1、論文名稱( 中文) RTX 毒素在創傷弧菌抗吞噬作用上所扮演的角色論文名稱( 英文) Role of Vibrio vulnificus RTX toxin in antiphagocytosis研究生( 中文) 陳怡璇研究生( 英文) Yi-Hsuan Chen中文摘要創傷弧菌是一種侵入性細菌病原，透過傷口感染或食入汙染海鮮，可造成嚴重的皮膚損傷與敗血症。本實驗室發現該菌所產生的一種稱為 RTX的細胞毒素可能藉由對抗吞噬細胞而促進創傷弧菌在感染部位的增殖，進而侵入血流。本論文研究進一步探討 RTX 毒素如何達成對抗吞噬細胞的功用。我們比較創傷弧菌野生株 YJ016 與其不產 RTX 的突變株
2、HL128 在殺死吞噬細胞、被吞噬細胞吞噬的數量、在吞噬細胞中的存活力等方面的差異。結果發現，YJ016 在 moi = 1 的條件下，與小鼠巨噬細胞株 RAW 264.7 細胞共同培養 90 分鐘時仍維持細胞膜的完整性。在此狀況下，YJ016 的總存活菌數比 HL128 高約 4 倍。而以 gentamicin 保護試驗或吖啶橙-結晶紫染色鏡檢法評估被吞入 RAW 264.7 細胞內的菌數，則 YJ016 比 HL128 低約 4 倍。不過，YJ016 與 HL128 被吞噬後，在RAW 264.7 細胞內的存活率皆相似。使用低溫 4或 cytochalasin D 抑制吞噬細胞吞噬作用，可
3、以看到 HL128 在細胞內菌量因此減少，並且以cytochalasin D 抑制吞噬細胞的吞噬能力後，可觀察到 HL128 的總存活數與 YJ016 相似。因此， RTX 可以保護創傷弧菌對抗巨噬細胞的吞噬作用，但對已被吞噬的細菌，則無提高其胞內存活力的作用。以創傷弧菌感染由小鼠腹腔回收的巨噬細胞，再以吖啶橙-結晶紫染色觀察細菌被吞噬的數量，得到與感染 RAW 264.7 細胞株時同樣的結果。另一方面，無莢膜突變株 JF046 感染 RAW 264.7 細胞後，總存活數與 HL128 相當，但胞內菌量則明顯較 HL128 為低。當我們進一步將 JF046 的 rtxA 基因刪除後所得到的不產
4、莢膜與 RTX 的雙突變株 YH001 在與 RAW 264.7 共同培養時，其總存活數顯著下降且胞內菌量甚至較 HL128 為高，顯示在無血清補體作用的條件下，RTX 對創傷弧菌抗吞噬細胞吞噬的能力比莢膜重要。 英文摘要Vibrio vulnificus, an invasive bacterial pathogen, causes severe skin lesions and septicemia in humans via wound infection or ingestion of contaminated seafood. Previous studies in this lab
5、oratory have shown that the RTX toxin of V. vulnificus promotes bacterial colonization at the infection site and subsequent invasion into the bloodstream by countering the infiltrating phagocytes. In this study we compared the total surviving and phagocytosed bacterial numbers, and the intracellular
6、 survival rate in phagocytes between the wild type strain, YJ016, and rtxA mutant, HL128, to understand how RTX contributes to antiphagocytosis. We found that when coincubated with the mouse macrophage cell line RAW 264.7 at a moi of 1 for 90 min, conditions in which the cells remained viable, YJ016
7、 survived about four-fold better than HL128. On the other hand, compared to YJ016, nearly four-fold more of HL128 were ingested into the RAW 264.7 cells in the gentamicin protection assay, which is consistent with the results of acridine orange-crystal violet stain for detecting the intracellular ba
8、cteria. Nevertheless, the phagocytosed bacteria of both YJ016 and HL128 were killed at similar rates. Inhibition of phagocytosis of RAW 264.7 cells by low temperature or cytochalasin D treatment resulted in reduced intracellular number of HL128, and the total survival of HL128 increased to the wild-
9、type level after cytochalasin D treatment of the phagocytes. These results suggest that RTX may prevent phagocytosis of V. vulnificus by the macrophages but have little contribution to the survival of phagocytosed bacteria. The phagocytosis of various V. vulnificus strains by the peritoneal macropha
10、ges was similar to that by the RAW264.7 cells when examined by acridine orange-crystal violet stain. The acapsular mutant, JF046, and HL128 survived equally well in the presence of RAW 264.7 cells, but much lower number of JF046 was phagocytosed. We further deleted rtxA from JF046 to obtain an acaps
11、ular, rtxA mutant, YH001. The survival of this double mutant in the presence of RAW 264.7 was significantly reduced, and the phagocytosed bacterial number of this mutant was even higher than that of HL128. These results suggest that RTX is more important than the capsule in protecting the unopsonized V. vulnificus from phagocytosis.
