JIA-2021-2061 CovS在马链球菌兽疫亚种毒力形成中的作用研究

doi:10.1016/j.jia.2022.08.109

Journal of Integrative Agriculture ›› 2023, Vol. 22 ›› Issue (2): 568-584.DOI: 10.1016/j.jia.2022.08.109

JIA-2021-2061 CovS在马链球菌兽疫亚种毒力形成中的作用研究

收稿日期:2021-12-07 接受日期:2022-04-21 出版日期:2023-02-20 发布日期:2022-04-21

The vital role of CovS in the establishment of Streptococcus equi subsp. zooepidemicus virulence

XU Bin^{1, 2}, MA Zhe^{1, 3}, ZHOU Hong¹, LIN Hui-xing¹, FAN Hong-jie^{1, 3}

¹ MOE Joint International Research Laboratory of Animal Health and Food Safety, College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, P.R.China
²Key Laboratory of Veterinary Biological Engineering and Technology of Ministry of Agriculture and Rural Affairs, National Research Center of Veterinary Biologicals Engineering and Technology, Institute of Veterinary Medicine, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, P.R.China
³Jiangsu Co-innovation Center for the Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou University, Yangzhou 225009, P.R.China

Received:2021-12-07 Accepted:2022-04-21 Online:2023-02-20 Published:2022-04-21
About author:XU Bin, E-mail: xubin9999@foxmail.com; Correspondence FAN Hong-jie, Tel/Fax: +86-25-84399592, E-mail: fhj@njau.edu.cn
Supported by:
This study was supported by the National Key Research and Development Program of China (2021YFD1800400), the National Natural Science Foundation of China (31872480), the Jiangsu Agriculture Science and Technology Innovation Fund, China (CX(19)2020), and the Priority Academic Program Development of Jiangsu Higher Education Institutions, China (PAPD).

摘要/Abstract

摘要：

目的：马链球菌兽疫亚种（Streptococcus equi subsp. zooepidemicus，SEZ）是一种人畜共患病病原，在我国主要引起猪链球菌病。本实验室前期研究发现了一株源于强毒株SEZ ATCC35246自然变异的弱毒株M35246。M35246表现为一个连续25基因的丢失和covS基因的功能丧失性突变。这是第一次发现在SEZ中的涉及covS的自然变异。涉及covS的自然变异是增强化脓链球菌致病性的关键，所以需要确定covS的自然变异是否对SEZ毒力具有相同的影响。本工作的目的是研究CovS在SEZ毒力形成中的作用，有助于研究SEZ的致病机制，特别是涉及SEZ毒力的转录调控机制。

方法：本研究通过转录组测序和DNA测序，确定了M35246中covS的碱基突变形式。在野生强毒株ATCC35246的基础上分别构建了25基因敲除株ΔPI和covS突变株McovS及对应的互补株。随后，本研究检测了ATCC35246、M35246、M35246 CcovS、McovS、CMcovS、ΔPI的生长能力、对上皮细胞HEp-2的黏附能力、对巨噬细胞Raw264.7的抗吞噬能力以及菌体荚膜含量；测定了ATCC35246、M35246、McovS、ΔPI对24种抗生素的敏感性、对小鼠的半数致死量和攻毒后的菌体脏器分布；进行了ATCC35246、M35246、McovS的比较转录组学分析。

结果：M35246中covS的变异导致其移码突变并造成提前翻译终止，且在基因N端形成终止子结构，遏制其转录。与ATCC35246相比，M35246和McovS的荚膜含量和抗吞噬能力显著降低。McovS对β-内酰胺类、氨基糖苷类、大环内酯类和林可酰胺类药物的敏感性显著高于ATCC35246。与ATCC35246相比，M35246、McovS和ΔPI对小鼠的半数致死量分别增加了10⁵、10⁵和5倍。用约2000倍ATCC35246半数致死量的剂量攻毒48小时后，M35246和McovS均不能从小鼠体内分离。转录组分析表明，McovS和ATCC35246之间存在668个显著差异表达的基因。相对于ATCC35246，McovS中与抗吞噬、荚膜形成、致病性和抗生素抗性有关的许多毒力因子编码基因和合成代谢相关基因显著下调。

结论：本文系统研究了SEZ CovS在细菌抗吞噬作用、荚膜形成、致病性、抗生素耐药性以及对各种重要毒力因子和关键代谢系统转录调控的作用。此外，转录组分析揭示了CovS在抗吞噬作用、荚膜形成、致病性和抗生素耐药性方面的调节机制。

创新性：该工作系统研究了参与SEZ致病性和抗生素耐药的调控因子，表明二元调控系统在不同细菌中调控的多样性，揭示CovS在SEZ毒力形成中起着至关重要的作用。

Abstract:

Streptococcus equi subsp. zooepidemicus (SEZ) is an important zoonotic agent. Here, a virulence-attenuated strain M35246 derived from natural variation of wild-type SEZ ATCC35246 was found. M35246 showed a deletion of 25 contiguous genes as well as a loss-of-function mutation in covS. Subsequently, a 25-gene-deleted strain (ΔPI), a covS-mutant strain (McovS), and relevant complementary strains were constructed and investigated. M35246 and McovS were significantly less encapsulated and exhibited poorer anti-phagocytic capacity compared to wild-type SEZ. McovS was significantly more sensitive to β-lactams, aminoglycosides, macrolides, and lincosamides than wild-type SEZ. M35246, McovS, and ΔPI exhibited an increase in median lethal dose (LD₅₀) in mice by 10⁵, 10⁵, and 5 times when compared to wild-type SEZ, respectively. Neither M35246 nor McovS were isolated from mice 48 h after being challenged with approximately 2 000 times the LD₅₀ of wild-type SEZ. Transcriptome analysis showed that 668 significantly differentially expressed genes existed between McovS and wild-type SEZ. Numerous virulence factor-encoding genes and anabolic-related genes in McovS that were involved in anti-phagocytosis, capsule formation, pathogenicity, and antibiotic resistance were downregulated significantly relative to the wild-type strain. This study revealed that the CovS plays a vital role in the establishment of SEZ virulence

Key words: Streptococcus equi subsp. zooepidemicus , covS , natural variation , virulence , regulation

. JIA-2021-2061 CovS在马链球菌兽疫亚种毒力形成中的作用研究[J]. Journal of Integrative Agriculture, 2023, 22(2): 568-584.

XU Bin, MA Zhe, ZHOU Hong, LIN Hui-xing, FAN Hong-jie. The vital role of CovS in the establishment of Streptococcus equi subsp. zooepidemicus virulence[J]. Journal of Integrative Agriculture, 2023, 22(2): 568-584.

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JIA-2021-2061 CovS在马链球菌兽疫亚种毒力形成中的作用研究

The vital role of CovS in the establishment of Streptococcus equi subsp. zooepidemicus virulence

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