流产布氏杆菌脂多糖突变株ΔrfbE和ΔrfbD的构建及其生物学特性研究
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摘要
布氏杆菌是兼性胞内生长的革兰氏阴性菌,可感染多种动物和人,引起布氏杆菌病。布氏杆菌的致病机制与其胞内存活、增殖有关。脂多糖(LPS)是布氏杆菌一种重要的毒力因子,完整的LPS对其胞内生存和毒力十分必要。rfbE (BAB1_0542)编码ABC型转运系统ATP酶,rfbD (BAB1_0543)编码ABC型转运系统运载体,这两个蛋白参与LPS的O-抗原从细胞膜内侧转运到周质侧。rfbE, rfbD基因突变,将导致O-抗原在胞内合成,但不能转运到周质侧,无法合成完整的LPS。
本研究通过同源重组的方法构建ΔrfbE和ΔrfbD突变株,通过比较突变株和野毒株的细菌表型、生长曲线、在小鼠巨噬细胞RAW264.7胞内生存能力、在BALB/c小鼠动物模型上的毒力情况,来探讨不完整LPS结构对布氏杆菌S2308生物学功能和毒力特性的影响；并通过监测ΔrfbE免疫后小鼠血清针对S2308产生的抗体效价、细胞因子表达,小鼠脾脏内MHC分子和共刺激分子在转录水平的表达,以及ΔrfbE免疫后小鼠对S2308攻毒产生的保护力,来评价ΔrfbE免疫保护性,探究其做为新型减毒活疫苗候选株的可能性。
结果显示,ΔrfbE和ΔrfbD突变株构建成功,rfbE基因(BAB1_0542)编码框内缺失446bp, rfbD基因(BAB1_0543)编码框内缺失668bp。同时用实时定量PCR方法在转录水平上验证ΔrfbE突变成功,对上下游基因功能没有影响。应用结晶紫染色和热凝集实验方法确定ΔrfbE和ΔrfbD均为粗糙型表型；生长曲线结果表明ArfbE突变株生长速度于36h后明显低于野毒株S2308(P<0.01),ΔrfbD突变株比野毒株S2308早8h到达平台期；ΔrfbE对大多数抗生素敏感,仅对红霉素不敏感,对表面活性剂Tween-20、Triton-X100不敏感,与野毒株S2308相比差异不显著；ΔrfbE和ΔrfbD突变株与野毒株S2308相比,在胞内的生存、增殖能力下降,在小鼠体内毒力减弱,而C2308ΔrfbE与野毒株S2308毒力一致。
此外,ArfbE免疫2周后针对S2308产生抗体效价明显低于S2308接种小鼠产生的效价(P<0.05),可以区分人为免疫和自然感染；ΔrfbE免疫小鼠1周后脾脏内MHCI的表达量明显上调(P<0.05),而野毒株S2308免疫小鼠1周后脾脏内MHCI的表达量上调不明显,突变株ΔrfbE相对于野毒株S2308在免疫早期引起更高水平的细胞免疫水平；ΔrfbE. S2308免疫后脾脏中共刺激分子CD40ΟCD80ΟCD86调节趋势一致,血清中细胞因子IL-2、 IL-4、IL-6、IL-10变化不明显,即ArfbE、S2308产生免疫分子的趋势一致；ΔrfbE免疫小鼠后对强毒的攻毒保护率为100%,与疫苗株RB51相似。因此,ΔrfbE可作为新型减毒活疫苗的候选株。
Brucella spp. is a Gram-negative, facultative intracellular bacterial pathogen of humans and other animals, causing Brucellosis. Its virulence depends on the survival and replication properties in host cells. Lipopolysaccharide (LPS) is an important virulence factor. The integrity of LPS molecule on the bacterial surface is one of the factors required for the intracellular survival of Brucella, and subsequently for virulence. rfbE (BAB1_0542) codes ABC transporter ATPase, and rfbD (BAB1_0543) codes acriflavin ABC transporter. Both the ABC proteins translocate the amino sugar-O-PS to the periplasmic side of the membrane. Inactivation of rfbE or rfbD gene made the integrity of LPS molecule destroyed.
