One of my favorite diseases is dental caries. I remember learning, as a wee microbiologist, that cavities were an infectious disease. The idea seemed revolutionary to me at the time. This idea is fairly routine in the world of dentistry nowadays, but it wasn’t always that way.
Hominids have suffered cavities throughout history, but at much lower rates than modern man does. There is a strong correlation of dietary sugar and caries, well illustrated by the decrease in cavities in countries with rationed sugar in WWII. The oral bacterium Streptococcus mutans ferments this sugar into lactic acid, which acidifies the environment directly next to the tooth enamel. Enamel is made of minerals such as calcium phosphate, which become soluble in acidic environments, and this erosion is what can eventually form a cavity. (This is a great example of the situational benefit of microbial metabolic byproducts – just last week, we covered lactic acid as one beneficial byproduct of the vaginal microbiome.)
To better understand how S. mutans causes caries, scientists have identified a number of virulence factors, including adherence, acid production, and acid tolerance, that help S. mutans cause this disease. Scientists hope to understand how these genes are regulated in the constantly changing environment of the oral cavity (imagine how things change when you drink soda or eat ice cream).
Previous work from Dr. Grace Spatafora’s lab found that metal deprivation affected virulence gene expression. Her lab studies SloR, an S. mutans protein that interacts with metal ion cofactors to activate genes involved in competence, antibiotic resistance, and adherence. The Spatafora lab collaborated with Dr. Arthur Glasfeld to characterize the mechanism of this modulation, and the results are now available in the latest issue of Journal of Bacteriology.
To test SloR-DNA interactions, the researchers used the promoter of sloABC, an operon coding for a metal transporter that is repressed by SloR. While the region of DNA recognized by SloR was known, the authors determined the 22 basepairs where SloR bound within this region, and further found the DNA would interact with either SloR dimers or double dimers. All protein-DNA interactions were abrogated by adding EDTA, a commonly used metal chelator – confirming that SloR requires a metal cofactor to act as a repressor.
How specific must the DNA sequence be for SloR to bind it? To test this, the authors systematically mutated each of the 22 nucleotides in the recognition region. They then looked by EMSA and qRT-PCR to investigate SloR-DNA binding and functional repression with these variant sequences. The results showed that several nucleotides in the SloR recognition sequence and flanking this sequence were required for full SloR repression, with three particular mutations having the strongest effect.
Finally, the authors used this data to generate a model predicting SloR-DNA binding. This shows the double-dimers binding (seen in the above experiments) around the -35 sequence, blocking transcription.
Why is it important to understand this protein-DNA interaction? Despite S. mutans causing one of the most common infectious diseases, our treatments of it remain relatively rudimentary: scraping out and cementing over infected regions. Studying metal scavenging will help dentists make recommendations on between-meal activities (since this is when the bacteria are most starved for metal cofactors). Understanding virulence regulation will also help dentists devise less invasive and more effective treatments. Imagine disabling S. mutans virulence gene expression by blocking SloR regulation using an additive in toothpaste – or even chewing gum!
-- Julie Wolf