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Animals infected with mycoplasma pneumoniae not only develop respiratory diseases, but also cause digestive diseases through the lung-gut axis mediated by the intestinal flora, and vice versa. Antimicrobial peptides are characterized by their bactericidal, anti-inflammatory, and intestinal flora-regulating properties. However, the effect of cecropin AD (CAD) against mycoplasma pneumonia remains unclear. To investigate the anti-inflammatory effect of CAD on mycoplasma pneumonia and the associated mechanism, mice were infected with Mycoplasma capricolum subsp. Capripneumoniae (Mccp) to elicit lung inflammation, followed by oral administration of CAD via gavage. The findings showed that mice receiving twice injections of 2.08 × 10 8 copies of Mccp suffered significant pathological damage to their lungs and colons. Additionally, there was a notable upsurge in inflammatory factors within the affected tissues. 16 S rDNA sequencing revealed alterations in the colonic microbiota, including a decrease in the abundance of beneficial bacteria such as Corynebacterium_glutamicum and Candidatus_Saccharimonas , and an increase in the abundance of potential pathogens like Lachnospiraceae_NK4A136_group and Escherichia-Shigella. As a result, there were abnormal rises in lipopolysaccharide (LPS) levels in both colonic content and blood. Moreover, CAD treatment reversed the microbial dysbiosis and decreased the LPS levels induced by Mccp, thereby suppressing the activation of the TLR-4/NF-κB pathway and the Fas/FasL-caspase-8/-3 pathway. Consequently, this significantly mitigated the morphological and functional damage to the lungs and colons caused by Mccp. The findings offer novel insights and approaches for the clinical management of Mccp infections.

Current antibiotics have limited action mode, which makes it difficult for the antibiotics dealing with the emergence of bacteria resisting the existing antibiotics. As a need for new bacteriolytic agents alternative to the antibiotics, AMPs have long been considered substitutes for the antibiotics. Cecropin B was expressed in a fusion form to six-histidine and SUMO tags in Escherichia coli. Six-histidine tag attached to SUMO was for purification of SUMO-cecropin B fusion proteins and removal of the SUMO tag from cecropin B. Chimeric gene was constructed into pKSEC1 vector that was designed to be functional in both Escherichia coli and chloroplast. To maximize translation of the fusion protein, sequences were codon-optimized. Four different constructs were tested for the level of expression and solubility, and the construct with a linker, 6xHisSUMO3xGly-cecropin B, showed the highest expression. In addition, cleavage of the SUMO tag by SUMOase in the three fusion constructs which have no linker sequence (3xGly, three glycines) was not as efficient as the construct with the linker between SUMO and cecropin B. The cleaved cecropin B showed bacteriolytic activity against Bacillus subtilis at a concentration of 0.0625 μg/μL, while cecropin B fused to SUMO had no activity at a higher concentration, 0.125 μg/μL. As an expression system for AMPs in prokaryotic hosts, the use of tag proteins and appropriate codon-optimization strategy can be employed and further genetic modification of the fusion construct should help the complete removal of the tag proteins from the AMP in the final step of purification.

The irrational use of antibiotics in veterinary practice can cause growth impairment and decrease production performance in young animals. In this study, the distinctive properties of cecropin AD (CAD), including its pathogen-killing and beneficial bacteria-promoting abilities, were used to enhance the growth of underdeveloped lambs. Fifty-four Hu sheep (female, 5 months, 19.78 ± 1.45 kg) were randomly assigned to three dietary treatments for 5 weeks: N-CON (basal diet), L-AMPs (basal diet + 0.05 g CAD/sheep/day), and H-AMPs (basal diet + 0.1 g CAD/sheep/day). Dry matter intake (DMI) was recorded daily. At the end, three sheep per group were slaughtered to analyze gut microbiota, morphology, and blood biochemistry. Results showed CAD improved daily gain and feed-to-gain ratio in impaired sheep. It enhanced liver (GLO, ALT, AST, GGT, ALP; p  < 0.03) and kidney function (BUN, CRE; p  < 0.02), and significantly increased rumen papillae width/height ( p  < 0.01), duodenal villi height ( p  < 0.05), and the villus-to-crypt ratio ( p  < 0.01). It also upregulated tight junction proteins (ZO-1, Occludin, Claudin-1) in the rumen wall ( p  < 0.05) and duodenal epithelium ( p  < 0.05). In addition, CAD enhanced the diversity (Shannon, F = 6.89, p  = 0.001) and abundance (Chao1, F = 8.32, p  = 0.002) of beneficial carbohydrate-fermenting bacteria (e.g., s_Rikenellaceae-RC9_gut_group_unclassified , g_Prevotella , o_Oscillospirales , g_ruminococcaceae_nk4a214_group , and g_Christensenellaceae_R-7_group ), while inhibiting non-fermenting ( g_Succiniclasticum ) and opportunistic pathogens ( c_Negativicutes , o_Veillonellales , and f_Veillonellaceae ). Consequently, in impaired sheep, CAD improved gastrointestinal tissue structure and barrier function, increased beneficial rumen flora, and thereby enhanced growth performance.

