 mM KH₂PO₄, 8 mM Na₂HPO₄, 0.14 M NaCl, 2 mM KCl [pH 7.4]) containing 1 mM phenylmethanesulfonylfluoride (PMSF). Glands were stored frozen in buffer at −20°C until use. Glands were transferred to a potter Elvehjem grinder containing the same buffer and homogenized. Then the mixture was centrifuged for 1 min at 10,000 × g. The protein concentration was determined by the Bradford procedure. Proteins were not further purified, to avoid inadvertently removing proteins of interest. Aliquots of supernatant (20 μg) were fractionated by electrophoresis in a 12.5% SDS-PAGE gel before transfer onto a nitrocellulose membrane according to the methods of Killiny et al. (32). After transfer, all steps were conducted under low agitation. Membranes were blocked in 10 ml of PBS with 6% nonfat dry milk and incubated with 2 ml of S. citri proteins (20 μg/ml) in PBS overnight at 4°C. S. citri proteins used as an overlay were prepared according to the methods described by Killiny et al. (32) with a few modifications. Disruption of the cells was performed by sonication (Vibracell sonicator; Sonics & Materials, Inc., Danbury, CT; rate of 40% pulses/s, 50 W, 4°C; 1 min of sonication and 1 min on ice alternatively, three times). After incubation, blots were washed with 50 ml of PBS buffer containing 0.1% Tween 20 (PBS-Tween) and incubated in 10 ml of PBS containing 1% nonfat dry milk (antibody buffer) with purified polyclonal IgGs against total S. citri proteins at a final concentration of 5 μg/ml for 1 h at room temperature (RT). After three washings with PBS-Tween, blots were incubated in 10 ml of antibody buffer with peroxidase-conjugated goat anti-rabbit IgGs (Sigma Aldrich) at a 1:50,000 dilution at RT for 1 h. Blots were then washed in PBS-Tween three times, followed by incubation with the substrate solution (Super Signal West Pico chemiluminescent substrate) according to the manufacturer's instructions (Pierce, Rockford, IL). Then blots were exposed on X-ray film.

Two micrograms of S. citri purified recombinant PGK was used in a far Western assay carried out to confirm interaction with leafhopper actin protein. The blot of His₆-tagged PGK was overlaid with 500 μg of total insect proteins. The experiment was conducted in a manner similar to that described above for the S. citri protein overlay assay except that anti-His monoclonal antibodies (MAb; Sigma) instead of S. citri polyclonal antibodies were used.

Immunofluorescence analysis of salivary glands.Salivary glands from infected or noninfected C. haematoceps leafhoppers were dissected and incubated in 500 μl of PBS containing 0.2% Triton X-100 at RT. All subsequent steps were performed in the same volume. Salivary glands were washed twice in PBS and immersed in fixative (4% paraformaldehyde in PBS plus 0.2% Triton X-100) overnight at 4°C. Then, fixed salivary glands were rinsed three times with PBS with 0.2% Triton X-100 and permeabilized with PBS containing 1% Triton X-100 (PBS-T) overnight at 4°C followed by an incubation in blocking buffer (PBS-T plus 1% bovine serum albumin [BSA]) for 1 h at RT. S. citri polyclonal antibodies diluted 1:500 in PBS containing 1% BSA were added for 2 h. They were washed in PBS and treated with Alexa 488-conjugated goat anti-rabbit IgG (Invitrogen) at a 1:200 dilution, and simultaneously F-actin was stained by Alexa 568-phalloidin (Invitrogen) at a 1:40 dilution in PBS containing 1% BSA for 1 h at RT. After three washings in PBS, they were then mounted with ProLong Gold antifade reagent (Invitrogen). The specificity of immunostaining was evaluated by omitting the antibodies against S. citri proteins. Immunofluorescence samples were imaged using a TCS SP2 upright Leica confocal laser scanning microscope, with a 40× water immersion or 63× oil immersion objective lens at 1,024 by 1,024 pixel resolution. The images were coded green (Alexa 488) and red (Alexa 568).

