摘要
生物膜(Biofilm)是细菌和其分泌的胞外基质在物体表面形成的高度组织化的多细胞结构,它是细菌生长过程中为适应生存环境而形成的一种与浮游细胞相对应的存在形式。一旦形成生物膜,细菌将具有极强的抗生素耐药性(比浮游细菌高100-1000倍)和逃避机体免疫系统攻击的能力。根据美国NIH的调查公告,80%以上的细菌性感染与生物膜有关。因此,如何防治细菌生物膜已成为人类面临的巨大挑战。
铜绿假单胞菌(Pseudomonas aeruginosa)是一种常见条件致病菌,可引起多种感染,其中呼吸道感染最为常见,近来铜绿假单胞菌的发病率呈明显上升趋势,且耐药性日趋严重。铜绿假单胞菌具有极强的环境适应能力和生物膜形成能力。宿主与病原菌的相互作用是医学和微生物学领域研究的前沿和热点,目前绝大多数研究集中在宿主如何感知病原菌的侵入和调节免疫系统清除病原菌方面。然而,关于病原体如何感知宿主免疫系统并相应改变自身表型逃脱免疫系统攻击的研究却很少。最近有人报道,铜绿假单胞菌能够利用其外膜蛋白OprF感知宿主细胞产生的免疫因子γ-干扰素,从而激活自身的群体感应系统来促进毒性因子的表达。本论文旨在研究铜绿假单胞菌是否能感知γ-干扰素促进其生物膜的形成,从而产生免疫逃逸。
本文建立了静态和动态铜绿假单胞菌生物膜模型,研究了γ-干扰素对铜绿假单胞菌生物膜形成的影响。结果表明,γ-干扰素能够显著地促进铜绿假单胞菌生物膜的形成。随着γ-干扰素浓度、作用时间和菌体密度的不同,对铜绿假单胞菌生物膜形成的作用也不同。在静态生物膜模型中,当γ-干扰素浓度为10ng/mL、处理6h和菌液OD600为1.8时,γ-干扰素对铜绿假单胞菌生物膜形成的促进作用最强,达到40%;在动态生物膜模型中,γ-干扰素浓度为10ng/mL和作用时间为72h时,效果最明显,生物膜的生物量和平均厚度分别增加了3倍和2倍。为了排除细菌生长代谢因素对生物膜形成的影响,我们检测了γ-干扰素对铜绿假单胞菌浮游菌生长的影响。结果证明,铜绿假单胞菌的生长代谢不受γ-干扰素的影响。
研究已发现,生物膜的形成和成熟与细菌群体感应系统有关。群体感应是细菌根据细胞密度变化进行基因表达调控的一种群体行为。为了探讨γ-干扰素促进铜绿假单胞菌生物膜形成的机制,利用群体感应系统rhlI突变菌株PDO100、lasI突变菌株PAO-JP1和rhlI / lasI双突变菌株PAO-MW,分别检测了γ-干扰素对其生物膜形成的影响。结果证明,γ-干扰素主要通过rhl群体感应系统促进铜绿假单胞菌生物膜形成。同时,γ-干扰素对铜绿假单胞菌外膜蛋白OprF缺失菌株的生物膜形成没有影响,表明OprF是γ-干扰素的受体。
综上所述,在病原菌与宿主之间的相互作用中,铜绿假单胞菌能够利用外膜蛋白OprF感知宿主细胞产生的γ-干扰素,激活自身的rhl群体感应系统,促进生物膜的形成,从而抵御宿主免疫系统的攻击,形成一种自身保护机制。本文从病原体的角度,阐明了病原菌如何根据宿主免疫系统的变化来调节自身的生存方式和产生免疫逃逸,为深入了解病原菌的致病机理和有效地防治细菌生物膜感染创造了条件。
Biofilms are highly structured, surface attached communities of bacterial cells enclosed in self-produced polymeric matrix. Their formation reflects an adaptive behavior that enables the bacterial cells to survive in the hostile environment. Biofilm formation by opportunistic pathogens is devastating because the bacteria in these structured communities can withstand host immune responses, and become more resistant to antibiotics (100-1000 folds higher than planktonic bacteria). According to the public announcement of National Institutes of Health (NIH), biofilms may be involved in up to 80 percent of human infections. Consequently, controlling of biofilms will play a crucial role in cure of bacterial infections.
