Novel small molecules that increase the susceptibility of Neisseria gonorrhoeae to cationic antimicrobial peptides by inhibiting lipid A phosphoethanolamine transferase.

OBJECTIVES: Neisseria gonorrhoeae is an exclusively human pathogen that commonly infects the urogenital tract resulting in gonorrhoea. Empirical treatment of gonorrhoea with antibiotics has led to multidrug resistance and the need for new therapeutics. Inactivation of lipooligosaccharide phosphoethanolamine transferase A (EptA), which attaches phosphoethanolamine to lipid A, results in attenuation of the pathogen in infection models. Small molecules that inhibit EptA are predicted to enhance natural clearance of gonococci via the human innate immune response. METHODS: A library of small-fragment compounds was tested for the ability to enhance susceptibility of the reference strain N. gonorrhoeae FA1090 to polymyxin B. The effect of these compounds on lipid A synthesis and viability in models of infection were tested. RESULTS: Three compounds, 135, 136 and 137, enhanced susceptibility of strain FA1090 to polymyxin B by 4-fold. Pre-treatment of bacterial cells with all three compounds resulted in enhanced killing by macrophages. Only lipid A from bacterial cells exposed to compound 137 showed a 17% reduction in the level of decoration of lipid A with phosphoethanolamine by MALDI-TOF MS analysis and reduced stimulation of cytokine responses in THP-1 cells. Binding of 137 occurred with higher affinity to purified EptA than the starting material, as determined by 1D saturation transfer difference NMR. Treatment of eight MDR strains with 137 increased susceptibility to polymyxin B in all cases. CONCLUSIONS: Small molecules have been designed that bind to EptA, inhibit addition of phosphoethanolamine to lipid A and can sensitize N. gonorrhoeae to killing by macrophages.

Emergence of a highly colistin-resistant Aeromonas jandaei clinical isolate harbouring four genes encoding phosphoethanolamine transferases in Nepal.

OBJECTIVES: This study aimed to describe a clinical isolate of Aeromonas jandaei (A. jandaei) in Nepal that harboured four types of genes encoding phosphoethanolamine transferases. METHODS: An isolate of colistin-resistant A. jandaei was obtained from a blood sample of an inpatient in a hospital in Nepal, and its complete genome sequence was determined. Escherichia coli (E. coli) and Aeromonas hydrophila (A. hydrophila) transformants expressing genes encoding novel phosphoethanolamine transferase variants were constructed and colistin-susceptibility profiles were determined. RESULTS: The isolate harboured four genes encoding phosphoethanolamine transferases on the chromosome, which were designated eptAv3.2, eptAv3.3, eptAv3.4 and eptAv7.2. The amino acid sequences of EptAv3.2, 3.3 and 3.4 were > 80% identical to MCR-3.1, and that of EptAv7.2 was > 79% identical to MCR-7.1. E. coli expressing eptAv3.2, 3.3 and 3.4 showed reduced susceptibility to colistin, whereas E. coli expressing eptAv7.2 did not. In contrast, A. hydrophila expressing eptAv7.2 showed reduced susceptibility to colistin, whereas A. hydrophila expressing eptAv3.2, 3.3 and 3.4 did not; eptAv3.3 and 3.4 formed a tandem structure. The genomic environments surrounding eptAv3.2, 3.3 and 3.4 were similar to Aeromonas veronii obtained from the effluent of a treatment plant in Japan in 2018. The genomic environment surrounding eptAv7.2 was similar to that of A. jandaei obtained from a chicken in the USA in 2019. CONCLUSIONS: The highly colistin-resistant A. jandaei clinical isolate harboured four chromosomal genes encoding phosphoethanolamine transferases, suggesting that Aeromonas spp. harbouring eptAv genes with strong similarities to mcr-3 and mcr-7 are emerging in medical settings as well as environments.

Diacylglycerol Kinase A Is Essential for Polymyxin Resistance Provided by EptA, MCR-1, and Other Lipid A Phosphoethanolamine Transferases.