Partial purification of S. citri proteins involved in an interaction with actin.Polyclonal antibodies against chicken actin (Sigma) were linked to protein A-Sepharose CL-4B according to the manufacturer's manual (GE Healthcare). All steps were conducted on an ÄKTA purifier liquid chromatography system (GE Healthcare). Noninfected leafhopper proteins were prepared as described above for salivary gland proteins. To trap leafhopper actin with antiactin antibodies, 1 mg of leafhopper proteins was loaded on the column and proteins bound nonspecifically to the actin column were eliminated with a 10-ml step using 2 M NaCl. Then, 500 μg of S. citri proteins, prepared as described in the previous section for far Western experiments, was loaded on the actin column. S. citri proteins bound to actin were eluted with a 0 to 2 M NaCl gradient (total volume, 20 ml). All the eluted proteins were subjected to SDS-PAGE, followed by gel staining or far Western analysis. Nonspecific binding of S. citri proteins on protein A-Sepharose and/or on antiactin antibodies was highlighted by a control experiment carried out without C. haematoceps proteins loaded on the column. S. citri proteins eluted from such columns were also subjected to SDS-PAGE and far Western blotting. Far Western experiments were carried out according to the conditions described above. Blots of eluted proteins were incubated with a mixture of C. haematoceps proteins, and the binding activities were revealed with polyclonal antibodies against actin. Gels intended for mass spectrometry analysis were stained with colloidal blue (38).

Protein identification by LC-MS/MS.Proteins of interest were excised from stained gel and digested with trypsin as previously described (31). The resulting digestion was used for peptide mass fingerprinting by liquid chromatography-tandem mass spectrometry (LC-MS/MS) as routinely performed on the proteomics platform of the University of Bordeaux 2, France (22).

Expression and purification of S. citri phosphoglycerate kinase recombinant protein. S. citri uses the UGA opal codon to incorporate tryptophan rather than a stop codon as in the universal genetic code (16). For PGK expression in Escherichia coli, the two opal codons contained in the gene were changed by site-directed mutagenesis by using overlap extension PCR (23). Primers used in the PCR are presented Table 1. The amplified product of 1,239 bp corresponding to the pgk gene, in which the two opal codons were replaced by TGG codons, was inserted in a plasmid (pBS) and the construct was used to transform E. coli DH10B. The gene of interest was then transferred into a plasmid pET28a(+) vector (Novagen), and 1 μg of the recombinant plasmid (pET28 FL) was used to transform E. coli DH10B. The desired sequence of the resulting plasmid (pET28 FL) was verified by sequencing the insert. Two micrograms of the pET28 FL plasmid bearing the two desired mutations was used to transform E. coli BL21(DE3). Transformants were selected on Luria-Bertani (LB) solid medium containing kanamycin (50 μg/ml) at 37°C.

To check the expression of the PGK fused to the N-terminal hexahistidine sequence under the control of an isopropyl-β-d-thiogalactopyranoside (IPTG)-inducible promoter, one transformant was cultivated in 500 ml of LB medium with kanamycin (30 μg/ml) until late log phase (optical density at 600 nm, 0.6). The recombinant His₆-tagged PGK protein was produced at 28°C in culture by adding 0.1 mM IPTG for 3 h. Bacterial cells were centrifuged at 7,000 × g for 10 min at 4°C, and then the pellet was resuspended in 10 ml of lysis buffer (50 mM Tris-HCl [pH 8.0], 0.3 M NaCl, 25 mM MgSO₄, 2.5 mM MnCl₂, 1 mM PMSF, and 100 unit/ml of DNase I). After 10 min of incubation at RT, lysozyme was added at 0.2 mg/ml for 10 min. The mixture was lysed by sonication (rate, 50% pulses/s; 50 W; 4°C; 1 min of sonication and 1 min on ice alternatively) until a clear lysate was obtained. The insoluble proteins were removed by subsequent centrifugation at 13,000 × g at 4°C for 10 min. The recombinant protein was purified by affinity chromatography using Ni²⁺-nitrilotriacetic acid (Ni-NTA) prepacked columns (Qiagen) followed by cation exchange chromatography (Mono Q columns; GE Healthcare) on the ÄKTA purifier liquid chromatography system (GE Healthcare) according to the manufacturer's instructions.

