@phdthesis{ElGohary2018, author = {Heba El Gohary}, title = {Functional characterization of a novel protease isolated from a mouse-adapted S. aureus strain}, journal = {Funktionelle Charakterisierung einer neuen Protease, die aus einem Maus-adaptierten S. aureus-Stamm isoliert wurde}, url = {https://nbn-resolving.org/urn:nbn:de:gbv:9-opus-25152}, pages = {106}, year = {2018}, abstract = {Background: The high incidence of methicillin-resistant Staphylococcus aureus (MRSA) strengthens the need for new effective antibiotics and a protective vaccine. Up till now, mainly human-adapted Staphylococcus aureus strains were used to study S. aureus pathogenicity in mouse models. However, it is known that S. aureus is highly host-specific. Recently, a mouse-adapted S. aureus strain, JSNZ, was identified. This strain could be a promising tool in developing more appropriate infection models. JSNZ produces high amounts of a putative extracellular protease, named JSNZ extracellular protease (Jep). Since the jep gene was only detected in S. aureus isolates from laboratory mice and wild small rodents and shrews, we hypothesize that Jep is important for colonization and infection in mice. The jep deletion mutant previously created by our collaborators from the University of Auckland, New Zealand, intriguingly showed a reduced survival and growth fitness in murine serum and whole blood as compared to the JSNZ wild type (WT) strain. Objective: To elucidate the role of Jep in the interaction between S. aureus and its host by comparing the impact of JSNZ WT with a mutant and a complement strain on the murine immune system. In addition, the elucidation of possible genetic factors behind host-adaptation of S. aureus strains isolated from wild rodents and shrews. Methods: A jep complemented strain was generated by chromosomal replacement. JSNZ WT, the jep mutant and the complement strain were subjected to functional assays (whole blood survival assay, coagulation assay). In addition, the genetic background that might confer host specificity was tested by staph array genotyping. Results: The mutant strain JSNZDjep was successfully complemented with the jep gene using a chromosomal integration approach. The WT strain and the complemented strain produced the Jep protein in comparable amounts. Unexpectedly, the complemented strains did not behave like the WT strain but rather like the mutant in a series of in vitro assays. Firstly, the growth of both the deletion mutant and the complemented strains was slightly reduced in TSB as compared to the WT strain. Secondly, the jep knockout strain showed a strongly reduced survival in murine whole blood compared to its wild type counterpart, but so did the complemented strain. Finally, the coagulation of murine plasma was less pronounced for the jep deletion mutant and the complemented strain as compared to the JSNZ WT. To exclude a defect in jep gene expression, we compared the amount of Jep expressed during growth in TSB medium for the three strains. The complemented strain produced Jep in a manner similar to the WT strain in a growth-phase dependent manner, suggesting that Jep expression was not affected during the creation of the complemented strain. The array data showed some differences in the genetic makeup between animal isolated strains and matched human strains. For example, while all animal isolates of the CC88 lacked the resistance mecA gene it was found in some human isolates of the same strain. Conclusion: In conclusion, our unidentified mutation created during the generation of the jep knock-out strain rather than the jep gene itself manipulated the murine immune response. The responsible gene and the underlying mechanisms remain to be clarified. Genetic profiling of S. aureus strains allowed us to obtain some valuable information including data about CC49, the most frequently isolated lineage in wild rodents and shrews where compared to the human isolates the murine strains showed clear signs of host adaptation. However, the analysis had several limitations including the small sample size.}, language = {en} }