Trueperella pyogenes pyolysin inhibits lipopolysaccharide-induced inflammatory response in endometrium stromal cells via autophagy- and ATF6-dependent mechanism

Abstract

Trueperella pyogenes (T. pyogenes) is a common opportunistic pathogen of many livestock and play an important regulation role during multibacterial infection and interaction with the host by its primary virulence factor pyolysin (PLO). The purpose of this study was to investigate the regulation role of PLO which serve as a combinational pathogen with lipopolysaccharide (LPS) during endometritis. In this study, the expression of bioactive recombinant PLO (rPLO) in a prokaryotic expression system and its purification are described. Moreover, we observed that rPLO inhibited the innate immune response triggered by LPS and that methyl-β-cyclodextrin (MBCD) abrogated this inhibitory effect in goat endometrium stromal cells (gESCs). Additionally, we show from pharmacological and genetic studies that rPLO-induced autophagy represses gene expression by inhibiting NLRP3 inflammasome activation. Importantly, this study reported that ATF6 serves as a primary regulator of the cellular inflammatory reaction to rPLO. Overall, these observations suggest that T. pyogenes PLO could create an immunosuppressive environment for other pathogens invasion by regulating cellular signaling pathways.

This is a preview of subscription content, access via your institution.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8

References

  1. 1.

    Billington SJ, Jost BH, Cuevas WA, Bright KR, Songer JG (1997) The Arcanobacterium (Actinomyces) pyogenes hemolysin, pyolysin, is a novel member of the thiol-activated cytolysin family. J Bacteriol 179(19):6100–6106. https://doi.org/10.1128/jb.179.19.6100-6106.1997

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  2. 2.

    Jost BH, Billington SJ (2005) Arcanobacterium pyogenes: molecular pathogenesis of an animal opportunist. Anton Leeuw Int J G 88(2):87–102. https://doi.org/10.1007/s10482-005-2316-5

    Article  Google Scholar 

  3. 3.

    Ribeiro MG, Risseti RM, Bolanos CAD, Caffaro KA, de Morais ACB, Lara GHB, Zamprogna TO, Paes AC, Listoni FJP, Franco MMJ (2015) Trueperella pyogenes multispecies infections in domestic animals: a retrospective study of 144 cases (2002 to 2012). Vet Q 35(2):82–87. https://doi.org/10.1080/01652176.2015.1022667

    CAS  Article  PubMed  Google Scholar 

  4. 4.

    Brodzki P, Bochniarz M, Brodzki A, Wrona Z, Wawron W (2014) Trueperella pyogenes and Escherichia coli as an etiological factor of endometritis in cows and the susceptibility of these bacteria to selected antibiotics. Pol J Vet Sci 17(4):657–664. https://doi.org/10.2478/pjvs-2014-0096

    CAS  Article  PubMed  Google Scholar 

  5. 5.

    Rogovskyy AS, Lawhon S, Kuczmanski K, Gillis DC, Wu J, Hurley H, Rogovska YV, Konganti K, Yang CY, Duncan K (2018) Phenotypic and genotypic characteristics of Trueperella pyogenes isolated from ruminants. J Vet Diagn Investig 30(3):348–353. https://doi.org/10.1177/1040638718762479

    Article  Google Scholar 

  6. 6.

    Wang ML, Liu MC, Xu J, An LG, Wang JF, Zhu YH (2018) Uterine microbiota of dairy cows with clinical and subclinical endometritis. Front Microbiol 9:2691. https://doi.org/10.3389/fmicb.2018.02691

    Article  PubMed  PubMed Central  Google Scholar 

  7. 7.

    Sheldon IM, Cronin J, Goetze L, Donofrio G, Schuberth HJ (2009) Defining postpartum uterine disease and the mechanisms of infection and immunity in the female reproductive tract in cattle. Biol Reprod 81(6):1025–1032. https://doi.org/10.1095/biolreprod.109.077370

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  8. 8.

    Risseti RM, Zastempowska E, Twaruzek M, Lassa H, Pantoja JCF, de Vargas APC, Guerra ST, Bolanos CAD, de Paula CL, Alves AC, Colhado BS, Portilho FVR, Tasca C, Lara GHB, Ribeiro MG (2017) Virulence markers associated with Trueperella pyogenes infections in livestock and companion animals. Lett Appl Microbiol 65(2):125–132. https://doi.org/10.1111/lam.12757

    CAS  Article  PubMed  Google Scholar 

  9. 9.

