scholarly journals Molecular Insights into Functional Differences between mcr-3- and mcr-1-Mediated Colistin Resistance

2018 ◽  
Vol 62 (9) ◽  
Author(s):  
Hui Li ◽  
Lu Yang ◽  
Zhihai Liu ◽  
Wenjuan Yin ◽  
Dejun Liu ◽  
...  
Keyword(s):  
Lipid A ◽  
Catalytic Mechanism ◽  
Sequence Divergence ◽  
Multidrug Resistant ◽  
Site Directed Mutagenesis ◽  
Head Group ◽  
Colistin Resistance ◽  
Flight Mass Spectrometry ◽  
Functional Differences

ABSTRACT The global emergence of plasmid-mediated colistin resistance genes mcr-1 and mcr-3 has threatened the role of the “last-resort” drug colistin in the defense against infections caused by multidrug-resistant Gram-negative bacteria. However, functional differences between these two genes in mediating colistin resistance remain poorly understood. Protein sequence alignment of MCR-3 and MCR-1 was therefore conducted in Clustal Omega to identify sequence divergence. The molecular recognition of lipid A head group phosphatidylethanolamine and MCR-3 enzyme was studied by homology modeling and molecular docking, with the catalytic mechanism of MCR-3 also being explored. Thr277 in MCR-3 was validated as the key amino acid residue responsible for the catalytic reaction using site-directed mutagenesis and was shown to act as a nucleophile. Lipid A modification induced by the MCR-3 and MCR-1 enzymes was confirmed by electrospray ionization–time of flight mass spectrometry. Far-UV circular dichroism spectra of the MCR-3 and MCR-1 enzymes suggested that MCR-3 was more thermostable than MCR-1, with a melting temperature of 66.19°C compared with 61.14°C for MCR-1. These data provided molecular insight into the functional differences between mcr-3 and mcr-1 in conferring colistin resistance.

scholarly journals Molecular survey of mcr1 and mcr2 plasmid mediated colistin resistance genes in Escherichia coli isolates of animal origin in Iran

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Kayhan Ilbeigi ◽  
Mahdi Askari Badouei ◽  
Hossein Vaezi ◽  
Hassan Zaheri ◽  
Sina Aghasharif ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Resistance Genes ◽  
Companion Animals ◽  
Multidrug Resistant ◽  
Animal Origin ◽  
Colistin Resistance ◽  
E Coli ◽  
High Level ◽  
Farming Industry

Abstract Objectives The emergence of colistin-resistant Enterobacteriaceae from human and animal sources is one of the major public health concerns as colistin is the last-resort antibiotic for treating infections caused by multidrug-resistant Gram-negative bacteria. We aimed to determine the prevalence of the prototype widespread colistin resistance genes (mcr-1 and mcr-2) among commensal and pathogenic Escherichia coli strains isolated from food-producing and companion animals in Iran. Results A total of 607 E. coli isolates which were previously collected from different animal sources between 2008 and 2016 used to uncover the possible presence of plasmid-mediated colistin resistance genes (mcr-1 and mcr-2) by PCR. Overall, our results could not confirm the presence of any mcr-1 or mcr-2 positive E. coli among the studied isolates. It is concluded that despite the important role of food-producing animals in transferring the antibiotic resistance, they were not the main source for carriage of mcr-1 and mcr-2 in Iran until 2016. This study suggests that the other mcr variants (mcr-3 to mcr-9) might be responsible for conferring colistin resistance in animal isolates in Iran. The possible linkage between pig farming industry and high level of mcr carriage in some countries needs to be clarified in future prospective studies.

scholarly journals Colistin heteroresistance and the involvement of the PmrAB regulatory system in Acinetobacter baumannii

2018 ◽  
Author(s):  
Yannick Charretier ◽  
Seydina M. Diene ◽  
Damien Baud ◽  
Sonia Chatellier ◽  
Emmanuelle Santiago-Allexant ◽  
...  
Keyword(s):  
Acinetobacter Baumannii ◽  
Population Analysis ◽  
Regulatory System ◽  
Clinical Problem ◽  
Multidrug Resistant ◽  
Regulatory Pathway ◽  
Colistin Resistance ◽  
Bacterial Killing ◽  
One Step

