Genetic relationship between pUB110 and antibiotic-resistant plasmids obtained from thermophilic bacilli

1985 ◽  
Vol 31 (7) ◽  
pp. 614-619 ◽  
Author(s):  
Takayuki Hoshino ◽  
Takayuki Ikeda ◽  
Kensuke Furukawa ◽  
Noboru Tomizuka
Keyword(s):  
Copy Number ◽  
Blot Hybridization ◽  
Host Cells ◽  
Dna Homology ◽  
Binding Region ◽  
Antibiotic Resistant ◽  
Nucleotidyl Transferase ◽  
Thermophilic Bacilli ◽  
Identical Gene

The molecular relationship between pUB110 (Kmr, 4.4 kilobases (kb)) and antibiotic-resistant plasmids from thermophilic bacilli, pTHT15 (Tcr, 4.5 kb) and pTHN1 (Kmr, 4.8 kb), were studied by blot hybridization. Extensive homology was observed between pUB110 and pTHT 15 at the region which includes the replication origin. Incompatibility studies revealed that pTHT 15 and pUB110 were slightly incompatible in Bacillus subtilis but that they were apparently compatible in B. stearothermophilus. This difference in incompatibility between pTHT15 and pUB110 in the two host cells might be due to a difference in the copy number of pTHT15 in the two organisms. From the results of blot hybridization, mode of kanamycin inactivation, and DNA sequencing, it was determined that pTHN1 encoded the identical gene for kanamycin nucleotidyl transferase as that of pUB110. All three plasmids pTHT15, pTHN1, and pUB110 shared a common DNA homology at the in vitro membrane-binding region.

Localization of a Vitronectin Binding Region of Plasminogen Activator Inhibitor-1

1995 ◽  
Vol 73 (05) ◽  
pp. 829-834 ◽  
Author(s):  
Jaya Padmanabhan ◽  
David C Sane
Keyword(s):  
Binding Site ◽  
Plasminogen Activator ◽  
Inhibitory Activity ◽  
Plasminogen Activator Inhibitor ◽  
Structural Homology ◽  
Binding Region ◽  
Plasminogen Activator Inhibitor 1 ◽  
Activator Inhibitor ◽  
Pai 1

SummaryThe PAI-1 binding site for VN was studied using two independent methods. PAI-1 was cleaved by Staph V8 protease, producing 8 fragments, only 2 of which bound to [125I]-VN. These fragments were predicted to overlap between residues 91-130. Since PAI-2 has structural homology to PAI-1, but does not bind to vitronectin, chimeras of PAI-1 and PAI-2 were constructed. Four chimeras, containing PAI-1 residues 1-70,1-105,1-114, and 1-167 were constructed and expressed in vitro. PAI-1, PAI-2, and all of the chimeras retained inhibitory activity for t-PA, but only the chimera containing PAI-1 residues 1-167 formed a complex with VN. Together, these results predict that the VN binding site of PAI-1 is between residues 115-130.

Mechanisms involved in effects of antimicrobial peptides, bactenectins ChBac3.4, ChBac5, and mini-ChBac7.5Nb, on bacterial cells

2017 ◽  
pp. 43-50
Author(s):  
О.В. Шамова ◽  
М.С. Жаркова ◽  
П.М. Копейкин ◽  
Д.С. Орлов ◽  
Е.А. Корнева
Keyword(s):  
Escherichia Coli ◽  
Antimicrobial Activity ◽  
Innate Immunity ◽  
Infectious Diseases ◽  
Metabolic Activity ◽  
Capra Hircus ◽  
Bacterial Cells ◽  
Antibiotic Resistant ◽  
Resistant Strains

