Characterization and Localization of Sol g 2.1 Protein from Solenopsis geminata Fire Ant Venom in the Central Nervous System of Injected Crickets (Acheta domestica).

Solenopsis geminata is recognized for containing the allergenic proteins Sol g 1, 2, 3, and 4 in its venom. Remarkably, Sol g 2.1 exhibits hydrophobic binding and has a high sequence identity (83.05%) with Sol i 2 from S. invicta. Notably, Sol g 2.1 acts as a mediator, causing paralysis in crickets. Given its structural resemblance and biological function, Sol g 2.1 may play a key role in transporting hydrophobic potent compounds, which induce paralysis by releasing the compounds through the insect's nervous system. To investigate this further, we constructed and characterized the recombinant Sol g 2.1 protein (rSol g 2.1), identified with LC-MS/MS. Circular dichroism spectroscopy was performed to reveal the structural features of the rSol g 2.1 protein. Furthermore, after treating crickets with S. geminata venom, immunofluorescence and immunoblotting results revealed that the Sol g 2.1 protein primarily localizes to the neuronal cell membrane of the brain and thoracic ganglia, with distribution areas related to octopaminergic neuron cell patterns. Based on protein-protein interaction predictions, we found that the Sol g 2.1 protein can interact with octopamine receptors (OctRs) in neuronal cell membranes, potentially mediating Sol g 2.1's localization within cricket central nervous systems. Here, we suggest that Sol g 2.1 may enhance paralysis in crickets by acting as carriers of active molecules and releasing them onto target cells through pH gradients. Future research should explore the binding properties of Sol g 2.1 with ligands, considering its potential as a transporter for active molecules targeting pest nervous systems, offering innovative pest control prospects.

Novel non-cytotoxic antimicrobial peptides WSKK11 and WSRR11 with potent activity against Cutibacterium acnes.

OBJECTIVES: Cutibacterium acnes is one of the common multifactorial causes that play an important role in the pathophysiology of acne vulgaris. We aimed to develop novel antimicrobial peptides for reduction of the hypercolonization. METHODS: Six cationic peptides were derived by de novo designation. The antimicrobial and cytotoxic activities of peptides were investigated. The peptide conformation was determined by circular dichroism spectrometry. The antimicrobial effects of peptides were evaluated using scanning electron microscopy (SEM), transmission electron microscopy (TEM) and DNA-binding ability assay. RESULTS: Among designed peptides, WSKK11 and WSRR11 were effective antimicrobials against C. acnes at MICs of 128 and 64 mg/L, respectively. The MICs of WSKK11 against Staphylococcus epidermidis, Staphylococcus aureus and Candida albicans were 8, 8 and 32 mg/L, while those of WSRR11 were 64, 32 and 32 mg/L, respectively. WSKK11 and WSRR11 were less toxic to human erythrocytes (<2%) and not toxic to macrophages, keratinocytes and fibroblasts up to 512 mg/L. WSKK11 and WSRR11 mostly revealed the conformation of the undefined or random coil structures under mimicked environmental conditions. The peptides affected cell surfaces and cell membranes of C. acnes as well as possibly translocating through the cell membrane, observed by a combination of SEM and TEM, respectively. WSKK11 and WSRR11 had the ability to bind bacterial DNA. CONCLUSIONS: The two novel antimicrobial peptides WSKK11 and WSRR11 are members of a new class of antimicrobial agents that could deal with acne problems. Therefore, the antimicrobial peptides may be promising novel active agents for dermatological, beauty and cosmeceutical applications.

Evaluation of TILI-2 as an Anti-Tyrosinase, Anti-Oxidative Agent and Its Role in Preventing Melanogenesis Using a Proteomics Approach.

There is a desire to develop new molecules that can combat hyperpigmentation. To this end, the N-terminal cysteine-containing heptapeptide TILI-2 has shown promising preliminary results. In this work, the mechanism by which it works was evaluated using a series of biochemical assays focusing on known biochemical pathways, followed by LC-MS/MS proteomics to discover pathways that have not been considered before. We demonstrate that TILI-2 is a competitive inhibitor of tyrosinase's monophenolase activity and it could potentially scavenge ABTS and DPPH radicals. It has a very low cytotoxicity up to 1400 µM against human fibroblast NFDH cells and macrophage-like RAW 264.7 cells. Our proteomics study revealed that another putative mechanism by which TILI-2 may reduce melanin production involves the disruption of the TGF-ß signaling pathway in mouse B16F1 cells. This result suggests that TILI-2 has potential scope to be used as a depigmenting agent.

