1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Spiroplamas are helical, cell wall-less bacteria belonging to the Class Mollicutes, a group of microorganisms phylogenetically related to low G+C, Gram-positive bacteria. Spiroplasma species are all found associated with arthropods and a few, including Spiroplasma citri are pathogenic to plant. Thus S. citri has the ability to colonize cells of two very distinct hosts, the plant and the insect vector. While spiroplasmal factors involved in transmission by the leafhopper Circulifer haematoceps have been identified, their specific contribution to invasion of insect cells is poorly understood. In this study we provide evidence that the lipoprotein spiralin plays a major role in the very early step of cell invasion. Confocal laser scanning immunomicroscopy revealed a relocalization of spiralin at the contact zone of adhering spiroplasmas. The implication of a role for spiralin in adhesion to insect cells was further supported by adhesion assays showing that a spiralin-less mutant was impaired in adhesion and that recombinant spiralin triggered adhesion of latex beads. We also showed that cytochalasin D induced changes in the surface-exposed glycoconjugates, as inferred from the lectin binding patterns, and specifically improved adhesion of S. citri wild-type but not of the spiralin-less mutant. These results indicate that cytochalasin D exposes insect cell receptors of spiralin that are masked in untreated cells. In addition, competitive adhesion assays with lectins strongly suggest spiralin to exhibit glycoconjugate binding properties similar to that of the Vicia villosa agglutinin (VVA) lectin.


  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Spiroplasmas are motile, helical bacteria belonging to the Class Mollicutes, a group of microorganisms having no cell wall though phylogenetically related to Gram-positive bacteria (Weisburg et al., 1989). Spiroplasmas are associated with a wide range of eukaryotic hosts, mostly insects and crustaceans both belonging to the Phylum Arthropoda, and thus, occupy diverse ecological niches. Some are commensals in the gut of leafhoppers and beetles (Konai et al., 1996; Ammar et al., 2011) while others behave as symbionts in Drosophila, weevil and aphid (Mateos et al., 2006; Herren et al., 2013; Łukasik et al., 2013; Merville et al., 2013) or pathogens in honeybee, Drosophila, moth, mosquito, crab and shrimp (Mouches et al., 1984; Clark et al., 1985; Humphery-Smith et al., 1991; Vazeille-Falcoz et al., 1994; Phillips and Humphery-Smith, 1995; Williamson et al., 1999; Anbutsu and Fukatsu, 2003; Wang et al., 2004; 2011; Nunan et al., 2005; Tabata et al., 2011). Therefore spiroplasmas are extensively studied for both their potential interest as biocontrol agents of insect pests (Anbutsu and Fukatsu, 2011; Lo et al., 2013) and their ability to confer insect resistance or tolerance to pathogenic fungi, parasitic nematodes and parasitoid wasps (Jaenike et al., 2010; Xie et al., 2010; 2011; Haselkorn et al., 2013; Łukasik et al., 2013).

Among the many spiroplasma species, three (Spiroplasma citri, S. kunkelii and S. phoeniceum) are pathogenic to plants. Plant pathogenic mollicutes, including phytoplasmas and spiroplasmas, are responsible for severe diseases affecting a wide variety of crops worldwide (Gasparich, 2010). They inhabit the phloem sieve tubes in which they are introduced by phloem-sap feeding insects. Thus, phytoplasmas and spiroplasmas multiply in two distinct hosts, the host plant and the insect vector.

Spiroplasma citri is the causative agent of citrus stubborn disease and horseradish brittle root disease (Saglio et al., 1971; Fletcher et al., 1981) and is transmitted from plant to plant by the leafhoppers Circulifer haematoceps (Fos et al., 1986) and Circulifer tenellus (Liu et al., 1983), in a persistent propagative manner. In contrast to phytoplasmas which could not be cultured so far, spiroplasmas and S. citri in particular has been cultured in cell-free medium since 1971 (Saglio et al., 1971; 1973). Over recent years S. citri has been made amenable to genetic manipulation providing new biological tools for studying the relationships of the spiroplasma with its insect and plant hosts (Bové et al., 2003; Duret et al., 2003; Breton et al., 2010; Renaudin et al., 2014). In addition to being a model organism for studying interactions of the various spiroplasma species with their arthropod hosts, the S. citri/C. haematoceps system also performs as a convenient model for deciphering at the molecular level the mechanisms of insect-transmission of phytoplasmas, which share the same ecological niches, phloem sap and insect haemolymph, but for which no pure culture and no mutants are available.

Successful transmission of S. citri requires the spiroplasmas to be acquired by the leafhopper vector feeding on the infected plant, multiply in the insect haemocoel and invade the insect organs before to be released along with saliva into a new plant through insect feeding. During this process, internalization of spiroplasmas into insect cells is an obligate step which requires intimate interactions, for both crossing the gut epithelium (acquisition) and invading the salivary gland cells (transmission). A remarkable feature of S. citri is its ability to undergo morphological changes from helical, in the phloem sap and insect haemolymph, to rounded shape when internalized into insect cells (Kwon et al., 1999), suggesting robust interactions between spiroplasmal and host cell membrane proteins during receptor-mediated endocytosis (Fletcher et al., 1998). Multiple components have been shown to be involved in transmission of S. citri by its leafhopper vector C. haematoceps (Duret et al., 2003; Boutareaud et al., 2004; Berho et al., 2006; Killiny et al., 2006; Breton et al., 2010; Labroussaa et al., 2011). Three of them, the phosphoglycerate kinase (PGK), the adhesin ScARP3d (S. citri adhesion-related protein 3d), and the lipoprotein spiralin, have been shown to directly interact with insect cell proteins in vitro or ex vivo. The PGK of S. citri GII3 was found to bind insect actin and competitive assays proved its contribution to the process of spiroplasma internalization into cultured insect cells (Labroussaa et al., 2010; 2011). The adhesin ScARP3d was shown to mediate adhesion and entry of spiroplasmas into the leafhopper cells through interaction with its surface-exposed, repeat domain (Béven et al., 2012). Finally, the lipoprotein spiralin of S. citri GII3, known to be required for efficient transmission of S. citri to periwinkle by C. haematoceps (Duret et al., 2003), was shown to act in vitro as a lectin binding insect glycoproteins although a limited set of lectins was used in this study (Killiny et al., 2005). However, investigation of the lectin-like activity of spiralin in vivo is required to assess the role of spiralin-glycoconjugates interactions in the process of insect cell invasion.