    Billington SJ, Songer JG, Jost BH (2001) Molecular characterization of the pore-forming toxin, pyolysin, a major virulence determinant of Arcanobacterium pyogenes. Vet Microbiol 82(3):261–274. https://doi.org/10.1016/S0378-1135(01)00373-X

    CAS  Article  PubMed  Google Scholar 

  10. 10.

    Dong WL, Liu L, Odah KA, Atiah LA, Gao YH, Kong LC, Ma HX (2019) Antimicrobial resistance and presence of virulence factor genes in Trueperella pyogenes isolated from pig lungs with pneumonia. Trop Anim Health Prod 51(7):2099–2103. https://doi.org/10.1007/s11250-019-01916-z

    Article  PubMed  Google Scholar 

  11. 11.

    Jost BH, Songer JG, Billington SJ (1999) An Arcanobacterium (Actinomyces) pyogenes mutant deficient in production of the pore-forming cytolysin pyolysin has reduced virulence. Infect Immun 67(4):1723–1728. https://doi.org/10.1016/S0928-8244(98)00155-2

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  12. 12.

    Griffin S, Healey GD, Sheldon IM (2018) Isoprenoids increase bovine endometrial stromal cell tolerance to the cholesterol-dependent cytolysin from Trueperella pyogenes. Biol Reprod 99(4):749–760. https://doi.org/10.1093/biolre/ioy099

    Article  PubMed  PubMed Central  Google Scholar 

  13. 13.

    Zhang WL, Wang HL, Wang B, Zhang Y, Hu YH, Ma B, Wang JW (2018) Replacing the 238th aspartic acid with an arginine impaired the oligomerization activity and inflammation-inducing property of pyolysin. Virulence 9(1):1112–1125. https://doi.org/10.1080/21505594.2018.1491256

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  14. 14.

    Amos MR, Healey GD, Goldstone RJ, Mahan SM, Duvel A, Schuberth HJ, Sandra O, Zieger P, Dieuzy-Labaye I, Smith DGE, Sheldon IM (2014) Differential endometrial cell sensitivity to a cholesterol-dependent cytolysin links Trueperella pyogenes to uterine disease in cattle. Biol Reprod 90(3):54. https://doi.org/10.1095/biolreprod.113.115972

    CAS  Article  PubMed  Google Scholar 

  15. 15.

    Griffin S, Preta G, Sheldon IM (2017) Inhibiting mevalonate pathway enzymes increases stromal cell resilience to a cholesterol-dependent cytolysin. Sci Rep 7:17050. https://doi.org/10.1038/s41598-017-17138-y

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  16. 16.

    McNeela EA, Burke A, Neill DR, Baxter C, Fernandes VE, Ferreira D, Smeaton S, El-Rachkidy R, McLoughlin RM, Mori A, Moran B, Fitzgerald KA, Tschopp J, Petrilli V, Andrew PW, Kadioglu A, Lavelle EC (2010) Pneumolysin activates the NLRP3 inflammasome and promotes proinflammatory cytokines independently of TLR4. PLoS Path 6(11):e1001191. https://doi.org/10.1371/journal.ppat.1001191

    CAS  Article  Google Scholar 

  17. 17.

    Malley R, Henneke P, Morse SC, Cieslewicz MJ, Lipsitch M, Thompson CM, Kurt-Jones E, Paton JC, Wessels MR, Golenbock DT (2003) Recognition of pneumolysin by toll-like receptor 4 confers resistance to pneumococcal infection. Proc Natl Acad Sci U S A 100(4):1966–1971. https://doi.org/10.1073/pnas.0435928100

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  18. 18.

    Sun P, Sun N, Yin W, Sun Y, Fan K, Guo J, Khan A, He Y, Li H (2019) Matrine inhibits IL-1β secretion in primary porcine alveolar macrophages through the MyD88/NF-κB pathway and NLRP3 inflammasome. Vet Res 50(1):53. https://doi.org/10.1186/s13567-019-0671-x

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  19. 19.

    Lamb CA, Yoshimori T, Tooze SA (2013) The autophagosome: origins unknown, biogenesis complex. Nat Rev Mol Cell Biol 14(12):759–774. https://doi.org/10.1038/nrm3696

    CAS  Article  PubMed  Google Scholar 

  20. 20.

