酵母表达肽抗生素hPAB-β及其抗金黄色葡萄球菌L型的活性分析
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摘要
肽抗生素(peptide antibiotics)是近年来发现的由生物体基因编码,具有抗生素样活性的小分子肽类物质,是宿主天然免疫的重要组成部分,与干扰素、补体等共同组成宿主的免疫防御系统。肽抗生素具有如下的生物学特点:多为一些小分子肽,由13-60个氨基酸组成,分子量在5kDa以下;具有两亲性结构;绝大多数带正电荷,富含精氨酸和赖氨酸残基,其所带有的强阳电荷是选择性作用于细菌胞膜的基础;具有广谱抗菌性,对革兰阳性细菌、革兰阴性细菌、真菌、病毒、寄生虫、癌细胞等都具有很好的活性。除了直接的抗菌功能外,肽抗生素还具有调节细胞增殖、诱导免疫、中和内毒素、促进伤口愈合和细胞因子释放等功能。此外肽抗生素独特的物理性“打孔”作用机制使细菌不易产生耐药性。传统抗生素的大量使用引起耐药菌株不断产生,因此寻找新的抗生素具有重要的现实意义。自1962年Kiss和Michl从铃蟾( Bombina variegata)皮肤分泌物中分离得到铃蟾肽抗生素(bombinin)以来,在短短的几十年中已从植物、昆虫、节肢动物、两栖动物、哺乳动物甚至人体内鉴定出近千种肽抗生素,或称为抗微生物肽(Antimicrobial peptide)。在临床耐药菌感染日益严峻的今天,肽抗生素的研究和开发利用无疑为人类抗感染治疗点燃了新的希望。
肽抗生素hPAB-β为本实验室构建的一种截短型人hBD-2突变体,保持了与天然hBD-2相似的生物学活性。在前期工作中,我们利用酵母表达系统的高产量和可分泌型表达的优点,成功构建并筛选了能分泌表达hPAB-β的酵母工程菌pPIC9K- hPAB-β/GS115,通过酵母高密度发酵表达的hPAB-β经纯化后具有良好的杀灭金黄色葡萄球菌和铜绿假单胞菌的活性。根据肽抗生素独特的杀菌机制,我们推测肽抗生素对那些胞质膜裸露的病原体(如带包膜的病毒、细菌L型等)有更好的杀灭活性。细菌L型是细菌细胞壁部分或完全缺失后形成的原生质体,其生长条件和药物敏感性都与原菌有较大不同,在临床上不易诊断,常漏诊误诊。许多细菌都可形成L-型,其感染与临床的许多慢性疾病有关,诊断和治疗相对困难,故寻找细菌L-型敏感的新型治疗剂成为近年来研究的热点。我们利用重组毕赤酵母进行高密度发酵表达肽抗生素hPAB-β,并根据肽抗生素的特性进行了纯化;利用低剂量抗生素(新青II)诱导金黄色葡萄球菌L-型,并对L型进行了鉴定;探讨了肽抗生素hPAB-β抗金黄色葡萄球菌L型的活性;建立了金黄色葡萄球菌L型感染大鼠间质性肺炎的动物模型,为后续检测肽抗生素hPAB-β的体内抗L型感染研究奠定了基础。主要实验方法和结果如下:
1.毕赤酵母高密度发酵表达肽抗生素hPAB-β
利用毕赤酵母易于高密度发酵的特性,在3.7L发酵罐中对重组了肽抗生素hPAB-β表达盒的pPIC9K-hPAB-β/GS115酵母优5工程菌进行高密度发酵,在种子接种后的18-24h后添加补料生长培养基,保持细菌生长,待菌体湿重生长至200~250g/L后停止流加补料3h,然后向罐中流加诱导培养基,用甲醇诱导目标基因的表达,在甲醇诱导期间,菌体生长缓慢,持续诱导60h后下罐。用Brandford法测得3次发酵液的总蛋白浓度分别为664.0 mg/L, 781.8 mg/L和721.3mg/L。用Bio-Rad公司Quantity One软件条带检测灰度分析工具对凝胶电泳后的蛋白条带进行分析,目的蛋白约占发酵上清液总蛋白的33.4%,故可知发酵上清中目的蛋白的表达量约为241.2±29.5 mg/L,实现了重组毕赤酵母高密度发酵表达肽抗生素hPAB-β的目标。
2.肽抗生素hPAB-β的纯化与鉴定
纯化的目的不仅仅是要获得高纯度的目标产物,还要使目标产物的生物学活性在纯化过程中不丧失,这就要根据目标分子与杂质的差异设计纯化路线,经过试验摸索加以优化。考虑到hPAB-β为一阳离子小肽(pI 9.001),我们设计了10kDa膜过滤、反相层析、阳离子交换、分子筛脱盐等纯化技术对目标肽进行纯化,最终从每升发酵液中纯化得到74.9±1.5 mg目标蛋白,纯度达到98%以上,总体回收效率为31.5%,实现了酵母表达肽抗生素hPAB-β的分离纯化。利用Bio-Rad公司DC Protein Assay Kit I试剂盒进行蛋白定量,最终测得目标产物浓度为30.55 mg/ml(约7.1 mmol/L),进一步利用琼脂扩散法检测了纯化产物对革兰阴、阳性细菌的杀灭活性,结果表明纯化产物具有生物学活性,为进一步开展hPAB-β的生物学功能研究奠定了基础。
3.金黄色葡萄球菌L型的诱导与鉴定
利用新青II纸片扩散法进行金黄色葡萄球菌L型的诱导,在高渗培养基的新青II纸片抑菌圈内有呈针尖样大小的颗粒状菌落,低倍镜下观察呈典型的“油煎蛋”样,革兰染色可见这些颗粒状菌落中的细菌为革兰阴性的大球形、短杆形、丝状等多形态,挑取单菌落接种于高渗液体培养基中培养后细菌呈沉淀生长。滤过实验证实诱导的L型细菌能通过0.45μm的滤器,在撤去新青II诱导剂后再培养数代,经血浆凝固酶和甘露醇发酵实验证实已完全回复为野生型金葡菌,透射电镜观察可见L型细菌细胞壁部分或完全缺失,菌体内结构疏松,少数呈巨型体,其胞壁全部脱落,最外层为脂质双分子层。上述实验表明经新青II诱导获得的能在高渗培养基中生长的细菌确为金葡菌L型,为后续探索肽抗生素hPAB-β抗细菌L型活性创造了前提。
