Cyclodipeptide oxidase is an enzyme filament.

Modified cyclic dipeptides represent a widespread class of secondary metabolites with diverse pharmacological activities, including antibacterial, antifungal, and antitumor. Here, we report the structural characterization of the Streptomyces noursei enzyme AlbAB, a cyclodipeptide oxidase (CDO) carrying out α,ß-dehydrogenations during the biosynthesis of the antibiotic albonoursin. We show that AlbAB is a megadalton heterooligomeric enzyme filament containing covalently bound flavin mononucleotide cofactors. We highlight that AlbAB filaments consist of alternating dimers of AlbA and AlbB and that enzyme activity is crucially dependent on filament formation. We show that AlbA-AlbB interactions are highly conserved suggesting that other CDO-like enzymes are likely enzyme filaments. As CDOs have been employed in the structural diversification of cyclic dipeptides, our results will be useful for future applications of CDOs in biocatalysis and chemoenzymatic synthesis.

Old role with new feature: T2SS ATPase as a cyclic-di-GMP receptor to regulate antibiotic production.

Cyclic di-GMP (c-di-GMP) is a crucial signaling molecule found extensively in bacteria, involved in the regulation of various physiological and biochemical processes such as biofilm formation, motility, and pathogenicity through binding to downstream receptors. However, the structural dissimilarity of c-di-GMP receptor proteins has hindered the discovery of many such proteins. In this study, we identified LspE, a homologous protein of the type II secretion system (T2SS) ATPase GspE in Lysobacter enzymogenes, as a receptor protein for c-di-GMP. We identified the more conservative c-di-GMP binding amino acid residues as K358 and T359, which differ from the previous reports, indicating that GspE proteins may represent a class of c-di-GMP receptor proteins. Additionally, we found that LspE in L. enzymogenes also possesses a novel role in regulating the production of the antifungal antibiotic HSAF. Further investigations revealed the critical involvement of both ATPase activity and c-di-GMP binding in LspE-mediated regulation of HSAF (Heat-Stable Antifungal Factor) production, with c-di-GMP binding having no impact on LspE's ATPase activity. This suggests that the control of HSAF production by LspE encompasses two distinct processes: c-di-GMP binding and the inherent ATPase activity of LspE. Overall, our study unraveled a new function for the conventional protein GspE of the T2SS as a c-di-GMP receptor protein and shed light on its role in regulating antibiotic production.IMPORTANCEThe c-di-GMP signaling pathway in bacteria is highly intricate. The identification and functional characterization of novel receptor proteins have posed a significant challenge in c-di-GMP research. The type II secretion system (T2SS) is a well-studied secretion system in bacteria. In this study, our findings revealed the ATPase GspE protein of the T2SS as a class of c-di-GMP receptor protein. Notably, we discovered its novel function in regulating the production of antifungal antibiotic HSAF in Lysobacter enzymogenes. Given that GspE may be a conserved c-di-GMP receptor protein, it is worthwhile for researchers to reevaluate its functional roles and mechanisms across diverse bacterial species.

Diverse Combinatorial Biosynthesis Strategies for C-H Functionalization of Anthracyclinones.

Streptomyces spp. are "nature's antibiotic factories" that produce valuable bioactive metabolites, such as the cytotoxic anthracycline polyketides. While the anthracyclines have hundreds of natural and chemically synthesized analogues, much of the chemical diversity stems from enzymatic modifications to the saccharide chains and, to a lesser extent, from alterations to the core scaffold. Previous work has resulted in the generation of a BioBricks synthetic biology toolbox in Streptomyces coelicolor M1152ΔmatAB that could produce aklavinone, 9-epi-aklavinone, auramycinone, and nogalamycinone. In this work, we extended the platform to generate oxidatively modified analogues via two crucial strategies. (i) We swapped the ketoreductase and first-ring cyclase enzymes for the aromatase cyclase from the mithramycin biosynthetic pathway in our polyketide synthase (PKS) cassettes to generate 2-hydroxylated analogues. (ii) Next, we engineered several multioxygenase cassettes to catalyze 11-hydroxylation, 1-hydroxylation, 10-hydroxylation, 10-decarboxylation, and 4-hydroxyl regioisomerization. We also developed improved plasmid vectors and S. coelicolor M1152ΔmatAB expression hosts to produce anthracyclinones. This work sets the stage for the combinatorial biosynthesis of bespoke anthracyclines using recombinant Streptomyces spp. hosts.

