Efficient in planta production of amidated antimicrobial peptides that are active against drug-resistant ESKAPE pathogens.

Antimicrobial peptides (AMPs) are promising next-generation antibiotics that can be used to combat drug-resistant pathogens. However, the high cost involved in AMP synthesis and their short plasma half-life render their clinical translation a challenge. To address these shortcomings, we report efficient production of bioactive amidated AMPs by transient expression of glycine-extended AMPs in Nicotiana benthamiana line expressing the mammalian enzyme peptidylglycine α-amidating mono-oxygenase (PAM). Cationic AMPs accumulate to substantial levels in PAM transgenic plants compare to nontransgenic N. benthamiana. Moreover, AMPs purified from plants exhibit robust killing activity against six highly virulent and antibiotic resistant ESKAPE pathogens, prevent their biofilm formation, analogous to their synthetic counterparts and synergize with antibiotics. We also perform a base case techno-economic analysis of our platform, demonstrating the potential economic advantages and scalability for industrial use. Taken together, our experimental data and techno-economic analysis demonstrate the potential use of plant chassis for large-scale production of clinical-grade AMPs.

Effect of silver nanoparticles on gene transcription of land snail Helix aspersa.

Silver nanoparticles (Ag-NPs) are extremely useful in a diverse range of consumer goods. However, their impact on the environment is still under research, especially regarding the mechanisms involved in their effect. Aiming to provide some insight, the present work analyzes the transcriptional activity of six genes (Hsp83, Hsp17.2, Hsp19.8, SOD Cu-Zn, Mn-SOD, and BPI) in the terrestrial snail Helix aspersa in the presence of different concentrations of Ag-NPs. The animals were exposed for seven days to Lactuca sativa soaked for one hour in different concentrations of Ag-NPs (20, 50, 100 mg/L). The results revealed that the highest concentration tested of Ag-NPs (100 mg/L) led to a statistically significant induction of the Hsp83 and BPI expression in the digestive gland compared to the control group. However, a trend to upregulation with no statistical significance was observed for all the genes in the digestive gland and the foot, while in the hemolymph, the trend was to downregulation. Ag-NPs affected the stress response and immunity under the tested conditions, although the impact was weak. It is necessary to explore longer exposure times to confirm that the effect can be maintained and impact on health. Our results highlight the usefulness of the terrestrial snail Helix aspersa as a bioindicator organism for silver nanoparticle pollution biomonitoring and, in particular, the use of molecular biomarkers of pollutant effect as candidates to be included in a multi-biomarker strategy.

Actinomadura graeca sp. nov.: A novel producer of the macrocyclic antibiotic zelkovamycin.

As part of a screening programme for antibiotic-producing bacteria, a novel Actinomadura species was discovered from a soil sample collected in Santorini, Greece. Preliminary 16S rRNA gene sequence comparisons highlighted Actinomadura macra as the most similar characterised species. However, whole-genome sequencing revealed an average nucleotide identity (ANI) value of 89% with A. macra, the highest among related species. Further phenotypic and chemotaxonomic analyses confirmed that the isolate represents a previously uncharacterised species in the genus Actinomadura, for which the name Actinomadura graeca sp. nov. is proposed (type strain 32-07T). The G+C content of A. graeca 32-07 is 72.36%. The cell wall contains DL-diaminopimelic acid, intracellular sugars are glucose, ribose and galactose, the predominant menaquinone is MK-9(H6), the major cellular lipid is phosphatidylinositol and fatty acids consist mainly of hexadecanoic acid. No mycolic acid was detected. Furthermore, A. graeca 32-07 has been confirmed as a novel producer of the non-ribosomal peptide antibiotic zelkovamycin and we report herein a provisional description of the unique biosynthetic gene cluster.

Construction of a Tandem Repeat Peptide Sequence with Pepsin Cutting Sites to Produce Recombinant α-Melanocyte-Stimulating Hormone.

