Antibiofilm Peptides Research Articles

Dual-function antimicrobial-antibiofilm peptide hybrid to tackle biofilm-forming Staphylococcus epidermidis

BackgroundDue to their resistance and difficulty in treatment, biofilm-associated infections are problematic among hospitalized patients globally and account for 60% of all bacterial infections in humans. Antibiofilm peptides have recently emerged as an alternative treatment since they can be effectively designed and exert a different mode of biofilm inhibition and eradication.MethodsA novel antibiofilm peptide, BiF, was designed from the conserved sequence of 18 α-helical antibiofilm peptides by template-assisted technique and its activity was improved by hybridization with a lipid binding motif (KILRR). Novel antibiofilm peptide derivatives were modified by substituting hydrophobic amino acids at positions 5 or 7, and both, with positively charged lysines (L5K, L7K). These peptide derivatives were tested for antibiofilm and antimicrobial activities against biofilm-forming Staphylococcus epidermidis and multiple other microbes using crystal violet and broth microdilution assays, respectively. To assess their impact on mammalian cells, the toxicity of peptides was determined through hemolysis and cytotoxicity assays. The stability of candidate peptide, BiF2_5K7K, was assessed in human serum and its secondary structure in bacterial membrane-like environments was analyzed using circular dichroism. The action of BiF2_5K7K on planktonic S. epidermidis and its effect on biofilm cell viability were assessed via viable counting assays. Its biofilm inhibition mechanism was investigated through confocal laser scanning microscopy and transcription analysis. Additionally, its ability to eradicate mature biofilms was examined using colony counting. Finally, a preliminary evaluation involved coating a catheter with BiF2_5K7K to assess its preventive efficacy against S. epidermidis biofilm formation on the catheter and its surrounding area.ResultsBiF2_5K7K, the modified antibiofilm peptide, exhibited dose-dependent antibiofilm activity against S. epidermidis. It inhibited biofilm formation at subinhibitory concentrations by altering S. epidermidis extracellular polysaccharide production and quorum-sensing gene expression. Additionally, it exhibited broad-spectrum antimicrobial activity and no significant hemolysis or toxicity against mammalian cell lines was observed. Its activity is retained when exposed to human serum. In bacterial membrane-like environments, this peptide formed an α-helix amphipathic structure. Within 4 h, a reduction in the number of S. epidermidis colonies was observed, demonstrating the fast action of this peptide. As a preliminary test, a BiF2_5K7K-coated catheter was able to prevent the development of S. epidermidis biofilm both on the catheter surface and in its surrounding area.ConclusionsDue to the safety and effectiveness of BiF2_5K7K, we suggest that this peptide be further developed to combat biofilm infections, particularly those of biofilm-forming S. epidermidis.

A potent candicidal peptide designed based on an encrypted peptide from a proteinase inhibitor

Antimicrobial peptides (AMP) represent an alternative in the treatment of fungal infections associated with countless deaths. Here, we report a new AMP, named KWI-19, which was designed based on a peptide encrypted in the sequence of an Inga laurina Kunitz-type inhibitor (ILTI). KWI-19 inhibited the growth of Candida species and acted as a fungicidal agent from 2.5 to 20 μmol L−1, also showing synergistic activity with amphotericin B. Kinetic assays showed that KWI-19 killed Candida tropicalis cells within 60 min. We also report the membrane-associated mechanisms of action of KWI-19 and its interaction with ergosterol. KWI-19 was also characterized as a potent antibiofilm peptide, with activity against C. tropicalis. Finally, non-toxicity was reported against Galleria mellonella larvae, thus strengthening the interest in all the bioactivities mentioned above. This study extends our knowledge on how AMPs can be engineered from peptides encrypted in larger proteins and their potential as candicidal agents.

