Antimicrobial Peptides Research Articles

Copper-Chelated Chitosan Microgels for the Selective Enrichment of Small Cationic Peptides

Copper-chelated chitosan microgels were investigated as an immobilized metal affinity chromatography (IMAC) phase for peptide separation. The copper-crosslinked chitosan beads were shown to strongly interact with a range of amino acids, in a wide range of pH and saline conditions. The beads exhibited an affinity that seemed to depend on the isoelectric point of the amino acid, with the extent of uptake increasing with decreasing isoelectric point. This selective interaction with anionic amino acids resulted in a significant relative enrichment of the supernatant solution in cationic amino acids. The beads were then studied as a novel fractionation system for complex milk hydrolysates. The copper chitosan beads selectively removed larger peptides from the hydrolysate aqueous solution, yielding a solution relatively enriched in medium and smaller peptides, which was characterized both quantitatively and qualitatively by size exclusion chromatography (SEC). Liquid chromatography–mass spectrometry (LCMS) work provided comprehensive data on a peptide sequence level and showed that a depletion of the anionic peptides by the beads resulted in a relative enrichment of the cationic peptides in the supernatant solution. It could be concluded that after fractionation a dramatic relative enrichment in respect to small- and medium-sized cationic peptides in the solution, characteristics that have been linked to bioactivities, such as anti-microbial and cell-penetrating properties. The results demonstrate the use of the chitosan copper gel bead system in lab scale fractionation of complex hydrolysate mixtures, with the potential to enhance milk hydrolysate bioactivity.

Gut bacterium Burkholderia cepacia (BsNLG8) and immune gene Defensin A contribute to the resistance against Nicotine-induced stress in Nilaparvata lugens (Stål)

Nicotine, a naturally occurring alkaloid found in tobacco, is a potent neurotoxin extensively used to control Nilaparvata lugens (Stål), a destructive insect pest of rice crops. The insect gut harbors a wide array of resident microorganisms that profoundly influence several biological processes, including host immunity. Maintaining an optimal gut microbiota and immune homeostasis requires a complex network of reciprocal regulatory interactions. However, the underlying molecular mechanisms driving these symbiotic exchanges, particularly between specific gut microbe and immunity, remain largely unknown in insects. Our previous investigations identified and isolated a nicotine-degrading Burkholderia cepacia strain (BsNLG8) with antifungal properties. Building on those findings, we found that nicotine intake significantly increased the abundance of a symbiotic bacteria BsNLG8, induced a stronger bacteriostatic effect in hemolymph, and enhanced the nicotine tolerance of N. lugens. Additionally, nicotine-induced antimicrobial peptides (AMPs) exhibited significant antibacterial effects against Staphylococcus aureus. We adopted RNA-seq to explore the underlying immunological mechanisms in nicotine-stressed N. lugens. Bioinformatic analyses identified numerous differentially expressed immune genes, including recognition/immune activation (GRPs and Toll) and AMPs (i.e., Defensin, Lugensin, lysozyme). Temporal expression profiling (12, 24, and 48 hours) of immune genes revealed pattern recognition proteins and immune effectors as primary responders to nicotine-induced stress. Defensin A, a broad-spectrum immunomodulatory cationic peptide, exhibited significantly high expression. RNA interference-mediated silencing of Defensin A reduced the survival, enhanced nicotine sensitivity of N. lugens to nicotine, and decreased the abundance of BsNLG8. The reintroduction of BsNLG8 improved the expression of immune genes, aiding nicotine resistance of N. lugens. Our findings indicate a potential reciprocal immunomodulatory interaction between Defensin A and BsNLG8 under nicotine stress. Moreover, this study offers novel and valuable insights for future research into enhancing nicotine-based pest management programs and developing alternative biocontrol methods involving the implication of insect symbionts.

Endogenous Antimicrobial-Immunomodulatory Molecules: Networking Biomolecules of Innate Immunity.

