The rise of multidrug-resistant (MDR) pathogens causes an increasing challenge to public health. Antimicrobial peptides are considered a possible solution to this problem. HBV core protein (HBc) contains an arginine-rich domain (ARD) at its C-terminus, which consists of 16 arginine residues separated into four clusters (ARD I to IV). In this study, we demonstrated that the peptide containing the full-length ARD I–IV (HBc147-183) has a broad-spectrum antimicrobial activity at micro-molar concentrations, including some MDR and colistin (polymyxin E)-resistant Acinetobacter baumannii. Furthermore, confocal fluorescence microscopy and SYTOX Green uptake assay indicated that this peptide killed Gram-negative and Gram-positive bacteria by membrane permeabilization or DNA binding. In addition, peptide ARD II–IV (HBc153-176) and ARD I–III (HBc147-167) were found to be necessary and sufficient for the activity against P. aeruginosa and K. peumoniae. The antimicrobial activity of HBc ARD peptides can be attenuated by the addition of LPS. HBc ARD peptide was shown to be capable of direct binding to the Lipid A of lipopolysaccharide (LPS) in several in vitro binding assays. Peptide ARD I–IV (HBc147-183) had no detectable cytotoxicity in various tissue culture systems and a mouse animal model. In the mouse model by intraperitoneal (i.p.) inoculation with Staphylococcus aureus, timely treatment by i.p. injection with ARD peptide resulted in 100-fold reduction of bacteria load in blood, liver and spleen, as well as 100% protection of inoculated animals from death. If peptide was injected when bacterial load in the blood reached its peak, the protection rate dropped to 40%. Similar results were observed in K. peumoniae using an IVIS imaging system. The finding of anti-microbial HBc ARD is discussed in the context of commensal gut microbiota, development of intrahepatic anti-viral immunity and establishment of chronic infection with HBV. Our current results suggested that HBc ARD could be a new promising antimicrobial peptide.
References
[1]
Hancock RE, Sahl HG (2006) Antimicrobial and host-defense peptides as new anti-infective therapeutic strategies. Nat Biotechnol 24: 1551–1557. doi: 10.1038/nbt1267
[2]
Brown KL, Hancock RE (2006) Cationic host defense (antimicrobial) peptides. Curr Opin Immunol 18: 24–30. doi: 10.1016/j.coi.2005.11.004
[3]
Peters BM, Shirtliff ME, Jabra-Rizk MA (2010) Antimicrobial peptides: primeval molecules or future drugs? PLoS Pathog 6: e1001067. doi: 10.1371/journal.ppat.1001067
[4]
Melo MN, Ferre R, Castanho MA (2009) Antimicrobial peptides: linking partition, activity and high membrane-bound concentrations. Nat Rev Microbiol 7: 245–250. doi: 10.1038/nrmicro2095
[5]
Zasloff M (2002) Antimicrobial peptides of multicellular organisms. Nature 415: 389–395. doi: 10.1038/415389a
[6]
Wang J, Wong ES, Whitley JC, Li J, Stringer JM, et al. (2011) Ancient antimicrobial peptides kill antibiotic-resistant pathogens: Australian mammals provide new options. PLoS One 6: e24030. doi: 10.1371/journal.pone.0024030
[7]
Mobarakai N, Quale JM, Landman D (1994) Bactericidal activities of peptide antibiotics against multidrug-resistant Enterococcus faecium. Antimicrob Agents Chemother 38: 385–387. doi: 10.1128/aac.38.2.385
[8]
Diz MS, Carvalho AO, Rodrigues R, Neves-Ferreira AG, Da Cunha M, et al. (2006) Antimicrobial peptides from chili pepper seeds causes yeast plasma membrane permeabilization and inhibits the acidification of the medium by yeast cells. Biochim Biophys Acta 1760: 1323–1332. doi: 10.1016/j.bbagen.2006.04.010
[9]
Moerman L, Bosteels S, Noppe W, Willems J, Clynen E, et al. (2002) Antibacterial and antifungal properties of alpha-helical, cationic peptides in the venom of scorpions from southern Africa. Eur J Biochem 269: 4799–4810. doi: 10.1046/j.1432-1033.2002.03177.x
[10]
Patrzykat A, Gallant JW, Seo JK, Pytyck J, Douglas SE (2003) Novel antimicrobial peptides derived from flatfish genes. Antimicrob Agents Chemother 47: 2464–2470. doi: 10.1128/aac.47.8.2464-2470.2003
[11]
Chen HC, Brown JH, Morell JL, Huang CM (1988) Synthetic magainin analogues with improved antimicrobial activity. FEBS Lett 236: 462–466. doi: 10.1016/0014-5793(88)80077-2
[12]
Sahl HG, Pag U, Bonness S, Wagner S, Antcheva N, et al. (2005) Mammalian defensins: structures and mechanism of antibiotic activity. J Leukoc Biol 77: 466–475.
