Boromycin has Rapid-Onset Antibiotic Activity Against Asexual and Sexual Blood Stages of Plasmodium falciparum

Boromycin is a boron-containing macrolide antibiotic produced by Streptomyces antibioticus with potent activity against certain viruses, Gram-positive bacteria and protozoan parasites. Most antimalarial antibiotics affect plasmodial organelles of prokaryotic origin and have a relatively slow onset of action. They are used for malaria prophylaxis and for the treatment of malaria when combined to a fast-acting drug. Despite the success of artemisinin combination therapies, the current gold standard treatment, new alternatives are constantly needed due to the ability of malaria parasites to become resistant to almost all drugs that are in heavy clinical use. In vitro antiplasmodial activity screens of tetracyclines (omadacycline, sarecycline, methacycline, demeclocycline, lymecycline, meclocycline), macrolides (oleandomycin, boromycin, josamycin, troleandomycin), and control drugs (chloroquine, clindamycin, doxycycline, minocycline, eravacycline) revealed boromycin as highly potent against Plasmodium falciparum and the zoonotic Plasmodium knowlesi. In contrast to tetracyclines, boromycin rapidly killed asexual stages of both Plasmodium species already at low concentrations (~ 1 nM) including multidrug resistant P. falciparum strains (Dd2, K1, 7G8). In addition, boromycin was active against P. falciparum stage V gametocytes at a low nanomolar range (IC50: 8.5 ± 3.6 nM). Assessment of the mode of action excluded the apicoplast as the main target. Although there was an ionophoric activity on potassium channels, the effect was too low to explain the drug´s antiplasmodial activity. Boromycin is a promising antimalarial candidate with activity against multiple life cycle stages of the parasite.

An Update on Molecular Tools for Genetic Engineering of Actinomycetes—The Source of Important Antibiotics and Other Valuable Compounds

The first antibiotic-producing actinomycete (Streptomyces antibioticus) was described by Waksman and Woodruff in 1940. This discovery initiated the “actinomycetes era”, in which several species were identified and demonstrated to be a great source of bioactive compounds. However, the remarkable group of microorganisms and their potential for the production of bioactive agents were only partially exploited. This is caused by the fact that the growth of many actinomycetes cannot be reproduced on artificial media at laboratory conditions. In addition, sequencing, genome mining and bioactivity screening disclosed that numerous biosynthetic gene clusters (BGCs), encoded in actinomycetes genomes are not expressed and thus, the respective potential products remain uncharacterized. Therefore, a lot of effort was put into the development of technologies that facilitate the access to actinomycetes genomes and activation of their biosynthetic pathways. In this review, we mainly focus on molecular tools and methods for genetic engineering of actinomycetes that have emerged in the field in the past five years (2015–2020). In addition, we highlight examples of successful application of the recently developed technologies in genetic engineering of actinomycetes for activation and/or improvement of the biosynthesis of secondary metabolites.

Optimization of culture conditions of Streptomyces antibioticus strain 1083 to improve the antimicrobial activity against Aeromonas hydrophila

Fish is a healthy, high protein and low fat food that encourages the health and growth of people, especially children. However, in fact fish is very sensitive to many diseases which affects the productivity and quality of fish. Therefore, identifying the cause of the diseases and finding preventive measures become an urgent task today. In the previous study, we isolated Streptomyces antibioticus strain 1083 that has the ability to antagonize Aeromonas hydrophila - a pathogenic bacterium in fish. Based on the obtained results, we continue to perform this study to determine optimal conditions for the culture of S. antibioticus strain 1083 in order to produce antimicrobial compounds against A. hydrophila. The production of antagonists by the strain 1083 was optimized by controlling the condition of different inoculations such as media, pH, temperature and incubation period. The results indicated that International Streptomyces Project 2 (ISP2) was the best medium for S. antibioticus strain 1083 to produce the highest antimicrobial activity against A. hydrophila with 32 mm in diameter of inhibited zone. The actinomycete strain 1083 could express the maximum antimicrobial activity when they were incubated in shaker incubator (200rpm) at 40oC with pH8 in 8 days. The ability of the actinomycete strain in antagonism against A. hydrophila was evaluated by adding different culture medium volume of S. antibioticus strain 1083. With adding 10% cultured solution volume of S. antibioticus strain 1083 into the culture medium of A. hydrophila, after 1 day of inoculation the number of pathogenic bacteria cells were completely eliminated.

