摘要：微生物角膜炎是屬一種很嚴重的眼部感染症，且常造成嚴重永久性的視力損害。而綠膿桿菌是造成微生物角膜炎中最常見的病菌之一，約占三分之一的病例。在綠膿桿菌角膜炎的致病機轉中綠膿桿菌的第三類分泌系統(Type three secretion system; T3SS) 是其重要的致病因子(virulencefactor)之一。此分泌系統會製造及分泌會傷害角膜細胞的外毒素(exotoxin)而造成角膜的發炎及破壞。這系統主要是會製造一種分泌管道(secretion apparatus)將製造的外毒素從細菌的細胞質穿過細菌的細胞膜、細胞壁及最後穿越宿主細胞的細胞膜進入宿主細胞質內造成宿主的破壞。目前已有四種綠膿桿菌T3SS 外毒素被發現: ExoS, ExoU, ExoT, 和ExoY. ExoS 是一種雙功能的外毒素同時具有GTPase-activating protein (GAP)及ADP ribosyl transferase (ADPRT) 兩種酵素活性。能分泌ExoS 的綠膿桿菌不會將宿主細胞馬上殺死而是可侵入到宿主細胞質內進行繁殖後再造成細胞破裂釋放出大量的細菌。這些綠膿桿菌則稱之為具侵入性表徵的菌種(invasive phenotype)。ExoU 是一種具phospholipase 功能的外毒素。因此能分泌此外毒素的綠膿桿菌菌株主要是在進入宿主細胞質後快速將細胞膜破壞和溶解使細胞在2 小時內死亡。具此特徵的綠膿桿菌菌株則稱之為細胞毒性菌株(cytotoxic phenotype)。目前研究顯示一般菌株很少同時能分泌ExoS 及ExoU 一般只能分泌其中一種，所以大部分的菌株只具有一種表徵。另外兩種外毒素ExoT 和ExoY 則幾乎所有菌株都具有。許多研究嘗試理解綠膿桿菌之T3SS 系統與各種人類感染性疾病機轉的關係，但對此系統的啟動(initiation)控制機轉及其對角膜上皮細胞的影響目前僅有初步的了解。目前研究發現綠膿桿菌角膜炎病人中細胞毒性菌株占較高比例且與多重藥物(multidrug)抗藥性有關。我們初步實驗結果發現侵入性表徵的菌種占多數。因此，此研究希望在第一年研究中將收集並了解台灣綠膿桿菌的菌株中各種表徵的比例及分析菌株的抗生素敏感性(antibiotic susceptibility)與菌株表徵的關係。另外也將分析菌株外毒素中的基因異質性(genetic heterogeneity)而了解是否有特定的菌株與臨床預後相關。第二年研究將進一步分析淚液中的鈣成分對T3SS 系統的影響。目前已知實驗中低鈣環境可促使T3SS 外毒素的分泌。另外臨床上也發現鈣會沉積於隱形眼鏡上可能使淚水中鈣成分降低。因此希望分析與比較有無配戴隱形眼鏡兔子淚水中的鈣成分對T3SS 系統的影響，借此部份解釋臨床上常見綠膿桿菌角膜炎多發生於配戴隱形眼鏡者的現象。此外我們也希分析不同菌株表徵對角膜上皮的屏障功能(barrier function)的影響尤其是與淚水的關係。第三年將利用T3SS基因宊變的菌株進一步了解對角膜上皮細胞的影響特別是針對ExoS 對細胞結構的影響。此研究的結果希望能使我們更加了解綠膿桿菌角膜炎的致病機轉以利未來開發治療或預防感染的新方向。
Abstract: Microbial infection of the cornea, also known as microbial keratitis, causes severe corneal inflammationthat could result in severe visual loss. The most commonly isolated pathogen found clinically wasPseudomonas aeruginosa which accounts for over one third of the cases. Among the various virulencefactors involved in the pathogenesis of pseudomonal keratitis, a secretion system known as type threesecretion system (T3SS) secretes toxins that damage the host cells. During the initiation of infection, P.aeruginosa creates a pore complex between the bacterium and the host cells delivering effector proteins orexotoxins into the host cell cytoplasm. To date, four effector proteins for P. aeruginosa has been identified:ExoS, ExoU, ExoT, and ExoY. ExoS is a bifunctional exotoxin with GTPase-activating protein (GAP)activity and ADP ribosyl transferase (ADPRT) activity. It results in an invasive phenotype of P. aeruginosacausing a relatively slower host cell death with intracellular invasion and possibly proliferation of bacterium.In contrast, ExoU expressing strains carries a cytotoxic phenotype that causes rapid host cell lysis due to itsphospholipase activity. These traits are mostly mutually exclusive in P. aeruginosa while ExoT and ExoY arealmost universally expressed in all strains. It has been previously reported that T3SS plays a critical role inthe pathogensis of pseudomonal keratitis. Previously, cytotoxic strains were reported to be more commonlyfound in patients with pseudomonal keratitis and were highly correlated with multidrug resistance.However, our preliminary results showed a higher distribution of invasive strains clinically. Thus, in thefirst year of this study, we will continue to collect and analyze the clinical strains of P. aeruginosa for itsgenotype and phenotype and compare the prevalence of invasive and cytotoxic strains involved in microbialinfection. The antibiotic susceptibility will also be compared among these strains to understand its effect inclinical outcomes. The genetic heterogeneity between these strains will also be analyzed to determinecorrelations between specific genetic polymorphism associated with clinical prognosis. During the secondyear, we hope to begin to understand the influence of tears fluid components specifically calciumconcentrations to T3SS of P. aeruginosa. Low calcium had been used in vitro to induce T3SS. Sincecalcium has also been known to deposit on contact lenses, it is possible that contact lens wearing might resultin low calcium level on tear fluid. We hope by determining the effect of contact lens wear on tear fluidcalcium concentration will help to explain for the higher incidence of pseudomonal keratitis among contactlens wearers. The effect of tear fluid on invasive or cytotoxic strains and corneal epithelial cell barrierfunction will also be determined. During the last year, we will obtain mutant strains with deletions in ExoS orits transcriptional activator ExsA and determine possible epithelial targets of ExoS in inducing disruption ofepithelial barrier function. Through the results of this study, we hope to understand role of T3SS inpseudomonal microbial keratitis in Taiwan, the importance of calcium level in tear fluid during P. aeruginosaT3SS activation, and interactions of pseudomonal T3SS with the ocular surface. The combined results fromthis extended research will hopefully bring new understanding in the pathogenesis of microbial keratitis dueto P. aeruginosa that will eventually provide novel strategies for its prevention and treatment.