The production of α-melanocyte-stimulating hormone (α-MSH), a peptide hormone composed of 13 amino acids, is attempted by recombinant expression using E. coli as the host. To achieve this aim, a synthetic gene containing eight tandem repeats of msh gene (8msh) was designed for ribosomal synthesis of 8 α-MSH. The merit of the strategy is to diminish the peptide toxicity against the host cell and to achieve a higher production yield. Pepsin cleavage sites are introduced between the peptides for enzymatic proteolysis to obtain the monomeric peptide of α-MSH. The constructed plasmid was transformed into different strains of E. coli hosts, and E. coli XL1-Blue with gene 8msh revealed the highest yield of 8 α-MSH. Although 8 α-MSH was fractionalized in the insoluble pellets after cell lysis, pepsin cleavage was able to produce soluble α-MSH peptide, as analyzed and confirmed by mass spectrometry and peptide activity assays. The production of α-MSH was quantified using HPLC with a yield of 42.9 mg/L of LB culture. This study demonstrates the feasibility of producing α-MSH using recombinant expression of tandem repeat gene. The production procedure involves minimal post-treatment and processing and can be scaled up for industrial application.

Responses of Mast Cells to Pathogens: Beneficial and Detrimental Roles.

Mast cells (MCs) are strategically located in tissues close to the external environment, being one of the first immune cells to interact with invading pathogens. They are long living effector cells equipped with different receptors that allow microbial recognition. Once activated, MCs release numerous biologically active mediators in the site of pathogen contact, which induce vascular endothelium modification, inflammation development and extracellular matrix remodeling. Efficient and direct antimicrobial mechanisms of MCs involve phagocytosis with oxidative and non-oxidative microbial destruction, extracellular trap formation, and the release of antimicrobial substances. MCs also contribute to host defense through the attraction and activation of phagocytic and inflammatory cells, shaping the innate and adaptive immune responses. However, as part of their response to pathogens and under an impaired, sustained, or systemic activation, MCs may contribute to tissue damage. This review will focus on the current knowledge about direct and indirect contribution of MCs to pathogen clearance. Antimicrobial mechanisms of MCs are addressed with special attention to signaling pathways involved and molecular weapons implicated. The role of MCs in a dysregulated host response that can increase morbidity and mortality is also reviewed and discussed, highlighting the complexity of MCs biology in the context of host-pathogen interactions.

Stress-Inducible Gene Atf3 Dictates a Dichotomous Macrophage Activity in Chemotherapy-Enhanced Lung Colonization.

Previously, we showed that chemotherapy paradoxically exacerbated cancer cell colonization at the secondary site in a manner dependent on Atf3, a stress-inducible gene, in the non-cancer host cells. Here, we present evidence that this phenotype is established at an early stage of colonization within days of cancer cell arrival. Using mouse breast cancer models, we showed that, in the wild-type (WT) lung, cyclophosphamide (CTX) increased the ability of the lung to retain cancer cells in the vascular bed. Although CTX did not change the WT lung to affect cancer cell extravasation or proliferation, it changed the lung macrophage to be pro-cancer, protecting cancer cells from death. This, combined with the initial increase in cell retention, resulted in higher lung colonization in CTX-treated than control-treated mice. In the Atf3 knockout (KO) lung, CTX also increased the ability of lung to retain cancer cells. However, the CTX-treated KO macrophage was highly cytotoxic to cancer cells, resulting in no increase in lung colonization-despite the initial increase in cell retention. In summary, the status of Atf3 dictates the dichotomous activity of macrophage: pro-cancer for CTX-treated WT macrophage but anti-cancer for the KO counterpart. This dichotomy provides a mechanistic explanation for CTX to exacerbate lung colonization in the WT but not Atf3 KO lung.

Recombinant production of Trx-Ib-AMP4 and Trx-E50-52 antimicrobial peptides and antimicrobial synergistic assessment on the treatment of methicillin-resistant Staphylococcus aureus under in vitro and in vivo situations.

PURPOSE: The production of alternative novel antimicrobial agents is considered an efficient way to cope with multidrug resistance among pathogenic bacteria. E50-52 and Ib-AMP4 antimicrobial peptides (AMPs) have illustrated great proven antibacterial effects. The aim of this study was recombinant production of these AMPs and investigation of their synergistic effects on methicillin-resistant Staphylococcus aureus (MRSA). METHOD: At first, the codon optimized sequences of the Ib-AMP4 (UniProt: 024006 (PRO_0000020721), and E50-52 (UniProtKB: P85148) were individually ligated into the pET-32α vector and transformed into E. coli. After the optimization of production and purification steps, the MIC (Minimum inhibitory concentration), time kill and growth kinetic tests of recombinant proteins were determined against MRSA. Finally, the in vivo wound healing efficiency was tested. RESULTS AND CONCLUSION: The recorded MIC of recombinant Trx-Ib-AMP4, Trx-E50-52 against MRSA bacterium were 0.375 and 0.0875 mg/mL respectively. The combination application of the produced AMPs by the checkerboard method confirmed their synergic activity. The results of the time-kill showed sharply decrease of the number of viable cells with over five time reductions in log10 CFU/mL by the combination of Trx-E50-52 and Trx-IbAMP4 at 2 × MIC within 240 min. The growth kinetic results confirmed the combination of Trx-E50-52 and Trx-IbAMP4 had much greater success in the reduction of over 50 % of MRSA suspensions' turbidity within the first hour. Wound healing assay and histological analysis of infected mice treated with Trx-Ib-AMP4 or Trx-E50-52 compared with those treated with a combination of Trx-Ib-AMP4 and Trx-E50-52 showed significant synergic effects.

