Original articles
Genzhu Wang, Liang Li, Xiukun Wang, Xue Li, Youwen Zhang, Jie Yu, Jiandong Jiang, Xuefu You, Yan Q. Xiong. Hypericin enhances β-lactam antibiotics activity by inhibiting sarA expression in methicillin-resistant Staphylococcus aureus[J]. Acta Pharmaceutica Sinica B, 2019, 9(6): 1174-1182

Hypericin enhances β-lactam antibiotics activity by inhibiting sarA expression in methicillin-resistant Staphylococcus aureus
Genzhu Wanga,b, Liang Lia, Xiukun Wangb, Xue Lib, Youwen Zhangb, Jie Yub, Jiandong Jiangb,d, Xuefu Youb, Yan Q. Xionga,c
a Los Angeles Biomedical Research Institute at Harbor-UCLA Medical Center, Torrance, CA 90502, USA;
b Beijing Key Laboratory of Antimicrobial Agents, Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China;
c Geffen School of Medicine at UCLA, Los Angeles, CA 90095, USA;
d State Key Laboratory of Bioactive Substances and Function of Natural Medicine, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China
Bacteremia is a life-threating syndrome often caused by methicillin-resistant Staphylococcus aureus (MRSA). Thus, there is an urgent need to develop novel approaches to successfully treat this infection. Staphylococcal accessory regulator A (SarA), a global virulence regulator, plays a critical role in pathogenesis and β-lactam antibiotic resistance in Staphylococcus aureus. Hypericin is believed to act as an antibiotic, antidepressant, antiviral and non-specific kinase inhibitor. In the current study, we investigated the impact of hypericin on β-lactam antibiotics susceptibility and mechanism(s) of its activity. We demonstrated that hypericin significantly decreased the minimum inhibitory concentrations of β-lactam antibiotics (e.g., oxacillin, cefazolin and nafcillin), biofilm formation and fibronectin binding in MRSA strain JE2. In addition, hypericin significantly reduced sarA expression, and subsequently decreased mecA, and virulence-related regulators (e.g., agr RNAⅢ) and genes (e.g., fnbA and hla) expression in the studied MRSA strain. Importantly, the in vitro synergistic effect of hypericin with β-lactam antibiotic (e.g., oxacillin) translated into in vivo therapeutic outcome in a murine MRSA bacteremia model. These findings suggest that hypericin plays an important role in abrogation of β-lactam resistance against MRSA through sarA inhibition, and may allow us to repurpose the use of β-lactam antibiotics, which are normally ineffective in the treatment of MRSA infections (e.g., oxacillin).
Key words:    Hypericin    β-lactam    MRSA    Synergistic effect    SarA   
Received: 2019-02-22     Revised: 2019-05-11
DOI: 10.1016/j.apsb.2019.05.002
Funds: This study was supported in part by CAMS Initiative for Innovative Medicine (grant numbers 2016-I2M-2-002 and 2016-I2M-3-014, China), National Mega-project for Innovative Drugs (grant number 2018ZX09721001, China), the National Science Foundation of China (grant number 81621064, China). We are very grateful to Dr. Ambrose Cheung at Dartmouth Medical School (Hanover, New Hampshire, USA) for providing the purified SarA protein.
Corresponding author: Xuefu You, Yan Q. Xiong     Email:xuefuyou@hotmail.com;yxiong@ucla.edu
Author description:
PDF(KB) Free
Genzhu Wang
Liang Li
Xiukun Wang
Xue Li
Youwen Zhang
Jie Yu
Jiandong Jiang
Xuefu You
Yan Q. Xiong

1. Bayer MG, Heinrichs JH, Cheung AL. The molecular architecture of the sar locus in Staphylococcus aureus. J Bacteriol 1996;178: 4563-70.
2. Wang L, Yang R, Yuan B, Liu Y, Liu C. The antiviral and antimicrobial activities of licorice, a widely-used Chinese herb. Acta Pharm Sin B 2015;5:310-5.
3. Boucher H, Miller LG, Razonable RR. Serious infections caused by methicillin-resistant Staphylococcus aureus. Clin Infect Dis 2010;51 Suppl 2:S183-97.
4. Cheung AL, Nishina KA, Trotonda MP, Tamber S. The SarA protein family of Staphylococcus aureus. Int J Biochem Cell Biol 2008;40: 355-61.
5. Abdelhady W, Bayer AS, Seidl K, Moormeier DE, Bayles KW, Cheung A, et al. Impact of vancomycin on sarA-mediated biofilm formation: role in persistent endovascular infections due to methicillin-resistant Staphylococcus aureus. J Infect Dis 2014;209: 1231-40.