Endogenous antimicrobial-immunomodulatory molecules (EAIMs) are essential to immune-mediated human health and evolution. Conventionally, antimicrobial peptides (AMPs) have been regarded as the dominant endogenous antimicrobial molecule; however, AMPs are not sufficient to account for the full spectrum of antimicrobial-immunomodulatory duality occurring within the human body. The threat posed by pathogenic microbes is pervasive with the capacity for widespread impact across many organ systems and multiple biochemical pathways; accordingly, the host needs the capacity to react with an equally diverse response. This can be attained by having EAIMs that traverse the full range of molecular size (small to large molecules) and structural diversity (including molecules other than peptides). This review identifies multiple molecules (peptide/protein, lipid, carbohydrate, nucleic acid, small organic molecule, and metallic cation) as EAIMs and discusses the possibility of cooperative, additive effects amongst the various EAIM classes during the host response to a microbial assault. This comprehensive consideration of the full molecular diversity of EAIMs enables the conclusion that EAIMs constitute a previously uncatalogued structurally diverse and collectively underappreciated immuno-active group of integrated molecular responders within the innate immune system's first line of defence.

Plant Protease Inhibitors as Emerging Antimicrobial Peptide Agents: A Comprehensive Review

Antimicrobial peptides (AMPs) are important mediator molecules of the innate defense mechanisms in a wide range of living organisms, including bacteria, mammals, and plants. Among them, peptide protease inhibitors (PPIs) from plants play a central role in their defense mechanisms by directly attacking pathogens or by modulating the plant’s defense response. The growing prevalence of microbial resistance to currently available antibiotics has intensified the interest concerning these molecules as novel antimicrobial agents. In this scenario, PPIs isolated from a variety of plants have shown potential in inhibiting the growth of pathogenic bacteria, protozoans, and fungal strains, either by interfering with essential biochemical or physiological processes or by altering the permeability of biological membranes of invading organisms. Moreover, these molecules are active inhibitors of a range of proteases, including aspartic, serine, and cysteine types, with some showing particular efficacy as trypsin and chymotrypsin inhibitors. In this review, we provide a comprehensive analysis of the potential of plant-derived PPIs as novel antimicrobial molecules, highlighting their broad-spectrum antimicrobial efficacy, specificity, and minimal toxicity. These natural compounds exhibit diverse mechanisms of action and often multifunctionality, positioning them as promising molecular scaffolds for developing new therapeutic antibacterial agents.

Phyllanthus emblica Fruit Improves Obesity by Reducing Appetite and Enhancing Mucosal Homeostasis via the Gut Microbiota-Brain-Liver Axis in HFD-Induced Leptin-Resistant Rats.

The impact of leptin resistance on intestinal mucosal barrier integrity, appetite regulation, and hepatic lipid metabolism through the microbiota-gut-brain-liver axis has yet to be determined. Water extract of Phyllanthus emblica L. fruit (WEPE) and its bioactive compound gallic acid (GA) effectively alleviated methylglyoxal (MG)-triggered leptin resistance in vitro. Therefore, this study investigated how WEPE and GA intervention relieve leptin resistance-associated dysfunction in the intestinal mucosa, appetite, and lipid accumulation through the microbiota-gut-brain-liver axis in high-fat diet (HFD)-fed rats. The results showed that WEPE and GA significantly reduced tissues (jejunum, brain, and liver) MG-evoked leptin resistance, malondialdehyde (MDA), proinflammatory cytokines, SOCS3, orexigenic neuropeptides, and lipid accumulation through increasing leptin receptor, tight junction proteins, antimicrobial peptides, anorexigenic neuropeptides, excretion of fecal triglyceride (TG), and short-chain fatty acids (SCFAs) via a positive correlation with the Allobaculum and Bifidobacterium microbiota. These novel findings suggest that WEPE holds the potential as a functional food ingredient for alleviating obesity and its complications.