The purified recombinant His₆-tagged PGK protein was tested with anti-His MAb and antiactin polyclonal antibodies in a Western blot experiment conducted as described previously (19).

Effect of PGK on S. citri attachment and invasion. (i) Attachment of S. citri to Ciha-1 cells.Monolayers of Ciha-1 cells grown in 24-well plates (approximately 2 × 10⁵ cells per well) were incubated with 900 μl of various concentrations of PGK from Saccharomyces cerevisiae (Sigma) or tagged PGK from S. citri in culture medium. Cells without PGK treatment were the positive controls, and uninfected cells served as negative controls. As additional controls, the effects of BSA (Sigma) and those of His₆-tagged eukaryotic initiation factor 4E (eIF4E) protein on S. citri adhesion were also assayed. Each assay condition was evaluated in triplicate. After incubation with proteins for 2 h at 32°C, the cells were infected with a 100-μl S. citri culture at a multiplicity of infection (MOI) of 15 to 30 for 4 h at 4°C. Normally, this temperature allows spiroplasma attachment to the cell surface but inhibits eukaryotic cell processes required for internalization. Following the incubation, the cells were then washed three times with 500 μl of Schneider's Drosophila medium to remove any spiroplasmas that had not attached to the monolayer. After trypsinization for 10 min at 32°C with TrypLE (Invitrogen), dilutions of the cells associated with adherent spiroplasmas were directly plated on solid SP4 medium. After incubation at 32°C for 1 week, the number of spiroplasma colonies on each plate was counted to estimate the number of Ciha-1 cells that had adherent spiroplasmas (4). In addition, treatment of spiroplasmas with trypsin alone in the absence of Ciha-1 cells had no effect on spiroplasma growth (18). The relative percentage of adhesion was calculated as follows: [(number of CFU for cells with protein treatment)/(number of CFU for untreated control cells)] × 100%. For statistical analysis, Student's t test was used when appropriate.

(ii) Spiroplasmal entry analysis.The effect of PGK treatment on the internalization of S. citri into Ciha-1 cells was determined as previously described using the gentamicin protection assay (4, 28). Until S. citri infection, all the steps were the same as those described above for attachment. Infection by 100 μl of S. citri culture at an MOI of 15 to 30 was carried out at 32°C for 18 h. The cells were thereafter washed three times with 500 μl of Schneider's Drosophila medium under low agitation to remove unbound bacteria. In order to eliminate bacteria that had attached but not internalized, cells were incubated with 1 ml of fresh culture medium containing 400 μg/ml of gentamicin (10 times the MIC) for 3 h at 32°C. Gentamicin was eliminated by three washes with 500 μl of Schneider's Drosophila medium followed by three additional washes with the same volume of PBS (1.54 mM KH₂PO₄, 155.17 mM NaCl, 2.71 mM Na₂HPO₄·7H₂O; pH 7.2). After washes, a 100-μl aliquot of the last wash was plated on SP4 medium to ensure that all extracellular bacteria had been killed (data not shown). Ciha-1 cells were trypsinized, plated on SP4 medium, and incubated at 32°C for 1 week. The number of colonies on each plate was counted to determine the number of cells in which S. citri was internalized.

Binding of His₆-tagged PGK to Ciha-1 cells.To determine whether the contact with Ciha-1 cells of His₆-tagged PGK, BSA, and His₆-tagged eiF4E has an effect on actin cytoskeleton, cells were grown on coverslips in 24-well plates and prepared by the same process described above until infection with S. citri. After the three washes in 500 μl of Schneider's Drosophila medium, cells on coverslips were fixed for 15 min in 500 μl of 4% formaldehyde at room temperature, washed three times with 500 μl of PBS, and permeabilized by incubation for 15 min with 500 μl of 0.1% Triton X-100 in PBS-1% BSA followed by another three times wash with 500 μl of PBS. Anti-His MAb at a 1:1,000 dilution was then added, followed by a secondary Alexa 514-conjugated rabbit anti-mouse antibody (1:200). Actin was stained with Alexa 568-phalloidin (1:40) and nuclei with 0.2 μg/ml of DAPI. The coverslips were rinsed once in water and were mounted onto slides as described above for salivary gland observations.

