布鲁氏菌入侵相关基因IalB缺失株的构建及生物学特性分析

Construction and characterization of ialB-deleted strain of Brucella suis

摘要:

入侵相关基因（invasion-associated locus B，ialB）的同源基因在布鲁氏菌所属的根瘤菌目中是广泛保守的，但其在布鲁氏菌中的功能研究几乎为空白。根据有限的报道资料，猜测ialB的功能可能与布鲁氏菌入侵细胞以及适应胞内环境胁迫有关。【目的】探究ialB在布鲁氏菌中的生物学功能，揭示其在布鲁氏菌黏附和入侵细胞以及胞内存活中的作用。【方法】以猪种布鲁氏菌S2株为亲本，运用同源重组的方法构建布鲁氏菌ialB缺失株ΔialB，并通过表达质粒转化的方法构建其回补株CΔialB，比较3种菌株的生长特性、对体外应激的敏感性；通过扫描电镜观察ialB缺失对布鲁氏菌形态的影响，通过实时荧光定量PCR检测3种菌株极性延长相关基因的表达；通过免疫荧光和平板计数的方法分析ialB缺失对布鲁氏菌黏附、入侵RAW264.7细胞以及胞内存活的影响。【结果】成功构建了菌株ΔialB和CΔialB；ΔialB与布鲁氏菌S2株相比，生长受限，活力降低，在酸应激、高渗应激、低渗应激、多黏菌素B应激条件下存活率降低，在氧化应激条件下存活率上升；而且，ΔialB的菌体形态发生改变，菌体变短，直径增加，极性延长相关基因mRNA水平的表达下调；此外，ialB缺失不影响布鲁氏菌黏附、入侵RAW264.7细胞的能力，但显著降低了布鲁氏菌胞内存活能力。【结论】入侵相关基因ialB在布鲁氏菌中具有调节细胞活性和菌体正常生长的功能，对布鲁氏菌抵抗外界环境刺激和在宿主细胞内的增殖具有重要作用。

关键词:布鲁氏菌;入侵相关基因ialB;基因缺失;RAW264.7细胞;生物学特性

Abstract:

The homologous genes of invasion-associated locus B (ialB) are conserved in Rhizobiales including Brucella spp., while little is known about this gene in Brucella. According to the limited reports, ialB of Brucella may be associated with the invasion into host cells and the adaptation to intracellular stress. [Objective] To explore the role of ialB gene in the Brucella adhering to and invading host cells and the intracellular survival of Brucella. [Methods] We constructed the ialB-deleted strain ΔialBof Brucella suis S2 by using homologous recombination method and the complemented strain CΔialB by transforming the expression plasmid containing the open reading frame of ialB into ΔialB. Subsequently, we compared the growth phenotype and stress resistance of different strains and assessed the effect of ialB deletion on the expression of genes associated with the polar cell elongation of Brucella. Additionally, we determined the effect of ialB deletion on the invasion and proliferation of Brucella in RAW264.7 murine macrophages. [Results] ΔialB and CΔialB were constructed successfully. Compared with B. suis S2, ΔialB showed suppressed growth and decreased cell viability. The deletion of ialB decreased the resistance of Brucella to acid stress, hypertonic stress, hypotonic stress, and polymyxin-B stress and increased the resistance to oxidative stress. Furthermore, the deletion changed the cell morphology of Brucella, which was manifested as shortened cell length and increased cell diameter. The mRNA levels of the genes associated with the polar cell elongation of Brucella were down-regulated in ΔialB. Compared with B. suis S2, deletion of ialB did not change the adhesion or invasion of Brucella and decreased the proliferation in RAW264.7 cells. [Conclusion] ialB is associated with the cell viability and polar growth of Brucella, playing a role in the stress resistance and proliferation of Brucella in host cells.

Key words:Brucella suisS2;invasion-associated locus B (ialB);gene deletion;RAW264.7 cells;biological characteristics