Recent developments in antibiotic contamination of animal products, soil, and water worldwide

Abstract Antimicrobial resistance (AMR), facilitated by antibiotic consumption, remains one of the biggest threats to global health and food security. The burgeoning AMR has an estimated forecast of 10 million deaths and 100 trillion USD economic losses annually worldwide by 2050 if no urgent actions are taken. The indiscriminate use of antibiotics in food animal production plays an expressive role in the AMR crisis. This paper compiles information regarding antibiotics and AMR in animals, animal-derived products, and agriculture-impacted environment. A holistic approach is needed to mitigate the burden of AMR within the context of human-animal-environment. Currently there are few approaches to this problem such as nanotechnology, anaerobic digestion, biochar composting, and alternatives to antibiotic treatments (like herbal plant extracts, probiotics, vaccines, enzymes, and antimicrobial peptides) have been developed. However, there are gaps in knowledge about AMR and areas for improvement are obvious. There is no a clear path to put an end to the persistent trends of AMR. Despite the trends for stricter regulation on the use of antibiotics worldwide, they find their way into food animal production, water, and soil as a result of misuses in many countries. We need to acknowledge the antibiotic contamination and/or AMR as a silent pandemic, and we are challenged to adopt a global approach to reducing and improving their use.

OA25 Transcriptome analysis of peripheral blood and renal tissue identifies genes that predict treatment response in proliferative lupus nephritis: a prospective observational study

Abstract Background/Aims Mechanisms contributing to non-response in lupus nephritis (LN) are not clear. Peripheral blood mononuclear cells (PBMCs) serve as easily available less invasive alternative to kidney biopsy in monitoring treatment response. However, it is unclear to what extent peripheral blood mirrors the inflamed kidney. This study aims to identify changes in functional pathways from PBMCs and renal tissues after cyclophosphamide treatment and to identify baseline genes that predict treatment response by RNA-sequencing. Methods RNA from PBMCs and renal biopsies was extracted from 16 treatment-naïve proliferative LN at baseline, 6-months and at the time of flare within 2 years of follow-up. Patients were classified as clinical-responders (CR) (n = 4) or non-responders (NR) (n = 8) at 6-months using EULAR/EDTA criteria. CR who developed renal flare on follow-up were considered FL (n = 4). RNA was sequenced on Illumina Novaseq-6000 platform. Healthy control datasets were taken from public databases. Differentially expressed genes (DEGs) and enriched pathways were analysed using Reactome database-v74. Comparisons for each group were performed before vs after treatment. Weighted gene co-expression network analysis (WGCNA) was used to identify baseline hub genes that predict treatment-response and ROC analysis was used to test their prognostic utility. Results Functional enrichment analysis identified neutrophil degranulation, autophagy, regulation of NF-κβ signalling, FCGR-dependent phagocytosis and TNF signalling upregulated and IFN-γ signalling and ECM molecules downregulated in PBMCs of CR. ECM molecules and proteoglycans, neddylation and BCR signalling were enriched in PBMCs of NR. In NR renal tissue, apoptosis, NF-κβ signalling, IKK complex formation upon TLR signalling and MAPK signalling were enriched. In FL PBMCs, neutrophil degranulation, ROS and RNS production in phagocytes, antimicrobial peptide and immune system pathways were downregulated. In FL renal tissue, glycogen metabolism, viral mRNA translation, cell-ECM interactions, cell-cell communications, apoptosis and ISG15 antiviral mechanism were downregulated. Clinical traits for WGCNA were BCR, BNR, BFL and HC. The most correlated modules and hub genes from baseline PBMC were: green (BCR- IFI27, CD24, AZU1, MT2A, SNRNP70), brown (BNR- AC090809.1, TENM2, NLGN1, LSAMP, DLGAP2, AP005230.1, TMEM132D, AL445255.1, LINC00536, AC013287.1, BX470102.1, TM4SF20) and blue module (BFL- AC092436.3, AC046158.3, LINC01147). From baseline renal tissues, the most correlated modules and genes were: green (BCR- AP003068.1, BX470102.1, AC009088.3, AC136475.1, AC073611.1, AP000757.2) and turquoise module (BNR & BFL: TRDN-AS1, AC107973.1, AL672167.1, AC020637.1, AL137100.3, AC018437.3, LINC01982, TPH2, AC136759). From PBMCs, ROC analysis showed that combinational model of TENM2, NLGN1 and AP005230.1 predicted NR (AUC-0.94; 95% CI: 0.80-1; p = 0.02) and AC092436.3 predicted FL (AUC-0.81; 95% CI: 0.53-1; p = 0.036). AP005230.1 from renal tissue also predicted NR (AUC-0.94; 95% CI: 0.82-1; p = 0.01). Conclusion Type I IFN and fibrosis pathways were enriched in NR and downregulated in CR and FL. Baseline PBMC markers TENM2, NLGN1 & AP005230.1 predicted NR and FL. AP005230.1 from PBMCs & renal tissue predicted NR. Disclosure S. Bulusu: None. A. Bavikatte: None. S. Shah: None. S. Murthy: None. C.M. Mariaselvam: None. C. Kavadichanda: None. S. Vembar: None. M. Thabah: None. V.S. Negi: None.