4.肽抗生素hPAB-β抗金黄色葡萄球菌L型的活性分析
利用重组毕赤酵母工程菌pPIC9K-hPAB-β/GS115进行高密度发酵,纯化,制备肽抗生素hPAB-β后。采用稀释法检测了重组hPAB-β对金黄色葡萄球菌L型的活性,在测定肽抗生素hPAB-β对金葡菌L型的抗菌活性时,我们选用了万古霉素,氯霉素,新青II作对照。结果发现,在相同摩尔浓度(500μmol/L~ 4μmol/L)条件下,4种抗生素对金葡菌野生型的抗菌活性由强到弱分别为:新青II>肽抗生素hPAB-β>万古霉素>氯霉素;而对金葡菌L型而言氯霉素>肽抗生素hPAB-β>万古霉素>新青II。可见金葡菌ATCC25923转变成L型后,其对抗生素的敏感性发生了明显改变,表现在对作用于细胞壁的抗生素——新青II的敏感性下降,而对氯霉素的敏感性增加。肽抗生素hPAB-β抗野生型的最小抑菌浓度(MIC)为8μmol/L(约34μg/ml),而对L型细菌的最小抑菌浓度(MIC)为32μmol/L(约137μg/ml)。实验结果显示肽抗生素hPAB-β抗L型细菌的抗菌活性较野生型相比差4倍,其原因可能是细菌L型的高渗培养基(含40 g/L NaCl)对肽抗生素的活性有一定影响。
5.金黄色葡萄球菌L型感染大鼠间质性肺炎动物模型的建立
实验中设置无菌与野生菌对照组,采用腹腔注射方法感染大鼠,细菌注射后常规饲养15天后颈动脉放血处死后解剖大鼠,通过大体观察可见感染组大鼠肺体积增大,色暗红,淤血,肺表面可见明显出血性病灶,血管充血扩张。组织块细菌培养能分离到L型细菌。炎症细胞计数可见感染组白细胞明显升高,金黄色葡萄球菌野生型感染组中性粒细胞升高而L型细菌组中性粒细胞变化不大,这可能与L型缺失细胞壁有一定关系。病理切片观察L型细菌感染大鼠后表现为间质性肺炎病变,弥漫性肺泡间质增生,肺泡腔变窄,间质内伴淋巴细胞和单核细胞浸润,局部地方肺泡腔有红细胞渗出,血管充血扩张,小支气管上皮增生,腔内红细胞渗出。表明成功建立L性细菌感染大鼠间质性肺炎动物模型,为后续检测肽抗生素hPAB-β的体内抗菌活性奠定了基础。
综上所述,本研究利用重组毕赤酵母高密度发酵表达肽抗生素hPAB-β,经10kDa膜过滤、反相层析、离子交换、分子筛层析等处理,从酵母发酵上清中纯化得高纯度目标产物74.9±1.5 mg/L,总体回收效率为31.5%,目的蛋白浓度为30.55 mg/ml(约7.1 mmol/L),该纯化产物具有杀灭革兰阴、阳性细菌的能力。采用新青II纸片法成功诱导金黄色葡萄球菌L型,通过形态学观察、滤过实验、回复实验、电镜观察鉴定为细菌L型。并用稀释法检测到肽抗生素hPAB-β对金葡菌L型有抗菌活性,其最小抑菌浓度(MIC)为32μmol/L(约137μg/ml)。成功建立了金黄色葡萄球菌L型感染大鼠间质性肺炎的动物模型,为后续深入探讨肽抗生素hPAB-β治疗L型细菌感染提供了实验依据,也为开展肽抗生素体内抗菌活性的检测奠定了基础。
Peptide antibiotics are small peptides that encoded by the genomic DNA of organisms. It is one of the important components of living creature’s natural immunity system. Peptide antibiotics generally contain 13-60 residues with molecular weight of <5kDa, possess a net positive charge and an amphipathic structure. Compared with conventional antibiotics, which are often produced by Actinomycetes and Molds, peptide antibiotics exert activity against a broad spectrum of microorganisms including Gram-positive and Gram-negative bacteria, fungi, parasites, viruses and even some cancer cells. Peptide antibiotics kill bacteria mainly by membrane- targeting pore–forming mechanisms, so it is difficult for a bacterium to develop resistance. Since the bombinin, the first peptide antibiotic discovered, was isolated from Bombina variegata skin in 1962, more than one thousand of antimicrobial peptides have been identified from plants, insects, arthropods, amphibians, mammals, and humans. The emergence of multiple-drug-resistant bacteria, fungi and parasites in recent two decades has fueled considerable interest in using endogenous antibiotic peptides as alternatives to treat infections.
