Transcription Of Antimicrobial Peptides Research Articles

Pleiotropic immunoregulation by growth-blocking peptide in Ostrinia furnacalis.

Insects rely on their innate immune system to eliminate pathogenic microbes. As a system component, cytokines transmit intercellular signals to control immune responses. Growth-blocking peptide (GBP) is a member of the stress-responsive peptide family of cytokines found in several orders of insects, including Drosophila. However, the physiological role of GBP in defence against pathogens is not thoroughly understood. In this study, we explored the functions of GBP in a lepidopteran pest, Ostrinia furnacalis. Injection of recombinant O. furnacalis GBP (OfGBP) precursor (proGBP) and chemically synthesised GBP significantly induced the transcription of antimicrobial peptides (AMPs) and other immunity-related genes including immune deficiency (IMD) and Dorsal. The level of OfGBP mRNA was upregulated after bacterial infection. Knockdown of OfGBP expression led to a decrease in IMD, Relish, MyD88 and Dorsal mRNA levels. OfGBP induced phenoloxidase activity and affected hemocyte behaviours in O. furnacalis larvae. In summary, GBP is a potent cytokine, effectively regulating AMP synthesis, melanization response and cellular immunity to eliminate invading pathogens.

Subcellular Translocation of Yorkie through the PRP4K-CRM1 Axis Regulates Antimicrobial Peptides Transcription and Defense against Bacterial Infection in Crab.

The Hippo signaling pathway plays important roles in innate immunity. In the current study, we found that bacterial infection did not influence mRNA and protein levels of yorkie (Yki), which is an important terminal molecule of the Hippo signaling pathway. However, bacterial infection promoted the translocation of Yki from the nucleus to the cytoplasm in Chinese mitten crab (Eriocheir sinensis), thus attenuating Yki-suppressed transcription of antimicrobial peptides through Cactus. Chromosome region maintenance 1 (CRM1)-silenced crab hemocytes significantly suppressed Yki translocation from the nucleus to the cytoplasm upon bacterial infection, resulting in significantly increased expression of Cactus, decreased expression of antimicrobial peptides, and higher bacterial susceptibility, which demonstrated the regulatory role of CRM1 in subcellular localization of Yki. However, RNA interference of Scalloped (Sd) exhibited no effect on the subcellular localization of Yki and its regulation of Cactus/antimicrobial peptides. Moreover, we elucidated that both CRM1 and Sd could interact with Yki and that the PRP4K-mediated phosphorylation of a conserved serine amino acid residue in the nuclear export signal of Yki is essential for interaction between Yki and CRM1; however, the phosphorylation did not affect the binding of Yki with Sd. We also found that bacterial infection significantly promoted the expression of PRP4K in hemocytes; RNA interference of PRP4K and phosphatase inhibitor suppressed Yki translocation from the nucleus to the cytoplasm, promoting Cactus expression and inhibiting antimicrobial peptide expression. Thus, subcellular localization of Yki regulates antibacterial infection through both PRP4K and CRM1 in crabs.

Priming European Sea Bass Female Broodstock Improves the Antimicrobial Immunity of Their Offspring.

Acquiring immunocompetence is essential in the development of fish embryos, as they are exposed to environmental pathogens even before they are fertilized. Despite the importance of the antimicrobial function as the first line of defense against foreign microorganisms, little knowledge is available about its role in larval development. In vertebrates, transgenerational immune priming influences the acquisition of immunocompetence of specimens, regulating the selective allocation of nongenetic resources to their progeny and modulating their development. In this work, we primed teleost European sea bass broodstock females with a viral protein expression vector in order to evaluate the innate immunity development of their offspring. Several antimicrobial functions, the pattern of expression of gene coding for different antimicrobial peptides (AMPs), and their protein levels, were evaluated in eggs and larvae during development. Our data determined the presence of antimicrobial proteins of maternal origin in eggs, and that female vaccination increases antimicrobial activities and the transcription and synthesis of AMPs during larval development.