Антимикробные пептиды (АМП) системы врожденного иммунитета - соединения, играющие важную роль в патогенезе инфекционных заболеваний, так как обладают свойством инактивировать широкий спектр патогенных бактерий, обеспечивая противомикробную защиту живых организмов. В настоящее время АМП рассматриваются как потенциальные соединения-корректоры инфекционной патологии, вызываемой антибиотикорезистентными бактериями (АБР). Цель данной работы состояла в изученим механизмов антибактериального действия трех пептидов, принадлежащих к семейству бактенецинов - ChBac3.4, ChBac5 и mini-ChBac7.5Nb. Эти химически синтезированные пептиды являются аналогами природных пролин-богатых АМП, обнаруженных в лейкоцитах домашней козы Capra hircus и проявляющих высокую антимикробную активность, в том числе и в отношении грамотрицательных АБР. Методы. Минимальные ингибирующие и минимальные бактерицидные концентрации пептидов (МИК и МБК) определяли методом серийных разведений в жидкой питательной среде с последующим высевом на плотную питательную среду. Эффекты пептидов на проницаемость цитоплазматической мембраны бактерий для хромогенного маркера исследовали с использованием генетически модифицированного штамма Escherichia coli ML35p. Действие бактенецинов на метаболическую активность бактерий изучали с применением маркера резазурина. Результаты. Показано, что все исследованные пептиды проявляют высокую антимикробную активность в отношении Escherichia coli ML35p и антибиотикоустойчивых штаммов Escherichia coli ESBL и Acinetobacter baumannii in vitro, но их действие на бактериальные клетки разное. Использован комплекс методик, позволяющих наблюдать в режиме реального времени динамику действия бактенецинов в различных концентрациях (включая их МИК и МБК) на барьерную функцию цитоплазматической мембраны и на интенсивность метаболизма бактериальных клеток, что дало возможность выявить различия в характере воздействия бактенецинов, отличающихся по структуре молекулы, на исследуемые микроорганизмы. Установлено, что действие каждого из трех исследованных бактенецинов в бактерицидных концентрациях отличается по эффективности нарушения целостности бактериальных мембран и в скорости подавления метаболизма клеток. Заключение. Полученная информация дополнит существующие фундаментальные представления о механизмах действия пролин-богатых пептидов врожденного иммунитета, а также послужит основой для биотехнологических исследований, направленных на разработку на базе этих соединений новых антибиотических препаратов для коррекции инфекционных заболеваний, вызываемых АБР и являющимися причинами тяжелых внутрибольничных инфекций. Antimicrobial peptides (AMPs) of the innate immunity are compounds that play an important role in pathogenesis of infectious diseases due to their ability to inactivate a broad array of pathogenic bacteria, thereby providing anti-microbial host defense. AMPs are currently considered promising compounds for treatment of infectious diseases caused by antibiotic-resistant bacteria. The aim of this study was to investigate molecular mechanisms of the antibacterial action of three peptides from the bactenecin family, ChBac3.4, ChBac5, and mini-ChBac7.5Nb. These chemically synthesized peptides are analogues of natural proline-rich AMPs previously discovered by the authors of the present study in leukocytes of the domestic goat, Capra hircus. These peptides exhibit a high antimicrobial activity, in particular, against antibiotic-resistant gram-negative bacteria. Methods. Minimum inhibitory and minimum bactericidal concentrations of the peptides (MIC and MBC) were determined using the broth microdilution assay followed by subculturing on agar plates. Effects of the AMPs on bacterial cytoplasmic membrane permeability for a chromogenic marker were explored using a genetically modified strain, Escherichia coli ML35p. The effect of bactenecins on bacterial metabolic activity was studied using a resazurin marker. Results. All the studied peptides showed a high in vitro antimicrobial activity against Escherichia coli ML35p and antibiotic-resistant strains, Escherichia coli ESBL and Acinetobacter baumannii, but differed in features of their action on bacterial cells. The used combination of techniques allowed the real-time monitoring of effects of bactenecin at different concentrations (including their MIC and MBC) on the cell membrane barrier function and metabolic activity of bacteria. The differences in effects of these three structurally different bactenecins on the studied microorganisms implied that these peptides at bactericidal concentrations differed in their capability for disintegrating bacterial cell membranes and rate of inhibiting bacterial metabolism. Conclusion. The obtained information will supplement the existing basic concepts on mechanisms involved in effects of proline-rich peptides of the innate immunity. This information will also stimulate biotechnological research aimed at development of new antibiotics for treatment of infectious diseases, such as severe in-hospital infections, caused by antibiotic-resistant strains.

