Dermaseptin B2 bioactive gene's potential for anticancer and anti-proliferative effect is linked to the regulation of the BAX/BBC3/AKT pathway.

Dermaseptin B2 (DrsB2) is an antimicrobial peptide with anticancer and angiostatic properties. We aimed to assess the in vitro inhibitory effect of pDNA/DrsB2 on the growth of breast cancer cells and its impact on the expression of genes involved in the BAX/BBC3/AKT pathway. The nucleic acid sequence of DrsB2 was artificially synthesized and inserted into the pcDNA3.1( +) Mammalian Expression Plasmid. PCR testing and enzyme digesting procedures evaluated the accuracy of cloning. The vectors were introduced into cells using LipofectamineTM2000 transfection reagent. The breast cancer cells were assessed by flow cytometry, MTT assessment, soft agar colony method, and wound healing investigation. The gene's transcription was evaluated using real-time PCR with a significance level of P < 0.05. The recombinant plasmid harboring the pDNA/DrsB2 vector was effectively produced, and the gene sequence showed absolute homogeneity (100% similarity) with the DrsB2 gene. The transfection effectiveness of MCF-7 and MCF-10A cells was 79% and 68%, respectively. The findings are measured using the growth inhibition 50% (GI50) metric, which indicates the concentration of pDNA/DrsB2 that stops 50% of cell growth. The proportions of early apoptosis, late apoptosis, necrosis, and viable MCF-7 cells in the pDNA/DrsB2 group were 40.50%, 2.31%, 1.69%, and 55.50%, respectively. The results showed a 100% increase in gene expression in programmed cell death following treatment with pDNA/DrsB2 (**P < 0.01). To summarize, the results described in this work offer new possibilities for treating cancer by targeting malignancies via pDNA/DrsB2 and activating the BAX/BBC3/AKT signaling pathways.

CXCR2, as a key regulatory gene of HDP-PG-1, maintains intestinal mucosal homeostasis.

The intestine defends against pathogenic microbial invasion via the secretion of host defense peptides (HDPs). Nutritional immunomodulation can stimulate the expression of endogenous HDPs and enhance the body's immune defense, representing a novel non-antibiotic strategy for disease prevention. The project aims to explore the regulatory mechanism of protegrin-1 (PG-1) expression using sodium phenylbutyrate (PBA) by omics sequencing technology and further investigate the role of key regulatory genes on intestinal health. The results showed that PBA promoted PG-1 expression in intestinal epithelial cells based on cell density through epidermal growth factor receptor (EGFR) and G protein-coupled receptor (GPR43). Transcriptome sequencing and microRNA sequencing revealed that C-X-C motif chemokine receptor 2 (CXCR2) exhibited interactions with PG-1. Pre-treatment cells with a CXCR2 inhibitor (SB225002) effectively suppressed the induction of PG-1 by PBA. Furthermore, SB225002 significantly suppressed the gene expression of HDPs in the jejunum of mice without influencing on the morphology, number of goblet cells, and proliferation of the intestine. CXCR2 inhibition significantly reduced the expression of HDPs during E. coli infection, and resulted in the edema of jejunal epithelial cells. The 16S rDNA analysis of cecal contents showed that the E. coli and SB225002 treatments changed gut microbiota diversity and composition at different taxonomic levels. Correlation analysis suggested a potential regulatory relationship between gut microbiota and HDPs. To that end, a gene involved in the HDP expression, CXCR2, has been identified in the study, which contributes to improving intestinal immune function. PBA may be used as a functional additive to regulate intestinal mucosal function, thereby enhancing the health of the intestinal and host.

Dectin-1/SYK Activation Induces Antimicrobial Peptide and Negative Regulator of NF-κB Signaling in Human Oral Epithelial Cells.

