A platform for deep sequence-activity mapping and engineering antimicrobial peptides

Developing potent antimicrobials, and platforms for their study and engineering, is critical as antibiotic resistance grows. A high-throughput method to quantify antimicrobial peptide and protein (AMP) activity across a broad continuum can elucidate sequence-activity landscapes and identify potent mutants. We developed a platform to perform sequence-activity mapping of AMPs via depletion (SAMP-Dep): a bacterial host culture is transformed with an AMP mutant library, induced to express AMPs, grown, and deep sequenced to quantify mutant frequency. The slope of mutant growth rate versus induction level indicates potency. Using SAMP-Dep, we screened 170,000 mutants of oncocin, a proline-rich AMP, for intracellular activity against Escherichia coli. Clonal validation of 36 mutants supported SAMP-Dep sensitivity and accuracy. The efficiency and accuracy of SAMP-Dep enabled mapping the oncocin sequence-activity space with remarkable detail and scale and guided focused, successful synthetic peptide library design, yielding a mutant with two-fold enhancement in both intracellular and extracellular activity.

Influence of Non-Thermal Atmospheric Pressure Plasma Jet on Extracellular Activity of α-Amylase in Aspergillus oryzae

In a previous study, we found that plasma can enhance spore germination and α-amylase secretion in A. oryzae, a beneficial fungus used in fermentation. To confirm this, in the current study, we investigated the effects of plasma on development and α-amylase secretion using an enlarged sample size and a different plasma source: a plasma jet. There was a ~10% (p < 0.01) increase in spore germination upon non-thermal atmospheric pressure plasma jet (NTAPPJ) treatment for 5 min and 10 min, as compared with the control (no plasma treatment). The activity of α-amylase detected in potato dextrose broth (PDB) media during incubation was significantly elevated in plasma-treated samples, with a more obvious increase upon 10 min and 15 min treatments and 24–96 h incubation periods. The levels of the oxidation reduction potential (ORP) and NOX (nitrogen oxide species) were higher in the plasma-treated samples than in the control samples, suggesting that these two variables could serve as standard indicators for enhancing α-amylase activity after plasma treatment. Genome sequencing analysis showed approximately 0.0016–0.0017% variations (changes in 596–655 base pairs out of a total of 37,912,014 base pairs) in the genomic DNA sequence of A. oryzae after plasma treatment. Our results suggest that NATPPJ can enhance the spore germination and extracellular activity of α-amylase, probably by increasing the levels of ORP and NOX to an optimum level.

Inhibiting cough by silencing large pore-expressing airway sensory neurons with a charged sodium channel blocker

Although multiple diseases of the respiratory system cause cough, there are few effective treatments for this common condition. We previously developed a strategy to treat pain and itch via the long-lasting inhibition of nociceptor sensory neurons with QX-314, a cationic sodium channel blocker that selectively enters only into activated nociceptors by permeating through the endogenous TRPV1 and TRPA1 large pore ion channels they express. In this study we design and characterize BW-031, a novel cationic compound with ~6-fold greater potency than QX-314 for inhibiting sodium channels when introduced inside cells and with minimal extracellular activity. We show that inhalation of aerosolized BW-031 effectively inhibits citric acid-induced cough in an allergic inflammation guinea pig cough model. These data support the use of charged sodium channel blockers for the selective inhibition of airway sensory neurons with activated large pore channels as a novel targeted therapy for treating cough.

Assessing the ability of Tigecycline and Meropenem to clear intra-macrophage Klebsiella pneumoniae

The emergence of hypervirulent Klebsiella pneumoniae of serotype K1 and K2 are a major cause of life-threatening, community-acquired infections. Recent studies demonstrated that Kp can persist for long periods of time within the spleen and liver and survive within macrophages. We aimed to explore whether two clinically relevant antimicrobials differed in their capacity to clear within-macrophage Kp. The mouse monocyte cell line J774a were used to model Kp macrophage infection (cultured in RPMI, 10% Fetal-bovine-serum, 37oC, 5%CO2). Cells were harvested and seeded in a 96-well plate at 2x106 cells/mL and incubated overnight. The following day, cells were infected with hypervirulent, K1 Kp (NTUH-K2044) for one hour at an MOI of 10. A 1-hour treatment with gentamicin and polymyxin-Bwas used to kill extracellular Kp. Cells were then washed, and incubated with serial 2-fold dilutions of meropenem and tigecycline for 6 hours. In parallel, a killing assay was performed with antibiotic, and Kp in cell culture media alone to compare the intracellular and extracellular activity of each antibiotic. We show that whilst the majority of the inoculum was resistant to phagocytosis, a small fraction of Kp were able to adhere to macrophages, enter the cell, and persist for up to 6h. Furthermore, we demonstrate that lower concentrations of tigecycline were required to inhibit intracellular Kp, compared with meropenem, which required concentrations in excess of the planktonic MIC to clear the intracellular niche. These data indicate that there is reason to re-examine the antimicrobial treatment regimens for hypervirulent Kp infection with a focus on intracellularly active drugs.

