Immune response and survival of Circulifer haematoceps to Spiroplasma citri infection requires expression of the gene hexamerin.

Spiroplasma citri is a cell wall-less bacterium that infects plants. It is transmitted by the leafhopper Circulifer haematoceps, which hosts this bacterium in the haemocel and insect tissues. Bacterial factors involved in spiroplasma colonization of the insect host have been identified, but the immune response of the leafhopper to S. citri infection remains unknown. In this study, we showed that C. haematoceps activates both humoral and cellular immune responses when challenged with bacteria. When infected by S. citri, C. haematoceps displayed a specific immune response, evidenced by activation of phagocytosis and upregulation of a gene encoding the protein hexamerin. S. citri infection also resulted in decreased phenoloxidase-like activity. Inhibition of hexamerin by RNA interference resulted in a significant reduction in phenoloxidase-like activity and increased mortality of infected leafhoppers. Therefore, the gene hexamerin is involved in S. citri control by interfering with insect phenoloxidase activity.

Immunotherapeutic potential of the immunodominant T-cell epitope of lipocalin allergen Bos d 2 and its analogues.

Lipocalin allergens, which contain most of the important animal-derived respiratory sensitizers, induce T helper type 2 (Th2) deviation, but the reasons for this are not clear. To explore the prospects for peptide-based allergen immunotherapy and to elucidate the characteristics of the immunodominant epitope of Bos d 2, BALB/c mice were immunized with a peptide containing the epitope, peptides containing its analogues, peptides from the corresponding regions of other lipocalin proteins, and peptides with a homologous sequence. We observed that murine spleen cells recognized the immunodominant epitope of Bos d 2, p127-142, in almost the same way as human Bos d 2-specific T cells did. Enzyme-linked immunosorbent spot-forming cell assay (ELISPOT) analyses showed that p127-142 and a corresponding peptide from horse Equ c 1 induced a Th2-deviated cellular response, whereas a homologous bacterial peptide from Spiroplasma citri induced a Th0-type response. Interestingly, the spleen cell response to the bacterial peptide and p127-142 was cross-reactive, that is, able to induce reciprocally the proliferation and cytokine production of primed spleen cells in vitro. More importantly, the peptides were able to skew the phenotype of T cells primed with the other peptide. Our results suggest that modified peptides can be useful in allergen immunotherapy.

Absence of plasmids encoding adhesion-related proteins in non-insect-transmissible strains of Spiroplasma citri.

In the plant-pathogenic mollicute Spiroplasma citri, spiralin is the major lipoprotein at the cell surface and is thought to be one of the components involved in the interactions of the spiroplasma with its insect vector. With the aim of identifying surface proteins other than spiralin, monoclonal antibodies (mAbs) were produced by immunization of mice with the spiralin-defective S. citri mutant GII3-9a2. mAb 10G3 was found to react with several polypeptides of 43-47 and 80-95 kDa, all of which were detected in the detergent phase after Triton X-114 partitioning of proteins. Mass spectrometry (MALDI-TOF) analyses of the two major polypeptides P47 and P80 of GII3-9a2, reacting with mAb 10G3, revealed that P47 was a processed product and represented the C-terminal moiety of P80. Search for sequence homologies revealed that P80 shared strong similarities with the S. citri adhesion-related protein P89 (Sarp1) of S. citri BR3, and is one (named Scarp4a) of the eight Scarps encoded by the S. citri GII-3 genome. The eight scarp genes are carried by plasmids pSci1-5. Western immunoblotting of proteins with mAb 10G3 revealed that, in contrast to the insect-transmissible S. citri strain GII-3, the non-insect-transmissible strains ASP-1, R8A2 and 44 did not express Scarps. Southern blot hybridization experiments indicated that these strains possessed no scarp genes, and did not carry plasmids pSci1-5. However, S. citri strain GII3-5, lacking pSci5, was still efficiently transmitted, showing that, in the genetic background of S. citri GII-3, the pSci5-encoded genes, and in particular scarp2b, 3b and 5a, are not essential for insect transmission. Whether plasmid-encoded genes are involved in transmission of S. citri by its leafhopper vector remains to be determined.