6. Balamurugan P, Praveen Krishna V, Bharath D, Lavanya R, Vairaprakash P, Adline Princy S. Staphylococcus aureus quorum regulator SarA targeted compound, 2-[(methylamino)methyl]phenol inhibits biofilm and down-regulates virulence genes. Front Microbiol 2017;8:1290.
7. Arya R, Ravikumar R, Santhosh RS, Princy SA. SarA based novel therapeutic candidate against Staphylococcus aureus associated with vascular graft infections. Front Microbiol 2015;6:416.
8. Cheung AL, Eberhardt KJ, Chung E, Yeaman MR, Sullam PM, Ramos M, et al. Diminished virulence of a sar-/agr-mutant of Staphylococcus aureus in the rabbit model of endocarditis. J Clin Investig 1994;94:1815-22.
9. Blevins JS, Elasri MO, Allmendinger SD, Beenken KE, Skinner RA, Thomas JR, et al. Role of sarA in the pathogenesis of Staphylococcus aureus musculoskeletal infection. Infect Immun 2003;71: 516-23.
10. Li L, Cheung A, Bayer AS, Chen L, Abdelhady W, Kreiswirth BN, et al. The global regulon sarA regulates β-lactam antibiotic resistance in methicillin-resistant Staphylococcus aureus in vitro and in endovascular infections. J Infect Dis 2016;214:1421-9.
11. Lawvere S, Mahoney MC. St. John’s wort. Am Fam Physician 2005; 72:2249-54.
12. Zhang J, Jiang C, Figueiro Longo JP, Azevedo RB, Zhang H, Muehlmann LA. An updated overview on the development of new photosensitizers for anticancer photodynamic therapy. Acta Pharm Sin B 2018;8:137-46.
13. Jacobson JM, Feinman L, Liebes L, Ostrow N, Koslowski V, Tobia A, et al. Pharmacokinetics, safety, and antiviral effects of hypericin, a derivative of St. John’s wort plant, in patients with chronic hepatitis C virus infection. Antimicrob Agents Chemother 2001;45:517-24.
14. Kashef N, Karami S, Djavid GE. Phototoxic effect of hypericin alone and in combination with acetylcysteine on Staphylococcus aureus biofilms. Photodiagn Photodyn Ther 2015;12:186-92.
15. Garcia I, Ballesta S, Gilaberte Y, Rezusta A, Pascual A. Antimicrobial photodynamic activity of hypericin against methicillin-susceptible and resistant Staphylococcus aureus biofilms. Future Microbiol 2015;10: 347-56.
16. Yow CM, Tang HM, Chu ES, Huang Z. Hypericin-mediated photodynamic antimicrobial effect on clinically isolated pathogens. Photochem Photobiol 2012;88:626-32.
17. Kairyte K, Lapinskas S, Gudelis V, Luksiene Z. Effective inactivation of food pathogens Listeria monocytogenes and Salmonella enterica by combined treatment of hypericin-based photosensitization and high power pulsed light. J Appl Microbiol 2012;112:1144-51.
18. Fey PD, Endres JL, Yajjala VK, Widhelm TJ, Boissy RJ, Bose JL, et al. A genetic resource for rapid and comprehensive phenotype screening of nonessential Staphylococcus aureus genes. mBio 2013;4: e00537-12.
19. Trotonda MP, Xiong YQ, Memmi G, Bayer AS, Cheung AL. Role of mgrA and sarA in methicillin-resistant Staphylococcus aureus autolysis and resistance to cell wall-active antibiotics. J Infect Dis 2009; 199:209-18.
20. Memmi G, Filipe SR, Pinho MG, Fu Z, Cheung A. Staphylococcus aureus PBP4 is essential for β-lactam resistance in communityacquired methicillin-resistant strains. Antimicrob Agents Chemother 2008;52:3955-66.
21. Lu X, Yang X, Li X, Lu Y, Ren Z, Zhao L, et al. In vitro activity of sodium new houttuyfonate alone and in combination with oxacillin or netilmicin against methicillin-resistant Staphylococcus aureus. PLoS One 2013;8:e68053.
22. White RL, Burgess DS, Manduru M, Bosso JA. Comparison of three different in vitro methods of detecting synergy: time-kill, checkerboard, and E test. Antimicrob Agents Chemother 1996;40:1914-8.
