Production Of Antibacterial Peptides Research Articles

Osteopontin Depletion in Non-haematopoietic Cells Improves Outcomes in Septic Mice by Enhancing Antimicrobial Peptide Production.

Osteopontin (Opn) depletion can improve septic outcomes, but the underlying mechanism remains unknown. In this study, we demonstrated that non-haematopoietic but not haematopoietic Opn depletion improved septic outcomes. Compared to wild-type (WT) mice, co-housed Opn-/- mice displayed enhanced production of antibacterial peptides (AMPs), decreased bacterial loads, and a distinct bacterial composition of gut microbiota. Fecal microbiota transplantation (FMT) and OPN neutralization assay showed that Opn depletion could reduce the bacterial loads and improve septic inflammation. By employing an intestinal organoid culture system, we proved that OPN neutralization in WT organoids could inactivate AKT and decrease FOXO3a phosphorylation, resulting in enhanced AMP production, whereas OPN treatment in OPN deficient organoids could activate AKT and increase FOXO3a phosphorylation, leading to reduced AMP production. Our findings identified OPN as a novel regulatory factor of AMP production to modulate bacterial loads and composition of gut microbiota, in turn affecting sepsis outcomes.

Assessment of bacteriocin production by clinical Pseudomonas aeruginosa isolates and their potential as therapeutic agents

IntroductionBacterial infections and the rising antimicrobial resistance pose a significant threat to public health. Pseudomonas aeruginosa produces bacteriocins like pyocins, especially S-type pyocins, which are promising for biological applications. This research focuses on clinical P. aeruginosa isolates to assess their bacteriocin production, inhibitory spectrum, chemical structure, antibacterial agents, and preservative potential.MethodsThe identification of P. aeruginosa was conducted through both phenotypic and molecular approaches. The inhibitory spectrum and antibacterial potential of the isolates were assessed. The kinetics of antibacterial peptide production were investigated, and the activity of bacteriocin was quantified in arbitrary units (AU ml−1). Physico-chemical characterization of the antibacterial peptides was performed. Molecular weight estimation was carried out using SDS–PAGE. qRT-PCR analysis was employed to validate the expression of the selected candidate gene.ResultThe antibacterial activity of P. aeruginosa was attributed to the secretion of bacteriocin compounds, which belong to the S-type pyocin family. The use of mitomycin C led to a significant 65.74% increase in pyocin production by these isolates. These S-type pyocins exhibited the ability to inhibit the growth of both Gram-negative (P. mirabilis and P. vulgaris) and Gram-positive (S. aureus, S. epidermidis, E. hirae, S. pyogenes, and S. mutans) bacteria. The molecular weight of S-type pyocin was 66 kDa, and its gene expression was confirmed through qRT-PCR.ConclusionThese findings suggest that S-type pyocin hold significant potential as therapeutic agents against pathogenic strains. The Physico-chemical resistance of S-type pyocin underscores its potential for broad applications in the pharmaceutical, hygiene, and food industries.

Exploring novel peptides in adzuki bean and mung bean hydrolysates with potent antibacterial activity

SummaryThis study delved into the antimicrobial potential of hydrolysates from adzuki bean and mung bean protein concentrates, created using Alcalase and Flavourzyme enzymes. Targeting common foodborne pathogens (Salmonella Typhimurium, Salmonella Enteritidis, Vibrio parahaemolyticus, Staphylococcus aureus, and Bacillus subtilis) and Escherichia coli, the research underscored the crucial role of peptidase in generating antibacterial peptides. Fractions F2 of Flavourzyme hydrolysed adzuki bean (ABF) and F4 of Flavourzyme hydrolysed mung bean (MBF) showed the highest antibacterial activity against S. Typhimurium and S. aureus, respectively, whereas F2 of Alcalase hydrolysed mung bean (MBA) exhibited very poor activity against S. aureus. TripleTOF LC–MS/MS analysis revealed small cationic antibacterial peptides, with lysine being prominent against S. aureus and proline–proline against S. Typhimurium. These findings highlighted the significant contribution of protease enzymes in fostering antibacterial peptide production, offering valuable insights for developing effective antimicrobial agents in the context of food safety.

