Macrophage-specific overexpression of antimicrobial peptide PR-39 inhibits intracellular growth of Salmonella enterica serovar Typhimurium in macrophage cells

Although purified and synthesised PR-39 shows potent antibacterial effects in vitro, its ability to kill intracellular bacteria in macrophages, which are a major cause of refractory intracellular infection, has not yet been demonstrated. Both to enhance its antimicrobial potential and to reduce systemic side effects, it would be desirable to deliver PR-39 into macrophage cells and to limit its activation to the site of infection. To address this issue, PR-39 DNA was inserted into the eukaryotic expression plasmid pIRES2-EGFP and the adenoviral vector Ad-MSP, from which PR-39 can be specifically expressed in macrophage cells from the macrophage-specific promoter. pIRES2-EGFP/PR39 and Ad-MSP/PR-39 were either transduced or infected into macrophage RAW264.7 cells for stable or transient expression of PR-39. PR-39 expression in macrophage cells was subsequently confirmed by reverse transcription polymerase chain reaction (RT-PCR) and immunocytochemistry. Furthermore, its antimicrobial activity in macrophage cells was evaluated by colony enumeration assay. Results showed that the macrophage-specific promoter could initiate targeted expression of PR-39 only in macrophage RAW264.7 cells. Moreover, either stable or transient expression of PR-39 in macrophage cells conferred enhanced antimicrobial activity against Salmonella enterica serovar Typhimurium. Our results have demonstrated that macrophage-specific expression of antimicrobial peptide PR-39 in macrophages could inhibit the growth of intracellular S. Typhimurium and indicated it to be a novel and promising approach for the control of refractory intracellular infection.