    Levine B, Kroemer G (2008) Autophagy in the pathogenesis of disease. Cell 132(1):27–42. https://doi.org/10.1016/j.cell.2007.12.018

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  21. 21.

    Levine B, Kroemer G (2019) Biological functions of autophagy genes: a disease perspective. Cell 176(1–2):11–42. https://doi.org/10.1016/j.cell.2018.09.048

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  22. 22.

    Tang DL, Kang R, Vanden Berghe T, Vandenabeele P, Kroemer G (2019) The molecular machinery of regulated cell death. Cell Res 29(5):347–364. https://doi.org/10.1038/s41422-019-0164-5

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  23. 23.

    Nakagawa I, Amano A, Mizushima N, Yamamoto A, Yamaguchi H, Kamimoto T, Nara A, Funao J, Nakata M, Tsuda K, Hamada S, Yoshimori T (2004) Autophagy defends cells against invading group a streptococcus. Science 306(5698):1037–1040. https://doi.org/10.1126/science.1103966

    CAS  Article  PubMed  Google Scholar 

  24. 24.

    Birmingham CL, Smith AC, Bakowski MA, Yoshimori T, Brumell JH (2006) Autophagy controls Salmonella infection in response to damage to the Salmonella-containing vacuole. J Biol Chem 281(16):11374–11383. https://doi.org/10.1074/jbc.M509157200

    CAS  Article  PubMed  Google Scholar 

  25. 25.

    Py BF, Lipinski MM, Yuan JY (2007) Autophagy limits Listeria monocytogenes intracellular growth in the early phase of primary infection. Autophagy 3(2):117–125. https://doi.org/10.4161/auto.3618

    CAS  Article  PubMed  Google Scholar 

  26. 26.

    Thurston TLM, Ryzhakov G, Bloor S, von Muhlinen N, Randow F (2009) The TBK1 adaptor and autophagy receptor NDP52 restricts the proliferation of ubiquitin-coated bacteria. Nat Immunol 10(11):1215–U1103. https://doi.org/10.1038/ni.1800

    Article  PubMed  Google Scholar 

  27. 27.

    Levine B (2005) Eating oneself and uninvited guests: autophagy-related pathways in cellular defense. Cell 120(2):159–162. https://doi.org/10.1016/j.cell.2005.01.005

    CAS  Article  PubMed  Google Scholar 

  28. 28.

    Preta G, Lotti V, Cronin JG, Sheldon IM (2015) Protective role of the dynamin inhibitor Dynasore against the cholesterol-dependent cytolysin of Trueperella pyogenes. FASEB J 29(4):1516–1528. https://doi.org/10.1096/fj.14-265207

    CAS  Article  PubMed  Google Scholar 

  29. 29.

    Chang YP, Ka SM, Hsu WH, Chen A, Chao LK, Lin CC, Hsieh CC, Chen MC, Chiu HW, Ho CL, Chiu YC, Liu ML, Hua KF (2015) Resveratrol inhibits NLRP3 inflammasome activation by preserving mitochondrial integrity and augmenting autophagy. J Cell Physiol 230(7):1567–1579. https://doi.org/10.1002/jcp.24903

    CAS  Article  PubMed  Google Scholar 

  30. 30.

    Shao BZ, Wei W, Ke P, Xu ZQ, Zhou JX, Liu C (2014) Activating cannabinoid receptor 2 alleviates pathogenesis of experimental autoimmune encephalomyelitis via activation of autophagy and inhibiting NLRP3 inflammasome. CNS Neurosci Ther 20(12):1021–1028. https://doi.org/10.1111/cns.12349

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  31. 31.

    Shao BZ, Xu ZQ, Han BZ, Su DF, Liu C (2015) NLRP3 inflammasome and its inhibitors: a review. Front Pharmacol 6:262. https://doi.org/10.3389/fphar.2015.00262

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  32. 32.

    Schroder K, Tschopp J (2010) The inflammasomes. Cell 140(6):821–832. https://doi.org/10.1016/j.cell.2010.01.040

    CAS  Article  PubMed  Google Scholar 

  33. 33.

    Williams A, Flavell RA, Eisenbarth SC (2010) The role of NOD-like receptors in shaping adaptive immunity. Curr Opin Immunol 22(1):34–40. https://doi.org/10.1016/j.coi.2010.01.004

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  34. 34.