Spiralin is the most abundant protein of the S. citri membrane as it could account for more than 20% of the total membrane protein content (Wróblewski et al., 1977; 1989). Spiralin can assemble into homomultimers at the spiroplasma surface (Wróblewski, 1981) and a ‘carpet’ model has been proposed in which spiralin organization would protect the spiroplasma membrane by covering most if not all the lipids present in the outer leaflet of the bilayer (Castano et al., 2002). In this study we show that spiralin is required for adhesion and entry of S. citri into cells of the C. haematoceps cell line Ciha-1 (Duret et al., 2010). During adhesion, spiralin relocalizes at the contact pole of spiroplasmas and acts as an adhesin with a VVA lectin-like activity.


  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Spiralin accumulates at the contact zone of spiroplasmas adhering to Ciha-1 cells

The morphological changes of S. citri GII3 infecting the Ciha-1 cell line were examined by TEM 1 h and 18 h after the spiroplasmas were added to the leafhopper cells. Transmission electron micrographs revealed spiroplasmas having different morphologies including regular helices similar to those observed in pure culture (Fig. 1Aa). Helical spiroplasmas were found to adhere to insect cells by one end and were only seen at the earlier stage post infection (1 h). At later stages, most spiroplasmas displayed an elongated pear-shaped, flask-shaped or ovoid morphology (Fig. 1Ab–d respectively). The images also showed tight contacts between the spiroplasma surface and the insect cell membrane (Fig. 1A, arrowheads). The finding that the proportion of spiroplasmas having helical morphology decreased continually with the time post infection suggests these morphological changes to be a dynamic process. Changing morphology from elongated to ovoid increases the contact area, and hence is expected to strengthen the attachment of the spiroplasmas to the host cells.


Figure 1. Morphological changes of S. citri during adhesion to the Ciha-1 cells and immunostaining showing accumulation of spiralin at the contact zone.

A. Transmission electron micrographs of spiroplasmas 1 h (a) and 18 h (b and d) after infection of Ciha-1 cells by S. citri GII3. a, helical spiroplasma attached by one end to the host cell; b and c, elongated spiroplasmas; d, round-shaped spiroplasmas. *, spiroplasmas; arrowheads indicate the contact zones between spiroplasmas and insect cells. Scale bar, 500 nm (a and d), 200 nm (b and c).

B. Immunofluorescent staining of isolated and adherent spiroplasmas. Insect cells were incubated with S. citri GII3 for 1 h, and after fixation, the spiroplasmas were labelled with anti-spiralin PAbs and Alexa 488-secondary antibodies. a to d, maximal (left) and average (right) projections of confocal images of S. citri GII3; a, isolated spiroplasma; b to d, spiroplasma images were divided into two distinct zones, the contact (CA) and distal (DA) areas.

C. Comparison of fluorescence intensity (integrated density per area) in the CA and DA of adherent S. citri GII3 spiroplasmas. Each bar represents the average value ± SD of 35 spiroplasmas.

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Such tight interactions of spiroplasmas with insect cells have been previously observed by TEM (Wayadande and Fletcher, 1998; Kwon et al., 1999). In many pathogenic bacteria, adherence to host cells is oriented and generally associated with localization of adhesins at the cell pole (Hogan and Kolter, 2002; Jones et al., 2003; Chiang et al., 2005). Therefore one may reasonably hypothesize that adhesion of S. citri to insect cells starts with the localization of adhesins at the contact zone of elongated cells. Spiralin has been shown to bind insect proteins in vitro (Killiny et al., 2005). To investigate whether spiralin was implicated in adhesion to insect cells ex vivo, distribution of spiralin at the spiroplasma surface was examined by immunofluorescent staining. Ciha-1 cells were infected by S. citri GII3 and incubated for 1 h before the spiroplasmas were labelled with anti-spiralin polyclonal antibodies (PAbs) and Alexa 488-secondary antibodies. Confocal laser scanning microscope (CLSM) observations of isolated spiroplasmas showed that the spiralin was uniformly distributed along the spiroplasmal surface (Fig. 1Ba). A similar distribution of spiralin has also been reported in isolated Spiroplasma melliferum by using TEM (Archer and Townsend, 1981; Townsend and Plaskitt, 1985). In contrast, when adhering to insect cells, the spiroplasmas clearly exhibited two distinct fluorescent areas: one with high fluorescence intensity corresponding to the spiroplasma end attached to the insect cells (which we named CA for Contact Area), the other with low fluorescence intensity and named distal area (DA) (Fig. 1Bb–d). The fluorescence intensity per area (Integrated density/area) was significantly higher in the CA than in the DA (Fig. 1C). The images obtained by maximal and average intensity projections yielded similar results. These data clearly showed that spiralin accumulates at the CA of spiroplasmas adhering to insect cells. They also indicated that spiralin relocalization takes place in the very early steps of spiroplasma adhesion to insect cells. Polar or patchy distribution of proteins, namely adhesins, is known to ensure robust attachment of pathogenic bacteria to their host cells. In the case of S. citri the finding that spiralin relocalized at the CA prompted us to investigate the role of this protein in the invasion of insect cells by the spiroplasmas.