Steroid hormone modulates the production of cathelicidin and human ß-defensins in lung epithelial cells and macrophages promoting Mycobacterium tuberculosis killing.

Several studies have documented the interaction between the immune and endocrine systems as an effective defense strategy against tuberculosis, involving the production of several molecules and immunological processes. In this study, we determined the effect of cortisol and dehydroepiandrosterone (DHEA) on the production of antimicrobial peptides such as cathelicidin and human ß-defensin (HBD) -2, and HBD-3 and their effect on intracellular growth of Mycobacterium tuberculosis (Mtb) in lung epithelial cells and macrophages. Our results showed that DHEA promotes the production of these antimicrobial peptides in infected cells, correlating with the decrease of Mtb bacilli loads. These results suggest the use of exogenous DHEA as an adjuvant for tuberculosis therapy.

Toll-like receptor activation of equine mesenchymal stromal cells to enhance antibacterial activity and immunomodulatory cytokine secretion.

OBJECTIVE: To evaluate effects of Toll-like and nucleotide-binding oligomerization domain (NOD)-like receptor (TLR, NLR) ligand stimulation of equine mesenchymal stromal cells (MSCs) on antibacterial and immunomodulatory properties in vitro. STUDY DESIGN: Controlled laboratory study. SAMPLE POPULATION: Equine bone-marrow-derived MSCs (three horses). METHODS: MSCs were stimulated with TLR (polyinosinic:polycytidylic acid [pIC] and lipopolysaccharide [LPS]) and NLR agonists (γ-d-Glu-mDAP [IE-DAP]) for 2 h, and plated at 1 × 105 cells/well 24 h. MSC-conditioned media (MSC-CM) were collected and assessed for antimicrobial peptide cathelicidin/LL-37 production, bactericidal action against multidrug-resistant planktonic and biofilm Staphylococcus aureus and neutrophil phagocytosis. Bacterial growth was measured by plating bacteria and counting viable colonies, reading culture absorbance, and live-dead staining with confocal microscopy imaging. Following initial comparison of activating stimuli, TLR3-agonist pIC protocols (cell density during activation and plating, culture time, %serum) were further optimized for bactericidal activity and secretion of interleukin-8 (IL-8), monocyte-chemoattractant-protein (MCP-1), and cathelicidin/LL37. RESULTS: MSCs stimulation with pIC (p = .004) and IE-DAP (p = .03) promoted increased bactericidal activity, evidenced by reduced viable planktonic colony counts. PIC stimulation (2 × 106 cells/ml, 2 h, 10 µg/ml) further suppressed biofilm formation (p = .001), enhanced neutrophil bacterial phagocytosis (p = .009), increased MCP-1 secretion (p < .0001), and enhanced cathelicidin/LL-37 production, which was apparent when serum concentration in media was reduced to 1% (p = .01) and 2.5% (p = .05). CONCLUSION: TLR-3 pIC MSCs activation was most effective to enhance antibacterial and cytokine responses, which were affected by serum reduction. CLINICAL SIGNIFICANCE: In vitro TLR-3 activation of equine MSCs tested here may be a strategy to improve antibacterial properties of MSCs to treat antibiotic-resistant infections.