BACKGROUND/AIM: Oral epithelial cells serve as the primary defense against microbial exposure in the oral cavity, including the fungus Candida albicans. Dectin-1 is crucial for recognition of ß-glucan in fungi. However, expression and function of Dectin-1 in oral epithelial cells remain unclear. MATERIALS AND METHODS: We assessed Dectin-1 expression in Ca9-22 (gingiva), HSC-2 (mouth), HSC-3 (tongue), and HSC-4 (tongue) human oral epithelial cells using flow cytometry and real-time polymerase chain reaction. Cell treated with ß-glucan-rich zymosan were evaluated using real-time polymerase chain reaction. Phosphorylation of spleen-associated tyrosine kinase (SYK) was analyzed by western blotting. RESULTS: Dectin-1 was expressed in all four cell types, with high expression in Ca9-22 and HSC-2. In Ca9-22 cells, exposure to ß-glucan-rich zymosan did not alter the mRNA expression of chemokines nor of interleukin (IL)6, IL8, IL1ß, IL17A, and IL17F. Zymosan induced the expression of antimicrobial peptides ß-defensin-1 and LL-37, but not S100 calcium-binding protein A8 (S100A8) and S100A9. Furthermore, the expression of cylindromatosis (CYLD), a negative regulator of nuclear factor kappa B (NF-κB) signaling, was induced. In HSC-2 cells, zymosan induced the expression of IL17A. The expression of tumor necrosis factor alpha-induced protein 3 (TNFAIP3), a negative regulator of NF-κB signaling, was also induced. Expression of other cytokines and antimicrobial peptides remained unchanged. Zymosan induced phosphorylation of SYK in Ca9-22 cells, as well as NF-κB. CONCLUSION: Oral epithelial cells express Dectin-1 and recognize ß-glucan, which activates SYK and induces the expression of antimicrobial peptides and negative regulators of NF-κB, potentially maintaining oral homeostasis.

Molecular characterization, expression and antibacterial function of a macin, HdMac, from Haliotis discus hannai.

Macins are a family of antimicrobial peptides, which play multiple roles in the elimination of invading pathogens. In the present study, a macin was cloned and characterized from Pacific abalone Haliotis discus hannai (Designated as HdMac). Analysis of the conserved domain suggested that HdMac was a new member of the macin family. In non-stimulated abalones, HdMac transcripts were constitutively expressed in all five tested tissues, especially in hemocytes. After Vibrio harveyi stimulation, the expression of HdMac mRNA in hemocytes was significantly up-regulated at 12 hr (P < 0.01). RNAi-mediated knockdown of HdMac transcripts affected the survival rates of abalone against V. harveyi. Moreover, recombinant protein of HdMac (rHdMac) exhibited high antibacterial activities against invading bacteria, especially for Vibrio anguillarum. In addition, rHdMac possessed binding activities towards glucan, lipopolysaccharides (LPS), and peptidoglycan (PGN), but not chitin in vitro. Membrane integrity analysis revealed that rHdMac could increase the membrane permeability of bacteria. Meanwhile, both the phagocytosis and chemotaxis ability of hemocytes could be significantly enhanced by rHdMac. Overall, the results showed that HdMac could function as a versatile molecule involved in immune responses of H. discus hannai.

An esculentin-1 homolog from a dark-spotted frog (Pelophylax nigromaculatus) possesses antibacterial and immunoregulatory properties.

BACKGROUND: Esculentin-1, initially discovered in the skin secretions of pool frogs (Pelophylax lessonae), has demonstrated broad-spectrum antimicrobial activity; however, its immunomodulatory properties have received little attention. RESULTS: In the present study, esculentin-1 cDNA was identified by analysing the skin transcriptome of the dark-spotted frog (Pelophylax nigromaculatus). Esculentin-1 from this species (esculentin-1PN) encompasses a signal peptide, an acidic spacer peptide, and a mature peptide. Sequence alignments with other amphibian esculentins-1 demonstrated conservation of the peptide, and phylogenetic tree analysis revealed its closest genetic affinity to esculentin-1P, derived from the Fukien gold-striped pond frog (Pelophylax fukienensis). Esculentin-1PN transcripts were observed in various tissues, with the skin exhibiting the highest mRNA levels. Synthetic esculentin-1PN demonstrated antibacterial activity against various pathogens, and esculentin-1PN exhibited bactericidal activity by disrupting cell membrane integrity and hydrolyzing genomic DNA. Esculentin-1PN did not stimulate chemotaxis in RAW264.7, a murine leukemic monocyte/macrophage cell line. However, it amplified the respiratory burst and augmented the pro-inflammatory cytokine gene (TNF-α and IL-1ß) expression in RAW264.7 cells. CONCLUSIONS: This novel finding highlights the immunomodulatory activity of esculentin-1PN on immune cells.