Peptide antibiotic hPAB-βis a kind of mutants of human hBD-2, which maintain the similar biological activity with hBD-2. In our previous study, we successfully constructed the engineered strain pPIC9K-hPAB-β/GS115, which can express the hPAB-β. Based on the membrane-targeting pore–forming mechanism of peptide antibiotics actions, we suppose that they may have a better activity against the L-form of bacteria. The L-forms are cell wall-deficient bacteria which are able to grow as spheroplasts or protoplasts. Most L-forms maintain their pathogenicities, often cause chroinc and relapsing infections. Multi-organic chronic interstitial inflammation is a main manifestation of L-forms infection, which is different with that of its parental form and results in difficulty in clinical diagnose. The drug sensitivity of L-forms to chemical reagents is also different from its partental strain, so it is important to find a new effective reagent against L-forms of bacteria infection. In this study, we firstly expressed and purified the peptide antibiotic hPAB-βby recombinant Pichia pastoris, then the L-form of Staphylococcus aureus was induced and its sensitivity to hPAB-βwas evaluated. The main experimental methods and results are as following:
1. High-density cell fermentation of Pichia pastoris for production of secreted hPAB-β
The high-cell density fermentation of pPIC9K-hPAB-β/GS115 strain You5 was performed in a 3.7 L fermentor. After the seeds grew 18-24h, added the fed growth medium to accelerate the growth of cell until the wet weight reached 200 ~ 250g/L, then added the methanol to induce the expression of the target gene. The fermentation was stopped 60h post induction and the total proteins in the fermentative supernatants were 664.0 mg/L, 781.8 mg/L and 721.3mg/L respectively (3 times of fermentation) determined by Brandford assay. The hPAB-βreached 33.4% of the supernatant total proteins analyzed by Quantity–one software (Bio-Rad) and the calculated amounts of expressed hPAB-βwere 241.2±29.5 mg/L.