Ultraviolet radiation, vitamin D, and COVID-19.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), has become pandemic on March 11th, 2020. COVID-19 has a range of symptoms that includes fever, fatigue, dry cough, aches, and labored breathing to acute respiratory distress and possibly death. Health systems and hospitals have been completely rearranged since March 2020 in order to limit the high rate of virus spreading. Hence, a great debate on deferrable visits and treatments including phototherapy for skin diseases is developing. In particular, as regards phototherapy very few data are currently available regarding the chance to continue it, even if it may be a useful resource for treating numerous dermatological patients. However, phototherapy has an immunosuppressive action possibly facilitating virus infection. In the context of COVID-19 infection risk it is important to pointed out whether sunlight, phototherapy and in particular ultraviolet radiation (UV-R) constitute or not a risk for patients. In this review we aimed to focus on the relationship between UV-R, sunlight, phototherapy, and viral infections particularly focusing on COVID-19.

Biosynthesis and Mechanism of Action of the Cell Wall Targeting Antibiotic Hypeptin.

Hypeptin is a cyclodepsipeptide antibiotic produced by Lysobacter sp. K5869, isolated from an environmental sample by the iChip technology, dedicated to the cultivation of previously uncultured microorganisms. Hypeptin shares structural features with teixobactin and exhibits potent activity against a broad spectrum of gram-positive pathogens. Using comprehensive in vivo and in vitro analyses, we show that hypeptin blocks bacterial cell wall biosynthesis by binding to multiple undecaprenyl pyrophosphate-containing biosynthesis intermediates, forming a stoichiometric 2:1 complex. Resistance to hypeptin did not readily develop in vitro. Analysis of the hypeptin biosynthetic gene cluster (BGC) supported a model for the synthesis of the octapeptide. Within the BGC, two hydroxylases were identified and characterized, responsible for the stereoselective ß-hydroxylation of four building blocks when bound to peptidyl carrier proteins. In vitro hydroxylation assays corroborate the biosynthetic hypothesis and lead to the proposal of a refined structure for hypeptin.

Inhibition of defensin A and cecropin A responses to dengue virus 1 infection in Aedes aegypti. / Inhibición de las respuestas de defensina A y cecropina A contra la infección del virus dengue 1 en Aedes aegypti

INTRODUCTION: It is essential to determine the interactions between viruses and mosquitoes to diminish dengue viral transmission. These interactions constitute a very complex system of highly regulated pathways known as the innate immune system of the mosquito, which produces antimicrobial peptides that act as effector molecules against bacterial and fungal infections. There is less information about such effects on virus infections. OBJECTIVE: To determine the expression of two antimicrobial peptide genes, defensin A and cecropin A, in Aedes aegypti mosquitoes infected with DENV-1. MATERIALS AND METHODS: We used the F1 generation of mosquitoes orally infected with DENV-1 and real-time PCR analysis to determine whether the defensin A and cecropin A genes played a role in controlling DENV-1 replication in Ae. aegypti. As a reference, we conducted similar experiments with the bacteria Escherichia coli. RESULTS: Basal levels of defensin A and cecropin A mRNA were expressed in uninfected mosquitoes at different times post-blood feeding. The infected mosquitoes experienced reduced expression of these mRNA by at least eightfold when compared to uninfected control mosquitoes at all times post-infection. In contrast with the behavior of DENV-1, results showed that bacterial infection produced up-regulation of defensin and cecropin genes; however, the induction of transcripts occurred at later times (15 days). CONCLUSION: DENV-1 virus inhibited the expression of defensin A and cecropin A genes in a wild Ae. aegypti population from Venezuela.

Introducción. Es esencial determinar las interacciones entre los virus y los mosquitos para disminuir la transmisión viral. Estas interacciones constituyen un sistema muy complejo y muy regulado conocido como sistema inmunitario innato del mosquito, el cual produce péptidos antimicrobianos, moléculas efectoras que funcionan contra las infecciones bacterianas y fúngicas; se tiene poca información de su acción sobre los virus. Objetivo. Determinar la expresión de dos genes AMP (defensina A y cecropina A) en mosquitos Aedes aegypti infectados con el virus DENV-1. Materiales y métodos. Se infectaron oralmente mosquitos de generación F1 con DENV-1 y mediante el análisis con PCR en tiempo real se determinó el potencial papel de los genes defensina A y cecropina A en el control de la replicación del DENV-1 en Ae. aegypti. Como referencia, se infectaron mosquitos con Escherichia coli. Resultados: Los mosquitos no infectados expresaron niveles basales de los ARNm de los genes defensina A y cecropina A en diversos momentos después de la alimentación. Los mosquitos infectados experimentaron una reducción, por lo menos, de ocho veces en la expresión de estos ARNm con respecto a los mosquitos de control en todo el periodo posterior a la alimentación. En contraste con el comportamiento del virus DENV-1, los resultados mostraron que la infección bacteriana produjo una regulación positiva de los genes defensina y cecropina; sin embargo, la inducción de los transcritos ocurrió tardíamente (15 días). Conclusión. El virus DENV-1 inhibió la expresión de los genes defensina A y cecropina A en una población silvestre de Ae. aegypti en Venezuela.

