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Special Issue:
细菌耐药性及兽药相关Bacterial resistance & Veterinary drug
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| The vital role of CovS in the establishment of Streptococcus equi subsp. zooepidemicus virulence |
| XU Bin1, 2, MA Zhe1, 3, ZHOU Hong1, LIN Hui-xing1, FAN Hong-jie1, 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
2 Key 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
3 Jiangsu Co-innovation Center for the Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou University, Yangzhou 225009, P.R.China
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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
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Received: 07 December 2021
Accepted: 21 April 2022
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| Fund:
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).
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| About author: XU Bin, E-mail: xubin9999@foxmail.com; Correspondence FAN Hong-jie, Tel/Fax: +86-25-84399592, E-mail: fhj@njau.edu.cn |
Cite this article:
XU Bin, MA Zhe, ZHOU Hong, LIN Hui-xing, FAN Hong-jie.
2023.
The vital role of CovS in the establishment of Streptococcus equi subsp. zooepidemicus virulence. Journal of Integrative Agriculture, 22(2): 568-584.
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Anders S, Huber W. 2010. Differential expression analysis for sequence count data. Genome Biology, 11, R106.
Anders S, Pyl P T, Huber W. 2015. HTSeq - A Python framework to work with high-throughput sequencing data. Bioinformatics, 31, 166–169.
Anzaldi L L, Skaar E P. 2010. Overcoming the heme paradox: Heme toxicity and tolerance in bacterial pathogens. Infection and Immunity, 78, 4977–4989.
Bao Y J, Liang Z, Mayfield J A, Lee S W, Ploplis V A, Castellino F J. 2015. CovRS-regulated transcriptome analysis of a hypervirulent M23 strain of group A Streptococcus pyogenes provides new insights into virulence determinants. Journal of Bacteriology, 197, 3191–3205.
Bisgaard M, Bojesen A M, Petersen M R, Christensen H. 2012. A major outbreak of Streptococcus equi subsp. zooepidemicus infections in free-range chickens is linked to horses. Avian Diseases, 56, 561–566.
Blumenkrantz N, Asboe-Hansen G. 1973. New method for quantitative determination of uronic acids. Analytical Biochemistry, 54, 484–489.
Boschwitz J S, Timoney J F. 1994. Characterization of the antiphagocytic activity of equine fibrinogen for Streptococcus equi subsp. equi. Microbial Pathogenesis, 17, 121.
Causey R C, Artiushin S C, Crowley I F, Weber J A, Homola A D, Kelley A, Stephenson L A, Opitz H M, Guilmain S, Timoney J F. 2010. Immunisation of the equine uterus against Streptococcus equi subspecies zooepidemicus using an intranasal attenuated Salmonella vector. Veterinary Journal, 184, 156–161.
Choby J E, Skaar E P. 2016. Heme synthesis and acquisition in bacterial pathogens. Journal of Molecular Biology, 428, 3408–3428.
Dmitriev A, Mohapatra S S, Chong P, Neely M, Biswas S, Biswas I. 2011. CovR-controlled global regulation of gene expression in Streptococcus mutans. PLoS ONE, 6, e20127.
Eyre D W, Kenkre J S, Bowler I C, McBride S J. 2010. Streptococcus equi subspecies zooepidemicus meningitis - A case report and review of the literature. European Journal of Clinical Microbiology and Infectious Diseases, 29, 1459–1463.
Fan H J, Tang F Y, Mao Y, Lu C P. 2009. Virulence and antigenicity of the szp-gene deleted Streptococcus equi ssp. zooepidemicus mutant in mice. Vaccine, 27, 56–61.
Feng Z. 1977. Outbreak of swine streptococcosis in Sichan province and identification of pathogen. Animal Husbandry Veternary Medicine Letter, 2, 7–12.
Garcia A F, Abe L M, Erdem G, Cortez C L, Kurahara D, Yamaga K. 2010. An insert in the covS gene distinguishes a pharyngeal and a blood isolate of Streptococcus pyogenes found in the same individual. Microbiology, 156, 3085–3095.
Ghosh D. 2012. Incorporating the empirical null hypothesis into the Benjamini-Hochberg procedure. Statistical Applications in Genetics and Molecular Biology, 4, 11.
Gotoh Y, Eguchi Y, Watanabe T, Okamoto S, Doi A, Utsumi R. 2010. Two-component signal transduction as potential drug targets in pathogenic bacteria. Current Opinion in Microbiology, 13, 232–239.
Graham M R, Smoot L M, Migliaccio C A, Virtaneva K, Sturdevant D E, Porcella S F, Federle M J, Adams G J, Scott J R, Musser J M. 2002. Virulence control in group A Streptococcus by a two-component gene regulatory system: Global expression profiling and in vivo infection modeling. Proceedings of the National Academy of Sciences of the United States of America, 99, 13855–13860.