The images were coded with blue (DAPI), green (Alexa 488), and red (Alexa 568).

In vitro protein interaction and in vivo colocalization of S. citri with host cytoskeleton.Salivary gland proteins from healthy leafhoppers separated by SDS-PAGE in one dimension were stained with colloidal blue (Fig. 1A, lane 1) or blotted onto nitrocellulose and probed with S. citri proteins. Binding levels were detected with polyclonal IgGs against total S. citri proteins. Spiroplasma proteins were found to bind to a set of insect salivary gland proteins having apparent molecular masses of 42, 35, 30, 27, and 25 kDa (Fig. 1A, lane 2). In the control experiment, in which S. citri proteins were omitted from the overlay assay, no leafhopper protein was recognized by polyclonal IgGs (Fig. 1A, lane 3). The protein band with a mobility of ∼42 kDa was excised from the stained gel, washed, and digested with trypsin prior to LC-MS/MS analysis. The MS spectra matched, in the NCBI nonredundant protein database, those of actin from the fruit fly (Drosophila melanogaster), brine shrimp (Artemia sp.), tapeworm (Diphyllobothrium dendriticum), and silk moth (Bombyx mori). The finding that actin was involved in an in vitro interaction with S. citri proteins suggested that in vivo an interaction between S. citri and the insect actin cytoskeleton may be involved in spiroplasma invasion of cells.

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FIG. 1.

In vitro protein interactions and in vivo colocalization of S. citri with the actin filaments in leafhopper salivary glands. (A) Far Western experiments: binding of S. citri proteins to C. haematoceps salivary gland proteins separated by SDS-PAGE. Lane 1, gel electrophoresis pattern of salivary gland proteins (20 μg) stained with colloidal blue. Lane 2, blot from salivary gland proteins probed with S. citri proteins (20 μg). Anti-S. citri polyclonal IgGs were used to detect bound spiroplasma proteins. Peroxidase-conjugated goat anti-rabbit IgGs were used as secondary antibodies, and detection was performed with chemiluminescent substrate. Arrows on the right indicate the five significant binding activities between S. citri proteins and insect salivary glands proteins with apparent molecular masses of 42, 35, 30, 27, and 25 kDa. Lane 3, control blot not probed with S. citri proteins but subjected to the same treatment as mentioned for lane 2. The relative molecular masses are indicated on the left. (B) Confocal images of noninfected and S. citri-infected C. haematoceps salivary glands. Fixed salivary glands were incubated with S. citri antibodies. Detection was carried out with Alexa 488-conjugated anti-rabbit IgG (green fluorescence). Actin filaments were stained with Alexa 568-phalloidin (red). Photos to the right are higher magnifications of the areas outlined in white boxes. Bars, 20 μm.

Thus, the far Western results prompted us to explore by confocal laser scanning microscopy the location of S. citri in salivary glands. In those infected with S. citri (Fig. 1B), spiroplasmas (green fluorescence) are preferentially present along the actin filaments (red color). Salivary glands from noninfected leafhoppers did not show spots of green fluorescence specific to spiroplasmas (Fig. 1B).

Identification of one S. citri protein interacting in vitro with actin.To determine the S. citri proteins interacting with actin, spiroplasma proteins were loaded on a column of leafhopper actin that was trapped by antiactin antibodies linked to protein A-Sepharose. The presence of actin in the mixture of leafhopper proteins used to prepare the column (Fig. 2A, lane 1) was confirmed by Western blotting using antiactin antibodies (Fig. 2A, lane 2).

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FIG. 2.