AbstractMultidrug-resistant Acinetobacter baumannii infection has recently emerged as a worldwide clinical problem and colistin is increasingly being used as last resort therapy. Despite its favorable bacterial killing, resistance and heteroresistance to colistin have been described. Mutations in the PmrAB regulatory pathway have been already associated with colistin resistance whereas the mechanisms for heteroresistance remain largely unknown. The purpose of the present study is to investigate the role of PmrAB in laboratory-selected mutants representative of global epidemic strains. During brief colistin exposure, colistin resistant and colistin heteroresistant mutants were selected in a one-step strategy. Population Analysis Profiling (PAP) was performed to confirm the suspected phenotype. Upon withdrawal of selective pressure, compensatory mutations were evaluated in another one-step strategy. A trans-complementation assay was designed to delineate the involvement of the PmrAB regulatory system using qPCR and PAP. Mutations in the PmrAB regulatory pathway were associated with colistin resistance and colistin heteroresistance as well. The transcomplementation assay provides a proof for the role played by changes in the PmrAB regulatory pathway. The level of colistin resistance is correlated to the level of expression of pmrC. The resistance phenotype was partially restored since the complemented strain became heteroresistant. This report shows the role of different mutations in the PmrAB regulatory pathway and warns on the development of colistin heteroresistance that could be present but not easily detected with routine testing.

scholarly journals Modification of Lipooligosaccharide with Phosphoethanolamine by LptA in Neisseria meningitidis Enhances Meningococcal Adhesion to Human Endothelial and Epithelial Cells

2008 ◽  
Vol 76 (12) ◽  
pp. 5777-5789 ◽  
Author(s):  
Hideyuki Takahashi ◽  
Russel W. Carlson ◽  
Artur Muszynski ◽  
Biswa Choudhury ◽  
Kwang Sik Kim ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Neisseria Meningitidis ◽  
Lipid A ◽  
Inner Core ◽  
Mass Spectrometry Analysis ◽  
Wild Type ◽  
Spectrometry Analysis ◽  
Flight Mass Spectrometry ◽  
Epithelial Cell Lines

ABSTRACT The lipooligosaccharide (LOS) of Neisseria meningitidis can be decorated with phosphoethanolamine (PEA) at the 4′ position of lipid A and at the O-3 and O-6 positions of the inner core of the heptose II residue. The biological role of PEA modification in N. meningitidis remains unclear. During the course of our studies to elucidate the pathogenicity of the ST-2032 (invasive) meningococcal clonal group, disruption of lptA, the gene that encodes the PEA transferase for 4′ lipid A, led to a approximately 10-fold decrease in N. meningitidis adhesion to four kinds of human endothelial and epithelial cell lines at an multiplicity of infection of 5,000. Complementation of the lptA gene in a ΔlptA mutant restored wild-type adherence. By matrix-assisted laser desorption ionization-time-of-flight mass spectrometry analysis, PEA was lost from the lipid A of the ΔlptA mutant compared to that of the wild-type strain. The effect of LptA on meningococcal adhesion was independent of other adhesins such as pili, Opc, Opa, and PilC but was inhibited by the presence of capsule. These results indicate that modification of LOS with PEA by LptA enhances meningococcal adhesion to human endothelial and epithelial cells in unencapsulated N. meningitidis.

scholarly journals Genetic and Functional Analyses of PptA, a Phospho-Form Transferase Targeting Type IV Pili in Neisseria gonorrhoeae

2007 ◽  
Vol 190 (1) ◽  
pp. 387-400 ◽  
Author(s):  
Cecilia L. Næssan ◽  
Wolfgang Egge-Jacobsen ◽  
Ryan W. Heiniger ◽  
Matthew C. Wolfgang ◽  
Finn Erik Aas ◽  
...  
Keyword(s):  
Neisseria Gonorrhoeae ◽  
Metal Binding ◽  
Catalytic Mechanism ◽  
Phylogenetic Analyses ◽  
Site Directed Mutagenesis ◽  
Head Group ◽  
Sequence Alignments ◽  
Type Iv ◽  
Covalent Modifications ◽  
Division Cell

ABSTRACT The PilE pilin subunit protein of Neisseria gonorrhoeae undergoes unique covalent modifications with phosphoethanolamine (PE) and phosphocholine (PC). The pilin phospho-form transferase A (PptA) protein, required for these modifications, shows sequence relatedness with and architectural similarities to lipopolysaccharide PE transferases. Here, we used regulated expression and mutagenesis as means to better define the relationships between PptA structure and function, as well as to probe the mechanisms by which other factors impact the system. We show here that pptA expression is coupled at the level of transcription to its distal gene, murF, in a division/cell wall gene operon and that PptA can act in a dose-dependent fashion in PilE phospho-form modification. Molecular modeling and site-directed mutagenesis provided the first direct evidence that PptA is a member of the alkaline phosphatase superfamily of metalloenzymes with similar metal-binding sites and conserved structural folds. Through phylogenetic analyses and sequence alignments, these conclusions were extended to include the lipopolysaccharide PE transferases, including members of the disparate Lpt6 subfamily, and the MdoB family of phosphoglycerol transferases. Each of these enzymes thus likely acts as a phospholipid head group transferase whose catalytic mechanism involves a trans-esterification step generating a protein-phospho-form ester intermediate. Coexpression of PptA with PilE in Pseudomonas aeruginosa resulted in high levels of PE modification but was not sufficient for PC modification. This and other findings show that PptA-associated PC modification is governed by as-yet-undefined ancillary factors unique to N. gonorrhoeae.

