Synthesis Of Antimicrobial Peptides Research Articles

Antimicrobial Peptides: Amphibian Host Defense Peptides.

Antimicrobial Peptides (AMPs) are one of the most common components of the innate immune system that protect multicellular organisms against microbial invasion. The vast majority of AMPs are isolated from the frog skin. Anuran (frogs and toads) skin contains abundant AMPs that can be developed therapeutically. Such peptides are a unique but diverse group of molecules. In general, more than 50% of the amino acid residues form the hydrophobic part of the molecule. Normally, there are no conserved structural motifs responsible for activity, although the vast majority of the AMPs are cationic due to the presence of multiple lysine residues; this cationicity has a close relationship with antibacterial activity. Notably, recent evidence suggests that synthesis of AMPs in frog skin may confer an advantage on a particular species, although they are not essential for survival. Frog skin AMPs exert potent activity against antibiotic-resistant bacteria, protozoa, yeasts, and fungi by permeating and destroying the plasma membrane and inactivating intracellular targets. Importantly, since they do not bind to a specific receptor, AMPs are less likely to induce resistance mechanisms. Currently, the best known amphibian AMPs are esculentins, brevinins, ranacyclins, ranatuerins, nigrocin-2, magainins, dermaseptins, bombinins, temporins, and japonicins-1 and -2, and palustrin-2. This review focuses on these frog skin AMPs and the mechanisms underlying their antimicrobial activity. We hope that this review will provide further information that will facilitate further study of AMPs and cast new light on novel and safer microbicides.

Identification of two LGBPs (isoform1 and isoform2) and their function in AMP expression and PO activation in male hepatopancreas

Two lipopolysaccharides (LPS) and β-1, 3-glucan binding protein (LGBP), designated as PcLGBP isoform1 and PcLGBP isoform2, respectively, were identified from Procambarus clarkii in this study. The full-length cDNA of PcLGBP isoform1 was 1308 bp containing an open reading frame (ORF) of 1113 bp encoding a protein of 370 amino acids. The full-length cDNA of PcLGBP isoform2 was 1440 bp containing an ORF of 1245 bp encoding a protein of 414 amino acids. Predicted PcLGBP isoform1 and PcLGBP isoform 2 proteins contained a signal peptide, a glycoside hydrolase domain, and a low-complexity region. The difference between the two LGBP isoforms was that PcLGBP isoform2 had 44 more amino acids behind the signal peptide than the PcLGBP isoform1. The PcLGBP isoform1 and PcLGBP isoform2 transcripts mainly expressed in the hepatopancreas in female and male crayfish. Moreover, the expression levels of the two genes in the hepatopancreas were higher in male than that in female crayfish. Upon being challenged with Vibrio parahaemolyticus or LPS, the expression levels of PcLGBP isoform1 and PcLGBP isoform2 in the hepatopancreas of female and male crayfish were most significantly up-regulated at different time points. The transcripts of anti-lipopolysaccharide factors (ALF5, ALF6, ALF8, and ALF9) and crustins (CRU1, CRU2, CRU3, and CRU4) were evidently down-regulated in the hepatopancreas of V. parahaemolyticus-challenged total PcLGBP (including PcLGBP isoform1 and PcLGBP isoform2)-silenced male crayfish. In addition, the phenoloxidase (PO) activity in the hepatopancreas of male crayfish was evidently higher than that of female crayfish. PcLGBP knock down could significantly decrease the PO activity in the hepatopancreas lysate (HLS) in male crayfish. The PO activity of male crayfish HLS was significantly increased when incubated with a mixture of recombinant LGBP protein and LPS or β-1, 3 glucan. We conclude that LGBP isoforms from P. clarkii function as a pattern recognition protein for recognizing and binding LPS and β-1, 3 glucan, and thus regulate the synthesis of antimicrobial peptides and activate the prophenoloxidase system.

