Two new studies on oral bacterium that causes lethal heart valve infection

S. sanguinis
False-colored transmission electron micrograph of Streptococcus sanguinis cells (purple ovals) encased within an infected heart valve in an animal model of infective endocarditis. Mutants lacking either the NrdEF ribonucleotide reductase or the NrdI protein required for manganese cofactor formation were unable to cause disease. Click on the image to see a larger version of it.

Two back-to-back studies published in The Journal of Biological Chemistry have provided significant insights into virulence of Streptococcus sanguinis, which causes a potentially lethal infection of heart valves.
Infective endocarditis occurs when the otherwise innocuous S. sanguinis, a Gram-positive, facultative aerobic, oral bacterium, enters the blood stream and colonizes vulnerable heart valves or endocardial tissue, an infection that proves to be lethal for more than 20 percent of patients. The severity of this disease and the lack of a vaccine for it make it imperative to understand the mechanism of virulence by S. sanguinis to facilitate the development of potent antimicrobial agents.
The studies in the JBC resulted from a collaboration between the labs led by Todd Kitten at Virginia Commonwealth University and JoAnne Stubbe at the Massachusetts Institute of Technology.
Two past observations prompted the research teams to examine the activity of the bacterium’s class Ib ribonucleotide reductase, or RNR. These essential enzymes rely on metallo-cofactors to convert ribonucleotides into deoxyribonucleotides, precursors for DNA replication and repair. In S. sanguinis, RNRs occur in two forms: the aerobic class Ib and the anaerobic class III.
The first observation was that deletion of a manganese transporter called SsaB drastically reduces the virulence of S. sanguinis and its ability to tolerate oxygen. So the researchers set out to “identify manganese-requiring proteins that would also be required for growth in oxygen,” explains Kitten. The second observation was that class Ib RNRs, along with an iron cofactor, also appear to employ a dimanganese-tyrosyl radical cofactor for in-vivo activity. “We wondered whether the oxygen-dependent class Ib RNR might be the manganese-requiring enzyme we were seeking,” Kitten says.
In the first study, the researchers demonstrated that the S. sanguinis RNR can not only self-assemble a diferric-tyrosyl radical in the presence of oxygen, but also assemble a dimanganese-tyrosyl radical, if provided with an additional enzyme called NrdI.

Todd Kitten
Joanne Stubbe

“In my view, the main contribution of the first study was that it identified all the components and established that the S. sanguinis RNR had the properties that were expected of it,” says Kitten. “We confirmed that RNRs behaved the way we thought they would in vitro, and we provided direct evidence about which components are required for RNR activity.”
Emboldened by those findings, the researchers in the second study created mutant strains of S. sanguinis lacking class Ib RNR or other RNR-related enzymes and tested those mutants for growth competency under aerobic and anaerobic conditions.
The authors reported that the mutants lacking the genes for synthesis of class Ib RNRs or the manganese cofactor were unable to grow aerobically (but grew normally under anaerobic conditions) or cause endocarditis in a rabbit model system. This phenotype, however, could be partially rescued by heterologous complementation with a class II RNR gene, which codes for an oxygen-independent, adenosylcobalamin-cofactored RNR.
These results allowed the authors to conclude that manganese was indeed critical for the proper function of RNRs and, consequently, for the virulence of S. sanguinis.
The work is significant because the results provide a novel target — manganese cofactored RNRs — for developing antimicrobial agents designed to treat infective endocarditis, and that is made even more promising by the fact that such RNRs do not exist in eukaryotes. The results also may answer the longstanding question of why some bacteria require manganese for oxygen tolerance and virulence.

Sapeck AgrawalSapeck Agrawal (sapeck.srivastava@ recently earned her Ph.D. in molecular microbiology and immunology from The Johns Hopkins University.