Computer-made peptide RQ18 acts as a dual antifungal and antibiofilm peptide though membrane-associated mechanisms of action

The spread of fungi resistant to conventional drugs has become a threatening problem. In this context, antimicrobial peptides (AMPs) have been considered as one of the main alternatives for controlling fungal infections. Here, we report the antifungal and antibiofilm activity and some clues about peptide RQ18's mechanism of action against Candida and Cryptococcus. This peptide inhibited yeast growth from 2.5 μM and killed all Candida tropicalis cells within 2 h incubation. Moreover, it showed a synergistic effect with antifungal agent the amphotericin b. RQ18 reduced biofilm formation and promoted C. tropicalis mature biofilms eradication. RQ18's mechanism of action involves fungal cell membrane damage, which was confirmed by the results of RQ18 in the presence of free ergosterol in the medium and fluorescence microscopy by Sytox green. No toxic effects were observed in murine macrophage cell lines and Galleria mellonella larvae, suggesting fungal target selectivity. Therefore, peptide RQ18 represents a promising strategy as a dual antifungal and antibiofilm agent that contributes to infection control without damaging mammalian cells.

Antibiofilm peptides enhance the corrosion resistance of titanium in the presence of Streptococcus mutans.

Titanium alloys have gained popularity in implant dentistry for the restoration of missing teeth and related hard tissues because of their biocompatibility and enhanced strength. However, titanium corrosion and infection caused by microbial biofilms remains a significant clinical challenge leading to implant failure. This study aimed to evaluate the effectiveness of antibiofilm peptides 1018 and DJK-5 on the corrosion resistance of titanium in the presence of Streptococcus mutans. Commercially pure titanium disks were prepared and used to form biofilms. The disks were randomly assigned to different treatment groups (exposed to S. mutans supplied with sucrose) including a positive control with untreated biofilms, peptides 1018 or DJK-5 at concentrations of 5μg/mL or 10μg/mL, and a negative control with no S. mutans. Dynamic biofilm growth and pH variation of all disks were measured after one or two treatment periods of 48h. After incubation, the dead bacterial proportion, surface morphology, and electrochemical behaviors of the disks were determined. The results showed that peptides 1018 and DJK-5 exhibited significantly higher dead bacterial proportions than the positive control group in a concentration dependent manner (p < 0.01), as well as far less defects in microstructure. DJK-5 at 10μg/mL killed 84.82% of biofilms and inhibited biofilm growth, preventing acidification due to S. mutans and maintaining a neutral pH. Potential polarization and electrochemical impedance spectroscopy data revealed that both peptides significantly reduced the corrosion and passive currents on titanium compared to titanium surfaces with untreated biofilms, and increased the resistance of the passive film (p < 0.05), with 10μg/mL of DJK-5 achieving the greatest effect. These findings demonstrated that antibiofilm peptides are effective in promoting corrosion resistance of titanium against S. mutans, suggesting a promising strategy to enhance the stability of dental implants by endowing them with antibiofilm and anticorrosion properties.

Effect of anti-biofilm peptide CRAMP-34 on the biofilms of Acinetobacter lwoffii derived from dairy cows.

Dairy mastitis is one of the most common diseases in dairy farming, and the formation of pathogenic bacteria biofilms may be an important reason why traditional antibiotic therapy fails to resolve some cases of dairy mastitis. We isolated and identified three strains of A. lwoffii were with strong biofilm forming ability from dairy cow mastitis samples from Chongqing dairy farms in China. In order to investigate the effect of novel anti-biofilm peptide CRAMP-34 on A.lwoffii biofilms, the anti-biofilm effect was evaluated by crystal violet staining, biofilms viable bacteria counting and confocal laser scanning microscopy (CLSM). In addition, transcriptome sequencing analysis, qRT-PCR and phenotypic verification were used to explore the mechanism of its action. The results showed that CRAMP-34 had a dose-dependent eradicating effect on A. lwoffii biofilms. Transcriptome sequencing analysis showed that 36 differentially expressed genes (11 up-regulated and 25 down-regulated) were detected after the intervention with the sub-inhibitory concentration of CRAMP-34. These differentially expressed genes may be related to enzyme synthesis, fimbriae, iron uptake system, capsular polysaccharide and other virulence factors through the functional analysis of differential genes. The results of subsequent bacterial motility and adhesion tests showed that the motility of A.lwoffii were enhanced after the intervention of CRAMP-34, but there was no significant change in adhesion. It was speculated that CRAMP-34 may promote the dispersion of biofilm bacteria by enhancing the motility of biofilm bacteria, thereby achieving the effect of eradicating biofilms. Therefore, these results, along with our other previous findings, suggest that CRAMP-34 holds promise as a new biofilm eradicator and deserves further research and development.

