Inhibition of RNase 7 by RNase inhibitor promotes inflammation and Staphylococcus aureus growth: Implications for atopic dermatitis.

BACKGROUND: The antimicrobial ribonuclease RNase 7 is abundantly expressed in the epidermis of lesional skin of atopic dermatitis (AD). Host RNase inhibitor (RI) binds to RNase 7 and blocks its ribonuclease activity. This study aimed to evaluate the impact of RNase 7-RI interactions on AD. METHODS: Cultured human primary keratinocytes, with siRNA-mediated downregulation of RNase 7 and RI, were stimulated with the synthetic RNA polyinosinic-polycytidylic acid (poly I:C). Induction of proinflammatory mediators was analyzed by real-time PCR and ELISA. RI expression in AD non-lesional and lesional skin biopsies and healthy controls was analyzed by real-time PCR and immunostaining. RI protein release in vivo on the AD skin surface was determined by western blot. Antimicrobial and ribonuclease assays were used to investigate the functional role of RI. RESULTS: RNase 7 inhibited the RNA-induced expression of proinflammatory mediators in keratinocytes. Accordingly, downregulation of RNase 7 in keratinocytes enhanced RNA-mediated induction of proinflammatory mediators, whereas downregulation of RI had the opposite effect. RI was released by damaged keratinocytes and epidermis. In vivo expression and release of RI on the skin surface were enhanced in lesional AD skin. Rinsing solution from the surface of lesional AD skin blocked the ribonuclease activity of RNase 7. The anti-Staphylococcus aureus activity of RNase 7 was abrogated by RI. CONCLUSIONS: Our data suggest a novel role of RI as a trigger factor of inflammation in AD by blocking the ribonuclease and antimicrobial activity of RNase 7, thereby enhancing RNA-mediated inflammation and S. aureus growth.

Separation and purification of antimicrobial substances from Paenibacillus polymyxa KH-19 and analysis of its physicochemical characterization.

Soft rot is one of the top ten most dangerous plant pathogens in agricultural production, storage, and transport, and the use of microorganisms and their metabolites to control soft rot is a current research hotspot. In this study, we identified the antimicrobial substance in the metabolite of Paenibacillus polymyxa KH-19, and determined that the antimicrobial substance of this strain was an active protein. The protein was completely precipitated at 40-60% ammonium sulphate saturation and showed good inhibitory effects against seven pathogenic bacteria including Pectobacterium carotovorum BC2 and seven pathogenic fungi including Pyricularia oryzae. The MIC of the protein was 51.563 µg/mL, temperature acid-base UV and light stability insensitive to protease, with high-temperature resistance. The antimicrobial protein was isolated and purified by DEAE-anion exchange column and Sephadex G-75 gel filtration chromatography, and the LC-MS/MS assay identified the protein as lysophosphatidyl esterase with a molecular weight of 25.255 kDa. The purified antimicrobial protein increased the inhibitory effect against P. carotovorum BC2, with the diameter of the circle of inhibition being 26.50 ± 0.915 mm. Bioinformatics analysis showed that the protein has the molecular formula of C1117H1732N316O338S5, encodes 224 amino acids, has an aliphatic index of 88.39, and belongs to the category of hydrophilic unstable proteins. The present study is the first report of an active protein with extreme thermoplastic and resistance to P. carotovorum BC2, which provides a reference for the preparation and application of the antimicrobial substances of P. polymyxa KH-19, as well as a theoretical basis for the study of the function of lysophosphodiesterase protein and its use as a microbial preparation.

The dynamics of colicin E9 release from Escherichia coli in native conditions.

Colicin (Col) plasmid contains colicin encoding genes arranged in an operon controlled by an SOS inducible promoter. Therefore, any external stresses to the host cell can induce the expression of the downstream genes in the Col operon, including a lysis gene. The lysis protein is involved in the extracellular release of colicin through lysis of the producer cells, which causes a decline in culture turbidity. However, it is not yet known that E. coli cells with the native pColE9-J plasmid hold the same level of cell death at the population level following a set of induced conditions. In this study, using a mitomycin C sensitivity assay along with a live dead staining method of detection, we showed that the native pColE9-J plasmid, which unusually carries an extended Col operon (ColE9) containing two lysis genes, did not confer a rapid decline in the culture turbidity following induction with mitomycin C. Interestingly a subset of the cells suffered perturbation of their outer membrane, which was not observed from single lysis mutant (∆celE or ∆celI) cells. This observed heterogeneity in the colicin E9 release leading to differential outer membrane perforation may bring a competitive advantage to these cells in a mixed population.