Partial purification and identification of the S. citri 44-kDa protein. (A) For purification of the actin-binding protein, a mixture of S. citri proteins was loaded on an actin column composed of leafhopper actin trapped by antiactin antibodies linked to protein A-Sepharose. A control experiment was carried out by omitting the leafhopper actin protein. Lane 1, the mixture of leafhopper proteins used to prepare the column was analyzed by SDS-PAGE and stained with colloidal blue. Lane 2, proteins extracted from whole insects were transferred onto nitrocellulose membranes. Saturated blots were probed with rabbit polyclonal antibodies against chicken actin, followed by immunological detection as previously described (19). The band at 42 kDa reflects the presence of actin in the protein mixture and the specificity of the antiactin antibody. Lane 3, S. citri proteins eluted from the antiactin protein A-Sepharose column were analyzed by SDS-PAGE. These proteins are linked to protein A-Sepharose or/and to actin antibodies (control experiment). Lane 4, S. citri proteins eluted from the actin column were analyzed by SDS-PAGE. Lane 5, binding of leafhopper proteins to S. citri proteins eluted from the actin column. The blot of S. citri proteins was probed with the mixture of leafhopper proteins containing actin. Binding was detected with rabbit antiactin antibodies followed by goat anti-rabbit antibodies labeled with peroxidase. Detection was performed with chemiluminescent substrate. Only one protein at 44 kDa was found to interact with leafhopper actin, corresponding with the apparent molecular mass of the additional band present in lane 4. (B) LC-MS/MS analysis of the 44-kDa protein identified phosphoglycerate kinase. Three distinct peptides matching the protein sequence are marked in bold. The underlined sequence in bold of 63 amino acids consists of four separate peptides overlap by several amino acids: KIGNSLLEVDKVEIAKT, KTFLAKGQGKIILPIDALEAPEFADVPAKV, KIILPIDALEAPEFADVPAKV, and KVTTGFDIDDGYMGLDIGPKT. Sequence coverage was 23.79%.

As shown in Fig. 2A, lane 3, S. citri proteins eluted from the antiactin-protein A-Sepharose column were those captured on Sepharose and/or on actin antibodies (control experiment). Figure 2A, lane 4, shows the profile of S. citri proteins eluted from the actin column. Comparison of the two protein patterns revealed one protein at 44 kDa present among the proteins bound to actin but absent in the control profile. To determine whether this protein displayed an affinity for actin, a far Western assay was performed using as overlay a mixture of whole leafhopper C. haematoceps proteins containing actin. A significant binding activity located at approximately 44 kDa was revealed by rabbit antiactin IgGs followed by goat anti-rabbit antibodies labeled with peroxidase (Fig. 2A, lane 5). The corresponding protein band was excised from the colloidal blue-stained gel (Fig. 2A, lane 4) and used for MS analysis. Two proteins of S. citri were identified, one with a molecular mass of 44.5 kDa and a second with a mass of 46 kDa. Seven identified peptides matched those of the PGK protein (44.5 kDa) annotated as SPICI 03-024 in the sequence of S. citri strain GII3 and covered 23.79% of the whole theoretical protein sequence (Fig. 2B). For the 46-kDa protein, nine peptides were identified as being a part of a lipoprotein (24%) annotated as SPICI 03-098 in the S. citri genome. Due to previous reports indicating that PGK has actin-binding properties, we decided to focus our attention on it.

In vitro and ex vivo interaction of S. citri tagged-PGK with leafhopper actin. (i) Purified tagged PGK binds leafhopper actin in a gel overlay assay.His₆-tagged PGK purified by Ni²⁺-NTA chromatography followed by cation exchange chromatography was analyzed by SDS-PAGE (Fig. 3, lane 1). As expected, only one protein band was observed at 44 kDa, and the presence of the tag was confirmed in a Western blot assay performed with anti-His MAb (Fig. 3B). To determine whether His₆-tagged PGK displays an affinity for actin, far Western assays were performed using a leafhopper protein mixture. One significant binding activity was revealed with rabbit antibodies against chicken actin (Fig. 3, lane 2), which suggested an interaction between the His₆-tagged PGK and actin protein present in the insect protein mixture (Fig. 3B). One additional control, to confirm that the binding signal in lane 2 was only due to the presence of actin in the insect preparation, revealed no reaction between the His₆-tagged PGK preparation and antiactin antibodies (Fig. 3B).