Relationship of Cytotoxic and Antimicrobial Effects of Triphenylphosphonium Conjugates with Various Quinone Derivatives.

Quinone derivatives of triphenylphosphonium have proven themselves to be effective geroprotectors and antioxidants that prevent oxidation of cell components with participation of active free radicals - peroxide (RO2·), alkoxy (RO·), and alkyl (R·) radicals, as well as reactive oxygen species (superoxide anion, singlet oxygen). Their most studied representatives are derivatives of plastoquinone (SkQ1) and ubiquinone (MitoQ), which in addition to antioxidant properties also have a strong antibacterial effect. In this study, we investigated antibacterial properties of other quinone derivatives based on decyltriphenylphosphonium (SkQ3, SkQT, and SkQThy). We have shown that they, just like SkQ1, inhibit growth of various Gram-positive bacteria at micromolar concentrations, while being less effective against Gram-negative bacteria, which is associated with recognition of the triphenylphosphonium derivatives by the main multidrug resistance (MDR) pump of Gram-negative bacteria, AcrAB-TolC. Antibacterial action of SkQ1 itself was found to be dependent on the number of bacterial cells. It is important to note that the cytotoxic effect of SkQ1 on mammalian cells was observed at higher concentrations than the antibacterial action, which can be explained by (i) the presence of a large number of membrane organelles, (ii) lower membrane potential, (iii) spatial separation of the processes of energy generation and transport, and (iv) differences in the composition of MDR pumps. Differences in the cytotoxic effects on different types of eukaryotic cells may be associated with the degree of membrane organelle development, energy status of the cell, and level of the MDR pump expression.

Antibacterial insights into alternariol and its derivative alternariol monomethyl ether produced by a marine fungus.

Alternaria alternata FB1 is a marine fungus identified as a candidate for plastic degradation in our previous study. This fungus has been recently shown to produce secondary metabolites with significant antimicrobial activity against various pathogens, including methicillin-resistant Staphylococcus aureus (MRSA) and the notorious aquaculture pathogen Vibrio anguillarum. The antibacterial compounds were purified and identified as alternariol (AOH) and its derivative, alternariol monomethyl ether (AME). We found that AOH and AME primarily inhibited pathogenic bacteria (MRSA or V. anguillarum) by disordering cell division and some other key physiological and biochemical processes. We further demonstrated that AOH could effectively inhibit the unwinding activity of MRSA topoisomerases, which are closely related to cell division and are the potential action target of AOH. The antibacterial activities of AOH and AME were verified by using zebrafish as the in vivo model. Notably, AOH and AME did not significantly affect the viability of normal human liver cells at concentrations that effectively inhibited MRSA or V. anguillarum. Finally, we developed the genetic operation system of A. alternata FB1 and blocked the biosynthesis of AME by knocking out omtI (encoding an O-methyl transferase), which facilitated A. alternata FB1 to only produce AOH. The development of this system in the marine fungus will accelerate the discovery of novel natural products and further bioactivity study.IMPORTANCEMore and more scientific reports indicate that alternariol (AOH) and its derivative alternariol monomethyl ether (AME) exhibit antibacterial activities. However, limited exploration of their detailed antibacterial mechanisms has been performed. In the present study, the antibacterial mechanisms of AOH and AME produced by the marine fungus Alternaria alternata FB1 were disclosed in vitro and in vivo. Given their low toxicity on the normal human liver cell line under the concentrations exhibiting significant antibacterial activity against different pathogens, AOH and AME are proposed to be good candidates for developing promising antibiotics against methicillin-resistant Staphylococcus aureus and Vibrio anguillarum. We also succeeded in blocking the biosynthesis of AME, which facilitated us to easily obtain pure AOH. Moreover, based on our previous results, A. alternata FB1 was shown to enable polyethylene degradation.

Antarctic marine sediment as a source of filamentous fungi-derived antimicrobial and antitumor compounds of pharmaceutical interest.

