鲍曼不动杆菌OMVs上调肺多功能蛋白聚糖致铜绿假单胞菌易感性增强及其机制研究

Being the most common pathogens identified from respiratory tract secretion, acinetobacter baumannii（AB） and pseudomonas aeruginosa（PA） can colonize in respiratory tract and become drug-resistant easily，and the fluxes and refluxes of these two pathogens lead a serious infective situation nowadays. We find that Versican, an extracellular matrix protein in lung, is increased by out-membrane microvesicles (OMVs) of AB. It has been reported that chondroitin sulfate (CS) side chain of Versican mediates adherence of PA and other pathogens. As a result, we postulate that OMVs of AB could enhance susceptibility towards PA due to increased CS concentration in lung. To verify this assumption, we design our experiments as follows: firstly, OMVs will be extracted and identified; secondly, severity of PA pneumonia will be observed to verify the modulating effect of OMVs; thirdly, we would inhibit Versican expression to confirm Versican mediates this effect and detect the increasing mechanisms of Versican. This study will ensure that AB forms the facilitative microenvironment for infection and existence of common pathogens in seriously illed people and the mechanisms involved, which would shed light on prevention and therapy of infections.

鲍曼不动杆菌和铜绿假单胞菌是危重患者最常见的呼吸道分离菌，因其易定植、多耐药，加之两者常此起彼伏，给临床处理带来了很大困难。前期研究中，我们发现鲍曼不动杆菌生物膜OMVs促进人体细胞外基质成分多功能蛋白聚糖（Versican）的表达增高。Versican含有的硫酸软骨素侧链（CS）是介导铜绿假单胞菌等多种病原体粘附的结构。因此，我们推测鲍曼不动杆菌通过分泌OMVs导致人体对铜绿假单胞菌易感性增加，而局部CS浓度提高是易感性增加的主要原因。为了验证这一假设，我们将首先提取鲍曼不动杆菌OMVs并鉴定，检测OMVs处理后小鼠铜绿假单胞菌肺炎的严重程度，确定OMVs对铜绿假单胞菌易感性的调节作用；明确Versican介导这一作用，及OMVs提高Versican表达的机制。本研究将明确生物膜内的鲍曼不动杆菌通过OMVs形成利于危重患者常见呼吸道感染致病菌生存的微环境及机制，为预防和治疗找到新靶标。

成膜生长（培养皿内）或浮游生长（培养基）的鲍曼不动杆菌均可分泌外膜微囊泡（OMVs），经过150000Xg超高速离心富集OMVs，聚丙烯酰胺凝胶电泳证实二者在蛋白表达上有所不同。稀释溶解后不同浓度的OMVs刺激人肺成纤维细胞及上皮细胞产生不同表达谱的细胞外基质蛋白。在上皮细胞beas-2中，成膜及浮游的OMVs明显抑制了versican蛋白的表达，成膜促进syndecan表达增多，在成纤维细胞HFL1中，成膜及浮游OMVs均促进versican表达，成膜OMVs促进decorin表达。细胞免疫荧光法检测到蛋白的表达增高。小鼠实验证实使用成膜OMVs进行小鼠气管内灌注，能够增加小鼠肺内syndecan和versican表达增高，并增加铜绿假单胞菌感染后小鼠死亡率。使用降解versican的酶处理小鼠，可部分抵消增高的死亡率，即说明versican介导了铜绿假单胞菌的粘附和感染。从而我们可以推论，成膜生长的OMVs升高了versican和syndecan的表达，介导了铜绿假单胞菌感染，造成了铜绿假单胞菌易感的细胞外环境。