As an enzootic pathogen, the Lyme disease bacterium Borrelia burgdorferi possesses multiple copies of chemotaxis proteins, including two chemotaxis histidine kinases (CHK), CheA1 and CheA2. Our previous study showed that CheA2 is a genuine CHK that is required for chemotaxis; however, the role of CheA1 remains mysterious. This report first compares the structural features that differentiate CheA1 and CheA2 and then provides evidence to show that CheA1 is an atypical CHK that controls the virulence of B. burgdorferi through modulating the stability of RpoS, a key transcriptional regulator of the spirochete. First, microscopic analyses using green-fluorescence-protein (GFP) tags reveal that CheA1 has a unique and dynamic cellular localization. Second, loss-of-function studies indicate that CheA1 is not required for chemotaxis in vitro despite sharing a high sequence and structural similarity to its counterparts from other bacteria. Third, mouse infection studies using needle inoculations show that a deletion mutant of CheA1 (cheA1mut) is able to establish systemic infection in immune-deficient mice but fails to do so in immune-competent mice albeit the mutant can survive at the inoculation site for up to 28 days. Tick and mouse infection studies further demonstrate that CheA1 is dispensable for tick colonization and acquisition but essential for tick transmission. Lastly, mechanistic studies combining immunoblotting, protein turnover, mutagenesis, and RNA-seq analyses reveal that depletion of CheA1 affects RpoS stability, leading to reduced expression of several RpoS-regulated virulence factors (i.e., OspC, BBK32, and DbpA), likely due to dysregulated clpX and lon protease expression. Bulk RNA-seq analysis of infected mouse skin tissues further show that cheA1mut fails to elicit mouse tnf-α, il-10, il-1β, and ccl2 expression, four important cytokines for Lyme disease development and B. burgdorferi transmigration. Collectively, these results reveal a unique role and regulatory mechanism of CheA1 in modulating virulence factor expression and add new insights into understanding the regulatory network of B. burgdorferi.

中文翻译：

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化学感应样组氨酸激酶对于体外趋化作用不是必需的，但通过调节 RpoS 的稳定性来调节伯氏疏螺旋体的毒力。

作为一种地方性病原体，莱姆病细菌伯氏疏螺旋体拥有多个趋化蛋白拷贝，包括两种趋化组氨酸激酶 (CHK)：CheA1 和 CheA2。我们之前的研究表明CheA2是趋化性所需的真正的CHK；然而，CheA1 的作用仍然神秘。该报告首先比较了区分CheA1和CheA2的结构特征，然后提供证据表明CheA1是一种非典型CHK，通过调节螺旋体关键转录调节因子RpoS的稳定性来控制伯氏疏螺旋体的毒力。首先，使用绿色荧光蛋白 (GFP) 标签的显微镜分析表明 CheA1 具有独特且动态的细胞定位。其次，功能丧失研究表明，尽管 CheA1 与其他细菌的对应物具有高度的序列和结构相似性，但 CheA1 并不是体外趋化所必需的。第三，使用针头接种的小鼠感染研究表明，CheA1 的缺失突变体 (cheA1mut) 能够在免疫缺陷的小鼠中建立全身感染，但在免疫功能正常的小鼠中却无法做到这一点，尽管该突变体可以在接种部位存活长达至 28 天。蜱和小鼠感染研究进一步表明，CheA1 对于蜱定植和获取来说是可有可无的，但对于蜱传播至关重要。最后，结合免疫印迹、蛋白质周转、诱变和 RNA-seq 分析的机制研究表明，CheA1 的缺失会影响 RpoS 稳定性，导致几种 RpoS 调节的毒力因子（即 OspC、BBK32 和 DbpA）的表达减少，这可能是由于clpX 和 lon 蛋白酶表达失调。对受感染小鼠皮肤组织的批量 RNA-seq 分析进一步表明，cheA1mut 无法引发小鼠 tnf-α、il-10、il-1β 和 ccl2 表达，这四种是莱姆病发展和伯氏疏螺旋体迁移的重要细胞因子。总的来说，这些结果揭示了 CheA1 在调节毒力因子表达中的独特作用和调节机制，并为理解伯氏疏螺旋体的调节网络提供了新的见解。