Gram-negative bacteria utilize glycerophospholipids (GPLs) as phospho-form donors to modify various surface structures. These modifications play important roles in bacterial fitness in diverse environments influencing cell motility, recognition by the host during infection, and antimicrobial resistance. A well-known example is the modification of the lipid A component of lipopolysaccharide by the phosphoethanolamine (pEtN) transferase EptA that utilizes phosphatidyethanoalmine (PE) as the phospho-form donor. Addition of pEtN to lipid A promotes resistance to cationic antimicrobial peptides (CAMPs), including the polymyxin antibiotics like colistin. A consequence of pEtN modification is the production of diacylglycerol (DAG) that must be recycled back into GPL synthesis via the diacylglycerol kinase A (DgkA). DgkA phosphorylates DAG forming phosphatidic acid, the precursor for GPL synthesis. Here we report that deletion of dgkA in polymyxin-resistant E. coli results in a severe reduction of pEtN modification and loss of antibiotic resistance. We demonstrate that inhibition of EptA is regulated posttranscriptionally and is not due to EptA degradation during DAG accumulation. We also show that the inhibition of lipid A modification by DAG is a conserved feature of different Gram-negative pEtN transferases. Altogether, our data suggests that inhibition of EptA activity during DAG accumulation likely prevents disruption of GPL synthesis helping to maintain cell envelope homeostasis. IMPORTANCE For Gram-negative bacteria, modification of a key surface structure known as lipopolysaccharide (LPS) is critical for resistance to cationic antimicrobial peptides, including the last-resort antibiotic polymyxin. One key enzyme that is critical for resistance is EptA that adds a positively charged residue to LPS, preventing polymyxin binding. Here we show that EptA can be posttranscriptionally regulated by a key cell envelope lipid leading to changes in antibiotic resistance.

Reduction trend of mcr-1 circulation in Emilia-Romagna Region, Italy.

This study aims to describe trends of mcr-positive Enterobacterales in humans based on laboratory surveillance with a defined catchment population. The data source is the Micro-RER surveillance system, established in Emilia-Romagna region (Italy), to monitor the trend of mcr resistance. Enterobacterales isolates from human clinical samples with minimum inhibitory concentration (MIC) ≥ 2 mg/L for colistin were sent to the study reference laboratory for the detection of mcr genes. Isolates prospectively collected in the period 2018-2020 were considered for the assessment of population rates and trends; further analyses were carried out for the evaluation of clonality and horizontal mcr gene transfer. Previous isolates from local laboratory collection were also described. In the period 2018-2020, 1164 isolates were sent to the reference laboratory, and 51 (4.4%) were confirmed as mcr-positive: 50 mcr-1 (42 Escherichia coli, 6 Klebsiella pneumoniae, 2 Salmonella enterica) and 1 mcr-4 (Enterobacter cloacae). The number of mcr-positive isolates dropped from 24 in the first half of 2018 to 3 in the whole of 2020 (trend p value < 0.001). Genomic analyses showed the predominant role of the horizontal transfer of mcr genes through plasmids or dissemination of transposable elements compared to clonal dissemination of mcr-positive microorganisms. The study results demonstrate a substantial decrease in the circulation of mcr-1 plasmid genes in Emilia-Romagna Region.

Molecular genetic characteristics of mcr-9-harbouring Salmonella enterica serotype Typhimurium isolated from raw milk.

Among the 10 reported mcr genes, mcr-9 was first identified in Salmonella enterica serotype Typhimurium, which is a leading cause of foodborne illness worldwide. However, information about the prevalence and genetic features of mcr-9 is still lacking, especially among food samples. This study reports the presence of mcr-9 in raw milk samples from China; the prevalence rate was low (0.83%, 1/120). mcr-9 was located on a transferable plasmid, and was stable in wild-type S. enterica. However, it had a biological fitness cost when transferred to an Escherichia coli recipient. Whole-genome sequencing revealed that mcr-9 was located on the IncHI2A-type plasmid, and was surrounded by IS903B and IS26 in its flanking regions. The mcr-9-carrying S. enterica 19SE belonged to ST26 and had a multi-drug-resistant phenotype. It was confirmed that mcr-9 did not mediate colistin resistance in this study, indicating that its transfer may not facilitate the dissemination of colistin resistance.

Global impact of mcr-1-positive Enterobacteriaceae bacteria on "one health".