PH- and Matrix Metalloproteinase-Responsive Multifunctional Bilayer Microneedles Platform for Treatment of Tinea Pedis.

Persistent foot odor and itchiness are common symptoms of tinea pedis, significantly disrupting the daily life of those affected. The cuticular barrier at the site of the tinea pedis is thickened, which impedes the effective penetration of antifungal agents. Additionally, fungi can migrate from the skin surface to deeper tissues, posing challenges in the current clinical treatment for tinea pedis. To effectively treat tinea pedis, we developed a platform of bilayer gelatin methacrylate (GelMA) microneedles (MNs) loaded with salicylic acid (SA) and FK13-a1 (SA/FK13-a1@GelMA MNs). SA/FK13-a1@GelMA MNs exhibit pH- and matrix metalloproteinase (MMP)-responsive properties for efficient drug delivery. The MNs are designed to deliver salicylic acid (SA) deep into the stratum corneum, softening the cuticle and creating microchannels. This process enables the antibacterial peptide FK13-a1 to penetrate through the stratum corneum barrier, facilitating intradermal diffusion and exerting antifungal and anti-inflammatory effects. In severe cases of tinea pedis, heightened local pH levels and MMP activity further accelerate drug release. Our research demonstrates that SA/FK13-a1@GelMA MNs are highly effective against Trichophyton mentagrophytes, Trichophyton rubrum, and Candida albicans. They also reduced stratum corneum thickness, fungal burden, and inflammation in a guinea pig model of tinea pedis induced by T. mentagrophytes. Furthermore, it was discovered that SA/FK13-a1@GelMA MNs exhibit excellent biocompatibility. These findings suggest that SA/FK13-a1@GelMA MNs have significant potential for the clinical treatment of tinea pedis as well as other fungal skin disorders.

Broad-spectrum activity of membranolytic cationic macrocyclic peptides against multi-drug resistant bacteria and fungi

The emergence of multidrug-resistant (MDR) strains causes severe problems in the treatment of microbial infections owing to limited treatment options. Antimicrobial peptides (AMPs) are drawing considerable attention as promising antibiotic alternative candidates to combat MDR bacterial and fungal infections. Herein, we present a series of small amphiphilic membrane-active cyclic peptides composed, in part, of various nongenetically encoded hydrophilic and hydrophobic amino acids. Notably, lead cyclic peptides 3b and 4b showed broad-spectrum activity against drug-resistant Gram-positive (MIC = 1.5–6.2 µg/mL) and Gram-negative (MIC = 12.5–25 µg/mL) bacteria, and fungi (MIC = 3.1–12.5 µg/mL). Furthermore, lead peptides displayed substantial antibiofilm action comparable to standard antibiotics. Hemolysis (HC50 = 230 µg/mL) and cytotoxicity (>70 % cell viability against four different mammalian cells at 100 µg/mL) assay results demonstrated the selective lethal action of 3b against microbes over mammalian cells. A calcein dye leakage experiment substantiated the membranolytic effect of 3b and 4b, which was further confirmed by scanning electron microscopy. The behavior of 3b and 4b in aqueous solution and interaction with phospholipid bilayers were assessed by employing nuclear magnetic resonance (NMR) spectroscopy in conjunction with molecular dynamics (MD) simulations, providing a solid structural basis for understanding their membranolytic action. Moreover, 3b exhibited stability in human blood plasma (t1/2 = 13 h) and demonstrated no signs of resistance development against antibiotic-resistant S. aureus and E. coli. These findings underscore the potential of these newly designed amphiphilic cyclic peptides as promising anti-infective agents, especially against Gram-positive bacteria.