An Integrated Proteomics and Bioinformatics Analysis of the Anticancer Properties of RT2 Antimicrobial Peptide on Human Colon Cancer (Caco-2) Cells.

New selective, efficacious chemotherapy agents are in demand as traditional drugs display side effects and face growing resistance upon continued administration. To this end, bioactive molecules such as peptides are attracting interest. RT2 is a cationic peptide that was used as an antimicrobial but is being repurposed for targeting cancer. In this work, we investigate the mechanism by which this peptide targets Caco-2 human colon cancer cells, one of the most prevalent and metastatic cancers. Combining label-free proteomics with bioinformatics data, our data explore over 1000 proteins to identify 133 proteins that are downregulated and 79 proteins that are upregulated upon treatment with RT2. These changes occur in a dose-dependent manner and suggest the former group are related to anticancer cell proliferation; the latter group is closely related to apoptosis levels. The mRNA levels of several genes (FGF8, PAPSS2, CDK12, LDHA, PRKCSH, CSE1L, STARD13, TLE3, and OGDHL) were quantified using RT-qPCR and were found to be in agreement with proteomic results. Collectively, the global change in Caco-2 cell protein abundance suggests that RT2 triggers multiple mechanisms, including cell proliferation reduction, apoptosis activation, and alteration of cancerous cell metabolism.

In Silico and In Vitro Structure-Activity Relationship of Mastoparan and Its Analogs.

Antimicrobial peptides are an important class of therapeutic agent used against a wide range of pathogens such as Gram-negative and Gram-positive bacteria, fungi, and viruses. Mastoparan (MpVT) is an α-helix and amphipathic tetradecapeptide obtained from Vespa tropica venom. This peptide exhibits antibacterial activity. In this work, we investigate the effect of amino acid substitutions and deletion of the first three C-terminal residues on the structure-activity relationship. In this in silico study, the predicted structure of MpVT and its analog have characteristic features of linear cationic peptides rich in hydrophobic and basic amino acids without disulfide bonds. The secondary structure and the biological activity of six designed analogs are studied. The biological activity assays show that the substitution of phenylalanine (MpVT1) results in a higher antibacterial activity than that of MpVT without increasing toxicity. The analogs with the first three deleted C-terminal residues showed decreased antibacterial and hemolytic activity. The CD (circular dichroism) spectra of these peptides show a high content α-helical conformation in the presence of 40% 2,2,2-trifluoroethanol (TFE). In conclusion, the first three C-terminal deletions reduced the length of the α-helix, explaining the decreased biological activity. MpVTs show that the hemolytic activity of mastoparan is correlated to mean hydrophobicity and mean hydrophobic moment. The position and spatial arrangement of specific hydrophobic residues on the non-polar face of α-helical AMPs may be crucial for the interaction of AMPs with cell membranes.

Novel Antimicrobial Peptides from a Cecropin-Like Region of Heteroscorpine-1 from Heterometrus laoticus Venom with Membrane Disruption Activity.

The increasing antimicrobial-resistant prevalence has become a severe health problem. It has led to the invention of a new antimicrobial agent such as antimicrobial peptides. Heteroscorpine-1 is an antimicrobial peptide that has the ability to kill many bacterial strains. It consists of 76 amino acid residues with a cecropin-like region in N-terminal and a defensin-like region in the C-terminal. The cecropin-like region from heteroscorpine-1 (CeHS-1) is similar to cecropin B, but it lost its glycine-proline hinge region. The bioinformatics prediction was used to help the designing of mutant peptides. The addition of glycine-proline hinge and positively charged amino acids, the deletion of negatively charged amino acids, and the optimization of the hydrophobicity of the peptide resulted in two mutant peptides, namely, CeHS-1 GP and CeHS-1 GPK. The new mutant peptide showed higher antimicrobial activity than the native peptide without increasing toxicity. The interaction of the peptides with the membrane showed that the peptides were capable of disrupting both the inner and outer bacterial cell membrane. Furthermore, the SEM analysis showed that the peptides created the pore in the bacterial cell membrane resulted in cell membrane disruption. In conclusion, the mutants of CeHS-1 had the potential to develop as novel antimicrobial peptides.