Polymyxins, especially polymyxin B and polymyxin E (colistin), are considered to be the last line of defence against infections caused by multi-drug-resistant (MDR) gram-negative bacteria such as carbapenem-resistant Enterobacteriaceae (CRE). However, the recent emergence and dissemination of the plasmid-mediated colistin resistance gene mcr-1 and its variants pose a serious challenge to public health and the livestock industry. This review describes the prevalence and dissemination of mcr-1-positive isolates from different sources, including animals (food animals, pet animals and wildlife), humans (healthy populations and patients) and the environment (farms, urban and rural communities and natural environments) based on existing epidemiological studies of mcr-1 and MCR-1-producing Enterobacteriaceae bacteria around the world. The major mechanisms of mcr-1 transmission across humans, animals and the environment are discussed.

Locations and contributions of the phosphotransferases EPT1 and CEPT1 to the biosynthesis of ethanolamine phospholipids.

The final step of the CDP-ethanolamine pathway is catalyzed by ethanolamine phosphotransferase 1 (EPT1) and choline/EPT1 (CEPT1). These enzymes are likely involved in the transfer of ethanolamine phosphate from CDP-ethanolamine to lipid acceptors such as 1,2-diacylglycerol (DAG) for PE production and 1-alkyl-2-acyl-glycerol (AAG) for the generation of 1-alkyl-2-acyl-glycerophosphoethanolamine. Here, we investigated the intracellular location and contribution to ethanolamine phospholipid (EP) biosynthesis of EPT1 and CEPT1 in HEK293 cells. Immunohistochemical analyses revealed that EPT1 localizes to the Golgi apparatus and CEPT1 to the ER. We created EPT1-, CEPT1-, and EPTI-CEPT1-deficient cells, and labeling of these cells with radio- or deuterium-labeled ethanolamine disclosed that EPT1 is more important for the de novo biosynthesis of 1-alkenyl-2-acyl-glycerophosphoethanolamine than is CEPT1. EPT1 also contributed to the synthesis of PE species containing the fatty acids 36:1, 36:4, 38:5, 38:4, 38:3, 40:6, 40:5, and 40:4. In contrast, CEPT1 was important for PE formation from shorter fatty acids such as 32:2, 32:1, 34:2, and 34:1. Brefeldin A treatment did not significantly affect the levels of the different PE species, indicating that the subcellular localization of the two enzymes is not responsible for their substrate preferences. In vitro enzymatic analysis revealed that EPT1 prefers AAG 16-20:4 > DAG 18:0-20:4 > DAG 16:0-18:1 = AAG 16-18:1 as lipid acceptors and that CEPT1 greatly prefers DAG 16:0-18:1 to other acceptors. These results suggest that EPT1 and CEPT1 differ in organelle location and are responsible for the biosynthesis of distinct EP species.

Lipid A Phosphoethanolamine Transferase: Regulation, Structure and Immune Response.

A wide variety of antibiotics are targeted to the bacterial membrane due to its unique arrangement and composition relative to the host mammalian membranes. By modification of their membranes, some gram-negative pathogens resist the action of antibiotics. Lipid A phosphoethanolamine transferase (EptA) is an intramembrane enzyme that modifies the lipid A portion of lipopolysaccharide/lipooligosaccharide by the addition of phosphoethanolamine. This modification reduces the overall net-negative charge of the outer membrane of some gram-negative bacteria, conferring resistance to polymyxin. This resistance mechanism has resulted in a global public health issue due to the increased use of polymyxin as last-resort antibiotic treatments against multi-drug-resistant pathogens. Studies show that, without EptA, pathogenic bacteria become more sensitive to polymyxin and to clearance by the host immune system, suggesting the importance of this target enzyme for the development of novel therapeutic agents. In this review, EptA will be discussed comprehensively. Specifically, this review will cover the regulation of eptA expression by the two component systems PmrA/PmrB and PhoP/PhoQ, the site of modification on lipid A, the structure and catalytic mechanism of EptA in comparison to MCR-1 and Escherichia coli alkaline phosphatase, and the host immune system's response to lipid A modification by EptA. The overarching aim of this review is to provide a comprehensive overview of polymyxin resistance mediated by EptA.