Discovery of new antibiotics using AI-guided spectroscopy and 3D drug-protein computer simulation technologies to combat MDR bacteria-associated mortality

Multidrug-resistant (MDR), extensively drug-resistant (XDR), and totally drug-resistant bacteria can cause sepsis and death in patients due to their ability to inactivate most antibiotics, including ampicillin, tetracycline, streptomycin, chloramphenicol, erythromycin, and ciprofloxacin. This paper aims to review recent advancements in synthetic antibiotics, lantibiotics, and phytoantibiotics and to present our research on phytoantibiotics, specifically focusing on CU1 and NU2. While third- and fifth-generation synthetic antibiotics such as meropenem, moxifloxacin, amikacin, and tigecycline are currently relied upon for treating MDR infections, research is underway to develop peptide antibiotics known as lantibiotics (e.g., nisins, bacteriocins, and salivaricins). Lantibiotics such as nisin-A and salivaricin-B have demonstrated efficacy in curing numerous MDR infections, while phytochemicals such as artemisinin and quinine have shown effectiveness against chloroquine-resistant Plasmodium falciparum infections (malaria). In our study, we utilized techniques such as mass spectroscopy, nuclear magnetic resonance, and Fourier transform infrared spectroscopy in conjunction with artificial intelligence (AI) and computer simulation technologies to determine the structure of phytochemicals. Our results revealed that CU1, derived from Cassia fistula bark ethanol extract, exhibits potent antibiotic activity against XDR bacteria by targeting the RNA polymerases of Escherichia coli and Mycobacterium tuberculosis. Consequently, our MDR-Cure extract containing CU1 represents a promising antibacterial Ayurvedic medicine specifically tailored for skin and nail infections. Similarly, NU2 poly-fluorophosphate-glycosides from Suregada multiflora roots ethanol extract exhibited strong inhibitory effects on XDR bacteria by targeting DNA topoisomerase I. Recently, many cyclic peptide antibiotics have been synthesized in vitro using computer-guided AI technologies to predict 3D drug-enzyme interactions and are currently undergoing clinical trials. Our ultimate goal is to combat XDR bacteria-associated deaths, which are predicted to escalate as we approach 2050.

Dissecting the respective roles of microbiota and host genetics in the susceptibility of Card9−/− mice to colitis

BackgroundThe etiology of inflammatory bowel disease (IBD) is unclear but involves both genetics and environmental factors, including the gut microbiota. Indeed, exacerbated activation of the gastrointestinal immune system toward the gut microbiota occurs in genetically susceptible hosts and under the influence of the environment. For instance, a majority of IBD susceptibility loci lie within genes involved in immune responses, such as caspase recruitment domain member 9 (Card9). However, the relative impacts of genotype versus microbiota on colitis susceptibility in the context of CARD9 deficiency remain unknown.ResultsCard9 gene directly contributes to recovery from dextran sodium sulfate (DSS)-induced colitis by inducing the colonic expression of the cytokine IL-22 and the antimicrobial peptides Reg3β and Reg3γ independently of the microbiota. On the other hand, Card9 is required for regulating the microbiota capacity to produce AhR ligands, which leads to the production of IL-22 in the colon, promoting recovery after colitis. In addition, cross-fostering experiments showed that 5 weeks after weaning, the microbiota transmitted from the nursing mother before weaning had a stronger impact on the tryptophan metabolism of the pups than the pups’ own genotype.ConclusionsThese results show the role of CARD9 and its effector IL-22 in mediating recovery from DSS-induced colitis in both microbiota-independent and microbiota-dependent manners. Card9 genotype modulates the microbiota metabolic capacity to produce AhR ligands, but this effect can be overridden by the implantation of a WT or “healthy” microbiota before weaning. It highlights the importance of the weaning reaction occurring between the immune system and microbiota for host metabolism and immune functions throughout life. A better understanding of the impact of genetics on microbiota metabolism is key to developing efficient therapeutic strategies for patients suffering from complex inflammatory disorders.-8ZHpF322gum1G2x3b5GvyVideo

Trypanosomes and Gut Microbiota Interactions in Triatomine bugs and Tsetse Flies: A vectorial perspective.

