Cooperativity of covalent attachment and ion exchange on alcalase immobilization using glutaraldehyde chemistry: Enzyme stabilization and improved proteolytic activity

Alcalase was scarcely immobilized on monoaminoethyl-N-aminoethyl (MANAE)-agarose beads at different pH values (<20% at pH 7). The enzyme did not immobilize on MANAE-agarose activated with glutaraldehyde at high ionic strength, suggesting a low reactivity of the enzyme with the support functionalized in this manner. However, the immobilization is relatively rapid when using low ionic strength and glutaraldehyde activated support. Using these conditions, the enzyme was immobilized at pH 5, 7, and 9, and in all cases, the activity vs. Boc-Ala-ONp decreased to around 50%. However, the activity vs. casein greatly depends on the immobilization pH, while at pH 5 it is also 50%, at pH 7 it is around 200%, and at pH 9 it is around 140%. All immobilized enzymes were significantly stabilized compared to the free enzyme when inactivated at pH 5, 7, or 9. The highest stability was always observed when the enzyme was immobilized at pH 9, and the worst stability occurred when the enzyme was immobilized at pH 5, in agreement with the reactivity of the amino groups of the enzyme. Stabilization was lower for the three preparations when the inactivation was performed at pH 5. Thus, this is a practical example on how the cooperative effect of ion exchange and covalent immobilization may be used to immobilize an enzyme when only one independent cause of immobilization is unable to immobilize the enzyme, while adjusting the immobilization pH leads to very different properties of the final immobilized enzyme preparation. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2768, 2019.     

Identification of Vaginal Lactobacilli with Potential Probiotic Properties Isolated from Women in North Lebanon

The aim of this work was to study the diversity of vaginal lactobacilli in Lebanese women and to evaluate the antagonism, hydrophobicity, and safety characteristics of these strains. This study was performed on samples from 135 women who visited a gynecology clinic in the north of Lebanon, between September 2012 and January 2013. From these samples, 53 different isolates of vaginal lactobacilli were collected from vaginal swabs and identified using biochemical and molecular methods. The use of genotypic Rep-PCR fingerprinting allowed for the organization of these isolates into 23 different groups. Seven of the isolated lactobacilli were antagonistic against the following vaginal pathogens: Gardnerella vaginalis CIP7074T, Staphylococcus aureus ATCC33862, Escherichia coli CIP103982, and Candida albicans ATCC10231. The antagonistic lactobacilli strains were then identified using 16S rDNA sequence. The data of this study show that the antagonistic lactobacilli were non-hemolytic, sensitive to most antibiotic tests, free of plasmid DNA, and exhibited interesting hydrophobicity and autoaggregation properties positioning them as potential candidates for probiotic design.     

A proteomic investigation of Fusobacterium nucleatum alkaline-induced biofilms

ABSTRACT: BACKGROUND: The Gram negative anaerobe Fusobacterium nucleatum has been implicated in the aetiology of periodontal diseases. Although frequently isolated from healthy dental plaque, its numbers and proportion increase in plaque associated with disease. One of the significant physico-chemical changes in the diseased gingival sulcus is increased environmental pH. When grown under controlled conditions in our laboratory, F. nucleatum subspecies polymorphum formed mono-culture biofilms when cultured at pH 8.2. Biofilm formation is a survival strategy for bacteria, often associated with altered physiology and increased virulence. A proteomic approach was used to understand the phenotypic changes in F. nucleatum cells associated with alkaline induced biofilms. The proteomic based identification of significantly altered proteins was verified where possible using additional methods including quantitative real-time PCR (qRT-PCR), enzyme assay, acidic end-product analysis, intracellular polyglucose assay and Western blotting. RESULTS: Of 421 proteins detected on two-dimensional electrophoresis gels, spot densities of 54 proteins varied significantly (p < 0.05) in F. nucleatum cultured at pH 8.2 compared to growth at pH 7.4. Proteins that were differentially produced in biofilm cells were associated with the functional classes; metabolic enzymes, transport, stress response and hypothetical proteins. Our results suggest that biofilm cells were more metabolically efficient than planktonic cells as changes to amino acid and glucose metabolism generated additional energy needed for survival in a sub-optimal environment. The intracellular concentration of stress response proteins including heat shock protein GroEL and recombinational protein RecA increased markedly in the alkaline environment. A significant finding was the increased abundance of an adhesin, Fusobacterial outer membrane protein A (FomA). This surface protein is known for its capacity to bind to a vast number of bacterial species and human epithelial cells and its increased abundance was associated with biofilm formation. CONCLUSION: This investigation identified a number of proteins that were significantly altered by F. nucleatum in response to alkaline conditions similar to those reported in diseased periodontal pockets. The results provide insight into the adaptive mechanisms used by F. nucleatum biofilms in response to pH increase in the host environment.     

