scholarly journals NADP+-Preferring d-Lactate Dehydrogenase from Sporolactobacillus inulinus

2015 ◽  
Vol 81 (18) ◽  
pp. 6294-6301 ◽  
Author(s):  
Lingfeng Zhu ◽  
Xiaoling Xu ◽  
Limin Wang ◽  
Hui Dong ◽  
Bo Yu ◽  
...  
Keyword(s):  
Lactate Dehydrogenase ◽  
Catalytic Mechanism ◽  
High Specificity ◽  
Biochemical Properties ◽  
Enzymatic System ◽  
Content Type ◽  
Wide Range ◽  
Lactate Dehydrogenases

ABSTRACTHydroxy acid dehydrogenases, includingl- andd-lactate dehydrogenases (L-LDH and D-LDH), are responsible for the stereospecific conversion of 2-keto acids to 2-hydroxyacids and extensively used in a wide range of biotechnological applications. A common feature of LDHs is their high specificity for NAD+as a cofactor. An LDH that could effectively use NADPH as a coenzyme could be an alternative enzymatic system for regeneration of the oxidized, phosphorylated cofactor. In this study, ad-lactate dehydrogenase from aSporolactobacillus inulinusstrain was found to use both NADH and NADPH with high efficiencies and with a preference for NADPH as its coenzyme, which is different from the coenzyme utilization of all previously reported LDHs. The biochemical properties of the D-LDH enzyme were determined by X-ray crystal structural characterization andin vivoandin vitroenzymatic activity analyses. The residue Asn174was demonstrated to be critical for NADPH utilization. Characterization of the biochemical properties of this enzyme will contribute to understanding of the catalytic mechanism and provide referential information for shifting the coenzyme utilization specificity of 2-hydroxyacid dehydrogenases.

scholarly journals In VivoEfficacy of Anuran Trypsin Inhibitory Peptides against Staphylococcal Skin Infection and the Impact of Peptide Cyclization

2015 ◽  
Vol 59 (4) ◽  
pp. 2113-2121 ◽  
Author(s):  
U. Malik ◽  
O. N. Silva ◽  
I. C. M. Fensterseifer ◽  
L. Y. Chan ◽  
R. J. Clark ◽  
...  
Keyword(s):  
Antimicrobial Agents ◽  
Cyclic Peptide ◽  
Sequence Similarity ◽  
Infection Model ◽  
Content Type ◽  
Wide Range ◽  
Inhibitory Activities ◽  
The Impact

ABSTRACTStaphylococcus aureusis a virulent pathogen that is responsible for a wide range of superficial and invasive infections. Its resistance to existing antimicrobial drugs is a global problem, and the development of novel antimicrobial agents is crucial. Antimicrobial peptides from natural resources offer potential as new treatments against staphylococcal infections. In the current study, we have examined the antimicrobial properties of peptides isolated from anuran skin secretions and cyclized synthetic analogues of these peptides. The structures of the peptides were elucidated by nuclear magnetic resonance (NMR) spectroscopy, revealing high structural and sequence similarity with each other and with sunflower trypsin inhibitor 1 (SFTI-1). SFTI-1 is an ultrastable cyclic peptide isolated from sunflower seeds that has subnanomolar trypsin inhibitory activity, and this scaffold offers pharmaceutically relevant characteristics. The five anuran peptides were nonhemolytic and noncytotoxic and had trypsin inhibitory activities similar to that of SFTI-1. They demonstrated weakin vitroinhibitory activities againstS. aureus, but several had strong antibacterial activities againstS. aureusin anin vivomurine wound infection model. pYR, an immunomodulatory peptide fromRana sevosa, was the most potent, with complete bacterial clearance at 3 mg · kg−1. Cyclization of the peptides improved their stability but was associated with a concomitant decrease in antimicrobial activity. In summary, these anuran peptides are promising as novel therapeutic agents for treating infections from a clinically resistant pathogen.