Tannic Acid-Stabilized Silver Nanoparticles Used in Biomedical Application as an Effective Antimelioidosis and Prolonged Efflux Pump Inhibitor against Melioidosis Causative Pathogen.

Burkholderia pseudomallei is the causative pathogen of melioidosis and this bacterium is resistant to several antibiotics. Silver nanoparticles (AgNPs) are an interesting agent to develop to solve this bacterial resistance. Here, we characterize and assess the antimelioidosis activity of AgNPs against these pathogenic bacteria. AgNPs were characterized and displayed a maximum absorption band at 420 nm with a spherical shape, being well-monodispersed and having high stability in solution. The average size of AgNPs is 7.99 ± 1.46 nm. The antibacterial efficacy of AgNPs was evaluated by broth microdilution. The bactericidal effect of AgNPs was further assessed by time-kill kinetics assay. Moreover, the effect of AgNPs on the inhibition of the established biofilm was investigated by the crystal violet method. In parallel, a study of the resistance induction development of B. pseudomallei towards AgNPs with efflux pump inhibiting effect was performed. We first found that AgNPs had strong antibacterial activity against both susceptible and ceftazidime-resistant (CAZ-resistant) strains, as well as being efficiently active against B. pseudomallei CAZ-resistant strains with a fast-killing mode via a bactericidal effect within 30 min. These AgNPs did not only kill planktonic bacteria in broth conditions, but also in established biofilm. Our findings first documented that the resistance development was not induced in B. pseudomallei toward AgNPs in the 30th passage. We found that AgNPs still showed an effective efflux pump inhibiting effect against these bacteria after prolonged exposure to AgNPs at sublethal concentrations. Thus, AgNPs have valuable properties for being a potent antimicrobial agent to solve the antibiotic resistance problem in pathogens.

The cationic cell-penetrating KT2 peptide promotes cell membrane defects and apoptosis with autophagy inhibition in human HCT 116 colon cancer cells.

The anticancer activity of cationic antimicrobial peptides (AMPs) has become more interesting because some AMPs have selective recognition against cancer cells. However, their antitumor properties and underlying mechanisms in cancer cells have not been clearly understood. In this study, we evaluated the effects of KT2 (lysine/tryptophan-rich AMP) on the cellular uptake and internalization mechanism, cell viability, surface charge of the cell membrane, membrane integrity, apoptotic cell death, and autophagy in human HCT 116 colon cancer cells. We found that KT2 interacted with the cell membrane of HCT 116 cells and was internalized into HCT 116 cells via clathrin-mediated and caveolae-mediated endocytosis mechanisms. The interaction of KT2 with cells caused cell membrane structure change, elevated membrane permeability, and KT2 also affected the lipid component. The results of atomic force microscopy showed cellular membrane defects of KT2-treated cells. The internalized KT2 induced nuclear condensation and apoptotic cell death. It elevated the apoptotic factor levels including those of cytochrome c and apoptosis-inducing factor. Furthermore, KT2 inhibited autophagy by the suppression of autophagy-related 5, autophagy-related 7, autophagy-related 16 like 1, and Beclin-1 proteins. In conclusion, these results revealed the cytotoxicity of cationic KT2 against HCT 116 cells and may help to clarify the interactions between cationic AMPs and cancer cells.

Identification of the first Crocodylus siamensis cathelicidin gene and RN15 peptide derived from cathelin domain exhibiting antibacterial activity.

Cathelicidins are effector molecules of vertebrate immunity that play vital roles against microbial invasion. They are widely identified in mammals, but few have been reported in Crocodilians, which are considered to be species with a powerful immune system. In the present study, we identified and characterized a novel cathelicidin from the blood of the Siamese crocodile, Crocodylus siamensis. A cDNA sequence (501 base pair) encoded a predicted 166-residue prepropeptide of C. siamensis cathelicidin (Cs-CATH), which comprised a 21-residue signal peptide, a 109-residue cathelin domain, and a 36-residue mature cathelicidin peptide. Multiple sequence alignment and phylogenetic analysis demonstrated that Cs-CATH shared a high degree of similarity with other crocodilian cathelicidins. Joint consideration of elastase cleavage site, physicochemical properties, and predicted secondary structure demonstrated that RN15 peptide is a candidate antimicrobial peptide derived from Cs-CATH. The synthetic RN15 peptide demonstrates antimicrobial activity against Gram-positive and Gram-negative bacteria. Scanning electron microscopy illustrated RN15-peptide-induced bacteria cells exhibited morphological change. Besides, RN15 peptide demonstrates low hemolytic activity against human erythrocytes and low cytotoxic activity against normal human dermal fibroblasts. This is the first cathelicidin identified from C. siamensis, and it is highlighted that its derived peptide from cathelin domain promises potent novel peptide antibiotics templates.

