title: Cloning, expression and purification of autolysin from methicillin-resistant Staphylococcus aureus: potency and challenge study in Balb/c mice.
abstract: Staphylococcus aureus (MRSA) is an opportunistic pathogen which causes a variety of clinical diseases and leads to high rates of morbidity and mortality. Development of an effective vaccine appears to be a useful strategy to control the infection. Here, the internal region of atl was cloned into the pET24a plasmid and expressed in E. coli BL21 (DE3). Cloning of atl was confirmed by colony-PCR, enzymatic digestion and sequencing. Protein expressed in E coli, BL21 DE3 and was confirmed with SDS-PAGE and western blot analysis. Subsequently, BALB/c mice were injected subcutaneously three times with 20μg of the recombinant autolysin. After Bleeding, autolysin-specific total IgG antibodies and isotypes were evaluated using ELISA. Opsonophagocytic killing assay was performed and experimental challenge was done by intraperitoneal injection with sub lethal doses of MRSA in mice and also survival rate was regularly monitored. Results showed that vaccinated mice could exhibit higher levels of autolysin-specific antibodies (P<0.0001) with a predominant IgG1 response versus control group. Results from in vitro experiments indicated that S. aureus opsonized with immunized-mice sera displayed significantly increased phagocytic uptake and effective intracellular killing versus non-immunized mice. The number of viable bacteria in the kidney of immunized mice showed 1000 times less than the control mice; additionally, an increased survival rate was found after immunization with the candidate vaccine versus control group. Results from this study demonstrated that the autolysin is a valuable target for the development of immunotherapeutic strategies against S. aureus and candidate vaccines.
authors.affiliation: Department of Microbiology, Faculty of Advanced Sciences and Technology, Pharmaceutical Sciences Branch, Islamic Azad University (IAUPS), Tehran, Iran. Electronic address: Haghighat.s@iaups.ac.ir.
authors.forename: Setareh
authors.initials: S
authors.lastname: Haghighat
authors.affiliation: Department of Mycobacteriology & Pulmonary Research, Microbiology Research center, Pasteur Institute of Iran, Tehran, Iran. Electronic address: D.siadat@gmail.com.
authors.forename: Seyed Davar
authors.initials: SD
authors.lastname: Siadat
authors.affiliation: Department of Pharmacology & Toxicology, Faculty of Pharmacy, Pharmaceutical Sciences Branch, Islamic Azad University (IAUPS), Tehran, Iran.
authors.forename: Seyed Mehdi Rezayat
authors.initials: SM
authors.lastname: Sorkhabadi
authors.affiliation: Department of Microbiology, Faculty of Basic Sciences, North Tehran Branch, Islamic Azad University, Tehran, Iran.
authors.forename: Abbas Akhavan
authors.initials: AA
authors.lastname: Sepahi
authors.affiliation: Department of Immunology, Pasteur Institute of Iran, Tehran, Iran.
authors.forename: Mehdi
authors.initials: M
authors.lastname: Mahdavi
mesh_terms: D000818:Animals; D000942:Antigens, Bacterial / Q000276:immunology*; D001426:Bacterial Proteins / Q000276:immunology; D015153:Blotting, Western; D003001:Cloning, Molecular; D004195:Disease Models, Animal; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D055624:Methicillin-Resistant Staphylococcus aureus / Q000276:immunology*; D051379:Mice; D008807:Mice, Inbred BALB C; D009238:N-Acetylmuramoyl-L-alanine Amidase / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification; D013203:Staphylococcal Infections / Q000276:immunology*; D013209:Staphylococcal Vaccines / Q000276:immunology*; D014614:Vaccines, Synthetic
chemical_list: D000942:Antigens, Bacterial; D001426:Bacterial Proteins; D013209:Staphylococcal Vaccines; D014614:Vaccines, Synthetic; D009238:N-Acetylmuramoyl-L-alanine Amidase
keywords: Autolysin; Methicillin-resistant Staphylococcus aureus; Recombinant vaccine