scholarly journals Exogenous and Endogenous Phosphoethanolamine Transferases Differently Affect Colistin Resistance and Fitness in Pseudomonas aeruginosa

2021 ◽  
Vol 12 ◽  
Author(s):  
Matteo Cervoni ◽  
Alessandra Lo Sciuto ◽  
Chiara Bianchini ◽  
Carmine Mancone ◽  
Francesco Imperi
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Lipid A ◽  
Cell Envelope ◽  
Resistance Mechanism ◽  
Multidrug Resistant ◽  
Transferase Activity ◽  
Colistin Resistance ◽  
Phosphoethanolamine Transferases ◽  
Positively Charged ◽  
Inducible Gene

Colistin represents a last-line treatment option for infections caused by multidrug resistant Gram-negative pathogens, including Pseudomonas aeruginosa. Colistin resistance generally involves the modification of the lipid A moiety of lipopolysaccharide (LPS) with positively charged molecules, namely phosphoethanolamine (PEtN) or 4-amino-4-deoxy-L-arabinose (Ara4N), that reduce colistin affinity for its target. Several lines of evidence highlighted lipid A aminoarabinosylation as the primary colistin resistance mechanism in P. aeruginosa, while the contribution of phosphoethanolamination remains elusive. PEtN modification can be due to either endogenous (chromosomally encoded) PEtN transferase(s) (e.g., EptA in P. aeruginosa) or plasmid borne MCR enzymes, commonly found in enterobacteria. By individually cloning eptA and mcr-1 into a plasmid for inducible gene expression, we demonstrated that MCR-1 and EptA have comparable PEtN transferase activity in P. aeruginosa and confer colistin resistance levels similar to those provided by lipid A aminoarabinosylation. Notably, EptA, but not MCR-1, negatively affects P. aeruginosa growth and, to a lesser extent, cell envelope integrity when expressed at high levels. Mutagenesis experiments revealed that PEtN transferase activity does not account for the noxious effects of EptA overexpression, that instead requires a C-terminal tail unique to P. aeruginosa EptA, whose function remains unknown. Overall, this study shows that both endogenous and exogenous PEtN transferases can promote colistin resistance in P. aeruginosa, and that PEtN and MCR-1 mediated resistance has no impact on growth and cell envelope homeostasis, suggesting that there may be no fitness barriers to the spread of mcr-1 in P. aeruginosa.

scholarly journals An amphipathic and cationic antimicrobial peptide kills colistin resistant Gram-negative pathogens in vivo

2021 ◽  
Author(s):  
Thomas T. Thomsen ◽  
Mette Kolpen ◽  
Vinoth Wigneswaran ◽  
Ulrik Kromann ◽  
Anna Ebbensgaard ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Lipid A ◽  
Multidrug Resistant ◽  
Colistin Resistance ◽  
Cationic Antimicrobial Peptide ◽  
Gram Negative ◽  
Excellent Activity ◽  
New Antibiotics ◽  
Charge Change

New antibiotics are needed against multidrug resistant Gram-negative pathogens that have compromised global health systems. Antimicrobial peptides are generally considered promising lead candidates for the next generation of antibiotics but have not fulfilled this expectation. Here we demonstrate activity of a cationic amphipathic undecapeptide (ChIP; Charge change Independent Peptide) against a wide panel of multidrug resistant Gram-negative pathogens. Importantly, the antimicrobial activity of ChIP is independent of the surface charge changes that confer colistin resistance through modification of Lipid A, while decreased activity of ChIP correlates with GlcN1 tri-acylation of Lipid A. In an in vivo peritonitis mouse model ChIP displays excellent activity against both colistin sensitive and resistant Escherichia coli and Acinetobacter baumannii strains.

scholarly journals Catalytic Mechanism of the Colistin Resistance Protein MCR-1

2020 ◽  
Author(s):  
Reynier Suardiaz ◽  
Emily Lythell ◽  
Philip Hinchliffed ◽  
Marc van der Kamp ◽  
James Spencer ◽  
...  
Keyword(s):  
Reaction Mechanism ◽  
Lipid A ◽  
Catalytic Mechanism ◽  
Second Step ◽  
Colistin Resistance ◽  
Membrane Bound ◽  
Resistance Protein ◽  
Phosphoethanolamine Transferases ◽  
Bacterial Lipid ◽  
Rate Limiting