Molecular Docking Approaches to Suggest the Anti-Mycobacterial Targets of Natural Products

2020 ◽  
Author(s):  
Rafael Baptista ◽  
Sumana Bhowmick ◽  
Shen Jianying ◽  
Luis Mur
Keyword(s):  
Natural Products ◽  
Molecular Docking ◽  
Free Energies ◽  
Antibiotic Resistant ◽  
Drug Lead ◽  
Bisbenzylisoquinoline Alkaloids ◽  
Docking Approach ◽  
Global Threat ◽  
Physicochemical And Structural Properties

Tuberculosis (TB) is a major global threat mostly due to the development of antibiotic resistant forms of Mycobacterium tuberculosis, the causal agent of the disease. Driven by the pressing need for new anti-mycobacterial agents, several natural products (NPs) have been shown to have in vitro activities against M. tuberculosis. The utility of any NP as a drug lead is augmented when the anti-mycobacterial target(s) is unknown. To suggest these, we used a molecular docking approach to predict the interactions of 53 selected anti-mycobacterial NPs against known ‘druggable’ mycobacterial targets ClpP1P2, DprE1, InhA, KasA, PanK, PknB and Pks13. The docking scores / binding free energies were predicted and calculated using AutoDock Vina along with physicochemical and structural properties of the NPs, using PaDEL descriptors. These were compared to the established inhibitor (control) drugs for each mycobacterial target. The specific interactions of the bisbenzylisoquinoline alkaloids 2-nortiliacorinine, tiliacorine and 13’-bromotiliacorinine against the targets PknB and DprE1 (-11.4, -10.9 and -9.8 kcal.mol-1 ; -12.7, -10.9 and -10.3 kcal.mol-1 , respectively) and the lignan αcubebin and Pks13 (-11.0 kcal.mol-1 ) had significantly superior docking scores compared to controls. Our approach can be used to suggest predicted targets for the NP to be validated experimentally but these in silico steps are likely to facilitate drug optimisation.

Novel Toxin-antitoxin System Xn-mazEF from Xenorhabdus nematophi-la: Identification, Characterization and Functional Exploration

2020 ◽  
Vol 16 ◽  
Author(s):  
Jogendra Singh Nim ◽  
Mohit Yadav ◽  
Lalit Kumar Gautam ◽  
Chaitali Ghosh ◽  
Shakti Sahi ◽  
...  
Keyword(s):  
Interaction Analysis ◽  
Functional Characterization ◽  
Host Cells ◽  
System Control ◽  
Xenorhabdus Nematophila ◽  
Functional Features ◽  
Dynamics Simulations ◽  
Species Specific ◽  
Rna Interaction

Background: Xenorhabdus nematophila maintains species-specific mutual interaction with nematodes of Steinernema genus. Type II Toxin Antitoxin (TA) systems, the mazEF TA system controls stress and programmed cell death in bacteria. Objective: This study elucidates the functional characterization of Xn-mazEF, a mazEF homolog in X. nematophila by computational and in vitro approaches. Methods: 3 D- structural models for Xn-MazE toxin and Xn-MazF antitoxin were generated, validated and characterized for protein - RNA interaction analysis. Further biological and cellular functions of Xn-MazF toxin were also predicted. Molecular dynamics simulations of 50ns for Xn-MazF toxin complexed with nucleic acid units (DU, RU, RC, and RU) were performed. The MazF toxin and complete MazEF operon were endogenously expressed and monitored for the killing of Escherichia coli host cells under arabinose induced tightly regulated system. Results: Upon induction, E. coli expressing toxin showed rapid killing within four hours and attained up to 65% growth inhibition, while the expression of the entire operon did not show significant killing. The observation suggests that the Xn-mazEF TA system control transcriptional regulation in X. nematophila and helps to manage stress or cause toxicity leading to programmed death of cells. Conclusion: The study provides insights into structural and functional features of novel toxin, XnMazF and provides an initial inference on control of X. nematophila growth regulated by TA systems.

