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Journal of Integrative Agriculture  2025, Vol. 24 Issue (12): 4760-4775    DOI: 10.1016/j.jia.2024.07.009
Animal Science · Veterinary Medicine Advanced Online Publication | Current Issue | Archive | Adv Search |
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 Pei 2, Hongjie Fan2, 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

 Highlights 
DnaK is required for SS2 to be antiphagocytosed by macrophages, enhancing bacterial pathogenicity.
DnaK could enter RAW264.7 cells, decreasing the mRNA and protein levels of SS2 endocytic receptor LRP1.
DnaK decreases LRP1 through PPARγ regulation and ubiquitin–proteasome system degradation. 

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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功能。前期本实验室SS2转座子突变文库中筛选到一株抗吞噬能力减弱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DnaKSS2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DnaKSS2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。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LRP1mRNA和蛋白水平。敲低LRP1 β(LRP1β)可显著降低巨噬细胞RAW264.7对SS2菌株ZY05719的吞噬率,提示LRP1SS2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.
Keywords:  Streptococcus suis serotype 2       DnaK        phagocytosis resistance        macrophages        LRP1  
Received: 20 February 2024   Accepted: 22 May 2024 Online: 06 July 2024  
Fund: 
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).
About author:  Qing Wang, E-mail: wangqing202008@ 163.com; #Correspondence Hongjie Fan, Tel: +86-25-84399592, E-mail: fhj@njau.edu.cn

Cite this article: 

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

Benga L, Fulde M, Neis C, Goethe R, Valentin-Weigand P. 2008. Polysaccharide capsule and suilysin contribute to extracellular survival of Streptococcus suis co-cultivated with primary porcine phagocytes. Veterinary Microbiology, 132, 211–219.

Bres E E, Faissner A. 2019. Low density receptor-related protein 1 interactions with the extracellular matrix: More than meets the eye. Frontiers in Cell and Developmental Biology7, 31.

Le Breton Y, Mistry P, Valdes K M, Quigley J, Kumar N, Tettelin H, McIver K S. 2013. Genome-wide identification of genes required for fitness of group A Streptococcus in human blood. Infection and Immunity81, 862–875.

Brown E J. 1986. The role of extracellular matrix proteins in the control of phagocytosis. Journal of Leukocyte Biology39, 579–591.

Castanie-Cornet M P, Bruel N, Genevaux P. 2014. Chaperone networking facilitates protein targeting to the bacterial cytoplasmic membrane. Biochimica et Biophysica Acta1843, 1442–1456.

Ceschin, D G, Sanchez M C, Chiabrando G A. 2009. Insulin induces the low density lipoprotein receptor-related protein 1 (LRP1) degradation by the proteasomal system in J774 macrophage-derived cells. Journal of Cellular Biochemistry106, 372–380.

Costales P, Castellano J, Revuelta-Lopez E, Cal R, Aledo R, Llampayas O, Nasarre L, Juarez C, Badimon L, Llorente-Cortes V. 2013. Lipopolysaccharide downregulates CD91/low-density lipoprotein receptor-related protein 1 expression through SREBP-1 overexpression in human macrophages. Atherosclerosis, 227, 79–88.

Emonard H, Theret L, Bennasroune A H, Dedieu S. 2014. Regulation of LRP-1 expression: Make the point. Pathologie Biologie62, 84–90.

Fang L, Shen H, Tang Y, Fang W. 2015. Superoxide dismutase of Streptococcus suis serotype 2 plays a role in anti-autophagic response by scavenging reactive oxygen species in infected macrophages. Veterinary Microbiology176, 328–336.

Fay A, Glickman M S. 2014. An essential nonredundant role for mycobacterial DnaK in native protein folding. PLoS Genetics10, e1004516.

Ganaie S S, Schwarz M M, McMillen C M, Price D A, Feng A X, Albe J R, Wang W J, Miersch S, Orvedahl A, Cole A R, Sentmanat M F, Mishra N, Boyles D A, Koenig Z T, Kujawa M R, Demers M A, Hoehl R M, Moyle A B, Wagner N D, Stubbs S H, et al. 2021. Lrp1 is a host entry factor for Rift Valley fever virus. Cell184, 5163–5178.e24.

Gauthier A, Vassiliou G, Benoist F, McPherson R. 2003. Adipocyte low density lipoprotein receptor-related protein gene expression and function is regulated by peroxisome proliferator-activated receptor gamma. The Journal of Biological Chemistry278, 11945–11953.

Griffin J E, Gawronski J D, Dejesus M A, Ioerger T R, Akerley B J, Sassetti C M. 2011. High-resolution phenotypic profiling defines genes essential for mycobacterial growth and cholesterol catabolism. PLoS Pathogens7, e1002251.