A humoral stress response protects Drosophila tissues from antimicrobial peptides.

7An efficient immune system must provide protection against a broad range of pathogens without causing excessive collateral tissue damage. While immune effectors have been well characterized, we know less about the resilience mechanisms protecting the host from its own immune response. Antimicrobial peptides (AMPs) are small, cationic peptides that contribute to innate defenses by targeting negatively charged membranes of microbes. While protective against pathogens, AMPs can be cytotoxic to host cells. Here, we reveal that a family of stress-induced proteins, the Turandots, protect the Drosophila respiratory system from AMPs, increasing resilience to stress. Flies lacking Turandot genes are susceptible to environmental stresses due to AMP-induced tracheal apoptosis. Turandot proteins bind to host cell membranes and mask negatively charged phospholipids, protecting them from cationic pore-forming AMPs. Collectively, these data demonstrate that Turandot stress proteins mitigate AMP cytotoxicity to host tissues and therefore improve their efficacy.

Expression of Toll-like receptors and host defence peptides in the cecum of chicken challenged with Eimeria tenella.

Chicken coccidiosis, caused by Eimeria protozoa, affects poultry farming. Toll-like receptors (TLRs) and host defence peptides (HDPs) help host innate immune responses to eliminate invading pathogens, but their roles in Eimeria tenella infection remain poorly understood. Herein, 14-day-old chickens were treated orally with 50,000 E. tenella oocysts and the cecum was dissected at different timepoints. mRNA expression of 10 chicken TLRs (chTLRs) and five HDPs was measured by quantitative real-time PCR. chTLR7 and chTLR15 were upregulated significantly at 3 h post-infection while other chTLRs were downregulated (p < .05). chTLR1a, chTLR1b, chTLR2b and chTLR4 peaked at 36 h post-infection, chTLR3, chTLR5 and chTLR15 peaked at 72 h post-infection and chTLR21 expression was highest among chTLRs, peaking at 48 h post-infection (p < 0.05). For HDPs, cathelicidin (CATH) 1 to 3 and B1 peaked at 48 h post-infection, liver-expressed antimicrobial peptide 2 peaked at 96 h post-infection, and CATH 2 expression was highest among HDPs. CATH2 and CATH3 were markedly upregulated at 3 h post-infection (p < .05). The results provide insight into innate immune molecules during E. tenella infection in chicken, and indicate that innate immune responses may mediate resistance to chicken coccidiosis.

Expression and purification of epinecidin-1 variant (Ac-Var-1) by acid cleavage.

The demand for massive quantities of therapeutic active antimicrobial peptides (AMPs) is high due to their potential as alternatives to antibiotics. However, each antimicrobial peptide has unique properties, necessitating distinct synthesis and purification strategies for their large-scale production. In this study, we bio-synthesized and purified a functional enhanced variant of the AMP epinecidin-1, known as Ac-Var-1 (acid-cleavable variant-1). To generate the active peptide, we cloned the gene for Ac-Var-1 with acid-cleavable site (aspartic acid-proline) into the pET-32a expression vector, purified the fusion protein by His tag enrichment chromatography, and performed acid cleavage to release the active Ac-Var-1 peptide. After acid cleavage, the active Ac-Var-1 was purified and characterized by SDS-PAGE and mass spectrometry. The results from both techniques provided confirmation of the intactness of the purified Ac-Var-1. The Ac-Var-1 inhibited the growth of pathogenic Escherichia coli and Staphylococcus aureus. KEY POINTS : • Epinecidin-1 is a well-known antimicrobial peptide having multipotential bioactivities. • Epinecidin-1 variant is developed via the site-directed mutagenesis method to improve its structural stability and bioactivity. • AC-Var-1 development is an economical and easy method to remove peptide from tag protein.