Background Mosquitoes are armed with physiologically active compounds to suppress the host immunity including host inflammatory reaction. However, the specific anti-inflammatory components in mosquitoes remain unknown. Results By searching for the immunomodulatory molecules from the mosquito Aedes aegypti (Diptera: Culicidae) at NCBI for anti-inflammatory function, five cecropins (for short in this study: Aeae Cec1, 2, 3, 4 and 5) were selected. Aeae Cec1-5 efficiently inhibited the expression of inducible nitric oxide synthase (iNOS), nitrite, tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and interleukin-6 (IL-6) in lipopolysaccharide (LPS)-stimulated mouse peritoneal macrophages and human peripheral blood mononuclear cells (PBMCs) with low toxicity to mammalian cells. Among the five analogues, Aeae Cec5 had the strongest anti-inflammatory activity, and generated an additive effect with other Aeae Cec peptides. In a mouse model of endotoxin shock, Aeae Cec1-5 effectively reduced TNF-α, IL-1β and IL-6 expression in lungs, serum and peritoneal lavage and correspondingly reduced lung damage and edema, with Aeae Cec5 showing the best protection. In mice infected with Escherichia coli or Pseudomonas aeruginosa , administration of Aeae Cec5 reduced the production of TNF-α, IL-1β and IL-6 and correspondingly reduced lung tissue damage. These effects of Ae. aegypti Aeae Cec1-5 were attributed to an efficient inhibition of the activation of mitogen-activated protein kinases (MAPKs) and transcriptional nuclear factor-κB (NF-κB) signaling pathways, as well as partial neutralization of LPS. Conclusions The current work characterized the specific anti-inflammatory agents in Ae. aegypti and provided Aeae Cec5 as a potent anti-endotoxin peptide that could serve as the basis for the development of anti-inflammatory therapy.

Cancer is a multifaceted health issue that affects people globally and it is considered one of the leading causes of death with a high percentage of victims worldwide. In recent years, research studies have uncovered great advances in cancer diagnosis and treatment. But, there are still major drawbacks of the conventional therapies used including severe side effects, toxicity, and drug resistance. That is why it is critical to develop new drugs with advantages like low cytotoxicity and no treatment resistance to the cancer cells. Antimicrobial peptides (AMPs) have recently attracted attention as a novel therapeutic strategy for the treatment of various cancers, targeting tumor cells with less toxicity to normal tissues. The aim of the study was to discover alternate treatments that do not lead to cancer resistance and have fewer side effects. Here, we report the effects induced by several AMPs, Melittin, Cecropin A, and a Cecropin A—Melittin hybrid, against two human colorectal cancer-derived spheroids. To study the effects of the peptides, cell viability was investigated using MTT, LDH, and ATP assays. Furthermore, cellular senescence and cell cycle were investigated. We found that using different concentrations of these peptides affected the spheroids, their structure being highly compromised by reducing cell viability, and the increase in ATP and LDH levels. Also, the cells are arrested in the G2/M phase leading to an increase in senescent cells. We show that Melittin and the hybrid are most effective against the 3D colorectal cancer cells compared to Cecropin A.