Novel antioxidant and anti-inflammatory peptides from the Siamese crocodile (Crocodylus siamensis) hemoglobin hydrolysate.

Novel antioxidant and anti-inflammatory peptides were isolated from hydrolysates of Siamese crocodile (Crocodylus siamensis) hemoglobin. C. siamensis hemoglobin hydrolysates (CHHs) were obtained by pepsin digestion at different incubation times (2, 4, 6, and 8 H) at 37 °C and subjected to antioxidant and anti-inflammatory activity assessment. CHH obtained by 2-H hydrolysis (2H-CHH) showed the highest anti-inflammatory activity with respect to decreasing nitric oxide (NO) production, whereas the strongest antioxidant activity was found for 6-H hydrolysis (6H-CHH) against nitric oxide radicals. To evaluate the anti-inflammatory and antioxidant activity of individual peptide components, 2H-CHH and 6H-CHH were purified by semipreparative HPLC. Peptide fraction P57 isolated from 6H-CHH was found to exhibit the highest nitric oxide radical inhibition activity (32.0%). Moreover, purification of 2H-CHH yielded peptide fraction P16, which displayed a high efficacy in decreasing NO production of macrophage RAW 264.7 cells (83.2%) and significantly reduced proinflammatory cytokines and inflammatory mediators interleukin-6 (IL-6), interleukin-1 beta (IL-1ß), and prostaglandin-E2 (PGE2 ) production to about 2.0, 0.3, and 1.9 ng/mL, respectively. Using LTQ orbitrap XL mass spectrometry, active peptide sequences were identified as antioxidant KIYFPHF (KF7), anti-inflammatory SAFNPHEKQ (SQ9), and IIHNEKVQAHGKKVL (IL15). Additionally, CHHs simulated gastric and intestinal in vitro digestion positively contributed to antioxidant and anti-inflammatory activity. Taken collectively, the results of this work demonstrate that CHHs contain several peptides with anti-inflammatory and antioxidant properties, which may prove valuable as treatment or supplement against diseases associated with inflammation and oxidative stress.

Antitumor activity of RT2 peptide derived from crocodile leukocyte peptide on human colon cancer xenografts in nude mice.

RT2, derived from the leukocyte peptide of Crocodylus siamensis, can kill human cervical cancer cells via apoptosis induction, but no evidence has shown in vivo. In this study, we investigated the antitumor effect of RT2 on human colon cancer xenografts in nude mice. Twenty-four mice were injected subcutaneously with human colon cancer HCT 116 cells. Eleven days after cancer cell implantation, the mice were treated with intratumoral injections of phosphate buffered saline (PBS) or RT2 (0.01, 0.1, and 1 mg/mouse) once every 2 days for a total of 5 times. The effect of a 10-day intratumoral injection of RT2 on body weight, biochemical, and hematological parameters in BALB/c mice showed no significant difference between the groups. Tumor volume showed a significant decrease only in the treatment group with RT2 (1 mg/mouse) at day 6 (P < .05), day 8 (P < .01), and day 10 (P < .01) after the first treatment. The protein expression levels of cleaved poly (ADP-ribose) polymerase (PARP), apoptosis-inducing factor (AIF), and the p53 tumor suppressor protein (p53) in xenograft tumors increased after treatment with RT2 (1 mg/mouse) compared to those in the PBS-injected group. Moreover, RT2 increased the expression of Endo G and Bcl-2 family proteins. Therefore, the peptide RT2 can inhibit tumor growth via the induction of apoptosis in an in vivo xenograft model.

Antimicrobial effects of novel peptides cOT2 and sOT2 derived from Crocodylus siamensis and Pelodiscus sinensis ovotransferrins.

