Chun FANG, Xiaowei FANG, Yu ZHANG, Chen WANG, Xiongyan LIANG, Yufang GU, Yuying YANG, Wei HU, Qingping LUO, Hui WU

Listeria monocytogenes’in Patojenitesinde Histidin Kinaz Geni yycG’nin Rolü

Gıda kaynaklı fırsatçı bir patojen olan Listeria monocytogenes, çevresel koşullara tepki olarak kendi özelliklerini başarılı bir şekilde değiştirebilir. L. monocytogenes’te, onaltı adet iki bileşenli sistemin (TCS) bakterilerin çeşitli durumları algılamasına ve adaptasyonuna katkıda bulunduğu belirlenmiştir. Önceki genomik çalışmamız, on altı TCS’den Lm850658 ve M7 arasındaki histidin kinaz gen yycG’de mutasyon gözlendiğini göstermiştir. YycFG TCS’nin diğer birçok Gram pozitif bakteride önemli roller oynadığı belirlenmiştir. Oysa L. monocytogenes’teki YycFG TCS’nin rolü hala tam olarak bilinmemektedir. Bu çalışmada, yycG’nin L. monositogenes’in patojenisitesinde rol oynayıp oynamadığını belirlemeyi amaçladık. Yaban tipi Lm850658 suşunu ve tamamlayıcı bir suşu (CΔyycG) temel alan bir histidin kinaz geni yycG silme suşu (ΔyycG) oluşturduk. YycG’nin silinmesinin büyüme kabiliyetini ve hareketliliğini önemli ölçüde bozduğunu, ancak in vitro hemoliz yeteneğini artırdığını bulduk. Ek olarak, hücre ve fare modelindeki enfeksiyon deneyleri, ΔyycG’nin enfeksiyon yeteneği ve virülansta önemli ölçüde kusurlu olduğunu gösterdi. ΔyycG’nin tüm bu fenotipleri, gen tamamlaması ile büyük ölçüde yaban tipi Lm850658 suşunun düzeyine tersine çevrilebilir. Hücre duvarı ilişkili ve salgılanan proteinler analizi, ΔyycG’nin salgılanan miktarının önemli ölçüde arttığını gösterdi. Western blot analizi, ΔyycG’nin salgılanan fraksiyonlarında; sırasıyla azalmış yapışma ve invazyon yeteneğinden ve artmış hemolitik aktiviteden sorumlu olabilecek Internalin protein B (InlB) ve Listerolysin O’nun (LLO) belirgin şekilde arttığını ortaya koydu. Histidin kinaz yycG’nin L. monocytogenes patojenitesinde önemli roller oynadığı ilk kez belirlendi. YycFG TCS’nin büyümeyi, hareketliliği, hücre yüzeyi proteinlerinin translokasyonunu ve virülansı nasıl değiştirdiğini belirlemek için ileri araştırmalara ihtiyaç vardır.

Roles of Histidine Kinase Gene yycG in the Pathogenicity of Listeria monocytogenes

As an opportunistic foodborne pathogen, Listeria monocytogenes could successfully switch self-characteristics in response to environmentalconditions. In L. monocytogenes, sixteen two-component systems (TCSs) have been found to contribute the bacteria to sensing and adapting tovarious conditions. Our previous genomic study showed that mutation was observed in the histidine kinase gene yycG between Lm850658 andM7 among the sixteen TCSs. The YycFG TCS was found playing important roles in many other Gram-positive bacteria. While the roles of YycFG TCSin L. monocytogenes remain poorly known. In this study, we aimed to determine whether yycG play roles in pathogenicity of L. monocytogenes. Wecreated a histidine kinase gene yycG deletion strain (ΔyycG) based on the wild type strain Lm850658 and a complemental strain (CΔyycG). We foundthe yycG deletion significantly impaired the growth ability and mobility, but enhanced the hemolysis ability in vitro. In addition, infection assays oncell and mice model showed that ΔyycG exhibited significantly defected in infection ability and virulence. All these phenotypes of the ΔyycG could bereversed largely to the levels of the wild type strain Lm850658 by gene complementation. Cell wall-associated and secreted protein analysis showedthat the secreted content of ΔyycG was significantly increased. And western blotting revealed that Internalin protein B (InlB) and Listerolysin O (LLO)was markedly increased in the secreted fractions of ΔyycG, which might be responsible for decreased adhesion and invasion ability and increasedhemolytic activity, respectively. Overall, we found the histidine kinase yycG played important roles in pathogenicity of L. monocytogenes for the firsttime. Further investigation is needed to explore how the YycFG TCS modulates the growth, mobility, cell surface proteins translocation and virulence.

