猪链球菌2型sntA基因缺失突变株的构建及生物学特性研究
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摘要
猪链球菌(Streptococcus suis, SS)是引起猪链球菌病的一种严重危害养猪业的细菌性传染病。根据其荚膜多糖成分的不同,将猪链球菌分为33个血清型(1～31、33型和1/2型)。血清2型是其中最常见、毒力最强的血清型。猪链球菌2型是一种人兽共患传染病病源,不仅影响养猪业的健康发展,而且对人类的健康造成危害。近来,对各种致病菌毒力蛋白和免疫保护性抗原的研究中,分泌蛋白和膜表面蛋白成为重点关注对象。目前研究较多的是SS2的分选酶催化反应锚定在细胞壁上的锚定蛋白,几乎所有的革兰氏阳性菌中都存在类似的蛋白锚定机制。这些被分选酶催化的锚定蛋白在C末端都含有LPXTG基序,其中X为任意氨基酸残基。据报道,SS2菌株中的SntA蛋白为膜表面蛋白,在C端有LPXTG基序,而且还含有三肽RGD基序,能与哺乳类动物细胞的整合素结合,而整合素是一种异质二聚体膜蛋白质,参与细胞与细胞黏附、细胞变异、迁移和连接到细胞外基质,SntA蛋白可能直接与整合素结合,参与宿主与病原菌的互作。SntA可能是SS2的一个毒力相关因子,因此本课题对sntA基因在SS2中的功能进行研究。
本课题以pSET4s质粒为穿梭载体成功构建了SS2菌株sntA基因缺失突变株(△sntA),比较了△sntA与野生型菌株的生长特性,发现两种菌株在对数生长期生长速度差异不明显,但△sntA更早进入生长平台期,限制了一定时间内细菌生长量的积累。比较△sntA与野生型菌株在Balb/c小鼠体内定植,试验结果表明△sntA在小鼠体内定植时间更长,sntA基因缺失后菌株的抗清除能力增强。以Hep-2细胞为模型,研究不同菌株对细胞的黏附特性,结果显示,sntA基因缺失后链球菌对Hep-2细胞的黏附能力与野生型菌株无明显差异,说明sntA基因的缺失不影响细菌对细胞的黏附能力。以Balb/c小鼠为模型,比较了△sntA与野生型菌株的毒力,结果显示△sntA的LD50(5×108)是野生型菌株LD50(2.5×108)的2倍,sntA基因缺失后菌株的毒力有所下降,但不显著。比较了SntA蛋白与猪链球菌2型灭活苗对Balb/c小鼠的免疫保护力,结果表明SntA蛋白具有免疫原性,用亲本菌株攻毒后,SntA蛋白免疫组显示了一定的免疫保护力,保护力明显高于Al(OH)3佐剂对照组,但不如猪链球菌2型灭活苗的保护力强。
Streptococcus suis is one of the most important swine pathogens. Based on the CPS antigens of S. Suis,33 serotypes (types 1-31,33 and 1/2)have been characterized. Streptococcus suis serotype 2(SS2) is the most commonly and most virulent of these serotypes. SS2 is an zoonotic agent, which is responsible for severe economic losses to the porcine industry, moreover, SS2 is the causative agent of serious infections in humans. Recently, in the study of the virulence of various pathogens and the immune protective antigen protein, secreted proteins and membrane proteins become to the focus. Currently, the membrane surface protein which is cleaved and catalyzed the transfer to cell wall by sortase attract moer attention, almost all gram-positive bacteria have a similar mechanism of protein anchored to the cell wall. These cell wall surface protein are covalently anchored to the cell wall by a mechanism that requiring a C-terminal anchoring motif, which constitute of a conserved amino acid sequence, Leu-Pro-X-Thr-Gly(LPXTG, where X is any amino acid). According to the previous literature, SS2 SntA is a membrane protein. Except the C-terminal motif, LPXTG, SntA protein contain another amino acid motif, a tripeptide RGD. The RGD motif binds to integrins of mammalian cells. Integrins are heterodimeric membrane proteins located on the surface of mammalian cells that participate in cell to cell adhesion and cellular differentiation, migration, and attachment to the extracellular matrix. The SntA protein may directly bind integrin and participate in host-pathogen interactions. That is, SntA may be one of the virulence factor of SS2. Thus, we investigate the function of sntA gene in SS2 in this article.