Characterization of Bioactive Compounds and Novel Proteins Derived from Promising Source Citrullus colocynthis along with In-Vitro and In-Vivo Activities.

Herbal products are preferable to synthetic medicines, and the use of traditional medicines is increasing day-by-day. The current study was designed to evaluate the potentials of bioactive compounds from Citrullus colocynthis by performing FTIR, HPLC, and GC-MS analyses, which explore the good concentration of the secondary metabolites, such as gallic acid (74.854 ppm), vanillic acid (122.616 ppm), and ferulic acid (101.045 ppm) with considerable bioactivities. Antimicrobial protein was estimated by performing SDS-PAGE, ranging from 15 to 70 kDa in all protein fractions. The current study also checked the cytotoxicity of the bioactive compounds in the active fraction of C. colocynthis, and to perform this activity, the groups of rats were arranged with 16 rats randomly divided into four groups (three experimental and one control) by administering various dosage of methanolic fractions in dose-dependent manner. Histopathology was conducted on the livers of the rats after 15 days of sacrifice under deep anesthesia. In liver cell slides examined at the maximum dose of 600 mg/kg, minimal morphological changes, such as slight ballooning, nuclear variation, vacuolar degeneration, and hydropic degeneration, were observed. Furthermore, the in silico analysis identified bioactive compounds as potential drug candidates.

Antimicrobial protein REG3A and signaling networks are predictive of stroke outcomes.

Regenerating Family Member 3 Alpha (REG3A) is a multifunctional protein with antimicrobial activity, and primarily secreted by the intestine and pancreas. Studies have shown an increased expression of REG3A in systemic inflammatory responses to acute injury and infection, but studies investigating REG3A during the pathogenesis of ischemic stroke are limited. The aims of this study were to examine the associations between arterial expression of REG3A and other arterial inflammatory proteins implicated in stroke pathogenesis, as well as associations between REG3A and markers of poor outcome for ischemic stroke. The University of Kentucky Blood and Clot Thrombectomy Registry and Collaboration (BACTRAC) protocol (clinicaltrials.gov NCT03153683) utilizes thrombectomy to isolate intracranial arterial blood (i.e. distal to thrombus) and systemic arterial blood (i.e. carotid). Samples were analyzed by Olink Proteomics for N = 42 subjects. Statistical analyses of plasma proteins included 2-sample t-tests, spearman and biserial correlations, and robust regression models to elucidate network signaling and association to clinical outcomes. Results indicated that levels of systemic REG3A were positively correlated with inflammatory proteins interleukin IL6 (R = 0.344, p = 0.030) and IL17C (R = 0.468, p = 0.002). 2-sided t- tests examining differences of systemic REG3A within quartiles of NIHSS admission score depicted significant differences between quartiles. Those with NIHSS scores corresponding to moderate and moderate-severe neurofunctional deficits had significantly higher levels of systemic REG3A compared to those with NIHSS scores corresponding to mild and mild-moderate neurofunctional deficits (p = 0.016). STRING analyses of proteins in each robust regression model demonstrated substantial networking between REG3A and other systemic proteins highly relevant to ischemic stroke. The present study provides novel data on systemic REG3A in the context of ischemic stroke. These results demonstrate the influential role of REG3A regarding surrogate functional and radiographic outcomes of stroke severity. Additionally, they provide novel insight into the role of REG3A and related proteins during the complex neuroinflammatory process of ischemic stroke. These data provide a foundation for future studies to investigate REG3A and related networking proteins as potential biomarkers with prognostic potential, as well as potential therapeutic targets.

Novel antimicrobial activity of protein produced by Streptomyces lividans TK24 against the phytopathogen Clavibacter michiganensis.