Two venom serpins from the parasitoid wasp Microplitis mediator inhibit the host prophenoloxidase activation and antimicrobial peptide synthesis

Endoparasitoid wasps inject venom proteins into the hemocoel of host insects to ensure survival, growth, and development of their progenies by blocking host immunity. We previously identified ten serine protease inhibitors of the serpin superfamily in venom of the endoparasitoid wasp, Microplitis mediator, but it is unclear how these inhibitors may interact with host immune serine proteases. In this study, we investigated the functions of two serpins, MmvSPN-1 and MmvSPN-2, in the regulation of humoral immune responses in two hosts, the oriental armyworm Pseudaletia separate and the cotton bollworm Helicoverpa armigera, by dsRNA knockdown and biochemical assays using recombinant proteins. Knockdown of the two serpins resulted in increases in prophenoloxidase (PPO) activation and antimicrobial peptide (AMP) production in the hosts. After injection into the host hemocoel, the recombinant serpins inhibited PPO activation and AMP transcription. Mass spectrometry analysis of the pull-downs and in vitro reconstitution experiments revealed that HacSP29, a clip-domain serine protease in H. armigera, is the target of these two serpins. Therefore, these two inhibitors in the wasp venom may protect eggs from attacks by melanization and AMPs in the host insects.

Antibacterial activity and immunomodulatory role of a proline-rich antimicrobial peptide SpPR-AMP1 against Vibrio campbellii infection in shrimp Litopenaeus vannamei

Antimicrobial peptides (AMPs) constitute one of the most promising sources of natural molecules used for the design of effective antimicrobial agents alternative to antibiotics. Previously, we have showed that a crab proline-rich AMP designated as SpPR-AMP1 is a potent AMP that exhibited antimicrobial activity against both Gram-positive and Gram-negative bacteria. Here, we demonstrated the importance of SpPR-AMP1 peptide in treating a virulent acute hepatopancreatic necrosis disease (AHPND) Vibrio campbellii VH-639 isolate and eliciting the innate immune response to counter the AHPND infection in shrimp Litopenaeus vannamei. SpPR-AMP1 exhibited a strong antimicrobial activity against V. campbellii VH-639 at MIC value of 0.195–0.39 μM. Scanning electron microscopy (SEM) revealed the membrane disruption potential of SpPR-AMP1 against the V. campbellii VH-639 cells. The in vivo effect of SpPR-AMP1 in shrimp L.vannamei was investigated and the results showed that SpPR-AMP1 was capable of modulating the innate immune response by stimulating the expression levels of AMP transcripts in shrimp hemocytes. Moreover, treatments with SpPR-AMP1 could promote the resistance of shrimp against V. campbellii VH-639 infection as demonstrated by a significant increase in shrimp survival rate and decrease in both the bacterial load and the expression levels of bacterial PirA and PirB toxin gene transcripts in the infected shrimp. These results suggest the potential of SpPR-AMP1 peptide with the combined antimicrobial and immunoenhancing capabilities as promising antimicrobial agent to treat V. campbellii VH-639 causing AHPND infection in shrimp aquaculture.

Severe Natural Outbreak of Cryptocaryon irritans in Gilthead Seabream Produces Leukocyte Mobilization and Innate Immunity at the Gill Tissue.

The protozoan parasite Cryptocaryon irritans causes marine white spot disease in a wide range of fish hosts, including gilthead seabream, a very sensitive species with great economic importance in the Mediterranean area. Thus, we aimed to evaluate the immunity of gilthead seabream after a severe natural outbreak of C. irritans. Morphological alterations and immune cell appearance in the gills were studied by light microscopy and immunohistochemical staining. The expression of several immune-related genes in the gills and head kidney were studied by qPCR, including inflammatory and immune cell markers, antimicrobial peptides (AMP), and cell-mediated cytotoxicity (CMC) molecules. Serum humoral innate immune activities were also assayed. Fish mortality reached 100% 8 days after the appearance of the C. irritans episode. Gill filaments were engrossed and packed without any space between filaments and included parasites and large numbers of undifferentiated and immune cells, namely acidophilic granulocytes. Our data suggest leukocyte mobilization from the head kidney, while the gills show the up-regulated transcription of inflammatory, AMPs, and CMC-related molecules. Meanwhile, only serum bactericidal activity was increased upon infection. A potent local innate immune response in the gills, probably orchestrated by AMPs and CMC, is triggered by a severe natural outbreak of C. irritans.

