Chun FANG, Xiaowei FANG, Xueyang CHEN, Chen WANG, Xiongyan LIANG, Yufang GU, Weihuan FANG, Yuying YANG

Evaluating the Contribution of Acid Resistance Systems and Probing the Different Roles of the Glutamate Decarboxylases of Listeria monocytogenes Under Acidic Conditions

Listeria monocytogenes is an important zoonotic foodborne pathogen, which can cause a severe invasive illness to susceptible humans and animals with high mortality. As L. monocytogenes is widely distributed in natural environments, the bacterium is easy to contaminate food processing facilities and the products to be ingested by host. But during the transition from a saprophyte to intracellular pathogen, one of the biggest challenge L. monocytogenes encounters is the acid stress. To combat the acidic environments, the bacterium developed several acid resistance systems, including acid tolerance response (ATR), F0F1-ATPase, glutamate decarboxylase (GAD), arginine deiminase (ADI) and agmatine deiminase (AgDI). In this study, we comprehensively evaluated the contributions of different acid resistance systems and explored the different roles of the three GAD components under acidic conditions. We found that the GadD2 of GAD system made the largest contribution to the survival of L. monocytogenes in artificial gastric juice (AGJ) and acidic brain heart infusion (BHI), which was followed by the global stress regulator SigB, GadD3 of GAD system, AguA1 of AgDI system and ArcA of ADI system. Transcription analysis showed that the mRNA level of the three GADs were consistent with their contribution to acid resistance. Similar results were observed in the other three representative strains EGDe, Lm850658 and M7. We further obtained the purified GADs and their poly-antibodies to demonstrate that the contribution of the three GADs were determined by the protein levels in L. monocytogenes. Further studies are needed to focus on the regulation of different expression of the GAD system.

Asidik Koşullar Altında Listeria monocytogenes’in Glutamat Dekarboksilazlarının Asit Direnç Sistemlerine Katkılarının Değerlendirilmesi ve Farklı Rollerinin Araştırılması

Listeria monocytogenes, duyarlı insan ve hayvanlarda yüksek ölüm oranı ile seyreden bulaşıcı hastalıklara neden olabilen, önemli bir gıda kaynaklı zoonotik patojendir. L. monocytogenes doğal ortamlarda yaygın olarak bulunduğundan, gıda işleme tesislerinin ve konakçı tarafından tüketilen ürünlerin bakteri ile kontaminasyonu kolaydır. Ancak bir saprofitten hücre içi patojene dönüşmesi sırasında, L. monocytogenes’in karşılaştığı en büyük güçlüklerden biri asit stresidir. Asidik ortamlarla savaşmak için, bakteri, asit tolerans yanıtı (ATR), F0F1-ATPase, glutamat dekarboksilaz (GAD), arginin deiminaz (ADI) ve agmatin deiminaz (AgDI) dahil olmak üzere çeşitli asit direnç sistemleri geliştirmiştir. Bu çalışmada, farklı asit direnç sistemlerinin katkıları kapsamlı bir şekilde değerlendirildi ve üç GAD bileşeninin asidik koşullar altında farklı rolleri araştırıldı. GAD sistemindeki GadD2’nin, L. monocytogenes’in yapay mide sıvısı (AGJ) ve asidik beyin kalp infüzyonunda (BHI) hayatta kalmasına en büyük katkıyı yaptığı ve bunu GAD sisteminden global stres regülatörü SigB, GadD3 ile AgDI sisteminden AguA1 ve ADI sisteminden ArcA’nın izlediği belirlendi. Transkripsiyon analizi, üç GAD’nin mRNA seviyesinin, asit direncine katkıları ile tutarlı olduğunu gösterdi. Benzer sonuçlar, diğer üç temsilci suş olan EGDe, Lm850658 ve M7’de de gözlendi. Ayrıca, üç GAD’nin katkısının, L. monocytogenes’teki protein seviyeleri tarafından belirlendiğini göstermek için saflaştırılmış GAD’ler ve bunların poliantikorlarını elde ettik. GAD sisteminin farklı ekspresyonlarının düzenlenme mekanizmasının anlaşılabilmesi için daha fazla çalışmaya ihtiyaç vardır.