ACIS, A Novel KepTide™, Binds to ACE-2 Receptor and Inhibits the Infection of SARS-CoV2 Virus in vitro in Primate Kidney Cells: Therapeutic Implications for COVID-19 (Preprint)

2020 ◽  
Author(s):  
Avik Sotira Scientific
Keyword(s):  
Social Life ◽  
Plaque Assay ◽  
Host Cells ◽  
Surface Glycoprotein ◽  
Crystallographic Structure ◽  
Therapeutic Implications ◽  
Biochemical Assays ◽  
Targeted Medicine

UNSTRUCTURED Coronavirus disease 2019 (COVID-19) is a severe acute respiratory syndrome (SARS) caused by a virus known as SARS-Coronavirus 2 (SARS-CoV2). Without a targeted-medicine, this disease has been causing a massive humanitarian crisis not only in terms of mortality, but also imposing a lasting damage to social life and economic progress of humankind. Therefore, an immediate therapeutic strategy needs to be intervened to mitigate this global crisis. Here, we report a novel KepTide™ (Knock-End Peptide) therapy that nullifies SARS-CoV2 infection. SARS-CoV2 employs its surface glycoprotein “spike” (S-glycoprotein) to interact with angiotensin converting enzyme-2 (ACE-2) receptor for its infection in host cells. Based on our in-silico-based homology modeling study validated with a recent X-ray crystallographic structure (PDB ID:6M0J), we have identified that a conserved motif of S-glycoprotein that intimately engages multiple hydrogen-bond (H-bond) interactions with ACE-2 enzyme. Accordingly, we designed a peptide, termed as ACIS (ACE-2 Inhibitory motif of Spike), that displayed significant affinity towards ACE-2 enzyme as confirmed by biochemical assays such as BLItz and fluorescence polarization assays. Interestingly, more than one biochemical modifications were adopted in ACIS in order to enhance the inhibitory action of ACIS and hence called as KEpTide™. Consequently, a monolayer invasion assay, plaque assay and dual immunofluorescence analysis further revealed that KEpTide™ efficiently mitigated the infection of SARS-CoV2 in vitro in VERO E6 cells. Finally, evaluating the relative abundance of ACIS in lungs and the potential side-effects in vivo in mice, our current study discovers a novel KepTide™ therapy that is safe, stable, and robust to attenuate the infection of SARS-CoV2 virus if administered intranasally. INTERNATIONAL REGISTERED REPORT RR2-https://doi.org/10.1101/2020.10.13.337584

scholarly journals Bromodomain and Extraterminal Protein Inhibitor, Apabetalone (RVX-208), Reduces ACE2 Expression and Attenuates SARS-Cov-2 Infection In Vitro

Biomedicines ◽  
2021 ◽  
Vol 9 (4) ◽  
pp. 437
Author(s):  
Dean Gilham ◽  
Audrey L. Smith ◽  
Li Fu ◽  
Dalia Y. Moore ◽  
Abenaya Muralidharan ◽  
...  
Keyword(s):  
Antiviral Agents ◽  
Host Cells ◽  
Protein Inhibitor ◽  
Angiotensin Converting Enzyme 2 ◽  
Dipeptidyl Peptidase 4 ◽  
Surface Receptors ◽  
Bet Inhibitor ◽  
Epigenetic Machinery ◽  
Unexplored Area

Effective therapeutics are urgently needed to counter infection and improve outcomes for patients suffering from COVID-19 and to combat this pandemic. Manipulation of epigenetic machinery to influence viral infectivity of host cells is a relatively unexplored area. The bromodomain and extraterminal (BET) family of epigenetic readers have been reported to modulate SARS-CoV-2 infection. Herein, we demonstrate apabetalone, the most clinical advanced BET inhibitor, downregulates expression of cell surface receptors involved in SARS-CoV-2 entry, including angiotensin-converting enzyme 2 (ACE2) and dipeptidyl-peptidase 4 (DPP4 or CD26) in SARS-CoV-2 permissive cells. Moreover, we show that apabetalone inhibits SARS-CoV-2 infection in vitro to levels comparable to those of antiviral agents. Taken together, our study supports further evaluation of apabetalone to treat COVID-19, either alone or in combination with emerging therapeutics.