In light of the increasing threat of bacterial drug resistance to human health on a global scale, research and development of antimicrobial peptides as a novel class of potent antibiotics has gained considerable attention. The present study focuses on the structural evaluation and membrane interaction of two new cationic antimicrobial peptides, cOT2 and sOT2, derived from Siamese crocodile (Crocodylus siamensis) and Chinese softshell turtle (Pelodiscus sinensis) ovotransferrins. Here, cOT1 (+3) and sOT1 (+3) were derived from reptile ovotransferrins by chromatographic purification and characterized by mass spectrometry and N-terminal sequencing analysis. In order to increase the antimicrobial efficacy, two novel peptides, cOT2 (+6) and sOT2 (+5), were designed and synthesized as "naturally-engineered" by primary amino acid sequence extension of cOT1 and sOT1, respectively. These rational designs of modified peptides were assayed in term of antimicrobial activity. These peptides display strong antimicrobial activity against several bacterial strains, e.g. Vibrio cholerae, Bacillus megaterium, and Bacillus pumilus TISTR 905, with MICs of 7-16.1µM. In terms of structural conformation in mimic environments, CD spectroscopic analysis of the secondary peptides structure features revealed fairly the similarity on α-helical content with magainin II. Hence, the modes of actions have been speculated as toroidal and carpet model. Furthermore, the disruption of intact bacterial cells induced by cOT2 and sOT2 was investigated by SEM and AFM. The results provided evidence that cOT2 and sOT2 have the potential to cause different morphological changes of bacterial cells and that these effects can be enhanced by increasing the peptide concentration.

Cationic amphipathic peptides KT2 and RT2 are taken up into bacterial cells and kill planktonic and biofilm bacteria.

We investigated the mechanisms of two tryptophan-rich antibacterial peptides (KT2 and RT2) obtained in a previous optimization screen for increased killing of both Gram-negative and Gram-positive bacteria pathogens. At their minimal inhibitory concentrations (MICs), these peptides completely killed cells of multidrug-resistant, enterohemorrhagic pathogen Escherichia coli O157:H7 within 1-5 min. In addition, both peptides exhibited anti-biofilm activity at sub-MIC levels. Indeed, these peptides prevented biofilm formation and triggered killing of cells in mature E. coli O157:H7 biofilms at 1 µM. Both peptides bound to bacterial surface LPS as assessed using the dansyl-polymyxin displacement assay, and were able to interact with the lipids of liposomes as determined by observing a tryptophan blue shift. Interestingly, even though these peptides were highly antimicrobial, they did not induce pore formation or aggregates in bacterial cell membranes. Instead these peptides readily penetrated into bacterial cells as determined by confocal microscopy of labeled peptides. DNA binding assays indicated that both peptides bound to DNA with higher affinity than the positive control peptide buforin II. We propose that cationic peptides KT2 and RT2 bind to negatively-charged LPS to enable self-promoted uptake and, subsequently interact with cytoplasmic membrane phospholipids through their hydrophobic domains enabling translocation across the bacterial membrane and entry into cells within minutes and binding to DNA and other cytoplasmic membrane. Due to their dual antimicrobial and anti-biofilm activities, these peptides may find use as an alternative to (or in conjunction with) conventional antibiotics to treat acute infections caused by planktonic bacteria and chronic, biofilm-related infections.

Effect of acyl chain length on therapeutic activity and mode of action of the CX-KYR-NH2 antimicrobial lipopeptide.

Peptide lipidation has proven to be an inexpensive and effective strategy for designing next-generation peptide-based drug compounds. In this study, the effect of the acyl chain length of ultrashort LiPs (CX-KYR-NH2; X=10, 12, 14 and 16) on their bacterial killing and membrane disruption kinetics was investigated. The geometric mean of the minimum inhibitory concentration (MIC) values for 4 pathogenic bacterial strains was 25 µM, with a selectivity index of 10.24 for C14-KYR-NH2. LiPs at all concentrations exhibited no cytotoxicity towards human erythrocytes, but towards Vero cells at 80 µM. All the LiPs adopted secondary structure in a membrane mimicking environment. C14-KYR-NH2 aggregated above 256 µM, while C16-KYR-NH2 did above 80 µM. All LiPs showed outer membrane permeabilization within 3 min after treatment, yet the extent and kinetics of inner membrane penetration and depolarization were dependent on the acyl chain length. Cell death subsequently occurred within 10 min, and killing activity appeared to correlate most with depolarization activity but not with outer or inner membrane permeability. AFM imaging of cells treated with C14-KYR-NH2 revealed rupture of the cell surface and cytosolic leakage depending on the length of incubation. This study highlights and follows the progression of events that occur during the membrane disintegration process over time, and determines the optimal amphipathicity of ultrashort LiPs with 12-14 carbon atoms for this membrane disrupting activity. The fast acting bactericidal properties of ultrashort LiPs with optimal chain lengths make them promising candidates for drug lead compounds.