Peptidome Profiling of Bubalus bubalis Urine and Assessment of Its Antimicrobial Activity against Mastitis-Causing Pathogens.

Urinary proteins have been studied quite exhaustively in the past, however, the small sized peptides have remained neglected for a long time in dairy cattle. These peptides are the products of systemic protein turnover, which are excreted out of the body and hence can serve as an important biomarker for various pathophysiologies. These peptides in other species of bovine have been reported to possess several bioactive properties. To investigate the urinary peptides in buffalo and simultaneously their bioactivities, we generated a peptidome profile from the urine of Murrah Buffaloes (n = 10). Urine samples were processed using <10 kDa MWCO filter and filtrate obtained was used for peptide extraction using Solid Phase Extraction (SPE). The nLC-MS/MS of the aqueous phase from ten animals resulted in the identification of 8165 peptides originating from 6041 parent proteins. We further analyzed these peptide sequences to identify bioactive peptides and classify them into anti-cancerous, anti-hypertensive, anti-microbial, and anti-inflammatory groups with a special emphasis on antimicrobial properties. With this in mind, we simultaneously conducted experiments to evaluate the antimicrobial properties of urinary aqueous extract on three pathogenic bacterial strains: S. aureus, E. coli, and S. agalactiae. The urinary peptides observed in the study are the result of the activity of possibly 76 proteases. The GO of these proteases showed the significant enrichment of the antibacterial peptide production. The total urinary peptide showed antimicrobial activity against the aforementioned pathogenic bacterial strains with no significant inhibitory effects against a buffalo mammary epithelial cell line. Just like our previous study in cows, the present study suggests the prime role of the antimicrobial peptides in the maintenance of the sterility of the urinary tract in buffalo by virtue of their amino acid composition.

Compartmentalized PGRP expression along the dipteran Bactrocera dorsalis gut forms a zone of protection for symbiotic bacteria.

All metazoan guts are subject to opposing pressures wherein the immune system must eliminate pathogens while tolerating the presence of symbiotic microbiota. The Imd pathway is an essential defense against invading pathogens in insect guts, but tolerance mechanisms are less understood. Here, we find PGRP-LB and PGRP-SB express mainly in the anterior and middle midgut in a similar pattern to symbiotic Enterobacteriaceae bacteria along the Bactrocera dorsalis gut. Knockdown of PGRP-LB and PGRP-SB enhances the expression of antimicrobial peptide genes and reduces Enterobacteriaceae numbers while increasing abundance of opportunistic pathogens. Microbiota numbers recover to normal levels after the RNAi effect subsided. In contrast, high expression of PGRP-LC in the foregut allows increased antibacterial peptide production to efficiently filter the entry of pathogens, protecting the symbiotic bacteria. Our study describes a mechanism by which regional expression of PGRPs construct a protective zone for symbiotic microbiota while maintaining the ability to fight pathogens.

Isolation and identification of kurthia gibsonii from paneer and study its antibacterial activity against intestinal pathogens

Kurthia is a bacterial genus from the Planococcaceae family and it is more closely related to members of Lactobacillus, Bacillus, Staphylococcus, and Streptococcus. These bacteria are usually found in meat, meat products, milk products, and produce antimicrobial peptides. Antibacterial activity of these peptides has not been reported against intestinal pathogens. This study shows the isolation and identification of Kurthia gibsonii from Paneer and study its antibacterial activity against intestinal pathogens. Kurthia gibsonii was isolated on De Mann Rogosa sharp agar for identification. Morphologically it is a Gram positive, non-motile, non-spore forming, and Rod-shaped bacteria. Probiotics characteristics of K. gibsonii was studied by Acid tolerance, Thermotolerance, Salt tolerance, and Proteolytic activity test. The optimal temperature for growth were 37℃, 45℃, and pH 7.0 respectively. It grew in the presence of 2%, 4%, and 6% NaCl. The organism was identified and confirmed by 16 S RNA sequencing. It was tested for production of Antibacterial peptide by Well Diffusion Assay. The Antibacterial peptide was isolated from Kurthia gibsonii by well plate assay and its extraction, purification was done by ammonium precipitation, Dialysis method, and Reverse phase High performance liquid chromatography method.

