Metagenomic analysis of hot spring soil for mining a novel thermostable enzybiotic.

The misuse and overuse of antibiotics have contributed to a rapid emergence of antibiotic-resistant bacterial pathogens. This global health threat underlines the urgent need for innovative and novel antimicrobials. Endolysins derived from bacteriophages or prophages constitute promising new antimicrobials (so-called enzybiotics), exhibiting the ability to break down bacterial peptidoglycan (PG). In the present work, metagenomic analysis of soil samples, collected from thermal springs, allowed the identification of a prophage-derived endolysin that belongs to the N-acetylmuramoyl-L-alanine amidase type 2 (NALAA-2) family and possesses a LysM (lysin motif) region as a cell wall binding domain (CWBD). The enzyme (Ami1) was cloned and expressed in Escherichia coli, and its bactericidal and lytic activity was characterized. The results indicate that Ami1 exhibits strong bactericidal and antimicrobial activity against a broad range of bacterial pathogens, as well as against isolated peptidoglycan (PG). Among the examined bacterial pathogens, Ami1 showed highest bactericidal activity against Staphylococcus aureus sand Staphylococcus epidermidis cells. Thermostability analysis revealed a melting temperature of 64.2 ± 0.6 °C. Overall, these findings support the potential that Ami1, as a broad spectrum antimicrobial agent, could be further assessed as enzybiotic for the effective treatment of bacterial infections. KEY POINTS: • Metagenomic analysis allowed the identification of a novel prophage endolysin • The endolysin belongs to type 2 amidase family with lysin motif region • The endolysin displays high thermostability and broad bactericidal spectrum.

Ligandability Assessment of Human Glutathione Transferase M1-1 Using Pesticides as Chemical Probes.

Glutathione transferases (GSTs; EC 2.5.1.18) form a group of multifunctional enzymes that are involved in phase II of the cellular detoxification mechanism and are associated with increased susceptibility to cancer development and resistance to anticancer drugs. The present study aims to evaluate the ligandability of the human GSTM1-1 isoenzyme (hGSTM1-1) using a broad range of structurally diverse pesticides as probes. The results revealed that hGSTM1-1, compared to other classes of GSTs, displays limited ligandability and ligand-binding promiscuity, as revealed by kinetic inhibition studies. Among all tested pesticides, the carbamate insecticide pirimicarb was identified as the strongest inhibitor towards hGSTM1-1. Kinetic inhibition analysis showed that pirimicarb behaved as a mixed-type inhibitor toward glutathione (GSH) and 1-chloro-2,4-dinitrobenzene (CDNB). To shine a light on the restricted hGSTM1-1 ligand-binding promiscuity, the ligand-free crystal structure of hGSTM1-1 was determined by X-ray crystallography at 1.59 Å-resolution. Comparative analysis of ligand-free structure with the available ligand-bound structures allowed for the study of the enzyme's plasticity and the induced-fit mechanism operated by hGSTM1-1. The results revealed important structural features of the H-site that contribute to xenobiotic-ligand binding and specificity. It was concluded that hGSTM1-1 interacts preferentially with one-ring aromatic compounds that bind at a discrete site which partially overlaps with the xenobiotic substrate binding site (H-site). The results of the study form a basis for the rational design of new drugs targeting hGSTM1-1.

Metaviromics analysis of marine biofilm reveals a glycoside hydrolase endolysin with high specificity towards Acinetobacter baumannii.

