Department of Microbiology, NYU Grossman School of Medicine, New York, New York, USA.
J Bacteriol. 2024 Oct 24;206(10):e0022224. doi: 10.1128/jb.00222-24. Epub 2024 Sep 17.
In , alginate biosynthesis gene expression is inhibited by the transmembrane anti-sigma factor MucA, which sequesters the AlgU sigma factor. Cell envelope stress initiates cleavage of the MucA periplasmic domain by site-1 protease AlgW, followed by further MucA degradation to release AlgU. However, after colonizing the lungs of people with cystic fibrosis, converts to a mucoid form that produces alginate constitutively. Mucoid isolates often have mutations, with the most common being , which truncates the periplasmic domain. MucA22 is degraded constitutively, and genetic studies suggested that the Prc protease is responsible. Some studies also suggested that Prc contributes to induction in strains with wild-type MucA, whereas others suggested the opposite. However, missing from all previous studies is a demonstration that Prc cleaves any protein directly, which leaves open the possibility that the effect of a null mutation is indirect. To address the ambiguities and shortfalls, we reevaluated the roles of AlgW and Prc as MucA and MucA22 site-1 proteases. analyses using three different assays and two different inducing conditions all suggested that AlgW is the only site-1 protease for wild-type MucA in any condition. In contrast, genetics suggested that AlgW or Prc act as MucA22 site-1 proteases in inducing conditions, whereas Prc is the only MucA22 site-1 protease in non-inducing conditions. For the first time, we also show that Prc is unable to degrade the periplasmic domain of wild-type MucA but does degrade the mutated periplasmic domain of MucA22 directly.
After colonizing the lungs of individuals with cystic fibrosis, undergoes mutagenic conversion to a mucoid form, worsening the prognosis. Most mucoid isolates have a truncated negative regulatory protein MucA, which leads to constitutive production of the extracellular polysaccharide alginate. The protease Prc has been implicated, but not shown, to degrade the most common MucA variant, MucA22, to trigger alginate production. This work provides the first demonstration that the molecular mechanism of Prc involvement is direct degradation of the MucA22 periplasmic domain and perhaps other truncated MucA variants as well. MucA truncation and degradation by Prc might be the predominant mechanism of mucoid conversion in cystic fibrosis infections, suggesting that Prc activity could be a useful therapeutic target.
在Alginate 生物合成基因表达受跨膜反西格玛因子 MucA 抑制，MucA 隔离 AlgU 西格玛因子。细胞包膜应激通过位点 1 蛋白酶 AlgW 引发 MucA 周质结构域的切割，随后进一步降解 MucA 以释放 AlgU。然而，在定植囊性纤维化患者的肺部后，转变成持续产生Alginate 的粘液形式。粘液分离株通常具有 突变，最常见的是 ，其截断周质结构域。MucA22 持续降解，遗传研究表明 Prc 蛋白酶负责。一些研究还表明，Prc 有助于诱导野生型 MucA 的菌株，而其他研究则表明相反。然而，以前的所有研究都没有证明 Prc 直接切割任何蛋白质，这使得 缺失突变的影响是间接的可能性仍然存在。为了解决这些模糊性和不足之处，我们重新评估了 AlgW 和 Prc 作为 MucA 和 MucA22 位点 1 蛋白酶的作用。使用三种不同的测定法和两种不同的诱导条件进行的 分析均表明，AlgW 是任何条件下野生型 MucA 的唯一位点 1 蛋白酶。相比之下，遗传学表明 AlgW 或 Prc 在诱导条件下充当 MucA22 位点 1 蛋白酶，而 Prc 是非诱导条件下唯一的 MucA22 位点 1 蛋白酶。我们首次表明，Prc 无法降解野生型 MucA 的周质结构域，但可直接降解 MucA22 的突变周质结构域。
定植在囊性纤维化个体的肺部后，转变成粘液形式的突变体，使预后恶化。大多数粘液分离株具有截断的负调控蛋白 MucA，导致细胞外多糖 Alginate 的持续产生。蛋白酶 Prc 已被牵连，但未被证明，可降解最常见的 MucA 变体 MucA22，触发 Alginate 产生。这项工作首次证明了 Prc 参与的分子机制是直接降解 MucA22 的周质结构域，也许还有其他截断的 MucA 变体。Prc 活性可能是囊性纤维化感染中粘液转化的主要机制，表明 Prc 活性可能是一种有用的治疗靶点。
