The Repetitive Domain of ScARP3d Triggers Entry of Spiroplasma citri into Cultured Cells of the Vector Circulifer haematoceps

Abstract

Spiroplasma citri is a plant pathogenic mollicute transmitted by the leafhopper vector Circulifer haematoceps. Successful transmission requires the spiroplasmas to cross the intestinal epithelium and salivary gland barriers through endocytosis mediated by receptor-ligand interactions. To characterize these interactions we studied the adhesion and invasion capabilities of a S. citri mutant using the Ciha-1 leafhopper cell line. S. citri GII3 wild-type contains 7 plasmids, 5 of which (pSci1 to 5) encode 8 related adhesins (ScARPs). As compared to the wild-type strain GII3, the S. citri mutant G/6 lacking pSci1 to 5 was affected in its ability to adhere and enter into the Ciha-1 cells. Proteolysis analyses, Triton X-114 partitioning and agglutination assays showed that the N-terminal part of ScARP3d, consisting of repeated sequences, was exposed to the spiroplasma surface whereas the C-terminal part was anchored into the membrane. Latex beads cytadherence assays showed the ScARP3d repeat domain (Rep3d) to be involved, and internalization of the Rep3d-coated beads to be actin-dependent. These data suggested that ScARP3d, via its Rep3d domain, was implicated in adhesion of S. citri GII3 to insect cells. Inhibition tests using anti-Rep3d antibodies and competitive assays with recombinant Rep3d both resulted in a decrease of insect cells invasion by the spiroplasmas. Unexpectedly, treatment of Ciha-1 cells with the actin polymerisation inhibitor cytochalasin D increased adhesion and consequently entry of S. citri GII3. For the ScARPs-less mutant G/6, only adhesion was enhanced though to a lesser extent following cytochalasin D treatment. All together these results strongly suggest a role of ScARPs, and particularly ScARP3d, in adhesion and invasion of the leafhopper cells by S. citri.

Invasion of insect cells bySpiroplasma citriinvolves spiralin relocalization and lectin/glycoconjugate-type interactions

“…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 ., ). 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).…”

“…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 ., ). 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 ., ).…”

“…However, due to the relative weakness of carbohydrate‐binding protein affinities for glycans (Dinglasan and Jacobs‐Lorena, ), 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 ., ).…”

“…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.…”

“…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 ., ). 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 ., ), 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 ., ).…”