scholarly journals A hyperthermoactive-Cas9 editing tool reveals the role of a unique arsenite methyltransferase in the arsenic resistance system of Thermus thermophilus HB27

2021 ◽  
Author(s):  
Giovanni Gallo ◽  
Ioannis Mougiakos ◽  
Mauricio Bianco ◽  
Miriam Carbonaro ◽  
Andrea Carpentieri ◽  
...  
Keyword(s):  
Catalytic Mechanism ◽  
Fluorescent Protein ◽  
Efflux Pump ◽  
Thermus Thermophilus ◽  
Yellow Fluorescent Protein ◽  
Arsenic Resistance ◽  
Wide Range ◽  
Resistance System

Arsenic detoxification systems can be found in a wide range of organisms, from bacteria to man. In a previous study, we discovered an arsenic-responsive transcriptional regulator in the thermophilic bacterium Thermus thermophilus HB27 (TtSmtB). Here, we characterize the arsenic resistance system of T. thermophilus in more detail. We employed TtSmtB-based pull-down assays with protein extracts from cultures treated with arsenate and arsenite to obtain an S-adenosylmethionine (SAM)-dependent arsenite methyltransferase (TtArsM). In vivo and in vitro analyses were performed to shed light on this new component of the arsenic resistance network and its peculiar catalytic mechanism. Heterologous expression of TtarsM in Escherichia coli resulted in arsenite detoxification at mesophilic temperatures. Although TtArsM does not contain a canonical arsenite binding site, the purified protein does catalyse SAM-dependent arsenite methylation. In addition, in vitro analyses confirmed the unique interaction between TtArsM and TtSmtB. Next, a highly efficient ThermoCas9-based genome-editing tool was developed to delete the TtArsM-encoding gene on the T. thermophilus genome, and to confirm its involvement in the arsenite detoxification system. Finally, the TtarsX efflux pump gene in the T. thermophilus ΔTtarsM genome was substituted by a gene, encoding a stabilised yellow fluorescent protein (sYFP), to create a sensitive genome-based bioreporter system for the detection of arsenic ions.

scholarly journals Cell-Fusing Agent Virus Reduces Arbovirus Dissemination in Aedes aegypti Mosquitoes In Vivo

2019 ◽  
Vol 93 (18) ◽  
Author(s):  
Artem Baidaliuk ◽  
Elliott F. Miot ◽  
Sebastian Lequime ◽  
Isabelle Moltini-Conclois ◽  
Fanny Delaigue ◽  
...  
Keyword(s):  
Aedes Aegypti ◽  
Dengue Virus ◽  
Cell Line ◽  
Content Type ◽  
Cells In Culture ◽  
Mosquito Cells ◽  
Wide Range ◽  
Natural Hosts

ABSTRACT Aedes aegypti mosquitoes are the main vectors of arthropod-borne viruses (arboviruses) of public health significance, such as the flaviviruses dengue virus (DENV) and Zika virus (ZIKV). Mosquitoes are also the natural hosts of a wide range of viruses that are insect specific, raising the question of their influence on arbovirus transmission in nature. Cell-fusing agent virus (CFAV) was the first described insect-specific flavivirus, initially discovered in an A. aegypti cell line and subsequently detected in natural A. aegypti populations. It was recently shown that DENV and the CFAV strain isolated from the A. aegypti cell line have mutually beneficial interactions in mosquito cells in culture. However, whether natural strains of CFAV and DENV interact in live mosquitoes is unknown. Using a wild-type CFAV isolate recently derived from Thai A. aegypti mosquitoes, we found that CFAV negatively interferes with both DENV type 1 and ZIKV in vitro and in vivo. For both arboviruses, prior infection by CFAV reduced the dissemination titer in mosquito head tissues. Our results indicate that the interactions observed between arboviruses and the CFAV strain derived from the cell line might not be a relevant model of the viral interference that we observed in vivo. Overall, our study supports the hypothesis that insect-specific flaviviruses may contribute to reduce the transmission of human-pathogenic flaviviruses. IMPORTANCE The mosquito Aedes aegypti carries several arthropod-borne viruses (arboviruses) that are pathogenic to humans, including dengue and Zika viruses. Interestingly, A. aegypti is also naturally infected with insect-only viruses, such as cell-fusing agent virus. Although interactions between cell-fusing agent virus and dengue virus have been documented in mosquito cells in culture, whether wild strains of cell-fusing agent virus interfere with arbovirus transmission by live mosquitoes was unknown. We used an experimental approach to demonstrate that cell-fusing agent virus infection reduces the propagation of dengue and Zika viruses in A. aegypti mosquitoes. These results support the idea that insect-only viruses in nature can modulate the ability of mosquitoes to carry arboviruses of medical significance and that they could possibly be manipulated to reduce arbovirus transmission.