Spiralin triggers adhesion of S. citri to Ciha-1 cells

To assess the role of spiralin in adhesion of S. citri to Ciha-1 cells, we compared the adhesion capability of the spiralin-less mutant GII3-9a3 to that of the wild-type strain GII3. Both strains yielded similar adhesion and invasion rates: 51% (± 7.7) and 49% (± 5.3) of Ciha-1 cells with adherent spiroplasmas, and 1.2% (± 0.06) and 0.8% (± 0.3) of infected cells for S. citri GII3 and GII3-9a3 respectively. However, in a previous study we had shown that adhesion of S. citri GII3 to Ciha-1 cells was optimum in the presence of 5 μg ml−1 cytochalasin D, an inhibitor of actin polymerization (Béven et al., 2012). Thus, we further compared adhesion capabilities of S. citri GII3, GII3-9a3 and GII3-9a3/pTC2 (GII3-9a3 complemented by transformation with the wild-type spiralin gene) in the presence of cytochalasin D. As a control we verified that cytochalasin D had no effect on viability (as determined by cfu counts), helical morphology and motility of the spiroplasmas (data not shown). As shown in Fig. 2A, treatment of the eukaryotic host cells with cytochalasin D significantly increased (two- to threefold increase) adhesion and entry of S. citri GII3 and GII3-9a3/pTC2 into Ciha-1 cells, whereas it had no significant effect on adhesion and entry of GII3-9a3 lacking spiralin. These results indicated that, in the presence of cytochalasin D, spiralin was required for the spiroplasmas to efficiently adhere and enter into insect cells.


Figure 2. Adhesion and entry of S. citri into Ciha-1 cells, and adhesion of spiralin-coated beads to the leafhopper cells.

A. Effect of cytochalasin D. Insect cells were incubated with S. citri GII3 (black bars), GII3-9a3 (white bars) or GII3-9a3/TC2 (grey bars) for 3 h in the presence (5) or absence (0) of 5 μg ml−1 cytochalasin D.

B. Inhibition assays. S. citri GII3 was pre-incubated with anti-spiralin PAbs (black bars) at the indicated dilutions before infection of cytochalasin D-treated Ciha-1 cells. Anti-fibril PAbs were used as the control (hatched bars).

C. Competitive assays. Ciha-1 cells were treated with 5 μg ml−1 cytochalasin D and pre-incubated with purified recombinant spiralin or BSA (hatched bars) at the indicated concentrations before to be infected with S. citri GII3.

*, significantly different from untreated cells (Student's test, P <0.05). Each bar represents the mean relative per cent ± SD of four distinct wells.

D. Adhesion of spiralin-coated beads. Ciha-1 cells were incubated with spiralin-coated beads (black bars) or BSA-coated beads (hatched bars) in the presence (5) or absence (0) of 5 μg ml−1 cytochalasin D. *, significantly different from Ciha-1 cells treated with cytochalasin D (Student's test, P <0.05). Each bar represents the mean relative per cent ± SD of three distinct experiments.

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The involvement of spiralin was further confirmed by adhesion and invasion inhibition assays using anti-spiralin polyclonal antibodies, and competitive assays with exogenous recombinant spiralin. To maximize the adhesion and invasion rates, experiments were performed in the presence of cytochalasin D. As a control, we previously verified that PAbs at the used concentrations did not induce aggregation of spiroplasmas that could have led to false cfu counts. Increasing concentrations of added anti-spiralin PAbs resulted in a concomitant reduction of both adhesion and entry of spiroplasmas into the insect cells whereas the anti-fibril PAbs used as the control had no effect (Fig. 2B). In competition experiments, adding exogenous recombinant spiralin had a significant though limited effect on the entry of S. citri GII3 into Ciha-1 cells (Fig. 2C). The highest protein concentration resulted in a 20% decrease of the percentage of infected cells, despite an increased adhesion rate.

To determine whether cytochalasin D treatment could have a direct effect on the interaction of spiralin with the insect cells, cytadherence assays were carried out using recombinant spiralin-coated latex beads. In the absence of cytochalasin D, adhesion rate of spiralin-coated beads to Ciha-1 cells (0.92 ± 0.27 beads per cell) was significantly higher than that of BSA-coated beads (0.64 ± 0.11 beads per cell) used as the control. In addition, whereas a significant increase was detected in the presence of cytochalasin D, no such an increase was detected with the control (Fig. 2D). These results, along with the finding that in contrast to the wild-type S. citri GII3 cytochalasin D treatment had no effect on adhesion of the spiralin-less mutant GII3-9a3, were consistent with the hypothesis that treatment with cytochalasin D increased adhesion of S. citri GII3 to Ciha-1 cells by uncovering spiralin-binding receptors that were masked in non-treated cells.

Cytochalasin D triggers changes in the lectin binding pattern of Ciha-1 cells

Drugs, such as cytochalasin D and colchicin, are known to alter the distribution and mobility of integral membrane proteins and lipids through the destruction of microfilaments and microtubules (Allen et al., 2006). From these observations we hypothesized that treatment of Ciha-1 cells with cytochalasin D might induce variations in the pattern of surface-exposed proteins, and glycoproteins particularly. To test this hypothesis, 20 different lectins (listed in Table 1) differing in their substrate specificity were used to determine the lectin binding profile of Ciha-1 cells in the presence and absence of cytochalasin D. Of the 20 lectins tested, 6 (DBA, PHA-E, PHA-L, PNA, PSA and UEA) did not bind the Ciha-1 cells, regardless of whether or not the cells were treated with cytochalasin D (Table 1). The finding that PNA did not bind Ciha-1 cells was consistent with the failure to purify glycoproteins from C. haematoceps using a PNA column (Killiny et al., 2005). The other 14 lectins all bound to the insect cells with different patterns and various intensities, indicating the diversity of carbohydrate residues exposed at the Ciha-1 cell surface. Finally, six lectins (GSL I, GSL II, LCA, RCA, VVA and WGA) exhibited a greater binding capacity in the presence of cytochalasin D than without it (Table 1), the highest fluorescence intensity being observed with the VVA lectin, which has an N-acetyl-galactosamine binding specificity. These results clearly showed that cytochalasin D treatment modified the lectin binding profile of Ciha-1 cells, unveiling lectin ligands that were not or poorly accessible in untreated cells.

