In Borrelia burgdorferi, BB0556 was annotated as a conserved hypothetical protein. We herein investigated gene expression and the importance of this protein during infection. Our data support that bb0556 forms an operon with five other genes. A transcriptional start site and the associated σ70‐type promoter were identified in the sequences upstream of bb0554, and luciferase reporter assays indicated that this promoter is functional in B. burgdorferi. Furthermore, the sequences upstream of bb0556 contain an internal promoter to drive gene expression. bb0556 expression was affected by various environmental factors such as changes in temperature, pH, and cell density when B. burgdorferi was grown in vitro. Surprisingly, significant differences were observed for bb0556 expression between B. burgdorferi strains B31‐A3 and CE162, likely due to the different cis‐ and trans‐acting factors in these strains. Moreover, bb0556 was found to be highly expressed by B. burgdorferi in infected mice tissues, suggesting that this gene plays an important role during animal infection. To test this hypothesis, we generated a bb0556 deletion mutant in a virulent bioluminescent B. burgdorferi strain. The mutant grew normally in the medium and displayed no defect in the resistance to environmental stresses such as reactive oxygen species, reactive nitrogen species, and osmotic stress. However, when the infectivity was compared between the mutant and its parental strain using in vivo bioluminescence imaging as well as analyses of spirochete recovery and bacterial burdens in animal tissues, our data showed that, contrary to the parental strain, the mutant was unable to infect mice. Complementation of bb0556 in cis fully restored the infectious phenotype to wild‐type levels. Taken together, our study demonstrates that the hypothetical protein BB0556 is a novel virulence factor essential for B. burgdorferi mammalian infection.

中文翻译：

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哺乳动物感染伯氏疏螺旋体时bb0556的表达及其作用分析


在伯氏疏螺旋体中，BB0556 被注释为保守的假设蛋白。我们在此研究了基因表达以及该蛋白质在感染过程中的重要性。我们的数据支持bb0556与其他五个基因形成操纵子。在bb0554上游的序列中鉴定出了转录起始位点和相关的 σ ⁷⁰型启动子，并且荧光素酶报告基因分析表明该启动子在伯氏疏螺旋体中具有功能。此外， bb0556上游的序列含有驱动基因表达的内部启动子。当伯氏疏螺旋体在体外生长时， bb0556 的表达受到各种环境因素的影响，例如温度、pH 值和细胞密度的变化。令人惊讶的是，在伯氏疏螺旋体菌株 B31-A3 和 CE162 之间观察到bb0556表达的显着差异，这可能是由于这些菌株中不同的顺式和反式作用因子。此外，发现bb0556在受感染的小鼠组织中被伯氏疏螺旋体高表达，表明该基因在动物感染过程中发挥着重要作用。为了检验这一假设，我们在有毒的生物发光伯氏疏螺旋体菌株中产生了bb0556缺失突变体。该突变体在培养基中生长正常，对活性氧、活性氮、渗透压等环境胁迫的抵抗能力没有缺陷。 然而，当使用体内生物发光成像以及对动物组织中螺旋体恢复和细菌负荷的分析来比较突变体与其亲本菌株之间的感染性时，我们的数据表明，与亲本菌株相反，突变体无法感染老鼠。顺式中bb0556的互补将感染表型完全恢复至野生型水平。综上所述，我们的研究表明，假设的蛋白质 BB0556 是伯氏疏螺旋体哺乳动物感染所必需的新型毒力因子。