scholarly journals New Triazole NT-a9 Has Potent Antifungal Efficacy against Cryptococcus neoformans In Vitro and In Vivo

2019 ◽  
Vol 64 (2) ◽  
Author(s):  
Ren-Yi Lu ◽  
Ting-Jun-Hong Ni ◽  
Jing Wu ◽  
Lan Yan ◽  
Quan-Zhen Lv ◽  
...  
Keyword(s):  
Cryptococcus Neoformans ◽  
Amphotericin B ◽  
Pathogenic Fungi ◽  
Control Group ◽  
Survival Times ◽  
Content Type ◽  
Fungal Burden ◽  
Wide Range

ABSTRACT In the past decades, the incidence of cryptococcosis has increased dramatically, which poses a new threat to human health. However, only a few drugs are available for the treatment of cryptococcosis. Here, we described a leading compound, NT-a9, an analogue of isavuconazole, that showed strong antifungal activities in vitro and in vivo. NT-a9 showed a wide range of activities against several pathogenic fungi in vitro, including Cryptococcus neoformans, Cryptococcus gattii, Candida albicans, Candida krusei, Candida tropicalis, Candida glabrata, and Candida parapsilosis, with MICs ranging from 0.002 to 1 μg/ml. In particular, NT-a9 exhibited excellent efficacy against C. neoformans, with a MIC as low as 0.002 μg/ml. NT-a9 treatment resulted in changes in the sterol contents in C. neoformans, similarly to fluconazole. In addition, NT-a9 possessed relatively low cytotoxicity and a high selectivity index. The in vivo efficacy of NT-a9 was assessed using a murine disseminated-cryptococcosis model. Mice were infected intravenously with 1.8 × 106 CFU of C. neoformans strain H99. In the survival study, NT-a9 significantly prolonged the survival times of mice compared with the survival times of the control group or the isavuconazole-, fluconazole-, or amphotericin B-treated groups. Of note, 4 and 8 mg/kg of body weight of NT-a9 rescued all the mice, with a survival rate of 100%. In the fungal-burden study, NT-a9 also significantly reduced the fungal burdens in brains and lungs, while fluconazole and amphotericin B only reduced the fungal burden in lungs. Taken together, these data suggested that NT-a9 is a promising antifungal candidate for the treatment of cryptococcosis infection.

scholarly journals A Toxic Environment: a Growing Understanding of How Microbial Communities Affect Escherichia coli O157:H7 Shiga Toxin Expression

2020 ◽  
Vol 86 (24) ◽  
Author(s):  
Erin M. Nawrocki ◽  
Hillary M. Mosso ◽  
Edward G. Dudley
Keyword(s):  
Escherichia Coli ◽  
Shiga Toxin ◽  
Microbial Interactions ◽  
Severe Illness ◽  
Content Type ◽  
E Coli ◽  
Wide Range ◽  
Lambdoid Prophage