    Bettigole SE, Glimcher LH (2015) Endoplasmic reticulum stress in immunity. Annu Rev Immunol 33:107–138. https://doi.org/10.1146/annurev-immunol-032414-112116

    CAS  Article  PubMed  Google Scholar 

  35. 35.

    Oakes SA, Papa FR (2015) The role of endoplasmic reticulum stress in human pathology. Annu Rev Pathol-Mech 10:173–194. https://doi.org/10.1146/annurev-pathol-012513-104649

    CAS  Article  Google Scholar 

  36. 36.

    Zhang KZ, Kaufman RJ (2008) From endoplasmic-reticulum stress to the inflammatory response. Nature 454(7203):455–462. https://doi.org/10.1038/nature07203

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  37. 37.

    Senft D, Ronai ZA (2015) UPR, autophagy, and mitochondria crosstalk underlies the ER stress response. Trends Biochem Sci 40(3):141–148. https://doi.org/10.1016/j.tibs.2015.01.002

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  38. 38.

    Zhang YY, Wang AH, Wu QX, Sheng HX, Jin YP (2010) Establishment and characteristics of immortal goat endometrial epithelial cells and stromal cells with hTERT. J Anim Vet Adv 9(21):2738–2747. https://doi.org/10.3923/javaa.2010.2738.2747

    CAS  Article  Google Scholar 

  39. 39.

    Yang DQ, Jiang TT, Liu JG, Hong J, Lin PF, Chen HT, Zhou D, Tang KQ, Wang AH, Jin YP (2018) Interferon-tau regulates prostaglandin release in goat endometrial stromal cells via JAB1-unfolded protein response pathway. Theriogenology 113:237–246. https://doi.org/10.1016/j.theriogenology.2018.03.007

    CAS  Article  PubMed  Google Scholar 

  40. 40.

    Sheldon IM, Noakes DE, Rycroft AN, Pfeiffer DU, Dobson H (2002) Influence of uterine bacterial contamination after parturition on ovarian dominant follicle selection and follicle growth and function in cattle. Reproduction 123(6):837–845. https://doi.org/10.1530/rep.0.1230837

    CAS  Article  PubMed  Google Scholar 

  41. 41.

    Yang B, Xue QH, Guo JN, Wang XP, Zhang YM, Guo KK, Li W, Chen SY, Xue TX, Qi XF, Wang JY (2020) Autophagy induction by the pathogen receptor NECTIN4 and sustained autophagy contribute to peste des petits ruminants virus infectivity. Autophagy 16(5):842–861. https://doi.org/10.1080/15548627.2019.1643184

    CAS  Article  PubMed  Google Scholar 

  42. 42.

    Chen FL, Lin PF, Li X, Sun J, Zhang Z, Du EQ, Wang AH, Jin YP (2014) Construction and expression of lentiviral vectors encoding recombinant mouse CREBZF in NIH 3T3 cells. Plasmid 76:24–31. https://doi.org/10.1016/j.plasmid.2014.08.004

    CAS  Article  PubMed  Google Scholar 

  43. 43.

    Leidal AM, Levine B, Debnath J (2018) Autophagy and the cell biology of age-related disease. Nat Cell Biol 20(12):1338–1348. https://doi.org/10.1038/s41556-018-0235-8

    CAS  Article  PubMed  Google Scholar 

  44. 44.

    Bischof LJ, Kao CY, Los FCO, Gonzalez MR, Shen ZX, Briggs SP, van der Goot FG, Aroian RV (2008) Activation of the unfolded protein response is required for defenses against bacterial pore-forming toxin in vivo. PLoS Path 4(10):e1000176. https://doi.org/10.1371/journal.ppat.1000176

    CAS  Article  Google Scholar 

  45. 45.

    Cui JX, Shao F (2011) Biochemistry and cell signaling taught by bacterial effectors. Trends Biochem Sci 36(10):532–540. https://doi.org/10.1016/j.tibs.2011.07.003

    CAS  Article  PubMed  Google Scholar 

  46. 46.

    Piersanti RL, Zimpel R, Molinari PCC, Dickson MJ, Ma ZX, Jeong KC, Santos JEP, Sheldon IM, Bromfield JJ (2019) A model of clinical endometritis in Holstein heifers using pathogenic Escherichia coli and Trueperella pyogenes. J Dairy Sci 102(3):2686–2697. https://doi.org/10.3168/jds.2018-15595

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  47. 47.