In recent years, antimicrobial peptides have received increased interest and are potential substitutes for antibiotics. However, natural antimicrobial peptides are always toxic to mammalian cells and usually exhibit weak antibacterial activity, which restrict their wide application. In this study, a novel antibacterial peptide named PEW300 was designed with three mutations to the parental peptide cecropin A. As predicted by bioinformatic programs, the positive charge of PEW300 increased from + 6 to + 9 compared with cecropin A, and the grand average of hydropathicity increased from − 0.084 to − 0.008. Expression of PEW300 resulted in serious inhibition of Escherichia coli BL21(DE3) cells, indicating designed PEW300 may have stronger antibacterial activity. A simple, fast, and low-cost approach without tedious protein purification steps was selected for the efficient production of PEW300 by fusion with ELK16 and about 7.38 μg/mg wet cell weight PEW300 was eventually obtained. Purified PEW300 exhibited strong antibacterial activity against various Gram-positive and Gram-negative bacteria which was enhanced four- to sevenfold compared with the parental peptide cecropin A. Besides, PEW300 had no hemolytic activity toward mammalian cells even at high concentration (224 ng/μl). PEW300 showed good stability in neutral and alkaline solutions. Moreover, PEW300 was thermally stable even at up to 100 °C and resistant to proteinase K, pepsin, snailase, and trypsin. The incubation with human serum had no effect on the antibacterial activity of PEW300. All these results demonstrated that PEW300 designed in this work may have good potential as a candidate pharmaceutical agent.

Resistance of pathogenic bacteria to standard antibiotics is an issue of great concern, and new treatments for bacterial infections are needed. Antimicrobial peptides (AMPs) are small, cationic, and amphipathic molecules expressed by metazoans that kill pathogens. They are a key part of the innate immune system in both vertebrates and invertebrates. Due to their low toxicity and broad antimicrobial activities, there has been increasing attention to their therapeutic usage. Our previous research demonstrated that four peptides-DAN1, DAN2, HOLO1 and LOUDEF1-derived from recently sequenced arthropod genomes exhibited potent antimicrobial effects in-vitro. In this study, we show that DAN2 protected 100% of mice when it was administered at a concentration of 20 mg/kg thirty minutes after the inoculation of a lethal dose of E. coli intraperitoneally. Lower concentrations of DAN2-10mg/kg and 5mg/kg protected more than 2/3s of the mice. All three dose levels reduced bacterial loads in blood and peritoneal fluid by 10-fold or more when counted six hours after bacterial challenge. We determined that DAN2 acts by compromising the integrity of the E. coli membrane. This study supports the potential of DAN2 peptide as a therapeutic agent for treating antibiotic resistant Gram-negative bacterial infections.

Background Several antimicrobial peptides (AMPs) belonging to the cecropin family have been identified from the salivary glands of different black fly species, however, the immunological functions for these molecules were poorly understood. Methods A novel cecropin-like antimicrobial peptide ( Siba Cec) was purified using reverse phase high-performance liquid chromatography (RP-HPLC) from the salivary glands of the black fly Simulium bannaense . The amino acid sequence of Siba Cec was determined by a combination method of automated Edman degradation and cDNA sequencing. The morphologic changes of Gram-negative bacteria Escherichia coli treated with Siba Cec were assessed by scanning electron microscopy (SEM). Quantitative PCR (qPCR) was performed to analyze the mRNA expression of the inducible NO synthase (iNOS) and pro-inflammatory cytokines. Nitric oxide (NO) generation was examined using a Griess assay and the secretion of pro-inflammatory cytokines was determined by an enzyme-linked immunosorbent assay (ELISA). The activation of extracellular signal-regulated kinase (ERK), p38, and the nuclear translocation of nuclear factor-kappaB (NF-κB) were assessed by Western blotting analysis. Circular dichroism (CD) spectroscopy was performed to evaluate the secondary structure of Siba Cec in solvent environment. Interaction of Siba Cec with lipopolysaccharide (LPS) was studied using fluorescein isothiocyanate (FITC)- conjugated LPS aggregates. Neutralization of LPS by Siba Cec was assayed with the chromogenic limulus amebocyte lysate (LAL) test. qPCR was also used to analyze the expression of Siba Cec mRNA in the salivary glands of insects after oral infection with the bacteria E.coli. Results Siba Cec possessed potent antimicrobial activity against Gram-negative bacteria, and showed low cytotoxicity toward mammalian cells. SEM analysis indicated that Siba Cec killed bacteria through the disruption of cell membrane integrity. Furthermore, Siba Cec significantly inhibited lipopolysaccharide (LPS)-induced production of NO and pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α), interferon-1β (IL-1β) and interferon-6 (IL-6) by blocking the activation of MAPKs and NF-κB signaling pathways. It mainly adopted an α-helix conformation in membrane-mimetic environments. Siba Cec could interact and neutralize LPS. Infection of black flies with bacteria caused an upregulation of the expression of Siba Cec. Conclusions These results demonstrated that in addition to the bactericidal capacity, Siba Cec can function as immune regulator, inhibiting host secretion of inflammatory factors.