Triatomines (kissing bugs) and tsetse flies (genus: Glossina) are natural vectors of Trypanosoma cruzi and Trypanosoma brucei, respectively. T. cruzi is the causative agent of Chagas disease, endemic in Latin America, while T. brucei causes African sleeping sickness disease in sub-Saharan Africa. Both triatomines and tsetse flies are host to a diverse community of gut microbiota that co-exist with the parasites in the gut. Evidence has shown that the gut microbiota of both vectors plays a key role in parasite development and transmission. However, knowledge on the mechanism involved in parasite-microbiota interaction remains limited and scanty. Here, we attempt to analyse Trypanosoma spp. and gut microbiota interactions in tsetse flies and triatomines, with a focus on understanding the possible mechanisms involved by reviewing published articles on the subject. We report that interactions between Trypanosoma spp. and gut microbiota can be both direct and indirect. In direct interactions, the gut microbiota directly affects the parasite via the formation of biofilms and the production of anti-parasitic molecules, while on the other hand, Trypanosoma spp. produces antimicrobial proteins to regulate gut microbiota of the vector. In indirect interactions, the parasite and gut bacteria affect each other through host vector-activated processes such as immunity and metabolism. Although we are beginning to understand how gut microbiota interacts with the Trypanosoma parasites, there is still a need for further studies on functional role of gut microbiota in parasite development to maximize the use of symbiotic bacteria in vector and parasite control.

Prediction of enzyme action for extraction of antimicrobial substances from <i>Sus scrofa</i> and <i>Bos taurus</i>

The study of antimicrobial compounds of animal origin, particularly antimicrobial peptides (AMPs), is a current research topic. However, extracting endogenous AMPs is a challenging process and requires the application of targeted enzymatic processing principles based on knowledge of the structure of prepropeptide molecules — precursors of AMPs. In this study, a search was conducted for antimicrobial peptides present in Sus scrofa and Bos taurus organisms, as well as their precursors, using The Antimicrobial Peptide Database and UniProtKB databases. In the amino acid sequences of prepropeptides, the sequences of the mature peptides were found, and cleavage sites for trypsin, bacterial collagenase (type I), and neutrophil elastase were determined. As a result of the search for antimicrobial compounds in The Antimicrobial Peptide Database, 18 antimicrobial peptides from Sus scrofa and 40 antimicrobial peptides from Bos taurus were identified. Based on the results of determining cleavage sites in AMP precursors, enzymes were ranked from less preferred to more preferred for AMP release as follows: bacterial collagenase (type I) ≤ trypsin < neutrophil elastase. This order is justified not only by the number of suitable cleavage sites and their accuracy but also by the action of enzymes within mature AMPs: it is important to consider that enzymes can “cut” the peptides themselves, thereby reducing their antimicrobial activity. The bioinformatics analysis conducted is applicable for both primary screening of raw material potential and determining of suitable enzymes for extracting antimicrobial compounds from Sus scrofa and Bos taurus organisms.

Hybridization of Honeybee and Frog Peptides: Antimicrobial Peptides Composed of Apidaecins and Temporins With Enhanced Activity and Production of Their Electrospun Nanofibers

ABSTRACTThe increased usage and misuse of antibiotics cause antimicrobial resistance, which makes it necessary to develop new antibiotic agents. Antimicrobial peptides (AMPs) could be a significant solution to addressing this issue. Antimicrobial temporin 1Ta and temporin 1Tb peptides, derived from frog skin secretions, exhibit notable activity against Gram (+) bacteria; on the other hand, apidaecin 1b peptide, obtained from honeybees, is effective against Gram (−) bacteria. In this study, hybrid AMPs were synthesized by merging temporin 1Ta and temporin 1Tb with apidaecin 1b to produce peptides that are active against a broad spectrum of microorganisms. Furthermore, the hybrid peptides were mixed with polyvinyl alcohol (PVA) polymer to produce bioactive nanofibers by electrospinning. The synthesized peptides and the nanofibers were tested on various Gram (+) and Gram (−) bacteria (E. coli ATCC 25922, P. aeruginosa ATCC 27853, S. aureus ATCC 29213, and E. faecalis ATCC 29212). As a result, the hybrid peptides were found to have a reasonable antibacterial effect on both bacterial groups. In addition, peptide‐containing nanofibers preserved the antibacterial activity as well. The hemolytic activities of the peptides were also examined, and it was found that the hybrid peptides were not lethal to red blood cells at a concentration of 16 μM.