Aktivitas Enzim Ekstraseluler dari Bakteri Endofit Tumbuhan Mangrove Avicennia marina

ABSTRAKBakteri endofit merupakan salah satu sumber penghasil senyawa ekstraseluler yaitu enzim. Enzim dari bakteri endofit lebih menguntungkan dan produksinya lebih cepat. Penelitian ini bertujuan untuk menganalisis kemampuan aktivitas enzim ekstraseluler dari bakteri endofit tumbuhan mangrove Avicennia marina. Metode penelitian yang digunakan yaitu eksploratif eksperimental, melakukan isolasi bakteri dari tumbuhan mangrove A. marina dan selanjutnya dilakukan uji potensi aktivitas enzim ekstraseluler dari isolat bakteri endofit. Hasil isolasi memperoleh tujuh isolat bakteri endofit dari tumbuhan A. marina, isolat endofit tersebut mampu menghasilkan aktivitas enzim ekstraseluler yaitu empat isolat menghasilkan enzim amilase, enam isolat menghasilkan protease, satu isolat menghasilkan selulase dan dua isolat menghasilkan gelatinase.Kata kunci: bakteri endofit; Avicennia marina; amilase; protease; selulase; gelatinase ABSTRACTEndophytic bacteria are one source that can produce extracellular compounds, namely enzymes. Enzymes from endophytic bacteria are more profitable and can produce faster. This study aims to analyze the ability of extracellular enzyme activity from endophytic bacteria in mangrove plants Avicennia marina. This research used experimental explorative method, isolating bacteria from mangrove plant A. marina and then testing the potential of enzyme extracellular activity from endophytic bacteria isolated. Isolating result obtained seven endophytic bacterial isolates from A. marina plants,this endophytic isolates are able to produce extracellular enzyme activity is four isolates can produced amylase enzyme, six isolated can produced protease, one isolated can produced cellulase and two isolated can produce gelatinase. Keywords: Endophytic bacteria; Avicennia marina; amylase, protease, cellulase, gelatinase

In Vitro and Intracellular Activities of Omadacycline against Legionella pneumophila

ABSTRACT Omadacycline is an aminomethylcycline antibiotic with in vitro activity against pathogens causing community-acquired bacterial pneumonia (CABP). This study investigated the activity of omadacycline against Legionella pneumophila strains isolated between 1995 and 2014 from nosocomial or community-acquired respiratory infections. Omadacycline exhibited extracellular activity similar to comparator antibiotics; intracellular penetrance was found by day 3 of omadacycline exposure. These results support the utility of omadacycline as an effective antibiotic for the treatment of CABP caused by L. pneumophila.

The commonness and difference among the Lactobacillus feruloyl esterases expressed in Escherichia coli

Background: Construction of recombinant Escherichia coli strains carrying feruloyl esterase genes for secretory expression offers an attractive way to facilitate enzyme purification and one-step produce ferulic acid from agricultural waste. It was found that the Lactobacillus crispatus feruloyl esterase could be secreted into extracellular environment of E. coli. Whether other Lactobacillus feruloyl esterases share the same secretory characteristic is worth investigation.Results: A total of ten feruloyl esterases derived from nine Lactobacillus species were used to analyze their commonness and compare their difference when heterologously expressed in E. coli BL21 (DE3). Extracellular activity determination showed all these Lactobacillus feruloyl esterases could be secreted out of E. coli cells. However, protein analysis indicated that they could be classified as three types. The first type presented a low secretion level, including feruloyl esterases derived from Lb. acidophilus and Lb. johnsonii. The second type showed a high secretion level, including feruloyl esterases derived from Lb. amylovorus, Lb. crispatus, Lb. gasseri and Lb. helveticus. The third type also behaved a high secretion level but easy degradation, including feruloyl esterases derived from Lb. farciminis, Lb. fermentum and Lb. reuteri. Moreover, these recombinant E. coli strains could directly release ferulic acid from de-starched wheat bran. Conclusions: Recombinant E. coli strains expressing feruloyl esterase of Lb. amylovorus, Lb. crispatus and Lb. helveticus displayed high secretion level and stable extracellular activity. Furthermore, the highest yield of ferulic acid was 140 µg on the basis of 0.1 g de-starched wheat bran after 72 h cultivation of E. coli expressing Lb. amylovorus feruloyl esterase. These results provided a solid basis for the production of feruloyl esterase and ferulic acid.

Recent advances in the development of legumain-selective chemical probes and peptide prodrugs

Legumain, which is also known as vacuolar processing enzyme (VPE) or asparaginyl endopeptidase (AEP), is a cysteine protease that was first discovered and characterized in the leguminous seeds of the moth bean in the early 1990s. Later, this enzyme was also detected in higher organisms, including eukaryotes. This pH-dependent protease displays the highest activity in acidic endolysosomal compartments; however, legumain also displays nuclear, cytosolic and extracellular activity when stabilized by other proteins or intramolecular complexes. Based on the results from over 25 years of research, this protease is involved in multiple cellular events, including protein degradation and antigen presentation. Moreover, when dysregulated, this protease contributes to the progression of several diseases, with cancer being the well-studied example. Research on legumain biology was undoubtedly facilitated by the use of small molecule chemical tools. Therefore, in this review, I present the historical perspectives and most current strategies for the development of small molecule substrates, inhibitors and activity-based probes for legumain. These tools are of paramount importance in elucidating the roles of legumain in multiple biological processes. Finally, as this enzyme appears to be a promising molecular target for anticancer therapies, the development of legumain-activated prodrugs is also described.