The Escherichia coli cellulose synthase subunit G (BcsG) is a Zn2+-dependent phosphoethanolamine transferase.

Bacterial biofilms are cellular communities that produce an adherent matrix. Exopolysaccharides are key structural components of this matrix and are required for the assembly and architecture of biofilms produced by a wide variety of microorganisms. The human bacterial pathogens Escherichia coli and Salmonella enterica produce a biofilm matrix composed primarily of the exopolysaccharide phosphoethanolamine (pEtN) cellulose. Once thought to be composed of only underivatized cellulose, the pEtN modification present in these matrices has been implicated in the overall architecture and integrity of the biofilm. However, an understanding of the mechanism underlying pEtN derivatization of the cellulose exopolysaccharide remains elusive. The bacterial cellulose synthase subunit G (BcsG) is a predicted inner membrane-localized metalloenzyme that has been proposed to catalyze the transfer of the pEtN group from membrane phospholipids to cellulose. Here we present evidence that the C-terminal domain of BcsG from E. coli (EcBcsGΔN) functions as a phosphoethanolamine transferase in vitro with substrate preference for cellulosic materials. Structural characterization of EcBcsGΔN revealed that it belongs to the alkaline phosphatase superfamily, contains a Zn2+ ion at its active center, and is structurally similar to characterized enzymes that confer colistin resistance in Gram-negative bacteria. Informed by our structural studies, we present a functional complementation experiment in E. coli AR3110, indicating that the activity of the BcsG C-terminal domain is essential for integrity of the pellicular biofilm. Furthermore, our results established a similar but distinct active-site architecture and catalytic mechanism shared between BcsG and the colistin resistance enzymes.

Reduced Fitness Costs of mcr-1.2 Compared to Mutated pmrB in Isogenic Colistin-Resistant KPC-3-Producing Klebsiella pneumoniae.

In the present study, we provide the results of a detailed genomic analysis and the growth characteristics of a colistin-resistant KPC-3-producing Klebsiella pneumoniae sequence type 512 (ST512) isolate (the colR-KPC3-KP isolate) with a mutated pmrB and isogenic isolates of colR-KPC3-KP with mcr-1.2 isolated from an immunocompromised patient. From 2014 to 2017, four colR-KPC3-KP isolates were detected in rectal swab samples collected from a pediatric hematology patient at the Azienda Ospedaliero-Universitaria Pisana in Pisa, Italy. Whole-genome sequencing was performed by MiSeq sequencing (Illumina). Growth experiments were performed using different concentrations of colistin. The growth lag phases both of an isolate harboring a deletion in pmrB and of clonal variants with mcr-1.2 were assessed by the use of real-time light-scattering measurements. In the first isolate (isolate 1000-pmrBΔ, recovered in September 2014), a 17-nucleotide deletion in pmrB was detected. In subsequent isolates, the mcr-1.2 gene associated with the plasmid pIncX4-AOUP was found, while pmrB was intact. Additionally, plasmid pIncQ-AOUP, harboring aminoglycoside resistance genes, was detected. The growth curves of the first three isolates were identical without colistin exposure; however, at higher concentrations of colistin, the growth curves of the isolate with a deletion in pmrB showed longer lag phases. We observed the replacement of mutated colR-KPC3-KP pmrB by isogenic isolates with multiple resistance plasmids, including mcr-1.2-carrying pIncX4, probably due to coselection under gentamicin treatment in a patient with prolonged colR-KPC3-KP carriage. The carriage of these isolates persisted in follow-up cultures. Coselection and the advantages in growth characteristics suggest that the plasmid-mediated resistance conferred by mcr has fewer fitness costs in colR-KPC3-KP than mutations in chromosomal pmrB, contributing to the success of this highly resistant hospital-adapted epidemiological lineage.IMPORTANCE Our study shows a successful prolonged human colonization by a colistin-resistant Klebsiella pneumoniae isolate harboring mcr-1.2 An intense antibiotic therapy contributed to the maintenance of this microorganism through the acquisition of new resistance genes. The isolates carrying mcr-1.2 showed fewer fitness costs than isogenic isolates with a pmrB mutation in the chromosome. Coselection and reduced fitness costs may explain the replacement of isolates with the pmrB mutation by other isolates and the ability of the microorganism to persist despite antibiotic treatment.

mcr-9, an Inducible Gene Encoding an Acquired Phosphoethanolamine Transferase in Escherichia coli, and Its Origin.

