Wolbachia isolation by fractioned centrifugationfrom Dirofilaria immitis for protein analysis
Objective : The biological roles of Wolbachia, a filarial intracellular bacteria, have promotedapplied researches in lymphatic filariasis. Analysis of Wolbachia proteinswill provide complementary information on the biology of Wolbachia, as wellas the immune mechanisms against filarial nematodes. We thereforedeveloped a Wolbachia isolation method from Dirofilaria immitis (dogheartworm) for protein analysis.Methods : Wolbachia bacteria were homogenized and isolated by fractionedcentrifugation by using 0.85% NaCl supplemented with varying concentrationof Nonidet P-40 (NP-40) as the homogenization buffer. The purity of Wolbachiaextracts were analyzed by sodium dodecyl sulfate polyacrylamide-gelelectrophoresis (SDS-PAGE), and by quantitative polymerase chain reaction(qPCR) targeting the single copy of the Wolbachia surface protein (wsp)gene. The contaminations from filarial extracts were analyzed by qPCRtargeting the filarial glutathione-S-transferase (gst) gene.Results : We were able to isolate Wolbachia bacteria from D. immitis with high purity.The higher concentration of NP-40 could increase the yield of bacterialisolation and decrease the host parasite contaminations.Conclusion : The homogenization buffer which provided the highest Wolbachia proteinconcentration, but the lowest filarial contamination, was 0.85% NaCl + 0.08%NP-40. This technique is likely to provide the most suitable methods forWolbachia isolation to facilitate the proteomic studies. However, ourpurification procedures do need validation in other applications, which requireintact Wolbachia DNA materials, and/or viable cells.
Faculty of Medicine, Chulalongkorn University
Sanprasert, V; Sungpradit, S; and Nuchprayoon, S.
"Wolbachia isolation by fractioned centrifugationfrom Dirofilaria immitis for protein analysis,"
Chulalongkorn Medical Journal: Vol. 54:
6, Article 2.
Available at: https://digital.car.chula.ac.th/clmjournal/vol54/iss6/2