scholarly journals An evolutionary approach to systematic discovery of novel deubiquitinases, applied to Legionella

2020 ◽  
Vol 3 (9) ◽  
pp. e202000838
Author(s):  
Thomas Hermanns ◽  
Ilka Woiwode ◽  
Ricardo FM Guerreiro ◽  
Robert Vogt ◽  
Michael Lammers ◽  
...  
Keyword(s):  
Legionella Pneumophila ◽  
Active Site ◽  
Pathogenic Bacteria ◽  
Markov Models ◽  
Substrate Selection ◽  
Deubiquitinating Enzymes ◽  
Protein Ubiquitination ◽  
Mammalian Genomes ◽  
Experimental Approaches ◽  
Ubiquitination System

Deubiquitinating enzymes (DUBs) are important regulators of the posttranslational protein ubiquitination system. Mammalian genomes encode about 100 different DUBs, which can be grouped into seven different classes. Members of other DUB classes are found in pathogenic bacteria, which use them to target the host defense. By combining bioinformatical and experimental approaches, we address the question if the known DUB families have a common evolutionary ancestry and share conserved features that set them apart from other proteases. By systematically comparing family-specific hidden Markov models, we uncovered distant relationships between established DUBs and other cysteine protease families. Most DUB families share a conserved aromatic residue linked to the active site, which restricts the cleavage of substrates with side chains at the S2 position, corresponding to Gly-75 in ubiquitin. By applying these criteria to Legionella pneumophila ORFs, we identified lpg1621 and lpg1148 as deubiquitinases, characterized their cleavage specificities, and confirmed the importance of the aromatic gatekeeper motif for substrate selection.

scholarly journals An evolutionary approach to systematic discovery of novel deubiquitinases, applied to Legionella

2020 ◽  
Author(s):  
Thomas Hermanns ◽  
Ilka Woiwode ◽  
Ricardo F. M. Guerreiro ◽  
Robert Vogt ◽  
Michael Lammers ◽  
...  
Keyword(s):  
Legionella Pneumophila ◽  
Active Site ◽  
Pathogenic Bacteria ◽  
Markov Models ◽  
Substrate Selection ◽  
Deubiquitinating Enzymes ◽  
Protein Ubiquitination ◽  
Mammalian Genomes ◽  
Experimental Approaches ◽  
Ubiquitination System

AbstractDeubiquitinating enzymes (DUBs) are important regulators of the posttranslational protein ubiquitination system. Mammalian genomes encode about hundred different DUBs, which can be grouped into seven different classes. Members of other DUB classes are found in pathogenic bacteria, which use them to target the host defense. By combining bioinformatical and experimental approaches, we address the question if the known DUB families have a common evolutionary ancestry and share conserved features that set them apart from other proteases. By systematically comparing family-specific Hidden-Markov-Models, we uncovered distant relationships between established DUBs and other cysteine protease families. Most DUB families share a conserved aromatic residue linked to the active site, which restricts the cleavage of substrates with sidechains at the S2 position, corresponding to Gly-75 in ubiquitin. By applying these criteria to Legionella pneumophila ORFs, we identified lpg1621 and lpg1148 as deubiquitinases, characterized their cleavage specificities, and confirmed the importance of the aromatic gatekeeper motif for substrate selection.

scholarly journals The Ubiquitination System within Bacterial Host–Pathogen Interactions

2021 ◽  
Vol 9 (3) ◽  
pp. 638
Author(s):  
Vera Vozandychova ◽  
Pavla Stojkova ◽  
Kamil Hercik ◽  
Pavel Rehulka ◽  
Jiri Stulik
Keyword(s):  
Host Cell ◽  
Legionella Pneumophila ◽  
Pathogenic Bacteria ◽  
Vaccine Development ◽  
Shigella Flexneri ◽  
Effector Proteins ◽  
Deubiquitinating Enzymes ◽  
Host Pathogen Interactions ◽  
Host Pathogen ◽  
Ubiquitination System