Improving Photocleavage Efficiency of Photocleavable Protein for Antimicrobial Peptide Histatin 1 Expression.

BACKGROUND: Antimicrobial peptides (AMPs) are promising alternative agents for antibiotics to overcome antibiotic resistance problems. But, it is difficult to produce large-scale antimicrobial research due to the toxicity towards expression hosts or degradation by peptidases in the host. Therefore, heterologous recombinant expression of antimicrobial peptides has always been a challenging issue. OBJECTIVES: To overcome toxicity to the expression host and low expression level, a new photocleavable protein fusion expression method for antimicrobial peptides is provided.3 Methods: Through directed evolution and high throughput screening, a photocleavable protein mutant R6-2-6-4 with a higher photocleavage efficiency was obtained. The DNA coding sequence of antimicrobial peptide Histatin 1 was fused within the sequence of R6-2-6-4 gene. The fusion gene was successfully expressed in Escherichia coli expression system. RESULTS: Antimicrobial peptide Histatin 1 could be successfully expressed and purified by fusing within PhoCl mutant R6-2-6-4. The antimicrobial activity was rarely affected, and the MIC value was 33 ug/mL, which was basically equivalent to 32 ug/mL of the chemically synthesized Histatin 1. After amplification in a 5 L fermenter, the expression of PhoCl mutant (R6-2-6-4)-Histatin1 improved up to 87.6 mg/L in fermenter, and Histatin1 obtained by photocleavage also could up to 11 mg/L. The prepared Histatin1 powder remained stable when stored at 4oC for up to 4 months without any degradation. In addition, the expression and photocleavage of ß -Defensin105 and Lysostaphin verified the certain universality of the PhoCl mutant fusion expression system. CONCLUSION: Antimicrobial peptides Histatin 1, ß -Defensin 105 and Lysostaphin were successfully expressed and purified by photocleavable protein mutant. This may provide a novel strategy to express and purify antimicrobial peptides in the Escherichia coli expression system.

Recombinant production of antimicrobial peptides in plants.

Classical plant breeding methods are limited in their ability to confer disease resistance on plants. However, in recent years, advancements in molecular breeding and biotechnological have provided new approaches to overcome these limitations and protect plants from disease. Antimicrobial peptides (AMPs) constitute promising agents that may be able to protect against infectious agents. Recently, peptides have been recombinantly produced in plants at scale and low cost. Because AMPs are less likely than conventional antimicrobials to elicit resistance of pathogenic bacteria, they open up exciting new avenues for agricultural applications. Here, we review recent advances in the design and production of bioactive recombinant AMPs that can effectively protect crop plants from diseases.

Occurrence and evolutionary conservation analysis of α-helical cationic amphiphilic segments in the human proteome.

The existence of encrypted fragments with antimicrobial activity in human proteins has been thoroughly demonstrated in the literature. Recently, algorithms for the large-scale identification of these segments in whole proteomes were developed, and the pervasiveness of this phenomenon was stated. These algorithms typically mine encrypted cationic and amphiphilic segments of proteins, which, when synthesized as individual polypeptide sequences, exert antimicrobial activity by membrane disruption. In the present report, the human reference proteome was submitted to the software kamal for the uncovering of protein segments that correspond to putative intragenic antimicrobial peptides (IAPs). The assessment of the identity of these segments, frequency, functional classes of parent proteins, structural relevance, and evolutionary conservation of amino acid residues within their corresponding proteins was conducted in silico. Additionally, the antimicrobial and anticancer activity of six selected synthetic peptides was evaluated. Our results indicate that cationic and amphiphilic segments can be found in 2% of all human proteins, but are more common in transmembrane and peripheral membrane proteins. These segments are surface-exposed basic patches whose amino acid residues present similar conservation scores to other residues with similar solvent accessibility. Moreover, the antimicrobial and anticancer activity of the synthetic putative IAP sequences was irrespective to whether these are associated to membranes in the cellular setting. Our study discusses these findings in light of the current understanding of encrypted peptide sequences, offering some insights into the relevance of these segments to the organism in the context of their harboring proteins or as separate polypeptide sequences.