<div> <div> <div> <p>The mcr-1 gene encodes a membrane-bound Zn2+-metalloenzyme, MCR-1, which catalyzes phosphoethanolamine transfer onto bacterial lipid A, making bacteria resistant to colistin, a last-resort antibiotic. Mechanistic understanding of this process remains incomplete. Here, we investigate possible catalytic pathways using DFT and ab initio calculations on cluster models and identify a complete two-step reaction mechanism. The first step, formation of a covalent phosphointermediate via trans-fer of phosphoethanolamine from a membrane phospholipid donor to the acceptor Thr285, is rate-limiting and proceeds with a single Zn2+ ion. The second step, transfer of the phosphoethanolamine group to lipid A, requires an additional Zn2+. The calculations suggest the involment of the Zn2+ orbitals directly in the reaction is limited, with the second Zn2+ acting to bind incoming lipid A and direct phosphoethanolamine addition. The new level of mechanistic detail obtained here, which distinguishes these enzymes from other phosphotransferases, will aid in the development of inhibitors specific to MCR-1 and related bacterial phosphoethanolamine transferases. </p> </div> </div> </div>

scholarly journals Detection of Colistin Resistance in Pseudomonas aeruginosa Using the MALDIxin Test on the Routine MALDI Biotyper Sirius Mass Spectrometer

2021 ◽  
Vol 12 ◽  
Author(s):  
Katy Jeannot ◽  
Katheryn Hagart ◽  
Laurent Dortet ◽  
Markus Kostrzewa ◽  
Alain Filloux ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Lipid A ◽  
Response Regulator ◽  
Multidrug Resistant ◽  
Positive Control ◽  
Colistin Resistance ◽  
Clinical Strains ◽  
Efficient Detection ◽  
Maldi Biotyper ◽  
Resistant Strains

Colistin is frequently a last resort treatment for Pseudomonas aeruginosa infections caused by multidrug-resistant (MDR) and extensively drug resistant (XDR) strains, and detection of colistin resistance is essential for the management of infected patients. Therefore, we evaluated the recently developed MALDIxin test for the detection of colistin resistance in P. aeruginosa clinical strains using the routine matrix-assisted laser desorption ionization (MALDI) Biotyper Sirius system. The test is based on the detection by mass spectrometry of modified lipid A by the addition of 4-amino-l-arabinose (l-ara4N) molecules on one or two phosphate groups, in strains resistant to colistin. Overproduction of l-Ara4N molecules is mainly due to the constitutive activation of the histidine kinase (PmrB) or the response regulator (PmrA) following an amino-acid substitution in clinical strains. The performance of the test was determined on a panel of 14 colistin-susceptible and 14 colistin-resistant P. aeruginosa clinical strains, the reference strain PAO1 and positive control mutants PmrB (V28G), PmrB (D172), PhoQ (D240–247), and ParR (M59I). In comparison with the broth microdilution (BMD) method, all the susceptible strains (n=14) and 8/14 colistin-resistant strains were detected in less than 1h, directly on whole bacteria. The remaining resistant strains (n=6) were all detected after a short pre-exposure (4h) to colistin before sample preparation. Validation of the method on a larger panel of strains will be the next step before its use in diagnostics laboratories. Our data showed that the MALDIxin test offers rapid and efficient detection of colistin resistant P. aeruginosa and is thus a valuable diagnostics tool to control the spread of these emerging resistant strains.

scholarly journals A Molecular Perspective on Colistin and Klebsiella pneumoniae: Mode of Action, Resistance Genetics, and Phenotypic Susceptibility

Diagnostics ◽  
2021 ◽  
Vol 11 (7) ◽  
pp. 1165
Author(s):  
Rita Elias ◽  
Aida Duarte ◽  
João Perdigão
Keyword(s):  
Klebsiella Pneumoniae ◽  
Mode Of Action ◽  
Lipid A ◽  
Drug Susceptibility ◽  
Multidrug Resistant ◽  
Resistance Mechanisms ◽  
Drug Susceptibility Testing ◽  
Gram Negative Bacteria ◽  
Colistin Resistance ◽  
Gram Negative

Klebsiella pneumoniae is a rod-shaped, encapsulated, Gram-negative bacteria associated with multiple nosocomial infections. Multidrug-resistant (MDR) K. pneumoniae strains have been increasing and the therapeutic options are increasingly limited. Colistin is a long-used, polycationic, heptapeptide that has regained attention due to its activity against Gram-negative bacteria, including the MDR K. pneumoniae strains. However, this antibiotic has a complex mode of action that is still under research along with numerous side-effects. The acquisition of colistin resistance is mainly associated with alteration of lipid A net charge through the addition of cationic groups synthesized by the gene products of a multi-genic regulatory network. Besides mutations in these chromosomal genes, colistin resistance can also be achieved through the acquisition of plasmid-encoded genes. Nevertheless, the diversity of molecular markers for colistin resistance along with some adverse colistin properties compromises the reliability of colistin-resistance monitorization methods. The present review is focused on the colistin action and molecular resistance mechanisms, along with specific limitations on drug susceptibility testing for K. pneumoniae.

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