The commensal Escherichia coli CEC15 reinforces intestinal defences in gnotobiotic mice and is protective in a chronic colitis mouse model

Escherichia coli is a regular inhabitant of the gut microbiota throughout life. However, its role in gut health is controversial. Here, we investigated the relationship between the commensal E. coli strain CEC15 (CEC), which we previously isolated, and the intestine in homeostatic and disease-prone settings. The impact of CEC was compared to that of the probiotic E. coli Nissle 1917 (Nissle) strain. The expression of ileal and colonic genes that play a key role in intestinal homeostasis was higher in CEC- and Nissle-mono-associated wild-type mice than in germfree mice. This included genes involved in the turnover of reactive oxygen species, antimicrobial peptide synthesis, and immune responses. The impact of CEC and Nissle on such gene expression was stronger in a disease-prone setting, i.e. in gnotobiotic IL10-deficient mice. In a chronic colitis model, CEC more strongly decreased signs of colitis severity (myeloperoxidase activity and CD3+ immune-cell infiltration) than Nissle. Thus, our study shows that CEC and Nissle contribute to increased expression of genes involved in the maintenance of gut homeostasis in homeostatic and inflammatory settings. We show that these E. coli strains, in particular CEC, can have a beneficial effect in a chronic colitis mouse model.

Characterization of immune-related PGRP gene expression and phenoloxidase activity in Cry1Ac-susceptible and -resistant Plutella xylostella (L.)

Peptidoglycan recognition proteins (PGRPs) are important recognition receptors which play a critical role in signal identification and transmission in Toll or immune deficiency (IMD) pathways, particularly when pathogens evade and circumvent reactive oxygen species. Antimicrobial peptides (AMPs) synthesis can be activated by these signals to further eliminate pathogens. In this study, we cloned and characterized three different PGRP genes in Plutella xylostella strains, DBM1Ac-S, DBM1Ac-R and a field strain (DBMF). The results showed that PGRP1 belongs to the PGRP-SA family, PGRP2 to PGRP-LB, and PGRP3 to PGRP-LF. Moreover, PGRP1 expressed the highest transcript level, followed by PGRP3 and PGRP2, in two tissues including the gut and the larval carcass tissues of the DBM1Ac-S strain. Furthermore, altered expression levels of PGRP1-3 genes were detected in both gut and carcass tissues. Moreover, the DBM1Ac-R strain had the highest phenol oxidase (PO) activity among these three strains. The characterization of PGRP gene expression and PO activity in DBM1Ac-S, DBM1Ac-R and DBM-F provides insights into their important physiological roles in the immune system of P. xylostella exposed to Bt Cry1Ac toxin.

Age-related modulation of the isthmic and uterine mucosal innate immune defense system in laying hens

Sustained production of good quality eggs for longer production cycles is a challenge for poultry farms. The impact of aging on the mucosal immune defense in the isthmus and uterus of hens, where the eggshell membrane and eggshell are formed, remains obscure. Thus, the aim of this study was to determine whether aging affects the mucosal tight junction (TJ) proteins, the synthesis of antimicrobial peptides including avian β-defensins (AvBDs) and cathelicidins (CATHs), and Toll-like receptors (TLRs) in the isthmus and uterus of laying hens. Young and aged White Leghorn laying hens (35 and 130 wk old, respectively) were used. Total RNA and protein contents were isolated from the isthmic and uterine mucosae of these hens to examine the expression of TJ proteins, AvBD, and CATH genes and AvBD proteins by the real-time polymerase chain reaction and western blotting. The results showed that the mRNA expression of TJ proteins, namely zonula occludin 2 in the isthmus and occludin in the uterus, was higher in aged hens than in young hens. Expression of 2 AvBD genes in the isthmus and 4 AvBD genes in the uterus was higher in aged hens than in young hens. However, the expression of AvBD proteins 1 and 11 was not altered by aging. Expressions of CATH genes were not affected by aging in the isthmus or uterus. Expression of TLR genes was higher in aged hens than in young hens in the isthmus, while their expression in the uterus was not affected by aging. It can be concluded that aged hens have a higher potential ability to express TJ proteins and AvBDs for mucosal defense in the isthmic and uterine mucosae than in young hens.

Anti-tumour effects of antimicrobial peptides, components of the innate immune system, against haematopoietic tumours in Drosophila mxc mutants.

