Characterization of proteins derived from Wolbachia of Brugia Malayi associated with proinflammatory responses in macrophage cell line

Abstract

Wolbachia are required for normal larval development and reproduction of filarial nematodes. The biological roles of Wolbachia have been promoted applied researches to use them as novel drug targets for treatment of human filarial diseases, including lymphatic filariasis. Moreover, Wolbachia may contribute to the inflammatory immune response in lymphatic filariasis patients. Analysis of proteins expressed by filarial nematode Wolbachia will provide important information on the biology of this endosymbiont, and will help validate candidate proinflammatory proteins of Wolbachia. In this study, we developed a Wolbachia isolation method from filarial nematodes, the dog heartworm (Dirofilaria immitis) and lymphatic filarial parasite (Brugia malayi), and characterized proteins derived from Wolbachia of B. malayi by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) coupled with matrix-assisted laser desorption/ ionization time of flight mass spectrometry (MALDI-TOF-MS). In addition, the role of recombinant Wolbachia surface protein (rWSP) of B. malayi Wolbachia in proinflammatory cytokine responses by the murine macrophages RAW 264.7 cell line was examined. The isolation protocol for the filarial nematode Wolbachia with the use of NP-40 at 0.04% was found to facilitate the protein analysis. Proteins of Wolbachia purified from D. immitis and B. malayi were defined specifically by Western blot analysis with anti-Wolbachia antibodies. Based on the immunoblot analysis, the 83-kDa, 48-kDa, 36-kDa, 26-kDa (WSP), 20-kDa and 17-kDa antigenic bands of B. malayi Wolbachia were detected. Moreover, the common antigens, WSP, 20-kDa, and 17-kDa, to both Wolbachia of D. immitis, and B. malayi were identified. The analysis of 2D-PAGE blots revealed 24 reactive spots specific for Wolbachia of B. malayi. The 6 WSP spots (26 kDa, pI 4.7-5.5), and additional 11 specific spots (20-62 kDa, pI 4.7-7.3) were identified on the Coomassie blue-stained gels. The WSP occurred in several spots different in pI values representing differently modified protein species that could be of biological relevance. The mapped B. malayi Wolbachia proteins will be useful for further structural and functional studies, which indicate their importance in biological functions of the Wolbachia, as well as in development and reproduction of filarial nematodes. The expression of proinflammatory cytokine mRNA in murine macrophage RAW 264.7 cells incubated with the rWSP of B. malayi Wolbachia was evaluated by real-time reverse transcriptase-polymerase chain reaction (real-time RT-PCR). Interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α mRNA from the murine macrophages were upregulated by the rWSP stimulation in a dose-dependant manner. When 9.0 μg/ml rWSP was supplemented to the cell cultures, highly enhanced production of IL-1β, IL-6, and TNF-α mRNA was detected, and comparable to Escherichia coli lipopolysaccharide (LPS) (0.1 μg/ml) induction. The induction of all cytokines was abolished by proteinase-K treatment of the rWSP. Our results indicated that the WSP represented proinflammatory activity that was capable of directly stimulating the transcription of the murine macrophage cell line RAW 264.7.