Antimicrobial peptides are innate immune molecules playing essential roles in insects, which lack the adaptive immune system. Insects possess Toll9, the innate pattern-recognition receptor highly similar to the mammalian Toll-like receptor 4 (TLR4), which is involved in recognizing lipopolysaccharide (LPS). TLR4 is an important therapeutic target, as it causes uncontrolled immune response in sepsis; therefore, identification of TLR4-targeting molecules is imperative. Papiliocin, an insect cecropin derived from the larvae of the swallowtail butterfly, possesses potent antibacterial activities against gram-negative bacteria. We investigated the molecular mechanism underlying the TLR4-antagonistic and antiseptic activities of papiliocin. Binding analysis, docking simulation, and flow cytometry showed that papiliocin inhibited LPS-induced TLR4 signaling by directly binding to TLR4/MD-2 and causing rapid dissociation of LPS from the TLR4/MD-2 complex. R13 and R16 in the N-terminal helix, conserved in insect cecropins, were the key binding sites at the TLR4/MD-2 interface, along with the flexible hinge region, which promoted the interaction of the hydrophobic carboxyl-terminal helix with the MD-2 pocket to competitively inhibit the LPS–TLR4/MD-2 interaction. Papiliocin, an antiendotoxin molecule and TLR4 inhibitor, rescued the pathology of Escherichia coli–induced sepsis in mice more effectively and with lower nephrotoxicity compared to polymyxin B. Our results provide insight into the key structural components and mechanism underlying the TLR4-antagonistic activities of papiliocin, which is essential for the innate immune response of the insect against microbial infection. Papiliocin may be useful for developing a multifunctional alternative to polymyxin B for treating gram-negative sepsis.

Background It has been reported that there are more than ten antimicrobial peptides (AMPs) belonging to the cecropin family in Musca domestica ; however, few of them have been identified, and the functions of the other molecules are poorly understood. Methods Sequences of the M. domestica cecropin family of genes were cloned from cDNA template, which was reverse-transcribed from total mRNA isolated from third-instar larvae of M. domestica that were challenged with pathogens. Sequence analysis was performed using DNAMAN comprehensive analysis software, and a molecular phylogenetic tree of the cecropin family was constructed using the Neighbour-Joining method in MEGA v.5.0 according to the mature peptide sequences. Antibacterial activity of the synthetic M. domestica cecropin protein was detected and the minimum inhibitory concentration (MIC) values were determined using broth microdilution techniques. Time-killing assays were performed on the Gram-negative bacteria, Acinetobacter baumannii , at the logarithmic or stabilizing stages of growth, and its morphological changes when treated with Cec4 were assessed by scanning electron microscopy (SEM) and detection of leakage of 260 nm absorbing material. Results Eleven cecropin family genes, namely Cec01 , Cec02 and Cec1-9 , show homology to the Cec form in a multigene family on the Scaffold18749 of M. domestica . In comparing the encoded cecropin protein sequences, most of them have the basic characteristics of the cecropin family, containing 19 conservative amino acid residues. To our knowledge, this is the first experimental demonstration that most genes in the Cec family are functional. Cec02, Cec1, Cec2, Cec5 and Cec7 have similar antibacterial spectra and antibacterial effects against Gram-negative bacteria, while Cec4 displays a more broad-spectrum of antimicrobial activity and has a very strong effect on A. baumannii . Cec4 eliminated A. baumannii in a rapid and concentration-dependent manner, with antibacterial effects within 24 h at 1× MIC and 2× MIC. Furthermore, SEM analysis and the leakage of 260 nm absorbing material detection indicated that Cec4 sterilized the bacteria through the disruption of cell membrane integrity. Conclusions Although there are more than ten cecropin genes related to M. domestica , some of them have no preferred antibacterial activity other than Cec4 against A. baumannii .