Ubiquitination of proteins, like phosphorylation and acetylation, is an important regulatory aspect influencing numerous and various cell processes, such as immune response signaling and autophagy. The study of ubiquitination has become essential to learning about host–pathogen interactions, and a better understanding of the detailed mechanisms through which pathogens affect ubiquitination processes in host cell will contribute to vaccine development and effective treatment of diseases. Pathogenic bacteria (e.g., Salmonella enterica, Legionella pneumophila and Shigella flexneri) encode many effector proteins, such as deubiquitinating enzymes (DUBs), targeting the host ubiquitin machinery and thus disrupting pertinent ubiquitin-dependent anti-bacterial response. We focus here upon the host ubiquitination system as an integral unit, its interconnection with the regulation of inflammation and autophagy, and primarily while examining pathogens manipulating the host ubiquitination system. Many bacterial effector proteins have already been described as being translocated into the host cell, where they directly regulate host defense processes. Due to their importance in pathogenic bacteria progression within the host, they are regarded as virulence factors essential for bacterial evasion. However, in some cases (e.g., Francisella tularensis) the host ubiquitination system is influenced by bacterial infection, although the responsible bacterial effectors are still unknown.

scholarly journals Structural and functional insights into esterase-mediated macrolide resistance

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Michał Zieliński ◽  
Jaeok Park ◽  
Barry Sleno ◽  
Albert M. Berghuis
Keyword(s):  
Active Site ◽  
Pathogenic Bacteria ◽  
Catalytic Mechanism ◽  
Three Dimensional ◽  
Resistance Mechanisms ◽  
Docking Studies ◽  
Macrolide Resistance ◽  
Flexible Docking ◽  
Sequence Identity ◽  
Substrate Spectrum

AbstractMacrolides are a class of antibiotics widely used in both medicine and agriculture. Unsurprisingly, as a consequence of their exensive usage a plethora of resistance mechanisms have been encountered in pathogenic bacteria. One of these resistance mechanisms entails the enzymatic cleavage of the macrolides’ macrolactone ring by erythromycin esterases (Eres). The most frequently identified Ere enzyme is EreA, which confers resistance to the majority of clinically used macrolides. Despite the role Eres play in macrolide resistance, research into this family enzymes has been sparse. Here, we report the first three-dimensional structures of an erythromycin esterase, EreC. EreC is an extremely close homologue of EreA, displaying more than 90% sequence identity. Two structures of this enzyme, in conjunction with in silico flexible docking studies and previously reported mutagenesis data allowed for the proposal of a detailed catalytic mechanism for the Ere family of enzymes, labeling them as metal-independent hydrolases. Also presented are substrate spectrum assays for different members of the Ere family. The results from these assays together with an examination of residue conservation for the macrolide binding site in Eres, suggests two distinct active site archetypes within the Ere enzyme family.

scholarly journals Antimicrobial activity of the volatile phase of essential oils and their constituents on Legionella pneumophila

2020 ◽  
Vol 14 (1) ◽  
pp. 54-61
Author(s):  
Lucia Bićanić ◽  
Silvestar Mežnarić ◽  
Ivana Gobin
Keyword(s):  
Essential Oil ◽  
Essential Oils ◽  
Legionella Pneumophila ◽  
Pathogenic Bacteria ◽  
Antimicrobial Properties ◽  
Model Organism ◽  
Public Health Problem ◽  
Volatile Components ◽  
Atypical Pneumonia ◽  
Pontiac Fever

Abstract Pathogenic bacteria of the genus Legionella cause atypical pneumonia known as Legionnaires’ disease and flu – like disease known as Pontiac fever. As pathogens of the respiratory system, these bacteria represent a public health problem and there is a need for examine new alternative ways to inactivate them. These bacteria live naturally in water and are transmitted by infectious aerosols. To purify the air, essential oils that show antimicrobial properties are widely used. The anti-Legionella activity of five exotic essential oils and five Mediterranean essential oils characteristic for coastal Croatia was examined. Model organism used in experiments was L. pneumophila (strain 130b). This experiment was conducting with modified version of sealed plate method using a BCYE medium. The exotic essential oil with highest anti-Legionella activity was Niaouli essential oil, and the best anti-Legionella activity among Mediterranean essential oils showed Immortelle essential oil. Anti- Legionella activity of four main chemical compounds was examined and compound that show significant highest anti-Legionella activity was α – pinene. Volatile components of essential oils have a great potential as anti-Legionella agents and further research are needed.