Promiscuous Installation of d-Amino Acids in Gene-Encoded Peptides.

d-Amino acids can have major effects on the structure, proteolytic stability, and bioactivity of peptides. Proteusin radical S-adenosyl methionine epimerases regioselectively install such residues in ribosomal peptides to generate peptides with the largest number of d-residues currently known in biomolecules. To study their utility in synthetic biology, we investigated the substrate tolerance and substrate-product relationships of the cyanobacterial model epimerase OspD using libraries of point mutants as well as distinct extended peptides that were fused to an N-terminal leader sequence. OspD was found to exhibit exceptional substrate promiscuity in E. coli, accepting 15 different amino acids and converting peptides with a broad range of compositions, secondary structures, and polarities. Diverse single and multiple epimerization patterns were identified that were dictated by the peptide sequence. The data suggest major potential in creating genetically encoded products previously inaccessible by synthetic biology.

Discovery and Biosynthetic Investigation of a New Antibacterial Dehydrated Non-Ribosomal Tripeptide.

Dehydroalanine (Dha) and dehydrobutyrine (Dhb) display considerable flexibility in a variety of chemical and biological reactions. Natural products containing Dha and/or Dhb residues are often found to display diverse biological activities. While the (Z) geometry is predominant in nature, only a handful of metabolites containing (E)-Dhb have been found thus far. Here we report discovery of a new antimicrobial peptide, albopeptide, through NMR analysis and chemical synthesis, which contains two contiguous unsaturated residues, Dha-(E)-Dhb. It displays narrow-spectrum activity against vancomycin-resistant Enterococcus faecium. In-vitro biochemical assays show that albopeptide originates from a noncanonical NRPS pathway featuring dehydration processes and catalysed by unusual condensation domains. Finally, we provide evidence of the occurrence of a previously untapped group of short unsaturated peptides in the bacterial kingdom, suggesting an important biological function in bacteria.

Antimicrobial Peptides: a New Frontier in Antifungal Therapy.

Invasive fungal infections in humans are generally associated with high mortality, making the choice of antifungal drug crucial for the outcome of the patient. The limited spectrum of antifungals available and the development of drug resistance represent the main concerns for the current antifungal treatments, requiring alternative strategies. Antimicrobial peptides (AMPs), expressed in several organisms and used as first-line defenses against microbial infections, have emerged as potential candidates for developing new antifungal therapies, characterized by negligible host toxicity and low resistance rates. Most of the current literature focuses on peptides with antibacterial activity, but there are fewer studies of their antifungal properties. This review focuses on AMPs with antifungal effects, including their in vitro and in vivo activities, with the biological repercussions on the fungal cells, when known. The classification of the peptides is based on their mode of action: although the majority of AMPs exert their activity through the interaction with membranes, other mechanisms have been identified, including cell wall inhibition and nucleic acid binding. In addition, antifungal compounds with unknown modes of action are also described. The elucidation of such mechanisms can be useful to identify novel drug targets and, possibly, to serve as the templates for the synthesis of new antimicrobial compounds with increased activity and reduced host toxicity.

IL-33 synergistically promotes the proliferation of lung cancer cells in vitro by inducing antibacterial peptide LL-37 and proinflammatory cytokines in macrophages.