Hampshire J B, Waibel A H. 1990. A novel objective function for improved phoneme recognition using time-delay neural networks. IEEE Transactions on Neural Networks, 1, 216–228.
Han H, Liu C, Wang Q, Xuan C, Zheng B, Tang J, Yan J, Zhang J, Li M, Cheng H. 2012. The two-component system Ihk/Irr contributes to the virulence of Streptococcus suis serotype 2 strain 05ZYH33 through alteration of the bacterial cell metabolism. Microbiology, 158, 1852–1866.
Holden M T G, Heather Z, Paillot R, Steward K F, Webb K, Ainslie F, Jourdan T, Bason N C, Holroyd N E, Mungall K. 2009. Genomic evidence for the evolution of Streptococcus equi: host restriction, increased virulence, and genetic exchange with human pathogens. PLoS Pathogens, 5, e1000346.
Horstmann N, Sahasrabhojane P, Saldaña M, Ajami N J, Flores A R, Sumby P, Liu C G, Yao H, Su X, Thompson E, Shelburne S A. 2015. Characterization of the effect of the histidine kinase CovS on response regulator phosphorylation in group A Streptococcus. Infection and Immunity, 83, 1068–1077.
Ikebe T, Ato M, Matsumura T, Hasegawa H, Sata T, Kobayashi K, Watanabe H. 2010. Highly frequent mutations in negative regulators of multiple virulence genes in group A streptococcal toxic shock syndrome isolates. PLoS Pathogen, 6, e1000832.
Jenkins A, Cote C, Twenhafel N, Merkel T, Bozue J, Welkos S. 2011. Role of purine biosynthesis in Bacillus anthracis pathogenesis and virulence. Infection and Immunity, 79, 153–166.
Kerdsin A, Chopjitt P, Hatrongjit R, Boueroy P, Gottschalk M. 2021. Zoonotic infection and clonal dissemination of Streptococcus equi subspecies zooepidemicus sequence type 194 isolated from humans in Thailand. Transboundary and Emerging Diseases, 69, e554–e565.
Kuusi M, Lahti E, Virolainen A, Hatakka M, Vuento R, Rantala L, Vuopio-Varkila J, Seuna E, Karppelin M, Hakkinen M, Takkinen J, Gindonis V, Siponen K, Huotari K. 2006. An outbreak of Streptococcus equi subspecies zooepidemicus associated with consumption of fresh goat cheese. BMC Infectious Diseases, 6, 36.
Li J, Tan C, Zhou Y, Fu S, Hu L, Hu J, Chen H, Bei W. 2011. The two-component regulatory system CiaRH contributes to the virulence of Streptococcus suis 2. Veterinary Microbiology, 148, 99.
Li M, Wang C, Feng Y, Pan X, Cheng G, Wang J, Ge J, Zheng F, Cao M, Dong Y. 2008. SalK/SalR, a two-component signal transduction system, is essential for full virulence of highly invasive Streptococcus suis serotype 2. PLoS ONE, 3, e2080.
Li Q, Zhang Y H, Du D C, Yu Y F, Zhang W. 2018. Characterization and functional analysis of PnuC that is involved in the oxidative stress tolerance and virulence of Streptococcus suis serotype 2. Veterinary Microbiology, 216, 198–206.
Lieberman H R. 1983. Estimating LD50 using the probit technique: A BASIC computer program. Drug and Chemical Toxicology, 6, 111–116.
Livak K J, Schmittgen T D. 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2(–delta delta C(T)) method. Methods, 25, 402–408.
Lobel L, Sigal N, Borovok I, Ruppin E, Herskovits A A. 2012. Integrative genomic analysis identifies isoleucine and CodY as regulators of Listeria monocytogenes virulence. PLoS Genetics, 8, e1002887.
Ma Z, Geng J N, Yi L, Xu B, Jia R Y, Li Y, Meng Q S, Fan H J, Hu S N. 2013. Insight into the specific virulence related genes and toxin-antitoxin virulent pathogenicity islands in swine streptococcosis pathogen Streptococcus equi ssp. zooepidemicus strain ATCC35246. BMC Genomics, 14, 1–14.
Masuno K, Okada R, Zhang Y, Isaka M, Tatsuno I, Shibata S, Hasegawa T. 2014. Simultaneous isolation of emm89-type Streptococcus pyogenes strains with a wild-type or mutated covS gene from a single streptococcal toxic shock syndrome patient. Journal of Medical Microbiology, 63, 504–507.
McClure R, Balasubramanian D, Sun Y, Bobrovskyy M, Sumby P, Genco C A, Vanderpool C K, Tjaden B. 2013. Computational analysis of bacterial RNA-Seq data. Nucleic Acids Research, 41, e140.
Meijerink M, Ferrando M L, Lammers G, Taverne N, Smith H E, Wells J M. 2012. Immunomodulatory effects of Streptococcus suis capsule type on human dendritic cell responses, phagocytosis and intracellular survival. PLoS ONE, 7, e35849.