ABSTRACT Enterohemorrhagic Escherichia coli (EHEC) strains, including E. coli O157:H7, cause severe illness in humans due to the production of Shiga toxin (Stx) and other virulence factors. Because Stx is coregulated with lambdoid prophage induction, its expression is especially susceptible to environmental cues. Infections with Stx-producing E. coli can be difficult to model due to the wide range of disease outcomes: some infections are relatively mild, while others have serious complications. Probiotic organisms, members of the gut microbiome, and organic acids can depress Stx production, in many cases by inhibiting the growth of EHEC strains. On the other hand, the factors currently known to amplify Stx act via their effect on the stx-converting phage. Here, we characterize two interactive mechanisms that increase Stx production by O157:H7 strains: first, direct interactions with phage-susceptible E. coli, and second, indirect amplification by secreted factors. Infection of susceptible strains by the stx-converting phage can expand the Stx-producing population in a human or animal host, and phage infection has been shown to modulate virulence in vitro and in vivo. Acellular factors, particularly colicins and microcins, can kill O157:H7 cells but may also trigger Stx expression in the process. Colicins, microcins, and other bacteriocins have diverse cellular targets, and many such molecules remain uncharacterized. The identification of additional Stx-amplifying microbial interactions will improve our understanding of E. coli O157:H7 infections and help elucidate the intricate regulation of pathogenicity in EHEC strains.

scholarly journals Phenolic Acids of Plant Origin—A Review on Their Antioxidant Activity In Vitro (O/W Emulsion Systems) Along with Their in Vivo Health Biochemical Properties

Foods ◽  
2020 ◽  
Vol 9 (4) ◽  
pp. 534 ◽  
Author(s):  
Sotirios Kiokias ◽  
Charalampos Proestos ◽  
Vassiliki Oreopoulou
Keyword(s):  
Phenolic Acids ◽  
Antioxidant Activities ◽  
Biochemical Properties ◽  
Potential Health ◽  
Wide Range ◽  
Literature Evidence ◽  
Emulsion Systems ◽  
Review Reports

Nature has generously offered a wide range of herbs (e.g., thyme, oregano, rosemary, sage, mint, basil) rich in many polyphenols and other phenolic compounds with strong antioxidant and biochemical properties. This paper focuses on several natural occurring phenolic acids (caffeic, carnosic, ferulic, gallic, p-coumaric, rosmarinic, vanillic) and first gives an overview of their most common natural plant sources. A summary of the recently reported antioxidant activities of the phenolic acids in o/w emulsions is also provided as an in vitro lipid-based model system. Exploring the interfacial activity of phenolic acids could help to further elucidate their potential health properties against oxidative stress conditions of biological membranes (such as lipoproteins). Finally, this review reports on the latest literature evidence concerning specific biochemical properties of the examined phenolic acids.

scholarly journals Two Proteins Form a Heteromeric Bacterial Self-Recognition Complex in Which Variable Subdomains Determine Allele-Restricted Binding

mBio ◽  
2015 ◽  
Vol 6 (3) ◽  
Author(s):  
Lia Cardarelli ◽  
Christina Saak ◽  
Karine A. Gibbs
Keyword(s):  
Molecular Recognition ◽  
Variable Region ◽  
Biochemical Properties ◽  
Binding Interaction ◽  
Content Type ◽  
Self Identity ◽  
Self Recognition ◽  
Recognition Determinant