Lung cancer is the primary cause of cancer-related deaths, and the persistent inflammation is inextricably linked with the lung cancer tumorigenesis. Pro-inflammatory cytokine interleukin-33 (IL-33) is able to serve as a potent modulator of cancer. Mounting evidence indicates IL-33 has significant effect on lung cancer progression by regulating host immune response, but the current opinions about the function and mechanism of IL-33 in lung cancer are still controversial. Meanwhile, antibacterial peptide LL-37 also exerts a momentous effect on immune responses to lung cancer. LL-37 is regarded as versatile, including antimicrobial activities, chemotaxis and immunoregulation. However, the immunomodulatory mechanism of IL-33 and LL-37 in lung cancer remains thoroughly not defined. Here, we determined the secretion of LL-37 was up-regulated in lung cancer serum samples. Similarly, the expression of CRAMP was enhancive in macrophages after co-cultured with lung cancer cells. Moreover, we expounded that IL-33 could up-regulate LL-37 secretion in macrophages, resulting in the massive releases of IL-6 and IL-1ß. Additionally, LL-37 cooperated with IL-33 to increase the phosphorylation of p38 MAPK and NF-κB p65 pathways, and augmented IL-6 and IL-1ß secretion, which resulting in the proliferation of lung cancer cells in vitro. In conclusion, our study identified that IL-33 aggravated the inflammation of lung cancer by increasing LL-37 expression in macrophages, thereby promoting lung cancer cell proliferation in vitro. It is contributed to our present understanding of the immunomodulatory relationship between pro-inflammatory cytokines and antibacterial peptides in the tumor immune response, and offer a novel perspective for controlling the progress of lung cancer.

MiR-196-5p involvement in selenium deficiency-induced immune damage via targeting of NFκBIA in the chicken trachea.

Dietary selenium (Se) deficiency can induce multifarious immune injury in tissues, accompanied by inflammation and a decreased expression of selenoproteins. The results of previous studies indicated that these issues are associated with Se-mediated microRNAs involved in immune regulation, although the specific mechanisms associated with these interactions have not been reported in the trachea of chickens. To explore the effects of Se deficiency in the trachea of chickens and the role of miR-196-5p, we established correlational models of tracheal injury in chickens. One hundred broilers were divided into four groups, including a control group (C group), a Se deficient group (L group), a lipopolysaccharide (LPS)-induced control group (C + LPS group) and a LPS-induced Se deficient group (L + LPS group). Light microscopy observations indicated that the infiltration of inflammatory cells was the major histopathological change caused by Se deficiency. Furthermore, ultrastructural observation of the tracheal epithelium and ciliary showed typical inflammatory signs owing to Se deficiency. We determined the targeting relationship between miR-196-5p and NFκBIA by bioinformatics analysis. In the case of Se deficiency, the changes were detected as follows: 19 selenoproteins showed different degrees of decrease (p < 0.05). Significant inhibition of both antimicrobial peptides and immunoglobulin production were observed (p < 0.05). IκB-α (NFκBIA) expression degraded with the increasing miR-196-5p (p < 0.05), and the NF-κB pathway was activated. Thereafter, we can see a significant increase in the mRNA levels of inflammatory cytokines-related genes (tumor necrosis factor (TNF)-α, inducible nitric oxide synthase (iNOS), cyclooxygenase (COX)-2, prostaglandin E (PTGE), interleukin (IL)-1ß, IL-6) and protein expression of NF-κB/iNOS pathway-related genes (NF-κB, iNOS, TNF-α, COX-2) (p < 0.05). The release of IL-2, interferon (IFN)-γ inhibited (p < 0.05) and the secretion of IL-4, IL-6 increased, suggesting the imbalance of Th1/Th2 (Th, helper T cell) cytokines. Compared to the control, the mRNA and protein expression levels of the anti-inflammatory system components with antioxidant activity (PPAR-γ/HO-1) were in an inhibitory state (p < 0.05). Antioxidases (SOD, CAT, GSH-Px) activities were suppressed. The activities of the peroxide markers (MDA, H2O2) were enhanced (p < 0.05). In addition, Se deficiency had a positive effect on the pathological changes of inflammation and the exceptional immunity in LPS-treated groups (p < 0.05). The results confirmed the relationship between miR-196-5p and NFκBIA in chickens, revealing that Se deficiency causes respiratory mucosal immune dysfunction via the miR-196-5p-NFκBIA axis, oxidative stress and inflammation. Moreover, Se deficiency exacerbates the inflammatory damage stimulated by LPS. Our work provides a theoretical basis for the prevention of tracheal injury owing to Se deficiency and can be used as a reference for comparative medicine. Furthermore, the targeted regulation of miR-196-5p and NFκBIA may contribute to the protection of the tracheal mucosa in chickens.

Does the Future of Antibiotics Lie in Secondary Metabolites Produced by Xenorhabdus spp.? A Review.