Multidrug-resistant (MDR) bacteria are a growing threat to the public health. Among them, the Gram-negative Acinetobacter baumannii is considered today as the most dangerous MDR pathogen. Phage-derived endolysins are peptidoglycan (PG) hydrolytic enzymes that can function as effective tools in the fight against MDR bacteria. In the present work, the viral diversity of a marine environmental sample (biofilm), formed near an industrial zone, was mined for the identification of a putative endolysin (AbLys2) that belongs to the glycoside hydrolase family 24 (GH24, EC 3.2.1.17). The coding sequence of AbLys2 was cloned and expressed in E. coli. The lytic activity and specificity of the recombinant enzyme were evaluated against suspensions of a range of Gram-positive and Gram-negative human pathogens using turbidity assays. AbLys2 displayed enhanced selectivity towards A. baumannii cells, compared to other bacteria. Kinetics analysis was carried out to characterize the dependence of its lytic activity on pH and showed that the enzyme exhibits its maximal activity at pH 5.5. Thermostability analysis showed that AbLys2 displays melting temperature Tm 47.1 °C. Florescence microscopy and cell viability assays established that AbLys2 is active towards live cultures of A. baumannii cells with an inhibitory concentration IC50 3.41 ± 0.09 µM. Molecular modeling allowed the prediction of important amino acid residues involved in catalysis. The results of the present study suggest that AbLys2 provides efficient lytic and antimicrobial activity towards A. baumannii cells and therefore is a promising new antimicrobial against this pathogen.

Microalgae as a Sustainable Source of Antioxidants in Animal Nutrition, Health and Livestock Development.

Microalgae are a renewable and sustainable source of bioactive compounds, such as essential amino acids, polyunsaturated fatty acids, and antioxidant compounds, that have been documented to have beneficial effects on nutrition and health. Among these natural products, the demand for natural antioxidants, as an alternative to synthetic antioxidants, has increased. The antioxidant activity of microalgae significantly varies between species and depends on growth conditions. In the last decade, microalgae have been explored in livestock animals as feed additives with the aim of improving both animals' health and performance as well as product quality and the environmental impact of livestock. These findings are highly dependent on the composition of microalgae strain and their amount in the diet. The use of carbohydrate-active enzymes can increase nutrient bioavailability as a consequence of recalcitrant microalgae cell wall degradation, making it a promising strategy for monogastric nutrition for improving livestock productivity. The use of microalgae as an alternative to conventional feedstuffs is becoming increasingly important due to food-feed competition, land degradation, water deprivation, and climate change. However, the cost-effective production and use of microalgae is a major challenge in the near future, and their cultivation technology should be improved by reducing production costs, thus increasing profitability.

Concluding the trilogy: The interaction of 2,2'-dihydroxy-benzophenones and their carbonyl N-analogues with human glutathione transferase M1-1 face to face with the P1-1 and A1-1 isoenzymes involved in MDR.

A series of 2,2'-dihydroxybenzophenones and their carbonyl N-analogues were studied as potential inhibitors against human glutathione transferase M1-1 (hGSTM1-1) purified from recombinant E. coli. Their screening revealed an inhibition against hGSTM1-1 within a range of 0-42% (25 µM). The IC50 values for the two stronger ones, 16 and 13, were 53.5 ± 5.6 µΜ and 28.5 ± 2.5 µΜ, respectively. The results were compared with earlier ones for isoenzymes hGSTP1-1 and hGSTA1-1 involved in MDR. All but one bind more strongly to A1-1, than M1-1 and P1-1, the latter being a poor binder. An order of potency A1-1 > > M1-1 >  P1-1 meritted 13, 14 and 16 as the most potent inhibitors with hGSTM1-1. Enzyme kinetics with hGSTM1-1 (Km(CDNB) 213 ± 10 µΜ and Km(GSH) 303 ± 11 µΜ) revealed a competitive modality for 16 (Ki(16)  = 22.3 ± 1.1 µΜ) and a mixed one for 13 versus CDNB (Ki(13)  = 33.3 ± 1.6 µM for the free enzyme and Ki(13) ' = 17.7 ± 1.7 µM for the enzyme-CDNB complex). 5- or 5'-Bromo- or phenyl-substituted (but not in combination) inhibitors, having a H-bonded oxime weakly acidic group of a small volume, are optimal candidates for binding hGSTM1-1. The outcome of the isoenzyme trilogy identified good binder leads for the investigated GSTs involved in MDR.