The plasmid-located mcr-9 gene, encoding a putative phosphoethanolamine transferase, was identified in a colistin-resistant human fecal Escherichia coli strain belonging to a very rare phylogroup, the D-ST69-O15:H6 clone. This MCR-9 protein shares 33% to 65% identity with the other plasmid-encoded MCR-type enzymes identified (MCR-1 to -8) that have been found as sources of acquired resistance to polymyxins in Enterobacteriaceae Analysis of the lipopolysaccharide of the MCR-9-producing isolate revealed a function similar to that of MCR-1 by adding a phosphoethanolamine group to lipid A and subsequently modifying the structure of the lipopolysaccharide. However, a minor impact on susceptibility to polymyxins was noticed once the mcr-9 gene was cloned and produced in an E. coli K-12-derived strain. Nevertheless, we showed here that subinhibitory concentrations of colistin induced the expression of the mcr-9 gene, leading to increased MIC levels. This inducible expression was mediated by a two-component regulatory system encoded by the qseC and qseB genes located downstream of mcr-9 Genetic analysis showed that the mcr-9 gene was carried by an IncHI2 plasmid. In silico analysis revealed that the plasmid-encoded MCR-9 shared significant amino acid identity (ca. 80%) with the chromosomally encoded MCR-like proteins from Buttiauxella spp. In particular, Buttiauxella gaviniae was found to harbor a gene encoding MCR-BG, sharing 84% identity with MCR-9. That gene was neither expressed nor inducible in its original host, which was fully susceptible to polymyxins. This work showed that mcr genes may circulate silently and remain undetected unless induced by colistin.

microRNA-16-5p promotes 3T3-L1 adipocyte differentiation through regulating EPT1.

Adipogenesis is an organized process of cellular differentiation by which pre-adipocytes differentiate towards mature adipocytes. miR-16-5p has been reported to be involved in cell proliferation, apoptosis, differentiation and angiogenesis. However little is known about miR-16-5p functional role in 3T3-L1 adipocyte differentiation. In this study, we found that miRNA-16-5p was significantly upregulated during 3T3-L1 preadipocytes differentiation towards mature adipocytes. Over-expression of miRNA-16-5p promoted mature adipocytes specific genes expression and fat droplet accumulation in vitro and in vivo. Meanwhile we have identified EPT1 as the target gene of miRNA-16-5p. Taken together, our data provided evidence to support that miRNA-16-5p promotes adipocyte differentiation by suppressing EPT1.

PmrC (EptA) and CptA Negatively Affect Outer Membrane Vesicle Production in Citrobacter rodentium.

Outer membrane vesicles (OMVs) are naturally produced by Gram-negative bacteria by a bulging of the outer membrane (OM) and subsequent release into the environment. By serving as vehicles for various cargos, including proteins, nucleic acids and small metabolites, OMVs are central to interbacterial interactions and both symbiotic and pathogenic host bacterial interactions. However, despite their importance, the mechanism of OMV formation remains unclear. Recent evidence indicates that covalent modifications of lipopolysaccharides (LPS) influence OMV biogenesis. Several enteric bacteria modify LPS with phosphoethanolamine (pEtN) using the iron-regulated PmrC (EptA) and CptA pEtN transferases. In wild-type Citrobacter rodentium, the presence of increasing subtoxic concentrations of iron was found to stimulate OMV production 4- to 9-fold above baseline. C. rodentium uses the two-component system PmrAB to sense and adapt to environmental iron. Compared to the wild type, the C. rodentium ΔpmrAB strain exhibited heightened OMV production at similar iron concentrations. PmrAB regulates transcription of pmrC (also known as eptA) and cptA OMV production in strains lacking either pmrC (eptA) or cptA was similarly increased in comparison to that of the wild type. Importantly, plasmid complementation of C. rodentium strains with either pmrC (eptA) or cptA resulted in a drastic inhibition of OMV production. Finally, we showed that ß-lactamase and CroP, two enzymes found in the C. rodentium periplasm and outer membrane (OM), respectively, are associated with OMVs. These data suggest a novel mechanism by which C. rodentium and possibly other Gram-negative bacteria can negatively affect OMV production through the PmrAB-regulated genes pmrC (eptA) and cptAIMPORTANCE Although OMVs secreted by Gram-negative bacteria fulfill multiple functions, the molecular mechanism of OMV biogenesis remains ill defined. Our group has previously shown that PmrC (also known as EptA) and CptA maintain OM integrity and provide resistance to iron toxicity and antibiotics in the murine pathogen Citrobacter rodentium In several enteric bacteria, these proteins modify the lipid A and core regions of lipopolysaccharide with phosphoethanolamine moieties. Here, we show that these proteins also repress OMV production in response to environmental iron in C. rodentium These data support the emerging understanding that lipopolysaccharide modifications are important regulators of OMV biogenesis in Gram-negative bacteria.

