| 摘要: | 創傷弧菌（Vibrio vulnificus）是一株生活在海水中的高致死率致病菌，且其發病到死亡往往
只要2-3 天，在臨床的病徵主要是食入含有創傷弧菌的海產所引發的敗血症，或是經由外傷感染，
包括海產養殖器具或海產刺傷所造成的組織潰爛。因為創傷弧菌好發於肝臟相關疾病以及免疫力
低弱的患者，並且台灣肝病患者比率相當高，再加上台灣養殖業興盛且近年來感染病例持續增加，
因此，尋找創傷弧菌主要的致病基因，以進一步加以控制並治療此致病菌所造成的傷害更是刻不
容緩。
血清是防禦外來微生物侵犯體內的重要防線，其中富含多種的殺菌蛋白（antimicrobial
peptides），包括血清補體（serum complement）、defensin 及polymyxin B….，致病菌之所以能感
染並侵入體內存活造成傷害，就必須具有抵抗這些殺菌蛋白殺菌作用的機制，菌體可能在外在環
境變化時如在含有殺菌蛋白(polymyxin B)的環境進行其基因及表現型的改變，以應付環境的變
化。我們發現大部分來自臨床的創傷弧菌在殺菌蛋白polymyxin B 的環境下都會形成菌體聚集的
現象，而環境菌株則較不易形成菌體聚集現象，並且這些形成菌體聚集現象的菌株明顯地比無菌
體聚集現象的菌株更具有殺菌蛋白的抗性能力，因此，由我們初步的結果顯示，創傷弧菌菌體聚
集的現象可能扮演菌體抵抗殺菌蛋白殺菌作用的重要機制，除此之外，菌體也可能由一些調節因
子來調節菌體的聚集現象。目前，有關創傷弧菌基因的研究也為數不少，包括夾膜、鐵吸收因子、
蛋白質分解酵素、及溶血蛋白都是可能的致病因子，然而，對於創傷弧菌在面對殺菌蛋白如
polymyxin B 時，菌體形成聚集的現象而產生抗殺菌作用的機制卻是一無所知，因此，我們試圖找
出創傷弧菌菌體聚集相關的基因，可進一步瞭解菌體聚集相關的基因在抗殺菌作用及感染過程的
角色。
目前我們由1,150 轉位子突變株進行菌體聚集實驗分析，找出5 株菌體聚集能力下降的轉位
子突變株，在本計畫中，我們準備進一步選殖這些菌體聚集基因，並分析其在殺菌蛋白polymyxin
B 及血清殺菌作用的角色，以期能對細菌感染控制有所助益。
Vibrio vulnificus, a highly virulent marine bacterium, is the causative agent of seafood-related diseases,
such as primary septicemia and wound infection in human, particularly in those with certain underlying
diseases especially in patients with chronic liver disease. Cases of V. vulnificus infections have been
reported from many areas of the world. Over the last decade, there has been a dramatic increase in the
number of cases due to V. vulnificus in the southern part of Taiwan. Because V. vulnificus often
contaminates the seafood and the consumption of seafood especially raw seafood is very popular, V.
vulnificus infection is apparently more prevalent on this island than in many areas of the world.
The bactericidal effect of serum is an important defense by the host against invading microorganisms.
The bactericidal mechanism often results from the killing actions of a group of cationic antimicrobial
peptide including serum complement, defensin and bactericidal/permeability-increasing protein (BPI).
They all knew to act by inserting in the bacterial cytoplasmic membrane to form pores resulting in
leakage of essential cellular components and death of the bacteria. Other cationic antimicrobial peptides
include melittin, magainins, polymyxin B, protamine sulfate, polylysin, and cecropins. In response to this
host defense, bacteria may evolve strategies to counter these antimicrobial peptides bactericidal effect.
Bacteria may lead to an increase in genetic and phenotypic variability under stress conditions including
antimicrobial peptide polymyxin B treatment. In our study, we found that most of clinical V. vulnificus
could form aggregated bacteria during antimicrobial polymyxin B treatment. In addition, non-aggregated
V. vulnificus strains isolated from enviroment were significantly more easily killed by polymyxin B than
were the aggregated strains. Our data suggested that aggregation play an important role in antimicrobial
peptide resistance during encountering antimicrobial peptides, and factor(s) of V. vulnificus may mediate
phenotypic variation in V. vulnificus aggregation.
Several bacterial products such as capsule polysaccharide (CPS), iron-sequestering systems,
metalloprotease, and type IV leader peptidase-N-methyltransferase have been suggested as the potential
virulence factors of V. vulnificus; however, a comprehensive analysis of gene(s) contributing bacteria
aggregation during polymyxin B treatment (or serum) has not been conducted. To address this deficiency
in knowledge, we continuously cloned genes for bacteria aggregation during polymyxin B treatment by
isolation and characterization of mutants of V. vulnificus with defects in their bacteria aggregation from
transposon mutant library. In the present, we already isolated 5 mutants with defects in their bacteria
aggregation during polymyxin B treatment from 1,150 transposon mutants and found that 2 mutants
exhibit opaque colony morphology and 3 mutants are translucent. In addition, we are going to study the
relationship between aggregation forming gene and the bactericidal effect of polymyxin B and serum by
analyzed the bacteria survival of wild type and bacteria aggregation mutant during polymyxin B and
serum treatment. |