Antarctica harbors a microbial diversity still poorly explored and of inestimable biotechnological value. Cold-adapted microorganisms can produce a diverse range of metabolites stable at low temperatures, making these compounds industrially interesting for biotechnological use. The present work investigated the biotechnological potential for antimicrobial and antitumor activity of filamentous fungi and bacteria isolated from marine sediment samples collected at Deception Island, Antarctica. A total of 89 microbial isolates were recovered from marine sediments and submitted to an initial screening for L-glutaminase with antitumoral activity and for antimicrobial metabolites. The isolates Pseudogymnoascus sp. FDG01, Pseudogymnoascus sp. FDG02, and Penicillium sp. FAD33 showed potential antiproliferative action against human pancreatic carcinoma cells while showing no toxic effect on non-tumor cells. The microbial extracts from unidentified three bacteria and four filamentous fungi showed antibacterial activity against at least one tested pathogenic bacterial strain. The isolate FDG01 inhibited four bacterial species, while the isolate FDG01 was active against Micrococcus luteus in the minimal inhibitory concentration of 0.015625 µg mL -1. The results pave the way for further optimization of enzyme production and characterization of enzymes and metabolites found and reaffirm Antarctic marine environments as a wealthy source of compounds potentially applicable in the healthcare and pharmaceutical industry.

Perception and protection: The role of Bce-modules in antimicrobial peptide resistance.

Continual synthesis and remodeling of the peptidoglycan layer surrounding Gram-positive cells is essential for their survival. Diverse antimicrobial peptides target the lipid intermediates involved in this process. To sense and counteract assault from antimicrobial peptides, low G + C content gram-positive bacteria (Firmicutes) have evolved membrane protein complexes known as Bce-modules. These complexes consist minimally of an ABC transporter and a two-component system that work in tandem to perceive and confer resistance against antimicrobial peptides. In this mini-review I highlight recent breakthroughs in comprehending the structure and function of these unusual membrane protein complexes, with a particular focus on the BceAB-RS system present in Bacillus subtilis.

Characterization of Chemical Interactions between Clinical Drugs and the Oral Bacterium, Corynebacterium matruchotii, via Bioactivity-HiTES.

In the field of natural product research, the rediscovery of already-known compounds is one of the significant issues hindering new drug development. Recently, an innovative approach called bioactivity-HiTES has been developed to overcome this limitation, and several new bioactive metabolites have been successfully characterized by this method. In this study, we applied bioactivity-HiTES to Corynebacterium matruchotii, the human oral bacterium, with 3120 clinical drugs as potential elicitors. As a result, we identified two cryptic metabolites, methylindole-3-acetate (MIAA) and indole-3-acetic acid (IAA), elicited by imidafenacin, a urinary antispasmodic drug approved by the Japanese Pharmaceuticals and Medical Devices Agency (PMDA). MIAA showed weak antibacterial activity against a pulmonary disease-causing Mycobacterium conceptionense with an IC50 value of 185.7 µM. Unexpectedly, we also found that C. matruchotii metabolized fludarabine phosphate, a USFDA-approved anticancer drug, to 2-fluoroadenine which displayed moderate antibacterial activity against both Bacillus subtilis and Escherichia coli, with IC50 values of 8.9 and 20.1 µM, respectively. Finally, acelarin, a prodrug of the anticancer drug gemcitabine, was found to exhibit unreported antibacterial activity against B. subtilis with an IC50 value of 33.6 µM through the bioactivity-HiTES method as well. These results indicate that bioactivity-HiTES can also be applied to discover biotransformed products in addition to finding cryptic metabolites in microbes.

Effect of Precursors and Their Regulators on the Biosynthesis of Antibiotics in Actinomycetes.

During the life activities of microorganisms, a variety of secondary metabolites are produced, including antimicrobials and antitumor drugs, which are widely used in clinical practice. In addition to exploring new antibiotics, this makes it one of the research priorities of Actinomycetes to effectively increase the yield of antibiotics in production strains by various means. Most antibiotic-producing strains have a variety of functional regulatory factors that regulate their growth, development, and secondary metabolite biosynthesis processes. Through the study of precursor substances in antibiotic biosynthesis, researchers have revealed the precursor biosynthesis process and the mechanism by which precursor synthesis regulators affect the biosynthesis of secondary metabolites, which can be used to obtain engineered strains with high antibiotic production. This paper summarizes the supply of antibiotic biosynthesis precursors and the progress of research on the role of regulators in the process of precursors in biosynthesis. This lays the foundation for the establishment of effective breeding methods to improve antibiotic yields through the manipulation of precursor synthesis genes and related regulators.