Targeting microbial resistance: Synthesis, antibacterial evaluation, DNA binding and modeling study of new chalcone-based dithiocarbamate derivatives.

New dithiocarbamate chalcone-based derivatives were synthesized, their structures were elucidated using different spectroscopic techniques. They were subjected to antimicrobial screening against selected Gram negative bacteria focusing on microbial resistance. Bacterial resistance was targeted via phosphoethanolamine transferase enzyme. Most of the synthesized compounds showed equal or higher activity to colistin standard. Compound 24 proved to be the most active candidate with MIC of 8 µg/ml against both Ps12 and K4 and MBC of 32 µg/ml against Ps12 and 16 µg/ml against K4 Molecular docking study showed that 20, 22, 24 and 25 had good binding affinity with active site residues via Thr280. DNA macromolecule was further targeted. Compounds 28 and 34 were recorded to have better DNA binding than doxurubucin with IC50 of 27.48 and 30.97 µg/ml respectively, suggesting that it could have a role in their higher antibacterial effect. Their docking into DNA has shown a clear intercalation matching with antibacterial data. Pharmacokinetics parameters of active compounds showed that they have better absorption through GIT.

Prevalence of the colistin resistance gene mcr-1 in colistin-resistant Escherichia coli and Klebsiella pneumoniae isolated from humans in Thailand.

OBJECTIVES: Historically, colistin has been considered a last-line therapeutic option against multidrug-resistant Gram-negative bacterial infections. However, chromosomally-encoded and plasmid-mediated colistin resistance is increasingly being reported worldwide. Spread of the plasmid-borne colistin resistance gene mcr-1 is of great concern since it can be transferred between bacteria. The aim of this study was to investigate the prevalence of mcr-1 in Escherichia coli and Klebsiella pneumoniae collected from human clinical specimens in Thailand during 2014-2017. METHODS: Minimum inhibitory concentrations (MICs) of colistin were determined by the broth microdilution method for 317 non-duplicate Enterobacteriaceae clinical isolates (37 E. coli and 280 K. pneumoniae). All isolates were screened for the mcr-1 gene by PCR. RESULTS: The colistin MIC50, MIC90 and MIC range for the 37 E. coli isolates were 0.5, 8 and 0.5-32mg/L, respectively. The mcr-1 gene was detected in 11 E. coli isolates (29.7%). Escherichia coli harbouring the mcr-1 gene had a colistin MIC range of 4-32mg/L. The colistin MIC50, MIC90, and MIC range for the 280 K. pneumoniae isolates were 32, >128, and 0.25 to >128mg/L, respectively. The mcr-1 gene was detected in 4 K. pneumoniae isolates (1.4%). Klebsiella pneumoniae harbouring the mcr-1 gene had a colistin MIC range of 4-64mg/L. CONCLUSIONS: This is the first report on the prevalence of the mcr-1 gene in colistin-resistant E. coli and K. pneumoniae isolated from humans in Thailand. These data provide added insight into the mechanism of colistin resistance among Enterobacteriaceae pathogens.

Emergence of High-Level Colistin Resistance in an Acinetobacter baumannii Clinical Isolate Mediated by Inactivation of the Global Regulator H-NS.