Minami M, Kamimura T, Isaka M, Tatsuno I, Ohta M, Hasegawa T. 2010. Clindamycin-induced CovS-mediated regulation of the production of virulent exoproteins streptolysin O, NAD glycohydrolase, and streptokinase in Streptococcus pyogenes. Antimicrobial Agents and Chemotherapy Journal Homepage, 54, 98–102.
Nygaard T K, Blouin G C, Liu M, Fukumura M, Olson J S, Fabian M, Dooley D M, Lei B. 2006a. The mechanism of direct heme transfer from the streptococcal cell surface protein Shp to HtsA of the HtsABC transporter. Journal of Biological Chemistry, 281, 20761–20771.
Nygaard T K, Liu M, McClure M J, Lei B. 2006b. Identification and characterization of the heme-binding proteins SeShp and SeHtsA of Streptococcus equi subspecies equi. BMC Microbiology, 6, 82.
Pan X Z, Ge J C, Ming L, Bo W, Wang C J, Jing W, Feng Y J, Yin Z M, Feng Z, Gong C. 2009. The orphan response regulator CovR: A globally negative modulator of virulence in Streptococcus suis serotype 2. Journal of Bacteriology, 191, 2601–2612.
Redlich S, Ribes S, Schütze S, Czesnik D, Nau R. 2012. Palmitoylethanolamide stimulates phagocytosis of Escherichia coli K1 and Streptococcus pneumoniae R6 by microglial cells. Journal of Neuroimmunology, 244, 32–34.
Takamatsu D, Osaki M, Sekizaki T. 2001a. Construction and characterization of Streptococcus suis–Escherichia coli shuttle cloning vectors. Plasmid, 45, 101–113.
Takamatsu D, Osaki M, Sekizaki T. 2001b. Thermosensitive suicide vectors for gene replacement in Streptococcus suis. Plasmid, 46, 140–148.
Tatsuno I, Okada R, Zhang Y, Isaka M, Hasegawa T. 2013. Partial loss of CovS function in Streptococcus pyogenes causes severe invasive disease. BMC Research Notes, 6, 126.
Velineni S, Timoney J F. 2013. Characterization and protective immunogenicity of the SzM protein of Streptococcus zooepidemicus NC78 from a clonal outbreak of equine respiratory disease. Clinical and Vaccine Immunology, 20, 1181–1188.
Watanabe T, Okada A, Gotoh Y, Utsumi R. 2008. Inhibitors targeting two-component signal transduction. Advances in Experimental Medicine and Biology, 631, 229–236.
Wei Z G, Fu Q, Chen Y S, Cong P Q, Xiao S Q, Mo D L, He Z Y, Liu X H. 2012. The capsule of Streptococcus equi ssp. zooepidemicus is a target for attenuation in vaccine development. Vaccine, 30, 4670–4675.
Xie C, Mao X, Huang J, Ding Y, Wu J, Dong S, Kong L, Gao G, Li C Y, Wei L. 2011. KOBAS 2.0: A web server for annotation and identification of enriched pathways and diseases. Nucleic Acids Research, 39, W316–W322.
Xu B, Pei X M, Su Y Q, Ma Z, Fan H J. 2016a. Capsule of Streptococcus equi subsp. zooepidemicus hampers the adherence and invasion of epithelial and endothelial cells and is attenuated during internalization. FEMS Microbiology Letter, 363, fnw164.
Xu B, Yang X Y, Zhang P, Ma Z, Lin H X, Fan H. 2016b. The arginine deiminase system facilitates environmental adaptability of Streptococcus equi ssp. zooepidemicus through pH adjustment. Research in Microbiology, 167, 403–412.
Xu B, Zhang P, Zhou H, Sun Y, Tang J S, Fan H J. 2019. Identification of novel genes associated with anti-phagocytic functions in Streptococcus equi subsp. zooepidemicus. Veterinary Microbiology, 233, 28–38.
Xu J, Fu S L, Liu M L, Xu Q X, Bei W C, Chen H C, Tan C. 2014. The two-component system NisK/NisR contributes to the virulence of Streptococcus suis serotype 2. Microbiological Research, 169, 541–546.
Yi L, Wang Y, Ma Z, Zhang H, Li Y, Zheng J X, Yang Y C, Lu C P, Fan H J. 2013a. Contribution of fibronectin-binding protein to pathogenesis of Streptococcus equi ssp. zooepidemicus. Pathogens and Disease, 67, 174–183.
Yi L, Wang Y, Ma Z, Zhang H, Xie H D, Yang Y C, Lu C P, Fan H J. 2013b. Identification of genes transcribed by Streptococcus equi ssp. zooepidemicus in infected porcine lung. Microbial Pathogenesis, 59–60, 7–12.
Zheng J X, Li Y, Zhang H, Fan H J, Lu C P. 2013. Identification and characterization of a novel hemolysis-related gene in Streptococcus suis serotype 2. PLoS ONE, 8, e74674.
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