Intermembrane rhomboid protease activity of Rothia mucilaginosa

Rothia mucilaginosa is a part of the normal oral microflora, which has been recognized as an endodontic pathogen and is also an emerging opportunistic pathogen in immune compromised hosts. In this study, we identified the rhomboid protease in R. mucilaginosa as a candidate virulence factor. A database search identified two rhomboid genes on the genome with open reading frames of 828 bp and 822 bp, respectively. The two genes are separated by 57 bp, suggesting that they constitute a bicistronic operon. Motifs conserved among rhomboid family proteins (HxxxN, GxSG, and GxxxG) were found in their deduced amino acid sequences. The two recombinant rhomboids and the rhomboid/liposome complexes (proteoliposomes) were prepared using a wheatgerm cell-free translation system, and their serine protease activities were assessed. The two rhomboids showed serine protease activity in the form of rhomboid/liposome complex, while the recombinant rhomboids alone showed little or no activity. On the supposition of host invasion, R. mucilaginosa was cultivated in medium with type I collagen, and the rhomboid mRNA levels were examined. Real-time reverse transcription polymerase chain reaction analysis revealed that the levels of the two rhomboid mRNAs increased slightly in culture with type I collagen, although the changes were not statistically significant. These results suggested that R. mucilaginosa possesses two active rhomboids with protease activity on the membrane, and the two rhomboids may be virulence factors involved in the response to host environmental conditions.