Electrospun L-Lysine/Amorphous Calcium Phosphate Loaded Core-Sheath Nanofibers for Managing Oral Biofilm Infections and Promoting Periodontal Tissue Repairment.

Introduction: Periodontitis, a chronic inflammatory disease prevalent worldwide, is primarily treated through GTR for tissue regeneration. The efficacy of GTR, however, remains uncertain due to potential infections and the intricate microenvironment of periodontal tissue. Herein, We developed a novel core-shell structure multifunctional membrane using a dual-drug-loaded coaxial electrospinning technique (Lys/ACP-CNF), contains L-lysine in the outer layer to aid in controlling biofilms after GTR regenerative surgery, and ACP in the inner layer to enhance osteogenic performance for accelerating alveolar bone repair. Methods: The biocompatibility and cell adhesion were evaluated through CCK-8 and fluorescence imaging, respectively. The antibacterial activity was assessed using a plate counting assay. ALP, ARS, and RT-qPCR were used to examine osteogenic differentiation. Additionally, an in vivo experiment was conducted on a rat model with acute periodontal defect and infection. Micro-CT and histological analysis were utilized to analyze the in vivo alveolar bone regeneration. Results: Structural and physicochemical characterization confirmed the successful construction of the core-shell fibrous structure. Additionally, the Lys/ACP-CNF showed strong antibacterial coaggregation effects and induced osteogenic differentiation of PDLSCs in vitro. The in vivo experiment confirmed that Lys/ACP-CNF promotes new bone formation. Conclusion: Lys/ACP-CNF rapidly exhibited excellent antibacterial activity, protected PDLSCs from infection, and was conducive to osteogenesis, demonstrating its potential application for clinical periodontal GTR surgery.

RAPP-containing arrest peptides induce translational stalling by short circuiting the ribosomal peptidyltransferase activity.

Arrest peptides containing RAPP (ArgAlaProPro) motifs have been discovered in both Gram-positive and Gram-negative bacteria, where they are thought to regulate expression of important protein localization machinery components. Here we determine cryo-EM structures of ribosomes stalled on RAPP arrest motifs in both Bacillus subtilis and Escherichia coli. Together with molecular dynamics simulations, our structures reveal that the RAPP motifs allow full accommodation of the A-site tRNA, but prevent the subsequent peptide bond from forming. Our data support a model where the RAP in the P-site interacts and stabilizes a single hydrogen atom on the Pro-tRNA in the A-site, thereby preventing an optimal geometry for the nucleophilic attack required for peptide bond formation to occur. This mechanism to short circuit the ribosomal peptidyltransferase activity is likely to operate for the majority of other RAPP-like arrest peptides found across diverse bacterial phylogenies.

Membrane fluidity homeostasis is required for tobramycin-enhanced biofilm in Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an opportunistic pathogen, which causes chronic infections, especially in cystic fibrosis (CF) patients where it colonizes the lungs via the build-up of biofilms. Tobramycin, an aminoglycoside, is often used to treat P. aeruginosa infections in CF patients. Tobramycin at sub-minimal inhibitory concentrations enhances both biofilm biomass and thickness in vitro; however, the mechanism(s) involved are still unknown. Herein, we show that tobramycin increases the expression and activity of SigX, an extracytoplasmic sigma factor known to be involved in the biosynthesis of membrane lipids and membrane fluidity homeostasis. The biofilm enhancement by tobramycin is not observed in a sigX mutant, and the sigX mutant displays increased membrane stiffness. Remarkably, the addition of polysorbate 80 increases membrane fluidity of sigX-mutant cells in biofilm, restoring the tobramycin-enhanced biofilm formation. Our results suggest the involvement of membrane fluidity homeostasis in biofilm development upon tobramycin exposure.IMPORTANCEPrevious studies have shown that sub-lethal concentrations of tobramycin led to an increase biofilm formation in the case of infections with the opportunistic pathogen Pseudomonas aeruginosa. We show that the mechanism involved in this phenotype relies on the cell envelope stress response, triggered by the extracytoplasmic sigma factor SigX. This phenotype was abolished in a sigX-mutant strain. Remarkably, we show that increasing the membrane fluidity of the mutant strain is sufficient to restore the effect of tobramycin. Altogether, our data suggest the involvement of membrane fluidity homeostasis in biofilm development upon tobramycin exposure.