A novel JNK induces innate immune response by activating the expression of antimicrobial peptides in Chinese mitten crab Eriocheir sinensis

c-Jun NH2-terminal kinase (JNK) is a member of mitogen-activated protein kinases (MAPKs) that participates in the regulation of various physiological and pathological processes. In this study, we identified a novel JNK (EsJNK) and determined the cDNA sequence of its isoform (EsJNK-a) from the Chinese mitten crab Eriocheir sinensis. The open reading frame (ORF) of EsJNK was predicted to encode 421 peptides with a serine/threonine protein kinase, a catalytic (S_TKc) domain, and a low complexity region. The ORF of EsJNK-a was 1380 bp encoding a protein with 459 amino acids, which was 38 amino acids more than that of EsJNK. The predicted tertiary structure of EsJNK was conserved and contained 15 α-helices and 10 β-sheets. Phylogenetic tree analysis revealed that EsJNK was clustered with the JNK homologs of other crustaceans. Quantitative real-time PCR assays showed that EsJNK was expressed in all the tissues examined, but it was relatively higher in hemocytes, muscles, and intestines. The expression of EsJNK mRNA in the hemocytes was upregulated by lipopolysaccharides and peptidoglycans, as well as by Staphylococcus aureus or Vibrio parahaemolyticus challenge. Functionally, after silencing EsJNK by siRNA in crabs, the expression levels of two antimicrobial peptides (AMPs), namely, anti-lipopolysaccharide factor and crustin, were significantly inhibited. The purified recombinant EsJNK protein with His-tag accelerated the elimination of the aforementioned bacteria in vivo. However, knockdown of EsJNK had an opposite effect. These findings suggested that EsJNK might be involved in the antibacterial immune defense of crabs by regulating the transcription of AMPs.

Immunological interactions of Chilo suppressalis Walker (Lepidoptera: Crambidae) with the native entomopathogenic fungi

Entomopathogenic fungi can attack many insect hosts and have been applied as the eco-friendly alternatives to synthetic chemicals for the control of pests. Insects have developed different defense systems encountering entomopathogens including humoral and cellular immune responses. In the present study, injection of some native entomopathogenic fungi to the Chilo suppressalis Walker larvae resulted in an enhancement of the cellular and antimicrobial defenses. The numbers of total and differential hemocytes increased rapidly in the first 3 and 6 h but those gradually reduced 12 and 24 h post-injections. The nodule formation and phenoloxidase activity increased at the time intervals after fungal infection. A similar trend was found in the transcription of antimicrobial peptides including attacin1 and 2, cecropin1 and 2, gallerimycin, defensin, lysozyme, and prophenoloxidase-activating proteinase-3 during infection fungi. In all cases, the target gene transcription was upper in the larvae injected by the fungi than that of control larvae. These results may elucidate better knowledge on the interaction of the fungi present in agroecosystems with the target insect pest.

Structural Basis for P450-Mediated Oxysterol Metabolism by Mycobacterium Tuberculosis

Mycobacterium tuberculosis (Mtb) infection is among top ten causes of death worldwide, and the number of drug-resistant strains is increasing. Mtb can avoid immune response, however the mechanisms are not fully investigated. Recently we have shown that Mtb cholesterol degrading enzymes, CYP124, CYP125, CYP142 and 3βHSD, metabolize a wide variety of human immune oxysterol messengers. The list includes: a) 25-hydroxy-cholesterol (agonist of LXR); b) 7β-hydroxy-cholesterol (agonist of orphan RORα/γ); c) 7α,25-dihydroxy-cholesterol (agonist of the EBI2); d) 20S-hydroxy-cholesterol (a ligand of Smoothened in the Hedgehog signaling pathway). CYP124 - the most potent among them - can also metabolize vitamin D3 (VD3) derivatives, which induces transcription of antimicrobial peptides - cathelicidin and β-defensin 2, through VDR. Here we give a structural basis for binding and oxidation of a wide variety of substrates by CYP124. Atomic resolution structures of CYP124, complexed with cholestenone (1.65 Å resolution), VD3 (1.3 Å) and 1α-hydroxy-VD3 (1.2 Å) reveal a site for oxidative attack during catalysis. In all the structures, binding pocket tightly locks aliphatic tails, leaving more degrees of freedom for positioning of the sterols A-rings. Moreover, SQ109 - a prospective antitubercular compound, which is structurally resembles the oxysterols and secosteroids, was also metabolized by CYP124, and we have solved 1.25 Å-resolution structure of the complex as well. Finally, we have also obtained high resolution crystal structures of CYP124, complex with two micromolar inhibitors, contributing to the drug design, based on the suppression of immune signal molecules degradation. This work was supported by a joint research grant of the Belarusian Republican Foundation for Fundamental Research (B20R-061) and the Russian Foundation for Basic Research (20-54-00005). VB and AM are supported by the Ministry of Science and Higher Education of the Russian Federation (agreement # 075-00337-20-03, project FSMG −2020-0003).