Huong V T, Ha N, Huy N T, Horby P, Nghia H D, Thiem V D, Zhu X, Hoa N T, Hien T T, Zamora J, Schultsz C, Wertheim H F, Hirayama K. 2014. Epidemiology, clinical manifestations, and outcomes of Streptococcus suis infection in humans. Emerging Infectioous Diseases20, 1105–1114.

Hyams C, Camberlein E, Cohen J M, Bax K, Brown J S. 2010. The Streptococcus pneumoniae capsule inhibits complement activity and neutrophil phagocytosis by multiple mechanisms. Infection and Immunity78, 704–715.

Kerdsin A. 2022. Human Streptococcus suis infections in Thailand: Epidemiology, clinical features, genotypes, and susceptibility. Tropical Medicine and Infectious Disease7, 359.

Kim J, Kim Y J, Kim J W. 2019. Bacterial clearance is enhanced by alpha 2,3-and alpha 2,6-sialyllactose via receptor-mediated endocytosis and phagocytosis. Infection and Immunity87, e00694–18.

Kim J S, Liu L, Vazquez-Torres A. 2021. The DnaK/DnaJ chaperone system enables RNA polymerase-DksA complex formation in Salmonella experiencing oxidative stress. mBio12, e03443–20.

Laatsch A, Panteli M, Sornsakrin M, Hoffzimmer B, Grewal T, Heeren J. 2012. Low density lipoprotein receptor-related protein 1 dependent endosomal trapping and recycling of apolipoprotein E. PLoS ONE7, e29385.

Lemos J A, Luzardo Y, Burne R A. 2007. Physiologic effects of forced down-regulation of dnaK and groEL expression in Streptococcus mutansJournal of Bacteriology, 189, 1582–1588.

Li Y, Wang J, Liu B B, Yu Y F, Yuan T, Wei Y N, Gan Y, Shao J, Shao G Q, Feng Z X, Tu Z G, Xiong Q Y. 2022. DnaK functions as a moonlighting protein on the surface of Mycoplasma hyorhinis cells. Frontiers in Microbiology13, 842058.

Liu H Z, Fu H, Jiang X W, Liao X Y, Yue M, Li X L, Fang W H. 2019. PrsA contributes to Streptococcus suis serotype 2 pathogenicity by modulating secretion of selected virulence factors. Veterinary Microbiology236, 108375.

Llorente-Cortes V, Costales P, Bernues J, Camino-Lopez S, Badimon L. 2006. Sterol regulatory element-binding protein-2 negatively regulates low density lipoprotein receptor-related protein transcription. Journal of Molecular Biology359, 950–960.

Mantuano E, Brifault C, Lam M S, Azmoon P, Gilder A S, Gonias S L. 2016. LDL receptor-related protein-1 regulates NF kappa B and microRNA–155 in macrophages to control the inflammatory response. Proceedings of the National Academy of Sciences of the United States of America113, 1369–1374.

Mao H, Lockyer P, Li L, Ballantyne C M, Patterson C, Xie L, Pi X. 2017. Endothelial LRP1 regulates metabolic responses by acting as a co-activator of PPARgamma. Nature Communications8, 14960.

May P. 2013. The low-density lipoprotein receptor-related protein 1 in inflammation. Current Opinion in Lipidology24, 134–137.

Mukouhara T, Arimoto T, Cho K, Yamamoto M, Igarashi T. 2011. Surface lipoprotein PpiA of Streptococcus mutans suppresses scavenger receptor MARCO-dependent phagocytosis by macrophages. Infection and Immunity79, 4933–4940.

Normile D. 2005. Infectious diseases. WHO probes deadliness of China’s pig-borne disease. Science309, 1308–1309.

Patel M, Morrow J, Maxfield F R, Strickland D K, Greenberg S, Tabas I. 2003. The cytoplasmic domain of the low density lipoprotein (LDL) receptor-related protein, but not that of the LDL receptor, triggers phagocytosis. The Journal of Biological Chemistry278, 44799–44807.

Pei X, Liu M, Zhou H, Fan H. 2020. Screening for phagocytosis resistance-related genes via a transposon mutant library of Streptococcus suis serotype 2. Virulence11, 825–838.

Pian Y, Gan S, Wang S, Guo J, Wang P, Zheng Y, Cai X, Jiang Y, Yuan Y. 2012. Fhb, a novel factor H-binding surface protein, contributes to the antiphagocytic ability and virulence of Streptococcus suisInfection and Immunity80, 2402–2413.

Pian Y, Wang P, Liu P, Zheng Y, Zhu L, Wang H, Xu B, Yuan Y, Jiang Y. 2015. Proteomics identification of novel fibrinogen-binding proteins of Streptococcus suis contributing to antiphagocytosis. Frontiers in Cellular and Infection Microbiology5, 19.