Comparison of recombinant α-hemoglobin from Crocodylus siamensis expressed in different cloning vectors and their biological properties.

Hemoglobin (Hb) is an important component in red blood cells of the vertebrate. It is a major respiratory protein with oxygen or carbon dioxide transport function. Hb has been reported to contain bioactive peptides which have antibacterial and antioxidant activities. In this study, the alpha-chain hemoglobin(αHb) gene of Crocodylus siamensis was cloned into the three different expression vectors and expressed in Escherichia coli BL21 (DE3). The recombinant αHb proteins from all constructs could be expressed and purified. The result from UV-visible absorption spectra showed a similar pattern of all recombinant proteins to the oxy-hemoglobin form of intact Hb. The different recombinant αHb could exhibit antioxidant activities. All recombinant proteins could inhibit the growth of Bacillus spp. Especially, most of the recombinant proteins could inhibit the growth of Bacillus amyloliquefaciens TISTR 1045 better than intact one. The result obtained from this study can provide us further information about the possibility using of αHb as a supplementary food.

A goose-type lysozyme from ostrich (Struthio camelus) egg white: multiple roles of His101 in its enzymatic reaction.

A goose-type lysozyme from ostrich egg white (OEL) was produced by Escherichia coli expression system, and the role of His101 of OEL in the enzymatic reaction was investigated by NMR spectroscopy, thermal unfolding, and theoretical modeling of the enzymatic hydrolysis of hexa-N-acetylchitohexaose, (GlcNAc)6. Although the binding of tri-N-acetylchitotriose, (GlcNAc)3, to OEL perturbed several backbone resonances in the (1)H-(15)N HSQC spectrum, the chemical shift of the backbone resonance of His101 was not significantly affected. However, apparent pKa values of His101 and Lys102 determined from the pH titration curves of the backbone chemical shifts were markedly shifted by (GlcNAc)3 binding. Thermal unfolding experiments and modeling study of (GlcNAc)6 hydrolysis using a His101-mutated OEL (H101A-OEL) revealed that the His101 mutation affected not only sugar residue affinities at subsites -3 and -2 but also the rate constant for bond cleavage. His101 appears to play multiple roles in the substrate binding and the catalytic reaction.

Proteomic analysis of crocodile white blood cells reveals insights into the mechanism of the innate immune system.

Crocodiles have a particularly powerful innate immune system because their blood contains high levels of antimicrobial peptides. They can survive injuries that would be fatal to other animals, and they are rarely afflicted with diseases. To better understand the crocodile's innate immune response, proteomic analysis was performed on the white blood cells (WBC) of an Aeromonas hydrophila-infected crocodile. Levels of WBC and red blood cells (RBC) rapidly increased within 1 h. In WBC, there were 109 up-regulated differentially expressed proteins (DEP) that were up-regulated. Fifty-nine DEPs dramatically increased expression from 1 h after inoculation, whereas 50 up-regulated DEPs rose after 24 h. The most abundant DEPs mainly had two biological functions, 1) gene expression regulators, for example, zinc finger proteins and histone H1 family, and 2) cell mechanical forces such as actin cytoskeleton proteins and microtubule-binding proteins. This finding illustrates the characteristic effective innate immune response mechanism of crocodiles that might occur via boosted transcription machinery proteins to accelerate cytoskeletal protein production for induction of phagocytosis, along with the increment of trafficking proteins to transport essential molecules for combating pathogens. The findings of this study provide new insights into the mechanisms of the crocodile's innate immune system.

Egg white hydrolysate peptides act as antimicrobial and anti-inflammatory agents for acne.