Antimicrobial proteins and peptides are an alternative to current antibiotics. Here, we report an antimicrobial activity in a low-molecular-weight protein secreted naturally by Streptomyces lividans TK24 when glucose or glycerol were used as carbon sources. The antimicrobial activity was demonstrated against Bacillus subtilis, Bacillus cereus, Kokuria rhizophila, Clostridium sporogenes and Clavibacter michiganensis, causal pathogen of tomato bacterial canker; one of the most destructive bacterial diseases of this crop. The protein fraction with antimicrobial activity was identified and quantified by LC-MS/MS. From a total of 155 proteins, 11 were found to be within the range of 11.3-13.9 kDa of which four proteins were selected by functional analysis as possibly responsible for the antimicrobial activity. Protein fractionation, correlation analysis between antimicrobial activity and abundance of selected proteins, as well as transcriptional expression analysis, indicate that 50S ribosomal protein L19 is the main candidate responsible for antimicrobial activity.

Analysis and Evaluation of the Flagellin Activity of Bacillus amyloliquefaciens Ba168 Antimicrobial Proteins against Penicillium expansum.

Blue mold caused by Penicillium expansum is one of the most common apple diseases, and it is becoming a serious threat in apple production. The strain Bacillus amyloliquefaciens Ba168 showed high levels of antimicrobial activity in our previous study. To analyze the antimicrobial protein of Ba168, a high-resolution LC-MS/MS proteomic analysis was performed. A total of 1155 proteins were identified from 5233 unique peptides. A total of 16 potential antimicrobial-activity-related proteins were identified; 10 of these proteins have direct antimicrobial effects, while 6 of these proteins are associated with the formation of antimicrobial substances. Then, an antifungal protein of Ba168 was isolated and purified by the sequential chromatography of DEAE Bio-sep FF anion exchange and Sephadex G-75. The single protein, named BP8-2, showed antifungal activity towards Penicillium expansum. The peptide mass fingerprinting of the protein band of BP8-2 had a high similarity with the amino acid sequences of flagellin protein. The results showed that BP8-2 significantly inhibited the growth of P. expansum and slowed the spread of apple blue mold. The results indicated that flagellin is one of the important antimicrobial substances from Ba168.

Identification of Multi-Potent Protein Subtilisin A from halophilic bacterium Bacillus firmus VE2.

Screening of halophiles with antimicrobial activity in saltpan soil samples from Nagapattinam district, Tamil Nadu, revealed isolate VE-2 as the most potent, identified as Bacillus firmus strain VE-2 through 16s rRNA gene sequencing. It had an optimum growth condition (OD 3.1) and antimicrobial protein (AMP) production (450 µg/mL) at 37 °C, pH 8, 25% NaCl, and 36 h incubation. SDS-PAGE analysis of the purified AMP showed the molecular weight of 36 kDa. HPLC analysis of the purified AMP showed different amino acids, such as asparagines, alanine, lysine, proline, threonine, glycine, cysteine, serine, aspartic acid leucine, and valine. Further characterization and identification using FT-IR, 2D-PAGE, MALDI-TOF, and in-silico analysis showed that the isolated AMP had the highest similarity to Subtilisin-A. It showed antibacterial activity against clinical bacterial pathogens like S. aureus, S. pyogenes, C. diphtheria, E. coli, and P. aeruginosa with the minimum inhibitory concentration (MIC) and the minimum bactericidal concentration of 2.5 µg/mL and 20 µg/mL and also against various fungal pathogens such as A. niger, A. flavus, C. albicans, C. tropicalis and C. parapsilosis with the MIC and minimum fungicidal concentrations of 1.25-80 µg/mL. The purified AMP had excellent antioxidant potential, showed a scavenging effect against DPPH and Nitric oxide radicals, and displayed anticancer activity against HeLa cell lines with the IC50 values 53 µg/mL. Hence, the purified bioactive antimicrobial peptides (AMP) could also be used in anticancer therapies.

Purification and stability analysis of antimicrobial proteins from Varuna litterata.