ABSTRACTThe innate immune response is the first line of defence against microbial infections. In Drosophila, two major pathways of the innate immune system (the Toll- and Imd-mediated pathways) induce the synthesis of antimicrobial peptides (AMPs) within the fat body. Recently, it has been reported that certain cationic AMPs exhibit selective cytotoxicity against human cancer cells; however, little is known about their anti-tumour effects. Drosophila mxcmbn1 mutants exhibit malignant hyperplasia in a larval haematopoietic organ called the lymph gland (LG). Here, using RNA-seq analysis, we found many immunoresponsive genes, including those encoding AMPs, to be upregulated in these mutants. Downregulation of these pathways by either a Toll or imd mutation enhanced the tumour phenotype of the mxc mutants. Conversely, ectopic expression of each of five different AMPs in the fat body significantly suppressed the LG hyperplasia phenotype in the mutants. Thus, we propose that the Drosophila innate immune system can suppress the progression of haematopoietic tumours by inducing AMP gene expression. Overexpression of any one of the five AMPs studied resulted in enhanced apoptosis in mutant LGs, whereas no apoptotic signals were detected in controls. We observed that two AMPs, Drosomycin and Defensin, were taken up by circulating haemocyte-like cells, which were associated with the LG regions and showed reduced cell-to-cell adhesion in the mutants. By contrast, the AMP Diptericin was directly localised at the tumour site without intermediating haemocytes. These results suggest that AMPs have a specific cytotoxic effect that enhances apoptosis exclusively in the tumour cells.

Calcitriol and polymorphism of the gene of the receptor vitamin D in the pathogenesis of perinatal infection (review)

The article presents literature data of the possible role of vitamin D (VD) and polymorphism of the gene of the vitamin D receptors (VDR) in the pathogenetic mechanisms of the formation of pregnancy complications associated with intrauterine infection. Information of the modern approach to the importance of the vitamin D / VD receptor system in the regulation of physiological processes associated with the presence of VD receptors in many organs and tissues is presented. Particular attention to the presence of calcitriol receptors directly in the organs of the reproductive system is paid, the action of which is realized in a genomic and non-genomic way.Information about participation of vitamin D in the regulation of the balance between specific and nonspecific immunity during pregnancy, on the regulation and proliferation of immunocompetent cells, on the presence of mitochondrial enzyme 1a-hydroxylase (CYP27B1) directly in the trophoblast and the placenta, where the extra-renal synthesis of the active metabolite VD is underlined. It is shown that calcitriol can regulate the synthesis of antimicrobial peptides, which play a key role in the innate immune response, and an increase calcitriol in the inflammatory focus as a protective reaction is considered. The main effect of calcitriol on the immune system is to simulate T-cell immunity.Data on possible mechanisms for regulating the activity of a cytokines with pro- and anti-inflammatory effects in the presence of calcitriol are presented. In the trophoblast under the influence of calcitriol phagocytosis and chemotaxis of macrophages are enhanced, by the following activation of the cathelicidin peptide with antimicrobial action, and the negative effect of proinflammatory cytokines is reduced. Separately, the problem of the immune adaptation of the pregnant woman’s under the vitamin D deficiency status and polymorphism of genes encoding VDR is presented. It is assumed that under the VD-deficiency status, the probability of the intrauterine infection really increases: further research to refine the assumptions is needed.

Immune response of pearl oysters to stress and diseases

AbstractPearl oysters are found in the marine ecosystem. Due to their frequent exposure to microbial pathogens and environmental perturbations, they have developed a multifaceted innate immune system with differentially expressed immune‐related genes in response to a range of stress via vast coordinated immune reactions, such as immune recognition, signal transduction, synthesis of antimicrobial peptides, encapsulation and phagocytosis of the circulating haemocytes. The species are farmed to produce pearls. With the increasing adoption of aquaculture techniques in pearl production, there is the need to understand their culture environment and their immune response mechanisms. Multi‐omic studies have documented responses to nuclei insertion operation, diseases and various environmental conditions in Pinctada fucata martensii (Dunker, 1880), Pinctada maxima (Jameson, 1901), Pinctada margaritifera and Pteria penguin (Roding, 1798). Among the various stressors, nuclei insertion operation has been identified as a prevailing factor modifying gene expression. Other factors include life stage, air exposure, temperature, salinity, metals and organic contaminants. This manuscript details the response mechanism of these pearl oysters to the various stressors.