Antimicrobial peptides in the seedling transcriptome of the tree legume Peltophorum dubium

Antimicrobial peptides (AMPs) play an essential role in plant defense against invading pathogens. Due to their biological properties, these molecules have been considered useful for drug development, as novel agents in disease therapeutics, applicable to both agriculture and medicine. New technologies of massive sequencing open opportunities to discover novel AMP encoding genes in wild plant species. This work aimed to identify cysteine-rich AMPs from Peltophorum dubium, a legume tree from South America. We performed whole-transcriptome sequencing of P. dubium seedlings followed by de novo transcriptome assembly, uncovering 78 AMP transcripts classified into five families: hevein-like, lipid-transfer proteins (LTPs), alpha hairpinins, defensins, and snakin/GASA (Giberellic Acid Stimulated in Arabidopsis) peptides. No transcripts with similarity to cyclotide or thionin genes were identified. Genomic DNA analysis by PCR confirmed the presence of 18 genes encoding six putative defensins and 12 snakin/GASA peptides and allowed the characterization of their exon-intron structure. The present work demonstrates that AMP prediction from a wild species is possible using RNA sequencing and de novo transcriptome assembly, regarding a starting point for studies focused on AMP gene evolution and expression. Moreover, this study allowed the detection of strong AMP candidates for drug development and novel biotechnological products.

Differential expression of akirin gene in black tiger shrimp Penaeus monodon in response to immunostimulant administration and infections with Vibrio harveyi and white spot syndrome virus

AbstractThe akirin gene, which is strictly localized in the nucleus, plays a critical role in regulating antimicrobial peptide transcription, and has parallel functions to NF‐κB signaling pathway in both vertebrates and invertebrates. In shrimp, the akirin gene is expressed as innate immunity in response to microbial infection. In the present study, expression of akirin gene in Penaeus monodon with respect to Vibrio harveyi and white spot syndrome virus (WSSV) infections and immunostimulant (β‐glucan) administration were investigated by quantitative polymerase chain reaction. The gene was expressed in various tissue samples of healthy shrimp. Maximum level of expression was immediately after V. harveyi infection, suggesting that it may be an early response gene. Gene expression was remarkably upregulated in the lymphoid organ, gill, and hepatopancreas, whereas downregulation was observed in hemocytes compared with the control. In the case of WSSV‐infected samples, the akirin gene was significantly downregulated in the lymphoid organ but there was no significant difference in expression pattern in hemocytes compared to the control. In gill tissue, maximum expression was observed after 2 hr of infection, the same in hepatopancreas. Experimental challenge of β‐glucan fed shrimp infected with V. harveyi and WSSV resulted in significant upregulation of akirin gene expression in lymphoid and gill tissue.

TmRelish is required for regulating the antimicrobial responses to Escherichia coli and Staphylococcus aureus in Tenebrio molitor

