Identification of two <i>Wolbachia</i> genes with cell proliferation-inhibitory activity in <i>Ostrinia</i> cultured cells

Wolbachia species are endosymbiotic bacteria that regulate the sexual development and reproduction of their insect hosts. Despite their biological importance, functional analyses of Wolbachia genes remain limited, largely due to the absence of a robust genetic manipulation system for these intracellular bacteria. Here, we examined the impact of w Fur, a Wolbachia strain originally isolated from Ostrinia furnacalis , on host cell proliferation. Inoculation of w Fur onto Wolbachia -free O. furnacalis cultured cells resulted in pronounced inhibition of cell proliferation, accompanied by cytotoxic effects. To identify the genetic determinants of this phenotype, we conducted functional screening of w Fur-derived genes in O. furnacalis cultured cells. This approach identified two candidate genes whose overexpression markedly suppressed cell proliferation. One gene, designated w52 , encodes a protein with strong sequence and structural homology to TomO (toxic manipulator of oogenesis), previously reported to inhibit Drosophila S2 cell proliferation. The second gene, designated w75 , encodes a putative membrane-associated protein widely conserved among Wolbachia genomes. Moreover, overexpression of w52 or w75 also impaired cell proliferation in Bombyx mori cultured cells, whereas no effect was observed in Spodoptera frugiperda Sf-9 cells. These results suggest that the cytostatic activity of these Wolbachia genes is likely to be insect species- and/or cell type-specific. IMPORTANCE Wolbachia are maternally inherited intracellular bacteria notable for their exceptionally broad distribution among insect species. Despite their importance, particularly as reproductive manipulators, only a limited number of studies have addressed the functions of Wolbachia genes, largely due to the absence of well-established transinfection systems and reliable genetic engineering tools for these intracellular bacteria. In this study, we developed a transinfection system using Ostrinia furnacalis cultured cells and its male-killing strain w Fur. Moreover, we identified two w Fur genes, whose expression inhibited cell proliferation and exhibited cytotoxicity, through an in-house functional screening system of w Fur genes. This strategy can be readily extended to investigations of other Wolbachia species, thereby contributing to a more comprehensive understanding of Wolbachia gene functions.