Table 1. Lectin binding patterns of Ciha-1 cells treated (+ cytoD) or not (− cytoD) with cytochalasin D (5 μg ml−1) as determined by fluorescence intensity
 IntDen per cellRelative % of IntDen per cell
Lectin nameAbbreviationPrimary sugar specificity− cytoD+ cytoD
  1. The Integrated intensity per cell (IntDen per cell) values correspond to the mean of integrated density of lectin fluorescence.

  2. Sugar abbreviations: Fuc, l-fucose; Gal, d-galactose; GalNAc, N-acetylgalactosamine; Glc, d-glucose; GlcNAc, N-acetylglucosamine, Man, mannose.

Concanavalin ACon AαMan, αGlc1.3 × 1065.8 × 10546 (± 40)
Dolichos biflorus agglutininDBAαGalNAc
Peanut agglutininPNAGalβ3GalNAc
Ricinus communis agglutininRCA IβGal9.2 × 1034.2 × 104460 (± 429)
Soybean agglutininSBAα > β GalNac5.8 × 1045.4 × 10494 (± 136)
Ulex europeanus agglutininUEA IαFuc
Wheat germ agglutininWGAα,β GlcNAc3.9 × 1041.1 × 105287 (± 180)
Griffonia (Bandeiraea) simplicifolia IGSL IαGalNAc, αGal6.3 × 1042.7 × 105426 (± 214)
Lens culinaris agglutininLCAαMan, αGlc1.6 × 1066.1 × 106393 (± 282)
Phaseolus vulgaris leucoagglutinin-EPHA-E



Phaseolus vulgaris leucoagglutinin-LPHA-LGalβ4GlcNAcβ6(GlcNAcβ2Manα3)Manα3
Pisum sativum agglutininPSAαMan, αGlc
Succinilated-wheat germ agglutinins-WGAα,β GlcNAc2.0 × 1063.4 × 106166 (± 77)
Griffonia (Bandeiraea) simplicifolia IIGSL IIα,β GlcNAc1.9 × 1051.1 × 106594 (± 614)
Datura stramonium lectinDSL(GlcNAc)2–47.9 × 1031.3 × 104168 (± 351)
Erythrina cristagalli lectinECLGalβ4GlcNAc7.9 × 1041.0 × 105127 (± 127)
JacalinJacalinGalβ3, GalNAc1.7 × 1052.3 × 105137 (± 106)
Lycopersicon esculentum lectinLEL(GlcNAc)2–41.5 × 1053.2 × 105209 (± 197)
Solanum tuberosum lectinSTL(GlcNAc)2–43.5 × 1052.8 × 10581 (± 70)
Vicia villosa agglutininVVAα,β GalNAc3.6 × 1044.6 × 1051289 (± 1464)

Distribution of glycoconjugates in salivary gland cells

In previous studies we have shown that the S. citri mutants affected in their transmissibility by C. haematoceps, and in particular the spiralin-less mutant GII3-9a3, were unable to cross the salivary gland barrier despite their ability to multiply in the haemolymph (Duret et al., 2003; Killiny et al., 2006). This observation suggested a reduced capability of the mutant to adhere to and/or enter into the salivary gland cells. Using fluorescent lectins with different sugar specificities, we checked whether the carbohydrates, the presence of which was revealed at the surface of Ciha-1 cells in the presence of cytochalasin D, were present at the surface of the salivary gland cells. Labelling salivary glands with fluorescent ConA, used as the control, yielded a strong fluorescent signal at the periphery of every lobule that certainly accounts for binding of ConA to glycoconjugates of the basal lamina (Fig. 3). The four lectins tested, WGA, LCA, GSLII and VVA, also recognized glycoconjugates at the surface of salivary gland cells, but only a few lobules were stained (Fig. 3). Moreover, fluorescent labelling appeared as scattered patches rather than uniform. Unlike ConA again, high fluorescence intensity was not only detected at the cell surface but also inside the salivary gland cells.


Figure 3. Staining of salivary glands of healthy C. haematoceps with fluorescent lectins. After PAF fixation in the presence of 0.2% Triton X-100, dissected salivary glands were incubated with 20 μg ml−1 FITC-labelled lectins, ConA, WGA, LCA, GSLII or VVA. Endogenous actin was stained with Alexa 568-phalloidin (red), glycoproteins with FITC-labelled lectins (green), and nuclei with DAPI (blue). Two optical x–y sections taken at the cell surface (top line) or at an intracellular location (bottom line) of the salivary gland are presented. Scale bar, 150 μm.

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Lectins inhibit adhesion of S. citri to Ciha-1 cells

To determine whether spiralin of S. citri could bind glycoconjugates present at the Ciha-1 cell surface, we carried out adhesion competitive assays with various lectins. The leafhopper cells were treated with cytochalasin D and incubated with the relevant lectin, before being exposed to S. citri GII3 or the spiralin-less mutant GII3-9a3. Adhesion of S. citri GII3 to Ciha-1 cells was found to decrease by 44 to 59% depending on the lectin, WGA, LCA or VVA, whereas no significant decrease was noticed in the presence of ConA or GSLII as the competitor (Fig. 4A, black bars). In the mutant GII3-9a3 also, adhesion was reduced by 42 to 72% in the presence of lectins. In this case however, competition was observed for WGA, LCA and GSLII but not VVA (Fig. 4A, white bars). These results indicated that one or several surface proteins of S. citri GII3 performed as lectins recognizing different glycosylated compounds of Ciha-1 cells and that spiralin acted as a VVA-type lectin. To strengthen the evidence that spiralin bound glycoconjugates recognized by the VVA lectin, adhesion competitive assays were carried out with the various lectins and spiralin-coated beads. A strong inhibition was observed with the VVA lectin (Fig. 4B). This result is consistent with the latex beads adhesion assays showing that treatment of Ciha-1 cells with cytochalasin D increased adhesion of spiralin-coated beads.