The bactericidal and antibiofilm effects of a lysine-substituted hybrid peptide, CM-10K14K, on biofilm-forming Staphylococcus epidermidis

Staphylococci, notably biofilm-forming Staphylococcus epidermidis, have been recognized as global nosocomial pathogens in medical device-related infections. Their potential to attach to and form biofilm on indwelling catheters are significant factors impeding conventional treatment. Due to their extensive antimicrobial and antibiofilm actions, antimicrobial peptides (AMPs) have attracted interest as promising alternative compounds for curing difficult-to-treat, biofilm-forming bacterial infections. Cecropin A-melittin or CM, a well-known hybrid peptide, exhibits broad-spectrum antimicrobial activity, however it also possesses high toxicity. In the current study, a series of hybrid CM derivatives was designed using an amino acid substitution strategy to explore potential antibacterial and antibiofilm peptides with low toxicity. Among the derivatives, CM-10K14K showed the least hemolysis along with potent antibacterial activity against biofilm-forming S. epidermidis (MICs = 3.91 μg/mL) and rapid killing after 15 min exposure (MBCs = 7.81 μg/mL). It can prevent the formation of S. epidermidis biofilm and also exhibited a dose-dependent eradication activity on mature or established S. epidermidis biofilm. In addition, it decreased the development of biofilm by surviving bacteria, and formation of biofilm on the surface of CM-10K14K-impregnated catheters. Released CM-10K14K decreased planktonic bacterial growth and inhibited biofilm formation by S. epidermidis in a dose-dependent manner for 6 and 24 h post-exposure. Impregnation of CM-10K14K prevented bacterial attachment on catheters and thus decreased formation of extensive biofilms. SEM images supported the antibiofilm activity of CM-10K14K. Flow cytometry analysis and TEM images demonstrated a membrane-active mechanism of CM-10K14K, inducing depolarization and permeabilization, and subsequent membrane rupture leading to cell death. The presence of an interaction with bacterial DNA was verified by gel retardation assay. These antibacterial and antibiofilm activities of CM-10K14K suggest its potential application to urinary catheters for prevention of biofilm-forming colonization or for treatment of medical devices infected with S. epidermidis.

Influence of a D-enantiomeric peptide on the anticorrosion ability of titanium with different surface roughness against Streptococcus mutans biofilms

ObjectiveTo investigate the effectiveness of a d-enantiomeric antibiofilm peptide (DJK-5) on the anticorrosion ability of titanium (Ti) with different surface roughness against Streptococcus mutans biofilms. MethodsCommercially pure Ti disks with machined (MA, smooth) or sandblasted + acid-etched (SLA, rough) surfaces were prepared and characterized. All disks were divided into three groups: a positive control (PC) group with S. mutans, a DJK-5-treated group, and a negative control (NC) group without S. mutans. Biofilm formation and corrosion on Ti surfaces were determined by confocal laser scanning microscopy and scanning electron microscopy after 2 and 6 days, and the electrochemical properties were evaluated. ResultsTen μg/mL of DJK-5 killed 83.3 % and 87.4 % of biofilms on SLA and MA Ti surfaces, respectively after 2 days, and 72.9 % and 77.7 % after 6 days, with more bacteria surviving on SLA surfaces with higher roughness (p < 0.05). DJK-5 treatment induced less surface defects with tiny pit corrosion than PC. DJK-5 treatment when compared to PC, led to electrochemical properties more reflecting NC surfaces, including significantly less negative corrosion potential, lower corrosion current, and higher passive film resistance (p < 0.05). SLA surfaces exhibited higher current density and lower resistance than MA surfaces (p < 0.05). ConclusionDJK-5 effectively enhanced the corrosion resistance of Ti with different surface roughness while killing S. mutans biofilms, and smooth surfaces were more susceptible to peptide treatment. Clinical significanceThe antibiofilm peptide is promising for promoting the anticorrosion ability of Ti against biofilms, thereby preventing biofilm-related infections.

