金黄色葡萄球菌Pfs的功能性研究及金葡萄感染的靶向性治疗
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摘要
金黄色葡萄球菌(简称金葡菌)是社区和医院感染发病和死亡的主要原因之一。金葡菌可在人体不同位点引发感染,从表皮的轻微性感染到深度、致命性的感染,如肺炎、心内膜炎、败血病、骨髓炎和其他转移性并发症等。随着各种耐药菌株的出现,及其生物膜相关感染的发生,金葡菌感染的治疗面临更加严峻的挑战,新的药物靶点的寻找迫在眉睫。新型药物靶点寻找的中心思想及重大挑战是能够寻找到一种抗感染药物,该药能在降低金葡菌的致病性及生物膜形成能力的同时对细菌却没有杀伤作用。这种药物的使用不会给耐药菌株创造选择性生长压力,能很好的解决传统药物耐药菌株出现的问题,并且能够很好的克服生物膜相关感染的内在性耐药问题。作为S-腺苷甲硫氨酸循环的重要组成部分,甲硫腺苷/S-腺苷高半胱氨酸核苷酶(Pfs)在生物甲基化、聚胺的生物合成、维生素的合成、细菌细胞间通信等重要的生理过程中起着关键性的作用。这里我们首次发现,Pfs在金葡菌的生物膜形成和致病性中起着重要的作用。金葡菌pfs基因的敲除导致了生物膜形成能力的下降,并同时降低了在Triton X-100诱导下的细菌自溶能力,在液体培养状态下细菌的团聚能力,及生物膜中的胞外DNA水平。因此我们推测,pfs基因对金葡菌生物膜形成能力的影响是通过降低自溶分泌的胞外DNA的水平实现的。另外,与同基因型的野生型菌株相比,pfs敲除菌株的胞外蛋白酶水平被下调了。根据对胞外蛋白酶谱的分析及对各生长时相RNA的实时定量PCR分析发现,其主要是由sspABC操纵子表达水平的大幅度下调所引起的。最后我们构建了小鼠动物模型以确定体外观察到的pfs敲除菌株的表型变化对其致病性的影响。实验表明,与其同基因型的野生型菌株相比,pfs敲除菌株在小鼠菌血症模型和表皮脓肿模型中的毒力大大下降了,并伴随着其体内增殖能力的降低。我们的数据表明,Pfs是金葡菌感染治疗的一个潜在药物靶点,该靶点的使用能同时达到降低细菌致病性和生物膜形成能力的目的。
除了耐药菌株的出现及生物膜相关感染以外,金葡菌感染的治疗还存在着其它一系列的问题。抗生素的非选择性杀伤作用导致抗生素在杀伤致病菌的同时,对人体的自然菌群造成了严重的破坏。人体的自然菌群在维持人体外环境稳定上起着关键性的作用,自然菌群的破坏会严重影响其营养、拮抗和免疫等生理作用,影响人体健康及人体对病原微生物的抵御能力。抗生素还可能会引起严重的耳毒性、肾毒性等副作用。另外通常使用的抗生素进入细胞的能力很差,这就使得胞内存活的细菌能够很好的逃避抗生素的杀伤,继而诱发慢性感染及复发性感染。随着金葡菌耐药菌株的出现,万古霉素成为了治疗耐药性金葡菌的最后一道防线。这里我们介绍了一种新型抗生素传输方法,目的是降低抗生素的毒副作用及改善其进入细胞的能力。我们利用了细菌感染部位形成的特殊微环境来设计对该微环境响应的纳米颗粒,实现药物选择性在病灶部位释放的目的。作为重要的致病因子,脂肪酶在细菌中的分布很广泛。于是我们设计合成了脂肪酶敏感的具有三层结构的纳米凝胶(TLN)作为抗生素的载体。这种三层结构的纳米凝胶是以脂肪酶敏感的聚己内酯(PCL)为中间层,聚乙二醇为壳,交联的聚磷酸酯为内核。聚磷酸酯内核可以包裹亲水药物。在水溶液中,PCL段塌缩,形成一层疏水的致密分子墙裹在聚磷酸酯的外围,阻碍药物的释放。在脂肪酶或脂肪酶分泌的细菌存在下,PCL分子墙会被脂肪酶所降解,导致药物释放,从而杀死细菌。另外,TLN将药物运送到细胞内,有效的杀伤胞内存活的细菌。这项技术可以用于选择性的输送多种抗生素以治疗脂肪分泌酶细菌引起的感染性疾病,为治疗胞内和胞外细菌引起的感染提供了一种新的、安全的、有效的和普遍适用的方法
Staphylococcus aureus is a major cause of infectious morbidity and mortality in community and hospital settings. This bacterium has the ability to cause a variety of infections in numerous ecological niches within the host, ranging from cutaneous infections to deep-seated infections such as pneumonia, endocarditis, septicaemia, osteomyelitis, and other metastatic complications. Nowadays, the universal biofilm formation and the incessant emergence of antibiotic resistant strains have created new challenges in the treatment of S. aureus infection. It highlights the urgent need for new agents for the treatment of S. aureus infection. It is a central goal and key challenge to develop an anti-infection agent capable of attenuation the virulence and biofilm formation ability of bacteria at the same time without killing them. In this way, the emergence of antibiotic resistant strains is assumed to be less significant, and the intrinsic resistance of biofilm-associated infections should be overcome. As an integral component of the S-adenosylmethionine pathway, methylthioadenosine/S-adenosylhomocysteine nucleosidase (Pfs) is predicted to be involved in methylation reactions, polyamine synthesis, vitamin synthesis, quorum sensing pathways, and so on. For the first time, we demonstrate a role of Pfs in biofilm formation and virulence of S. aureus. The pfs mutation decreases the biofilm formation ability of S. aureus, and correspondingly the pfs mutation decreases Triton X-100induced autolysis, clumping ability in liquid culture, and extracellular DNA level in biofilm. It is suspected that the decreased biofilm formation of the pfs mutant is associated with the decreased extracellular DNA level in biofilm, which is released through autolysis. Compared to the isogenic wild