Identification of a novel, small, conserved hypothetical protein involved in Brucella abortus virulence by modifying the expression of multiple genes
Identification of a novel, small, conserved hypothetical protein involved in Brucella abortus virulence by modifying the expression of multiple genes
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通过修饰多个基因的表达来鉴定与流产布鲁氏菌毒力有关的新型、小型、保守的假设蛋白
DOI：
10.1111/tbed.13028
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发表时间：
2019-01-01
影响因子：
4.3
通讯作者：
Yu, Shengqing
中科院分区：
文献类型：
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作者：
Tian, Mingxing;Lian, Zhengmin;Yu, Shengqing
布鲁氏菌病是世界范围内一种重要的人畜共患病，由布鲁氏菌属（Brucella spp.）引起，布鲁氏菌是兼性胞内菌，没有经典的毒力因子，因为其毒力取决于在宿主细胞内入侵和复制的能力。在本研究中，我们鉴定出一个新基因bab_RS22045，它在根瘤菌目中编码一种高度保守的小蛋白。为了研究该基因的作用，构建了缺失突变体和互补菌株。毒力测试表明，bab_RS22045对布鲁氏菌的毒力是必需的，并被命名为毒力相关假定蛋白VhpA。细胞感染实验结果表明，vhpA与布鲁氏菌对HeLa细胞的黏附和入侵以及在RAW264.7细胞内的进一步胞内存活无关。敏感性测试结果显示，vhpA突变体对过氧化氢、多粘菌素B和硝普钠的敏感性与野生型（WT）菌株相似。有趣的是，RNA - seq分析表明，vhpA基因的缺失影响了多个布鲁氏菌基因的表达模式，并且通过实时定量PCR分析进一步证实了主要的4个上调基因和5个下调基因。随后，构建了一系列过表达菌株，毒力测试表明，4个上调基因（bab_RS17930、bab_RS17925、bab_RS26460和bab_RS30050）的过表达显著降低了野生型菌株的毒力，并且vhpA突变体中bab_RS18680的过表达部分恢复了毒力，这表明vhpA通过改变多个基因的表达模式在布鲁氏菌毒力中起重要作用。此外，异源互补分析表明，尽管VhpA在根瘤菌目中是一种高度保守的蛋白，但苜蓿中华根瘤菌（Sinorhizobium meliloti）和根癌农杆菌（Agrobacterium tumefaciens）的同源vhpA基因不能恢复vhpA突变体的毒力。总体而言，鉴定出一个与牛布鲁氏菌（B. abortus）毒力相关的新的、小的假定基因，这凸显了小编码基因在布鲁氏菌毒力中的作用。
Brucellosis is an important zoonotic disease worldwide, caused by Brucella spp., which are facultative intracellular bacteria with no classic virulence factors, as virulence is dependent on the ability to invade and replicate within host cells. In this study, we identified a novel gene bab_RS22045 that encodes a small highly conserved protein in Rhizobiales. To investigate the role of this gene, a deletion mutant and complement strain were constructed. Virulence testing showed that bab_RS22045 is necessary for Brucella virulence, and was designated as virulence-related hypothetical protein, VhpA. The results of a cell infection experiment showed that vhpA was not associated with Brucella adherence to and invasion of HeLa cells, or further intracellular survival within RAW264.7 cells. The results of sensitivity testing showed the vhpA mutant had similar sensitivity to hydrogen peroxide, polymyxin B, and sodium nitroprusside as the wild-type (WT) strain. Interestingly, RNA-seq analysis showed that deletion of the vhpA gene affected the expression patterns of multiple Brucella genes, and the main four up-regulated genes and five down-regulated genes were further confirmed using quantitative real-time PCR analysis. Subsequently, a series of over-expression strains were constructed, and virulence testing showed that over-expression of four up-regulated genes (bab_RS17930, bab_RS17925, bab_RS26460, and bab_RS30050) significantly reduced virulence of the WT strain, and over-expression of bab_RS18680 in the vhpA mutant partially restored virulence, suggesting that vhpA plays an important role in Brucella virulence by changing the expression patterns of multiple genes. Additionally, heterogeneous complementary analysis showed that the homologous vhpA genes of Sinorhizobium meliloti and Agrobacterium tumefaciens could not restore virulence of the vhpA mutant, although VhpA is a highly conserved protein in Rhizobiales. Overall, a novel, small, hypothetical gene was identified that is associated with B. abortus virulence, which highlights the roles of small encoding genes in Brucella virulence.