College of Veterinary Medicine, Hunan Agricultural University, Changsha 410125, China.
Hunan Institute of Animal and Veterinary Science, Changsha 410131, China.
Vet Microbiol. 2024 Nov;298:110263. doi: 10.1016/j.vetmic.2024.110263. Epub 2024 Sep 24.
The thioredoxin (Trx) system plays a vital role in protecting against oxidative stress and ensures correct disulfide bonding to maintain protein function. Our previous research demonstrated that TrxA of Streptococcus suis Serotype 2 (SS2), a clinical strain from the lung of a diseased pig, contributes to virulence but is not involved in antioxidative stress. In this study, we identified another gene in the Trx family, TrxC, which encodes a protein of 104 amino acids with a CGDC active motif and 22.4 % amino acid sequence homology with TrxA. Unlike the TrxA, TrxC mutant strains were more susceptible to oxidative stresses induced by hydrogen peroxide and paraquat. In vitro experiments, the survival rate of the TrxC deletion mutant in RAW264.7 macrophages was only one-eighth of that of TrxA mutant strains. Transcriptome analysis revealed that autophagy-related genes were significantly upregulated in the TrxC mutant compared to those in the wild-type or TrxA mutant strains. Co-localization of LC3 puncta with TrxC was confirmed using laser confocal microscopy, and autophagy-related indicators were quantified using western blotting. Autophagy deficiency induced by ATG5 knockout significantly increased SS2 survival rate, especially in TrxC mutant strains. For the upstream signal regulation pathways, we found ΔTrxC strains regulate autophagy by activation of PI3K/Akt/mTOR signaling in RAW264.7 macrophages. In the Akt1-overexpressing cell line, ΔTrxC infection significantly decreased the autophagic response and promoted ΔTrxC mutant strain survival, while inhibition of Akt with MK2206 resulted in reduced ΔTrxC mutant strain survival and enhance the autophagic response. Furthermore, loss of TrxC increased the activity of MSR1, thereby inducing cellular autophagy and phagocytosis. Our data demonstrate that TrxC of SS2 contributes to virulence by inducing antioxidative stress and inhibits autophagy via the PI3K-Akt-mTOR pathway in macrophages, with MSR1 acting as a key factor in controlling infection.
硫氧还蛋白（Trx）系统在抵御氧化应激中起着至关重要的作用，并确保正确的二硫键形成以维持蛋白质功能。我们之前的研究表明，猪链球菌 2 型（SS2）临床分离株的 TrxA 有助于毒力，但不参与抗氧化应激。在这项研究中，我们鉴定了 Trx 家族中的另一个基因 TrxC，它编码一个由 104 个氨基酸组成的蛋白质，具有 CGDC 活性基序，与 TrxA 的氨基酸序列同源性为 22.4%。与 TrxA 不同，TrxC 突变株对过氧化氢和百草枯诱导的氧化应激更敏感。体外实验中，TrxC 缺失突变株在 RAW264.7 巨噬细胞中的存活率仅为 TrxA 突变株的八分之一。转录组分析显示，与野生型或 TrxA 突变株相比，TrxC 突变株中自噬相关基因显著上调。激光共聚焦显微镜证实了 LC3 斑点与 TrxC 的共定位，并用 Western blot 定量了自噬相关指标。ATG5 敲除诱导的自噬缺陷显著增加了 SS2 的存活率，特别是在 TrxC 突变株中。对于上游信号调节途径，我们发现 ΔTrxC 菌株通过激活 RAW264.7 巨噬细胞中的 PI3K/Akt/mTOR 信号通路来调节自噬。在 Akt1 过表达细胞系中，ΔTrxC 感染显著降低了自噬反应并促进了 ΔTrxC 突变株的存活，而用 MK2206 抑制 Akt 导致 ΔTrxC 突变株的存活减少并增强了自噬反应。此外，TrxC 的缺失增加了 MSR1 的活性，从而诱导细胞自噬和吞噬作用。我们的数据表明，SS2 的 TrxC 通过诱导抗氧化应激和通过 PI3K-Akt-mTOR 通路抑制巨噬细胞中的自噬来促进毒力，MSR1 是控制感染的关键因素。