Colistin is a crucial last-line drug used for the treatment of life-threatening infections caused by multidrug-resistant strains of the Gram-negative bacterium Acinetobacter baumannii However, colistin-resistant A. baumannii isolates can still be isolated following failed colistin therapy. Resistance is most often mediated by the addition of phosphoethanolamine (pEtN) to lipid A by PmrC, following missense mutations in the pmrCAB operon encoding PmrC and the two-component signal transduction system PmrA/PmrB. We recovered a pair of A. baumannii isolates from a single patient before (6009-1) and after (6009-2) failed colistin treatment. These strains displayed low and very high levels of colistin resistance (MICs, 8 to 16 µg/ml and 128 µg/ml), respectively. To understand how increased colistin resistance arose, we sequenced the genome of each isolate, which revealed that 6009-2 had an extra copy of the insertion sequence element ISAba125 within a gene encoding an H-NS family transcriptional regulator. To confirm the role of H-NS in colistin resistance, we generated an hns deletion mutant in 6009-1 and showed that colistin resistance increased upon the deletion of hns We also provided 6009-2 with an intact copy of hns and showed that the strain was no longer resistant to high concentrations of colistin. Transcriptomic analysis of the clinical isolates identified more than 150 genes as being differentially expressed in the colistin-resistant hns mutant 6009-2. Importantly, the expression of eptA, encoding a second lipid A-specific pEtN transferase but not pmrC, was increased in the hns mutant. This is the first time an H-NS family transcriptional regulator has been associated with a pEtN transferase and colistin resistance.

An Evolutionarily Conserved Mechanism for Intrinsic and Transferable Polymyxin Resistance.

Polymyxins, a family of cationic antimicrobial cyclic peptides, act as a last line of defense against severe infections by Gram-negative pathogens with carbapenem resistance. In addition to the intrinsic resistance to polymyxin E (colistin) conferred by Neisseria eptA, the plasmid-borne mobilized colistin resistance gene mcr-1 has been disseminated globally since the first discovery in Southern China, in late 2015. However, the molecular mechanisms for both intrinsic and transferable resistance to colistin remain largely unknown. Here, we aim to address this gap in the knowledge of these proteins. Structural and functional analyses of EptA and MCR-1 and -2 have defined a conserved 12-residue cavity that is required for the entry of the lipid substrate, phosphatidylethanolamine (PE). The in vitro and in vivo data together have allowed us to visualize the similarities in catalytic activity shared by EptA and MCR-1 and -2. The expression of either EptA or MCR-1 or -2 is shown to remodel the surface of enteric bacteria (e.g., Escherichia coli, Salmonella enterica, Klebsiella pneumoniae, etc.), rendering them resistant to colistin. The parallels in the PE substrate-binding cavities among EptA, MCR-1, and MCR-2 provide a comprehensive understanding of both intrinsic and transferable colistin resistance. Domain swapping between EptA and MCR-1 and -2 reveals that the two domains (transmembrane [TM] region and phosphoethanolamine [PEA] transferase) are not functionally exchangeable. Taken together, the results represent a common mechanism for intrinsic and transferable PEA resistance to polymyxin, a last-resort antibiotic against multidrug-resistant pathogens.IMPORTANCE EptA and MCR-1 and -2 remodel the outer membrane, rendering bacteria resistant to colistin, a final resort against carbapenem-resistant pathogens. Structural and functional analyses of EptA and MCR-1 and -2 reveal parallel PE lipid substrate-recognizing cavities, which explains intrinsic and transferable colistin resistance in gut bacteria. A similar mechanism is proposed for the catalytic activities of EptA and MCR-1 and -2. Together, they constitute a common mechanism for intrinsic and transferable polymyxin resistance.

Chromosomally encoded and plasmid-mediated polymyxins resistance in Acinetobacter baumannii: a huge public health threat.