The involvement of CgCaspase-8-2 in regulating the expressions of cytokines, antibacterial peptide and autophagy-related genes in oysters

Caspase-8 has been reported to be involved not only in apoptosis, but also in many other important immune response processes, such as inflammation and autophagy. In the present study, the open reading frame of CgCaspase-8-2 was cloned from the Pacific oyster Crassostrea gigas, which was of 2160 bp encoding 737 amino acids. There were two death effector domains (DEDs) and a cysteine aspartase cysteine structural (CASc) domain in the deduced amino acid sequences of CgCaspase-8-2. The mRNA expressions of CgCaspase-8-2 in haemocytes and gills all increased significantly after Vibrio splendidus stimulation at 3 h, 6 h, and 24 h. The cleaved CgCaspase-8-2 protein was observed in haemocytes at 3 h after V. splendidus stimulation and the expression of CgCaspase-8-2 protein was relatively higher in granulocytes, compared with that in agranulocytes. In CgCaspase-8-2-RNAi oysters, the mRNA expressions of CgIL17s (CgIL17-1, −2, −3, −4, −6), CgTNF, CgIFNLP and CgBigDef1 all decreased significantly at 12 h after V. splendidus stimulation. Meanwhile, the mRNA expressions of CgATG5 and CgBeclin1 decreased significantly at 12 h after V. splendidus stimulation, while CgBcl2 increased significantly. These results indicated that CgCaspase-8-2 was involved in not only the regulation of cytokine and antibacterial peptide production, but also autophagy-related gene expressions.

&lt;i&gt;Helicobacter pylori&lt;/i&gt; infection and inflammatory bowel diseases

Helicobacter pylori is detected in the human intestine on average in 35% of clinical cases, but the question about its etiopathogenetic role in intestinal diseases has not been fully investigated. Many scientists study a relationship between the H. pylori persistence and development of various bowel diseases. Diverse viewpoints have been proposed regarding a potential link between H. pylori and inflammatory bowel diseases (IBD). Here we review the data from domestic and foreign studies aimed at examining potential role of H. pylori both as a trigger and protector resulting in the pathogenetic alterations leading to developing Crohn‘s disease and ulcerative colitis. The former is favored by the hypothesis wherein H. pylori may trigger IBD due to potential connection between extragastric infection and its direct damaging action as well as indirect effects contributing to the initiation of oxidative stress, autoimmune aggression and development of intestinal dysbiosis. In addition, the effects of enterohepatic Helicobacter spp. promoting IBD pathogenesis are discussed. The mechanisms underlying the protective role of H. pylori infection may be driven via differentially expressed acute and/or chronic local inflammatory mucosal response able to downmodulate systemic immune responses and suppress autoimmune reactions, as well as skewing host immune response from a pro-inflammatory Th1/Th17 cell-mediated towards regulatory T-cell response. Moreover, it was found that H. pylori may induce production of antibacterial peptides counteracting potentially pathogenic bacteria involved in IBD pathogenesis. In particular, it was found that IBD patients are dominated with moderate active antral gastritis coupled to atrophy, with the peak intensity observed in patients under 30 years of age. Intensity of intestinal metaplasia in the gastric mucosa of IBD patients accounted for by the duration of the disease course. Basal IBD therapy with 5-aminosalicylic acid lowers severity and activity of gastritis, degree of atrophy as well as magnitude H. pylori invasion in the gastric mucosa. There is evidence that 5-aminosalicylic acid-containing drugs may result in a so-called “spontaneous eradication” of H. pylori infection. Extended investigations are required to examine a role of H. pylori in IBD pathogenesis.

