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Invertebrate Microbiology

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

Fabien Labroussaa, Nathalie Arricau-Bouvery, Marie-Pierre Dubrana, Colette Saillard
Fabien Labroussaa
INRA et Université Victor Ségalen Bordeaux 2, UMR 1090 Génomique Diversité Pouvoir Pathogène, 71 Avenue Edouard Bourlaux BP 81, F-33883 Villenave d'Ornon, France
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Nathalie Arricau-Bouvery
INRA et Université Victor Ségalen Bordeaux 2, UMR 1090 Génomique Diversité Pouvoir Pathogène, 71 Avenue Edouard Bourlaux BP 81, F-33883 Villenave d'Ornon, France
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Marie-Pierre Dubrana
INRA et Université Victor Ségalen Bordeaux 2, UMR 1090 Génomique Diversité Pouvoir Pathogène, 71 Avenue Edouard Bourlaux BP 81, F-33883 Villenave d'Ornon, France
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Colette Saillard
INRA et Université Victor Ségalen Bordeaux 2, UMR 1090 Génomique Diversité Pouvoir Pathogène, 71 Avenue Edouard Bourlaux BP 81, F-33883 Villenave d'Ornon, France
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  • For correspondence: saillard@bordeaux.inra.fr
DOI: 10.1128/AEM.02384-09
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ABSTRACT

Transmission of the phytopathogenic mollicutes, spiroplasmas, and phytoplasmas by their insect vectors mainly depends on their ability to pass through gut cells, to multiply in various tissues, and to traverse the salivary gland cells. The passage of these different barriers suggests molecular interactions between the plant mollicute and the insect vector that regulate transmission. In the present study, we focused on the interaction between Spiroplasma citri and its leafhopper vector, Circulifer haematoceps. An in vitro protein overlay assay identified five significant binding activities between S. citri proteins and insect host proteins from salivary glands. One insect protein involved in one binding activity was identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS) as actin. Confocal microscopy observations of infected salivary glands revealed that spiroplasmas colocated with the host actin filaments. An S. citri actin-binding protein of 44 kDa was isolated by affinity chromatography and identified by LC-MS/MS as phosphoglycerate kinase (PGK). To investigate the role of the PGK-actin interaction, we performed competitive binding and internalization assays on leafhopper cultured cell lines (Ciha-1) in which His6-tagged PGK from S. citri or purified PGK from Saccharomyces cerevisiae was added prior to the addition of S. citri inoculum. The results suggested that exogenous PGK has no effect on spiroplasmal attachment to leafhopper cell surfaces but inhibits S. citri internalization, demonstrating that the process leading to internalization of S. citri in eukaryotic cells requires the presence of PGK. PGK, regardless of origin, reduced the entry of spiroplasmas into Ciha-1 cells in a dose-dependent manner.

Phloem-feeding leafhoppers transmit plant pathogenic mollicutes, spiroplasmas, and phytoplasmas from plant to plant in a persistent propagative manner (26, 43). These phytopathogenic mollicutes are restricted to phloem and to certain vector tissues; thus, their vectors are phloem sap-feeding specialists. After being ingested from plant phloem by their insect vectors, they traverse the insect gut wall, move into the hemolymph, where they multiply, and invade the salivary glands (20, 33, 34, 36). During their movements in the insect vector until its transmission to a new host plant, spiroplasmas and phytoplasmas must traverse two major physical barriers, namely, the insect intestine and the salivary gland (35, 53). Until now, little was known about the molecular and cellular interactions contributing to the crossing of these physical barriers. Several lines of evidence suggest that host-pathogen interactions could be a prerequisite for invasion and colonization of insect vector organs (2, 48, 53). For human and animal pathogenic mollicutes, it is well established that successful colonization of the host cells requires adhesion as the first step. This event is mediated by surface proteins, and among these proteins adhesins play an important role (8, 44). Recently, it was reported that an antigenic membrane protein (Amp) of onion yellow phytoplasma interacts with the insect microfilament complex and that interaction plays an important role in determining the insect vector specificity (48). Several other immunodominant membrane proteins from various phytoplasmas have been mentioned in the literature as candidates for involvement in host-phytoplasma interactions (29, 30).