Effect of Antimicrobial Peptides Divergicin M35 and Nisin A on <Emphasis Type="Italic">Listeria monocytogenes</Emphasis> LSD530 Potassium Channels

The aim of this work was to study the effect of antimicrobial peptides: divergicin M35 and nisin A on Listeria monocytogenes LSD 530 potassium (K⁺) channels: ATP-sensitive (K_ATP), calcium-activated (BK_Ca), and depolarization-activated (K_v) types. Increase on K⁺ efflux and inhibition of cellular growth were observed after adding K⁺ channel activators pinacidil, NS1619, and cromakalim to divergicin M35. Increase in K⁺ efflux from log-phase cells was about 18 ± 1.1, 11 ± 0.63, and nmol mg⁻¹ of cell dry weight (CDW) for pinacidil and NS1619, respectively, over the efflux obtained with divergicin M35 alone. Increases in K⁺ efflux obtained by adding the same K⁺ channel activators to nisin A fit a completely different profile. Divergicin M35 activates K⁺ channels, particularly of the K_v and BK_Ca types and to a lesser extent the K_ATP type, causing K⁺ efflux and consequently cell death.     

The early pregnancy placenta foreshadows DNA methylation alterations of solid tumors

The placenta relies on phenotypes that are characteristic of cancer to successfully implant the embryo in the uterus during early pregnancy. Notably, it has to invade its host tissues, promote angiogenesis—while surviving hypoxia—, and escape the immune system. Similarities in DNA methylation patterns between the placenta and cancers suggest that common epigenetic mechanisms may be involved in regulating these behaviors. We show here that megabase-scale patterns of hypomethylation distinguish first from third trimester chorionic villi in the placenta, and that these patterns mirror those that distinguish many tumors from corresponding normal tissues. We confirmed these findings in villous cytotrophoblasts isolated from the placenta and identified a time window at the end of the first trimester, when these cells come into contact with maternal blood, as the likely time period for the methylome alterations. Furthermore, the large genomic regions affected by these patterns of hypomethylation encompass genes involved in pathways related to epithelial-mesenchymal transition, immune response, and inflammation. Analyses of expression profiles corresponding to genes in these hypomethylated regions in colon adenocarcinoma tumors point to networks of differentially expressed genes previously implicated in carcinogenesis and placentogenesis, where nuclear factor kappa B is a key hub. Taken together, our results suggest the existence of epigenetic switches involving large-scale changes of methylation in the placenta during pregnancy and in tumors during neoplastic transformation. The characterization of such epigenetic switches might lead to the identification of biomarkers and drug targets in oncology as well as in obstetrics and gynecology.     

Essential Oil of Thymus munbyanus subsp. coloratus from Algeria: Chemotypification and in vitro Biological Activities

Thymus munbyanus subsp. coloratus (Lamiaceae) is a small shrub endemic to Algeria and Morocco where is found in lawns, rockeries and mountainous regions. From a phytochemical point of view this taxon has never been characterized. In this work we have analysed the chemical compositions of the essential oils obtained from inflorescences and vegetative parts by GC/MS. A new chemotype, i.e. borneol‐chemotype, was characterized for the first time in the species. Furthermore, we assessed the biological activities of essential oils, namely the antioxidant, antimicrobial and cytotoxicity on tumor cells that were evaluated by the DPPH, ABTS, and FRAP, disc diffusion, and MTT methods, respectively. Biological assays highlighted a moderate inhibitory effect on Staphylococcus aureus, Escherichia coli and Candida albicans (inhibition zone diameter in the range 9 – 10 mm), and noteworthy cytotoxicity on A375 human melanoma cells (IC₅₀ of 46.95 μg/ml).     

An integrated approach reveals how lipo‐chitooligosaccharides interact with the lysin motif receptor‐like kinase MtLYR3