Controlling Candida: immune regulation of commensal fungi in the gut.

The intestinal microbiome harbors fungi that pose a significant risk to human health as opportunistic pathogens and drivers of inflammation. Inflammatory and autoimmune diseases are associated with dysbiotic fungal communities and the expansion of potentially pathogenic fungi. The gut is also the main reservoir for disseminated fungal infections. Immune interactions are critical for preventing commensal fungi from becoming pathogenic. Significant strides have been made in defining innate and adaptive immune pathways that regulate intestinal fungi, and these discoveries have coincided with advancements in our understanding of the fungal molecular pathways and effectors involved in both commensal colonization and pathogenesis within the gut. In this review, we will discuss immune interactions important for regulating commensal fungi, with a focus on how specific cell types and effectors interact with fungi to limit their colonization or pathogenic potential. This will include how innate and adaptive immune pathways target fungi and orchestrate antifungal immune responses, in addition to how secreted immune effectors, such as mucus and antimicrobial peptides, regulate fungal colonization and inhibit pathogenic potential. These immune interactions will be framed around our current understanding of the fungal effectors and pathways regulating colonization and pathogenesis within this niche. Finally, we highlight important unexplored mechanisms by which the immune system regulates commensal fungi in the gut.

Self-assembly of spin-labeled antimicrobial peptides magainin 2 and PGLa in lipid bilayers

The cationic antimicrobial peptides PGLa and magainin 2 (Mag2) are known for their antimicrobial activity and synergistic enhancement in antimicrobial and membrane leakage assays. Further use of peptides in combinatory therapy requires knowledge of the mechanisms of action of both individual peptides and their mixtures. Here, electron paramagnetic resonance (EPR), double electron-electron resonance (DEER, also known as PELDOR) and electron spin echo envelope modulation (ESEEM) spectroscopies were applied to study self-assembly and localization of spin-labeled PGLa and Mag2 in POPE/POPG membranes with a wide range of peptide/lipid ratios (P/L) from ∼1/1500 to 1/50. EPR and DEER data showed that both peptides tend to organize in clusters, which occurs already at the lowest peptide/lipid molar ratio of 1/1500 (0.067 mol%). For individual peptides, these clusters are quite dense with intermolecular distances of the order of ∼2 nm. In the presence of a synergistic peptide partner, these homo-clusters are transformed into lipid-diluted hetero-clusters. These clusters are characterized by a local surface density that is several times higher than expected from a random distribution. ESEEM data indicate a slightly different insertion depth of peptides in hetero-clusters when compared to homo-clusters.

Corallococcus senghenyddensis sp. nov., a myxobacterium with potent antimicrobial activity.

Corallococcus species are diverse in the natural environment with ten new Corallococcus species having been characterised in just the last five years. As well as being an abundant myxobacterial genus they produce several secondary metabolites, including Corallopyronin, Corramycin, Coralmycin and Corallorazine. We isolated a novel strain Corallococcus spp RDP092CA from soil in South Wales, United Kingdom using Candida albicans as prey bait and characterised its predatory activities against pathogenic bacteria and yeast. The size of the RDP092CA genome was 8.5 Mb with a G+C content of 71.4%. Phylogenetically, RDP092CA is closely related to Corallococcus interemptor, Corallococcus coralloides and Corallococcus exiguus. However, genome average nucleotide identity and digital DNA-DNA hybridisation values are lower than 95% and 70% when compared to those type strains, implying it belongs to a novel species. The RDP092CA genome harbours seven types of biosynthetic gene clusters (BGCs) and 152 predicted antimicrobial peptides. In predation assays, RDP092CA showed good predatory activity against Escherichia coli, Pseudomonas aeruginosa, Citrobacter freundii, Staphylococcus aureus but not against Enterococcus faecalis. It also showed good antibiofilm activity against all five bacteria in biofilm assays. Antifungal activity against eight Candida spp. was variable, with particularly good activity against Meyerozyma guillermondii DSM 6381. Antimicrobial peptide RDP092CA_120 exhibited potent antibiofilm activity with>50% inhibition and>60% dispersion of biofilms at concentrations down to 1μg/ml. We propose that strain RDP092CA represents a novel species with promising antimicrobial activities, Corallococcus senghenyddensis sp. nov (=NBRC 116490T=CCOS 2109T), based on morphological, biochemical, and genomic features.