Ten marine species, including different crabs, bivalve molluscs, and fish intestines were selected to screen the natural antimicrobial protein or peptide as they are enriched with various microorganisms. The crude extract from Varuna litterata, a marine crab which is used as a raw material in the preparation of pickled crabs in Chaoshan area of China, was proved to have a potent bacteriostatic effect against gram-negative bacterium (Escherichia coli) and gram-positive bacterium(Staphylococcus aureus) compared with other marine species. The crude proteins of Varunalitterata were salted-out for preliminary purification and further purified by gel filtration (Sephadex G-150) or anion exchange (DEAE-cellulose 52) chromatographic column. An increase in the antimicrobial activity was noted with the increase in the purity level of the protein. A relatively pure protein was eventually obtained, which was determined to be belonging to the hemocyanin family based on the mass spectrometric data analysis. The purified proteins solution (1 mg/ml) from Varuna litterata exhibited similar antimicrobial activity to that of gentamycin sulfate (0.2 mg/ml), which were relatively stable in a certain pH or temperature range. A structure-activity relationship of the purified hemocyanin was determined based on the interaction of hemocyanin and different chromatographic medium, which revealed that the integrated hexamers played a remarkable role in its bacteriostatic activity. Moreover, the phenoloxidase activity of hemocyanin from Varuna litterata was found as the underlying cause of its antimicrobial potential.

Isolation of a broad spectrum antimicrobial producing thermophilic Bacillus and characterization of its antimicrobial protein.

The hot spring water of Atri in India was believed to have disease curing property. An antibacterial producing organism was isolated and identified as Bacillus paralicheniformis by morphology, microscopy, and 16S-rRNA. Its secretion inhibited bacteria, yeast, and fungus in well-diffusion-method. The secreted antimicrobial was a 16.74 kDa protein homologous of chicken-lysozyme-C. The novel lysozyme's activities were recorded under different parameters. It was active from pH 5-9 and endured up to 60 °C for 120 min. Complete cell wall lysis of S. flexneri and P. aeruginosa was observed under a microscope at 4500× with a minimum inhibitory concentration of 7.8 µg/ml, while others required a higher dose, i.e., 13 µg/ml, and 20 µg/ml for E.coli and S. typhimurium, respectively. The discovered lysozyme has the extraordinary potential to lyse Gram-positive bacteria, yeast, fungus, and more efficiently lyse chick-lysozyme-C resistant lipopolysaccharide rich Gram-negative bacteria's outer cell wall.

Toposome: Source of antimicrobial molecules in the gonads of the sea urchin Lytechinus variegatus (Lamarck, 1816).

Sea urchins live in a challenging environment that requires rapid and efficient responses against pathogens and invaders. This response may be also important in reproductive processes once males and females release their gametes into water. In addition, the gonads are organs with dual function: reproductive organ and nutrient reserve, therefore it needs efficient protective mechanisms to preserve the nutrients as well as the reproductive cells. The aim of this study was to evaluate the presence and characterize antimicrobial molecules in the male and female gonads of the sea urchin Lytechinus variegatus. Through HPLC purification, antimicrobial activity test and mass spectrometry several antimicrobial molecules were found in the gonads of both gender. Computational in silico analyses showed that they are fragments of a glycoprotein called toposome, also known as major yolk protein (MYP) which is one of the major proteins found in the gonads. Although different functions have been reported for this protein, this is the first description of a direct antimicrobial activity in Lytechinus variegatus. The results indicate that when undergoing proteolysis the toposome generates different fragments with antimicrobial activity which may indicate the importance of a rapid defense response strategy against invading microorganisms in the gonads used by both males and females sea urchins.

An LCI-like protein APC2 protects ginseng root from Fusarium solani infection.

AIMS: We aimed to purify an antimicrobial protein from Bacillus amyloliquefaciens FS6 culture supernatant, verify its antimicrobial activity against Fusarium solani and evaluate its biocontrol potential for ginseng root rot. METHODS AND RESULTS: The antimicrobial protein was purified from FS6 culture supernatant using ammonium sulphate precipitation, anion exchange and gel chromatography. Based on mass spectrometry results, the purified protein was identified as an antimicrobial protein of the LCI family and was designated APC2 . The APC2 recombinant protein expressed in Escherichia coli (BL21) significantly inhibited F. solani and decreased the infection and spread of F. solani in ginseng root. An overexpressing APC2 strain FS6-APC2 was constructed and shown to have enhanced antimicrobial activity compared to the wild-type strain FS6. CONCLUSIONS: The APC2 protein shows strong antimicrobial activity against F. solani, reduces the incidence and severity of ginseng root rot caused by F. solani and exhibits a great biocontrol potential. SIGNIFICANCE AND IMPACT OF THE STUDY: This study reports the inhibitory activity of APC2 protein (LCI family) against F. solani and its protective efficacy on ginseng root rot. These findings provide a scientific basis for future research on the biocontrol mechanism, as well as the development and application of FS6.