scholarly journals The presence and absence of periplasmic rings in bacterial flagellar motors correlates with stator type

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Mohammed Kaplan ◽  
Debnath Ghosal ◽  
Poorna Subramanian ◽  
Catherine M Oikonomou ◽  
Andreas Kjaer ◽  
...  
Keyword(s):  
Legionella Pneumophila ◽  
Pathogenic Bacteria ◽  
Bioinformatics Analysis ◽  
Cell Envelope ◽  
Shewanella Oneidensis ◽  
Flagellar Motor ◽  
Flagellar Motors ◽  
A Cell ◽  
Animal Hosts

The bacterial flagellar motor, a cell-envelope-embedded macromolecular machine that functions as a cellular propeller, exhibits significant structural variability between species. Different torque-generating stator modules allow motors to operate in different pH, salt or viscosity levels. How such diversity evolved is unknown. Here, we use electron cryo-tomography to determine the in situ macromolecular structures of three Gammaproteobacteria motors: Legionella pneumophila, Pseudomonas aeruginosa, and Shewanella oneidensis, providing the first views of intact motors with dual stator systems. Complementing our imaging with bioinformatics analysis, we find a correlation between the motor’s stator system and its structural elaboration. Motors with a single H+-driven stator have only the core periplasmic P- and L-rings; those with dual H+-driven stators have an elaborated P-ring; and motors with Na+ or Na+/H+-driven stators have both their P- and L-rings embellished. Our results suggest an evolution of structural elaboration that may have enabled pathogenic bacteria to colonize higher-viscosity environments in animal hosts.

The PPIase Active Site of Legionella pneumophila Mip Protein Is Involved in the Infection of Eukaryotic Host Cells

2003 ◽  
Vol 384 (1) ◽  
Author(s):  
J.H. Helbig ◽  
B. König ◽  
H. Knospe ◽  
B. Bubert ◽  
C. Yu ◽  
...  
Keyword(s):  
Legionella Pneumophila ◽  
Active Site ◽  
Host Cells

scholarly journals Sensitivity of Vermamoeba (Hartmannella) vermiformis cysts to conventional disinfectants and protease

2014 ◽  
Vol 13 (2) ◽  
pp. 302-310 ◽  
Author(s):  
Emilie Fouque ◽  
Yann Héchard ◽  
Philippe Hartemann ◽  
Philippe Humeau ◽  
Marie-Cécile Trouilhé
Keyword(s):  
Legionella Pneumophila ◽  
Pathogenic Bacteria ◽  
Hot Water ◽  
Water Networks ◽  
Important Health ◽  
Hartmannella Vermiformis ◽  
Important Health Issue ◽  
Few Data ◽  
First Time

Vermamoeba vermiformis is a free-living amoeba (FLA) widely distributed in the environment, known to colonize hot water networks and to be the reservoir of pathogenic bacteria such as Legionella pneumophila. FLA are partly resistant to biocides, especially in their cyst form. The control of V. vermiformis in hot water networks represents an important health issue, but there are very few data on their resistance to disinfection treatments. The sensitivity of cysts of two strains of V. vermiformis to three disinfectants frequently used in hot water networks (chlorine, heat shock, peracetic acid (PAA) mixed with hydrogen peroxide (H2O2)) was investigated. In vitro, several concentrations of biocides, temperatures and exposure times according to the French regulation were tested. Cysts were fully inactivated by the following conditions: 15 mg/L of chlorine for 10 min; 60 °C for 30 min; and 0.5 g/L equivalent H2O2 of PAA mixed with H2O2 for 30 min. For the first time, the strong efficacy of subtilisin (0.625 U/mL for 24 h), a protease, to inactivate the V. vermiformis cysts has been demonstrated. It suggests that novel approaches may be efficient for disinfection processes. Finally, V. vermifomis cysts were sensitive to all the tested treatments and appeared to be more sensitive than Acanthamoeba cysts.