Perspectives in Searching Antimicrobial Peptides (AMPs) Produced by the Microbiota.

Changes in the structure and function of the microbiota are associated with various human diseases. These microbial changes can be mediated by antimicrobial peptides (AMPs), small peptides produced by the host and their microbiota, which play a crucial role in host-bacteria co-evolution. Thus, by studying AMPs produced by the microbiota (microbial AMPs), we can better understand the interactions between host and bacteria in microbiome homeostasis. Additionally, microbial AMPs are a new source of compounds against pathogenic and multi-resistant bacteria. Further, the growing accessibility to metagenomic and metatranscriptomic datasets presents an opportunity to discover new microbial AMPs. This review examines the structural properties of microbiota-derived AMPs, their molecular action mechanisms, genomic organization, and strategies for their identification in any microbiome data as well as experimental testing. Overall, we provided a comprehensive overview of this important topic from the microbial perspective.

Molecular hybridization strategy for tuning bioactive peptide function.

The physicochemical and structural properties of antimicrobial peptides (AMPs) determine their mechanism of action and biological function. However, the development of AMPs as therapeutic drugs has been traditionally limited by their toxicity for human cells. Tuning the physicochemical properties of such molecules may abolish toxicity and yield synthetic molecules displaying optimal safety profiles and enhanced antimicrobial activity. Here, natural peptides were modified to improve their activity by the hybridization of sequences from two different active peptide sequences. Hybrid AMPs (hAMPs) were generated by combining the amphipathic faces of the highly toxic peptide VmCT1, derived from scorpion venom, with parts of four other naturally occurring peptides having high antimicrobial activity and low toxicity against human cells. This strategy led to the design of seven synthetic bioactive variants, all of which preserved their structure and presented increased antimicrobial activity (3.1-128 µmol L-1). Five of the peptides (three being hAMPs) presented high antiplasmodial at 0.8 µmol L-1, and virtually no undesired toxic effects against red blood cells. In sum, we demonstrate that peptide hybridization is an effective strategy for redirecting biological activity to generate novel bioactive molecules with desired properties.

Resistance to host antimicrobial peptides mediates resilience of gut commensals during infection and aging in Drosophila.

Resilience to short-term perturbations, like inflammation, is a fundamental feature of microbiota, yet the underlying mechanisms of microbiota resilience are incompletely understood. Here, we show that Lactiplantibacillus plantarum, a major Drosophila commensal, stably colonizes the fruit fly gut during infection and is resistant to Drosophila antimicrobial peptides (AMPs). By transposon screening, we identified L. plantarum mutants sensitive to AMPs. These mutants were impaired in peptidoglycan O-acetylation or teichoic acid D-alanylation, resulting in increased negative cell surface charge and higher affinity to cationic AMPs. AMP-sensitive mutants were cleared from the gut after infection and aging-induced gut inflammation in wild-type, but not in AMP-deficient flies, suggesting that resistance to host AMPs is essential for commensal resilience in an inflamed gut environment. Thus, our work reveals that in addition to the host immune tolerance to the microbiota, commensal-encoded resilience mechanisms are necessary to maintain the stable association between host and microbiota during inflammation.

Antibacterial peptide PMAP-37(F34-R), expressed in Pichia pastoris, is effective against pathogenic bacteria and preserves plums.