[13]
Harder J, Bartels J, Christophers E, Schroder JM (1997) A peptide antibiotic from human skin. Nature 387: 861. doi: 10.1038/43088
[14]
Agerberth B, Lee JY, Bergman T, Carlquist M, Boman HG, et al. (1991) Amino acid sequence of PR-39. Isolation from pig intestine of a new member of the family of proline-arginine-rich antibacterial peptides. Eur J Biochem 202: 849–854. doi: 10.1111/j.1432-1033.1991.tb16442.x
[15]
Groot F, Sanders RW, ter Brake O, Nazmi K, Veerman EC, et al. (2006) Histatin 5-derived peptide with improved fungicidal properties enhances human immunodeficiency virus type 1 replication by promoting viral entry. J Virol 80: 9236–9243. doi: 10.1128/jvi.00796-06
[16]
Lesmes LP, Bohorquez MY, Carreno LF, Patarroyo ME, Lozano JM (2009) A C-terminal cationic fragment derived from an arginine-rich peptide exhibits in vitro antibacterial and anti-plasmodial activities governed by its secondary structure properties. Peptides 30: 2150–2160. doi: 10.1016/j.peptides.2009.08.011
[17]
Scocchi M, Tossi A, Gennaro R (2011) Proline-rich antimicrobial peptides: converging to a non-lytic mechanism of action. Cell Mol Life Sci 68: 2317–2330. doi: 10.1007/s00018-011-0721-7
[18]
Block TM, Guo H, Guo JT (2007) Molecular virology of hepatitis B virus for clinicians. Clin Liver Dis 11: 685–706, vii. doi: 10.1016/j.cld.2007.08.002
[19]
Thomas D, Zoulim F (2012) New challenges in viral hepatitis. Gut 61 Suppl 1: i1–5. doi: 10.1136/gutjnl-2012-302122
[20]
Hirsch RC, Lavine JE, Chang LJ, Varmus HE, Ganem D (1990) Polymerase gene products of hepatitis B viruses are required for genomic RNA packaging as wel as for reverse transcription. Nature 344: 552–555. doi: 10.1038/344552a0
[21]
Beck J, Nassal M (2007) Hepatitis B virus replication. World J Gastroenterol 13: 48–64.
[22]
Seeger C, Zoulim F, Mason WS (2007) Hepadnaviruses. In: Knipe DM, Howley P, Griffin DE, Lamb RA, Martin MA et al.., editors. Fields Virology. Philadelphia: Lippincott Williams & Wildins. pp 2977–3029.
[23]
Birnbaum F, Nassal M (1990) Hepatitis B virus nucleocapsid assembly: primary structure requirements in the core protein. J Virol 64: 3319–3330.
[24]
Nassal M (1992) The arginine-rich domain of the hepatitis B virus core protein is required for pregenome encapsidation and productive viral positive-strand DNA synthesis but not for virus assembly. J Virol 66: 4107–4116.