In-depth analysis of the treatment effect and synergistic mechanism of TanReQing injection on clinical multi-drug resistant Pseudomonas aeruginosa.

Antibiotic resistance is a recognized and concerning public health issue. Gram-negative bacilli, such as Pseudomonas aeruginosa (P. aeruginosa), are notorious for their rapid development of drug resistance, leading to treatment failures. TanReQing injection (TRQ) was chosen to explore its pharmacological mechanisms against clinical multidrug-resistant P. aeruginosa (MDR-PA), given its antibacterial and anti-inflammatory properties. We revealed the expression of proteins and genes in P. aeruginosa after co-culture with TRQ. This study developed an assessment method to evaluate clinical resistance of P. aeruginosa using MALDI-TOF MS identification and Biotyper database searching techniques. Additionally, it combined MIC determination to investigate changes in MDR-PA treated by TRQ. TRQ effectively reduced the MICs of ceftazidime and cefoperazone and enhanced the confidence scores of MDR-PA as identified by mass spectrometry. Using this evaluation method, the fingerprints of standard P. aeruginosa and MDR-PA were compared, and the characteristic peptide sequence (Seq-PA No. 1) associated with flagellum was found. The phenotypic experiments were conducted to confirm the effect of TRQ on the motility and adhesion of P. aeruginosa. A combination of co-immunoprecipitation and proteome analysis was employed, and 16 proteins were significantly differentially expressed and identified as potential candidates for investigating the mechanism of inhibiting resistance in P. aeruginosa treated by TRQ. The candidates were verified by quantitative real-time PCR analysis, and TRQ may affect these core proteins (MexA, MexB, OprM, OprF, OTCase, IDH, and ASL) that influence resistance of P. aeruginosa. The combination of multiple methods helps elucidate the synergistic mechanism of TRQ in overcoming resistance of P. aeruginosa.IMPORTANCEPseudomonas aeruginosa is an opportunistic pathogen closely associated with various life-threatening acute and chronic infections. The presence of antimicrobial resistance and multidrug resistance in P. aeruginosa infections significantly complicates antibiotic treatment. The expression of ß-lactamase, efflux systems such as MexAB-OprM, and outer membrane permeability are considered to have the greatest impact on the sensitivity of P. aeruginosa. The study used a method to assess the clinical resistance of P. aeruginosa using matrix-assisted laser desorption ionization time of flight mass spectrometry identification and Biotyper database search techniques. TanReQing injection (TRQ) effectively reduced the MICs of ceftazidime and cefoperazone in multidrug-resistant P. aeruginosa (MDR-PA) and improved the confidence scores for co-cultured MDR-PA. The study found a characteristic peptide sequence for distinguishing whether P. aeruginosa is resistant. Through co-immunoprecipitation and proteome analysis, we explored the mechanism of TRQ overcoming resistance of P. aeruginosa.

A biomedical perspective of pyocyanin from Pseudomonas aeruginosa: its applications and challenges.

Pyocyanin is a bioactive pigment produced by Pseudomonas aeruginosa. It is an important virulence factor that plays a critical role in P. aeruginosa infections as a redox-active secondary metabolite and a quorum sensing (QS) signaling molecule. Pyocyanin production from chorismic acid requires the involvement of two homologous operons, phz1 and phz2, which are activated by QS regulatory proteins. Pyocyanin inhibits the proliferation of bacterial, fungal, and mammalian cells by inducing oxidative stress due to which it acts as a potent antibacterial, antifungal, and anticancer agent. Its potential role as a neuroprotectant needs further exploration. However, pyocyanin exacerbates the damaging effects of nosocomial infections caused by P. aeruginosa in immunocompromised individuals. Further, cystic fibrosis (CF) patients are highly susceptible to persistent P. aeruginosa infections in the respiratory system. The bacterial cells form colonies and three interconnected QS networks-pqs, las, and rhl-get activated, thus stimulating the cells to produce pyocyanin which exacerbates pulmonary complications. As an opportunistic pathogen, P. aeruginosa produces pyocyanin to impede the recovery of injuries like burn wounds through its anti-proliferative activity. Moreover, pyocyanin plays a vital role in compounding P. aeruginosa infections by promoting biofilm formation. This review begins with a brief description of the characteristics of pyocyanin, its activity, and the different aspects of its production including its biosynthesis, the role of QS, and the effect of environmental factors. It then goes on to explore the potential applications of pyocyanin as a biotherapeutic molecule while also highlighting the biomedical challenges and limitations that it presents.

