EcDBS1R6: A novel cationic antimicrobial peptide derived from a signal peptide sequence

BackgroundBacterial infections represent a major worldwide health problem the antimicrobial peptides (AMPs) have been considered as potential alternative agents for treating these infections. Here we demonstrated the antimicrobial activity of EcDBS1R6, a peptide derived from a signal peptide sequence of Escherichia coli that we previously turned into an AMP by making changes through the Joker algorithm.MethodsAntimicrobial activity was measured by broth microdilution method. Membrane integrity was measured using fluorescent probes and through scanning electron microscopy imaging. A sliding window of truncated peptides was used to determine the EcDBS1R6 active core. Molecular dynamics in TFE/water environment was used to assess the EcDBS1R6 structure.ResultsSignal peptides are known to naturally interact with membranes; however, the modifications introduced by Joker transformed this peptide into a membrane-active agent capable of killing bacteria. The C-terminus was unable to fold into an α-helix whereas its fragments showed poor or no antimicrobial activity, suggesting that the EcDBS1R6 antibacterial core was located at the helical N-terminus, corresponding to the signal peptide portion of the parent peptide.ConclusionThe strategy of transforming signal peptides into AMPs appears to be promising and could be used to produce novel antimicrobial agents.General significanceThe process of transforming an inactive signal peptide into an antimicrobial peptide could open a new venue for creating new AMPs derived from signal peptides.     

Outer Membrane Protein I of Pseudomonas aeruginosa Is a Target of Cationic Antimicrobial Peptide/Protein

Cationic antimicrobial peptides/proteins (AMPs) are important components of the host innate defense mechanisms against invading microorganisms. Here we demonstrate that OprI (outer membrane protein I) of Pseudomonas aeruginosa is responsible for its susceptibility to human ribonuclease 7 (hRNase 7) and α-helical cationic AMPs, instead of surface lipopolysaccharide, which is the initial binding site of cationic AMPs. The antimicrobial activities of hRNase 7 and α-helical cationic AMPs against P. aeruginosa were inhibited by the addition of exogenous OprI or anti-OprI antibody. On modification and internalization of OprI by hRNase 7 into cytosol, the bacterial membrane became permeable to metabolites. The lipoprotein was predicted to consist of an extended loop at the N terminus for hRNase 7/lipopolysaccharide binding, a trimeric α-helix, and a lysine residue at the C terminus for cell wall anchoring. Our findings highlight a novel mechanism of antimicrobial activity and document a previously unexplored target of α-helical cationic AMPs, which may be used for screening drugs to treat antibiotic-resistant bacterial infection.     

Discovery of new inhibitors against both NF-κB and osteoclastogenesis from in-house library with α, β-unsaturated-enone fragment

The α,β-unsaturated-enone contained natural products have been reported showing NF-κB inhibition effect. It is well known that NF-κB inhibitors can also be used to inhibit osteoclastogenesis. In a continual discovery new agents for anti-osteoclastogenesis, 8 different type compounds with α,β-unsaturated-enone fragments from our in-house library were evaluated for NF-κB inhibition and anti-osteoclastogenesis. Experimental results indicated five compounds exhibited inhibition of NF-κB signal pathway. Among them, one compound ((E)-2-(4-fluorobenzylidene)-3,4-dihydronaphthalen-1(2H)-one, 6a) simultaneously inhibits both osteoclastogenesis and NF-κB signal pathway. Furthermore, 12 compounds with similar scaffold with 6a were tested for anti-osteoclastogenesis. As a result, 9 compounds inhibited both NF-κB and osteoclastogenesis. Among them, compound 6b is the most potent inhibitor against NF-κB (IC₅₀ = 2.09 μM) and osteoclast differentiation (IC₅₀ = 0.86 μM). Further studies show that compound 6b blocks the phosphorylation of both p65 and IκBα, and suppresses NF-κB targeted gene expression without interfering MAPKs and PI3K/Akt signal transduction pathways. This study demonstrates that we can identify promising synthesized compounds with new scaffolds as therapeutic solutions against osteoclastogenesis inspired by the privileged fragment derived from natural leads.     