Development of an Antimicrobial Peptide SAAP‐148‐Functionalized Supramolecular Coating on Titanium to Prevent Biomaterial‐Associated Infections

AbstractTitanium implants are widely used in medicine but have a risk of biomaterial‐associated infection (BAI), of which traditional antibiotic‐based treatment is affected by resistance. Antimicrobial peptides (AMPs) are used to successfully kill antibiotic‐resistant bacteria. Herein, a supramolecular coating for titanium implants is developed which presents the synthetic antimicrobial and antibiofilm peptide SAAP‐148 via supramolecular interactions using ureido‐pyrimidinone supramolecular units (UPy‐SAAP‐148GG). Material characterization of dropcast coatings shows the presence of UPy‐SAAP‐148GG at the surface. The supramolecular immobilized peptide remains antimicrobially active in dropcast polymer films and can successfully kill (antibiotic‐resistant) Staphylococcus aureus, Acinetobacter baumannii, and Escherichia coli. Minor toxicity for human dermal fibroblasts is observed, with a reduced cell attachment after 24 h. Subsequently, a dipcoat coating on titanium implants is developed and tested in vivo in a subcutaneous implant infection mouse model with S. aureus administered locally on the implant before implantation to mimic contamination during surgery. The supramolecular coating containing 5 mol% of UPy‐SAAP‐148GG significantly prevents colonization of the implant surface as well as of the surrounding tissue, with no signs of toxicity. This shows that supramolecular AMP coatings on titanium are eminently suitable to prevent BAI.

Design of a machine learning-aided screening framework for antibiofilm peptides

Biofilms are formed by multicellular colonies of microorganisms that are protected by hard extracellular matrices. Eradication of biofilms is a challenging task due to their recalcitrant nature and thus biofilm formation poses a global threat to public health. In this regard, antibiofilm peptides are a promising class of therapeutics that are active against biofilms. However, large-scale experimental screening and testing of peptides for antibiofilm activity is a resource-intensive task. In this study, a machine learning-aided design framework is proposed to aid in screening of antibiofilm peptides. An SVM-based binary classification model is developed using amino acid compositions, sequence, and physicochemical properties of peptides as independent features. The physicochemical property-based model developed in this study achieved the highest accuracy of 97.9%, which is found to be substantially higher than the other feature representation techniques. The explainability of this model is performed using SHAP analysis. Results obtained show that amphiphilicity, aliphaticity and cationicity have positive correlation whereas steric parameter, length, and volume have negative correlation with antibiofilm activity of peptides. The developed model can be accessed freely via web tool: AntiBFP.

Complex Networks Analyses of Antibiofilm Peptides: An Emerging Tool for Next-Generation Antimicrobials' Discovery.

Microbial biofilms cause several environmental and industrial issues, even affecting human health. Although they have long represented a threat due to their resistance to antibiotics, there are currently no approved antibiofilm agents for clinical treatments. The multi-functionality of antimicrobial peptides (AMPs), including their antibiofilm activity and their potential to target multiple microbes, has motivated the synthesis of AMPs and their relatives for developing antibiofilm agents for clinical purposes. Antibiofilm peptides (ABFPs) have been organized in databases that have allowed the building of prediction tools which have assisted in the discovery/design of new antibiofilm agents. However, the complex network approach has not yet been explored as an assistant tool for this aim. Herein, a kind of similarity network called the half-space proximal network (HSPN) is applied to represent/analyze the chemical space of ABFPs, aiming to identify privileged scaffolds for the development of next-generation antimicrobials that are able to target both planktonic and biofilm microbial forms. Such analyses also considered the metadata associated with the ABFPs, such as origin, other activities, targets, etc., in which the relationships were projected by multilayer networks called metadata networks (METNs). From the complex networks' mining, a reduced but informative set of 66 ABFPs was extracted, representing the original antibiofilm space. This subset contained the most central to atypical ABFPs, some of them having the desired properties for developing next-generation antimicrobials. Therefore, this subset is advisable for assisting the search for/design of both new antibiofilms and antimicrobial agents. The provided ABFP motifs list, discovered within the HSPN communities, is also useful for the same purpose.

Physical and Functional Characterization of PLGA Nanoparticles Containing the Antimicrobial Peptide SAAP-148.

