創傷弧菌是一種藉由傷口或食入受汙染之海鮮，感染人類的侵入性革蘭氏陰性病原菌，可造成嚴重的皮膚損傷與敗血症。我們實驗室已發現該菌所產生的RTX細胞毒素在對抗巨噬細胞之吞噬作用上扮演重要角色，然而，此毒素如何造成吞噬作用之缺陷，目前仍不清楚。為了探討RTX毒素如何參與抗吞噬作用，我們必須瞭解此毒素的分泌機制，以及當細菌感染宿主細胞之後，此巨型毒素是否被切割，和此毒素會位於宿主細胞的何處。本研究中，我初步純化出包含RID domain的RTX2366-2928胜肽鏈，並以此胜肽鏈免疫小鼠，製備anti-RID抗血清。以此抗血清進行西方墨點分析生長於LB培養基之創傷弧菌野生株的全細胞萃取液，在130至170 kDa的區間能偵測到RTX專一性的bands。將野生株與HEp-2細胞共同培養後，再收取其全細胞萃取液進行西方墨點分析，發現RTX專一性的bands數目更多，分布的範圍也更廣，顯示當RTX毒素被運送穿越細菌，再與宿主細胞接觸的過程中，此毒素可能進一步被切割。然而，在霍亂弧菌已被發現參與RTX自我切割的CPD domain，在本研究中，則發現並非為創傷弧菌RTX的切割或活性所必須。以西方墨點分析構成第一型分泌系統之成員(包含RtxB、RtxD和TolC) 的突變株，發現其RTX毒素會累積於細胞膜和細胞質中。Anti-RID抗血清在免疫螢光染色之實驗中，於未感染細菌之HEp-2細胞組別和以不產RTX的突變株感染之組別，都有非專一性訊號。此顯示若使用此anti-RID 抗血清於免疫螢光染色實驗以偵測RTX毒素，此抗血清之專一性需加以改善。

Vibrio vulnificus, a gram-negative invasive bacterial pathogen, causes severe skin lesions and septicemia via wound infection or ingestion of contaminated seafood. We have previously shown that the RTX (repeat in toxin) cytotoxin is an important virulence factor of V. vulnificus protecting the organism from engulfment by the phagocytes. However, how this toxin leads to impairment of phagocytosis is unclear. To explore how RTX participates in antiphagocytosis, we need to understand how this toxin is secreted, whether this enormous toxin is processed, and where this toxin is located in the infected host cell. In this study, I have purified a peptide, RTX2366-2928, that contains the Rho-inactivation domain (RID), and generated the antiserum against this peptide in mice. Western blotting with this anti-RID antiserum revealed the RTX-specific bands ranging from 130 kDa to 170 kDa in the whole cell lysate of a wild-type strain. A different RTX banding pattern with a wider range of molecular weight was detected in the whole cell lysate of the wild-type strain coincubated with HEp-2 cells. These suggest that the RTX toxin might be processed during transport across the bacterial cell wall and interaction with the host cell. However, the CPD (cysteine protease domain) domain, which has been implicated in RTX autoprocessing in V. cholerae, of RTX is found in this study not required for the processing or activity of RTX in V. vulnificus. The RTX toxin accumulated in cell membrane, and cytoplasm of the mutants defective in various components, including RtxB, RtxD and TolC, of Type I secretion system. The anti-RID antiserum gave rise to non-specific signals in the uninfected HEp-2 cells or those infected by the RTX-deficient V. vulnificus mutant examined by immunofluorescent staining. This indicates that the specificity of anti-RID antiserum needs to be improved if it is to be used in immunofluorescent staining to detect the RTX toxin.

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