Kuru quickly sought the collaborative support of one of the world's leading Chlamydiae researchers, Anthony Maurelli of the U.S. government's Uniformed Services University of the Health Sciences, and forwarded the new FDAA probes to Maurelli's laboratory in Bethesda, Md.
"When the FDAAs failed to label peptidoglycan in Chlamydiae, my postdoc George Liechti and I were obviously disappointed. But we really believed that Chlamydiae made peptidoglycan so we encouraged Erkin to come up with a new labeling method," Maurelli said. "We hypothesized that enzymes unique to synthesizing peptidoglycan in Chlamydiae may not have recognized the FDAA probes, so we needed Erkin to develop an alternative labeling strategy."
"It turned out the strategy Erkin needed was already in his tool box," VanNieuwenhze said. "As an alternative labeling strategy, we had synthesized a different type of probe, this time mimicking the last two amino acids of the peptidoglycan peptide sequence."
The IU team, with chemistry support from graduate student Edward Hall and postdoctoral researcher Alvin Kalinda, linked the two amino acids (D-alanine-D-alanine, or dipeptide) to chemical groups that could be selectively functionalized with fluorescent probes, after their incorporation into peptidoglycan, by a very specific chemical reaction called click chemistry. The clickable dipeptides were used and validated with a number of other bacterial species to show they were incorporated in peptidoglycan, and the new probes were forwarded to Maurelli's team.
When the new dipeptide probes were applied to cells infected with Chlamydia trachomatis, it worked immediately, said Liechti, a postdoctoral researcher in Maurelli's lab who conducted the experiments.
"The chlamydia inside the infected cells lit up with green fluorescent peptidoglycan rings in the middle of the red-stained bacterial cells," Liechti said.
|Contact: Stephen Chaplin|