Synthetic antimicrobial and antibiofilm peptide (SAAP-148) commits significant antimicrobial activities against antimicrobial resistant (AMR) planktonic bacteria and biofilms. However, SAAP-148 is limited by its low selectivity index, i.e., ratio between cytotoxicity and antimicrobial activity, as well as its bioavailability at infection sites. We hypothesized that formulation of SAAP-148 in PLGA nanoparticles (SAAP-148 NPs) improves the selectivity index due to the sustained local release of the peptide. The aim of this study was to investigate the physical and functional characteristics of SAAP-148 NPs and to compare the selectivity index of the formulated peptide with that of the peptide in solution. SAAP-148 NPs displayed favorable physiochemical properties [size = 94.1 ± 23 nm, polydispersity index (PDI) = 0.08 ± 0.1, surface charge = 1.65 ± 0.1 mV, and encapsulation efficiency (EE) = 86.7 ± 0.3%] and sustained release of peptide for up to 21 days in PBS at 37 °C. The antibacterial and cytotoxicity studies showed that the selectivity index for SAAP-148 NPs was drastically increased, by 10-fold, regarding AMR Staphylococcus aureus and 20-fold regarding AMR Acinetobacter baumannii after 4 h. Interestingly, the antibiofilm activity of SAAP-148 NPs against AMR S. aureus and A. baumannii gradually increased overtime, suggesting a dose-effect relationship based on the peptide's in vitro release profile. Using 3D human skin equivalents (HSEs), dual drug SAAP-148 NPs and the novel antibiotic halicin NPs provided a stronger antibacterial response against planktonic and cell-associated bacteria than SAAP-148 NPs but not halicin NPs after 24 h. Confocal laser scanning microscopy revealed the presence of SAAP-148 NPs on the top layers of the skin models in close proximity to AMR S. aureus at 24 h. Overall, SAAP-148 NPs present a promising yet challenging approach for further development as treatment against bacterial infections.

Encapsulation of SAAP-148 in Octenyl Succinic Anhydride-Modified Hyaluronic Acid Nanogels for Treatment of Skin Wound Infections

Chronic wound infections colonized by bacteria are becoming more difficult to treat with current antibiotics due to the development of antimicrobial resistance (AMR) as well as biofilm and persister cell formation. Synthetic antibacterial and antibiofilm peptide (SAAP)-148 is an excellent alternative for treatment of such infections but suffers from limitations related to its cationic peptidic nature and thus instability and possible cytotoxicity, resulting in a narrow therapeutic window. Here, we evaluated SAAP-148 encapsulation in nanogels composed of octenyl succinic anhydride (OSA)-modified hyaluronic acid (HA) to circumvent these limitations. SAAP-148 was efficiently (>98%) encapsulated with high drug loading (23%), resulting in monodispersed anionic OSA-HA nanogels with sizes ranging 204-253 nm. Nanogel lyophilization in presence of polyvinyl alcohol maintained their sizes and morphology. SAAP-148 was sustainedly released from lyophilized nanogels (37-41% in 72 h) upon reconstitution. Lyophilized SAAP-148-loaded nanogels showed similar antimicrobial activity as SAAP-148 against planktonic and biofilm-residing AMR Staphylococcus aureus and Acinetobacter baumannii. Importantly, formulated SAAP-148 showed reduced cytotoxicity against human erythrocytes, primary human skin fibroblasts and human keratinocytes. Additionally, lyophilized SAAP-148-loaded nanogels eradicated AMR S. aureus and A. baumannii colonizing a 3D human epidermal model, without inducing any cytotoxicity in contrast to SAAP-148. These findings indicate that OSA-HA nanogels increase SAAP-148's therapeutic potential for treatment of skin wound infections.