Spiroplasma citri, the first phytopathogenic mollicute available in culture (45), has emerged as an outstanding model for studying spiroplasma interactions with its two hosts: the periwinkle plant and the insect vector Circulifer haematoceps. Following observations of membrane-bound cytoplasmic vesicles of midgut epithelium and salivary gland cells, S. citri was hypothesized to cross these physical barriers by receptor-mediated cell endocytosis (3, 33, 39). Several S. citri protein candidates have been identified as involved in transmission and, for a few of them, in an interaction with leafhopper vector proteins. Spiralin, the most abundant membrane protein, was suspected to be involved in the transmission for two reasons: (i) a S. citri spiralinless mutant was less effective in its transmissibility (19); (ii) spiralin acted in vitro as a lectin able to bind to glycoproteins of insect vectors and therefore might function as a ligand able to interact with leafhopper receptors (32). In addition, the ability of S. citri to be transmitted by C. haematoceps is clearly affected by disruption of a gene predicted to encode a lipoprotein with homology to a solute-binding protein of an ABC transporter (14). The proteome of nontransmissible S. citri strains specifically lacks adhesion-related proteins (ScARPs) and the membrane-associated protein P32 present in the proteome of transmissible strains (12, 13, 31). These proteins are encoded by plasmids pSci1 to -6 (46), which are present only in transmissible strains, and ScARPs share strong similarities with the adhesion-related protein SARP1 of S. citri strain BR3, in which the presence has been correlated to the ability for the spiroplasma to adhere to insect cells in vitro (9, 55). The specific interactions of S. citri with eukaryotic cells remain to be elucidated, but a combination of the effects of several proteins or a complex would be necessary to explain the invasion of a variety of host cell types by S. citri (33).

Nevertheless, in the last sequence of events involved in insect vector transmission, the first contact and recognition for the efficient penetration of the salivary gland cells represents an essential step. In the present study, confocal images of infected salivary glands show the localization of S. citri cells along the actin filaments. We report the results of the first attempt to decipher the role of the spiroplasma's phosphoglycerate kinase (PGK) in the internalization of S. citri in its insect vector's cells.

MATERIALS AND METHODS

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.

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TABLE 1.

Primers used for site-directed mutagenesis of S. citri PGKa

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).

RESULTS

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.

FIG. 1.
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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).

FIG. 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).

FIG. 3.
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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.

FIG. 4.
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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.

FIG. 5.
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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.

FIG. 6.
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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).

DISCUSSION

Salivary gland invasion is an essential step of the phytopathogenic mollicutes life cycle in their leafhopper vectors. Fluorescence microscopy of S. citri-infected salivary glands showed localization of the spiroplasmas along the actin filament. These results are in good agreement with those obtained with “Candidatus Phytoplasma asteris” (OY strain) (48) in which the surface immunodominant membrane protein (Amp) forms a complex with three leafhopper proteins involved in a microfilament complex. According to the authors, these interactions determined the insect-vector specificity of the phytoplasmas and played a role in the transmission.

The preferential location of spiroplasmas along actin filaments suggested that invasion in the host cells could involve interactions between S. citri proteins and the cytoskeletal actin. These occurrences were not completely unexpected, as involvement of host cell actin in bacterial invasion has been demonstrated in a number of bacterial pathogens, such as species of Shigella (24), Salmonella (56), and Streptococcus (40, 51). It has been often reported that surface proteins are implicated in the interaction between bacteria and host microfilaments (17, 25). Our microscopic analysis highlighted the presence of actin-binding protein(s) on the membrane proteins of S. citri.

One candidate protein identified was PGK. PGK is a transferase enzyme used in the seventh step of glycolysis. It transfers a phosphate group from 1,3-biphosphoglycerate to ADP, forming ATP and 3-phosphoglycerate. It was at first somewhat surprising to find an interaction between host cell actin and PGK, a glycolytic enzyme classically described as a cytoplasmic protein. Only one copy of the gene is present in the S. citri genome sequence, and the deduced amino acid sequence of the protein has no N-terminal signal peptide and does not possess a C-terminal anchor sequence or typical transmembrane domain required for protein insertion and folding in membranes. Yet, proteomic analysis of membrane protein fractions has identified PGK, in a two-dimensional SDS-PAGE, as an integral or peripheral membrane protein (M. P. Dubrana, personal communication). Nevertheless, there is precedent for microbial surface proteins without transmembrane domains, such as enolase (10, 54), glyceraldehyde-3-phosphate dehydrogenase (11), and 6-phosphogluconate-dehydrogenase (49). Moreover, it is not the first time that PGK was found associated with a membrane. A proteomic analysis of Streptococcus agalactiae identified PGK as a major outer surface protein (27), and in the opportunistic pathogen Candida albicans several lines of evidence support the conclusion that PGK is present at the outer surface of the cell membrane and cell wall as well as in the cytoplasm as expected (1). Despite the assigned function of PGK, it may well have some other roles to play in bacteria. The finding that the glycolytic enzyme PGK of S. citri interacts with eukaryotic actin is supported by earlier studies that provided evidence that several glycolytic enzymes from rabbit muscle can be bound in vitro to actin while retaining the enzymatic activity (5). Studies of actin recruitment to the site of streptococcal attachment to cells suggest that streptococcus PGK acts as an actin-binding protein (15, 51).