Phillyrin (KD-1) exerts anti-viral and anti-inflammatory activities against novel coronavirus (SARS-CoV-2) and human coronavirus 229E (HCoV-229E) by suppressing the nuclear factor kappa B (NF-κB) signaling pathway

BackgroundSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has extensively and rapidly spread in the world, causing an outbreak of acute infectious pneumonia. However, no specific antiviral drugs or vaccines can be used. Phillyrin (KD-1), a representative ingredient of Forsythia suspensa, possesses anti-inflammatory, anti-oxidant, and antiviral activities. However, little is known about the antiviral abilities and mechanism of KD-1 against SARS-CoV-2 and human coronavirus 229E (HCoV-229E).PurposeThe study was designed to investigate the antiviral and anti-inflammatory activities of KD-1 against the novel SARS-CoV-2 and HCoV-229E and its potential effect in regulating host immune response in vitro.MethodsThe antiviral activities of KD-1 against SARS-CoV-2 and HCoV-229E were assessed in Vero E6 cells using cytopathic effect and plaque-reduction assay. Proinflammatory cytokine expression levels upon infection with SARS-CoV-2 and HCoV-229E infection in Huh-7 cells were measured by real-time quantitative PCR assays. Western blot assay was used to determine the protein expression of nuclear factor kappa B (NF-κB) p65, p-NF-κB p65, IκBα, and p-IκBα in Huh-7 cells, which are the key targets of the NF-κB pathway.ResultsKD-1 could significantly inhibit SARS-CoV-2 and HCoV-229E replication in vitro. KD-1 could also markedly reduce the production of proinflammatory cytokines (TNF-α, IL-6, IL-1β, MCP-1, and IP-10) at the mRNA levels. Moreover, KD-1 could significantly reduce the protein expression of p-NF-κB p65, NF-κB p65, and p-IκBα, while increasing the expression of IκBα in Huh-7 cells.ConclusionsKD-1 could significantly inhibit virus proliferation in vitro, the up-regulated expression of proinflammatory cytokines induced by SARS-CoV-2 and HCoV-229E by regulating the activity of the NF-кB signaling pathway. Our findings indicated that KD-1 protected against virus attack and can thus be used as a novel strategy for controlling the coronavirus disease 2019.     

Antimicrobial Activities of Novel Peptides Derived from Defensin Genes of <Emphasis Type="Italic">Brassica hybrid</Emphasis> cv Pule

Plant defensins are small and basic antimicrobial peptides characterized by conserved cysteine stabilizing structure with α-helix and triple strand antiparallel β-sheet. In the present study, two novel defensin genes, designated as BhDef1 and BhDef2, was isolated from Brassica hybrid cv Pule, a native unexplored Brassicaceae species found in Thailand. The full-length cDNA of BhDef1 and BhDef2 were 240 and 258 bp encoding a 79 and 85 amino acid residues with 29 and 25 amino acid signal peptide at N-terminal, respectively. The putative BhDef1 and BhDef2 mature proteins showed significant similarity to other Brassicaceae defensins. Their secondary structure comprises of one α-helix and a triple stranded β-sheet stabilized by four disulphide bridges of eight cysteines. BhDef1 and BhDef2 also contain a highly conserved γ-core and α-core motif exhibiting antifungal activity against Colletotrichum gloeosporioides causing anthracnose disease. Six out of eight synthetic BhDef peptide derivatives showed antibacterial activity against both gram-positive bacteria and gram-negative bacteria used in this study. BhDef14, the derivative of BhDef1, showed the highest activity against two test pathogenic bacteria. This activity could probably due to a net positively charge and alpha-helical conformation which are known as the key determinant for the bacterial membrane disruption. To our knowledge, this is the first report on defensin genes isolated from B. hybrid cv Pule. The synthetic peptides designed from their sequences showed antifungal and antibacterial activity.     

Conformational Flexibility Determines Selectivity and Antibacterial, Antiplasmodial, and Anticancer Potency of Cationic ��-Helical Peptides

We used a combination of fluorescence, circular dichroism (CD), and NMR spectroscopies in conjunction with size exclusion chromatography to help rationalize the relative antibacterial, antiplasmodial, and cytotoxic activities of a series of proline-free and proline-containing model antimicrobial peptides (AMPs) in terms of their structural properties. When compared with proline-free analogs, proline-containing peptides had greater activity against Gram-negative bacteria, two mammalian cancer cell lines, and intraerythrocytic Plasmodium falciparum, which they were capable of killing without causing hemolysis. In contrast, incorporation of proline did not have a consistent effect on peptide activity against Mycobacterium tuberculosis. In membrane-mimicking environments, structures with high α-helix content were adopted by both proline-free and proline-containing peptides. In solution, AMPs generally adopted disordered structures unless their sequences comprised more hydrophobic amino acids or until coordinating phosphate ions were added. Proline-containing peptides resisted ordering induced by either method. The roles of the angle subtended by positively charged amino acids and the positioning of the proline residues were also investigated. Careful positioning of proline residues in AMP sequences is required to enable the peptide to resist ordering and maintain optimal antibacterial activity, whereas varying the angle subtended by positively charged amino acids can attenuate hemolytic potential albeit with a modest reduction in potency. Maintaining conformational flexibility improves AMP potency and selectivity toward bacterial, plasmodial, and cancerous cells while enabling the targeting of intracellular pathogens.     