Why is it worth remembering the lung microbiome in ICU patients?

In recent years commensal microorganisms are not just "passive occupants", but important element of homeostasis. There are numerous reports documenting the composition and role of the gut, skin or vagina microbiome but the role of commensal orga-nisms living in the lungs is relatively unknown. Pulmonary microbiome impact on the immune response of the host organism and may indicate new therapeutic directions. Lung microbiome, by modulating the expression of innate immunity genes, causes an increase in the concentration of interleukin (IL)-5, IL-10, interferon γ and C-C motif chemokine ligand 11, affects the toll-like receptor-4-dependent response of pulmonary macrophages and modulate the production of antibacterial peptides contained in the mucus. It is documented that disorders of the lung microbiome contribute to asthma or chronic obstructive pulmonary disease. However it is known that pulmonary dysbiosis also occurs in critically ill patients. It is possible, therefore, that microbiota-targeted therapy may constitute the future therapeutic direction in ICU.

Purification of Bioactive Peptide with Antimicrobial Properties Produced by Saccharomyces cerevisiae.

A variety of organisms produce bioactive peptides that express inhibition activity against other organisms. Saccharomyces cerevisiae is considered the best example of a unicellular organism that is useful for studying peptide production. In this study, an antibacterial peptide was produced and isolated from Saccharomyces cerevisiae (Baker’s yeast) by an ultrafiltration process (two membranes with cut-offs of 2 and 10 kDa) and purified using the ÄKTA Pure 25 system. Antibacterial peptide activity was characterized and examined against four bacterial strains including Gram-positive and Gram-negative bacteria. The optimum condition for yeast growth and antibacterial peptide production against both Escherichia. coli and Klebsiella aerogenes was 25–30 °C within a 48 h period. The isolated peptide had a molecular weight of 9770 Da, was thermostable at 50–90 °C for 30 min, and tolerated a pH range of 5–7 at 4 °C and 25 °C during the first 24 h, making this isolated antibacterial peptides suitable for use in sterilization and thermal processes, which are very important aspect in food production. The isolated antibacterial peptide caused a rapid and steady decline in the number of viable cells from 2 to 2.3 log units of gram-negative strains and from 1.5 to 1.8 log units of gram-positive strains during 24 h of incubation. The isolated antibacterial peptide from Saccharomyces cerevisiae may present a potential biopreservative compound in the food industry exhibiting inhibition activity against gram-negative and gram-positive bacteria.

Gut commensal bacteria show beneficial properties as wildlife probiotics

Probiotics are noninvasive, environmentally friendly alternatives for reducing infectious diseases in wildlife species. Our aim in the present study was to evaluate the potential of gut commensals such as lactic acid bacteria (LAB) as wildlife probiotics. The LAB selected for our analyses were isolated from European badgers (Meles meles), a wildlife reservoir of bovine tuberculosis, and comprised four different genera: Enterococcus, Weissella, Pediococcus, and Lactobacillus. The enterococci displayed a phenotype and genotype that included the production of antibacterial peptides and stimulation of antiviral responses, as well as the presence of virulence and antibiotic resistance genes; Weissella showed antimycobacterial activity owing to their ability to produce lactate and ethanol; and lactobacilli and pediococci modulated proinflammatory phagocytic responses that associate with protection against pathogens, responses that coincide with the presence of immunomodulatory markers in their genomes. Although both lactobacilli and pediococci showed resistance to antibiotics, this was naturally acquired, and almost all isolates demonstrated a phylogenetic relationship with isolates from food and healthy animals. Our results show that LAB display probiotic benefits that depend on the genus, and that lactobacilli and pediococci are probably the most obvious candidates as probiotics against infectious diseases in wildlife because of their food-grade status and ability to modulate protective innate immune responses.