A simple method to generate antibacterial peptides by alkaline hydrolysis of hen egg whites is reported. The method reproducibly generates short peptides with molecular weight of less than 14.4 kDa that exhibit low to no cytotoxicity on RAW 264.7 macrophage cells, but do inhibit the bacterial growth of Cutibacterium acnes (C. acnes), Staphylococcus aureus (S. aureus) and antibiotic-resistant S. aureus (MRSA), while also reducing nitric oxide production from heat-killed C. acnes-treated RAW 264.7 cells. Peptidomics revealed at least thirty peptides within the complex mixture, of which eight were evaluated individually. Three peptides (PK8, EE9 and RP8) were potent anti-inflammation and antibacterial agents, but notably the complex egg white hydrolysate (EWH) was more effective than the individual peptides. Electron microscopy suggests the antibacterial mechanism of both the hydrolysate and the selected peptides is through disruption of the cell membrane of C. acnes. These findings suggest that EWH and EWH-derived peptides are promising candidates for infection and inflammation treatment, particularly in managing acne and combating antibiotic-resistant bacteria like MRSA.

Rational Design of RN15m4 Cathelin Domain-Based Peptides from Siamese Crocodile Cathelicidin Improves Antimicrobial Activity.

Antimicrobial peptides are becoming a new generation of antibiotics due to their therapeutic potential and ability to decrease drug-resistant bacteria development. Cathelicidins are known as effective peptides of vertebrate immunity that play crucial roles in the defensive strategy against pathogens. To improve its potency, the RN15 antibacterial peptide derived from the cathelin domain of Crocodylus siamensis cathelicidin has been modified and its antimicrobial properties investigated. Peptides were derived by template-based and physicochemical designation. The RN15 derivative peptides were predicted through their structure modeling, antimicrobial potency, and peptide-membrane calculation. The antimicrobial and cytotoxic activities of candidate peptides were investigated. Simultaneous consideration of physicochemical characteristics, secondary structure modeling, and the result of antimicrobial peptide tools prediction indicated that RN15m4 peptide was a candidate derivative antimicrobial peptide. The RN15m4 peptide expresses antimicrobial activity against most Gram-positive and Gram-negative bacteria and fungi with a lower minimum inhibition concentration (MIC) than the parent peptide. Besides, the time-killing assay shows that the designed peptide performed its ability to quickly kill bacteria better than the original peptide. Scanning electron microscopy (SEM) displayed the destruction of the bacterial cell membrane caused by the RN15m4 peptide. In addition, the RN15m4 peptide exhibits low hemolytic activity and low cytotoxic activity as good as the template peptide. The RN15m4 peptide performs a range of antimicrobial activities with low cell toxicity. Our study has illustrated the combination approach to peptide design for potent antibiotic peptide discovery.

Rational design and characterization of cell-selective antimicrobial peptides based on a bioactive peptide from Crocodylus siamensis hemoglobin.

Antimicrobial resistance is a growing health concern. Antimicrobial peptides are a potential solution because they bypass conventional drug resistance mechanisms. Previously, we isolated a peptide from Crocodylus siamensis hemoglobin hydrolysate, which has antimicrobial activity and identified the main peptide from this mixture (QL17). The objective of this work was to evaluate and rationally modify QL17 in order to: (1) control its mechanism of action through bacterial membrane disruption; (2) improve its antimicrobial activity; and (3) ensure it has low cytotoxicity against normal eukaryotic cells. QL17 was rationally designed using physicochemical and template-based methods. These new peptide variants were assessed for: (1) their in vitro inhibition of microbial growth, (2) their cytotoxicity against normal cells, (3) their selectivity for microbes, and (4) the mode of action against bacteria using scanning electron microscopy (SEM), transmission electron microscopy (TEM) and confocal microscopy. The results indicate that all designed peptides have more potent antimicrobial efficacy than QL17 and IL15 peptides. However, only the most rationally modified peptides showed strong antimicrobial activity and minimal toxicity against normal cells. In particular, IL15.3 (hydrophobicity of 47% and net charge of + 6) was a potent antimicrobial agent (MIC = 4-12 µg/mL; MBC = 6-25 µg/mL) and displayed excellent selectivity for microbes (cf. human cells) via FACS assays. Microscopy confirmed that IL15.3 acts against bacteria by disrupting the cell membrane integrity and penetrating into the membrane. This causes the release of intracellular content into the outer environment leading to the death of bacteria. Moreover, IL15.3 can also interact with DNA suggesting it could have dual mode of action. Overall, a novel variant of QL17 is described that increases antimicrobial activity by over 1000-fold (~ 5 µg/mL MIC) and has minimal cytotoxicity. It may have applications in clinical use to treat and safeguard against bacteria.