C-Terminal PEGylation Improves SAAP-148 Peptide’s Immunomodulatory Activities

Synthetic antibacterial and anti-biofilm peptide (SAAP)-148 was developed to combat bacterial infections not effectively treatable with current antibiotics. SAAP-148 is highly effective against antimicrobial-resistant bacteria without inducing resistance; however, challenges for further development of SAAP-148 include its cytotoxicity and short circulation half-life. To circumvent these drawbacks, a library of SAAP-148 linked to polyethylene glycol (PEG) groups of various lengths was synthesized and screened for in vitro antibacterial activity and hemolytic activity. Results indicated that PEGylated SAAP-148 variants combine antibacterial activities with reduced hemolysis compared to SAAP-148. Interestingly, proinflammatory immunomodulatory activities of SAAP-148 were enhanced upon C-terminal PEGylation, with SAAP-148-PEG27 showing the most effect. SAAP-148-PEG27 enhanced SAAP-148’s capacity to chemoattract human neutrophils and was able to more efficiently (re)direct M-CSF-induced monocyte-macrophage differentiation toward type 1 macrophages as opposed to SAAP-148. Furthermore, dendritic cells with a stronger mature expression profile were produced if monocytes were exposed to SAAP-148-PEG27 during monocyte-immature dendritic cell differentiation in comparison to SAAP-148. Parameters that influenced the immunomodulatory activities of the peptide-PEG conjugate include (i) the length of the PEG group, (ii) the position of PEG conjugation, and (iii) the peptide sequence. Together, these results indicate that SAAP-148-PEG27 is highly effective in redirecting monocyte-macrophage differentiation toward a proinflammatory phenotype and promoting monocyte-mature dendritic cell development. Therefore, SAAP-148-PEG27 may be a promising agent to modulate inadequate immune responses in case of tumors and chronically infected wounds.

Peptide Stability Is Important but Not a General Requirement for Antimicrobial and Antibiofilm Activity In Vitro and In Vivo.

Peptide stability to proteases has been a major requirement for developing peptide therapeutics. This study investigates the effects of peptide stability on antimicrobial and antibiofilm activity under various conditions. For this purpose, two human cathelicidin-derived peptides differing in stability to proteases were utilized. While GF-17, a peptide derived from the major antimicrobial region of human LL-37, can be rapidly cleaved by proteases, the engineered peptide 17BIPHE2 is resistant to multiple proteases. In the standard antimicrobial susceptibility, killing kinetics, and membrane permeabilization assays conducted in vitro using planktonic bacteria, these two peptides displayed similar potency. The two peptides were also similarly active against methicillin-resistant Staphylococcus aureus (MRSA) USA300 prior to biofilm formation. However, 17BIPHE2 was superior to GF-17 in disrupting preformed biofilms probably due to both enhanced stability and slightly higher DNA binding capacity. In a wax moth model, 17BIPHE2 better protected insects from MRSA infection-caused death than GF-17, consistent with the slower degradation of 17BIPHE2 than GF-17. Here, peptide antimicrobial activity was found to be critical for in vivo efficacy. When incorporated in the nanofiber/microneedle delivery device, GF-17 and 17BIPHE2 displayed a similar effect in eliminating MRSA in murine chronic wounds, underscoring the advantage of nanofibers in protecting the peptide from degradation. Since nanoformulation can ease the requirement of peptide stability, it opens the door to a direct use of natural peptides or their cocktails for antimicrobial treatment, accelerating the search of effective antibiofilm peptides to treat chronic wounds.

Polypharmacological Cell-Penetrating Peptides from Venomous Marine Animals Based on Immunomodulating, Antimicrobial, and Anticancer Properties.

Complex pathological diseases, such as cancer, infection, and Alzheimer's, need to be targeted by multipronged curative. Various omics technologies, with a high rate of data generation, demand artificial intelligence to translate these data into druggable targets. In this study, 82 marine venomous animal species were retrieved, and 3505 cryptic cell-penetrating peptides (CPPs) were identified in their toxins. A total of 279 safe peptides were further analyzed for antimicrobial, anticancer, and immunomodulatory characteristics. Protease-resistant CPPs with endosomal-escape ability in Hydrophis hardwickii, nuclear-localizing peptides in Scorpaena plumieri, and mitochondrial-targeting peptides from Synanceia horrida were suitable for compartmental drug delivery. A broad-spectrum S. horrida-derived antimicrobial peptide with a high binding-affinity to bacterial membranes was an antigen-presenting cell (APC) stimulator that primes cytokine release and naïve T-cell maturation simultaneously. While antibiofilm and wound-healing peptides were detected in Synanceia verrucosa, APC epitopes as universal adjuvants for antiviral vaccination were in Pterois volitans and Conus monile. Conus pennaceus-derived anticancer peptides showed antiangiogenic and IL-2-inducing properties with moderate BBB-permeation and were defined to be a tumor-homing peptide (THP) with the ability to inhibit programmed death ligand-1 (PDL-1). Isoforms of RGD-containing peptides with innate antiangiogenic characteristics were in Conus tessulatus for tumor targeting. Inhibitors of neuropilin-1 in C. pennaceus are proposed for imaging probes or therapeutic delivery. A Conus betulinus cryptic peptide, with BBB-permeation, mitochondrial-targeting, and antioxidant capacity, was a stimulator of anti-inflammatory cytokines and non-inducer of proinflammation proposed for Alzheimer's. Conclusively, we have considered the dynamic interaction of cells, their microenvironment, and proportional-orchestrating-host- immune pathways by multi-target-directed CPPs resembling single-molecule polypharmacology. This strategy might fill the therapeutic gap in complex resistant disorders and increase the candidates' clinical-translation chance.