Identification of a novel proline-rich antimicrobial protein from the hemolymph of Antheraea mylitta.

Antimicrobial proteins (AMPs) are small, cationic proteins that exhibit activity against bacteria, viruses, parasites, fungi as well as boost host-specific innate immune responses. Insects produce these AMPs in the fat body and hemocytes, and release them into the hemolymph upon microbial infection. Hemolymph was collected from the bacterially immunized fifth instar larvae of tasar silkworm, Antheraea mylitta, and an AMP was purified by organic solvent extraction followed by size exclusion and reverse-phase high-pressure liquid chromatography. The purity of AMP was confirmed by thin-layer chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. The molecular mass was determined by matrix-assisted laser desorption ionization-time of flight mass spectrometry as 14 kDa, and hence designated as AmAMP14. Peptide mass fingerprinting of trypsin-digested AmAMP14 followed by de novo sequencing of one peptide fragment by tandem mass spectrometry analysis revealed the amino acid sequences as CTSPKQCLPPCK. No homology was found in the database search and indicates it as a novel AMP. The minimum inhibitory concentration of the purified AmAMP14 was determined against Escherichia coli, Staphylococcus aureus, and Candida albicans as 30, 60, and 30 µg/ml, respectively. Electron microscopic examination of the AmAMP14-treated cells revealed membrane damage and release of cytoplasmic contents. All these results suggest the production of a novel 14 kDa AMP in the hemolymph of A. mylitta to provide defense against microbial infection.

Antimicrobial properties of L-amino acid oxidase: biochemical features and biomedical applications.

Mucus layer that covers the body surface of various animal functions as a defense barrier against microbes, environmental xenobiotics, and predators. Previous studies have reported that L-amino acid oxidase (LAAO), present in several animal fluids, has potent properties against pathogenic bacteria, viruses, and parasites. LAAO catalyzes the oxidative deamination of specific L-amino acids with the generation of hydrogen peroxide and L-amino acid metabolites. Further, the generated hydrogen peroxide is involved in oxidation (direct effect) while the metabolites activate immune responses (indirect effect). Therefore, LAAO exhibits two different mechanisms of bioactivation. Previously, we described the selective, specific, and local oxidative and potent antibacterial actions of various LAAOs as potential therapeutic strategies. In this review, we focus on their biochemical features, enzymatic regulations, and biomedical applications with a view of describing their probable role as biochemical agents and biomarkers for microbial infections, cancer, and autoimmune-mediated diseases. We consider that LAAOs hold implications in biomedicine owing to their antimicrobial activity wherein they can be used in treatment of infectious diseases and as diagnostic biomarkers in the above-mentioned diseased conditions. KEY POINTS: •Focus on biochemical features, enzymatic regulation, and biomedical applications of LAAOs. •Mechanisms of antimicrobial activity, inflammatory regulation, and immune responses of LAAOs. •Potential biomedical application as an antimicrobial and anti-infection agent, and disease biomarker.

High cell-density fermentation, expression and purification of bacteriophage lysin TSPphg, a thermostable antimicrobial protein from extremophilic Thermus bacteriophage TSP4.

Recently, high cell-density (HCD) cultivation has become an important tool for production of many microbial products. However, to the best of our knowledge, no study regarding HCD fermentation, overproduction and purification of thermostable bacteriophage lysin has been reported. Here, by employing a glucose-limited fed-batch strategy, we performed high density fermentation of the host Escherichia coli BL21(DE3) cells, compared the efficiency of high pressure homogenization, ultrasonication and thermolysis in bacterial cell disruption after HCD cultivation, and purified TSPphg, a thermostable lysin derived from extremophilic bacteriophage TSP4. On the 20-L scale, the overproduction level of TSPphg was up to 67.8 ± 0.7%. In total, we obtained a broth titer of 3322.8 ± 26 mg/L TSPphg with a purity of 95.5 ± 0.7% from a bacterial cell mass of 86.3 ± 4.9 g/L after 26 h of fermentation. The overall productivity of TSPphg was 127.8 ± 1 mg/L/h. Additionally, the antimicrobial activity of purified TSPphg against both Gram-negative (Escherichia coli O157) and Gram-positive (Staphylococcus aureus) pathogenic bacteria was further confirmed by scanning electron microscope analysis. Summarily, for the first time, we have established a relatively stable and efficient HCD cultivation and purification process for recovery of thermostable lysins from extremophilic Thermus bacteriophages. Our results provide insights into the strategies for time-saving and cost-effective production of antimicrobial proteins to replace or supplement antibiotics in the current age of mounting antibiotic resistance.