Effects of Tetraselmis subcordiformis (Wille) Butcher with antimicrobial peptides as feed additives on gut microbial community and immunity of turbot (Scophthalmus maximus L.)

Effects of Tetraselmis subcordiformis (Wille) Butcher with antimicrobial peptides as feed additives on gut microbial community and immunity of turbot (Scophthalmus maximus L.)

The role of morphine- and fentanyl-induced impairment of intestinal epithelial antibacterial activity in dysbiosis and its impact on the microbiota-gut-brain axis.

Recent evidence suggests that chronic exposure to opioid analgesics such as morphine disrupts the intestinal epithelial layer and causes intestinal dysbiosis. Depleting gut bacteria can preclude the development of tolerance to opioid-induced antinociception, suggesting an important role of the gut-brain axis in mediating opioid effects. The mechanism underlying opioid-induced dysbiosis, however, remains unclear. Host-produced antimicrobial peptides (AMPs) are critical for the integrity of the intestinal epithelial barrier as they prevent the pathogenesis of the enteric microbiota. Here, we report that chronic morphine or fentanyl exposure reduces the antimicrobial activity in the ileum, resulting in changes in the composition of bacteria. Fecal samples from morphine-treated mice had increased levels of Akkermansia muciniphila with a shift in the abundance ratio of Firmicutes and Bacteroidetes. Fecal microbial transplant (FMT) from morphine-naïve mice or oral supplementation with butyrate restored (a) the antimicrobial activity, (b) the expression of the antimicrobial peptide, Reg3γ, (c) prevented the increase in intestinal permeability and (d) prevented the development of antinociceptive tolerance in morphine-dependent mice. Improved epithelial barrier function with FMT or butyrate prevented the enrichment of the mucin-degrading A. muciniphila in morphine-dependent mice. These data implicate impairment of the antimicrobial activity of the intestinal epithelium as a mechanism by which opioids disrupt the microbiota-gut-brain axis.

Pan-neuronal expression of human mutant SOD1 in Drosophila impairs survival and motor performance, induces early neuroinflammation and chromosome aberrations

Several mutations in the SOD1 gene encoding for the antioxidant enzyme Superoxide Dismutase 1, are associated with amyotrophic lateral sclerosis, a rare and devastating disease characterized by motor neuron degeneration and patients' death within 2–5 years from diagnosis. Motor neuron loss and related symptomatology manifest mostly in adult life and, to date, there is still a gap of knowledge on the precise cellular and molecular events preceding neurodegeneration.To deepen our awareness of the early phases of the disease, we leveraged two Drosophila melanogaster models pan-neuronally expressing either the mutation A4V or G85R of the human gene SOD1 (hSOD1A4V or hSOD1G85R).We demonstrate that pan-neuronal expression of the hSOD1A4V or hSOD1G85R pathogenic construct impairs survival and motor performance in transgenic flies. Moreover, protein and transcript analysis on fly heads indicates that mutant hSOD1 induction stimulates the glial marker Repo, up-regulates the IMD/Toll immune pathways through antimicrobial peptides and interferes with oxidative metabolism. Finally, cytological analysis of larval brains demonstrates hSOD1-induced chromosome aberrations. Of note, these parameters are found modulated in a timeframe when neurodegeneration is not detected.The novelty of our work is twofold: we have expressed for the first time hSOD1 mutations in all neurons of Drosophila and confirmed some ALS-related pathological phenotypes in these flies, confirming the power of SOD1 mutations in generating ALS-like phenotypes. Moreover, we have related SOD1 pathogenesis to chromosome aberrations and antimicrobial peptides up-regulation. These findings were unexplored in the SOD1-ALS field.