2. Purification and biological activity determination of recombinant hPAB-β
The aim of the purification is not only to get the pure products, but also to keep the bioactivity of the protein during the process of purification. The hPAB-βis a small cationic peptide with a pI of 9.001, so we designed a procedure of 10kDa membrane ultrafiltration, reverse phase chromatogryphy, cation exchange and gel filtration to purify hPAB-βfrom the fermentative supernatants of recombinant Pichia pastoris. The final recovery was 74.9±1.5 mg/L with a purity of 98%. The overall recovery rate was 31.5% after a 4-step purification process. The concentration of hPAB-βstock solution was 30.55 mg/ml (about 7.1 mmol/L) and it had high antibacterial activity against Staphylococcus aureus and Escherichia coli determined with agar diffusion method.
3. Induction and identification of L-form of Staphylococcus aureus
The L-form of Staphylococcus aureus strain ATCC25923 was induced by oxacillin paper disk assay. The typical“fried egg”like colonies appeared in oxacillin inhibition zone 3 days post culture at 37℃. The short rod-shaped, filamentous and other pleomorphic Gram-negative cells were seen under the microscope after Gram staining. The L-form could permeated through a 0.45μm filter. After remove the inducer (oxacillin), the L-form of Staphylococcus aureus could reversed back to the wild type after 3~5 passages in high osmotic LB meidum. The most cell walls of the induced L-forms were partial absent and a few of them were completely lost observed under the transmission electron microscope (TEM). These results showed that the L-forms of Staphylococcus aureus were successfully induced with oxacillin.
4. Analysis the antibacterial activity of hPAB-βagainst the L-form of Staphylococcus aureus
The antibacterial activity of hPAB-βagainst L-form of Staphylococcus aureus was performed by serial dilution method in a 96-well plate. Three other antibiotics (oxacillin, vancomycin and chloramphenicol) were chosen as controls. Among the four kinds of antibiotics, oxacillin exhibits the highest antibacterial activity against Staphylococcus aureus wild-type, hPAB-βranked the second and chloramphenicol held the last. When against the L-form, the activity ranked from strong to weak was chloramphenicol, peptide antibiotics hPAB-β, vancomycin and oxacillin. Our results shew that the sensitivity to antibiotics was shifted when a wild type of Staphylococcus aureus changed to a L-form. The minimum inhibitory concentration (MIC) of peptide antibiotics hPAB-βagainst the wild type was 8μmol/L (about 34μg/ml), while against the L-form was 32μmol/L (about 137μg/ml), which was 4 times higher than that against the wild type. The reason may be the effect of hypertonic medium (containing 40g/L NaCl in culturing the bacterial L-form) on the activity of peptide antibiotics.
5. Establish the animal model infected by the L-form of Staphylococcus aureus.
The morphological changes of mouse lung infected by L-form bacteria including larger volume, color dull red, congestion, pulmonary clearly visible and vascular congestion expansion. Through the inflammatory cell count, we can see that the number of WBC significantly increased. The number of neutrophil is increased in the infect group of wild-type bacteria while the L-form bacteria infect group didn’t have obiviously change. The probable reason may be the lack of cell wall in the cells of L-form. After HE staining, the infected lung tissue indicated interstitial pneumonia lesions, interstitial alveolar hyperplasia, alveolar space narrowing, interstitial infiltration of lymphocytes and mononuclear cells, red blood cells effusion in alveolar cavity, vascular congestion expansion, small bronchial epithelial hyperplasia and intraluminal red blood cells leaking. The result showed that we successfully establish the animal model of mouse interstitial pneumonia.