Relish, a transcription factor, is a critical downstream component of the immune deficiency (Imd) pathway and regulates host defense against bacterial infection by mediating antimicrobial peptide (AMP) synthesis. Understanding the immunological function of the mealworm beetle, Tenebrio molitor Relish (TmRelish) will be instructive in understanding insect immunity. In the present study, full-length ORF of TmRelish was retrieved from T. molitor-expressed sequence tags and RNA-seq database. The predicted TmRelish amino acid sequence contained an N-terminal Rel-homology domain; an Ig-like, plexin, and transcription factor domain; ankyrin repeat motifs; a nuclear localization signal; and a C-terminal death domain and shared the highly conserved structure of the Relish proteins of other insect species. TmRelish mRNA was detected in all developmental stages of the insect; however, the highest levels were detected in the larval gut tissue and adult hemocytes. TmRelish mRNA level was upregulated in the fat body, hemocyte, and gut tissue 9 h after infection of T. molitor larvae by the gram-negative bacteria, Escherichia coli. Furthermore, TmRelish knockdown led to significantly higher mortality of the E. coli-infected larvae, and significantly lower mortality of larvae infected with Staphylococcus aureus or Candida albicans. To elucidate the possible cause of mortality, we measured AMP transcription in the fat body, hemocytes, gut, and Malpighian tubules (MTs) of T. molitor larvae. TmRelish knockdown suppressed the expression of nine AMP genes in the larval fat body and gut tissue during E. coli infection, suggesting that TmRelish positively regulates AMP expression in both immune-related tissues, in response to E. coli challenge. Furthermore, negative regulation of some AMPs by TmRelish in the MTs, gut and hemocytes in response to C. albicans infection suggests a crosstalk between the Toll and Imd pathways.

EsGPCR89 regulates cerebral antimicrobial peptides through hemocytes in Eriocheir sinensis

G protein-coupled receptors (GPCRs) are important transmembrane receptors that participate in diverse physiological processes including metabolism, cell growth and immune processes by transmitting extracellular signals to intracellular effectors. In this study, a gene belonging to the GPCR family was cloned from Eriocheir sinensis and named EsGPCR89. The full-length gene includes an open reading frame (ORF) of 465 amino acid residues, and bioinformatic analysis confirmed the high conservation between species. EsGPCR89 was detected in various tissues of E. sinensis, and was up-regulated in brain following Staphylococcus aureus infection. Expression levels of cerebral antimicrobial peptides (AMPs) were also up-regulated following bacterial challenge, reflecting their function in cerebral immunity. Additionally, EsGPCR89 silencing in hemocytes by RNA interference, down-regulated AMPs in brain after S. aureus infection. Moreover, through Immunisation assay and Polyacrylamide gel electrophoresis (SDS-PAGE) experiments, we could infer that bacterially infected hemocytes released effectors under the regulation of EsGPCR89, thereby activating transcription of cerebral AMPs. These results demonstrate that EsGPCR89 plays important roles in cerebral antimicrobial function via hemocytes.

Identification and characterization of three novel antimicrobial peptides from Acipenser dabryanus

Antimicrobial peptides (AMPs) play essential roles in the innate immune system to protect against a wide variety of pathogens in aquatic environments. In this study, three very important AMPs, cathelicidin, hepcidin and defensin, were identified in the critically endangered Acipenser dabryanus. The full-length cDNA sequences of these three AMPs were identified from transcriptome sequencing and the rapid amplification of cDNA ends (RACE) technique. Phylogenetic analysis showed that cathelicidin formed a clade with the other members of the cathelicidin family, and similar results were obtained for hepcidin. The A. dabryanus β-defensin belonged to the fish class 2 β-defensins. A tissue distribution study showed that the three AMP transcripts could be detected constitutively in various tissues. The highest expression levels of cathelicidin and hepcidin were found in the liver, while defensin was primarily expressed in the skin. Bacterial challenge in vivo revealed significant changes in the gene expression of the three AMPs at both mucosal sites and systemic sites. Striking upregulation of cathelicidin and hepcidin was observed in the skin at 12 h post-challenge, with increases of more than 7000-fold and 1000-fold, respectively, compared to the control, and the expression of defensin mRNA was remarkably elevated in the hindgut (by 230-fold at 6 h post-challenge). Moreover, according to the expression profiles of the AMPs post-challenge, we found that the mucosal immune response occurred earlier than the systemic immune response following bacterial infection. Our results suggest that these three novel AMPs may play important roles in the innate immune system of A. dabryanus to protect against invading pathogens, especially during the mucosal immune response.