Rapid and efficient production of cecropin A antibacterial peptide in Escherichia coli by fusion with a self-aggregating protein

BackgroundCecropin A (CeA), a natural cationic antimicrobial peptide, exerts potent antimicrobial activity against a broad spectrum of Gram-positive and Gram-negative bacteria, making it an attractive candidate substitute for antimicrobials. However, the low production rate and cumbersome, expensive processes required for both its recombinant and chemical synthesis have seriously hindered the exploitation and application of CeA. Here, we utilized a short β-structured self-aggregating protein, ELK16, as a fusion partner of CeA, which allowed the efficient production of high-purity CeA antibacterial peptide with a simple inexpensive process.ResultsIn this study, three different approaches to the production of CeA peptide were investigated: an affinity tag (His-tag)-fused protein expression system (AT-HIS system), a cell-free protein expression system (CF system), and a self-assembling peptide (ELK16)-fused protein expression system (SA-ELK16 system). In the AT-HIS and CF systems, the CeA peptide was obtained with purities of 92.1% and 90.4%, respectively, using one or more affinity-chromatographic purification steps. The procedures were tedious and costly, with CeA yields of only 0.41 and 0.93 μg/mg wet cell weight, respectively. Surprisingly, in the SA-ELK16 system, about 6.2 μg/mg wet cell weight of high-purity (approximately 99.8%) CeA peptide was obtained with a simple low-cost process including steps such as centrifugation and acetic acid treatment. An antimicrobial test showed that the high-purity CeA produced in this study had the same antimicrobial activity as synthetic CeA peptide.ConclusionsIn this study, we designed a suitable expression system (SA-ELK16 system) for the production of the antibacterial peptide CeA and compared it with two other protein expression systems. A high yield of high-purity CeA peptide was obtained with the SA-ELK16 system, which greatly reduced the cost and time required for downstream processing. This system may provide a platform for the laboratory scale production of the CeA antibacterial peptide.

Novel antibacterial peptides induced by probiotics in Hermetia illucens (Diptera: Stratiomyidae) larvae

AbstractThere is a need to discover new therapeutic substances due to the emergence of deadly infectious diseases and various antibiotic resistance. We focused on the larvae that are utilized as a medical insect for the treatment of skin damage in Europe and America. This study was to investigate the pharmacological activities of novel antibacterial peptides isolated from Hermetia illucens larvae against the Klebsiella pneumoniae and Shigella dysenteriae. The larvae were immunized by probiotics (Lactobacillus casei) for 24 h. The hemolymph from the immunized larvae was fractionated through reverse‐phase chromatography. Peptides were purified using HPLC and the coomassie blue staining, and identified using Nano‐LC‐ESI‐MS/MS system. Antibacterial activities of the peptides were evaluated by turbidometric assay, liquid broth dilution assay, resazurin assay, and agar disk diffusion method. The minimum inhibitory concentrations (MICs) of the peptides were measured as 150 μg/mL through the turbidometric, liquid broth dilution, and resazurin assays. The peptides effectively inhibited their growth/proliferation as well as the survival rate of the tested bacteria. Furthermore, the immunized larvae exhibited overexpression of the peptides compared to non‐immunized larvae. These results demonstrate that the peptides induced by H. illucens exert strong antibacterial activity against Gram‐negative bacteria. The results suggest that the activation of the humoral immunity induced by immunization functioned to enhance the production of antibacterial peptides from the insect and their antibacterial properties. This study indicates the potential of the peptides produced from larvae as antibacterial peptide substance for the development of novel antibacterial drugs.