Fluorescence-based active site probes for profiling deubiquitinating enzymes

2012 ◽  
Vol 10 (17) ◽  
pp. 3379 ◽  
Author(s):  
Joanna F. McGouran ◽  
Holger B. Kramer ◽  
Mukram M. Mackeen ◽  
Katalin di Gleria ◽  
Mikael Altun ◽  
...  
Keyword(s):  
Active Site ◽  
Deubiquitinating Enzymes

scholarly journals His224 Alters the R2 Drug Binding Site and Phe218 Influences the Catalytic Efficiency of the Metallo-β-Lactamase VIM-7

2014 ◽  
Vol 58 (8) ◽  
pp. 4826-4836 ◽  
Author(s):  
Hanna-Kirsti S. Leiros ◽  
Susann Skagseth ◽  
Kine Susann Waade Edvardsen ◽  
Marit Sjo Lorentzen ◽  
Gro Elin Kjæreng Bjerga ◽  
...  
Keyword(s):  
Hydrogen Bonding ◽  
Active Site ◽  
Pathogenic Bacteria ◽  
Catalytic Efficiency ◽  
Drug Binding ◽  
Side Chain ◽  
Wild Type ◽  
Drug Binding Site ◽  
Chain Conformations ◽  
Positively Charged

ABSTRACTMetallo-β-lactamases (MBLs) are the causative mechanism for resistance to β-lactams, including carbapenems, in many Gram-negative pathogenic bacteria. One important family of MBLs is the Verona integron-encoded MBLs (VIM). In this study, the importance of residues Asp120, Phe218, and His224 in the most divergent VIM variant, VIM-7, was investigated to better understand the roles of these residues in VIM enzymes through mutations, enzyme kinetics, crystal structures, thermostability, and docking experiments. The tVIM-7-D120A mutant with a tobacco etch virus (TEV) cleavage site was enzymatically inactive, and its structure showed the presence of only the Zn1 ion. The mutant was less thermostable, with a melting temperature (Tm) of 48.5°C, compared to 55.3°C for the wild-type tVIM-7. In the F218Y mutant, a hydrogen bonding cluster was established involving residues Asn70, Asp84, and Arg121. The tVIM-7-F218Y mutant had enhanced activity compared to wild-type tVIM-7, and a slightly higherTm(57.1°C) was observed, most likely due to the hydrogen bonding cluster. Furthermore, the introduction of two additional hydrogen bonds adjacent to the active site in the tVIM-7-H224Y mutant gave a higher thermostability (Tm, 62.9°C) and increased enzymatic activity compared to those of the wild-type tVIM-7. Docking of ceftazidime in to the active site of tVIM-7, tVIM-7-H224Y, and VIM-7-F218Y revealed that the side-chain conformations of residue 224 and Arg228 in the L3 loop and Tyr67 in the L1 loop all influence possible substrate binding conformations. In conclusion, the residue composition of the L3 loop, as shown with the single H224Y mutation, is important for activity particularly toward the positively charged cephalosporins like cefepime and ceftazidime.

Substrate interaction dynamics and oxygen control in the active site of thymidylate synthase ThyX

2014 ◽  
Vol 459 (1) ◽  
pp. 37-45 ◽  
Author(s):  
Hubert F. Becker ◽  
Kamel Djaout ◽  
Isabelle Lamarre ◽  
Jonathan E. Ulmer ◽  
Delphine Schaming ◽  
...  
Keyword(s):  
Thymidylate Synthase ◽  
Active Site ◽  
Oxidase Activity ◽  
Pathogenic Bacteria ◽  
Oxygen Control ◽  
Time Resolved ◽  
Time Resolved Spectroscopy ◽  
Substrate Interaction ◽  
Interaction Dynamics

Thymidylate synthase ThyX is required for synthesis of thymine in many pathogenic bacteria. Using time-resolved spectroscopy of the flavin cofactor, we unravelled the interplay between the three native substrates and show how harmful oxidase activity is avoided in aerobic environments.

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