[25]
Summers J, Mason WS (1982) Replication of the genome of a hepatitis B–like virus by reverse transcription of an RNA intermediate. Cell 29: 403–415. doi: 10.1016/0092-8674(82)90157-x
[26]
Chua PK, Tang FM, Huang JY, Suen CS, Shih C (2010) Testing the balanced electrostatic interaction hypothesis of hepatitis B virus DNA synthesis by using an in vivo charge rebalance approach. J Virol 84: 2340–2351. doi: 10.1128/jvi.01666-09
[27]
Newman M, Chua PK, Tang FM, Su PY, Shih C (2009) Testing an electrostatic interaction hypothesis of hepatitis B virus capsid stability by using an in vitro capsid disassembly/reassembly system. J Virol 83: 10616–10626. doi: 10.1128/jvi.00749-09
[28]
Li HC, Huang EY, Su PY, Wu SY, Yang CC, et al. (2010) Nuclear export and import of human hepatitis B virus capsid protein and particles. PLoS Pathog 6: e1001162. doi: 10.1371/journal.ppat.1001162
[29]
Aspedon A, Groisman EA (1996) The antibacterial action of protamine: evidence for disruption of cytoplasmic membrane energization in Salmonella typhimurium. Microbiology 142(Pt 12): 3389–3397. doi: 10.1099/13500872-142-12-3389
[30]
Huang YC, Lin YM, Chang TW, Wu SJ, Lee YS, et al. (2007) The flexible and clustered lysine residues of human ribonuclease 7 are critical for membrane permeability and antimicrobial activity. J Biol Chem 282: 4626–4633. doi: 10.1074/jbc.m607321200
[31]
Chang KC, Lin MF, Lin NT, Wu WJ, Kuo HY, et al. (2012) Clonal spread of multidrug-resistant Acinetobacter baumannii in eastern Taiwan. J Microbiol Immunol Infect 45: 37–42. doi: 10.1016/j.jmii.2011.09.019
[32]
Falagas ME, Koletsi PK, Bliziotis IA (2006) The diversity of definitions of multidrug-resistant (MDR) and pandrug-resistant (PDR) Acinetobacter baumannii and Pseudomonas aeruginosa. J Med Microbiol 55: 1619–1629. doi: 10.1099/jmm.0.46747-0
[33]
CLSI (2009) Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically. 8th edition. Informational supplement: M07-A08. Wayne (Pennsylvania): CLSI.
[34]
Aramwit P, Kanokpanont S, Nakpheng T, Srichana T (2010) The effect of sericin from various extraction methods on cell viability and collagen production. Int J Mol Sci 11: 2200–2211. doi: 10.3390/ijms11052200
[35]
Chen CP, Chou JC, Liu BR, Chang M, Lee HJ (2007) Transfection and expression of plasmid DNA in plant cells by an arginine-rich intracellular delivery peptide without protoplast preparation. FEBS Lett 581: 1891–1897. doi: 10.1016/j.febslet.2007.03.076
[36]
Fernandez L, Alvarez-Ortega C, Wiegand I, Olivares J, Kocincova D, et al. (2013) Characterization of the Polymyxin B Resistome of Pseudomonas aeruginosa. Antimicrob Agents Chemother 57: 110–119. doi: 10.1128/aac.01583-12
[37]
Wang Z, Wang G (2004) APD: the Antimicrobial Peptide Database. Nucleic Acids Res 32: D590–592. doi: 10.1093/nar/gkh025
[38]
Noskin GA, Rubin RJ, Schentag JJ, Kluytmans J, Hedblom EC, et al. (2005) The burden of Staphylococcus aureus infections on hospitals in the United States: an analysis of the 2000 and 2001 Nationwide Inpatient Sample Database. Arch Intern Med 165: 1756–1761. doi: 10.1001/archinte.165.15.1756
[39]
Hancock RE (1998) Resistance mechanisms in Pseudomonas aeruginosa and other nonfermentative gram-negative bacteria. Clin Infect Dis 27 Suppl 1: S93–99. doi: 10.1086/514909
[40]
Evans ME, Feola DJ, Rapp RP (1999) Polymyxin B sulfate and colistin: old antibiotics for emerging multiresistant gram-negative bacteria. Ann Pharmacother 33: 960–967. doi: 10.1345/aph.18426