Changes in immune responses of Helicoverpa armigera Hübner followed by feeding on Knotgrass, Polygonum persicaria agglutinin.

There is no study implying the effect of plant lectins on insect immune elements in both challenged and non-challenged conditions with entomopathogenic agents. Lectins may bind to immune receptors on the surface of insect hemocytes, thus inducing or even disabling common immune functions including hemocyte counts, nodulation/encapsulation, phenoloxidase activity, and synthesis of antimicrobial peptides. In the present study, effect of Polygonum persicaria L. agglutinin (PPA) on immune responses of Helicoverpa armigera Hübner was investigated by feeding artificial diet treated to the larvae. Subsequently hemocyte count and expression of some immune-related genes were considered for analyses. The two groups of larvae including control and PPA-treated (1%) were divided into four subgroups of intact, Tween-80 injected, latex-bead injected and Beauveria bassiana-injected. Except for intact larvae, the highest numbers of total and differential hemocyte counts were recorded 12 hr postinjection, however, the PPA-fed larvae showed a significantly lower hemocyte counts compared to control. The number of nodules in PPA-fed larvae was significantly lower than control, but the injected larvae of both control and PPA showed the highest nodulation 24 hr postinjection. Although the highest activity of phenoloxidase was observed 12 and 24 hr postinjection but its activity significantly decreased in PPA-fed larvae compared to control. Gene expression of antimicrobial peptides including attacin, cecropin, and peptidoglycan receptor proteins were significantly decreased in artificial diet-fed larvae containing PPA and then injected by B. bassiana spores and latex bead compared to control. These results clearly indicate adverse effects of PPA on immune responses in H. armigera.

Transcriptomic insight into antimicrobial peptide factors involved in the prophylactic immunity of crowded Mythimna separata larvae

Similar to pathogenic infection, a high population density alters insect prophylactic immunity. Antimicrobial peptides (AMPs) are known to play critical roles in an insect's humoral immune response to microbial infection. We applied RNA sequencing to investigate differential gene expression levels in fat body and hemocyte samples from larvae reared in high- (10 larvae per jar) and low-density (1 larva per jar) conditions; the samples exhibited density-dependent prophylaxis. A number of AMP molecule-related proteins were annotated for the first time from 145,439 assembled unigenes from M. separata larvae. The transcript levels of AMP molecules such as gloverin-, defensin-, cecropin-, lebocin- and attacin-related unigenes were increased with the prophylactic immunity of high-density larvae. The pattern recognition receptor peptidoglycan recognition protein (PGRP), a key protein in the synthesis of AMPs in IMD- and Toll pathway-related unigenes, was also upregulated in the larvae from the high-density group. The resultant transcriptomic database was validated by the transcript levels of four selected AMP genes quantified from the high- and low-density larval groups with quantitative real-time PCR. The antimicrobial activity against gram-positive Staphylococcus aureus and Bacillus subtilis and gram-negative Edwardsiella ictaluri and Vibrio anguillarum in the hemolymph of larvae from the high-density group was significantly higher than that of larvae from the low-density group. Our findings provide the first insight into the role of AMP genes in the mechanisms of density-dependent prophylaxis in M. separata and provide new insight into the control of M. separata with biopesticides.

Antimicrobial Peptides: Recent Insights on Biotechnological Interventions and Future Perspectives.

With the unprecedented rise of drug-resistant pathogens, particularly antibiotic-resistant bacteria, and no new antibiotics in the pipeline over the last three decades, the issue of antimicrobial resistance has emerged as a critical public health threat. Antimicrobial Peptides (AMP) have garnered interest as a viable solution to this grave issue and are being explored for their potential antimicrobial applications. Given their low bioavailability in nature, tailoring new AMPs or strategizing approaches for increasing the yield of AMPs, therefore, becomes pertinent.The present review focuses on biotechnological interventions directed towards enhanced AMP syn-thesis and revisits existing genetic engineering and synthetic biology strategies for production of AMPs. This review further underscores the importance and potential applications of advanced gene editing technologies for the synthesis of novel AMPs in future.