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1. 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

2. Chen M, Cheng J, Zhang J, Chen Y, Zeng H, Xue L, Lei T, Pang R, Wu S, Wu H, Zhang S, Wei X, Zhang Y, Ding Y, Wu Q: Isolation, potential virulence, and population diversity of Listeria monocytogenes from meat and meat products in China. Front Microbiol, 10:946, 2019. DOI: 10.3389/ fmicb.2019.00946

3. Roberts AJ, Wiedmann M: Pathogen, host and environmental factors contributing to the pathogenesis of listeriosis. Cell Mol Life Sci, 60 (5): 904918, 2003. DOI: 10.1007/s00018-003-2225-6

4. Scallan E, Hoekstra RM, Angulo FJ, Tauxe RV, Widdowson MA, Roy SL, Jones JL, Griffin PM: Foodborne illness acquired in the United States--major pathogens. Emerg Infect Dis, 17 (1): 7-15, 2011. DOI: 10.3201/ eid1701.P11101

5. Johansson J, Freitag NE: Regulation of Listeria monocytogenes virulence. Microbiol Spectr, 7 (4), 2019. DOI: 10.1128/microbiolspec.GPP30064-2019

6. Nguyen BN, Peterson BN, Portnoy DA: Listeriolysin O: A phagosomespecific cytolysin revisited. Cell Microbiol, 21 (3): e12988, 2019. DOI: 10.1111/cmi.12988

7. Ireton K, Rigano LA, Polle L, Schubert WD: Molecular mechanism of protrusion formation during cell-to-cell spread of Listeria. Front Cell Infect Microbiol, 4:21, 2014. DOI: 10.3389/fcimb.2014.00021

8. Smith JL, Liu Y, Paoli GC: How does Listeria monocytogenes combat acid conditions? Can J Microbiol, 59 (3): 141-152, 2013. DOI: 10.1139/cjm2012-0392

9. Ryan S, Hill C, Gahan CG: Acid stress responses in Listeria monocytogenes. Adv Appl Microbiol, 65, 67-91, 2008. DOI: 10.1016/S0065-2164(08)00603-5

10. Ryan S, Begley M, Gahan CGM, Hill C: Molecular characterization of the arginine deiminase system in Listeria monocytogenes: regulation and role in acid tolerance. Environ Microbiol, 11 (2): 432-445, 2009. DOI: 10.1111/j.1462-2920.2008.01782.x

11. Cheng C, Chen J, Fang C, Xia Y, Shan Y, Liu Y, Wen G, Song H, Fang W: Listeria monocytogenes aguA1, but Not aguA2, Encodes a Functional Agmatine Deiminase: Biochemical Characterization of its catalytic properties and roles in acid tolerance. J Biol Chem, 288 (37): 26606-26615, 2013. DOI: 10.1074/jbc.M113.477380

12. Cotter PD, Gahan CG, Hill C: A glutamate decarboxylase system protects Listeria monocytogenes in gastric fluid. Mol Microbiol, 40 (2): 465475, 2001.

13. Abram F, Starr E, Karatzas KA, Matlawska-Wasowska K, Boyd A, Wiedmann M, Boor KJ, Connally D, O’Byrne CP: Identification of components of the sigma B regulon in Listeria monocytogenes that contribute to acid and salt tolerance. Appl Environ Microbiol, 74 (22): 68486858, 2008. DOI: 10.1128/AEM.00442-08

14. Chen J, Fang C, Zheng T, Zhu N, Bei Y, Fang W: Genomic presence of gadD1 glutamate decarboxylase correlates with the organization of ascBdapE internalin cluster in Listeria monocytogenes. Foodborne Pathog Dis, 9 (2): 175-178, 2012. DOI: 10.1089/fpd.2011.1022

15. Cheng C, Chen J, Shan Y, Fang C, Liu Y, Xia Y, Song H, Fang W: Listeria monocytogenes ArcA contributes to acid tolerance. J Med Microbiol, 62 (6): 813-821, 2013. DOI: 10.1099/jmm.0.055145-0