In conclusion, peptide antibiotic hPAB-βwas expressed in high-cell density fermentation of recombinant Pichia pastoris. The typical L-forms of Staphylococcus aureus strain ATCC25923 were induced by oxacillin paper disk diffusion method and their morphological characters, colonies formation, filtering features, ultra structure and reversion phenomenon were investigated. The antibacterial activity of peptide antibiotics hPAB-βagainst the L-form of Staphylococcus aureus was determined by serial dilution method and the minimum inhibitory concentration (MIC) was 32μmol/L (approximately 137μg/ml). A animal model of interstitial pneumonia lesions infectd by the L-form of Staphylococcus aureus was successfully established. Our study lay down a good foundation for futher evaluation of peptide antibiotics hPAB-βas a potential therapic agent against clinical infection of Staphylococcus aureus L-forms.
肽抗生素hPAB-β为本实验室构建的一种截短型人hBD-2突变体,保持了与天然hBD-2相似的生物学活性。在前期工作中,我们利用酵母表达系统的高产量和可分泌型表达的优点,成功构建并筛选了能分泌表达hPAB-β的酵母工程菌pPIC9K- hPAB-β/GS115,通过酵母高密度发酵表达的hPAB-β经纯化后具有良好的杀灭金黄色葡萄球菌和铜绿假单胞菌的活性。根据肽抗生素独特的杀菌机制,我们推测肽抗生素对那些胞质膜裸露的病原体(如带包膜的病毒、细菌L型等)有更好的杀灭活性。细菌L型是细菌细胞壁部分或完全缺失后形成的原生质体,其生长条件和药物敏感性都与原菌有较大不同,在临床上不易诊断,常漏诊误诊。许多细菌都可形成L-型,其感染与临床的许多慢性疾病有关,诊断和治疗相对困难,故寻找细菌L-型敏感的新型治疗剂成为近年来研究的热点。我们利用重组毕赤酵母进行高密度发酵表达肽抗生素hPAB-β,并根据肽抗生素的特性进行了纯化;利用低剂量抗生素(新青II)诱导金黄色葡萄球菌L-型,并对L型进行了鉴定;探讨了肽抗生素hPAB-β抗金黄色葡萄球菌L型的活性;建立了金黄色葡萄球菌L型感染大鼠间质性肺炎的动物模型,为后续检测肽抗生素hPAB-β的体内抗L型感染研究奠定了基础。主要实验方法和结果如下:
1.毕赤酵母高密度发酵表达肽抗生素hPAB-β
利用毕赤酵母易于高密度发酵的特性,在3.7L发酵罐中对重组了肽抗生素hPAB-β表达盒的pPIC9K-hPAB-β/GS115酵母优5工程菌进行高密度发酵,在种子接种后的18-24h后添加补料生长培养基,保持细菌生长,待菌体湿重生长至200~250g/L后停止流加补料3h,然后向罐中流加诱导培养基,用甲醇诱导目标基因的表达,在甲醇诱导期间,菌体生长缓慢,持续诱导60h后下罐。用Brandford法测得3次发酵液的总蛋白浓度分别为664.0 mg/L, 781.8 mg/L和721.3mg/L。用Bio-Rad公司Quantity One软件条带检测灰度分析工具对凝胶电泳后的蛋白条带进行分析,目的蛋白约占发酵上清液总蛋白的33.4%,故可知发酵上清中目的蛋白的表达量约为241.2±29.5 mg/L,实现了重组毕赤酵母高密度发酵表达肽抗生素hPAB-β的目标。
2.肽抗生素hPAB-β的纯化与鉴定
纯化的目的不仅仅是要获得高纯度的目标产物,还要使目标产物的生物学活性在纯化过程中不丧失,这就要根据目标分子与杂质的差异设计纯化路线,经过试验摸索加以优化。考虑到hPAB-β为一阳离子小肽(pI 9.001),我们设计了10kDa膜过滤、反相层析、阳离子交换、分子筛脱盐等纯化技术对目标肽进行纯化,最终从每升发酵液中纯化得到74.9±1.5 mg目标蛋白,纯度达到98%以上,总体回收效率为31.5%,实现了酵母表达肽抗生素hPAB-β的分离纯化。利用Bio-Rad公司DC Protein Assay Kit I试剂盒进行蛋白定量,最终测得目标产物浓度为30.55 mg/ml(约7.1 mmol/L),进一步利用琼脂扩散法检测了纯化产物对革兰阴、阳性细菌的杀灭活性,结果表明纯化产物具有生物学活性,为进一步开展hPAB-β的生物学功能研究奠定了基础。
3.金黄色葡萄球菌L型的诱导与鉴定