Acinetobacter baumannii is an opportunistic pathogen associated with nosocomial and community infections of great clinical relevance. Its ability to rapidly develop resistance to antimicrobials, especially carbapenems, has re-boosted the prescription and use of polymyxins. However, the emergence of strains resistant to these antimicrobials is becoming a critical issue in several regions of the world because very few of currently available antibiotics are effective in these cases. This review summarizes the most up-to-date knowledge about chromosomally encoded and plasmid-mediated polymyxins resistance in A. baumannii. Different mechanisms are employed by A. baumannii to overcome the antibacterial effects of polymyxins. Modification of the outer membrane through phosphoethanolamine addition, loss of lipopolysaccharide, symmetric rupture, metabolic changes affecting osmoprotective amino acids, and overexpression of efflux pumps are involved in this process. Several genetic elements modulate these mechanisms, but only three of them have been described so far in A. baumannii clinical isolates such as mutations in pmrCAB, lpxACD, and lpsB. Elucidation of genotypic profiles and resistance mechanisms are necessary for control and fight against resistance to polymyxins in A. baumannii, thereby protecting this class for future treatment.

EPT1 (selenoprotein I) is critical for the neural development and maintenance of plasmalogen in humans.

Ethanolamine phosphotransferase (EPT)1, also known as selenoprotein 1 (SELENOI), is an enzyme that transfers phosphoethanolamine from cytidine diphosphate-ethanolamine to lipid acceptors to produce ethanolamine glycerophospholipids, such as diacyl-linked phosphatidylethanolamine (PE) and ether-linked plasmalogen [1-alkenyl-2-acyl-glycerophosphoethanolamine (plasmenyl-PE)]. However, to date there has been no analysis of the metabolomic consequences of the mutation of EPT1 on the concentration of ethanolamine glycerophospholipids in mammalian cells. We studied a patient with severe complicated hereditary spastic paraplegia, sensorineural-deafness, blindness, and seizures. Neuroimaging revealed hypomyelination, followed by brain atrophy mainly in the cerebellum and brainstem. Using whole exome sequencing, we identified a novel EPT1 mutation (exon skipping). In vitro EPT activity, as well as the rate of biosynthesis of ethanolamine glycerophospholipids, was markedly reduced in cultures of the patient's skin fibroblasts. Quantification of phospholipids by LC-MS/MS demonstrated reduced levels of several PE species with polyunsaturated fatty acids, such as 38:6, 38:4, 40:6, 40:5, and 40:4. Notably, most plasmenyl-PE species were significantly decreased in the patient's cells, whereas most plasmanylcholine [1-alkyl-2-acyl-glycerophosphocholine (plasmanyl-PC)] species were increased. Similar findings regarding decreased plasmenyl-PE and increased plasmanyl-PC were obtained using EPT1-KO HeLa cells. Our data demonstrate for the first time the indispensable role of EPT1 in the myelination process and neurodevelopment, and in the maintenance of normal homeostasis of ether-linked phospholipids in humans.

Characterization of Two Novel Lipopolysaccharide Phosphoethanolamine Transferases in Pasteurella multocida and Their Role in Resistance to Cathelicidin-2.

The lipopolysaccharide (LPS) produced by the Gram-negative bacterial pathogen Pasteurella multocida has phosphoethanolamine (PEtn) residues attached to lipid A, 3-deoxy-d-manno-octulosonic acid (Kdo), heptose, and galactose. In this report, we show that PEtn is transferred to lipid A by the P. multocida EptA homologue, PetL, and is transferred to galactose by a novel PEtn transferase that is unique to P. multocida called PetG. Transcriptomic analyses indicated that petL expression was positively regulated by the global regulator Fis and negatively regulated by an Hfq-dependent small RNA. Importantly, we have identified a novel PEtn transferase called PetK that is responsible for PEtn addition to the single Kdo molecule (Kdo1), directly linked to lipid A in the P. multocida glycoform A LPS. In vitro assays showed that the presence of a functional petL and petK, and therefore the presence of PEtn on lipid A and Kdo1, was essential for resistance to the cationic, antimicrobial peptide cathelicidin-2. The importance of PEtn on Kdo1 and the identification of the transferase responsible for this addition have not previously been shown. Phylogenetic analysis revealed that PetK is the first representative of a new family of predicted PEtn transferases. The PetK family consists of uncharacterized proteins from a range of Gram-negative bacteria that produce LPS glycoforms with only one Kdo molecule, including pathogenic species within the genera Vibrio, Bordetella, and Haemophilus We predict that many of these bacteria will require the addition of PEtn to Kdo for maximum protection against host antimicrobial peptides.