綠膿桿菌為一株伺機性感染之病原菌，藉由肺部、傷口、尿道進行感染並造成全身性的菌血症。其能夠分泌許多蛋白酶，在感染過程中扮演重要的角色。其中，彈性蛋白酶 B (LasB) 為金屬蛋白酶，它能夠分解人體組織中的蛋白質，例如彈性蛋白和膠原蛋白；彈性蛋白酶 A (LasA) 為肽鏈內切酶，它能夠切割含有甘氨酸的蛋白質及彈性蛋白；蛋白酶 IV (PrpL) 為賴氨酸肽鏈內切酶，其會分解纖維蛋白原、纖維蛋白溶酶原以及一些與免疫防禦系統相關的蛋白質。這些蛋白酶已經被證實在綠膿桿菌感染初期扮演重要的角色，但它們之間的交互作用以及對其他外分泌蛋白的調節機制仍不清楚。在本研究中，我們分析突變株 ΔlasA、ΔlasB、ΔprpL以及野生型 B136-33 之外分泌蛋白體 (secretome)，以了解 LasA、LasB 及 PrpL 對外分泌蛋白的調節。首先以聚丙烯醯胺膠體電泳解析細菌的外分泌蛋白質，再以基質輔助激光解吸電離飛行時間質譜 (MALDI-TOF MS) 和液相層析串聯質譜 (LC-MS/MS) 進行蛋白分析與鑑定。結果顯示，許多分泌性胜肽酶、外膜脂蛋白和A型鞭毛蛋白都會受到 LasA、LasB 及 PrpL其中一種的調節。此外，ΔlasB 突菌株之溶血能力、酪蛋白酶活性以及生物膜形成能力均降低，而綠膿素 (pyocyanin) 生成量增加。在 ΔlasA 和 ΔprpL 中，螢光鐵載體 (pyoverdine) 生成量皆降低。綠膿桿菌 B136-33、ΔlasA、ΔlasB 和 ΔprpL 之培養上清液皆能夠誘導巨噬細胞 RAW264.7 之 IL-1β、IFN-γ 以及 TNF-α產生；其中，以 ΔlasA 和 ΔprpL 之培養上清液刺激巨噬細胞時，其會產生較少的TNF-α。綜合以上結果，綠膿桿菌的 LasA、LasB 和 PrpL 不僅是重要的致病因子，同時也扮演調控的角色，對於其他毒力因子以及本身的調節是不可缺少的。本研究提供了分泌蛋白酶之間的相互作用及調節之重要資訊，以用於改善綠膿桿菌感染症的治療。

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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