Entry of Spiroplasma citri into Circulifer haematoceps cells involves interaction between spiroplasma phosphoglycerate kinase and leafhopper actin

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 His(6)-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.

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.

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%.

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.

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.

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.

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.