利用新青II纸片扩散法进行金黄色葡萄球菌L型的诱导,在高渗培养基的新青II纸片抑菌圈内有呈针尖样大小的颗粒状菌落,低倍镜下观察呈典型的“油煎蛋”样,革兰染色可见这些颗粒状菌落中的细菌为革兰阴性的大球形、短杆形、丝状等多形态,挑取单菌落接种于高渗液体培养基中培养后细菌呈沉淀生长。滤过实验证实诱导的L型细菌能通过0.45μm的滤器,在撤去新青II诱导剂后再培养数代,经血浆凝固酶和甘露醇发酵实验证实已完全回复为野生型金葡菌,透射电镜观察可见L型细菌细胞壁部分或完全缺失,菌体内结构疏松,少数呈巨型体,其胞壁全部脱落,最外层为脂质双分子层。上述实验表明经新青II诱导获得的能在高渗培养基中生长的细菌确为金葡菌L型,为后续探索肽抗生素hPAB-β抗细菌L型活性创造了前提。
4.肽抗生素hPAB-β抗金黄色葡萄球菌L型的活性分析
利用重组毕赤酵母工程菌pPIC9K-hPAB-β/GS115进行高密度发酵,纯化,制备肽抗生素hPAB-β后。采用稀释法检测了重组hPAB-β对金黄色葡萄球菌L型的活性,在测定肽抗生素hPAB-β对金葡菌L型的抗菌活性时,我们选用了万古霉素,氯霉素,新青II作对照。结果发现,在相同摩尔浓度(500μmol/L~ 4μmol/L)条件下,4种抗生素对金葡菌野生型的抗菌活性由强到弱分别为:新青II>肽抗生素hPAB-β>万古霉素>氯霉素;而对金葡菌L型而言氯霉素>肽抗生素hPAB-β>万古霉素>新青II。可见金葡菌ATCC25923转变成L型后,其对抗生素的敏感性发生了明显改变,表现在对作用于细胞壁的抗生素——新青II的敏感性下降,而对氯霉素的敏感性增加。肽抗生素hPAB-β抗野生型的最小抑菌浓度(MIC)为8μmol/L(约34μg/ml),而对L型细菌的最小抑菌浓度(MIC)为32μmol/L(约137μg/ml)。实验结果显示肽抗生素hPAB-β抗L型细菌的抗菌活性较野生型相比差4倍,其原因可能是细菌L型的高渗培养基(含40 g/L NaCl)对肽抗生素的活性有一定影响。
5.金黄色葡萄球菌L型感染大鼠间质性肺炎动物模型的建立
实验中设置无菌与野生菌对照组,采用腹腔注射方法感染大鼠,细菌注射后常规饲养15天后颈动脉放血处死后解剖大鼠,通过大体观察可见感染组大鼠肺体积增大,色暗红,淤血,肺表面可见明显出血性病灶,血管充血扩张。组织块细菌培养能分离到L型细菌。炎症细胞计数可见感染组白细胞明显升高,金黄色葡萄球菌野生型感染组中性粒细胞升高而L型细菌组中性粒细胞变化不大,这可能与L型缺失细胞壁有一定关系。病理切片观察L型细菌感染大鼠后表现为间质性肺炎病变,弥漫性肺泡间质增生,肺泡腔变窄,间质内伴淋巴细胞和单核细胞浸润,局部地方肺泡腔有红细胞渗出,血管充血扩张,小支气管上皮增生,腔内红细胞渗出。表明成功建立L性细菌感染大鼠间质性肺炎动物模型,为后续检测肽抗生素hPAB-β的体内抗菌活性奠定了基础。
综上所述,本研究利用重组毕赤酵母高密度发酵表达肽抗生素hPAB-β,经10kDa膜过滤、反相层析、离子交换、分子筛层析等处理,从酵母发酵上清中纯化得高纯度目标产物74.9±1.5 mg/L,总体回收效率为31.5%,目的蛋白浓度为30.55 mg/ml(约7.1 mmol/L),该纯化产物具有杀灭革兰阴、阳性细菌的能力。采用新青II纸片法成功诱导金黄色葡萄球菌L型,通过形态学观察、滤过实验、回复实验、电镜观察鉴定为细菌L型。并用稀释法检测到肽抗生素hPAB-β对金葡菌L型有抗菌活性,其最小抑菌浓度(MIC)为32μmol/L(约137μg/ml)。成功建立了金黄色葡萄球菌L型感染大鼠间质性肺炎的动物模型,为后续深入探讨肽抗生素hPAB-β治疗L型细菌感染提供了实验依据,也为开展肽抗生素体内抗菌活性的检测奠定了基础。
Peptide antibiotics are small peptides that encoded by the genomic DNA of organisms. It is one of the important components of living creature’s natural immunity system. Peptide antibiotics generally contain 13-60 residues with molecular weight of <5kDa, possess a net positive charge and an amphipathic structure. Compared with conventional antibiotics, which are often produced by Actinomycetes and Molds, peptide antibiotics exert activity against a broad spectrum of microorganisms including Gram-positive and Gram-negative bacteria, fungi, parasites, viruses and even some cancer cells. Peptide antibiotics kill bacteria mainly by membrane- targeting pore–forming mechanisms, so it is difficult for a bacterium to develop resistance. Since the bombinin, the first peptide antibiotic discovered, was isolated from Bombina variegata skin in 1962, more than one thousand of antimicrobial peptides have been identified from plants, insects, arthropods, amphibians, mammals, and humans. The emergence of multiple-drug-resistant bacteria, fungi and parasites in recent two decades has fueled considerable interest in using endogenous antibiotic peptides as alternatives to treat infections.