In the study, homologous recombination was used to construct B. abortus LPS mutants of ArfbE and ArfbD respectively. The phenotype, growth rate, survival ability in macrophages RAW264.7and mice were compared with wild strain S2308to demonstrate the importance of integrity LPS on the biological and virulence of B. abortus.In addition, ArfbE was used to immunize the BALB/c mice, the antibody titers against S2308and the cytokine production in the sera as well as the expression of MHC and co-stimulatory molecules in the spleens were investigated. Furthermore, the protection against the challenge of virulent wild type strain S2308was investigated to evaluate the potential usage of vaccine candidate.
The results showed that the construction of ArfbE and ArfbD are successful. The rfbE (BAB1_0542) of B. abortus strain2308was inactivated by deleting a446bp fragment from the gene, and the rfbD (BAB1_0543) of B. abortus strain2308was inactivated by deleting a668bp fragment from the gene. Real time PCR analysis further confirmed the inactivation of rfbE with no polar effect on the transcription of its upstream and downstream genes. Both ArfbE and ArfbD were identified as a rough phenotype strain using heat agglutination test and crystal violet staining, but the growth rate of ArfbE is much lower than S2308after36h cultured (P<0.01) and the ArfbD arrived stationary phase8h earlier than S2308. ArfbE as well as S2308is sensitive to most of the antibiotics except Erythromycin, Tween-20and Triton-X100. Moreover, both mutants of ArfbE and ArfbD showed attenuated virulence in RAW264.7macrophages in vitro and in BALB/c mice in vivo. Complementation of ArfbE recovered the smooth phenotype of the bacteria and the complemented strain C2308ArfbE survived in BALB/c mice as shown of wild type strain S2308.
Animal experiments showed that immunization of ArfbE generated similar levels of cytokines IL-2, IL-4, IL-6and IL-10, compared with infection of S2308, however, the antibody titers against S2308were significantly lower than that of S2308immunized mice (P<0.05). The expression of MHC I in transcriptional level in ArfbE immunized mice spleens is significantly higher than control group at the early stage of infection (P<0.05), and the mutant strain ArfbE protected immunized mice from challenge with the virulent wild type strain S2308, which suggests that the mutant strain ArfbE could be used as a vaccine candidate in future studies.
本研究通过同源重组的方法构建ΔrfbE和ΔrfbD突变株,通过比较突变株和野毒株的细菌表型、生长曲线、在小鼠巨噬细胞RAW264.7胞内生存能力、在BALB/c小鼠动物模型上的毒力情况,来探讨不完整LPS结构对布氏杆菌S2308生物学功能和毒力特性的影响；并通过监测ΔrfbE免疫后小鼠血清针对S2308产生的抗体效价、细胞因子表达,小鼠脾脏内MHC分子和共刺激分子在转录水平的表达,以及ΔrfbE免疫后小鼠对S2308攻毒产生的保护力,来评价ΔrfbE免疫保护性,探究其做为新型减毒活疫苗候选株的可能性。
结果显示,ΔrfbE和ΔrfbD突变株构建成功,rfbE基因(BAB1_0542)编码框内缺失446bp, rfbD基因(BAB1_0543)编码框内缺失668bp。同时用实时定量PCR方法在转录水平上验证ΔrfbE突变成功,对上下游基因功能没有影响。应用结晶紫染色和热凝集实验方法确定ΔrfbE和ΔrfbD均为粗糙型表型；生长曲线结果表明ArfbE突变株生长速度于36h后明显低于野毒株S2308(P<0.01),ΔrfbD突变株比野毒株S2308早8h到达平台期；ΔrfbE对大多数抗生素敏感,仅对红霉素不敏感,对表面活性剂Tween-20、Triton-X100不敏感,与野毒株S2308相比差异不显著；ΔrfbE和ΔrfbD突变株与野毒株S2308相比,在胞内的生存、增殖能力下降,在小鼠体内毒力减弱,而C2308ΔrfbE与野毒株S2308毒力一致。
此外,ArfbE免疫2周后针对S2308产生抗体效价明显低于S2308接种小鼠产生的效价(P<0.05),可以区分人为免疫和自然感染；ΔrfbE免疫小鼠1周后脾脏内MHCI的表达量明显上调(P<0.05),而野毒株S2308免疫小鼠1周后脾脏内MHCI的表达量上调不明显,突变株ΔrfbE相对于野毒株S2308在免疫早期引起更高水平的细胞免疫水平；ΔrfbE. S2308免疫后脾脏中共刺激分子CD40ΟCD80ΟCD86调节趋势一致,血清中细胞因子IL-2、 IL-4、IL-6、IL-10变化不明显,即ArfbE、S2308产生免疫分子的趋势一致；ΔrfbE免疫小鼠后对强毒的攻毒保护率为100%,与疫苗株RB51相似。因此,ΔrfbE可作为新型减毒活疫苗的候选株。