ABSTRACT Self- versus nonself-recognition in bacteria has been described recently through genetic analyses in multiple systems; however, understanding of the biochemical properties and mechanisms of recognition-determinant proteins remains limited. Here we extend the molecular and biochemical understanding of two recognition-determinant proteins in bacteria. We have found that a heterotypic complex is formed between two bacterial self-recognition proteins, IdsD and IdsE, the genes of which have been shown to genetically encode the determinants for strain-specific identity in the opportunistic bacterial pathogen Proteus mirabilis. This IdsD-IdsE complex forms independently of other P. mirabilis-encoded self-recognition proteins. We have also shown that the binding between IdsD and IdsE is strain- and allele-specific. The specificity for interactions is encoded within a predicted membrane-spanning subdomain within each protein that contains stretches of unique amino acids in each P. mirabilis variant. Finally, we have demonstrated that this in vitro IdsD-IdsE binding interaction correlates to in vivo population identity, suggesting that the binding interactions between IdsD and IdsE are part of a cellular pathway that underpins self-recognition behavior in P. mirabilis and drives bacterial population sociality. IMPORTANCE Here we demonstrate that two proteins, the genes of which were genetically shown to encode determinants of self-identity in bacteria, bind in vitro in an allele-restricted interaction, suggesting that molecular recognition between these two proteins is a mechanism underpinning self-recognition behaviors in P. mirabilis. Binding specificity in each protein is encapsulated in a variable region subdomain that is predicted to span the membrane, suggesting that the interaction occurs in the cell envelope. Furthermore, conversion of binding affinities in vitro correlates with conversion of self-identity in vivo, suggesting that this molecular recognition might help to drive population behaviors.

scholarly journals Expression of a Toll Signaling Regulator Serpin in a Mycoinsecticide for Increased Virulence

2014 ◽  
Vol 80 (15) ◽  
pp. 4531-4539 ◽  
Author(s):  
Linzhi Yang ◽  
Nemat O. Keyhani ◽  
Guirong Tang ◽  
Chuang Tian ◽  
Ruipeng Lu ◽  
...  
Keyword(s):  
Entomopathogenic Fungi ◽  
Galleria Mellonella ◽  
Lethal Dose ◽  
Defense Responses ◽  
Content Type ◽  
Greater Wax Moth ◽  
Wide Range ◽  
New Strategy

ABSTRACTSerpins are ubiquitously distributed serine protease inhibitors that covalently bind to target proteases to exert their activities. Serpins regulate a wide range of activities, particularly those in which protease-mediated cascades are active. TheDrosophila melanogasterserpin Spn43Ac negatively controls the Toll pathway that is activated in response to fungal infection. The entomopathogenic fungusBeauveria bassianaoffers an environmentally friendly alternative to chemical pesticides for insect control. However, the use of mycoinsecticides remains limited in part due to issues of efficacy (low virulence) and the recalcitrance of the targets (due to strong immune responses). Since Spn43Ac acts to inhibit Toll-mediated activation of defense responses, we explored the feasibility of a new strategy to engineer entomopathogenic fungi with increased virulence by expression of Spn43Ac in the fungus. Compared to the 50% lethal dose (LD50) for the wild-type parent, the LD50ofB. bassianaexpressing Spn43Ac (strain Bb::S43Ac-1) was reduced ∼3-fold, and the median lethal time against the greater wax moth (Galleria mellonella) was decreased by ∼24%, with the more rapid proliferation of hyphal bodies being seen in the host hemolymph.In vitroandin vivoassays showed inhibition of phenoloxidase (PO) activation in the presence of Spn43Ac, with Spn43Ac-mediated suppression of activation by chymotrypsin, trypsin, laminarin, and lipopolysaccharide occurring in the following order: chymotrypsin and trypsin > laminarin > lipopolysaccharide. Expression of Spn43Ac had no effect on the activity of the endogenousB. bassiana-derived cuticle-degrading protease (CDEP-1). These results expand our understanding of Spn43Ac function and confirm that suppression of insect immune system defenses represents a feasible approach to engineering entomopathogenic fungi for greater efficacy.

scholarly journals A fluorescent sensor for spatiotemporally resolved endocannabinoid dynamics in vitro and in vivo

2020 ◽  
Author(s):  
Ao Dong ◽  
Kaikai He ◽  
Barna Dudok ◽  
Jordan S Farrell ◽  
Wuqiang Guan ◽  
...  
Keyword(s):  
Membrane Trafficking ◽  
Basolateral Amygdala ◽  
Fluorescent Sensor ◽  
Brain Slices ◽  
High Specificity ◽  
Cultured Neurons ◽  
Spatiotemporal Resolution ◽  
Wide Range