Influence of Whey Protein Concentrate on the Production of Antibacterial Peptides Derived from Fermented Milk by Lactic Acid Bacteria

In the study, growth, proteolysis and antimicrobial activity of lactic acid bacteria were evaluated in skim milk medium supplemented with different concentration of whey protein concentrate (WPC 70). Lactobacillus helveticus (V3) showed maximum pH reduction with 1% WPC. Lactobacillus rhamnosus (NS4) also produced maximum lactic acid production and viable cells counts at 1 and 1.5% WPC, respectively. However, V3 showed maximum proteolytic activity with 1.5% WPC. Streptococcus thermophilus (MD2) was found to exhibit maximum antimicrobial activity with 1.5% WPC. Peptides formed during fermentation were purified by RP-HPLC and identified using RP-LC/MS analysis. Antimicrobial peptide was identified as lactoferrin, which was found in fermented milk supplemented with 1.5% WPC by NS4.

Purification and Biochemical Characterization of a Neutral Serine Protease from Trichoderma harzianum. Use in Antibacterial Peptide Production from a Fish By-Product Hydrolysate.

This study reports the purification and biochemical characterization of an extracellular neutral protease from the fungus Trichoderma harzianum. The protease (Th-Protease) was purified from the culture supernatant to homogeneity by a three-step procedure with 14.2% recovery and 9.06-fold increase in specific activity. The purified enzyme appeared as a single protein band after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with a molecular mass of about 20kDa. The optimum pH and temperature for the proteolytic activity were pH 7.0 and 40°C, respectively. The enzyme was then investigated for its potential application in the production of antibacterial peptides. Interestingly, Scorpaena notata viscera protein hydrolysate prepared using the purified serine protease (Th-Protease) showed remarkable in vitro antibacterial activities. A peptide with a high antibacterial activity was further purified by a three-step procedure, and its sequence was identified as FPIGMGHGSRPA. The result of this study offers a promising alternative to produce natural antibacterial peptides from fish protein hydrolysate.

Functional relevance of intestinal epithelial cells in inflammatory bowel disease.

The intestinal epithelium constitutes a physical barrier between inner and outer side of our body. It also functions as a "hub" which connects factors that determine the development of inflammatory bowel disease, such as microbiota, susceptibility genes, and host immune response. Accordingly, recent studies have implicated and further featured the role of intestinal epithelial cell dysfunction in the pathophysiology of inflammatory bowel disease. For example, mucin producing goblet cells are usually "depleted" in ulcerative colitis patients. Studies have shown that those goblet cells exhibit various immune-regulatory functions in addition to mucin production, such as antigen presentation or cytokine production. Paneth cells are another key cell lineage that has been deeply implicated in the pathophysiology of Crohn's disease. Several susceptibility genes for Crohn's disease may lead to impairment of anti-bacterial peptide production and secretion by Paneth cells. Also, other susceptibility genes may determine the survival of Paneth cells, which leads to reduced Paneth cell function in the patient small intestinal mucosa. Further studies may reveal other unexpected roles of the intestinal epithelium in the pathophysiology of inflammatory bowel disease, and may help to develop alternative therapies targeted to intestinal epithelial cell functions.

Valorization of cruor slaughterhouse by-product by enzymatic hydrolysis for the production of antibacterial peptides: focus on α 1-32 family peptides mechanism and kinetics modeling.

Bovine hemoglobin is the major component of the cruor (slaughterhouse by-product) and can be considered as an important source of active peptides that could be obtained by pepsic hydrolysis. The kinetics of appearance and disappearance of several antibacterial peptides from α 1-32 family during hydrolysis of synthesized α 1-32 peptide, of purified bovine hemoglobin and of cruor was studied, and reaction scheme for the hydrolysis of α 1-32 family peptides from these three sources was determined. On this basis, a mathematical model was proposed to predict the concentration of each peptide of interest of this family depending on hydrolysis time, and also on temperature (in the range 15-37 °C), pH (in the range 3.5-5.5) and enzyme to substrate ratio (in the range 1/50-1/200 for the synthesized peptide and 1/5-1/20 for purified bovine hemoglobin and cruor). Apparent rate constants of reactions were determined by applying the model on a set of experimental data and it was shown that they depended on the temperature according to Arrhenius's law, that their dependence on the pH was linear, and that enzyme to substrate ratio influence was limited (in the studied range).