23. Odds FC. Synergy, antagonism, and what the chequerboard puts between them. J Antimicrob Chemother 2003;52:1.
24. Belley A, Neesham-Grenon E, Arhin FF, McKay GA, Parr Jr TR, Moeck G. Assessment by timeekill methodology of the synergistic effects of oritavancin in combination with other antimicrobial agents against Staphylococcus aureus. Antimicrob Agents Chemother 2008; 52:3820-2.
25. Abdelhady W, Bayer AS, Seidl K, Nast CC, Kiedrowski MR, Horswill AR, et al. Reduced vancomycin susceptibility in an in vitro catheter-related biofilm model correlates with poor therapeutic outcomes in experimental endocarditis due to methicillin-resistant Staphylococcus aureus. Antimicrob Agents Chemother 2013;57: 1447-54.
26. Seidl K, Chen L, Bayer AS, Hady WA, Kreiswirth BN, Xiong YQ. Relationship of agr expression and function with virulence and vancomycin treatment outcomes in experimental endocarditis due to methicillin-resistant Staphylococcus aureus. Antimicrob Agents Chemother 2011;55:5631-9.
27. Karlsson A, Saravia-Otten P, Tegmark K, Morfeldt E, Arvidson S. Decreased amounts of cell wall-associated protein A and fibronectinbinding proteins in Staphylococcus aureus sarA mutants due to upregulation of extracellular proteases. Infect Immun 2001;69:4742-8.
28. Oliveira DC, de Lencastre H. Multiplex PCR strategy for rapid identification of structural types and variants of the mec element in methicillin-resistant Staphylococcus aureus. Antimicrob Agents Chemother 2002;46:2155-61.
29. Ster C, Gilbert FB, Cochard T, Poutrel B. Transcriptional profiles of regulatory and virulence factors of Staphylococcus aureus of bovine origin: oxygen impact and strain-to-strain variations. Mol Cell Probes 2005;19:227-35.
30. Chien Y, Manna AC, Projan SJ, Cheung AL. SarA, a global regulator of virulence determinants in Staphylococcus aureus, binds to a conserved motif essential for sar-dependent gene regulation. J Biol Chem 1999;274:37169-76.
31. Oliveira DC, de Lencastre H. Methicillin-resistance in Staphylococcus aureus is not affected by the overexpression in trans of the mecA gene repressor: a surprising observation. PLoS One 2011;6:e23287.
32. Wang G, Pang J, Hu X, Nie T, Lu X, Li X, et al. Daphnetin: a novel anti-Helicobacter pylori agent. Int J Mol Sci 2019;20. pii: E850.
33. Zhou Z, Wang X, Zhang H, Sun J, Zheng L, Liu H, et al. Chromopeptide A, a highly cytotoxic depsipeptide from the marine sedimentderived bacterium Chromobacterium sp. HS-13-94. Acta Pharm Sin B 2015;5:62-6.
34. Wang R, Braughton KR, Kretschmer D, Bach TH, Queck SY, Li M, et al. Identification of novel cytolytic peptides as key virulence determinants for community-associated MRSA. Nat Med 2007;13: 1510-4.
35. Wang Q, Lv Y, Pang J, Li X, Lu X, Wang X, et al. In vitro and in vivo activity of D-serine in combination with β-lactam antibiotics against methicillin-resistant Staphylococcus aureus. Acta Pharm Sin B 2019; 9:496-504.
36. Blank M, Lavie G, Mandel M, Hazan S, Orenstein A, Meruelo D, et al. Antimetastatic activity of the photodynamic agent hypericin in the dark. Int J Cancer 2004;111:596-603.
37. Jiang W, Li B, Zheng X, Liu X, Cen Y, Li J, et al. Artesunate in combination with oxacillin protect sepsis model mice challenged with lethal live methicillin-resistant Staphylococcus aureus (MRSA) via its inhibition on proinflammatory cytokines release and enhancement on antibacterial activity of oxacillin. Int Immunopharmacol 2011;11: 1065-73.
38. Waters EM, Rudkin JK, Coughlan S, Clair GC, Adkins JN, Gore S, et al. Redeploying β-lactam antibiotics as a novel antivirulence strategy for the treatment of methicillin-resistant Staphylococcus aureus infections. J Infect Dis 2017;215:80-7.
39. Fridkin SK, Hageman JC, Morrison M, Sanza LT, Como-Sabetti K, Jernigan JA, et al. Methicillin-resistant Staphylococcus aureus disease in three communities. N Engl J Med 2005;352:1436-44.
40. CDC. Vancomycin-resistant Staphylococcus aureusdPennsylvania. MMWR Morb Mortal Wkly Rep 2002 2002;51:902.
41. Marty FM, Yeh WW, Wennersten CB, Venkataraman L, Albano E, Alyea EP, et al. Emergence of a clinical daptomycin-resistant Staphylococcus aureus isolate during treatment of methicillin-resistant Staphylococcus aureus bacteremia and osteomyelitis. J Clin Microbiol 2006;44:595-7.