Stepwise identification of potent antimicrobial peptides from human genome

The increasing incidence of hospital acquired infections caused by antibiotic resistant pathogens has led to an increase in morbidity and mortality, finding alternative antibiotics unaffected by resistance mechanisms is fundamentally important for treating this problem. Naturally occurring proteins usually carry short peptide fragments that exhibit noticeable biological activity against a wide variety of microorganisms such as bacteria, fungi and protozoa. Traditional discovery of such antimicrobially active fragments (i.e. antimicrobial peptides, AMPs) from protein repertoire is either random or led by chance. Here, we report the use of a rational protocol that combines in silico prediction and in vitro assay to identify potential AMPs with high activity and low toxicity from the entire human genome. In the procedure, a three-step inference strategy is first proposed to perform genome-wide analysis to infer AMPs in a high-throughput manner. By employing this strategy we are able to screen more than one million peptide candidates generated from various human proteins, from which we identify four highly promising samples, and subsequently their antibacterial activity on five strains as well as cytotoxicity on human myoblasts are tested experimentally. As a consequence, two high-activity, low-toxicity peptides are discovered, which could be used as the structural basis to further develop new antibiotics. In addition, from 1491 known AMPs we also derive a quantitative measure called antibacterial propensity index (API) for 20 naturally occurring amino acids, which shows a significant allometric correlation with the theoretical minimal inhibitory concentration of putative peptides against Gram-positive and Gram-negative bacteria. This study may provide a proof-of-concept paradigm for the genome-wide discovery of novel antimicrobial peptides by using a combination of in silico and in vitro analyses.     

Broad antiviral and anti-inflammatory activity of Qingwenjiere mixture against SARS-CoV-2 and other human coronavirus infections

BackgroundQingwenjiere Mixture (QJM) is a traditional Chinese medicine (TCM) that has been shown to have remarkable clinical efficacy against COVID-19. However, little is known about the antiviral and anti-inflammatory activities of QJM against a wider range of human coronavirus (HCoV) strains.PurposeThe study aims to investigate the antiviral and anti-inflammatory activities of QJM, as well as the underlying mechanisms against HCoV infections.MethodsThe chemical compositions from QJM were analyzed by LC-MS. The inhibitory effect of QJM on infections of HCoV-OC43, HCoV-229E, HCoV-NL63, and SARS-CoV-2 was evaluated in HRT-18 cells, Huh7 cells, LLC-MK2 cells, and Vero-E6 cells, respectively, by using cytopathic effect (CPE) inhibition assay or RT-qPCR detection of viral n, s, or RdRp/Hel genes. The expression of pro-inflammatory cytokines induced by HCoV-OC43, HCoV-229E, and SARS-CoV-2, as well as the host ace2 gene was also determined by RT-qPCR assay. Furthermore, the expression of key molecules in the NF-κB/MAPKs signaling pathways was determined by western blot.ResultsIn alcohol-extraction groups of QJM and reference decoction pieces, 53 similar ion peaks were identified, the majority of which were phenylpropanoids, iridoids, and flavonoids. In addition, QJM reduced CPE caused by HCoVs and the expression of viral n genes or N protein. Pretreatment with QJM also exerted inhibitory effect on viral n gene expression. QJM also inhibited the expression of RdRp/Hel and s genes of SARS-CoV-2, as well as the host ace2 gene. Besides, QJM markedly reduced virus-induced mRNA expression of a panel of pro-inflammatory cytokines, such as IL-6, CXCL-8/IL-8, CXCL-10/IP-10, CCL-5/RANTES, TNF-α, IFN-α, CCL-2/MCP-1, CXCL-9/MIG, and IL1-α. We further showed that QJM inhibited the phosphorylation of NF-κB p65, and JNK, ERK 1/2, and p38 MAPKs in HCoV-OC43-infected HRT-18 cells.ConclusionsQJM has broad antiviral and anti-inflammatory activity against both common and newly emerged HCoVs possibly by inhibiting the activation of key components in NF-κB/MAPKs signaling pathway. QJM also has a prevention effect against HCoV infections and inhibits the host receptor required for virus entry. These results indicate that QJM may have the therapeutic potential in the treatment of diseases caused by a broad range of HCoVs.     