type strain, the pfs mutant strain displayed a decreased production of extracellular proteases. Through the zyogram analysis of extracellular protease and the transcription analysis along the growth phases, it is shown that the decreased extracellular protease activity was correlated with a dramatic decrease in the expression of the sspABC operon. Finally, mouse infection models were constructed to investigate the significance of these observations in vitro to disease pathogenesis. The mouse models of sepsis and subcutaneous abscesses indicated the pfs mutant strain displayed highly impaired virulence. The decreased virulence of the pfs mutant strain is correspondence with decreased proliferation in vivo. Our data suggested that Pfs is a potential novel target for anti-infection therapy capable of attenuation the virulence and biofilm formation of S. aureus at the same time.
Besides the emergence of antibiotic resistant strains and the biofilm related infection, there are other issues in S. aureus infection treatment. The unselected killing of antibacterial agent makes destructions to the commensal microflora of the human body, which is crucial for maintaining stability of the body external environment. The destruction of commensal microflora is proved to be harmful, and would decrease the resistant ability of the human body to pathogenic microorganism. The side effects of antibiotics also include the ototoxicity, renal toxicity and so on. Another issue is that the antibiotics exhibit poor penetration into cells. The intracellular bacteria can evade from the bactericidal action of antibiotics, leading to infection recurrence and chronic infection. Vancomycin has become the last defence of anti-infection therapy of the resistant strains of5. aureus. Here we report a new strategy for differential delivery of antibiotics to bacterial infection sites for decreasing the side effects and increasing the penetration ability into cells of antibiotics. The unique micro environment of the infection site was utilized for the design of microenviroment-response nanoparticles, enabling the on-demand release of antibiotics. As an important virulence factor, lipases are wildly distributed in bacteria. We designed the lipase-sensitive polymeric triple-layered nanogel (TLN) as the carrier of antibiotics. The TLN contains lipase-senstive poly (ε-caprolactone)(PCL) interlayer between the cross-linked polyphosphoester core and the shell of poly (ethylene glycol). The hydrophilic antibiotics can reside in the polyphosphoester core. The hydrophobic PCL segments collapsed and surrounded the polyphosphoester core, forming a hydrophobic and compact molecular fence in aqueous solution which prevented antibiotic release from the polyphosphoester core prior to reaching bacterial infection sites. However, once the TLN sensed the lipase or lipase-secreting bacteria, the PCL fence of the TLN degraded to release the antibiotic and kill the bacteria. The TLN further delivered the drug into bacteria-infected cells and efficiently released the drug to kill intracellular bacteria. This technique can be generalized to selectively deliver a variety of antibiotics for the treatment of various infections caused by lipase-secreting bacteria and thus provides a new, safe, effective, and universal approach for the treatment of extracellular and intracellular bacterial infections.