Figure 4. Adhesion of S. citri to the Ciha-1 cells and to the salivary glands of C. haematoceps.

A. Competitive adhesion of S. citri GII3 in the presence of lectins. Insect cells were pre-treated with 5 μg ml−1 cytochalasin D and incubated with 20 μg ml−1 of the selected lectin prior infection with S. citri GII3 (black bars) or GII3-9a3 (white bars) for 3 h. *, significantly different from untreated Ciha-1 cells (Student's test, P <0.05). Each bar represents the mean relative per cent ± SD of four distinct wells.

B. Competitive adhesion of spiralin-coated latex beads in the presence of lectins. Ciha-1 cells treated with 5 μg ml−1 cytochalasin D were incubated with lectins ConA, WGA, LCA, GSLII or VVA prior to adding the spiralin-coated beads. Each box represents the median (bolt line) and the quartiles (25%/75%) of three independent measurements. *, significantly different from adhesion in the absence of lectin (Student's test, P <0.01).

C. Confocal immunofluorescence microscopy of S. citri spiroplasmas associated to C. haematoceps salivary glands. Three weeks after injection of S. citri GII3 to C. haematoceps leafhoppers, salivary glands were dissected and stained with anti-S. citri GII3 PAbs and Alexa 633-secondary antibodies (green), Alexa 568-phalloidin (red) and DAPI (blue). Scale bar, 144 μm.

D. In vivo colocalization of S. citri GII3 with salivary gland cell glycoconjugates as revealed by fluorescent lectins. Three weeks after injection of S. citri GII3 to C. haematoceps, salivary glands were prepared as above to label spiroplasmas and further stained with the fluorescent lectins WGA, LCA, GLSII and VVA as indicated at the top of the figure. For each lectin, the figure shows spiroplasmas, glycoconjugates, actin and cell nuclei respectively coded white (Alexa 633), green (FITC-lectins), red (Alexa 568) and blue (DAPI) of the same salivary gland. Images of the bottom line show the overlays. Scale bar 150 μm.

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To confirm the involvement of S. citri-glycoconjugates interactions in vivo, salivary glands of S. citri-infected leafhoppers were analysed by CLSM after double-labelling of spiroplasmas and glycoconjugates as described in Experimental procedures. Three weeks after infection, spiroplasmas were attached to salivary gland cells of several lobules (Fig. 4C). The merged images revealed the colocalization of glycoconjugates labelled with the four lectins (WGA, LCA, GLSII and VVA) and the spiroplasmas attached to the cells (Fig. 4D).


  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

In this study we showed that relocalization of spiralin at the contact pole was associated with adhesion to insect cells in culture. Attachment of pathogenic bacteria to their host cells often relies on specific distribution of proteins including adhesins. As for example in human pathogenic mollicutes, attachment of Mycoplasma pneumoniae to the host respiratory epithelium is mediated by the so-called terminal organelle (Razin and Jacobs, 1992). This small membrane-bound extension is characterized by a central electron-dense core and a set of trans-membrane and membrane-associated proteins involved in cytadherence, gliding motility and cell division (Krause and Balish, 2004; Hasselbring et al., 2006). The spatial distribution of the P1 adhesin in particular is essential to its functional capacity for receptor recognition (Cloward and Krause, 2011). In Streptococcus pyogenes, the fibronectin-binding protein SfbI is a surface protein that specifically localizes at the poles of the bacterium (Ozeri et al., 2001). In these two cases however, specific location of the adhesins pre-exists and does not result from the contact between the bacteria and the host cells. In contrast, there are examples in which relocalization of membrane proteins such as eukaryotic cell receptors is triggered by bacteria/host cell interactions. For example, β1-integrins are recruited to the entry site of S. pyogenes and match the position of the SfbI protein present at the bacterial surface (Ozeri et al., 2001). Lateral movement of proteins in the membrane lipid bilayer has also been described in Bacillus subtilis, in which the sporulation protein YuaG forms spots that move in the membrane following a spiralling track (Donovan and Bramkamp, 2009). Also, in Flavobacterium johnsoniae, SprB is a cell-surface adhesin, of which the left-handed helical movement is required for efficient gliding (Nakane et al., 2013).

In the case of S. citri, comparing the wild-type and spiralin-less mutant strains in adhesion as well as in competition and inhibition assays proved spiralin to be an adhesin. In these competition assays, adding purified recombinant spiralin resulted in an increased adhesion of spiroplasmas to Ciha-1 cells, despite a decreased entry. A similar situation was previously reported in the case of adhesin ScARP3d (Béven et al., 2012). In these former experiments, competition with a recombinant protein corresponding to the ScARP3d repeat domain also resulted in an increased adhesion and a reduced rate of spiroplasma entry (Béven et al., 2012). In the case of spiralin, which is known to form homomultimers (Wróblewski, 1981), the adhesion increase could result from spiralin-spiralin interactions in addition to the interactions of spiralin with the host cell receptors.