Expression and Characterization of <i>Manduca sexta</i> Stress Responsive Peptide-1; An Inducer of Antimicrobial Peptide Synthesis

In response to stress conditions such as wounding or infections in insects, several short peptides are processed to act as cytokines that induce AMP gene expression. To study their structure-activity relationship, immune inducibility, tissue specificity, stress responsiveness, and development relatedness, we chemically synthesized Manduca sexta stress response peptide-1, a 25-residue peptide with one disulfide bond (SRP1: FGVRVGTCPSGYVRRGTFCFPDDDY). Upon injection of the SRP1 into naïve larvae, several antimicrobial peptide genes were expressed at higher levels. The mRNA levels of SRP1 increased significantly in hemocytes and fat body after larvae were challenged with a mixture of bacteria and β-1,3-glucan. The expression patterns of SRP1 and its target genes are somewhat different from SRP2's, suggesting overlapping yet distinct functions. We elucidated the 3D structure of SRP1 in solution by two-dimensional 1H-1H NMR spectroscopy. The tertiary structure of SRP1 consists of two short β-strands at Y12-R15 and F18-F20, one type-II β-turn at R15-F18 in its well-defined core and is stabilized by a covalent disulfide bond between C8 and C19. The conformational ensemble of SRP1 from extensive atomistic simulation in explicit solvent (with 3.0 μs total effective sampling) shows high consistency with experimental intramolecular NOEs of the core region. The SRP1 core adopts a fold similar to the carboxyl-terminal subdomain of epidermal growth factor (EGF), suggesting that SRP1 may interact with EGF receptor-like molecules to trigger its biological function.

The Influence of Habitat and Phylogeny on the Skin Microbiome of Amphibians in Guatemala and Mexico.

Microbial symbionts are increasingly recognized as playing a critical role in organismal health across a wide range of hosts. Amphibians are unique hosts in that their skin helps to regulate the exchange of water, ions, and gases, and it plays an active role in defense against pathogens through the synthesis of anti-microbial peptides. The microbiome of amphibian skin includes a diverse community of bacteria known to defend against pathogens, including the global pandemic lineage of Batrachochytrium dendrobatidis associated with mass amphibian die-offs. The relative influence of host phylogeny and environment in determining the composition of the amphibian skin microbiome remains poorly understood. We collected skin swabs from montane amphibians in Mexico and Guatemala, focusing on two genera of plethodontid salamanders and one genus of frogs. We used high throughput sequencing to characterize the skin bacterial microbiome and tested the impact of phylogeny and habitat on bacterial diversity. Our results show that phylogenetic history strongly influences the diversity and community structure of the total bacterial microbiome at higher taxonomic levels (between orders), but on lower scales (within genera and species), the effect of habitat predominates. These results add to a growing consensus that habitat exerts a strong effect on microbiome structure and composition, particularly at shallow phylogenetic scales.

Roles of the inflammasome in the gut‑liver axis (Review).

The gut-liver axis connects the liver with the intestine via bile acid metabolism. Bile acid dysregulation leads to intestinal dysbiosis, that allows enterogenous pathogenic bacteria, including Gram-negative bacteria and their products lipopolysaccharide (LPS), into the liver via the portal vein, triggering inflammation in the liver. The inflammasome serves as an intracellular pattern recognition receptor that detects pathogens or danger signals and mediates innate immunity in the liver or gut. Specifically, the NACHT, LRR and PYD domains-containing protein (NLRP)6 inflammasome maintains intestinal microbial balance, by promoting interleukin (IL)-18-dependent antimicrobial peptide synthesis and mucus secretion from goblet cells. The NLRP3 inflammasome, in contrast, primarily induces IL-1β and aggravates inflammatory liver injury. Furthermore, the NLRP3 inflammasome affects the epithelial integrity of cholangiocytes by inducing the production of pro-inflammatory cytokines. In addition, bile acids, including deoxycholic acid and chenodeoxycholic acid, are able to activate the NLRP3 inflammasome in macrophages; however, bile acids have the potential to exert the opposite role by interacting with the membrane-bound Takeda G-protein receptor 5 or by activating nuclear farnesoid-X receptor. Therefore, further investigation of the molecular mechanisms underlying the inflammasome, involved in the gut-liver axis, may provide important insights into the identification of a potential therapeutic target for the treatment of liver and gut diseases. The present review discusses the roles of the inflammasome in the gut-liver axis, and the emerging associations between the inflammasome and the intestinal microbiota or the bile acids in the gut-liver axis.