[41]
Fernandez L, Gooderham WJ, Bains M, McPhee JB, Wiegand I, et al. (2010) Adaptive resistance to the “last hope” antibiotics polymyxin B and colistin in Pseudomonas aeruginosa is mediated by the novel two-component regulatory system ParR-ParS. Antimicrob Agents Chemother 54: 3372–3382. doi: 10.1128/aac.00242-10
[42]
McPhee JB, Lewenza S, Hancock RE (2003) Cationic antimicrobial peptides activate a two-component regulatory system, PmrA-PmrB, that regulates resistance to polymyxin B and cationic antimicrobial peptides in Pseudomonas aeruginosa. Mol Microbiol 50: 205–217. doi: 10.1046/j.1365-2958.2003.03673.x
[43]
Fernandez L, Jenssen H, Bains M, Wiegand I, Gooderham WJ, et al. (2012) The two-component system CprRS senses cationic peptides and triggers adaptive resistance in Pseudomonas aeruginosa independently of ParRS. Antimicrob Agents Chemother 56: 6212–6222. doi: 10.1128/aac.01530-12
[44]
Bulet P, Urge L, Ohresser S, Hetru C, Otvos L Jr (1996) Enlarged scale chemical synthesis and range of activity of drosocin, an O-glycosylated antibacterial peptide of Drosophila. Eur J Biochem 238: 64–69. doi: 10.1111/j.1432-1033.1996.0064q.x
[45]
Otvos L Jr (2002) The short proline-rich antibacterial peptide family. Cell Mol Life Sci 59: 1138–1150. doi: 10.1007/s00018-002-8493-8
[46]
Pini A, Falciani C, Mantengoli E, Bindi S, Brunetti J, et al. (2010) A novel tetrabranched antimicrobial peptide that neutralizes bacterial lipopolysaccharide and prevents septic shock in vivo. FASEB J 24: 1015–1022. doi: 10.1096/fj.09-145474
[47]
Lin YM, Wu SJ, Chang TW, Wang CF, Suen CS, et al. (2010) Outer membrane protein I of Pseudomonas aeruginosa is a target of cationic antimicrobial peptide/protein. J Biol Chem 285: 8985–8994. doi: 10.1074/jbc.m109.078725
[48]
Chang TW, Lin YM, Wang CF, Liao YD (2012) Outer Membrane Lipoprotein Lpp Is Gram-negative Bacterial Cell Surface Receptor for Cationic Antimicrobial Peptides. J Biol Chem 287: 418–428. doi: 10.1074/jbc.m111.290361
[49]
Miller SI, Ernst RK, Bader MW (2005) LPS, TLR4 and infectious disease diversity. Nat Rev Microbiol 3: 36–46. doi: 10.1038/nrmicro1068
[50]
Kulshin VA, Zahringer U, Lindner B, Jager KE, Dmitriev BA, et al. (1991) Structural characterization of the lipid A component of Pseudomonas aeruginosa wild-type and rough mutant lipopolysaccharides. Eur J Biochem 198: 697–704. doi: 10.1111/j.1432-1033.1991.tb16069.x
[51]
Schneider T, Kruse T, Wimmer R, Wiedemann I, Sass V, et al. (2010) Plectasin, a fungal defensin, targets the bacterial cell wall precursor Lipid II. Science 328: 1168–1172. doi: 10.1126/science.1185723
[52]
Kashyap DR, Wang M, Liu LH, Boons GJ, Gupta D, et al. (2011) Peptidoglycan recognition proteins kill bacteria by activating protein-sensing two-component systems. Nat Med 17: 676–683. doi: 10.1038/nm.2357
[53]
Park CB, Kim HS, Kim SC (1998) Mechanism of action of the antimicrobial peptide buforin II: buforin II kills microorganisms by penetrating the cell membrane and inhibiting cellular functions. Biochem Biophys Res Commun 244: 253–257. doi: 10.1006/bbrc.1998.8159
[54]
Perez F, Joliot A, Bloch-Gallego E, Zahraoui A, Triller A, et al. (1992) Antennapedia homeobox as a signal for the cellular internalization and nuclear addressing of a small exogenous peptide. J Cell Sci 102(Pt 4): 717–722.
