Dr. Martin Chalfie is a co-recipient of the 2008 Nobel Prize in Chemistry for his groundbreaking work on green fluorescent protein (GFP) which has become essential for visualizing a variety of cellular processes with breathtaking, glow-in-the-dark clarity. However, the GFP and its variants required oxygen to work their magic, and that limited greatly their applicability to anaerobic, or oxygen-phobic microorganisms. For scientists who study the oral biofilm, that can be a big problem. Porphyromonas gingivalis, Prevotella intermedia, Fusobacterium spp, Peptostreptococcus, Eubacterium—all are anaerobic, and all are associated with chronic periodontitis. Following these bugs and their metabolic activities in real-time would provide a much-needed tool to track the onset and progression of chronic periodontitis.
In the April 2011 issue of the journal PLoS ONE, a team of National Institute of Dental and Craniofacial Research (NIDCR)-supported scientists reports that it has the solution. They demonstrated for the first time in P gingivalis that a strictly anaerobic microorganism can be genetically engineered to express a GFP probe. In this case, the probe is an adaptation of the recently discovered oxygen-independent flavin mononucleotide fluorescent proteins. Their natural fluorescence derives not from jellyfish, the original source of GFP, but from blue light-sensing proteins of the bacteria Bacillus subtilis and Pseudomonas putida. In their initial in vitro experiments, the scientists could directly track green protein-expressing P gingivalis as it invaded living human gingival epithelial cells. The scientists could distinguish in which cellular compartments copies of the bacterium colocalized, a microscopy term for an overlap of fluorescent tags that would indicate their close spatial proximity. The researchers also found the bioengineered bacteria were equally adept as natural P gingivalis at invading the gingival cells and proliferating within them. Based on their initial work, the scientists said they are confident the technique will be effective in additional cell types and to track the movement of P gingivalis and other anaerobes in animal models.
(Source: NIDCR, Science News in Brief, July 13, 2011)