Pseudomonas aeruginosa, a versatile Gram-negative opportunistic human pathogen, is the most prevalent bacterial pathogens in infectious diseases, such as respiratory tract infections, bacteremia, pneumonia, urinary tract infections, cystic fibrosis, infections of burn, catheter infections and infections of heart valve. P. aeruginosa has shown multi-drug resistance that always causes failure in clinical therapy and persistent and chronic infections. So, infections with P. aeruginosa are a major health problem worldwide, being associated with significant morbidity and mortality. The host-pathogen interaction has become a hot-spot for research of medicine and microbiology. At present, most of the studies are involved in how hosts sense pathogen’s invading and regulate immune system to clear the bacteria away, whereas it is not clear how pathogens sense host immune response and alter itself phenotype to evade immune system. More recently, it is reported that IFN-γbinds to an outer membrane protein in P. aeruginosa, OprF, resulting in the expression of a quorum-sensing dependent virulence determinant, the PA-I lectin by triggering QS system of itself. This study is aimed to clarify whether P. aeruginosa can sense IFN-γand enhance the formation of biofilm to escape from host defense.
To determine whether IFN-γis capable of influcening P. aeruginosa biofilm formation, biofilm assay of P. aeruginosa under static and dynamic culture condition with or without IFN-γwere carried out. The results indicated that IFN-γcan promote the formation of P. aeruginosa biofilm dramatically. Furthermore, there was distinct influence on P. aeruginosa biofilm formation with various concentrations of IFN-γ, incubation time and cell densities. The results of the static biofilm assay indicated that the formation of P. aeruginosa biofilms was increased by 40% when the concention of IFN-γwas 10 ng/ml, cultured 6 hours and at high cell densities (OD600=1.8). And in the dynamic flow-cell conditions, significant enhancement of biofilm development, 4.0-fold as assessed by total biomass, 3.0-fold as assessed by average thickness, was observed when cultivated with 10 ng/ml of IFN-γfor 72 h. Moreover, IFN-γin concentrations used for this study had no effect on growth of P. aeruginosa PAO1. Therefore, IFN-γworked specifically on biofilm formation and not indirectly by disruption of primary metabolic functions.
Previous studies have suggested that quorum-sensing (QS) is involved in both the initiation and the maturation of P. aeruginosa biofilm. Quorum-sensing system is the behavior of the entire community that tune gene expression and regulation according to bacteria cell densities, which is widespread and involves complex networks that serve as fine-tuner of the performance of diverse behaviors. To further investigate the mechanism underlying the effect of IFN-γon the formation of P. aeruginosa biofilm, PAO1 and three strains with mutations in different QS system (PAO-JP1, PDO100, PAO-MW) were used to study the distinction among the effect of IFN-γon their biofilm formation in this study. The results showed that this influence was abrogated in strains PDO100 and PAO-MW, which are rhlI-deficient mutants of P. aeruginosa PAO1, suggesting that the rhl QS system plays a crucial role in biofilm formation response to exogenous IFN-γ. Furthermore, the results showed that IFN-γhad no effect on the formation of oprF-deficient strain biofilm, suggesting that OprF is the receptor of IFN-γ.
In conclusion, P. aeruginosa can sense the IFN-γproduction of host cells using the major outer-membrane protein OprF specifically, and then IFN-γ-OprF signal activate rhl QS system to promote the formation of P. aeruginosa biofilm. This study clarified the mechanism underlying pathogens how to sense host immune response and alter itself lifestyle to evade immune system, which may provide new insight into the mechanisms of P. aeruginosa chronic infection and may also provide a new therapeutic strategy.
引文
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