Identification and Characterization of Novel Antimicrobial Peptide from <Emphasis Type="Italic">Hippocampus comes</Emphasis> by In Silico and Experimental Studies

Antimicrobial peptides (AMPs) have attracted attentions as a novel antimicrobial agent because of their unique activity against microbes. In the present study, we described a new, previously unreported AMP, moronecidin-like peptide, from Hippocampus comes and compared its antimicrobial activity with moronecidin from hybrid striped bass. Antibacterial assay indicated that gram-positive bacteria were more sensitive to moronecidin and moronecidin-like compared with gram-negative bacteria. Furthermore, both AMPs were found to exhibit effective antifungal activity. Comparative analysis of the antimicrobial activity revealed that moronecidin-like peptide has higher activity against Acinetobacter baumannii and Staphylococcus epidermidis relative to moronecidin. Both moronecidin-like and moronecidin peptides retained their antibacterial activity in physiological pH and salt concentration. The time-killing assay showed that the AMPs completely killed A. baumannii and S. epidermidis isolates after 1 and 5 h at five- and tenfold above their corresponding MICs, respectively. Anti-biofilm assay demonstrated that peptides were able to inhibit 50% of biofilm formation at sub-MIC of 1/8 MIC. Furthermore, moronecidin-like significantly inhibited biofilm formation more than moronecidin at 1/16 MIC. Collectively, our results revealed that antimicrobial and anti-biofilm activities of moronecidin-like are comparable to moronecidin. In addition, the hemolytic and cytotoxic activities of moronecidin-like were lower than those of moronecidin, suggesting it as a potential novel therapeutic agent, and a template to design new therapeutic AMPs.     

NIK and Cot cooperate to trigger NF-kappaB p65 phosphorylation

The serine/threonine kinase Cot triggers NF-κB-dependent transactivation and activation of various MAPKinases. Here we identify Cot as a novel p65 interacting protein kinase. Cot expression induces p65 phosphorylation at serines 536 and 468 in dependence from its kinase function. Accordingly, shRNA-mediated knockdown of Cot expression interferes with TNF-induced NF-κB-dependent gene expression. Also the C-terminally truncated, oncogenic form of Cot is able to trigger p65 phosphorylation. In vitro kinase assays and dominant negative mutants revealed that NIK functions downstream of Cot to mediate p65 phosphorylation.     

Fast and potent bactericidal membrane lytic activity of PaDBS1R1, a novel cationic antimicrobial peptide

Antimicrobial peptides (AMPs) are promising candidates for the development of future antibiotics. In an attempt to increase the efficacy of therapeutic AMPs, computer-based design methods appear as a reliable strategy. In this study, we evaluated the antimicrobial efficiency and mechanism of action of a novel designed AMP named PaDBS1R1, previously designed by means of the Joker algorithm, using a fragment of the ribosomal protein L39E from the archaeon Pyrobaculum aerophilum as a template. PaDBS1R1 displayed low micromolar broad-spectrum antimicrobial activity against Gram-negative (MIC of 1.5?μM) and Gram-positive (MIC of 3?μM) bacteria, including carbapenem-resistant Klebsiella pneumoniae (MIC of 6.25?μM) and methicillin-resistant Staphylococcus aureus (MIC of 12.5?μM), without cytotoxicity towards HEK-293 cells. In addition, membrane permeabilization and depolarization assays, combined with time-kill studies and FEG-SEM imaging, indicated a fast membrane permeation and further leakage of intracellular content. Biophysical studies with lipid vesicles show a preference of PaDBS1R1 for Gram-negative bacteria-like membranes. We investigated the three-dimensional structure of PaDBS1R1 by CD and NMR analyses. Our results suggest that PaDBS1R1 adopts an amphipathic α-helix upon interacting with hydrophobic environments, after an initial electrostatic interaction with negative charges, suggesting a membrane lytic effect. This study reveals that PaDBS1R1 has potential application in antibiotic therapy.