Influence of differential protein levels of feed on production performance and immune response of pacific white leg shrimp in a biofloc–based system

A130-day culture experiment was performed to compare the effects of rearing Litopenaeus vannamei in a biofloc system and conventional autotrophic system, supplemented with diets having graded protein levels on growth performance, non-specific immune response and immunomodulatory activity. The experiment groups consisted of four protein-level treatments with four replicates each, and was performed in 16 experimental units (7.5-Tcapacity) stocked with juvenile L. vannamei (initial average weight 1.48 ± 0.4 g) at a density of 150 juvenile shrimps /m3. Biofloc-based groups fed with supplementary feed containing varying levels of crude protein (CP) BFT40 (40% CP), BFT32 (32% CP) and BFT24 (24% CP), and a control group (CP 40%), were maintained in an autotrophic system. At the end of the experimental period, there was an improvement of 32.6–52.6% in terms of productivity, 22–27.6% average body weight; 8.7–19.6% survival rate and 10–31% feed conversion ratio (FCR) in shrimp maintained in biofloc systems as compared to control. Similarly, the protein efficiency ratio (PER) in biofloc treatment groups was32 to 83% higher than control. The shrimp also showed higher resistance to disease in the biofloc treatment when challenged with pathogenic Vibrio parahaemolyticus, as survival of juveniles was significantly higher (p < 0.05) in the biofloc treatments than in the control group. The total hemocyte count and proPhenoloxidase activity were significantly increased in the biofloc treatments when compared to control. The mRNA profiling of important immune genes in the shrimps showed higher levels of expression in the 32 and 40% CP feed treatments. Superoxide dismutase (SOD) mRNA transcript levels were highly up-regulated in the BFT40 treatment (7-fold), moderately upregulated in the BFT32 treatment (2-fold), and downregulated in the BFT24 treatment (0.9-fold). Transcription of antimicrobial peptides like crustin showed significant up-regulation in BFT40 (10.31-fold) followed by BFT32 (6.11-fold) and BFT24 (1.07-fold). Likewise, other immune genes, such as MnSOD, hemocyanin, proPhenoloxidase (proPO), peroxinectin (PX) and serine protease (SP) showed similar trends, indicating immunomodulation in the biofloc groups. We concluded that the biofloc system has the potential to improve growth performance, immune response and disease resistance in Pacific white shrimp in spite of the low-protein supplemented diet.

Interleukin-6/Stat3 signaling has an essential role in the host antimicrobial response to urinary tract infection

The signaling networks regulating antimicrobial activity during urinary tract infection (UTI) are incompletely understood. Interleukin-6 (IL-6) levels increase with UTI severity, but the specific contributions of IL-6 to host immunity against bacterial uropathogens are unknown. To clarify this we tested whether IL-6 activates the Stat3 transcription factor, to drive a program of antimicrobial peptide gene expression in infected urothelium during UTI. Transurethral inoculation of uropathogenic Escherichia coli led to IL-6 secretion, urothelial Stat3 phosphorylation, and activation of antimicrobial peptide transcription, in a Toll-like receptor 4-dependent manner in a murine model of cystitis. Recombinant IL-6 elicited Stat3 phosphorylation in primary urothelial cells invitro, and systemic IL-6 administration promoted urothelial Stat3 phosphorylation and antimicrobial peptide expression invivo. IL-6 deficiency led to decreased urothelial Stat3 phosphorylation and antimicrobial peptide mRNA expression following UTI, a finding mirrored by conditional Stat3 deletion. Deficiency in IL-6 or Stat3 was associated with increased formation of intracellular bacterial communities, and exogenous IL-6 reversed this phenotype in IL-6 knockout mice. Moreover, chronic IL-6 depletion led to increased renal bacterial burden and severe pyelonephritis in C3H/HeOuJ mice. Thus, IL-6/Stat3 signaling drives a transcriptional program of antimicrobial gene expression in infected urothelium, with key roles in limiting epithelial invasion and ascending infection.

High-Throughput Identification of Antimicrobial Peptides from Amphibious Mudskippers.

Widespread existence of antimicrobial peptides (AMPs) has been reported in various animals with comprehensive biological activities, which is consistent with the important roles of AMPs as the first line of host defense system. However, no big-data-based analysis on AMPs from any fish species is available. In this study, we identified 507 AMP transcripts on the basis of our previously reported genomes and transcriptomes of two representative amphibious mudskippers, Boleophthalmus pectinirostris (BP) and Periophthalmus magnuspinnatus (PM). The former is predominantly aquatic with less time out of water, while the latter is primarily terrestrial with extended periods of time on land. Within these identified AMPs, 449 sequences are novel; 15 were reported in BP previously; 48 are identically overlapped between BP and PM; 94 were validated by mass spectrometry. Moreover, most AMPs presented differential tissue transcription patterns in the two mudskippers. Interestingly, we discovered two AMPs, hemoglobin β1 and amylin, with high inhibitions on Micrococcus luteus. In conclusion, our high-throughput screening strategy based on genomic and transcriptomic data opens an efficient pathway to discover new antimicrobial peptides for ongoing development of marine drugs.