Resistin-like molecule beta, a colonic epithelial protein, exhibits antimicrobial activity against Staphylococcus aureus including methicillin-resistant strains.

PURPOSE: Resistin-like molecule beta (RELMß) is a small cysteine-rich protein secreted by colonic epithelial cells. RELMß mRNA and protein expressions are dramatically induced by bacterial exposure in germ-free mice. We hypothesized that RELMß has antimicrobial activity. METHODS: The antimicrobial activity of RELMß was screened by an agar spot test and confirmed by a liquid broth test. The amount of RELMß in human stools was semi-quantified by Western blot analysis. The induction of RELMß mRNA and protein expression by bacteria was measured by quantitative RT-PCR using LS174T cells. Electron microscopic immunohistochemistry was performed using polyclonal anti-RELMß antibody. RESULTS: RELMß showed antimicrobial activity against S. aureus and all MRSAs examined in a dose- and pH-dependent fashion. Western blot study showed that the amount of RELMß in healthy human stools was comparable to that exhibiting antimicrobial activity in vitro. Both RELMß mRNA and protein expression were induced by heat-inactivated S. aureus, but not by E. coli in LS174T cells. Electron microscopic immunohistochemistry showed that RELMß bound to the cell surface of S. aureus, followed by destruction of the bacterial cytoplasm. CONCLUSIONS: RELMß is a colonic antimicrobial protein and its antibacterial activity is species selective. Because RELMß is abundant in healthy human stool, RELMß may modulate gut flora.

Almond allergens: update and perspective on identification and characterization.

Almond (Prunus dulcis) is not only widely used as a human food as a result of its flavor, nutrients, and health benefits, but it is also one of the most likely tree nuts to trigger allergies. Almond allergens, however, have not been studied as extensively as those of peanuts and other selected tree nuts. This review provides an update of the molecular properties of almond allergens to clarify some confusion about the identities of almond allergens and our perspective on characterizing putative almond allergens. At present, the following almond allergens have been designated by the World Health Organization/International Union of Immunological Societies Allergen Nomenclature Sub-Committee: Pru du 3 (a non-specific lipid transfer protein 1, nsLTP1), Pru du 4 (a profilin), Pru du 5 (60S acidic ribosomal protein 2), Pru du 6 (an 11S legumin known as prunin) and Pru du 8 (an antimicrobial protein with cC3C repeats). Besides, almond vicilin and almond γ-conglutin have been identified as food allergens, although further characterization of these allergens is still of interest. In addition, almond 2S albumin was reported as a food allergen as a result of the misidentification of Pru du 8. Two more almond proteins have been called allergens based on their sequence homology with known food allergens and their 'membership' in relevant protein families that contain allergens in many species. These include the pathogenesis related-10 protein (referred to as Pru du 1) and the thaumatin-like protein (referred to as Pru du 2). Almonds thus have five known food allergens and five more likely ones that need to be investigated further. Published 2020. This article is a U.S. Government work and is in the public domain in the USA.

The antimicrobial activity of protein elicitor AMEP412 against Streptomyces scabiei.