Breaking Iron Homeostasis: Iron Capturing Nanocomposites for Combating Bacterial Biofilm.

Given the scarcity of novel antibiotics, the eradication of bacterial biofilm infections poses formidable challenges. Upon bacterial infection, the host restricts Fe ions, which are crucial for bacterial growth and maintenance. Having coevolved with the host, bacteria developed adaptive pathways like the hemin-uptake system to avoid iron deficiency. Inspired by this, we propose a novel strategy, termed iron nutritional immunity therapy (INIT), utilizing Ga-CT@P nanocomposites constructed with gallium, copper-doped tetrakis (4-carboxyphenyl) porphyrin (TCPP) metal-organic framework, and polyamine-amine polymer dots, to target bacterial iron intakes and starve them. Owing to the similarity between iron/hemin and gallium/TCPP, gallium-incorporated porphyrin potentially deceives bacteria into uptaking gallium ions and concurrently extracts iron ions from the surrounding bacteria milieu through the porphyrin ring. This strategy orchestrates a "give and take" approach for Ga3+/Fe3+ exchange. Simultaneously, polymer dots can impede bacterial iron metabolism and serve as real-time fluorescent iron-sensing probes to continuously monitor dynamic iron restriction status. INIT based on Ga-CT@P nanocomposites induced long-term iron starvation, which affected iron-sulfur cluster biogenesis and carbohydrate metabolism, ultimately facilitating biofilm eradication and tissue regeneration. Therefore, this study presents an innovative antibacterial strategy from a nutritional perspective that sheds light on refractory bacterial infection treatment and its future clinical application.

GNAT toxin may have a potential role in Pseudomonas aeruginosa persistence: an in vitro and in silico study.

Aims: Persistent cells are primarily responsible for developing antibiotic resistance and the recurrence of Pseudomonas aeruginosa. This study investigated the possible role of GNAT toxin in persistence. Materials & methods: P. aeruginosa was exposed to five MIC concentrations of ciprofloxacin. The expression levels of target genes were assessed. The GNAT/HTH system was bioinformatically studied, and an inhibitory peptide was designed to disrupt this system. Results: Ciprofloxacin can induce bacterial persistence. There was a significant increase in the expression of the GNAT toxin during the persistence state. A structural study of the GNAT/HTH system determined that an inhibitory peptide could be designed to block this system effectively. Conclusion: The GNAT/HTH system shows promise as a novel therapeutic target for combating P. aeruginosa infections.

Antibiotics are used to treat infections caused by bacteria. Over time, some of these infections have become more difficult to treat. This is because the bacteria can slow their growth and tolerate the antibiotic, known as persistence. It is important to find new ways to treat infections caused by persistent bacteria. This study researched a toxin­antitoxin system, called GNAT/HTH, that may play a role in bacterial persistence. This system could be a target for new antibiotics.

S100A12 protein as a porcine health status biomarker when quantified in saliva samples.

The S100A12 protein was validated as a biomarker of health status in porcine saliva samples using a semi-quantitative approach based on Western blotting in four healthy and sixteen diseased animals, and in four animals with severe respiratory disease during three days of antibiotic therapy. Afterwards, a non-competitive sandwich immunoassay was then developed, validated, and used to quantify S100A12 in clinical porcine samples, using 14 healthy and 25 diseased pigs. Finally, the S100A12 concentrations in the saliva of ten pigs with respiratory disease were monitored during antibiotic therapy. Diseased animals showed higher concentrations of S100A12 than healthy animals, and the high concentrations of S100A12 in pigs with respiratory distress were reduced after antimicrobial therapy. The assay developed showed good precision and accuracy, as well as a low limit of detection of 3.19 ng/mL. It was possible to store saliva samples at -20 °C, or even at 4 °C, for two weeks before analysis without losing the validity of the results. The concentrations of S100A12 observed in serum and saliva samples showed a moderately positive association with a correlation coefficient of 0.48. The concentrations of the new validated biomarker S100A12 are highly associated with the novel salivary biomarker of inflammation, adenosine deaminase, and moderately to highly associated with the total oxidant status. The results reported in this study provide a new way of evaluating inflammatory diseases in pigs using saliva samples, which should be further explored for disease prevention and monitoring in the field.