Peptide antibiotic hPAB-βis a kind of mutants of human hBD-2, which maintain the similar biological activity with hBD-2. In our previous study, we successfully constructed the engineered strain pPIC9K-hPAB-β/GS115, which can express the hPAB-β. Based on the membrane-targeting pore–forming mechanism of peptide antibiotics actions, we suppose that they may have a better activity against the L-form of bacteria. The L-forms are cell wall-deficient bacteria which are able to grow as spheroplasts or protoplasts. Most L-forms maintain their pathogenicities, often cause chroinc and relapsing infections. Multi-organic chronic interstitial inflammation is a main manifestation of L-forms infection, which is different with that of its parental form and results in difficulty in clinical diagnose. The drug sensitivity of L-forms to chemical reagents is also different from its partental strain, so it is important to find a new effective reagent against L-forms of bacteria infection. In this study, we firstly expressed and purified the peptide antibiotic hPAB-βby recombinant Pichia pastoris, then the L-form of Staphylococcus aureus was induced and its sensitivity to hPAB-βwas evaluated. The main experimental methods and results are as following:
1. High-density cell fermentation of Pichia pastoris for production of secreted hPAB-β
The high-cell density fermentation of pPIC9K-hPAB-β/GS115 strain You5 was performed in a 3.7 L fermentor. After the seeds grew 18-24h, added the fed growth medium to accelerate the growth of cell until the wet weight reached 200 ~ 250g/L, then added the methanol to induce the expression of the target gene. The fermentation was stopped 60h post induction and the total proteins in the fermentative supernatants were 664.0 mg/L, 781.8 mg/L and 721.3mg/L respectively (3 times of fermentation) determined by Brandford assay. The hPAB-βreached 33.4% of the supernatant total proteins analyzed by Quantity–one software (Bio-Rad) and the calculated amounts of expressed hPAB-βwere 241.2±29.5 mg/L.