BACKGROUND: Recently, researchers have focused on the search for alternatives to conventional antibiotics. Antimicrobial peptides are small bioactive peptides that regulate immune activation and have antibacterial activity with a reduced risk of bacterial resistance. Porcine myeloid antibacterial peptide 37 (PMAP-37) is a small-molecule peptide with broad-spectrum antibacterial activity isolated from pig bone marrow, and PMAP-37(F34-R) is its analogue. In this study, PMAP-37(F34-R) was recombinantly expressed in Pichia pastoris, and the recombinant peptide was further investigated for its antibacterial properties, mechanism and preservative in plums. RESULTS: To obtain a Pichia pastoris strain expressing PMAP-37(F34-R), we constructed a plasmid expressing recombinant PMAP-37(F34-R) (pPICZα-PMAP-37(F34-R)-A) and introduced it into Pichia pastoris. Finally, we obtained a highly active recombinant peptide, PMAP-37(F34-R), which inhibited the activity of both Gram-positive and Gram-negative bacteria. The minimum inhibitory concentration is 0.12-0.24 µg/mL, and it can destroy the integrity of the cell membrane, leading to cell lysis. It has good stability and is not easily affected by the external environment. Hemolysis experiments showed that 0.06 µg/mL-0.36 µg/mL PMAP-37(F34-R) had lower hemolysis ability to mammalian cells, and the hemolysis rate was below 1.5%. Additionally, 0.36 µg/mL PMAP-37(F34-R) showed a good preservative effect in plums. The decay and weight loss rates of the treated samples were significantly lower than those of the control group, and the respiratory intensity of the fruit was delayed in the experimental group. CONCLUSIONS: In this study, we constructed a recombinant Pichia pastoris strain, which is a promising candidate for extending the shelf life of fruits and has potential applications in the development of new preservatives.

Effective Healing of Staphylococcus aureus-Infected Wounds in Pig Cathelicidin Protegrin-1-Overexpressing Transgenic Mice.

Antimicrobial peptides (AMPs) are promising alternatives to existing treatments for multidrug-resistant bacteria-infected wounds. Therefore, the effect of protegrin-1 (PG1), a potent porcine AMP with broad-spectrum activity, on wound healing was evaluated. PG1-overexpressing transgenic mice were used as an in vivo model to evaluate its healing efficiency against Staphylococcus aureus-infected (106 colony forming units) wounds. We analyzed the wounds under four specific conditions in the presence or absence of antibiotic treatment. We observed the resolution of bacterial infection and formation of neo-epithelium in S. aureus-infected wounds of the mice, even without antibiotic treatment, whereas all wild-type mice with bacterial infection died within 8 to 10 days due to uncontrolled bacterial proliferation. Interestingly, the wound area on day 7 was smaller (p < 0.01) in PG1 transgenic mice than that in the other groups, including antibiotic-treated mice, suggesting that PG1 exerts biological effects other than bactericidal effect. Additionally, we observed that the treatment of primary epidermal keratinocytes with recombinant PG1 enhanced cell migration in in vitro scratch and cell migration assays. This study contributes to the understanding of broad-spectrum endogenous cathelicidins with potent antimicrobial activities, such as PG1, on wound healing. Furthermore, our findings suggest that PG1 is a potent therapeutic candidate for wound healing.

Antimicrobial peptides from Bacillus spp. and strategies to enhance their yield.

Antibiotic resistance is a growing concern that is affecting public health globally. The search for alternative antimicrobial agents has become increasingly important. Antimicrobial peptides (AMPs) produced by Bacillus spp. have emerged as a promising alternative to antibiotics, due to their broad-spectrum antimicrobial activity against resistant pathogens. In this review, we provide an overview of Bacillus-derived AMPs, including their classification into ribosomal (bacteriocins) and non-ribosomal peptides (lipopeptides and polyketides). Additionally, we delve into the molecular mechanisms of AMP production and describe the key biosynthetic gene clusters involved. Despite their potential, the low yield of AMPs produced under normal laboratory conditions remains a challenge to large-scale production. This review thus concludes with a comprehensive summary of recent studies aimed at enhancing the productivity of Bacillus-derived AMPs. In addition to medium optimization and genetic manipulation, various molecular strategies have been explored to increase the production of recombinant antimicrobial peptides (AMPs). These include the selection of appropriate expression systems, the engineering of expression promoters, and metabolic engineering. Bacillus-derived AMPs offer great potential as alternative antimicrobial agents, and this review provides valuable insights on the strategies to enhance their production yield, which may have significant implications for combating antibiotic resistance. KEY POINTS: • Bacillus-derived AMP is a potential alternative therapy for resistant pathogens • Bacillus produces two main classes of AMPs: ribosomal and non-ribosomal peptides • AMP yield can be enhanced using culture optimization and molecular approaches.