scholarly journals Effective decellularisation of human saphenous veins for biocompatible arterial tissue engineering applications: Bench optimisation and feasibility in vivo testing

2021 ◽  
Vol 12 ◽  
pp. 204173142098752
Author(s):  
Nadiah S Sulaiman ◽  
Andrew R Bond ◽  
Vito D Bruno ◽  
John Joseph ◽  
Jason L Johnson ◽  
...  
Keyword(s):  
Tissue Engineering ◽  
Mechanical Strength ◽  
Vascular Tissue ◽  
Sodium Dodecyl ◽  
Host Cells ◽  
Replacement Model ◽  
In Vivo Testing ◽  
Vascular Scaffolds

Human saphenous vein (hSV) and synthetic grafts are commonly used conduits in vascular grafting, despite high failure rates. Decellularising hSVs (D-hSVs) to produce vascular scaffolds might be an effective alternative. We assessed the effectiveness of a detergent-based method using 0% to 1% sodium dodecyl sulphate (SDS) to decellularise hSV. Decellularisation effectiveness was measured in vitro by nuclear counting, DNA content, residual cell viability, extracellular matrix integrity and mechanical strength. Cytotoxicity was assessed on human and porcine cells. The most effective SDS concentration was used to prepare D-hSV grafts that underwent preliminary in vivo testing using a porcine carotid artery replacement model. Effective decellularisation was achieved with 0.01% SDS, and D-hSVs were biocompatible after seeding. In vivo xeno-transplantation confirmed excellent mechanical strength and biocompatibility with recruitment of host cells without mechanical failure, and a 50% patency rate at 4-weeks. We have developed a simple biocompatible methodology to effectively decellularise hSVs. This could enhance vascular tissue engineering toward future clinical applications.

scholarly journals Lipoproteins Are Responsible for the Pro-Inflammatory Property of Staphylococcus aureus Extracellular Vesicles

2021 ◽  
Vol 22 (13) ◽  
pp. 7099
Author(s):  
Pradeep Kumar Kopparapu ◽  
Meghshree Deshmukh ◽  
Zhicheng Hu ◽  
Majd Mohammad ◽  
Marco Maugeri ◽  
...  
Keyword(s):  
Extracellular Vesicles ◽  
Inflammatory Responses ◽  
Surface Proteins ◽  
Host Cells ◽  
Immune Stimulation ◽  
Domains Of Life ◽  
Major Surface ◽  
Staphylococcal Aureus

Staphylococcal aureus (S. aureus), a Gram-positive bacteria, is known to cause various infections. Extracellular vesicles (EVs) are a heterogeneous array of membranous structures secreted by cells from all three domains of life, i.e., eukaryotes, bacteria, and archaea. Bacterial EVs are implied to be involved in both bacteria–bacteria and bacteria–host interactions during infections. It is still unclear how S. aureus EVs interact with host cells and induce inflammatory responses. In this study, EVs were isolated from S. aureus and mutant strains deficient in either prelipoprotein lipidation (Δlgt) or major surface proteins (ΔsrtAB). Their immunostimulatory capacities were assessed both in vitro and in vivo. We found that S. aureus EVs induced pro-inflammatory responses both in vitro and in vivo. However, this activity was dependent on lipidated lipoproteins (Lpp), since EVs isolated from the Δlgt showed no stimulation. On the other hand, EVs isolated from the ΔsrtAB mutant showed full immune stimulation, indicating the cell wall anchoring of surface proteins did not play a role in immune stimulation. The immune stimulation of S. aureus EVs was mediated mainly by monocytes/macrophages and was TLR2 dependent. In this study, we demonstrated that not only free Lpp but also EV-imbedded Lpp had high pro-inflammatory activity.