An Invertebrate Host to Study Fungal Infections, Mycotoxins and Antifungal Drugs: Tenebrio molitor.

Faced with ethical conflict and social pressure, researchers have increasingly chosen to use alternative models over vertebrates in their research. Since the innate immune system is evolutionarily conserved in insects, the use of these animals in research is gaining ground. This review discusses Tenebrio molitor as a potential model host for the study of pathogenic fungi. Larvae of T. molitor are known as cereal pests and, in addition, are widely used as animal and human feed. A number of studies on mechanisms of the humoral system, especially in the synthesis of antimicrobial peptides, which have similar characteristics to vertebrates, have been performed. These studies demonstrate the potential of T. molitor larvae as a model host that can be used to study fungal virulence, mycotoxin effects, host immune responses to fungal infection, and the action of antifungal compounds.

Two novel serine proteases from Scylla paramamosain involved in the synthesis of anti-lipopolysaccharide factors and activation of prophenoloxidase system

Serine proteases (SPs) are important in various immune responses, including prophenoloxidase (proPO) activation, antimicrobial peptides (AMPs) synthesis, and hemolymph coagulation in invertebrates. In this study, SP3 and SP5 of mud crab (Scylla paramamosain) were studied. SP3 and SP5 were expressed in all examined tissues (mainly in hemocytes), and are associated with the immune responses of mud crab to Vibrio parahemolyticus and Staphylococcus aureus, as well as interacted with TRAF6, and are involved in the activation of anti-lipopolysaccharide factors (ALFs) probably through the TLR/NF-κB pathway. Depletion of SP3 inhibited the expression of ALF1, ALF2, ALF3, and ALF6, while knockdown of SP5 significantly decreased ALF5, and ALF6. Furthermore, both SP5 and TRAF6 regulated the PO activity in the hemolymph of mud crab. Overexpression assay showed that both SP3 and SP5 could enhance the promoter activities of ALFs in mud crab. Taken together, the results of this study indicate that SP3 and SP5 might play important roles in the immune system of mud crab against pathogen invasion.

Antibacterial Activity and Mechanism of Action of Bovine Lactoferricin Derivatives with Symmetrical Amino Acid Sequences.

In recent years, the overuse of antibiotics has become very serious. Many pathogenic bacteria have become resistant to them, with serious potential health consequences. Thus, it is urgent that we develop new antibiotic drugs. Antimicrobial peptides (AMPs) are important endogenous antibacterial molecules that contribute to immunity. Most have spectral antibacterial properties and do not confer drug resistance. In this paper, an 11-residue peptide (LFcinB18–28) with a sequence of KCRRWQWRMKK was modified by amino acid substitution to form a symmetrical amino acid sequence. The antibacterial activities and mechanisms of action of engineered peptides including KW-WK (KWRRWQWRRWK), FP-PF (FPRRWQWRRPF), FW-WF (FWRRWQWRRWF), and KK-KK (KKRRWQWRRKK) were investigated. The four engineered peptides could more effectively inhibit bacteria than the original peptide, LFcinB18–28. This suggested that a symmetrical amino acid sequence might enhance the antibacterial activity of AMPs. However, only peptides KW-WK, FP-PF, and KK-KK were safe; FW-WF displayed hemolytic activity. The engineered peptides shared cationic and amphipathic characteristics that facilitated interactions with the anionic microbial membranes, leading to disruption of membrane integrity and permeabilizing microbial membranes, resulting in cell death. Therefore, a symmetrical amino acid sequence and related structural parameters offer an alternative approach to the design of AMPs. This will provide a scientific basis for the design and synthesis of new AMPs.