Nematicidal potential of Streptomyces antibioticus strain M7 against Meloidogyne incognita

Meloidogyne spp. are microscopic, obligatory endoparasites with worldwide distribution which cause severe damage to agricultural crops. The present study revealed the nematicidal activity of Streptomyces antibioticus strain M7 against Meloidogyne incognita. The culture supernatant of the isolate caused 100% J2 mortality after 24 h and inhibited egg hatching (only 3%). In addition, the nematicidal activity of actinomycins V, X2 and D purified from strain M7 was also checked. In vitro studies displayed 97.0–99.0% juvenile mortality and 28.0–44.0% egg hatching after 168 h at 240 µg/ml of actinomycin, with LD50 (lethal dose) values of 28–120 µg/ml. In vivo study further validated the nematicidal activity of strain M7, where nematode infested tomato plants treated with culture supernatant/cells/solvent extract showed reduction in root galls and egg masses per plant by 50.0–62.06% and 53.48–76.74%, respectively, and significantly enhanced the shoot length (54.67–76.39%), root length (36.45–64.88%), shoot fresh weight (111–171.77%), root fresh weight (120–163.33%), shoot dry weight (54.45–145.45%), and root dry weight (100–133.3%) over the nematode infested plants treated with water. Furthermore, tomato plants treated with cells/culture supernatant/extract of strain M7 without nematode infestation also showed significant increase in various plant growth parameters. Thus, the outcome of the study revealed the potential of S. antibioticus strain M7 and actinomycins produced from it to be developed as safe nematicidal agents to control the root knot nematodes, and to increase the crop yield.

Tat-Dependent Heterologous Secretion of Recombinant Tyrosinase by Pseudomonas fluorescens Is Aided by a Translationally Fused Caddie Protein

ABSTRACT Tyrosinase is a monooxygenase that catalyzes both the hydroxylation of p-hydroxyphenyl moieties to o-catechols and the oxidation of o-catechols to o-quinones. Apart from its critical functionality in melanogenesis and the synthesis of various neurotransmitters, this enzyme is also used in a variety of biotechnological applications, most notably mediating covalent cross-linking between polymers containing p-hydroxyphenyl groups, forming a hydrogel. Tyrosinases from the genus Streptomyces are usually secreted as a complex with their caddie protein. In this study, we report an increased secretion efficiency observed when the Streptomyces antibioticus tyrosinase gene melC2 was introduced into Pseudomonas fluorescens along with its caddie protein gene melC1, which has the DNA sequence for the Tat (twin-arginine translocation) signal. IMPORTANCE We observed that the S. antibioticus extracellular tyrosinase secretion level was even higher in its nonnatural translationally conjugated fusion protein form than in the natural complex of two separated polypeptides. The results of this study demonstrate that tyrosinase-expressing P. fluorescens can be a stable source of bacterial tyrosinase through exploiting the secretory machinery of P. fluorescens.

Acyl chain that matters: introducing sn-2 acyl chain preference to a phospholipase D by protein engineering

Phospholipase D (PLD) is an enzyme widely used for enzymatic synthesis of structured phospholipids (PLs) with modified head groups. These PLs are mainly used as food supplements and liposome ingredients. Still, there is a need for an enzyme that discriminates between PLs and lysoPLs, for specific detection of lysoPLs in various specimens and enzymatic synthesis of certain PLs from a mixed substrate. To meet this demand, we aimed at altering sn-2 acyl chain recognition of a PLD, leading to a variant enzyme preferably reacting on lysoPLs, by protein engineering. Based on the crystal structure of Streptomyces antibioticus PLD, W166 was targeted for saturation mutagenesis due to its strong interaction with the sn-2 acyl chain of the PL. Screening result pointed at W166R and W166K PLDs to selectively react on lysophosphatidylcholine (lysoPC), while not on PC. These variants showed a negative correlation between activity and sn-2 chain length of PL substrates. This behavior was not observed in the wild-type (WT)-PLD. Kinetic analysis revealed that the W166R and W166K variants have 7–10 times higher preference to lysoPC compared to the WT-PLD. Additionally, W166R PLD showed detectable activity toward glycero-3-phosphocholine, unlike the WT-PLD. Applicability of the lysoPC-preferring PLD was demonstrated by detection of lysoPC in the mixed PC/lysoPC sample and by the synthesis of cyclic phosphatidic acid. Structure model analyses supported the experimental findings and provided a basis for the structure model-based hypothesis on the observed behavior of the enzymes.