Mechanisms associated with Staphylococcus aureus and Acanthamoeba Interactions, and Therapeutic applications"
Staphylococcus aureus is a major cause of infections within hospitals. Its spread among patients in hospitals is one of the major problems that require urgent attention. Staphylococcus aureus has the ability to interact with other microorganisms such as the free-living amoebae Acanthamoeba which acts as a host for various pathogens. This interaction is highly complex and may be beneficial to one of them or both. The main aims of this project were to study the interactions of Acanthamoeba castellanii with methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-sensitive Staphylococcus aureus (MSSA), and to understand the molecular mechanisms involved. A large variety of molecules play a role in growth, function, migration and apoptosis. The role of Phosphoinositide 3-kinase (PI3K) in Acanthamoeba/bacteria interaction was studied through the use of inhibitors such as LY294002 and wortmannin, while the involvement of protein tyrosine kinase was studied through genistein and protein tyrosine phosphatase by sodium orthovandate (Na3VO4). The role of serine proteases of Acanthamoeba was investigated by using its inhibitor Phenylmethylsulfonyl fluoride (PMSF). The results showed that MRSA exhibited more invasive abilities towards Acanthamoeba, when compared with MSSA. In addition, inhibition of PI3K and tyrosine phosphatase subsequently resulted in the reduction of MRSA and MSSA invasion. However, PMSF increased bacterial uptake by Acanthamoeba. Two dimensional gel electrophoresis was performed to separate and fractionate the Acanthamoeba conditioned medium and the Universal Protein Resource Uniprot database was used for the analysis and identification of protein for the forty three spots identified. This study further stresses the need for antimicrobial drugs that are effective without inflicting damage upon the host, as a result of the resistant nature of MRSA towards a variety of antibiotics. One possible method that can be used is Photodynaic Therapy, due to its effectiveness against microorganisms in a localised infected area, through the use of local combined photosensitizers and light. In addition, to simplify the interaction process, heat-killed fluorescein isothiocyanate-labelled (FITC)-labelled MRSA and MSSA were used. The findings suggest that trophozoites Acanthamoeba trophozoites uptake of MRSA was significantly higher than MSSA which may act as "vehicle" that facilitates the spread of MRSA to susceptible hosts. This study showed that photodynamic therapy had a significant impact on bacterial survival. Photosensitizers m-Tetra (hydroxyphenyl) chlorin (m-THPC) and psoralen were very effective in eradicating bacteria. In antimicrobial assays, natural product eugenol derivatives demonstrated significant inhibitory activity on the growth of MRSA and exhibited dose-dependent cytotoxic effects on Acanthamoeba trophozoites. Future work should continue to identify more molecular mechanisms of Acanthamoeba interaction with MRSA and finding new strategies for effective antimicrobial therapies.
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