Expression and characterization of the new antimicrobial peptide AP138L-arg26 anti Staphylococcus aureus.

The low activity and yield of antimicrobial peptides (AMPs) are pressing problems. The improvement of activity and yield through modification and heterologous expression, a potential way to solve the problem, is a research hot-pot. In this work, a new plectasin-derived variant L-type AP138 (AP138L-arg26) was constructed for the study of recombination expression and druggablity. As a result, the total protein concentration of AP138L-arg26 was 3.1 mg/mL in Pichia pastoris X-33 supernatant after 5 days of induction expression in a 5-L fermenter. The recombinant peptide AP138L-arg26 has potential antibacterial activity against selected standard and clinical Gram-positive bacteria (G+, minimum inhibitory concentration (MIC) 2-16 µg/mL) and high stability under different conditions (temperature, pH, ion concentration) and 2 × MIC of AP138L-arg26 could rapidly kill Staphylococcus aureus (S. aureus) (> 99.99%) within 1.5 h. It showed a high safety in vivo and in vivo and a long post-antibiotic effect (PAE, 1.91 h) compared with vancomycin (1.2 h). Furthermore, the bactericidal mechanism was revealed from two dimensions related to its disruption of the cell membrane resulting in intracellular potassium leakage (2.5-fold higher than control), and an increase in intracellular adenosine triphosphate (ATP), and reactive oxygen species (ROS), the decrease of lactate dehydrogenase (LDH) and further intervening metabolism in S. aureus. These results indicate that AP138L-arg26 as a new peptide candidate could be used for more in-depth development in the future. KEY POINTS: • The AP138L-arg26 was expressed in the P. pastoris expression system with high yield • The AP138 L-arg26 showed high stability and safety in vitro and in vivo • The AP138L-arg26 killed S. aureus by affecting cell membranes and metabolism.

Marine Streptomyces-Derived Novel Alkaloids Discovered in the Past Decade.

Natural alkaloids originating from actinomycetes and synthetic derivatives have always been among the important suppliers of small-molecule drugs. Among their biological sources, Streptomyces is the highest and most extensively researched genus. Marine-derived Streptomyces strains harbor unconventional metabolic pathways and have been demonstrated to be efficient producers of biologically active alkaloids; more than 60% of these compounds exhibit valuable activity such as antibacterial, antitumor, anti-inflammatory activities. This review comprehensively summarizes novel alkaloids produced by marine Streptomyces discovered in the past decade, focusing on their structural features, biological activity, and pharmacological mechanisms. Future perspectives on the discovery and development of novel alkaloids from marine Streptomyces are also provided.

The enigmatic mode of action of the lantibiotic epilancin 15X.

Epilancin 15X is a lantibiotic that has an antimicrobial activity in the nanomolar concentration range towards Staphylococcus simulans. Such low MICs usually imply that these peptides employ a mechanism of action (MoA) involving high affinity targets. Here we studied this MoA by using epilancin 15X's ability to dissipate the membrane potential of intact S. simulans cells. These membrane depolarization assays showed that treatment of the bacteria by antibiotics known to affect the bacterial cell wall synthesis pathway decreased the membrane depolarization effects of epilancin 15X. Disruption of the Lipid II cycle in intact bacteria using several methods led to a decrease in the activity of epilancin 15X. Antagonism-based experiments on 96-well plate and agar diffusion plate pointed towards a possible interaction between epilancin 15X and Lipid II and this was confirmed by Circular Dichroism (CD) based experiments. However, this interaction did not lead to a detectable effect on either carboxyfluorescein (CF) leakage or proton permeability. All experiments point to the involvement of a phosphodiester-containing target within a polyisoprene-based biosynthesis pathway, yet the exact identity of the target remains obscure so far.