Expression of Diverse Streptococcal Multiple Peptide Resistance Factors and Lipid Hydrolase in Streptococcus Mitis

Streptococcus agalactiae (Group B Streptococcus; GBS) is a gram-positive pathogen that colonizes the gastrointestinal and lower genital tracts. In GBS, the multiple peptide resistance factor (MprF) synthesizes a novel lipid, lysyl-glucosyl-diacylglycerol (Lys-Glc-DAG), and the well-known lipid lysyl-phosphatidylglycerol (Lys-PG). Lys-PG reduces the negative charge of the membrane, protecting bacteria from cationic antimicrobial peptides (CAMPs). Additionally, GBS encodes a predicted alpha-beta hydrolase upstream of mprF. In Enterococcus faecium, this hydrolase is responsible for the turnover of Lys-PG. This project has three aims: to determine whether other streptococcal MprF proteins also synthesize Lys-Glc-DAG and/or Lys-PG; the impact of Lys-Glc-DAG and Lys-PG production on Streptococcus mitis survival in low pH; and whether the GBS hydrolase is responsible for the turnover of both Lys-Glc-DAG and Lys-PG. S. mitis was chosen as a heterologous host for this study since it does not natively encode mprF and does not natively synthesize Lys-Glc-DAG or Lys-PG. Candidate MprF proteins from other streptococcal species (S. downei and S. ferus) with high identity to GBS MprF were identified by BLASTp. These genes and the GBS hydrolase gene were inserted into a plasmid using Gibson assembly and transformed into S. mitis. This study found that the expression of S. ferus mprF and S. downei mprF in S. mitis conferred synthesis of Lys-Glc-DAG. Significantly, S. ferus MprF synthesized Lys-Glc-DAG at a similar level to GBS MprF. Previous research found that expression of S. salivarius mprF in S. mitis conferred synthesis of Lys-PG also at a similar level to GBS MprF. The production of Lys-Glc-DAG and/or Lys-PG in S. mitis through the utilization of plasmids expressing the different mprFs did not increase the survival of S. mitis in low pH. Finally, preliminary investigation of the GBS hydrolase and E. faecium hydrolase through a cell lysate assay did not show turnover of Lys-Glc-DAG and/or Lys-PG, demonstrating that methods for investigating hydrolase activity require refinement.