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1. Pizarro-Cerda J, Cossart P: Listeria monocytogenes: Cell biology of invasion and intracellular growth. Microbiol Spectr, 6 (6), 6(6):GPP3-0013- 2018, 2018. DOI: 10.1128/microbiolspec.GPP3-0013-2018
2. Freitag NE, Port GC, Miner MD: Listeria monocytogenes - from saprophyte to intracellular pathogen. Nat Rev Microbiol, 7 (9): 623-628, 2009. DOI: 10.1038/nrmicro2171
3. Radoshevich L, Cossart P: Listeria monocytogenes: Towards a complete picture of its physiology and pathogenesis. Nat Rev Microbiol, 16 (1): 32- 46, 2018. DOI: 10.1038/nrmicro.2017.126
4. Pizarro-Cerda J, Kuhbacher A, Cossart P: Entry of Listeria monocytogenes in mammalian epithelial cells: An updated view. Cold Spring Harb Perspect Med, 2 (11):a010009, 2012. DOI: 10.1101/cshperspect.a010009
5. Fang C, Cao T, Cheng C, Xia Y, Shan Y, Xin Y, Guo N, Li X, Song H, Fang W: Activation of PrfA results in overexpression of virulence factors but does not rescue the pathogenicity of Listeria monocytogenes M7. J Med Microbiol, 64, 818-827, 2015. DOI: 10.1099/jmm.0.000101
6. Forster BM, Marquis H: Protein transport across the cell wall of monoderm Gram-positive bacteria. Mol Microbiol, 84 (3): 405-413, 2012. DOI: 10.1111/j.1365-2958.2012.08040.x
7. Desvaux M, Hebraud M: The protein secretion systems in Listeria: inside out bacterial virulence. FEMS Microbiol Rev, 30 (5): 774-805, 2006. DOI: 10.1111/j.1574-6976.2006.00035.x
8. Desvaux M, Hebraud M, Talon R, Henderson IR: Secretion and subcellular localizations of bacterial proteins: A semantic awareness issue. Trends Microbiol, 17 (4): 139-145, 2009. DOI: 10.1016/j.tim.2009.01.004
9. Cahoon LA, Freitag NE: Listeria monocytogenes virulence factor secretion: Don’t leave the cell without a chaperone. Front Cell Infect Microbiol, 4: 13, 2014. DOI: 10.3389/fcimb.2014.00013
10. Scott JR, Barnett TC: Surface proteins of gram-positive bacteria and how they get there. Annu Rev Microbiol, 60, 397-423, 2006. DOI: 10.1146/ annurev.micro.60.080805.142256
11. Stock AM, Robinson VL, Goudreau PN: Two-component signal transduction. Annu Rev Biochem, 69, 183-215, 2000. DOI: 10.1146/annurev. biochem.69.1.183
12. Williams T, Bauer S, Beier D, Kuhn M: Construction and characterization of Listeria monocytogenes mutants with in-frame deletions in the response regulator genes identified in the genome sequence. Infect Immun, 73 (5): 3152-3159, 2005. DOI: 10.1128/IAI.73.5.3152-3159.2005
13. Xu T, Wu Y, Lin Z, Bertram R, Gotz F, Zhang Y, Qu D: Identification of genes controlled by the essential YycFG two-component system reveals a role for biofilm modulation in Staphylococcus epidermidis. Front Microbiol, 8:724, 2017. DOI: 10.3389/fmicb.2017.00724