N‐acetylglucosamine containing compounds acting as pathogenic or symbiotic signals are perceived by plant‐specific Lysin Motif Receptor‐Like Kinases (LysM‐RLKs). The molecular mechanisms of this perception are not fully understood, notably those of lipo‐chitooligosaccharides (LCOs) produced during root endosymbioses with nitrogen‐fixing bacteria or arbuscular mycorrhizal fungi. In Medicago truncatula, we previously identified the LysM‐RLK LYR3 (MtLYR3) as a specific LCO‐binding protein. We also showed that the absence of LCO binding to LYR3 of the non‐mycorrhizal Lupinus angustifolius, (LanLYR3), was related to LysM3, which differs from that of MtLYR3 by several amino acids and, particularly, by a critical tyrosine residue absent in LanLYR3. Here, we aimed to define the LCO binding site of MtLYR3 by using molecular modelling and simulation approaches, combined with site‐directed mutagenesis and LCO binding experiments. 3D models of MtLYR3 and LanLYR3 ectodomains were built, and homology modelling and molecular dynamics (MD) simulations were performed. Molecular docking and MD simulation on the LysM3 identified potential key residues for LCO binding. We highlighted by steered MD simulations that in addition to the critical tyrosine, two other residues were important for LCO binding in MtLYR3. Substitution of these residues in LanLYR3‐LysM3 by those of MtLYR3‐LysM3 allowed the recovery of high‐affinity LCO binding in experimental radioligand‐binding assays. An analysis of selective constraints revealed that the critical tyrosine has experienced positive selection pressure and is absent in some LYR3 proteins. These findings now pave the way to uncover the functional significance of this specific evolutionary pattern.     

The continuing story of class IIa bacteriocins.

Many bacteria produce antimicrobial peptides, which are also referred to as peptide bacteriocins. The class IIa bacteriocins, often designated pediocin-like bacteriocins, constitute the most dominant group of antimicrobial peptides produced by lactic acid bacteria. The bacteriocins that belong to this class are structurally related and kill target cells by membrane permeabilization. Despite their structural similarity, class IIa bacteriocins display different target cell specificities. In the search for new antibiotic substances, the class IIa bacteriocins have been identified as promising new candidates and have thus received much attention. They kill some pathogenic bacteria (e.g., Listeria) with high efficiency, and they constitute a good model system for structure-function analyses of antimicrobial peptides in general. This review focuses on class IIa bacteriocins, especially on their structure, function, mode of action, biosynthesis, bacteriocin immunity, and current food applications. The genetics and biosynthesis of class IIa bacteriocins are well understood. The bacteriocins are ribosomally synthesized with an N-terminal leader sequence, which is cleaved off upon secretion. After externalization, the class IIa bacteriocins attach to potential target cells and, through electrostatic and hydrophobic interactions, subsequently permeabilize the cell membrane of sensitive cells. Recent observations suggest that a chiral interaction and possibly the presence of a mannose permease protein on the target cell surface are required for a bacteria to be sensitive to class IIa bacteriocins. There is also substantial evidence that the C-terminal half penetrates into the target cell membrane, and it plays an important role in determining the target cell specificity of these bacteriocins. Immunity proteins protect the bacteriocin producer from the bacteriocin it secretes. The three-dimensional structures of two class IIa immunity proteins have been determined, and it has been shown that the C-terminal halves of these cytosolic four-helix bundle proteins specify which class IIa bacteriocin they protect against.     

The behaviour of Fusobacterium nucleatum chemostat-grown in glucose- and amino acid-based chemically defined media

Fusobacterium nucleatum is a Gram-negative anaerobe, found in a number of different areas of human and animal bodies as part of the resident microbiota. However, it also appears to be involved in polymicrobial infections in such sites. It occurs in the oral cavity where it is a prominent member of various bacterial consortia associated with periodontal diseases. Like most fusobacteria, it derives energy via the fermentation of amino acids which it can obtain through the dissimilation of small peptides. However, the role of simple carbohydrates, such as glucose, in its growth and metabolism are not well understood. Accordingly, the aim of the present study was to study the behaviour ofF. nucleatum grown anaerobically in continuous culture in two different chemically-defined media (CDM); one containing only amino acids as the energy source, the other containing glucose as the predominant energy provider. At various dilution rates the culture were assayed for dry weight, intracellular polyglucose (IP) content, residual amino acids and glucose and acidic metabolic end-products. In the carbohydrate-free CDM the acidic end-products were a constant acetate : butyrate : formate of 1.5 : 1 : 0.4. The values of Y(max)amino acids, maximum yield of bacteria per mol of amino acids consumed, for two strains were estimated to be 15.2 and 18.6 g dry wt/mol, respectively. Them(amino acids), maintenance energy requirement for growth on amino acids, for the two strains was 0.81 and 0.94 mmol/g dry wt/h, respectively. Growth of one strain in the glucose-based CDM gave an estimated Y(max)glucose of 67.2 and an m(glucose) of 0.38; the acidic end-products were a fairly constant acetate : butyrate : formate : lactate of 0.7 : 1 : 0.3 : 2.5. Only at low growth rates, and then only in small amount, was IP produced in this medium. Overall, it was concluded that the occurrence of F. nucleatum in widely-differing oral niches may be explained, at least in part, by its metabolic versatility.