猪链球菌2型DnaK通过降低RAW264.7巨噬细胞内吞受体LRP1蛋白水平参与抗吞噬

doi:10.1016/j.jia.2024.07.009

Journal of Integrative Agriculture ›› 2025, Vol. 24 ›› Issue (12): 4760-4775.DOI: 10.1016/j.jia.2024.07.009

猪链球菌2型DnaK通过降低RAW264.7巨噬细胞内吞受体LRP1蛋白水平参与抗吞噬

收稿日期:2024-02-20 修回日期:2024-07-06 接受日期:2024-05-22 出版日期:2025-12-20 发布日期:2025-11-13

DnaK of Streptococcus suis serotype 2 contributes to phagocytosis resistance by decreasing endocytic receptor LRP1 protein levels in RAW264.7 macrophages

Qing Wang¹, Guangbin Bao¹, Shinuo Fan¹, Xiaomeng Pei², Hongjie Fan^{2, 3#}

¹ Anhui Province Key Laboratory of Veterinary Pathobiology and Disease Control, College of Veterinary Medicine, Anhui Agricultural University, Hefei 230036, China
² Ministry of Education Joint International Research Laboratory of Animal Health and Food Safety, College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, China
³ College of Animal Science, Anhui Science and Technology University, Fengyang 233100, China

Received:2024-02-20 Revised:2024-07-06 Accepted:2024-05-22 Online:2025-12-20 Published:2025-11-13
About author:Qing Wang, E-mail: wangqing202008@ 163.com; #Correspondence Hongjie Fan, Tel: +86-25-84399592, E-mail: fhj@njau.edu.cn
Supported by:
This study was funded by the National Key Research and Development Program of China (2021YFD1800400), the National Natural Science Foundation of China (32373018), Jiangsu Agriculture Science and Technology Innovation Fund, China (CX(23)1029), the Excellent Research Innovation Team in Universities in Anhui Province, China (2022AH010088), and the Shennong Scholar Project of Anhui Agricultural University, China (rc392101).

摘要/Abstract

摘要：

猪链球菌血清型2（Streptococcus suis serotype 2, SS2）是一种重要的人畜共患病病原，可引起猪败血症和人链球菌中毒性休克样综合征（STSLS）等急性感染，这表明SS2可逃避先天免疫。巨噬细胞能够直接吞噬和杀死病原体，在先天免疫系统中发挥重要的功能。前期，本实验室从SS2转座子突变文库中筛选到一株抗吞噬能力减弱的dnaK突变株，但dnaK参与抗吞噬的具体机制尚不清楚。本研究旨在明确DnaK在SS2抗吞噬中的作用，从而拓展对DnaK功能的认知，并有助于阐明SS2的抗吞噬机制。在本研究中，通过构建dnaK基因缺失株和互补株，我们进一步证明了DnaK是SS2抗巨噬细胞吞噬和在恶劣环境下存活所必需的。小鼠攻毒实验表明，DnaK促进了菌血症和SS2的全身系统性传播，增强了SS2的致病性。Western blot、免疫荧光实验和透射电镜观察结果显示，DnaK可由SS2分泌，并能进入RAW264.7巨噬细胞中。然后，通过RNA测序技术（RNA- seq）鉴定了DnaK调控的内吞受体LRP1。我们发现，DnaK降低了LRP1的mRNA和蛋白水平。敲低LRP1 β链（LRP1β）可显著降低巨噬细胞RAW264.7对SS2菌株ZY05719的吞噬率，提示LRP1是SS2的吞噬受体。此外，抑制剂处理实验显示，DnaK通过转录因子PPARγ和泛素-蛋白酶体系统两种途径降低LRP1蛋白水平。由于DnaK蛋白序列在链球菌中高度保守，我们推测在其他链球菌中可能存在类似的抗吞噬机制。综上所述，DnaK通过降低巨噬细胞中LRP1蛋白水平参与了SS2的抗吞噬。本研究为SS2的抗吞噬机制提供了新的见解，有助于进一步阐明SS2的致病机制。

Abstract:

Streptococcus suis serotype 2 (SS2) is a zoonotic pathogen that can cause acute infection, such as septicemia in pigs and streptococcal toxic shock-like syndrome (STSLS) in humans, indicating that SS2 can evade innate immunity. Macrophages perform essential antimicrobial functions in the innate immune system by engulfing and killing pathogens. Previously, a dnaK mutant strain that showed impaired phagocytosis resistance ability was screened from the transposon mutant library of SS2, but the specific mechanism is unclear. In this study, we further demonstrated that DnaK was required for SS2 to be antiphagocytosed by macrophages and survive in adverse environments. A mouse challenge experiment indicated that DnaK promoted bacteremia and systemic dissemination of SS2, enhancing bacterial pathogenicity. Western blot and immunofluorescence results indicated that DnaK could be secreted by SS2 and was able to enter RAW264.7 macrophages. Then, the endocytic receptor LRP1 regulated by DnaK was identified through RNA sequencing (RNA-Seq). We found that DnaK decreased both the mRNA and protein levels of LRP1. Knockdown of the LRP1 β-chain (LRP1β) significantly decreased the phagocytosis rate of the SS2 strain ZY05719, suggesting that LRP1 is a phagocytic receptor of SS2. Furthermore, inhibitor treatment assays revealed that DnaK decreased LRP1 protein levels through the transcription factor PPARγ and the ubiquitin–proteasome system. In summary, DnaK contributes to the phagocytosis resistance of SS2 by decreasing LRP1 protein levels in macrophages, providing new insights into the antiphagocytosis mechanisms of SS2 and helping to understand its pathogenesis.

Key words: Streptococcus suis serotype 2 , DnaK , phagocytosis resistance , macrophages , LRP1

Qing Wang, Guangbin Bao, Shinuo Fan, Xiaomeng Pei , Hongjie Fan. 猪链球菌2型DnaK通过降低RAW264.7巨噬细胞内吞受体LRP1蛋白水平参与抗吞噬[J]. Journal of Integrative Agriculture, 2025, 24(12): 4760-4775.

Qing Wang, Guangbin Bao, Shinuo Fan, Xiaomeng Pei, Hongjie Fan. DnaK of Streptococcus suis serotype 2 contributes to phagocytosis resistance by decreasing endocytic receptor LRP1 protein levels in RAW264.7 macrophages[J]. Journal of Integrative Agriculture, 2025, 24(12): 4760-4775.

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猪链球菌2型DnaK通过降低RAW264.7巨噬细胞内吞受体LRP1蛋白水平参与抗吞噬

DnaK of Streptococcus suis serotype 2 contributes to phagocytosis resistance by decreasing endocytic receptor LRP1 protein levels in RAW264.7 macrophages

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