14. Iwata Y, Satou K, Tsuzuku H, Furuichi K, Senda Y, Sakai-Takemori Y, Wada T, Fujita S, Miyake T, Yasuda H, Sakai N, Kitajima S, Toyama T, Shinozaki Y, Sagara A, Miyagawa T, Hara A, Shimizu M, Kamikawa Y, Kaneko S, Wada T: Down-regulation of the two-component system and cell-wall biosynthesis-related genes was associated with the reversion to daptomycin susceptibility in daptomycin non-susceptible methicillinresistant Staphylococcus aureus. Eur J Clin Microbiol Infect Dis, 36 (10): 1839-1845, 2017. DOI: 10.1007/s10096-017-2999-3
15. Mohedano ML, Amblar M, de la Fuente A, Wells JM, Lopez P: The Response regulator yycf inhibits expression of the fatty acid biosynthesis repressor FabT in Streptococcus pneumoniae. Front Microbiol, 7:1326, 2016. DOI: 10.3389/fmicb.2016.01326
16. Liu H, Zhao Y, Zhao D, Gong T, Wu Y, Han H, Xu T, Peschel A, Han S, Qu D: Antibacterial and anti-biofilm activities of thiazolidione derivatives against clinical staphylococcus strains. Emerg Microbes Infect, 4 (1):e1, 2015. DOI: 10.1038/emi.2015.1
17. Vidaillac C, Gardete S, Tewhey R, Sakoulas G, Kaatz GW, Rose WE, Tomasz A, Rybak MJ: Alternative mutational pathways to intermediate resistance to vancomycin in methicillin-resistant Staphylococcus aureus. J Infect Dis, 208 (1): 67-74, 2013. DOI: 10.1093/infdis/jit127
18. Fukushima T, Furihata I, Emmins R, Daniel RA, Hoch JA, Szurmant H: A role for the essential YycG sensor histidine kinase in sensing cell division. Mol Microbiol, 79 (2): 503-522, 2011. DOI: 10.1111/j.1365- 2958.2010.07464.x
19. Pontinen A, Markkula A, Lindstrom M, Korkeala H: Two-componentsystem histidine kinases involved in growth of Listeria monocytogenes EGD-e at low temperatures. Appl Environ Microbiol, 81 (12): 3994-4004, 2015. DOI: 10.1128/AEM.00626-15
20. Fang C, Fang X, Chen X, Wang C, Liang X, Gu Y, Fang W, Yang Y: Evaluating the contribution of acid resistance systems and probing the different roles of the glutamate decarboxylases of Listeria monocytogenes under acidic conditions. Kafkas Univ Vet Fak Derg, 26 (2): 231-238, 2020. DOI: 10.9775/kvfd.2019.22784
21. Cheng C, Jiang L, Ma T, Wang H, Han X, Sun J, Yang Y, Chen Z, Yu H, Hang Y, Liu F, Wang B, Fang W, Huang H, Fang C, Cai C, Freitag N, Song H: Carboxyl-terminal residues n478 and v479 required for the cytolytic activity of listeriolysin o play a critical role in Listeria monocytogenes pathogenicity. Front Immunol, 8:1439, 2017. DOI: 10.3389/ fimmu.2017.01439
22. Fang C, Cao T, Shan Y, Xia Y, Xin Y, Cheng C, Song H, Bowman J, Li X, Zhou X, Fang W: Comparative genomic analysis reveals that the 20K and 38K prophages in Listeria monocytogenes serovar 4a strains Lm850658 and M7 contribute to genetic diversity but not to virulence. J Microbiol Biotechnol, 26 (1): 197-206, 2016. DOI: 10.4014/jmb.1504.04075
23. Fang C, Chen X, Liang X, Fang X, Gao K, Chen J, Gu Y, Yang Y: The Effect of single amino acid substitution in SecA2 on protein translocation and pathogenicity of Listeria monocytogenes. Kafkas Univ Vet Fak Derg, 25 (5): 665-672, 2019. DOI: 10.9775/kvfd.2018.21558