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FIG. 3.

In vitro interaction between S. citri His₆-tagged PGK and leafhopper actin. (A) Lane 1, His₆-tagged PGK purified by Ni²⁺-NTA chromatography followed by cation exchange chromatography was analyzed by SDS-PAGE and stained with colloidal blue. Lane 2, a far Western assay was performed using a leafhopper protein mixture with actin as the overlay. Polyclonal antiactin antibodies were used to detect an interaction. Peroxidase-conjugated goat anti-rabbit IgGs were used as secondary antibodies, and detection was performed with chemiluminescent substrate. (B) Purified S. citri His₆-tagged PGK and insect proteins used in the far Western assays were tested with anti-His MAb and antiactin antiserum. M, molecular mass marker; relative molecular masses are indicated on the left.

(ii) Purified His₆-tagged PGK colocates with actin of leafhopper Ciha-1 cells.To see the potential effect of PGK protein on the host cell actin cytoskeleton, about 2 × 10⁵ Ciha-1 cells in insect culture medium were incubated with various quantities of tagged PGK for 2 h at 32°C. As a control, Ciha-1 cells were treated with BSA, at concentration equal to that of PGK. As an additional control, the effect of an unrelated His₆-tagged protein (eIF4E) on Ciha-1 cells was also assayed. Compared to untreated control cells (Fig. 4A), Ciha-1 cells treated for 2 h at 32°C with 400 μg/ml of BSA (Fig. 4B) or tagged eIF4E (Fig. 4C) showed no alteration in the cell cytoskeleton. In the three cases, the cells were of similar size, showed no obvious morphological differences, and the actin filaments stained with phalloidin were clearly visible. These observations suggested that exogenous proteins and in particular the polyhistidine tag have no effect on the Ciha-1 cells. When the cells were treated with His₆-tagged PGK (400 μg for 2 × 10⁵ cells), faint cellular changes were observed and the major alterations were on the actin filaments (Fig. 4D). The immunofluorescent staining allows the detection of distinct spots of aggregated tagged PGK (Fig. 4E) colocated with actin filaments (Fig. 4F). These observations confirm the interaction observed in vitro between PGK and actin.

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FIG. 4.

Colocalization of purified S. citri His₆-tagged PGK with actin filaments in leafhopper cells (Ciha-1). Confocal images show Ciha-1 cells untreated (A) or treated for 2 h with various proteins at 400 μg/ml, BSA (B), His₆-tagged eIF4E protein (C), and S. citri His₆-tagged PGK (D). The three images in panels D, E, and F represent the same area. Cells were stained for nuclei with 4′,6-diamidino-2-phenylindole (blue). Cellular actin was stained with Alexa 568-phalloidin (red; A, B, C, and D). (E) Detection of His₆-tagged PGK was performed with anti-His MAb serum followed by a secondary Alexa 514-conjugated rabbit anti-mouse antibody (green). (F) Merged immunofluorescent images from those in panels D and E. The coincidence of PGK and F-actin staining appears in yellow. Bars, 8 μm.

Treatment of Ciha-1 cells with tagged PGK has no effect on spiroplasma attachment.If spiroplasmal PGK is involved in adhesion to host cell surfaces, adding exogenous PGK to Ciha-1 cells should competitively inhibit the attachment of S. citri to this cell line. We took advantage of the PGK sequence conservation among species to include in our experiment the commercially prepared PGK from S. cerevisiae (hereafter referred to as ScPGK). Cells without PGK treatment were the positive controls, and cells treated prior to infection with S. citri with BSA and His₆-tagged eIF4E were also included as a control. As seen in Fig. 5, no difference was observed between the number of CFU obtained with untreated cells and those obtained with cells preincubated with 50 or 400 μg/ml of BSA or with 400 μg/ml of His₆-tagged eIF4E. Preincubation of Ciha-1 cells with either ScPGK (10, 25, 40, or 50 μg/ml) or recombinant His₆-tagged PGK from S. citri (10, 25, 40, 50, 100, 200, or 400 μg/ml) prior to incubation with spiroplasmas had no effect on the attachment of S. citri to Ciha-1 cells. The percentage of cells with attaching spiroplasmas varied between 80 and 100%, whatever the concentration and origin of PGK. These results suggest that the interaction between PGK and actin is not involved in the attachment of S. citri to insect cells.