In this paper, we report the antimicrobial activity of AMEP412 (a protein elicitor from Bacillus subtilis) against Streptomyces scabiei, which is the potato common scab pathogen. The purified protein samples showed an obvious inhibition zone on an S. scabiei agar plate, and the minimum inhibition concentration detected was 50 µg mL-1. The fluorescence localization assay revealed that AMEP412 could bind to aerial mycelia and spores. The stability test showed that AMEP412 was stable at 60 °C for 30 min and in pH values from 5.0 to 10.0. Its antimicrobial activity was not sensitive to metal cations. However, its activity declined by 23% when treated with Proteinase K, and was completely abrogated with Tween 80 treatment. Three antimicrobial peptides (GS21, GY20 and GY23) were identified from AMEP412, which further verified its antimicrobial activity. This research reveals the antimicrobial function of AMEP412, which not only enriches the function of the protein elicitor, but also provides a candidate for the biocontrol of potato common scab.

Validation and Stabilization of a Prophage Lysin of Clostridium perfringens by Using Yeast Surface Display and Coevolutionary Models.

Bacteriophage lysins are compelling antimicrobial proteins whose biotechnological utility and evolvability would be aided by elevated stability. Lysin catalytic domains, which evolved as modular entities distinct from cell wall binding domains, can be classified into one of several families with highly conserved structure and function, many of which contain thousands of annotated homologous sequences. Motivated by the quality of these evolutionary data, the performance of generative protein models incorporating coevolutionary information was analyzed to predict the stability of variants in a collection of 9,749 multimutants across 10 libraries diversified at different regions of a putative lysin from a prophage region of a Clostridium perfringens genome. Protein stability was assessed via a yeast surface display assay with accompanying high-throughput sequencing. Statistical fitness of mutant sequences, derived from second-order Potts models inferred with different levels of sequence homolog information, was predictive of experimental stability with areas under the curve (AUCs) ranging from 0.78 to 0.85. To extract an experimentally derived model of stability, a logistic model with site-wise score contributions was regressed on the collection of multimutants. This achieved a cross-validated classification performance of 0.95. Using this experimentally derived model, 5 designs incorporating 5 or 6 mutations from multiple libraries were constructed. All designs retained enzymatic activity, with 4 of 5 increasing the melting temperature and with the highest-performing design achieving an improvement of +4°C.IMPORTANCE Bacteriophage lysins exhibit high specificity and activity toward host bacteria with which the phage coevolved. These properties of lysins make them attractive for use as antimicrobials. Although there has been significant effort to develop platforms for rapid lysin engineering, there have been numerous shortcomings when pursuing the ultrahigh throughput necessary for the discovery of rare combinations of mutations to improve performance. In addition to validation of a putative lysin and stabilization thereof, the experimental and computational methods presented here offer a new avenue for improving protein stability and are easily scalable to analysis of tens of millions of mutations in single experiments.

Functional evaluation of a monotreme-specific antimicrobial protein, EchAMP, against experimentally induced mastitis in transgenic mice.

EchAMP, the tenth most abundant transcript expressed in the mammary gland of echidna, has in vitro broad-spectrum antibacterial effects. However, the effects of EchAMP on mastitis, a condition where inflammation is triggered following mammary gland infection, has not been investigated. To investigate the impact of EchAMP against mastitis, EchAMP transgenic mice were generated. In antibacterial assays, the whey fractions of milk from transgenic mice significantly reduced growth of Staphylococcus aureus, Bacillus subtilis, Escherichia coli and Pseudomonas aeruginosa compared with whey fractions from wildtype mice. Furthermore, a mastitis model created by infecting mammary gland with these four bacterial strains displayed a significant reduction in bacterial load in transgenic mice injected with S. aureus and B. subtilis. On further confirmation, histomorphologic analysis showed absence of necrosis and cell infiltration in the mammary glands of transgenic mice. To understand the role of EchAMP against inflammation, we employed an LPS-injected mastitis mouse model. LPS is known to induce phopshorylation of NF-κB and MAPK pathways, which in turn activate downstream proinflammatory signaling mediators, to promote inflammation. In LPS-treated EchAMP transgenic mice, phosphorylation levels of NF-κB, p38 and ERK1/2 were significantly downregulated. Furthermore, in mammary gland of transgenic mice, there was a significant downregulation of mRNA levels of proinflammatory cytokines, namely TNF-α, IL-6 and IL-1ß. Taken together, these data suggest that EchAMP has an antiinflammatory response and is effective against S. aureus and B. subtilis. We suggest that EchAMP may be a potential prophylactic protein against mastitis in dairy animals by expressing this gene in their mammary gland.