When the S. citri His6-tagged PGK was in contact with Ciha-1 cells, it was found to be colocated with actin filaments. Although actin is mainly an intracellular constituent, this protein has also been reported to be present on the cell surface of various cells (37). Fluorescent spots of tagged PGK are probably located at the surface of cells, and so this protein is not in the Ciha-1 cells. To determine if the PGK-actin interaction contributes to adhesion and/or internalization during S. citri infection, the ability of PGK to inhibit spiroplasma adhesion or/and internalization was tested in a competitive binding and invasion assay. Our results suggested that PGKs from spiroplasmas and from S. cerevisiae do not interfere with adhesion of S. citri to Ciha-1 cells, although actin has been reported to be present on the cell surface of various cells (37). However, both PGKs inhibited internalization into Ciha-1 cells in a dose-dependent manner. The difference observed between the two PGK proteins could be due to a difference in the protein conformations. PGK from S. cerevisiae is a nonrecombinant commercial protein, whereas spiroplasmal PGK is a tagged protein, and the polyhistidine tag could slightly modify the conformation of the protein interaction with the actin. Moreover, during expression in E. coli, posttransductional modifications may also disturb the conformation of the protein and modify the enzymatic activity. As in group B streptococci, these results suggest that the PGK-actin interaction plays a role in the internalization but not in the attachment to the cells (15). Interestingly, streptococcal internalization into eukaryotic cells is completely abolished by PGK from S. cerevisiae, but the experiment was not conducted with the streptococci PGK. Others have reported that several bacterial glycolytic enzymes, including glyceraldehyde-3-phosphate dehydrogenase (42, 47), enolase (41), and PGK (15), bind to host cell cytoskeletal proteins like fibronectin, myosin, and actin. In Mycoplasma fermentans, the glycolytic enzyme α-enolase is a membrane-associated protein located on the cell surface that binds to plasminogen (54), as in Streptococcus pneumoniae (10). One other human mycoplasma, Mycoplasma pneumoniae, has been reported to bind fibronectin through cytoplasmic proteins with well-known biological functions, elongation factor Tu and pyruvate dehydrogenase E1 β-subunit (6, 7).

The identification in this work of PGK, well known to be a glycolytic enzyme present in the cytosol, as an actin-binding protein associated with the membrane was unexpected and indicates secondary functions for PGK. In the invasion process, the early steps in adhesion could involve interactions between specific proteins exposed at the spiroplasma surface, such as spiralin, Sc76, ScARPs, and receptors on the surface of the host cells. The lipoprotein annotated SPICI 03-098, detected as a candidate for interaction with actin protein, could belong to this group of S. citri proteins putatively involved in the transmission. These early steps would be important in determining the insect-vector specificity. Once the specificity is established, late steps in the invasion process would not necessarily be specific. Taken together, our results show that the interaction between the two highly conserved proteins, PGK and actin, might constitute one of these late steps involved in internalization of S. citri into cells. In this respect, it was hypothesized that PGK might play a role in the transmission. To test this hypothesis, the experimental transmission of mutants generated by inactivation of the S. citri pgk gene through homologous recombination is currently under investigation.

ACKNOWLEDGMENTS

We give special thanks to Michel Castroviejo and Laure Béven for thoughtful advice and technical suggestions for protein purification. Bénédicte Doublet kindly provided the His6-tagged eIF4E.

This work was supported with funding from INRA and Université Victor Ségalen Bordeaux 2. F.L. was supported by a Ph.D. fellowship from the Ministère de l'Enseignement Supérieur et de la Recherche.

FOOTNOTES

    • Received 1 October 2009.
    • Accepted 18 January 2010.
  • Copyright © 2010 American Society for Microbiology

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Entry of Spiroplasma citri into Circulifer haematoceps Cells Involves Interaction between Spiroplasma Phosphoglycerate Kinase and Leafhopper Actin
Fabien Labroussaa, Nathalie Arricau-Bouvery, Marie-Pierre Dubrana, Colette Saillard
Applied and Environmental Microbiology Mar 2010, 76 (6) 1879-1886; DOI: 10.1128/AEM.02384-09

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Entry of Spiroplasma citri into Circulifer haematoceps Cells Involves Interaction between Spiroplasma Phosphoglycerate Kinase and Leafhopper Actin
Fabien Labroussaa, Nathalie Arricau-Bouvery, Marie-Pierre Dubrana, Colette Saillard
Applied and Environmental Microbiology Mar 2010, 76 (6) 1879-1886; DOI: 10.1128/AEM.02384-09
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KEYWORDS

Actins
Hemiptera
Phosphoglycerate Kinase
Protein Interaction Mapping
Spiroplasma citri

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