除了耐药菌株的出现及生物膜相关感染以外,金葡菌感染的治疗还存在着其它一系列的问题。抗生素的非选择性杀伤作用导致抗生素在杀伤致病菌的同时,对人体的自然菌群造成了严重的破坏。人体的自然菌群在维持人体外环境稳定上起着关键性的作用,自然菌群的破坏会严重影响其营养、拮抗和免疫等生理作用,影响人体健康及人体对病原微生物的抵御能力。抗生素还可能会引起严重的耳毒性、肾毒性等副作用。另外通常使用的抗生素进入细胞的能力很差,这就使得胞内存活的细菌能够很好的逃避抗生素的杀伤,继而诱发慢性感染及复发性感染。随着金葡菌耐药菌株的出现,万古霉素成为了治疗耐药性金葡菌的最后一道防线。这里我们介绍了一种新型抗生素传输方法,目的是降低抗生素的毒副作用及改善其进入细胞的能力。我们利用了细菌感染部位形成的特殊微环境来设计对该微环境响应的纳米颗粒,实现药物选择性在病灶部位释放的目的。作为重要的致病因子,脂肪酶在细菌中的分布很广泛。于是我们设计合成了脂肪酶敏感的具有三层结构的纳米凝胶(TLN)作为抗生素的载体。这种三层结构的纳米凝胶是以脂肪酶敏感的聚己内酯(PCL)为中间层,聚乙二醇为壳,交联的聚磷酸酯为内核。聚磷酸酯内核可以包裹亲水药物。在水溶液中,PCL段塌缩,形成一层疏水的致密分子墙裹在聚磷酸酯的外围,阻碍药物的释放。在脂肪酶或脂肪酶分泌的细菌存在下,PCL分子墙会被脂肪酶所降解,导致药物释放,从而杀死细菌。另外,TLN将药物运送到细胞内,有效的杀伤胞内存活的细菌。这项技术可以用于选择性的输送多种抗生素以治疗脂肪分泌酶细菌引起的感染性疾病,为治疗胞内和胞外细菌引起的感染提供了一种新的、安全的、有效的和普遍适用的方法
Staphylococcus aureus is a major cause of infectious morbidity and mortality in community and hospital settings. This bacterium has the ability to cause a variety of infections in numerous ecological niches within the host, ranging from cutaneous infections to deep-seated infections such as pneumonia, endocarditis, septicaemia, osteomyelitis, and other metastatic complications. Nowadays, the universal biofilm formation and the incessant emergence of antibiotic resistant strains have created new challenges in the treatment of S. aureus infection. It highlights the urgent need for new agents for the treatment of S. aureus infection. It is a central goal and key challenge to develop an anti-infection agent capable of attenuation the virulence and biofilm formation ability of bacteria at the same time without killing them. In this way, the emergence of antibiotic resistant strains is assumed to be less significant, and the intrinsic resistance of biofilm-associated infections should be overcome. As an integral component of the S-adenosylmethionine pathway, methylthioadenosine/S-adenosylhomocysteine nucleosidase (Pfs) is predicted to be involved in methylation reactions, polyamine synthesis, vitamin synthesis, quorum sensing pathways, and so on. For the first time, we demonstrate a role of Pfs in biofilm formation and virulence of S. aureus. The pfs mutation decreases the biofilm formation ability of S. aureus, and correspondingly the pfs mutation decreases Triton X-100induced autolysis, clumping ability in liquid culture, and extracellular DNA level in biofilm. It is suspected that the decreased biofilm formation of the pfs mutant is associated with the decreased extracellular DNA level in biofilm, which is released through autolysis. Compared to the isogenic wild type strain, the pfs mutant strain displayed a decreased production of extracellular proteases. Through the zyogram analysis of extracellular protease and the transcription analysis along the growth phases, it is shown that the decreased extracellular protease activity was correlated with a dramatic decrease in the expression of the sspABC operon. Finally, mouse infection models were constructed to investigate the significance of these observations in vitro to disease pathogenesis. The mouse models of sepsis and subcutaneous abscesses indicated the pfs mutant strain displayed highly impaired virulence. The decreased virulence of the pfs mutant strain is correspondence with decreased proliferation in vivo. Our data suggested that Pfs is a potential novel target for anti-infection therapy capable of attenuation the virulence and biofilm formation of S. aureus at the same time.
Besides the emergence of antibiotic resistant strains and the biofilm related infection, there are other issues in S. aureus infection treatment. The unselected killing of antibacterial agent makes destructions to the commensal microflora of the human body, which is crucial for maintaining stability of the body external environment. The destruction of commensal microflora is proved to be harmful, and would decrease the resistant ability of the human body to pathogenic microorganism. The side effects of antibiotics also include the ototoxicity, renal toxicity and so on. Another issue is that the antibiotics exhibit poor penetration into cells. The intracellular bacteria can evade from the bactericidal action of antibiotics, leading to infection recurrence and chronic infection. Vancomycin has become the last defence of anti-infection therapy of the resistant strains of5. aureus. Here we report a new strategy for differential delivery of antibiotics to bacterial infection sites for decreasing the side effects and increasing the penetration ability into cells of antibiotics. The unique micro environment of the infection site was utilized for the design of microenviroment-response nanoparticles, enabling the on-demand release of antibiotics. As an important virulence factor, lipases are wildly distributed in bacteria. We designed the lipase-sensitive polymeric triple-layered nanogel (TLN) as the carrier of antibiotics. The TLN contains lipase-senstive poly (ε-caprolactone)(PCL) interlayer between the cross-linked polyphosphoester core and the shell of poly (ethylene glycol). The hydrophilic antibiotics can reside in the polyphosphoester core. The hydrophobic PCL segments collapsed and surrounded the polyphosphoester core, forming a hydrophobic and compact molecular fence in aqueous solution which prevented antibiotic release from the polyphosphoester core prior to reaching bacterial infection sites. However, once the TLN sensed the lipase or lipase-secreting bacteria, the PCL fence of the TLN degraded to release the antibiotic and kill the bacteria. The TLN further delivered the drug into bacteria-infected cells and efficiently released the drug to kill intracellular bacteria. This technique can be generalized to selectively deliver a variety of antibiotics for the treatment of various infections caused by lipase-secreting bacteria and thus provides a new, safe, effective, and universal approach for the treatment of extracellular and intracellular bacterial infections.
引文
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