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FIG. 5.

Effect of PGK treatment on S. citri attachment to Ciha-1 cells. Monolayers of Ciha-1 cells were incubated for 2 h at 32°C with various amounts of PGK from S. cerevisiae, and His₆-tagged PGK from S. citri, BSA, or His₆-tagged eIF4E. After incubation, the cells were infected with S. citri at a MOI of 15 to 30 for 4 h at 4°C. Untreated cells infected under the same conditions were the positive controls. Then, the cells were washed and plated on SP4 solid medium. After spiroplasma growth at 32°C, the number of colonies was counted to evaluate the number of cells associated with adherent spiroplasmas. Each value represents the mean of two independent triplicate assays. Vertical lines represent standard error of the mean. Student's t test was used, and no statistical differences were found. Black bars, untreated cells; bars with diagonal hatching, S. cerevisiae PGK; brick-filled bars, S. citri His₆-tagged PGK; white bars, controls.

Treatment with PGK from S. citri or S. cerevisiae inhibits S. citri internalization.Ciha-1 cells were infected with S. citri, and the percentage of internalized bacteria was determined in a gentamicin protection assay. This assay involves the use of gentamicin to kill any noninternalized spiroplasma after an infection period of 18 h. As shown in Fig. 6, the entry levels of S. citri in the untreated Ciha-1 cells and in cells incubated with BSA (50 or 400 μg/ml) appeared to be comparable even at the highest concentration. Treatment with tagged eIF4E at a concentration of 400 μg/ml did not affect the number of spiroplasmas able to invade the cells; invasion was not reduced by any more than 10%. This result supports the conclusion that the polyhistidine tag does not inhibit the entrance of S. citri in the cells.

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FIG. 6.

Inhibition of S. citri invasion into Ciha-1 cells by PGK treatment. Monolayers of Ciha-1 cells were incubated for 2 h at 32°C with various amounts of PGK from S. cerevisiae, His₆-tagged PGK from S. citri, BSA, or His₆-tagged eIF4E prior to infection with S. citri. Following infection with S. citri at a MOI of 15 to 30 for 18 h at 32°C, the cells were treated with gentamicin (400 μg/ml) for 3 h at 32°C to kill attached spiroplasmas. Then, the cells were trypsinized and plated on SP4 medium for counting the infected cells. Cells without protein treatment before infection were the positive controls. Each value represents the mean of two independent triplicate assays. Vertical lines represent standard errors of the means. *, significant differences (P < 0.001; Student's t test) between S. citri internalization with PGK, BSA, and eIF4E treatment and S. citri internalization without any protein treatment. Black bars, untreated cells; bars with diagonal hatching, S. cerevisiae PGK; brick-filled bars, S. citri His₆-tagged PGK; white bars, controls.

S. citri showed an ∼20-fold decrease in its ability to invade Ciha-1 cells treated with ScPGK at a concentration of 50 μg/ml compared to untreated cells (P < 0.001) (Fig. 6). With the same concentration of S. citri His₆-tagged PGK, the entry of spiroplasmas in Ciha-1 cells was not noticeably affected. Higher concentrations significantly diminished invasion by S. citri but less than with ScPGK. An S. citri His₆-tagged PGK concentration of 50 μg/ml reduced entry of spiroplasmas into Ciha-1 cells by 10%, at 100 μg/ml by 30% (P < 0.001), at 200 μg/ml by 50% (P < 0.001), and at 400 μg/ml by 70% (P < 0.001).