| Summary: | 碩士 === 國立清華大學 === 分子醫學研究所 === 103 === Pseudomonas aeruginosa is an opportunist pathogen that can cause lung infection, severe wound infection, urinary tract infections and bacteremia. It secretes several proteases that are play critical roles for infection. Elastase B (LasB) is a metalloprotease, which can degrade proteins from human tissues such as elastin and collagen. Elastase A (LasA) is a endopeptidase, which can cleave glycine-containing proteins and elastin. Protease IV (PrpL) is a lysyl endopeptidase that can digest fibrinogen, plasminogen and several proteins which belonging to immune defense system. These proteases have been demonstrated to play critical roles in initial infection of P. aeruginosa, but interactions between them and modulation of secretome by these proteases are not clear. In this study, we analyzed the secretome of ∆lasA, ∆lasB, ∆prpL, and wild type B136-33 and find out modulation of secretome by LasA, LasB, PrpL. The secreted proteins were resolved by SDS-PAGE, and subsequently identified using MALDI-TOF MS and LC-MS/MS. The result showed that a number of secreted peptidases, outer membrane lipoproteins and A-type flagellin were modulated by one of three proteases. In addition, the hemolysis activity, caseinase activity and biofilm formation were decreased, while pyocyanin production was increased in the ∆lasB strain. Pyoverdine production was decreased in both ∆lasA and ∆prpL strains. P. aeruginosa wild type B136-33 and ∆lasA, ∆lasB and ∆prpL were able to induce cytokines IL-1β, IFN-γ and TNF-α production from macrophages RAW 264.7. Among these cytokines, the production of TNF-α was decreased after macrophage was induced by ∆lasA and ∆prpL culture supernatant. Taken together, the study indicates that LasB and LasA and PrpL not only are the virulence factors of P. aeruginosa, but also are modulators, essential for processing other virulence components and their own selves. This study provides useful information for understanding the interactions among secreted proteases, and for improving the treatment of P. aeruginosa infection diseases.
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