In eukaryotic cells, surface-exposed and membrane-associated proteins are not homogenously distributed but instead organized in clusters. This organization and/or the clustering of micro-domains relies on interactions with and dynamic rearrangement of the cytoskeleton (Head et al., 2014). Cortical actin filaments form a scaffold that through its reorganization directly influences the spatial distribution of cell membrane proteins. For example, actin filaments are involved in lateral movement of glycosylphosphatidylinositol-anchored proteins and thereby contribute to many cellular processes. This phenomenon is an active process implying short dynamic actin filaments (Gowrishankar et al., 2012). Consequently, destruction of microfilaments changes the distribution of integrated membrane proteins (Allen et al., 2006). Likewise, in Ciha-1 cells treated with cytochalasin D, lectin binding assays revealed changes in the distribution of glycoconjugates at the cell surface. As a result, lectins GSLI and II, LCA, RCA, VVA and WGA bound the treated cells more efficiently.

Competitive adhesion assays with lectins showed that at least four distinct lectin/glycoconjugate pairs contribute to the adhesion of S. citri GII3 to the Ciha-1 cells. In previous studies, protein overlay assays revealed interactions between proteins of S. citri GII3 and several glycoproteins of C. haematoceps (Killiny et al., 2005). In these former in vitro experiments, positive interactions were detected between insect glycoproteins purified from ConA, WGA and LCA columns, and spiralin-enriched extracts. In agreement with these results, spiroplasma adhesion was found to strongly decrease in the presence of lectins WGA and LCA. These data indicate that S. citri GII3 possesses a set of adhesins with various lectin activities. In insects, a glycocalyx is present in both the intestine and the salivary glands. Diversity of glycoconjugates at the surface of salivary glands has been reported in Aedes and Rhodnius (Basseri et al., 2002; Nascimento et al., 2010). Differential distribution of glycoconjugates among the salivary gland cells of C. haematoceps is consistent with previous data indicating that cells from the two lobes showed different staining features, ultrastructures and morphologies (Wayadande et al., 1997). In arthropods, interactions with the host glycoconjugates most frequently mediate the first step of infection, regardless of the pathogens, viruses bacteria or protozoans (Dinglasan and Jacobs-Lorena, 2005). As an example, several carbohydrates-binding proteins have been associated with the primary adhesion of the plant pathogenic bacterium Xylella fastidiosa to intestinal epithelium cells of the leafhopper host (Killiny et al., 2012). Likewise, adhesion of the protozoan Blastocrithidia culicis to the salivary gland cells of Aedes aegypti is dependent of insect glycoconjugates (Nascimento et al., 2010). In this study, scanning electron and immunofluorescence microscopy images revealed that the protozoan did not preferentially adhere to specific regions of the salivary glands. A similar situation was observed in the plant pathogenic spiroplasmas S. citri (this study) and S. kunkelii (Ammar and Hogenhout, 2005).

In conclusion, spiralin is important for S. citri GII3 to adhere and invade insect cells in culture, and relocalization of spiralin at the contact zone seems to be a prerequisite for internalization of the spiroplasmas. To our knowledge, this is the first report of the recruitment of a bacterial adhesin at the contact zone during adhesion to eukaryotic host cells. Adhesion of spiralin to insect cells is mediated by a lectin/carbohydrate-like interaction. However, due to the relative weakness of carbohydrate-binding protein affinities for glycans (Dinglasan and Jacobs-Lorena, 2005), the strong adhesion of S. citri to insect cells probably requires additional adhesin/receptor interactions following the initial glycan/spiralin recognition event. In good agreement with this statement, we have previously reported the contribution of ScARP3d in adhesion of S. citri to the Ciha-1 cells (Béven et al., 2012).

Experimental procedures

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Spiroplasmas, plasmids, leafhoppers and insect cell line

Spiroplasma citri GII3 wild-type has been isolated from the leafhopper vector C. haematoceps captured in Morocco (Vignault et al., 1980). Spiroplasmas were grown at 32°C in SP4 medium from which fresh yeast extract was omitted (Whitcomb, 1983). The spiralin-less mutant GII3-9a3 is identical to S. citri GII3-9a2 obtained by site-directed mutagenesis (Duret et al., 2003) except that, in contrast to GII3-9a2 lacking pSci5, it contains the whole set of plasmids pSci1 to pSci6. The S. citri strain GII3-9a3/pTC2 was obtained by transformation of S. citri GII3-9a3 with plasmid pTC2 carrying the wild-type spiralin gene. To construct pTC2, the spiralin gene of S. citri GII3 (including the transcription promoter) was PCR amplified with primers SR5 (5′-GGTCAGATCTAGCCAGAATTAAAAGTTAGTG-3′) and SR30 (5′-TATATGGATCCTAGTTGATATTCTAAGATTG-3′), and the BglII+BamHI (Promega, Madison, WI, USA) digested fragment was inserted into the BglII-linearized pSRCAT (Duret et al., 2005) to yield pSRCAT-SpiG. Plasmid pTC2 was obtained by combining the BamHI-linearized pSRCAT-SpiG to the BglII-linearized, S. citri plasmid pSci21NT (Breton et al., 2008), and introduced into S. citri GII3-9a3 by electrotransformation (Stamburski et al., 1991). Spiroplasmal transformants were selected and grown in the presence of chloramphenicol 5 μg ml−1 (Sigma-Aldrich, St. Louis, MO, USA). Production of spiralin in these transformants was confirmed by Western immunoblotting with anti-spiralin monoclonal antibody FD1 according to standard procedures (Duret et al., 2003) (Fig. S1).

Circulifer haematoceps leafhoppers were reared on stock (Mattiola incana) plants in insect-proof cages at 30°C. Microinjection of S. citri GII3 into C. haematoceps has been described previously (Foissac et al., 1996). Injected leafhoppers were maintained for 3 weeks on stock plants before salivary glands were dissected.

The non-phagocytic cell line Ciha-1 established from embryos of the leafhopper vector C. haematoceps and its infection by S. citri have been described previously (Duret et al., 2010).

