A transwell assay method to evaluate Borrelia burgdorferi sensu stricto migratory chemoattraction toward tick saliva proteins.

ABSTRACT

We developed a transwell assay to quantify migration of the Lyme disease agent, Borrelia burgdorferi sensu stricto (s.s.), toward Ixodes scapularis salivary gland proteins. The assay was designed to assess B. burgdorferi s.s. migration upward against gravity through a transwell polycarbonate membrane overlaid with 6% gelatin. Borreliae that channeled into the upper transwell chamber in response to test proteins were enumerated by flow cytometry. The transwell assay measured chemoattractant activity for B. burgdorferi s.s. from salivary gland extract (SGE) harvested from nymphal ticks during bloodmeal engorgement on mice 42 h post-attachment and saliva collected from adult ticks. Additionally, SGE protein fractions separated by size exclusion chromatography demonstrated various levels of chemoattractant activity in the transwell assay. Sialostatin L, and Salp-like proteins 9 and 11 were identified by mass spectrometry in SGE fractions that exhibited elevated activity. Recombinant forms of these proteins were tested in the transwell assay and showed positive chemoattractant properties compared to controls and another tick protein, S15A. These results were reproducible providing evidence that the transwell assay is a useful method for continuing investigations to find tick saliva components instrumental in driving B. burgdorferi s.s. chemotaxis.