    Taniguchi K, Karin M (2018) NF-kappa B, inflammation, immunity and cancer: coming of age. Nat Rev Immunol 18(5):309–324. https://doi.org/10.1038/nri.2017.142

    CAS  Article  PubMed  Google Scholar 

  48. 48.

    Li HT, Xu H, Zhou Y, Zhang J, Long CZ, Li SQ, Chen S, Zhou JM, Shao F (2007) The phosphothreonine lyase activity of a bacterial type III effector family. Science 315(5814):1000–1003. https://doi.org/10.1126/science.1138960

    CAS  Article  PubMed  Google Scholar 

  49. 49.

    Mizushima N, Yoshimori T, Levine B (2010) Methods in mammalian autophagy research. Cell 140(3):313–326. https://doi.org/10.1016/j.cell.2010.01.028

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  50. 50.

    Ge JN, Gong YN, Xu Y, Shao F (2012) Preventing bacterial DNA release and absent in melanoma 2 inflammasome activation by a Legionella effector functioning in membrane trafficking. Proc Natl Acad Sci U S A 109(16):6193–6198. https://doi.org/10.1073/pnas.1117490109

    Article  PubMed  PubMed Central  Google Scholar 

  51. 51.

    Jiang Q, Chen S, Ren WK, Liu G, Yao K, Wu GY, Yin YL (2017) Escherichia coli aggravates endoplasmic reticulum stress and triggers CHOP-dependent apoptosis in weaned pigs. Amino Acids 49(12):2073–2082. https://doi.org/10.1007/s00726-017-2492-4

    CAS  Article  PubMed  Google Scholar 

  52. 52.

    Quan K, Li SC, Wang DD, Shi Y, Yang ZX, Song JP, Tian YL, Liu YJ, Fan ZY, Zhu W (2018) Berberine attenuates macrophages infiltration in intracranial aneurysms potentially through FAK/Grp78/UPR Axis. Front Pharmacol 9:565. https://doi.org/10.3389/fphar.2018.00565

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  53. 53.

    Zhang HS, Chen Y, Fan L, Xi QL, Wu GH, Li XX, Yuan TL, He SQ, Yu Y, Shao ML, Liu Y, Bai CG, Ling ZQ, Li M, Liu Y, Fang J (2015) The endoplasmic reticulum stress sensor IRE1 alpha in intestinal epithelial cells is essential for protecting against colitis. J Biol Chem 290(24):15327–15336. https://doi.org/10.1074/jbc.M114.633560

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  54. 54.

    Appenzeller-Herzog C, Hall MN (2012) Bidirectional crosstalk between endoplasmic reticulum stress and mTOR signaling. Trends Cell Biol 22(5):274–282. https://doi.org/10.1016/j.tcb.2012.02.006

    CAS  Article  PubMed  Google Scholar 

Download references

Acknowledgments

We sincerely thank Professor Wei Liu and Associate Professor Keqiong Tang for providing help when we designed the study.

Funding

This study was supported by National Natural Science Foundation of China, Grant Number: 31772817.

Author information

Affiliations

Authors

Corresponding author

Correspondence to Yaping Jin.

Ethics declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Additional information

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Responsible Editor: Cristiano Gallina Moreira

Supplementary information

Figure S

Construction of the recombinant vector and purification of the fusion protein. A. The gene fragment encoding mature T. pyogenes PLO was cloned by PCR. B. The PLO gene fragment was connected to the prokaryotic expression vector pET-32a (+). Lane 1 shows PLO gene amplification from recombinant vector pET-32a-PLO. Lane 2 shows the positive control with the T. pyogenes genome as the PCR template. Lane 3 shows the negative control. C. Purified protein was authenticated by Coomassie brilliant blue staining (DOCX 370 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Qi, M., Liu, J., Jiang, Q. et al. Trueperella pyogenes pyolysin inhibits lipopolysaccharide-induced inflammatory response in endometrium stromal cells via autophagy- and ATF6-dependent mechanism. Braz J Microbiol (2021). https://doi.org/10.1007/s42770-021-00422-5

Download citation

Keywords

  • Pyolysin
  • LPS
  • Costimulation
  • Autophagy
  • ATF6