Department of Comparative Biomedical Sciences, College of Veterinary Medicine, Mississippi State University, Mississippi State, Mississippi, USA.
Department of Animal and Dairy Sciences, Mississippi State University, Mississippi State, Mississippi, USA.
J Bacteriol. 2024 Jul 25;206(7):e0003324. doi: 10.1128/jb.00033-24. Epub 2024 Jun 20.
is a foodborne bacterial pathogen that causes listeriosis. Positive regulatory factor A (PrfA) is a pleiotropic master activator of virulence genes of that becomes active upon the entry of the bacterium into the cytosol of infected cells. can survive and multiply at low temperatures; this is accomplished through the maintenance of appropriate membrane fluidity via branched-chain fatty acid (BCFA) synthesis. Branched-chain α-keto acid dehydrogenase (BKD), which is composed of four polypeptides encoded by , , , and , is known to play a vital role in BCFA biosynthesis. Here, we constructed BKD-deficient strains by in-frame deletion of , , , and genes. To determine the role in and , mouse model challenges, plaque assay in murine L2 fibroblast, and intracellular replication in J744A.1 macrophage were conducted. BKD-deficient strains exhibited defects in BCFA composition, virulence, and PrfA-regulon function within the host cells. Transcriptomics analysis revealed that the transcript level of the PrfA-regulon was lower in Δ strain than those in the wild-type. This study demonstrates that strains lacking BKD complex components were defective in PrfA-regulon function, and full activation of wild-type may not occur within host cells in the absence of BKD. Further study will investigate the consequences of BKD deletion on PrfA function through altering BCFA catabolism.IMPORTANCE is the causative agent of listeriosis, a disease with a high mortality rate. In this study, we have shown that the deletion of BKD can impact the function of PrfA and the PrfA-regulon. The production of virulence proteins within host cells is necessary for to promote its intracellular survival and is likely dependent on membrane integrity. We thus report a link between membrane integrity and the function of PrfA. This knowledge will increase our understanding of pathogenesis, which may provide insight into the development of antimicrobial agents.
李斯特菌是一种食源性细菌病原体，可导致李斯特菌病。正调控因子 A（PrfA）是李斯特菌毒力基因的多效主激活因子，在细菌进入感染细胞的细胞质时变得活跃。李斯特菌可以在低温下存活和繁殖；这是通过通过分支链脂肪酸（BCFA）合成来维持适当的膜流动性来实现的。分支链α-酮酸脱氢酶（BKD）由 、 、 和 四个多肽组成，已知在 BCFA 生物合成中发挥重要作用。在这里，我们通过框内缺失 、 、 和 基因构建了 BKD 缺陷型 菌株。为了确定在宿主细胞中的作用和 ，进行了小鼠模型挑战、鼠 L2 成纤维细胞中的噬菌斑测定和 J744A.1 巨噬细胞中的细胞内复制。BKD 缺陷型菌株在 BCFA 组成、毒力和宿主细胞中 PrfA 调节子功能方面表现出缺陷。转录组学分析显示，Δ 菌株的 PrfA 调节子转录水平低于野生型。这项研究表明，缺乏 BKD 复合物成分的 菌株在 PrfA 调节子功能上存在缺陷，并且在没有 BKD 的情况下，野生型 可能不会在宿主细胞内完全激活。进一步的研究将通过改变 BCFA 分解代谢来研究 BKD 缺失对 PrfA 功能的影响。
重要李斯特菌是李斯特菌病的病原体，该病死亡率很高。在这项研究中，我们表明 BKD 的缺失会影响 PrfA 和 PrfA 调节子的功能。在宿主细胞内产生毒力蛋白对于李斯特菌促进其细胞内存活是必要的，并且可能依赖于膜完整性。因此，我们报告了 膜完整性与 PrfA 功能之间的联系。这一知识将增加我们对李斯特菌发病机制的理解，这可能为开发抗菌剂提供启示。
