Immunity studies in cholera
Investigator: Wagner Montor, Ph.D.
Collaborators: John Mekalanos (Harvard Medical School), Peter Yoon (Harvard Medical School), Ann Thanawastien (Harvard Medical School)
Together with the Mekalanos lab, we have developed a high throughput method for screening for proteins that trigger toll-like receptor response in cells. We have screened the entire V. cholerae proteome and identified approximately 13 proteins that trigger an innate immune response. One of these has been characterized in detail as acting through TLR4 that is independent of the LPS response.
V. cholerae proteins expressed in vitro are active in biological assays.
A. Six different proteins were produced alone or together as a pool. After in vitro protein production, 10 ul of each RRL mixture and its serial dilutions were added to treat A549 reporter cells for 4 h. Luciferase-based NF-kB activation was shown in the bottom. Squares shown in red indicate positive NF-kB activation. FlaD, Flagellin D; FlaC, Flagellin C; NAR, nitrate reductase; OmpA, outer membrane A; FNR, anaerobic transcriptional activator; TRFac, transcription factor.
B. Western blot analysis of in vitro-synthesized proteins.