Expression and purification of recombinant protein His6-spiralin

The DNA fragment (682 bp) corresponding to the extracellular region (amino acids C24-G241) was PCR amplified from S. citri GII3 genomic DNA with primers SpiF2Nd (5′-GGT TGC ATA TGT GTA ATA AAA CTG-3′) and SpiRH (5′-GAA AAA GCT TAA TTC TTA TCC TGC-3′); the introduced restriction sites are in bold. The obtained DNA fragment was digested by NdeI and HindIII (Promega) and ligated to the pET28a(+) vector (Novagen, Merck Millipore, Darmstadt, Germany) digested by NdeI+HindIII. The mixture was used to transform Escherichia coli DH10B (Thermo Fisher Scientific, Carlsbad, CA, USA) and the transformants were selected on LB medium containing 50 μg ml−1 kanamycin (Sigma-Aldrich). The recombinant plasmid pET-spi containing the spiralin insert was verified by sequencing.

Expression and purification of the S. citri GII3 spiralin tagged with hexahistidine (His6) at its N-terminus were carried out under native conditions as previously described (Labroussaa et al., 2010). The purified His6-spiralin was desalted using a PD-10 column (GE Healthcare, Little Chalfont, UK). Protein concentration was estimated by the Bradford procedure (Bradford, 1976). Purification was confirmed by 12.5% SDS-PAGE and Western immunoblotting using rabbit anti-spiralin PAbs (Wróblewski et al., 1977) as primary antibodies (Fig. S1) essentially as described in Béven et al. (2012). Rabbit anti-spiralin PAbs were a kind gift from Pr. H. Wróblewski, University of Rennes I, France.

Immunofluorescent staining of spiroplasmas

Spiroplasma citri infected Ciha-1 cells were washed in Schneider's Drosophila medium (Thermo Fisher Scientific) and fixed at room temperature for 15 min with 4% paraformaldehyde (PAF) in PBS. Spiroplasmas were stained with 1:500 anti-spiralin polyclonal antibodies (PAbs) using Alexa 488- or 633-conjugate goat anti-rabbit antibodies (Thermo Fisher Scientific) as the secondary antibody according to the previously reported procedures (Duret et al., 2010). Immunofluorescent samples were imaged using a TCS SP2 upright Leica confocal laser scanning microscope (CLSM) equipped with a 63× oil immersion objective (70 nm pixel size). Fluorochromes were detected sequentially frame by frame with the acousto-optical tunable filter system using excitation laser lines 405 nm (DAPI), 488 nm [Alexa 488, fluorescent beads (see below)], 543 nm (Alexa 568) and 633 nm (Alexa 633). Emission wavelengths ranges were 410–485 nm (DAPI), 507–553 nm (Alexa 488, fluorescent beads), 588–650 nm (Alexa 568) and 645–700 nm (Alexa 633).

The spiroplasma-associated fluorescence was quantified from confocal stack projections (Maximal and Average projections with Leica software) for each spiroplasmal acquisition with the free software package ImageJ ( Maximal intensity projection creates an output image each of whose pixels contains the maximum value over all images in the stack at each pixel location, and average intensity projection outputs an image wherein each pixel stores average intensity over all images in the stack at the corresponding pixel location. Integrated Density (sum of the values of the pixels in the selection) was determined in the cell contact area (CA) as well as in the distal area (DA) of each spiroplasma (Intden/Area). CA and DA were differentiated on the basis of their apparent morphology, rounded for CA and elongated for DA.

Transmission electron microscopy

Ciha-1 cells were grown on coverslips in 24-well plates and incubated with S. citri at an moi (multiplicity of infection) of 30 for 1 h and 18 h at 32°C in cell culture medium. Sample fixation in glutaraldehyde and post-fixation in osmium tetroxide, dehydratation in ethanol, and inclusion in Epon resin were carried out as described previously (Duret et al., 2010; Béven et al., 2012). Micrographs were taken at 80 kV on a FEI CM10 transmission electron microscope equipped with an AMT XR60 digital camera (Elexience, Verrières-le-buisson, France).

Ciha-1 cells adhesion and invasion assays

Binding and internalization of spiroplasmas into Ciha-1 cells were determined essentially as described previously (Duret et al., 2010). In brief, Ciha-1 cells (approximately 105 per well) were inoculated with spiroplasmas at an moi of 30–50 and incubated at 32°C for 3 h. After removal of unbound spiroplasmas and three washing with 1 ml Schneider's Drosophila medium and two with 1 ml Dulbecco's phosphate-buffered saline (Eurobio, Courtaboeuf, France) for 5 min each, the cells were trypsinized with TrypLE (Thermo Fisher Scientific) for 5 min at 32°C and serial dilutions were plated onto SP4 containing 1% noble agar (Whitcomb, 1983) for cfu counting, cfu counts corresponding to the number of cells with adherent and/or internalized spiroplasmas. The number of infected cells was determined by a gentamicin protection assay. Gentamicin (Sigma-Aldrich) treatment (400 μg ml−1 for 3 h) was applied 3 h after adding the spiroplasmas. After washing to remove the antibiotic and trypsinization, the cells were plated to determine the number of infected cells, i.e. cells with internalized spiroplasmas surviving the gentamicin treatment. The number of cells with adherent spiroplasmas was calculated by subtracting cfu counts of infected cells (with internalized spiroplasmas) from those of cells with associated (adherent plus internalized) spiroplasmas. Each experiment was performed in four distinct wells and was repeated four times.