The project constructed the sntA gene deletion mutant strain(△sntA) of SS2 using plasmit pSET4s successfully. Compared the growth characteristics between△sntA and the wild strain, we found that the growth of two strains are similar in the logarithmic growth phase, and when△sntA grow into the plateau phase, the wild strain still in the logarithmic growth. Thus,△sntA limit the accumulation of bacteria quantity. Compared the colonization of△sntA and the wild strain in mice, we found that the colonization time of△sntA was longer, the anti-clear ability of△sntA has increased. Hep-2 cells as a model to study the different cell adhesion properties between△sntA and the wild atrain, via flow cytometry analysis, the results show that sntA gene deletion mutant's capacity of the adhesion to Hep-2 cells is similar to the wild strain, and inactivated the sntA gene can't alter the cellular adhesion ability. Compared the virulence of AsntA and the wild strain to the Balb/c mice, the outcome expressed that△sntA has a certain degree of decline, the LD50 value was 5×108CFU for△sntA, and the LD50 value of the wild strain was 2.5×108CFU.△sntA was attenuated at least two fold. Balb/c mice as a model to compared the immunity protective of SntA protein and the inactivated vaccine of SS2, the results indicate that SntA protein has a certain immune protection on the Balb/c mice, but it not as good as the protection of the inactivated vaccine of SS2.
本课题以pSET4s质粒为穿梭载体成功构建了SS2菌株sntA基因缺失突变株(△sntA),比较了△sntA与野生型菌株的生长特性,发现两种菌株在对数生长期生长速度差异不明显,但△sntA更早进入生长平台期,限制了一定时间内细菌生长量的积累。比较△sntA与野生型菌株在Balb/c小鼠体内定植,试验结果表明△sntA在小鼠体内定植时间更长,sntA基因缺失后菌株的抗清除能力增强。以Hep-2细胞为模型,研究不同菌株对细胞的黏附特性,结果显示,sntA基因缺失后链球菌对Hep-2细胞的黏附能力与野生型菌株无明显差异,说明sntA基因的缺失不影响细菌对细胞的黏附能力。以Balb/c小鼠为模型,比较了△sntA与野生型菌株的毒力,结果显示△sntA的LD50(5×108)是野生型菌株LD50(2.5×108)的2倍,sntA基因缺失后菌株的毒力有所下降,但不显著。比较了SntA蛋白与猪链球菌2型灭活苗对Balb/c小鼠的免疫保护力,结果表明SntA蛋白具有免疫原性,用亲本菌株攻毒后,SntA蛋白免疫组显示了一定的免疫保护力,保护力明显高于Al(OH)3佐剂对照组,但不如猪链球菌2型灭活苗的保护力强。
Streptococcus suis is one of the most important swine pathogens. Based on the CPS antigens of S. Suis,33 serotypes (types 1-31,33 and 1/2)have been characterized. Streptococcus suis serotype 2(SS2) is the most commonly and most virulent of these serotypes. SS2 is an zoonotic agent, which is responsible for severe economic losses to the porcine industry, moreover, SS2 is the causative agent of serious infections in humans. Recently, in the study of the virulence of various pathogens and the immune protective antigen protein, secreted proteins and membrane proteins become to the focus. Currently, the membrane surface protein which is cleaved and catalyzed the transfer to cell wall by sortase attract moer attention, almost all gram-positive bacteria have a similar mechanism of protein anchored to the cell wall. These cell wall surface protein are covalently anchored to the cell wall by a mechanism that requiring a C-terminal anchoring motif, which constitute of a conserved amino acid sequence, Leu-Pro-X-Thr-Gly(LPXTG, where X is any amino acid). According to the previous literature, SS2 SntA is a membrane protein. Except the C-terminal motif, LPXTG, SntA protein contain another amino acid motif, a tripeptide RGD. The RGD motif binds to integrins of mammalian cells. Integrins are heterodimeric membrane proteins located on the surface of mammalian cells that participate in cell to cell adhesion and cellular differentiation, migration, and attachment to the extracellular matrix. The SntA protein may directly bind integrin and participate in host-pathogen interactions. That is, SntA may be one of the virulence factor of SS2. Thus, we investigate the function of sntA gene in SS2 in this article.
The project constructed the sntA gene deletion mutant strain(△sntA) of SS2 using plasmit pSET4s successfully. Compared the growth characteristics between△sntA and the wild strain, we found that the growth of two strains are similar in the logarithmic growth phase, and when△sntA grow into the plateau phase, the wild strain still in the logarithmic growth. Thus,△sntA limit the accumulation of bacteria quantity. Compared the colonization of△sntA and the wild strain in mice, we found that the colonization time of△sntA was longer, the anti-clear ability of△sntA has increased. Hep-2 cells as a model to study the different cell adhesion properties between△sntA and the wild atrain, via flow cytometry analysis, the results show that sntA gene deletion mutant's capacity of the adhesion to Hep-2 cells is similar to the wild strain, and inactivated the sntA gene can't alter the cellular adhesion ability. Compared the virulence of AsntA and the wild strain to the Balb/c mice, the outcome expressed that△sntA has a certain degree of decline, the LD50 value was 5×108CFU for△sntA, and the LD50 value of the wild strain was 2.5×108CFU.△sntA was attenuated at least two fold. Balb/c mice as a model to compared the immunity protective of SntA protein and the inactivated vaccine of SS2, the results indicate that SntA protein has a certain immune protection on the Balb/c mice, but it not as good as the protection of the inactivated vaccine of SS2.
引文
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