Wolbachia isolation by fractioned centrifugation from Dirofilaria immitis for protein analysis

Abstract

Objective : The biological roles of Wolbachia, a filarial intracellular bacteria, have promoted applied researches in lymphatic filariasis. Analysis of Wolbachia proteins will provide complementary information on the biology of Wolbachia, as well as the immune mechanisms against filarial nematodes. We therefore developed a Wolbachia isolation method from Dirofilaria immitis (dog heartworm) for protein analysis.

Methods : Wolbachia bacteria were homogenized and isolated by fractioned centrifugation by using 0.85% NaCl supplemented with varying concentration of Nonidet P-40 (NP-40) as the homogenization buffer. The purity of Wolbachia extracts were analyzed by sodium dodecyl sulfate polyacrylamide-gel electrophoresis (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 qPCR targeting 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 bacterial isolation and decrease the host parasite contaminations.

Conclusion : The homogenization buffer which provided the highest Wolbachia protein concentration, but the lowest filarial contamination, was 0.85% NaCl + 0.08% NP-40. This technique is likely to provide the most suitable methods for Wolbachia isolation to facilitate the proteomic studies. However, our purification procedures do need validation in other applications, which require intact Wolbachia DNA materials, and/or viable cells.