42. He M, Shao L, Liu Q, Li J, Lin H, Jing L, et al. Mechanism of synergy between SIPI-8294 and β-lactam antibiotics against methicillin-resistant Staphylococcus aureus. Lett Appl Microbiol 2016;63:3-10.
43. Sakoulas G, Gold HS, Venkataraman L, DeGirolami PC, Eliopoulos GM, Qian Q. Methicillin-resistant Staphylococcus aureus: comparison of susceptibility testing methods and analysis of mecApositive susceptible strains. J Clin Microbiol 2001;39:3946-51.
44. Mirani ZA, Aziz M, Khan SI. Small colony variants have a major role in stability and persistence of Staphylococcus aureus biofilms. J Antibiot (Tokyo) 2015;68:98-105.
45. Beenken KE, Blevins JS, Smeltzer MS. Mutation of sarA in Staphylococcus aureus limits biofilm formation. Infect Immun 2003;71: 4206-11.
46. Atwood DN, Loughran AJ, Courtney AP, Anthony AC, Meeker DG, Spencer HJ, et al. Comparative impact of diverse regulatory loci on Staphylococcus aureus biofilm formation. Microbiology 2015;4: 436-51.
47. Houston P, Rowe SE, Pozzi C, Waters EM, O’Gara JP. Essential role for the major autolysin in the fibronectin-binding protein-mediated Staphylococcus aureus biofilm phenotype. Infect Immun 2011;79: 1153-65.
48. Valle J, Toledo-Arana A, Berasain C, Ghigo JM, Amorena B, Penades JR, et al. SarA and not sigmaB is essential for biofilm development by Staphylococcus aureus. Mol Microbiol 2003;48: 1075-87.
49. Trotonda MP, Manna AC, Cheung AL, Lasa I, Penades JR. SarA positively controls bap-dependent biofilm formation in Staphylococcus aureus. J Bacteriol 2005;187:5790-8.
50. O’Neill E, Pozzi C, Houston P, Smyth D, Humphreys H, Robinson DA, et al. Association between methicillin susceptibility and biofilm regulation in Staphylococcus aureus isolates from devicerelated infections. J Clin Microbiol 2007;45:1379-88.
51. Cheung AL, Manna AC. Role of the distal sarA promoters in SarA expression in Staphylococcus aureus. Infect Immun 2005;73: 4391-4.
52. Dunman PM, Murphy E, Haney S, Palacios D, Tucker-Kellogg G, Wu S, et al. Transcription profiling-based identification of Staphylococcus aureus genes regulated by the agr and/or sarA loci. J Bacteriol 2001;183:7341-53.
53. Wolz C, Pohlmann-Dietze P, Steinhuber A, Chien YT, Manna A, van Wamel W, et al. Agr-independent regulation of fibronectin-binding protein(s) by the regulatory locus sar in Staphylococcus aureus. Mol Microbiol 2000;36:230-43.
54. Xiong YQ, Bayer AS, Yeaman MR, van Wamel W, Manna AC, Cheung AL. Impacts of sarA and agr in Staphylococcus aureus strain Newman on fibronectin-binding protein A gene expression and fibronectin adherence capacity in vitro and in experimental infective endocarditis. Infect Immun 2004;72:1832-6.
55. Viedma E, Perez-Montarelo D, Villa J, Munoz-Gallego I, Larrosa N, Fernandez-Hidalgo N, et al. Sub-inhibitory concentrations of oxacillin modify the expression of agr locus in Staphylococcus aureus clinical strains belonging to different clonal complexes. BMC Infect Dis 2018; 18:177.
56. Rudkin JK, Laabei M, Edwards AM, Joo HS, Otto M, Lennon KL, et al. Oxacillin alters the toxin expression profile of communityassociated methicillin-resistant Staphylococcus aureus. Antimicrob Agents Chemother 2014;58:1100-7.
Similar articles:
1.Qing Wang, Yuemeng Lv, Jing Pang, Xue Li, Xi Lu, Xiukun Wang, Xinxin Hu, Tongying Nie, Xinyi Yang, Yan Q. Xiong, Jiandong Jiang, Congran Li, Xuefu You.In vitro and in vivo activity of D-serine in combination with β-lactam antibiotics against methicillin-resistant Staphylococcus aureus[J]. Acta Pharmaceutica Sinica B, 2019,9(3): 496-504