Manipulation of the silkworm immune system by a metalloprotease from the pathogenic bacterium Pseudomonas aeruginosa

Antimicrobial peptide (AMP) production and melanization are two key humoral immune responses in insects. Induced synthesis of AMPs results from Toll and IMD signal transduction whereas melanization depends on prophenoloxidase (PPO) activation system. During invasion, pathogens produce toxins and other virulent factors to counteract host immune responses. Here we show that the pathways leading to PPO activation and AMP synthesis in the silkworm Bombyx mori are affected by a metalloprotease, named elastase B, secreted by Pseudomonas aeruginosa (PAO1). The metalloprotease gene (lasB) was expressed shortly after PAO1 cells had been injected into the larval silkworm hemocoel, leading to an increase of elastase activity. Injection of the purified PAO1 elastase B into silkworm hemolymph compromised PPO activation. In contrast, the protease caused a level increase of gloverin, an AMP in the hemolymph. To verify our results obtained using the purified elastase B, we infected B. mori with PAO1 ΔlasB mutant and found that PO activity in hemolymph of the PAO1 ΔlasB-infected larvae was significantly higher than that in the wild type-infected. The mutant-inhabited hemolymph had lower levels of gloverin and antimicrobial activity. PAO1 ΔlasB showed a decreased viability in the silkworm hemolymph whereas the host had a lower mortality. In addition, the effects caused by the ΔlasB mutant were restored by a complementary strain. These data collectively indicated that the elastase B produced by PAO1 is an important virulent factor that manipulates the silkworm immune system during infection.

Evolutionary ecology of parasitic fungi and their host insects

Abstract The coevolution of parasites and their hosts is accompanied by rapid reciprocal adaptations. Recent studies focusing on the evolutionary ecology of parasitic fungi and their host insects have revealed an unexpectedly complex molecular arms race. Insects recognize the presence of invading fungi when molecular patterns associated with the fungal cell wall bind to host receptors and respond by inducing the synthesis of proteinase inhibitors and antifungal peptides such as metchnikowin. Parasitic fungi can in turn sense the presence of these host-derived defense molecules and respond by synthesizing chymotrypsin and metalloproteases to digest them. As a counter-response, insects have evolved the ability to perceive the presence of microbial metalloproteinases, inducing the synthesis of further antimicrobial peptides and the insect metalloproteinase inhibitor to regulate these virulence-associated fungal proteinases. These specific responses and counter-responses indicate that coevolution has resulted in communication between fungal parasites and their insect hosts, which was recently shown to involve transcriptional reprogramming controlled by histone acetylation/deacetylation and DNA methylation. Consequently, the reciprocal adaptations associated with host–parasite coevolution can be expanded beyond the genetic level to encompass epigenetic mechanisms.

A clip domain serine protease stimulates melanization activation and expression of antimicrobial peptides in the Chinese oak silkworm, Antheraea pernyi

Abstract Clip domain serine proteases (Clips) mediate various biological processes, especially in insect immune responses such as prophenoloxidase (proPO) activation and Toll pathway initiation. In the present study, we cloned and studied the functions of a clip domain serine protease from Antheraea pernyi (ApSnake). ApSnake contains a typical clip domain and a trypsin-like serine protease domain. It shares high similarity with serine protease snake in Bombyx mori, and groups into the CLIPC family. ApSnake mRNA expressions were detected in all tested tissues, particularly high in the hemocytes. Challenged with four different microorganisms (Escherichia coli, Beauveria bassiana, Micrococcus luteus or Nuclear polyhedrosis virus), the expressions of ApSnake mRNA were induced significantly compared with control, particularly challenged by B. bassiana and M. luteus. Recombinant ApSnake protein stimulated melanization reaction and prophenoloxidase activation in A. pernyi hemolymph, but had no effect on phenoloxidase activity. Injection of recombinant ApSnake into A. pernyi larvae increased the transcript levels of proPO and antimicrobial peptides (AMPs) in hemocytes significantly. Our results suggested that ApSnake might be a modulating protein that both stimulate the melanization activation process and AMPs synthesis, and plays an important role in the innate immunity of A. pernyi.