[55]
Vives E, Brodin P, Lebleu B (1997) A truncated HIV-1 Tat protein basic domain rapidly translocates through the plasma membrane and accumulates in the cell nucleus. J Biol Chem 272: 16010–16017. doi: 10.1074/jbc.272.25.16010
[56]
Futaki S, Suzuki T, Ohashi W, Yagami T, Tanaka S, et al. (2001) Arginine-rich peptides. An abundant source of membrane-permeable peptides having potential as carriers for intracellular protein delivery. J Biol Chem 276: 5836–5840. doi: 10.1074/jbc.m007540200
[57]
Tosteson MT, Holmes SJ, Razin M, Tosteson DC (1985) Melittin lysis of red cells. J Membr Biol 87: 35–44. doi: 10.1007/bf01870697
[58]
Duerkop BA, Vaishnava S, Hooper LV (2009) Immune responses to the microbiota at the intestinal mucosal surface. Immunity 31: 368–376. doi: 10.1016/j.immuni.2009.08.009
[59]
Cerf-Bensussan N, Gaboriau-Routhiau V (2010) The immune system and the gut microbiota: friends or foes? Nat Rev Immunol 10: 735–744. doi: 10.1038/nri2850
[60]
Abt MC, Osborne LC, Monticelli LA, Doering TA, Alenghat T, et al. (2012) Commensal bacteria calibrate the activation threshold of innate antiviral immunity. Immunity 37: 158–170. doi: 10.1016/j.immuni.2012.04.011
[61]
Ostman S, Rask C, Wold AE, Hultkrantz S, Telemo E (2006) Impaired regulatory T cell function in germ-free mice. Eur J Immunol 36: 2336–2346. doi: 10.1002/eji.200535244
[62]
Ivanov II, Frutos Rde L, Manel N, Yoshinaga K, Rifkin DB, et al. (2008) Specific microbiota direct the differentiation of IL-17-producing T-helper cells in the mucosa of the small intestine. Cell Host Microbe 4: 337–349. doi: 10.1016/j.chom.2008.09.009
[63]
Wu HJ, Ivanov II, Darce J, Hattori K, Shima T, et al. (2010) Gut-residing segmented filamentous bacteria drive autoimmune arthritis via T helper 17 cells. Immunity 32: 815–827. doi: 10.1016/j.immuni.2010.06.001
[64]
Chinen T, Volchkov PY, Chervonsky AV, Rudensky AY (2010) A critical role for regulatory T cell-mediated control of inflammation in the absence of commensal microbiota. J Exp Med 207: 2323–2330. doi: 10.1084/jem.20101235
[65]
Cirera I, Bauer TM, Navasa M, Vila J, Grande L, et al. (2001) Bacterial translocation of enteric organisms in patients with cirrhosis. J Hepatol 34: 32–37. doi: 10.1016/s0168-8278(00)00013-1
Tandon P, Garcia-Tsao G (2008) Bacterial infections, sepsis, and multiorgan failure in cirrhosis. Semin Liver Dis 28: 26–42. doi: 10.1055/s-2008-1040319
[68]
Huang LR, Wu HL, Chen PJ, Chen DS (2006) An immunocompetent mouse model for the tolerance of human chronic hepatitis B virus infection. Proc Natl Acad Sci U S A 103: 17862–17867. doi: 10.1073/pnas.0608578103