Host Response of Human Epidermis to Methicillin-Resistant Staphylococcus aureus Biofilm Infection and Synthetic Antibiofilm Peptide Treatment.

Bacterial biofilm infections associated with wounded skin are prevalent, recalcitrant, and in urgent need of treatments. Additionally, host responses in the skin to biofilm infections are not well understood. Here we employed a human organoid skin model to explore the transcriptomic changes of thermally-injured epidermis to methicillin-resistant Staphylococcus aureus (MRSA) biofilm colonization. MRSA biofilm impaired skin barrier function, enhanced extracellular matrix remodelling, elicited inflammatory responses including IL-17, IL-12 family and IL-6 family interleukin signalling, and modulated skin metabolism. Synthetic antibiofilm peptide DJK-5 effectively diminished MRSA biofilm and associated skin inflammation in wounded human ex vivo skin. In the epidermis, DJK-5 shifted the overall skin transcriptome towards homeostasis including modulating the biofilm induced inflammatory response, promoting the skin DNA repair function, and downregulating MRSA invasion of thermally damaged skin. These data clarified the underlying immunopathogenesis of biofilm infections and revealed the intrinsic promise of synthetic peptides in reducing inflammation and biofilm infections.

Molecular regulation of conditioning film formation and quorum quenching in sulfate reducing bacteria.

Sensing surface topography, an upsurge of signaling biomolecules, and upholding cellular homeostasis are the rate-limiting spatio-temporal events in microbial attachment and biofilm formation. Initially, a set of highly specialized proteins, viz. conditioning protein, directs the irreversible attachment of the microbes. Later signaling molecules, viz. autoinducer, take over the cellular communication phenomenon, resulting in a mature microbial biofilm. The mandatory release of conditioning proteins and autoinducers corroborated the existence of two independent mechanisms operating sequentially for biofilm development. However, both these mechanisms are significantly affected by the availability of the cofactor, e.g., Copper (Cu). Generally, the Cu concentration beyond threshold levels is detrimental to the anaerobes except for a few species of sulfate-reducing bacteria (SRB). Remarkably SRB has developed intricate ways to resist and thrive in the presence of Cu by activating numerous genes responsible for modifying the presence of more toxic Cu(I) to Cu(II) within the periplasm, followed by their export through the outer membrane. Therefore, the determinants of Cu toxicity, sequestration, and transportation are reconnoitered for their contribution towards microbial adaptations and biofilm formation. The mechanistic details revealing Cu as a quorum quencher (QQ) are provided in addition to the three pathways involved in the dissolution of cellular communications. This review articulates the Machine Learning based data curing and data processing for designing novel anti-biofilm peptides and for an in-depth understanding of QQ mechanisms. A pioneering data set has been mined and presented on the functional properties of the QQ homolog in Oleidesulfovibrio alaskensis G20 and residues regulating the multicopper oxidase properties in SRB.

In Silico Identification of Potential Insect Peptides against Biofilm-Producing Staphylococcus aureus.