Endocannabinoids (eCBs) are retrograde neuromodulators that play an important role in a wide range of physiological processes; however, the release and in vivo dynamics of eCBs remain largely unknown, due in part to a lack of suitable probes capable of detecting eCBs with sufficient spatiotemporal resolution. Here, we developed a new eCB sensor called GRABeCB2.0. This genetically encoded sensor consists of the human CB1 cannabinoid receptor fused to circular-permutated EGFP, providing cell membrane trafficking, second-resolution kinetics, high specificity for eCBs, and a robust fluorescence response at physiological eCB concentrations. Using the GRABeCB2.0 sensor, we monitored evoked changes in eCB dynamics in both cultured neurons and acute brain slices. Interestingly, in cultured neurons we also observed spontaneous compartmental eCB transients that spanned a distance of approximately 11 μm, suggesting constrained, localized eCB signaling. Moreover, by expressing GRABeCB2.0 in the mouse brain, we readily observed foot shock-elicited and running-triggered eCB transients in the basolateral amygdala and hippocampus, respectively. Lastly, we used GRABeCB2.0 in a mouse seizure model and observed a spreading wave of eCB release that followed a Ca2+ wave through the hippocampus. Thus, GRABeCB2.0 is a robust new probe for measuring the dynamics of eCB release under both physiological and pathological conditions.

scholarly journals Genome Integration and Excision by a New Streptomyces Bacteriophage, ϕJoe

2016 ◽  
Vol 83 (5) ◽  
Author(s):  
Paul C. M. Fogg ◽  
Joshua A. Haley ◽  
W. Marshall Stark ◽  
Margaret C. M. Smith
Keyword(s):  
Synthetic Biology ◽  
High Efficiency ◽  
Dna Recombination ◽  
Streptomyces Venezuelae ◽  
Site Specific ◽  
Content Type ◽  
Wide Range ◽  
Serine Integrase

ABSTRACT Bacteriophages are the source of many valuable tools for molecular biology and genetic manipulation. In Streptomyces, most DNA cloning vectors are based on serine integrase site-specific DNA recombination systems derived from phage. Because of their efficiency and simplicity, serine integrases are also used for diverse synthetic biology applications. Here, we present the genome of a new Streptomyces phage, ϕJoe, and investigate the conditions for integration and excision of the ϕJoe genome. ϕJoe belongs to the largest Streptomyces phage cluster (R4-like) and encodes a serine integrase. The attB site from Streptomyces venezuelae was used efficiently by an integrating plasmid, pCMF92, constructed using the ϕJoe int-attP locus. The attB site for ϕJoe integrase was occupied in several Streptomyces genomes, including that of S. coelicolor, by a mobile element that varies in gene content and size between host species. Serine integrases require a phage-encoded recombination directionality factor (RDF) to activate the excision reaction. The ϕJoe RDF was identified, and its function was confirmed in vivo. Both the integrase and RDF were active in in vitro recombination assays. The ϕJoe site-specific recombination system is likely to be an important addition to the synthetic biology and genome engineering toolbox. IMPORTANCE Streptomyces spp. are prolific producers of secondary metabolites, including many clinically useful antibiotics. Bacteriophage-derived integrases are important tools for genetic engineering, as they enable integration of heterologous DNA into the Streptomyces chromosome with ease and high efficiency. Recently, researchers have been applying phage integrases for a variety of applications in synthetic biology, including rapid assembly of novel combinations of genes, biosensors, and biocomputing. An important requirement for optimal experimental design and predictability when using integrases, however, is the need for multiple enzymes with different specificities for their integration sites. In order to provide a broad platform of integrases, we identified and validated the integrase from a newly isolated Streptomyces phage, ϕJoe. ϕJoe integrase is active in vitro and in vivo. The specific recognition site for integration is present in a wide range of different actinobacteria, including Streptomyces venezuelae, an emerging model bacterium in Streptomyces research.

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