Identification of a Novel Antimicrobial Peptide from Human Hepatitis B Virus Core Protein Arginine-Rich Domain (ARD)

Abstract

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.

Novel Engineered Peptides of a Phage Lysin as Effective Antimicrobials against Multidrug-Resistant Acinetobacter baumannii

“…In addition, we fused eight amino acids (SQSRESQC) to the C terminus of P307 and yielded P307 SQ-8C (Table 1). This modification was based on the observation of an earlier study showing that the lack of these eight amino acids reduces the activity spectrum of the antimicrobial peptide from hepatitis B virus core protein (28). We also generated two additional modifications of P307 SQ-8C : scrambling the sequence of the last eight amino acids to CSQRQSES (P307 CS-8 ); and changing the last amino acid from C to A (P307 SQ-8A ) ( Table 1).…”

“…The permeability of bacterial membrane upon peptide treatment was measured by SYTOX green uptake (28). Briefly, overnight cultures of bacteria were washed and resuspended to ϳ10 7 CFU/ml in 50 mM Tris-HCl (pH 7.5).…”

“…The hemolytic assays were conducted as previously described (28). Briefly, human blood was gathered in an EDTA tube, and red blood cells (RBCs) were collected through low-speed centrifugation.…”

Nucleic Acid Chaperone Activity Associated with the Arginine-Rich Domain of Human Hepatitis B Virus Core Protein

“…This result is consistent with our previous studies of R-to-K substitutions in HBc ARD (32). difficulty in testing this hypothesis in HBV by in vivo experiments is related to the multiple overlapping functions of the ARD domain in the life cycle of HBV, including capsid assembly/disassembly (29,62), RNA encapsidation (10,11,30,32), core protein trafficking (57,58,(64)(65)(66), DNA synthesis (9-13, 32, 62, 63, 67), and antimicrobial activity (68). Given such an extreme degree of functional complexity associated with a very short HBc ARD domain (HBc 147-183), it is conceivable that attempts to dissociate nucleic acid chaperone activity of HBc ARD from its many other reported functions in vivo will be very challenging in the future.…”

Utilisation of peptides against microbial infections – a review

“…They showed that a peptide containing the complete ARD I-IV (HBc 147-183) acts antimicrobially at micromolar concentrations against some multidrugresistant pathogens, including colistin-polymyxin E-resistant Acinetobacter baumannii [43]. It was also determined that the peptides ARD II-IV (HBc 153-176) and ARD I-III (HBc 147-167) showed activity against P. aeruginosa and K. pneumoniae.…”