For antibody inhibition assays, spiroplasmas were pre-incubated with anti-spiralin PAbs or anti-fibril PAbs (recognizing the cytoplasmic fibril protein) at 32°C for 1 h before being added to the Ciha-1 cells pre-treated with 5 μg ml−1 cytochalasin D for 1 h. Rabbit anti-fibril PAbs were kindly provided by Dr D.L. Williamson, State University of New York, USA. For binding competitive assays, Ciha-1 cells were treated with 5 μg ml−1 cytochalasin D for 1 h, and then incubated at 32°C for 1 h with various concentrations of purified spiralin, or 20 μg ml−1 of the selected lectin (Vector Laboratories, Nanterre, France) before being inoculated with spiroplasmas. In this case, cytochalasin D was maintained in the medium during incubation of Ciha-1 cells with spiralin or lectin, and during further incubation with spiroplasmas. The number of cells with associated spiroplasmas was determined 3 h post infection. The relative percentages of cells with adherent spiroplasmas and infected cells were calculated for each strain, separately. Each experiment was performed in four distinct wells and was repeated four times.

Coated latex beads binding assays

The yellow-green fluorescent and carboxylate-modified latex beads (4 × 109 beads of 1 μm diameter) (Sigma-Aldrich) were covalently coated with 64 μg of recombinant spiralin or BSA according to the supplier's instructions. About 105 Ciha-1 cells cultivated on coverslips in 24-well plates were incubated with 2·106 coated latex beads in Schneider's Drosophila medium for 1 h at 32°C. All further steps were carried out at room temperature. After three washes in 0.5 ml Schneider's Drosophila medium for 5 min, the cells were fixed for 15 min with 4% paraformaldehyde in PBS and the spiralin-coated beads were incubated with anti-spiralin PAbs diluted 1:500 in PBS-BSA solution (PBS containing 1% BSA) for 30 min. After three washes with PBS, the cells were incubated for 30 min with Alexa 633-conjugated goat anti-rabbit antibodies (Thermo Fisher Scientific) diluted 1:200 in PBS-BSA buffer. After the cell nuclei were stained with 1 μg ml−1 DAPI for 5 min (for cell counting) the samples were mounted in the anti-fading ProLong Gold Reagent (Thermo Fisher Scientific) and immunofluorescent samples were analysed with a fluorescence microscope (Nikon Eclipse E800) at 40× magnification. For each experiment 20 to 25 fields with approximately 100 cells per field were observed randomly. The area of single beads was calculated field by field from confocal stack projections for each acquisition with the free software package ImageJ ( The average area of the beads was used to calculate the number of beads per field that was then divided by the number of cells per field.

The percentage of adherent coated beads was also determined in the presence of cytochalasin D and lectins (see below). The cells were first pre-incubated with cytochalasin D and lectins for 1 h and then addition of the spiralin-coated latex beads was performed as described above.

Determination of lectin binding profiles

Ciha-1 cells treated or not with cytochalasin D were fixed with paraformaldehyde as described above, and incubated with the selected fluorescent FITC-conjugated lectins (Vector Laboratories; Table 1) for 1 h. After the cell nuclei were stained with DAPI (for cell number determination) the samples were examined using a fluorescence microscope (Nikon Eclipse E800) at 60× magnification. For each sample, fluorescence intensity was calculated from 30 different fields using the free software package ImageJ ( To avoid possible interference of fluorescent background only areas with pixel values higher than 50% of the maximum value were selected. For each lectin the same threshold was applied to both non-treated and cytochalasin D-treated cells. The Integrated Density was then calculated from this selection and was indicated per cell. The experiments were repeated three times.

Groups of 10 salivary glands were fixed with 4% paraformaldehyde in PBS plus 0.2% Triton X-100 overnight at 4°C. Organs were washed in PBS, blocked for 10 min with BSA 1% in PBS (PBS-BSA), and then incubated for 1 h in the presence of 20 μg ml−1 of one of the following FITC-conjugated lectins: ConA, WGA, LCA, GLSII and VVA in PBS plus Alexa 568-phalloidin (Invitrogen), used to stain the actin filaments of the Ciha-1 cells, at room temperature. After washing, nuclei were stained with DAPI and the salivary glands were mounted with ProLong Gold antifade reagent (Thermo Fisher Scientific). Immunofluorescent samples were imaged using CLSM with a 20× water immersion objective lens with pixel size comprised between of 500 nm and 700 nm.

Immunofluorescence of infected salivary glands

Three weeks after injection of S. citri GII3 into leafhoppers, the salivary glands were excised, fixed and blocked as above. After washing, they were incubated with a 1:500 dilution of anti-S. citri polyclonal antibodies in PBS-BSA, washed and then incubated with Alexa 633-conjugated goat anti-rabbit IgG (Thermo Fisher Scientific) at a 1:200 dilution. F-actin, glycoconjugates and nuclei were stained as above; immunofluorescent samples were mounted with ProLong Gold antifade reagent and observed as above.

Statistical analyses

For purposes of statistical evaluation the Student's t-test for comparing two samples was used. The similarities of deviations were checked with the anova and Fisher's tests. The results of the statistical analyses using Student's t-test were considered significant if their corresponding P-values were less than 0.05.


  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

We are grateful to L. Brocard and C. Cheniclet of the Bordeaux Imaging Center for advices in the use of the ImageJ software, and C. Garcion for improvement of the manuscript. The authors gratefully acknowledge K. Guionneaud and D. Lacaze for insect rearing and the Structure Fédérative de Recherche ‘Biologie Intégrative et Ecologie’ for financial support.


  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information
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Supporting Information

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Fig. S1. Gel electrophoresis and Western immunoblot analysis of spiralin in S. citri.

A. Coomassie-stained 12.5% SDS-PAGE gel of whole-cell lysates of S. citri GII3, the spiralin-less mutant GII3-9a3, and the complemented mutant GII3-9a3/pTC2.

B. Western immunoblot analysis of whole-cell lysates of S. citri GII3, the spiralin-less mutant GII3-9a3, and the complemented mutant GII3-9a3/pTC2. Proteins were probed using anti-spiralin PAbs.

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