2. Purification and biological activity determination of recombinant hPAB-β
The aim of the purification is not only to get the pure products, but also to keep the bioactivity of the protein during the process of purification. The hPAB-βis a small cationic peptide with a pI of 9.001, so we designed a procedure of 10kDa membrane ultrafiltration, reverse phase chromatogryphy, cation exchange and gel filtration to purify hPAB-βfrom the fermentative supernatants of recombinant Pichia pastoris. The final recovery was 74.9±1.5 mg/L with a purity of 98%. The overall recovery rate was 31.5% after a 4-step purification process. The concentration of hPAB-βstock solution was 30.55 mg/ml (about 7.1 mmol/L) and it had high antibacterial activity against Staphylococcus aureus and Escherichia coli determined with agar diffusion method.
3. Induction and identification of L-form of Staphylococcus aureus
The L-form of Staphylococcus aureus strain ATCC25923 was induced by oxacillin paper disk assay. The typical“fried egg”like colonies appeared in oxacillin inhibition zone 3 days post culture at 37℃. The short rod-shaped, filamentous and other pleomorphic Gram-negative cells were seen under the microscope after Gram staining. The L-form could permeated through a 0.45μm filter. After remove the inducer (oxacillin), the L-form of Staphylococcus aureus could reversed back to the wild type after 3~5 passages in high osmotic LB meidum. The most cell walls of the induced L-forms were partial absent and a few of them were completely lost observed under the transmission electron microscope (TEM). These results showed that the L-forms of Staphylococcus aureus were successfully induced with oxacillin.
4. Analysis the antibacterial activity of hPAB-βagainst the L-form of Staphylococcus aureus
The antibacterial activity of hPAB-βagainst L-form of Staphylococcus aureus was performed by serial dilution method in a 96-well plate. Three other antibiotics (oxacillin, vancomycin and chloramphenicol) were chosen as controls. Among the four kinds of antibiotics, oxacillin exhibits the highest antibacterial activity against Staphylococcus aureus wild-type, hPAB-βranked the second and chloramphenicol held the last. When against the L-form, the activity ranked from strong to weak was chloramphenicol, peptide antibiotics hPAB-β, vancomycin and oxacillin. Our results shew that the sensitivity to antibiotics was shifted when a wild type of Staphylococcus aureus changed to a L-form. The minimum inhibitory concentration (MIC) of peptide antibiotics hPAB-βagainst the wild type was 8μmol/L (about 34μg/ml), while against the L-form was 32μmol/L (about 137μg/ml), which was 4 times higher than that against the wild type. The reason may be the effect of hypertonic medium (containing 40g/L NaCl in culturing the bacterial L-form) on the activity of peptide antibiotics.
5. Establish the animal model infected by the L-form of Staphylococcus aureus.
The morphological changes of mouse lung infected by L-form bacteria including larger volume, color dull red, congestion, pulmonary clearly visible and vascular congestion expansion. Through the inflammatory cell count, we can see that the number of WBC significantly increased. The number of neutrophil is increased in the infect group of wild-type bacteria while the L-form bacteria infect group didn’t have obiviously change. The probable reason may be the lack of cell wall in the cells of L-form. After HE staining, the infected lung tissue indicated interstitial pneumonia lesions, interstitial alveolar hyperplasia, alveolar space narrowing, interstitial infiltration of lymphocytes and mononuclear cells, red blood cells effusion in alveolar cavity, vascular congestion expansion, small bronchial epithelial hyperplasia and intraluminal red blood cells leaking. The result showed that we successfully establish the animal model of mouse interstitial pneumonia.
In conclusion, peptide antibiotic hPAB-βwas expressed in high-cell density fermentation of recombinant Pichia pastoris. The typical L-forms of Staphylococcus aureus strain ATCC25923 were induced by oxacillin paper disk diffusion method and their morphological characters, colonies formation, filtering features, ultra structure and reversion phenomenon were investigated. The antibacterial activity of peptide antibiotics hPAB-βagainst the L-form of Staphylococcus aureus was determined by serial dilution method and the minimum inhibitory concentration (MIC) was 32μmol/L (approximately 137μg/ml). A animal model of interstitial pneumonia lesions infectd by the L-form of Staphylococcus aureus was successfully established. Our study lay down a good foundation for futher evaluation of peptide antibiotics hPAB-βas a potential therapic agent against clinical infection of Staphylococcus aureus L-forms.
引文
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