24. Pontinen A, Lindstrom M, Skurnik M, Korkeala H: Screening of the two-component-system histidine kinases of Listeria monocytogenes EGD-e. LiaS is needed for growth under heat, acid, alkali, osmotic, ethanol and oxidative stresses. Food Microbiol, 65, 36-43, 2017. DOI: 10.1016/j. fm.2017.01.018
25. Cemma M, Lam GY, Stockli M, Higgins DE, Brumell JH: Strainspecific interactions of Listeria monocytogenes with the autophagy system in host cells. PLoS One, 10 (5): e0125856, 2015. DOI: 10.1371/journal. pone.0125856
26. Bisicchia P, Noone D, Lioliou E, Howell A, Quigley S, Jensen T, Jarmer H, Devine KM: The essential YycFG two-component system controls cell wall metabolism in Bacillus subtilis. Mol Microbiol, 65 (1): 180- 200, 2007. DOI: 10.1111/j.1365-2958.2007.05782.x
27. Campeotto I, Percy MG, MacDonald JT, Forster A, Freemont PS, Grundling A: Structural and mechanistic insight into the Listeria monocytogenes two-enzyme lipoteichoic acid synthesis system. J Biol Chem, 289 (41): 28054-28069, 2014. DOI: 10.1074/jbc.M114.590570
28. Osborne SE, Brumell JH: Listeriolysin O: From bazooka to Swiss army knife. Philos Trans R Soc Lond B Biol Sci, 372:20160222, 2017. DOI: 10.1098/ rstb.2016.0222
29. Han X, Yu R, Ji L, Zhen D, Tao S, Li S, Sun Y, Huang L, Feng Z, Li X, Han G, Schmidt M, Han L: InlB-mediated Listeria monocytogenes internalization requires a balanced phospholipase D activity maintained through phospho-cofilin. Mol Microbiol, 81 (4): 860-880, 2011. DOI: 10.1111/ j.1365-2958.2011.07726.x
30. Mungunsukh O, Lee YH, Marquez AP, Cecchi F, Bottaro DP, Day RM: A tandem repeat of a fragment of Listeria monocytogenes internalin B protein induces cell survival and proliferation. Am J Physiol Lung Cell Mol Physiol, 299 (6): L905-L914, 2010. DOI: 10.1152/ajplung.00094.2010
31. Vadia S, Arnett E, Haghighat AC, Wilson-Kubalek EM, Tweten RK, Seveau S: The pore-forming toxin listeriolysin O mediates a novel entry pathway of L. monocytogenes into human hepatocytes. PLoS Pathog, 7 (11): e1002356, 2011. DOI: 10.1371/journal.ppat.1002356
32. Nguyen BN, Peterson BN, Portnoy DA: Listeriolysin O: A phagosomespecific cytolysin revisited. Cell Microbiol, 21 (3): e12988, 2019. DOI: 10.1111/cmi.12988
33. Johansson J, Freitag NE: Regulation of Listeria monocytogenes virulence. Microbiol Spectr, 7 (4): GPP3-0064-2019, 2019. DOI: 10.1128/ microbiolspec.GPP3-0064-2019
34. Schnupf P, Hofmann J, Norseen J, Glomski IJ, Schwartzstein H, Decatur AL: Regulated translation of listeriolysin O controls virulence of Listeria monocytogenes. Mol Microbiol, 61 (4): 999-1012, 2006. DOI: 10.1111/j.1365-2958.2006.05286.x
35. Lety MA, Frehel C, Dubail I, Beretti JL, Kayal S, Berche P, Charbit A: Identification of a PEST-like motif in listeriolysin O required for phagosomal escape and for virulence in Listeria monocytogenes. Mol Microbiol, 39 (5): 1124-1139, 2001. DOI: 10.1111/j.1365-2958.2001.02281.x