16. 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

17. 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

18. 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

19. Cheng C, Dong Z, Han X, Wang H, Jiang L, Sun J, Yang Y, Ma T, Shao C, Wang X, Chen Z, Fang W, Freitag NE, Huang H, Song H: Thioredoxin a is essential for motility and contributes to host infection of Listeria monocytogenes via redox interactions. Front Cell Infect Microbiol, 7:287, 2017. DOI: 10.3389/fcimb.2017.00287

20. 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

21. Gahan CGM, Hill C: Listeria monocytogenes: Survival and adaptation in the gastrointestinal tract. Front Cell Infect Microbiol, 4:9, 2014. DOI: 10.3389/fcimb.2014.00009

22. Feehily C, Finnerty A, Casey PG, Hill C, Gahan CG, O’Byrne CP, Karatzas KAG: Divergent evolution of the activity and regulation of the glutamate decarboxylase systems in Listeria monocytogenes EGD-e and 10403S: Roles in virulence and acid tolerance. PLoS One, 9 (11): e112649, 2014. DOI: 10.1371/journal.pone.0112649

23. Karatzas KAG, Brennan O, Heavin S, Morrissey J, O’Byrne CP: Intracellular accumulation of high levels of gamma-aminobutyrate by Listeria monocytogenes 10403S in response to low pH: uncoupling of gamma-aminobutyrate synthesis from efflux in a chemically defined medium. Appl Environ Microbiol, 76 (11): 3529-3537, 2010. DOI: 10.1128/ AEM.03063-09

24. Cheng C, Dong Z, Han X, Sun J, Wang H, Jiang L, Yang Y, Ma T, Chen Z, Yu J, Fang W, Song H: Listeria monocytogenes 10403S arginine repressor ArgR finely tunes arginine metabolism regulation under acidic conditions. Front Microbiol, 8:145, 2017. DOI: 10.3389/fmicb. 2017.00145

25. Cotter PD, Gahan CGM, Hill C: Analysis of the role of the Listeria monocytogenes F0F1 -AtPase operon in the acid tolerance response. Int J Food Microbiol, 60 (2-3): 137-146, 2000. DOI: 10.1016/S01681605(00)00305-6

26. Datta AR, Benjamin MM: Factors controlling acid tolerance of Listeria monocytogenes: Effects of nisin and other ionophores. Appl Environ Microbiol, 63 (10): 4123-4126, 1997.

27. Chen J, Chen F, Cheng C, Fang W: Prevalence of the lmo0036-0043 gene cluster encoding arginine deiminase and agmatine deiminase systems in Listeria monocytogenes. New Microbiol, 36 (2): 187-192, 2013.

28. Karatzas KA, Suur L, O’Byrne CP: Characterisation of the intracellular-glutamate decarboxylase system: Analysis of its function, transcription and role in the acid resistance of various strains of Listeria monocytogenes. Appl Environ Microbiol, 78 (10): 3571-3579, 2012. DOI: 10.1128/AEM.00227-12

29. NicAogain K, O’Byrne CP: The Role of stress and stress adaptations in determining the fate of the bacterial pathogen Listeria monocytogenes in the food chain. Front Microbiol, 7:1865, 2016. DOI: 10.3389/fmicb. 2016.01865

30. Pizarro-Cerda J, Cossart P: Listeria monocytogenes: Cell biology of invasion and intracellular growth. Microbiol Spectr, 6 (6), 2018. DOI: 10.1128/microbiolspec.GPP3-0013-2018

31. Kazmierczak MJ, Mithoe SC, Boor KJ, Wiedmann M: Listeria monocytogenes σB regulates stress response and virulence functions. J Bacteriol, 185 (19): 5722-5734, 2003.

32. Cotter PD, Ryan S, Gahan CG, Hill C: Presence of GadD1 glutamate decarboxylase in selected Listeria monocytogenes strains is associated with an ability to grow at low pH. Appl Environ Microbiol, 71 (6): 28322839, 2005. DOI: 10.1128/AEM.71.6.2832-2839.2005

33. Bowman JP, Hages E, Nilsson RE, Kocharunchitt C, Ross T: Investigation of the Listeria monocytogenes scott A acid tolerance response and associated physiological and phenotypic features via whole proteome analysis. J Proteome Res, 11 (4): 2409-2426, 2012. DOI: 10.1021/pr201137c