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Journal of Proteome Research
Quantitative Proteomic Analyses of Molecular Mechanisms Associated with Cytoplasmic Incompatibility in Drosophila melanogaster Induced by Wolbachia Lin-Ling Yuan1‡, Xiulan Chen2‡, Qiong Zong1‡, Ting Zhao1, Jia-Lin Wang1,Ya Zheng1, Ming Zhang1, Zailong Wang3, Jeremy C. Brownlie4, Fuquan Yang2*, Yu-Feng Wang1* 1 Hubei Key laboratory of genetic regulation and integrative biology, School of Life Sciences, Central China Normal University, Wuhan 430079, P. R. China, 2 Key Laboratory of Protein and Peptide Pharmaceuticals & Laboratory of Proteomics, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, P. R. China, 3 Novartis Pharmaceutics, East Hanover, NJ, 07936, USA, 4 School of Natural Science, Griffith University, Nathan, QLD 4111, Australia
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ABSTRACT: To investigate the molecular mechanisms of cytoplasmic incompatibility (CI) induced by Wolbachia bacteria in Drosophila melanogaster, we applied an iTRAQ-based quantitative proteomic assay to identify differentially expressed proteins extracted from spermathecae and seminal receptacles (SSR) of uninfected females mated with either 1d Wolbachia-uninfected (1T) or infected males (1W), or 5d infected males (5W). A total of 1317 proteins were quantified; 83 proteins were identified as having at least a 1.5 fold change in expression when compared 1W with 1T. Differentially expressed proteins were related to metabolism, immunity, and reproduction. Wolbachia changed the expression of seminal fluid proteins (Sfps). Wolbachia may disrupt the abundance of proteins in SSR by affecting ubiquitinproteasome mediated proteolysis. Knocking down two Sfp genes (CG9334, CG2668) in Wolbachia-free males resulted in significantly lower embryonic hatch rates with a phenotype of chromatin bridges. Wolbachia-infected females may rescue the hatch rates. This suggests that the changed expression of some Sfps may be one of the mechanisms of CI induced by Wolbachia. This study provides a panel of candidate proteins that may be involved in the interaction between Wolbachia and their insect hosts, and through future functional studies, may help to elucidate the underlying mechanisms of Wolbachia-induced CI.
KEYWORDS: Drosophila melanogaster, Wolbachia, iTRAQ, quantitative proteomics, spermatheca, seminal receptacle.
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Journal of Proteome Research
INTRODUCTION First discovered in 1924 by Hertig and Wolbach in mosquito ovaries,1 Wolbachia are endosymbiotic bacteria that infect a wide range of invertebrates including up to 40% of arthropod species, as well as filarial nematodes.2,3 As Wolbachia are not infectious, but are maternally transmitted, these bacteria have evolved several strategies such as male killing, feminization, parthenogenesis and cytoplasmic incompatibility (CI) to manipulate the reproduction of insect hosts. These manipulations seek to either increase the proportion of Wolbachia infected females within the population, or provide a reproductive benefit to Wolbachia infected females when compared to their uninfected counterparts. CI is the most common reproductive modification induced by Wolbachia and results in embryonic lethality when Wolbachia-infected males are crossed with uninfected females or females infected with a different strain of Wolbachia.2 As female hosts infected with the same strain of Wolbachia can rescue embryonic lethality associated with CI, these females have higher reproductive output when compared to uninfected females. While much is known about the effects of CI at a population level,4 the molecular mechanisms by which Wolbachia induce CI are still unclear, though several studies suggest that Wolbachia could modify host sperm. CI is interpreted to involve a modification-rescue system whereby Wolbachia in infected males modify the sperm, while the same strain of Wolbachia must be present in the egg to rescue the modification, otherwise the embryonic development will arrest at an early stage (for review see 5
). Cytologically, CI is observed as a delay in the nuclear envelope breakdown of the paternal
nucleus, which induces improper condensation of paternal chromatin in the embryo, typically leading to embryonic death.6,7 Landmann et al. have demonstrated that Wolbachia-infection
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delays the deposition of the replication-independent histone H3.3/H4 complex in the male pronucleus,8 which could explain the chromosome condensation and segregation defects observed in incompatible crosses. Through in vitro and in vivo analysis, Xi et al. demonstrated an interaction of Ance (coding for Angiotensin Converting Enzyme) with the CI mechanism in Drosophila.9 Our previous study suggested that decreased expression of Hira, encoding a chaperone of H3.3, in male Drosophila is causally linked to the CI strength induced by Wolbachia.10 We have also previously compared the transcriptional profiles of Wolbachiainfected and uninfected 3rd instar larval testes by microarray and identified 296 genes that have at least a 1.5 fold change (≥1.5 fold changes, q-value 20 to remove all the low scoring matches, which ensures a 95% confidence for peptide assignment. The global false discovery rate (FDR) calculated from the target-decoy search for the four dataset was all