Biofilm-producing Staphylococcus aureus (SA) strains are frequently found in medical environments, from surgical/ wound sites, medical devices. These biofilms reduce the efficacy of applied antibiotics during the treatment of several infections, such as cystic fibrosis, endocarditis, or urinary tract infections. Thus, the development of potential therapeutic agents to destroy the extra protective biofilm layers or to inhibit the biofilm-producing enzymes is urgently needed. Advanced and cost-effective bioinformatics tools are advantageous in locating and speeding up the selection of antibiofilm candidates. Based on the potential drug characteristics, we have selected one-hundred thirty-three antibacterial peptides derived from insects to assess for their antibiofilm potency via molecular docking against five putative biofilm formation and regulated target enzymes: the staphylococcal accessory regulator A or SarA (PDB ID: 2FRH), 4,4'-diapophytoene synthase or CrtM (PDB ID: 2ZCQ), clumping factor A or ClfA (PDB ID: 1N67) and serine-aspartate repeat protein C or SdrC (PDB ID: 6LXH) and sortase A or SrtA (PDB ID: 1T2W) of SA bacterium. In this study, molecular docking was performed using HPEPDOCK and HDOCK servers, and molecular interactions were examined using BIOVIA Discovery Studio Visualizer-2019. The docking score (kcal/mol) range of five promising antibiofilm peptides against five targets was recorded as follows: diptericin A (-215.52 to -303.31), defensin (-201.11 to -301.92), imcroporin (-212.08 to -287.64), mucroporin (-228.72 to -286.76), apidaecin II (-203.90 to -280.20). Among these five, imcroporin and mucroporin were 13 % each, while defensin contained only 1 % of positive net charged residues (Arg+Lys) projected through ProtParam and NetWheels tools. Similarly, imcroporin, mucroporin and apidaecin II were 50 %, while defensin carried 21.05 % of hydrophobic residues predicted by the tool PEPTIDE. 2.0. Most of the peptides exhibited potential characteristics to inhibit S. aureus-biofilm formation via disrupting the cell membrane and cytoplasmic integrity. In summary, the proposed hypothesis can be considered a cost-effective platform for selecting the most promising bioactive drug candidates within a limited timeframe with a greater chance of success in experimental and clinical studies.

Synthetic Peptides against Plant Pathogenic Bacteria.

The control of plant diseases caused by bacteria that seriously compromise crop productivity around the world is still one of the most important challenges in food security. Integrated approaches for disease control generally lack plant protection products with high efficacy and low environmental and health adverse effects. Functional peptides, either from natural sources or synthetic, are considered as novel candidates to develop biopesticides. Synthetic peptides can be obtained based on the structure of natural compounds or de novo designed, considering the features of antimicrobial peptides. The advantage of this approach is that analogues can be conveniently prepared, enabling the identification of sequences with improved biological properties. Several peptide libraries have been designed and synthetized, and the best sequences showed strong bactericidal activity against important plant pathogenic bacteria, with a good profile of biodegradability and low toxicity. Among these sequences, there are bacteriolytic or antibiofilm peptides that work against the target bacteria, plant defense elicitor peptides, and multifunctional peptides that display several of these properties. Here, we report the research performed by our groups during the last twenty years, as well as our ongoing work. We also highlight those peptides that can be used as candidates to develop novel biopesticides, and the main challenges and prospects.

Combination of Six Individual Derivatives of the Pom-1 Antibiofilm Peptide Doubles Their Efficacy against Invasive and Multi-Resistant Clinical Isolates of the Pathogenic Yeast Candida albicans

In previous studies, derivatives of the peptide Pom-1, which was originally extracted from the freshwater mollusk Pomacea poeyana, showed an exceptional ability to specifically inhibit biofilm formation of the laboratory strain ATCC 90028 as a model strain of the pathogenic yeast Candida albicans. In follow-up, here, we demonstrate that the derivatives Pom-1A to Pom-1F are also active against biofilms of invasive clinical C. albicans isolates, including strains resistant against fluconazole and/or amphotericin B. However, efficacy varied strongly between the isolates, as indicated by large deviations in the experiments. This lack of robustness could be efficiently bypassed by using mixtures of all peptides. These mixed peptide preparations were active against biofilm formation of all the isolates with uniform efficacies, and the total peptide concentration could be halved compared to the original MIC of the individual peptides (2.5 µg/mL). Moreover, mixing the individual peptides restored the antifungal effect of fluconazole against fluconazole-resistant isolates even at 50% of the standard therapeutic concentration. Without having elucidated the reason for these synergistic effects of the peptides yet, both the gain of efficacy and the considerable increase in efficiency by combining the peptides indicate that Pom-1 and its derivatives in suitable formulations may play an important role as new antibiofilm antimycotics in the fight against invasive clinical infections with (multi-) resistant C. albicans.