Evaluation of the PBP2 transglycosylase region of Staphylococcus aureus as a target for immunotherapeutic approaches.

Infections caused by Staphylococcus aureus are increasingly prevalent, and treatment has become more difficult due to the emergence of strains that are resistant to multiple drugs, such as methicillin-resistant Staphylococcus aureus (MRSA). Penicillin-binding proteins (PBPs) are essential enzymes in peptidoglycan biosynthesis. Only found in bacteria, they are an excellent target for the development of bacterial control strategies. S. aureus has 4 PBPs, and only PBP2 has transglycosylation activity, making it a good model to evaluate whether the inactivation of the transglycosylase domain (PBP2t) could lead to bacterial death. (His6)-tagged PBP2t was purified from the E. coli cell lysate using Ni-charged resin, and ELISA and immunoblotting assays demonstrated that PBP2t is immunogenic. Flow cytometry analysis was performed to verify the binding of polyclonal antibodies to the bacterial cell surface. In order to verify the ability to provide protection, immunized mice were challenged with a sublethal dose of MRSA, and the bacterial loads in kidneys and spleen were evaluated. A reduction of 2-2.5 logs was seen in organs from immunized mice compared with the negative controls in two independent assays (p < 0.01). Our results demonstrate that the PBP2t is a promising target for the development of novel antimicrobial strategies, but further testing should be performed to validate the protection conferred by immunization with this protein.

Cloning and expression of protease ClpP from Streptococcus pneumoniae in Escherichia coli: study of the influence of kanamycin and IPTG concentration on cell growth, recombinant protein production and plasmid stability.

Infections caused by Streptococcus pneumoniae are one of the main causes of death around the world. In order to address this problem, investigations are being made into the development of a protein-based vaccine. The aims of this study were to clone and express ClpP, a protein from S. pneumoniae serotype 14 in Escherichia coli, to optimize protein expression by using experimental design and to study plasmid segregation in the system. ClpP was cloned into the pET28b vector and expressed in E. coli BL21 Star (DE3). Protein expression was optimized by using central composite design, varying the inducer (IPTG) and kanamycin concentration, with a subsequent analysis being made of the concentration of heterologous protein, cell growth and the fraction of plasmid-bearing cells. In all the experiments, approximately the same concentration of ClpP was expressed in its soluble form, with a mean of 240.4mg/L at the center point. Neither the IPTG concentration nor the kanamycin concentration was found to have any statistically significant influence on protein expression. Also, higher IPTG concentrations were found to have a negative effect on cell growth and plasmid stability. Plasmid segregation was identified in the system under all the concentrations studied. Using statistical analysis, it was possible to ascertain that the procedures for determining plasmid stability (serial dilution and colony counting) were reproducible. It was concluded that the inducer concentration could be reduced tenfold and the antibiotic eliminated from the system without significantly affecting expression levels and with the positive effect of reducing costs.