Brucella spp. is a Gram-negative, facultative intracellular bacterial pathogen of humans and other animals, causing Brucellosis. Its virulence depends on the survival and replication properties in host cells. Lipopolysaccharide (LPS) is an important virulence factor. The integrity of LPS molecule on the bacterial surface is one of the factors required for the intracellular survival of Brucella, and subsequently for virulence. rfbE (BAB1_0542) codes ABC transporter ATPase, and rfbD (BAB1_0543) codes acriflavin ABC transporter. Both the ABC proteins translocate the amino sugar-O-PS to the periplasmic side of the membrane. Inactivation of rfbE or rfbD gene made the integrity of LPS molecule destroyed.
In the study, homologous recombination was used to construct B. abortus LPS mutants of ArfbE and ArfbD respectively. The phenotype, growth rate, survival ability in macrophages RAW264.7and mice were compared with wild strain S2308to demonstrate the importance of integrity LPS on the biological and virulence of B. abortus.In addition, ArfbE was used to immunize the BALB/c mice, the antibody titers against S2308and the cytokine production in the sera as well as the expression of MHC and co-stimulatory molecules in the spleens were investigated. Furthermore, the protection against the challenge of virulent wild type strain S2308was investigated to evaluate the potential usage of vaccine candidate.
The results showed that the construction of ArfbE and ArfbD are successful. The rfbE (BAB1_0542) of B. abortus strain2308was inactivated by deleting a446bp fragment from the gene, and the rfbD (BAB1_0543) of B. abortus strain2308was inactivated by deleting a668bp fragment from the gene. Real time PCR analysis further confirmed the inactivation of rfbE with no polar effect on the transcription of its upstream and downstream genes. Both ArfbE and ArfbD were identified as a rough phenotype strain using heat agglutination test and crystal violet staining, but the growth rate of ArfbE is much lower than S2308after36h cultured (P<0.01) and the ArfbD arrived stationary phase8h earlier than S2308. ArfbE as well as S2308is sensitive to most of the antibiotics except Erythromycin, Tween-20and Triton-X100. Moreover, both mutants of ArfbE and ArfbD showed attenuated virulence in RAW264.7macrophages in vitro and in BALB/c mice in vivo. Complementation of ArfbE recovered the smooth phenotype of the bacteria and the complemented strain C2308ArfbE survived in BALB/c mice as shown of wild type strain S2308.
Animal experiments showed that immunization of ArfbE generated similar levels of cytokines IL-2, IL-4, IL-6and IL-10, compared with infection of S2308, however, the antibody titers against S2308were significantly lower than that of S2308immunized mice (P<0.05). The expression of MHC I in transcriptional level in ArfbE immunized mice spleens is significantly higher than control group at the early stage of infection (P<0.05), and the mutant strain ArfbE protected immunized mice from challenge with the virulent wild type strain S2308, which suggests that the mutant strain ArfbE could be used as a vaccine candidate in future studies.
引文
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