scholarly journals Structural comparison of CD163 SRCR5 from different species sheds some light on its involvement in porcine reproductive and respiratory syndrome virus-2 infection in vitro

2021 ◽  
Vol 52 (1) ◽  
Author(s):  
Hongfang Ma ◽  
Rui Li ◽  
Longguang Jiang ◽  
Songlin Qiao ◽  
Xin-xin Chen ◽  
...  
Keyword(s):  
Scavenger Receptor ◽  
Host Cells ◽  
Respiratory Syndrome Virus ◽  
Swine Industry ◽  
Prrs Virus ◽  
Rich Domain ◽  
Serious Disease ◽  
The One ◽  
And Control

AbstractPorcine reproductive and respiratory syndrome (PRRS) is a serious disease burdening global swine industry. Infection by its etiological agent, PRRS virus (PRRSV), shows a highly restricted tropism of host cells and has been demonstrated to be mediated by an essential scavenger receptor (SR) CD163. CD163 fifth SR cysteine-rich domain (SRCR5) is further proven to play a crucial role during viral infection. Despite intense research, the involvement of CD163 SRCR5 in PRRSV infection remains to be elucidated. In the current study, we prepared recombinant monkey CD163 (moCD163) SRCR5 and human CD163-like homolog (hCD163L1) SRCR8, and determined their crystal structures. After comparison with the previously reported crystal structure of porcine CD163 (pCD163) SRCR5, these structures showed almost identical structural folds but significantly different surface electrostatic potentials. Based on these differences, we carried out mutational research to identify that the charged residue at position 534 in association with the one at position 561 were important for PRRSV-2 infection in vitro. Altogether the current work sheds some light on CD163-mediated PRRSV-2 infection and deepens our understanding of the viral pathogenesis, which will provide clues for prevention and control of PRRS.

scholarly journals Stereotypic neutralizing VH antibodies against SARS-CoV-2 spike protein receptor binding domain in COVID-19 patients and healthy individuals

2021 ◽  
pp. eabd6990
Author(s):  
Sang Il Kim ◽  
Jinsung Noh ◽  
Sujeong Kim ◽  
Younggeun Choi ◽  
Duck Kyun Yoo ◽  
...  
Keyword(s):  
B Cells ◽  
Receptor Binding ◽  
Neutralizing Antibodies ◽  
De Novo ◽  
Binding Domain ◽  
Host Cells ◽  
Spike Protein ◽  
Receptor Binding Domain ◽  
Healthy Individuals

Stereotypic antibody clonotypes exist in healthy individuals and may provide protective immunity against viral infections by neutralization. We observed that 13 out of 17 patients with COVID-19 had stereotypic variable heavy chain (VH) antibody clonotypes directed against the receptor-binding domain (RBD) of SARS-CoV-2 spike protein. These antibody clonotypes were comprised of immunoglobulin heavy variable (IGHV)3-53 or IGHV3-66 and immunoglobulin heavy joining (IGHJ)6 genes. These clonotypes included IgM, IgG3, IgG1, IgA1, IgG2, and IgA2 subtypes and had minimal somatic mutations, which suggested swift class switching after SARS-CoV-2 infection. The different immunoglobulin heavy variable chains were paired with diverse light chains resulting in binding to the RBD of SARS-CoV-2 spike protein. Human antibodies specific for the RBD can neutralize SARS-CoV-2 by inhibiting entry into host cells. We observed that one of these stereotypic neutralizing antibodies could inhibit viral replication in vitro using a clinical isolate of SARS-CoV-2. We also found that these VH clonotypes existed in six out of 10 healthy individuals, with IgM isotypes predominating. These findings suggest that stereotypic clonotypes can develop de novo from naïve B cells and not from memory B cells established from prior exposure to similar viruses. The expeditious and stereotypic expansion of these clonotypes may have occurred in patients infected with SARS-CoV-2 because they were already present.

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