Current Topics on Vitamin D. The effects of vitamin D on the immune system

Various kinds of immune cells-including macrophages, dendritic cells, T cells and B cells- express the vitamin D receptor and 1α-hydroxylase (CYP27B1), the enzyme necessary for the conversion of circulating 25-hydroxyvitamin D into its active form, 1,25-dihydroxyvitamin D. It suggests that vitamin D has a regulatory role on innate and adaptive immune responses. Vitamin D has been recently shown to promote antimicrobial responses through the production of antibacterial peptides, and stimulation of the autophagic activity in macrophages. Recent epidemiological evidence indicates a significant association between vitamin D deficiency and an increased incidence of several infectious diseases. Here, we review the essential roles of vitamin D in modulating the immune system and discuss the protective effects of vitamin D supplementation in diverse infections.

Gastric LTi cells promote lymphoid follicle formation but are limited by IRAK-M and do not alter microbial growth.

Lymphoid tissue inducer (LTi) cells are activated by accessory cell IL-23, and promote lymphoid tissue genesis and antibacterial peptide production by the mucosal epithelium. We investigated the role of LTi cells in the gastric mucosa in the context of microbial infection. Mice deficient in IRAK-M, a negative regulator of TLR signaling, were investigated for increased LTi cell activity, and antibody mediated LTi cell depletion was used to analyze LTi cell dependent antimicrobial activity. H. pylori infected IRAK-M deficient mice developed increased gastric IL-17 and lymphoid follicles compared to wild type mice. LTi cells were present in naive and infected mice, with increased numbers in IRAK-M deficient mice by two weeks. Helicobacter and Candida infection of LTi cell depleted rag1−/− mice demonstrated LTi-dependent increases in calprotectin but not RegIII proteins. However, pathogen and commensal microbiota populations remained unchanged in the presence or absence of LTi cell function. These data demonstrate LTi cells are present in the stomach and promote lymphoid follicle formation in response to infection, but are limited by IRAK-M expression. Additionally, LTi cell mediated antimicrobial peptide production at the gastric epithelium is less efficacious at protecting against microbial pathogens than has been reported for other tissues.

Tweedle cuticular protein BmCPT1 is involved in innate immunity by participating in recognition of Escherichia coli

Bombyx mori, a lepidopteran insect, is one of the earliest models for pattern recognition of Gram-negative bacteria, which may induce the IMD pathway for production of antibacterial peptides. So far, several recognition proteins have been reported in B. mori. However, the connection between pattern recognition of Gram negative bacteria and activation of BmRelish1, a transcription factor controlled by the IMD pathway remains largely unknown. In the present study, we identify BmCPT1, a cuticle protein bearing a Tweedle domain. Its gene expression is co-regulated by NF-kappaB and juvenile hormone signals. BmCPT1 is induced by Escherichia coli in fat bodies and hemocytes, but is constitutively expressed in the epidermis. In vitro binding assays indicate that BmCPT1 protein recognizes and binds to E. coli peptidoglycan. Post-transcriptionally modified BmCPT1 in the hemolymph binds to E. coli cells through interactions with peptidoglycan recognition protein-5 (BmPGRP5) and lipopolysaccharide binding protein (BmLBP). Transgenic overexpression of BmCPT1 causes the upregulated expression of BmRelish1 and clear induction of two gloverin genes. Therefore, BmCPT1 may work along with BmPGRP-S5 and BmLBP to recognize E. coli in the hemolymph and indirectly activate BmRelish1 to induce antimicrobial peptide synthesis.