Infection of the Circulifer haematoceps cell line Ciha-1 by Spiroplasma citri: the non-insect-transmissible strain 44 is impaired in invasion

Abstract

Successful transmission of Spiroplasma citri by its leafhopper vector requires a specific interaction between the spiroplasma surface and the insect cells. With the aim of studying these interactions at the cellular and molecular levels, a cell line, named Ciha-1, was established using embryonic tissues from the eggs of the S. citri natural vector Circulifer haematoceps. This is the first report, to our knowledge, of a cell line for this leafhopper species and of its successful infection by the insect-transmissible strain S. citri GII3. Adherence of the spiroplasmas to the cultured Ciha-1 cells was studied by c.f.u. counts and by electron microscopy. Entry of the spiroplasmas into the insect cells was analysed quantitatively by gentamicin protection assays and qualitatively by double immunofluorescence microscopy. Spiroplasmas were detected within the cell cytoplasm as early as 1 h after inoculation and survived at least 2 days inside the cells. Comparing the insect-transmissible GII3 and non-insect-transmissible 44 strains revealed that adherence to and entry into Ciha-1 cells of S. citri 44 were significantly less efficient than those of S. citri GII3.

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

“…The non-phagocytic cell line Ciha-1 established from C. haematoceps [12] was cultured at 32°C as described previously [12].…”

“…Successful transmission requires the spiroplasmas to pass through enterocytes of the mid-gut epithelium, multiply to high population in the hemocoel, and invade the salivary glands to be released in the saliva duct [7], [8]. Transmission electron microscopy (TEM) studies investigating invasion of insect cells in vivo and ex vivo both revealed invagination of the cell membrane in close contact with the spiroplasmas [9], [10], [11], [12], [13]. Together with the presence of spiroplasmas within membrane-bound cytoplasmic vesicles of insect cells [11], these observations strongly suggest an endocytosis mechanism mediated by receptor-ligand interactions [8].…”

“…However evidence of a direct interaction of ScARPs with the insect cell membrane has not been established. Previously, we showed that S. citri 44, a non-insect-transmissible strain lacking ScARPs, was impaired in its ability to invade the leafhopper cell line Ciha-1 [12]. This apparent correlation between the ability of the spiroplasma to invade insect cells ex vivo and its ability to be transmitted by the leafhopper vector prompted us to further use the Ciha-1 cell line to explore the implication of ScARPs in the internalization process, with the aim to better understand the function of these adhesins in insect transmission of S. citri .…”

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

“…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)] ϫ 100%.…”

Sequences Essential for Transmission of Spiroplasma citri by Its Leafhopper Vector, Circulifer haematoceps , Revealed by Plasmid Curing and Replacement Based on Incompatibility

“…In these studies, protein overlay assays revealed that two membrane-associated proteins, a lipoprotein and the glycolytic enzyme phosphoglycerate kinase (PGK), interacted with actin in vitro. By use of the Ciha-1 leafhopper cell culture system (11), it was shown that PGK, by specifically binding to actin, was involved in internalization of spiroplasmas into insect cells (25). In contrast to these membraneassociated proteins, the pSci6_06 protein was predicted to be cytosolic, making a direct interaction with leafhopper cells unlikely.…”