Antimicrobial Peptides in the Global Microbiome: Biosynthetic Genes and Resistance Determinants.

Antimicrobial peptides are a promising new class of antimicrobials that could address the antibiotic resistance crisis, which poses a major threat to human health. These peptides are present in all kingdoms of life, but especially in microorganisms, having multiple origins in diverse taxa. To date, there has been no global study on the diversity of antimicrobial peptides, the hosts in which these occur, and the potential for resistance to these agents. Here, we investigated the diversity and number of antimicrobial peptides in four main habitats (aquatic, terrestrial, human, and engineered) by analyzing 52,515 metagenome-assembled genomes. The number of antimicrobial peptides was higher in the human gut microbiome than in other habitats, and most hosts of antimicrobial peptides were habitat-specific. The relative abundance of genes that confer resistance to antimicrobial peptides varied between habitats and was generally low, except for the built environment and on human skin. The horizontal transfer of potential resistance genes among these habitats was probably constrained by ecological barriers. We systematically quantified the risk of each resistance determinant to human health and found that nearly half of them pose a threat, especially those that confer resistance to multiple AMPs and polymyxin B. Our results help identify the biosynthetic potential of antimicrobial peptides in the global microbiome, further identifying peptides with a low risk of developing resistance.

The effect of combined knockdowns of Attacins on survival and bacterial load in Tenebrio molitor.

Introduction: Upon infection, insect hosts simultaneously express a cocktail of antimicrobial peptides (AMPs) which can impede pathogen colonization and increase host fitness. It has been proposed that such a cocktail might be adaptive if the effects of co-expressed AMPs are greater than the sum of individual activities. This could potentially prevent the evolution of bacterial resistance. However, in vivo studies on AMPs in combination are scarce. Attacins are one of the relatively large AMP families, which show anti-Gram-negative activity in vitro. Material and methods: Here, we used RNA interference (RNAi) to silence three members of the Attacin family genes in the mealworm beetle, Tenebrio molitor: (TmAttacin1a (TmAtt1a), TmAttacin1b (TmAtt1b), and TmAttacin2 (TmAtt2) both individually and in combination. We then infected T. molitor with the Gram negative entomopathogen Pseudomonas entomophila. Results: We found that survival of the beetles was only affected by the knockdown of TmAttacin1b, TmAttacin2 and the knockdown of all three Attacins together. Triple knockdown, rather than individual or double knockdowns of AMPs, changes the temporal dynamics of their efficiency in controlling the colonization of P. entomophila in the insect body. Discussion: More precisely, AMP gene expression influences P. entomophila load early in the infection process, resulting in differences in host survival. Our results highlight the importance of studying AMP-interactions in vivo.

Unveiling the Impact of Gene Presence/Absence Variation in Driving Inter-Individual Sequence Diversity within the CRP-I Gene Family in Mytilus spp.

Mussels (Mytilus spp.) tolerate infections much better than other species living in the same marine coastal environment thanks to a highly efficient innate immune system, which exploits a remarkable diversification of effector molecules involved in mucosal and humoral responses. Among these, antimicrobial peptides (AMPs) are subjected to massive gene presence/absence variation (PAV), endowing each individual with a potentially unique repertoire of defense molecules. The unavailability of a chromosome-scale assembly has so far prevented a comprehensive evaluation of the genomic arrangement of AMP-encoding loci, preventing an accurate ascertainment of the orthology/paralogy relationships among sequence variants. Here, we characterized the CRP-I gene cluster in the blue mussel Mytilus edulis, which includes about 50 paralogous genes and pseudogenes, mostly packed in a small genomic region within chromosome 5. We further reported the occurrence of widespread PAV within this family in the Mytilus species complex and provided evidence that CRP-I peptides likely adopt a knottin fold. We functionally characterized the synthetic peptide sCRP-I H1, assessing the presence of biological activities consistent with other knottins, revealing that mussel CRP-I peptides are unlikely to act as antimicrobial agents or protease inhibitors, even though they may be used as defense molecules against infections from eukaryotic parasites.