The over-prescription of antibiotics for treatment of infections is primarily to blame for the increase in bacterial resistance. Added to the problem is the slow rate at which novel antibiotics are discovered and the many processes that need to be followed to classify antimicrobials safe for medical use. Xenorhabdus spp. of the family Enterobacteriaceae, mutualistically associated with entomopathogenic nematodes of the genus Steinernema, produce a variety of antibacterial peptides, including bacteriocins, depsipeptides, xenocoumacins and PAX (peptide antimicrobial-Xenorhabdus) peptides, plus additional secondary metabolites with antibacterial and antifungal activity. The secondary metabolites of some strains are active against protozoa and a few have anti-carcinogenic properties. It is thus not surprising that nematodes invaded by a single strain of a Xenorhabdus species are not infected by other microorganisms. In this review, the antimicrobial compounds produced by Xenorhabdus spp. are listed and the gene clusters involved in synthesis of these secondary metabolites are discussed. We also review growth conditions required for increased production of antimicrobial compounds.

Fungal-Associated Molecules Induce Key Genes Involved in the Biosynthesis of the Antifungal Secondary Metabolites Nunamycin and Nunapeptin in the Biocontrol Strain Pseudomonas fluorescens In5.

Pseudomonas fluorescens In5 synthesizes the antifungal cyclic lipopeptides (CLPs) nunamycin and nunapeptin, which are similar in structure and genetic organization to the pseudomonas-derived phytotoxins syringomycin and syringopeptin. Regulation of syringomycin and syringopeptin is dependent on the two-component global regulatory system GacS-GacA and the SalA, SyrF, and SyrG transcription factors, which activate syringomycin synthesis in response to plant signal molecules. Previously, we demonstrated that a specific transcription factor, NunF, positively regulates the synthesis of nunamycin and nunapeptin in P. fluorescens In5 and that the nunF gene is upregulated by fungal-associated molecules. This study focused on further unravelling the complex regulation governing CLP synthesis in P. fluorescens In5. Promoter fusions were used to show that the specific activator NunF is dependent on the global regulator of secondary metabolism GacA and is regulated by fungal-associated molecules and low temperatures. In contrast, GacA is stimulated by plant signal molecules leading to the hypothesis that P. fluorescens is a hyphosphere-associated bacterium carrying transcription factor genes that respond to signals indicating the presence of fungi and oomycetes. Based on these findings, we present a model for how synthesis of nunamycin and nunapeptin is regulated by fungal- and oomycete-associated molecules.IMPORTANCE Cyclic lipopeptide (CLP) synthesis gene clusters in pseudomonads display a high degree of synteny, and the structures of the peptides synthesized are very similar. Accordingly, the genomic island encoding the synthesis of syringomycin and syringopeptin in P. syringae pv. syringae closely resembles that of P. fluorescens In5, which contains genes coding for synthesis of the antifungal and anti-oomycete peptides nunamycin and nunapeptin, respectively. However, the regulation of syringomycin and syringopeptin synthesis is different from that of nunamycin and nunapeptin synthesis. While CLP synthesis in the plant pathogen P. syringae pv. syringae is induced by plant signal molecules, such compounds do not significantly influence synthesis of nunamycin and nunapeptin in P. fluorescens In5. Instead, fungal-associated molecules positively regulate antifungal peptide synthesis in P. fluorescens In5, while the synthesis of the global regulator GacA in P. fluorescens In5 is positively regulated by plant signal molecules but not fungal-associated molecules.

Maltose Induced Expression of Cecropin AD by SUMO Technology in Bacillus subtilis WB800N.

Cecropin AD (CAD) is a hybrid peptide composed of 37 amino acids with the characters of strong antibacterial, antitumor properties and no hemolytic activity, which was regarded as a promising antibiotic candidate. Thus, a safe method to produce Cecropin AD is necessary to be found. In the study, Bacillus subtilis WB800N was employed as host strain. The CAD coding sequence fused with the signal peptide of SPsacB, the 6 × His gene and the gene of small ubiquitin-like modifier were cloned into the maltose-inducible vector pGJ148. Under the induction by 6% maltose, the recombinant fusion protein was successfully expressed and detected in culture substrate. An optimized amount (26.4 mg/L) of the recombinant CAD was purified of culture supernatant. After purification and digestion, the recombinant CAD was harvested about 4.5 mg/L with a purity of 93%. Recombinant CAD exhibited similar antimicrobial activity with synthetic CAD. This shows that the production of CAD in maltose-induced Bacillus subtilis expression system is a relatively safe method, which is vital for the application of CAD in animal husbandry production.