The GCN2-ATF4 Signaling Pathway Induces 4E-BP to Bias Translation and Boost Antimicrobial Peptide Synthesis in Response to Bacterial Infection

Bacterial infection often leads to suppression of mRNA translation, but hosts are nonetheless able to express immune response genes through as yet unknown mechanisms. Here, we use a Drosophila model to demonstrate that antimicrobial peptide (AMP) production during infection is paradoxically stimulated by the inhibitor of cap-dependent translation, 4E-BP (eIF4E-binding protein; encoded by the Thor gene). We found that 4E-BP is induced upon infection with pathogenic bacteria by the stress-response transcription factor ATF4 and its upstream kinase, GCN2. Loss of gcn2, atf4, or 4e-bp compromised immunity. While AMP transcription is unaffected in 4e-bp mutants, AMP protein levels are substantially reduced. The 5' UTRs of AMPs score positive in cap-independent translation assays, andthis cap-independent activity is enhanced by 4E-BP. These results are corroborated invivo using transgenic 5' UTR reporters. These observations indicate that ATF4-induced 4e-bp contributes to innate immunity by biasing mRNA translation toward cap-independent mechanisms, thus enhancing AMP synthesis.

Ocean acidification and pathogen exposure modulate the immune response of the edible mussel Mytilus chilensis

Ocean acidification (OA) is one of the main consequences of increasing atmospheric carbon dioxide (CO2), impacting key biological processes of marine organisms such as development, growth and immune response. However, there are scarce studies on the influence of OA on marine invertebrates' ability to cope with pathogens. This study evaluated the single and combined effects of OA and bacterial infection on the transcription expression of genes related to antioxidant system, antimicrobial peptides and pattern recognition receptors in the edible mussel Mytilus chilensis. Individuals of M. chilensis were exposed during 60 days at two concentrations of pCO2 (550 and 1200 μatm) representing respectively current and future scenario of OA and were then injected with the pathogenic bacterium Vibrio anguillarum. Results evidenced an immunomodulation following the OA exposure with an up-regulation of C-type Lectin and Mytilin B and a down-regulation of Myticin A and PGRP. This immunomodulation pattern is partially counteracted after challenge with V. anguillarum with a down-regulation of the C-type lectin and Mytilin B and the up-regulation of Myticin A. In turn, these results evidence that pCO2-driven OA scenarios might triggers specific immune-related genes at early stages of infection, promoting the transcription of antimicrobial peptides and patterns recognition receptors. This study provides new evidence of how the immune response of bivalves is modulated by higher CO2 conditions in the ocean, as well one factor for the resilience of marine population upon global change scenarios.

Differential sensitivity to infections and antimicrobial peptide-mediated immune response in four silkworm strains with different geographical origin

The domesticated silkworm Bombyx mori has an innate immune system, whose main effectors are the antimicrobial peptides (AMPs). Silkworm strains are commonly grouped into four geographical types (Japanese, Chinese, European and Tropical) and are generally characterised by a variable susceptibility to infections. To clarify the genetic and molecular mechanisms on which the different responses to infections are based, we exposed one silkworm strain for each geographical area to oral infections with the silkworm pathogens Enterococcus mundtii or Serratia marcescens. We detected a differential susceptibility to both bacteria, with the European strain displaying the lowest sensitivity to E. mundtii and the Indian one to S. marcescens. We found that all the strains were able to activate the AMP response against E. mundtii. However, the highest tolerance of the European strain appeared to be related to the specific composition of its AMP cocktail, containing more effective variants such as a peculiar Cecropin B6 isoform. The resistance of the Indian strain to S. marcescens seemed to be associated with its prompt capability to activate the systemic transcription of AMPs. These data suggest that B. mori strains with distinct genetic backgrounds employ different strategies to counteract bacterial infections, whose efficacy appears to be pathogen-dependent.