Rondon Ortiz A N, Cardenas C L L, Martinez Malaga J, Gonzales Urday A L, Gugnani K S, Bohlke M, Maher T J, Pino Figueroa A J. 2017. High concentrations of rosiglitazone reduce mRNA and protein levels of LRP1 in HepG2 cells. Frontiers in Pharmacology8, 772.

Schoellkopf J, Mueller T, Hippchen L, Mueller T, Reuten R, Backofen R, Orth J, Schmidt G. 2022. Genome wide CRISPR screen for Pasteurella multocida toxin (PMT) binding proteins reveals LDL receptor related protein 1 (LRP1) as crucial cellular receptor. PLoS Pathogens18, e1010781.

Schorch B, Song S, van Diemen F R, Bock H H, May P, Herz J, Brummelkamp T R, Papatheodorou P, Aktories K. 2014. LRP1 is a receptor for Clostridium perfringens TpeL toxin indicating a two-receptor model of clostridial glycosylating toxins. Proceedings of the National Academy of Sciences of the United States of America111, 6431–6436.

Schottelndreier D, Langejurgen A, Lindner R, Genth H. 2020. Low density lipoprotein receptor-related protein-1 (LRP1) is involved in the uptake of Clostridioides difficile toxin A and serves as an internalizing receptor. Frontiers in Cellular and Infection Microbiology10, 565465.

Segura M, Fittipaldi N, Calzas C, Gottschalk M. 2017. Critical Streptococcus suis virulence factors: Are they all really critical? Trends in Microbiology25, 585–599.

Segura M, Gottschalk M, Olivier M. 2004. Encapsulated Streptococcus suis inhibits activation of signaling pathways involved in phagocytosis. Infection and Immunity72, 5322–5330.

Singh V K, Syring M, Singh A, Singhal K, Dalecki A, Johansson T. 2012. An insight into the significance of the DnaK heat shock system in Staphylococcus aureusInternational Journal of Medical Microbiology302, 242–252.

Singh V K, Utaida S, Jackson L S, Jayaswal R K, Wilkinson B J, Chamberlain N R. 2007. Role for dnaK locus in tolerance of multiple stresses in Staphylococcus aureusMicrobiology153, 3162–3173.

Sun-Wang J L, Ivanova S, Zorzano A. 2020. The dialogue between the ubiquitin-proteasome system and autophagy: Implications in ageing. Ageing Research Reviews64, 101203.

Takamatsu D, Osaki M, Sekizaki T. 2001. Thermosensitive suicide vectors for gene replacement in Streptococcus suisPlasmid, 46, 140–148.

Tomoyasu T, Tabata A, Imaki H, Tsuruno K, Miyazaki A, Sonomoto K, Whiley R A, Nagamune H. 2012. Role of Streptococcus intermedius DnaK chaperone system in stress tolerance and pathogenicity. Cell Stress & Chaperones17, 41–55.

Wang S, Lyu C, Duan G, Meng F, Yang Y, Yu Y, He X, Wang Z, Gottschalk M, Li G, Cai X, Wang G. 2020. Streptococcus suis Serotype 2 infection causes host immunomodulation through induction of thymic atrophy. Infection and Immunity88, e00950–19.

Xia X J, Qin W H, Zhu H L, Wang X, Jiang J Q, Hu J H. 2019. How Streptococcus suis serotype 2 attempts to avoid attack by host immune defenses. Journal of MicrobiologyImmunology and Infection52, 516–525.

Xie F, Zan Y, Zhang Y, Zheng N, Yan Q, Zhang W, Zhang H, Jin M, Chen F, Zhang X, Liu S. 2019. The cysteine protease ApdS from Streptococcus suis promotes evasion of innate immune defenses by cleaving the antimicrobial peptide cathelicidin LL-37. The Journal of Biological Chemistry, 294, 17962–17977.

Xing P, Liao Z, Ren Z, Zhao J, Song F, Wang G, Chen K, Yang J. 2016. Roles of low-density lipoprotein receptor-related protein 1 in tumors. Chinese Journal of Cancer35, 6.

Xu B, Zhang P, Li W, Liu R, Tang J, Fan H. 2017. hsdS, belonging to the type I restriction-modification system, contributes to the Streptococcus suis serotype 2 survival ability in phagocytes. Frontiers in Microbiology8, 1524.

Yahiro K, Satoh M, Nakano M, Hisatsune J, Isomoto H, Sap J, Suzuki H, Nomura F, Noda M, Moss J, Hirayama T. 2012. Low-density lipoprotein receptor-related protein-1 (LRP1) mediates autophagy and apoptosis caused by Helicobacter pylori VacA. The Journal of Biological Chemistry287, 31104–31115.

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