Investigation of the Interplay between Macrophages and Drug-Resistant Staphylococcus Aureus upon Antibiotic Treatment

Project: National Science and Technology CouncilNational Science and Technology Council Academic Grants

Project Details


Staphylococcus aureus has long been recognized as a major cause of healthcare- associated infections. We previously hypothesized that treatment with antibiotics may act as environmental stresses to induce σB, a stress response transcription factor, in antibiotic- resistant strains. We were especially interested in the stress response in vancomycin-resistant S. aureus (VRSA) strains treated with vancomycin. Our recent findings indicate that vancomycin induced σB activity led to the alternation of downstream virulence genes in VRSA strains, as well as the increase in cytotoxicity. We then observed that more vancomycin-treated VRSA cells were engulfed than untreated VRSA and persisted intracellularly in macrophages for several days followed by the lysis of macrophages. The untreated VRSA was cleared quickly upon phagocytosis by macrophages. An increase in TNF-α, as well as a decrease in IL-1βcytokine secretion was also observed in macrophages upon infection by vancomycin-treated VRSA. We hypothesize that phagocytosis of VRSA by macrophages is enhanced upon vancomycin treatment but the intracellular killing of the ingested bacteria is suppressed thereafter. The mechanisms underlying may reflect to our previous findings that the expression of fnbAB (encoding fibronectin binding proteins; FnBPs) and hla (encoding α-hemolysin) is altered through the activation of σB triggered by vancomycin. These possibilities will be investigated using different mutant strains (fnbAB, hla, or both) to evaluate their capacity to trigger the phagocytosis and intracellular killing in the presence/absence of vancomycin. The impairment of intracellular killing of ingested VRSA upon vancomycin treatment will be studied by investigating the maturation and function of phagosomes and phagolysosomes in macrophages. Whether the intracellularly persisted VRSA enhances the apoptosis of macrophages will be investigated by detecting biochemical markers using Western blotting, as well as quantitative assay by flow cytometry. Activation of pro-inflammatory cytokine response is triggered by MyD88-mediated signaling through the ligands binding to various receptors thereby inducing the activation of NF-κB, and increased expression of IL-1R1 accessory protein, TNF-α and IL-1β.The discordant expression of TNF-α and IL-1βin our case suggests that the function of caspase-1, a protease that is essential in cleaving pro- IL-1βto active form, is impaired. Activation of caspase-1 depends on the formation of inflammasome through the recognition of conserved microbial structures. Whether the intracellularly persisted VRSA impairs the inflammasome formation will be investigated by measuring the mRNA and protein expression levels of inflammasome-associated genes and proteins using quantitative real-time PCR and Western blotting, respectively. Secretion of pro-inflammatory cytokines by macrophages upon infection by different VRSA mutant strains will be quantified using ELISA assay to determine bacterial factors involving the alteration of cytokine secretion. We anticipate that our study will provide new insights into the interaction between macrophages and drug-resistant bacteria upon improper antibiotic treatment.

Project IDs

Project ID:PC10308-0672
External Project ID:MOST103-2320-B182-022
Effective start/end date01/08/1431/07/15


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