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Genes encoding lipid A modifying phosphoethanolamine transferases (PETs) are genetically diverse and can confer resistance to colistin and antimicrobial peptides. To better understand the functional diversity of PETs, we characterized three canonical mobile colistin resistance ( mcr ) alleles ( mcr-1 , -3 , -9 ), one intrinsic pet ( eptA ), and two mcr -like genes ( petB , petC ) in Escherichia coli . Using an isogenic expression system, we show that mcr-1 and mcr-3 confer similar phenotypes of decreased colistin susceptibility with low fitness costs. mcr-9 , which is phylogenetically closely related to mcr-3 , and eptA only provide fitness advantages in the presence of sub-inhibitory concentrations of colistin and significantly reduce fitness in media without colistin. PET-B and PET-C were phenotypically distinct from bonafide PETs; neither impacted colistin susceptibility nor caused considerable fitness cost. Strikingly, we found for the first time that different PETs selectively modify different phosphates of lipid A; MCR-1, MCR-3, and PET-C selectively modify the 4'-phosphate, whereas MCR-9 and EptA modify the 1-phosphate. However, 4'-phosphate modifications facilitated by MCR-1 and -3 are associated with lowered colistin susceptibility and low toxicity. Our results suggest that PETs have a wide phenotypic diversity and that increased colistin resistance is associated with specific lipid A modification patterns that have been largely unexplored thus far., Importance: Rising levels of resistance to increasing numbers of antimicrobials have led to the revival of last resort antibiotic colistin. Unfortunately, resistance to colistin is also spreading in the form of mcr genes, making it essential to (i) improve the identification of resistant bacteria to allow clinicians to prescribe effective drug regimens and (ii) develop new combination therapies effective at targeting resistant bacteria. Our results demonstrate that PETs, including MCR variants, are site-selective in Escherichia coli and that site-selectivity correlates with the level of susceptibility and fitness costs conferred by certain PETs. Site selectivity associated with a given PET may not only help predict colistin resistance phenotypes but may also provide an avenue to (i) improve drug regimens and (ii) develop new combination therapies to better combat colistin-resistant bacteria., Competing Interests: M.W. holds stocks for Neogen and is a paid consultant for BioMerieux and Neogen.

SUMMARYLipopolysaccharides (LPS) are an integral part of the outer membrane of Gram-negative bacteria and play essential structural and functional roles in maintaining membrane integrity as well as in stress response and virulence. LPS comprises a membrane-anchored lipid A group, a sugar-based core region, and an O-antigen formed by repeating oligosaccharide units. 3-Deoxy-D- manno -octulosonic acid-lipid A (Kdo 2 -lipid A) is the minimum LPS component required for bacterial survival. While LPS modifications are not essential, they play multifaceted roles in stress response and host-pathogen interactions. Gram-negative bacteria encode several distinct LPS-modifying phosphoethanolamine transferases (PET) that add phosphoethanolamine (pEtN) to lipid A or the core region of LPS. The pet genes differ in their genomic locations, regulation mechanisms, and modification targets of the encoded enzyme, consistent with their various roles in different growth niches and under varied stress conditions. The discovery of mobile colistin resistance genes, which represent lipid A-modifying pet genes that are encoded on mobile elements and associated with resistance to the last-resort antibiotic colistin, has led to substantial interest in PETs and pEtN-modified LPS over the last decade. Here, we will review the current knowledge of the functional diversity of pEtN-based LPS modifications, including possible roles in niche-specific fitness advantages and resistance to host-produced antimicrobial peptides, and discuss how the genetic and structural diversities of PETs may impact their function. An improved understanding of the PET group will further enhance our comprehension of the stress response and virulence of Gram-negative bacteria and help contextualize host-pathogen interactions., Competing Interests: The authors declare no conflict of interest.

The phosphoethanolamine transferase EptA utilizes phosphatidylethanolamine (PE) in the bacterial cell membrane to modify the structure of lipopolysaccharide, thereby conferring antimicrobial resistance on Gram-negative pathogens. Previous studies have indicated that excessive consumption of PE can disrupt the cell membrane, leading to cell death. This implies the presence of a regulatory mechanism for EptA catalysis to maintain a balance between antimicrobial resistance and bacterial growth. Through microsecond-scale all-atom molecular dynamics simulations, we demonstrate that membrane lipid homeostasis modulates the conformational transition and catalytic activation of EptA. The conformation of EptA oscillates between closed and open states, ensuring the precise spatiotemporal sequence of substrates binding. Interestingly, the conformation of EptA is significantly influenced by its surrounding lipid microenvironment, particularly the PE proportion in the membrane. PE-rich membrane conditions initiate and stabilize the open conformation of EptA through both orthosteric and allosteric effects. Importantly, the reaction mediated by EptA gradually depletes PE in the membrane, ultimately hindering its conformational transition and catalytic activation. These findings collectively establish a self-promoted model, illustrating the regulatory mechanism of EptA during the development of antibiotic resistance., Competing Interests: Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

The emergence of plasmid-encoded colistin resistance mechanisms, MCR-1, a phosphoethanolamine transferase, rendered colistin ineffective as last resort antibiotic against severe infections caused by clinical Gram-negative bacterial pathogens. Through screening FDA-approved drug library, we identified two structurally similar compounds, namely cetylpyridinium chloride (CET) and domiphen bromide (DOM), which potentiated colistin activity in both colistin-resistant and susceptible Enterobacterales. These compounds were found to insert their long carbon chain to a hydrophobic pocket of bacterial phosphoethanolamine transferases including MCR-1, competitively blocking the binding of lipid A tail for substrate recognition and modification, resulting in the increase of bacterial sensitivity to colistin. In addition, these compounds were also found to dissipate bacterial membrane potential leading to the increase of bacterial sensitivity to colistin. Importantly, combinational use of DOM with colistin exhibited remarkable protection of test animals against infections by colistin-resistant bacteria in both mouse thigh infection and sepsis models. For mice infected by colistin-susceptible bacteria, the combinational use of DOM and colistin enable us to use lower dose of colistin to for efficient treatment. These properties render DOM excellent adjuvant candidates that help transform colistin into a highly potent antimicrobial agent for treatment of colistin-resistant Gram-negative bacterial infections and allowed us to use of a much lower dosage of colistin to reduce its toxicity against colistin-susceptible bacterial infection such as carbapenem-resistant Enterobacterales., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier GmbH.)

Selenoprotein I (Selenoi) is highly expressed in liver and plays a key role in lipid metabolism as a phosphatidylethanolamine (PE) synthase. However, the precise function of Selenoi in the liver remains elusive. In the study, we generated hepatocyte-specific Selenoi conditional knockout (cKO) mice on a high-fat diet to identify the physiological function of Selenoi. The cKO group exhibited a significant increase in body weight, with a 15.6% and 13.7% increase in fat accumulation in white adipose tissue (WAT) and the liver, respectively. Downregulation of the lipolysis-related protein (p-Hsl) and upregulation of the adipogenesis-related protein (Fasn) were observed in the liver of cKO mice. The cKO group also showed decreased oxygen consumption (VO 2 ), carbon dioxide production (VCO 2 ), and energy expenditure (p < .05). Moreover, various metabolites of the steroid hormone synthesis pathway were affected in the liver of cKO mice. A potential cascade of Selenoi-phosphatidylethanolamine-steroid hormone synthesis might serve as a core mechanism that links hepatocyte-specific Selenoi cKO to biochemical and molecular reactions. In conclusion, we revealed that Selenoi inhibits body fat accumulation and hepatic steatosis and elevates energy consumption; this protein could also be considered a therapeutic target for such related diseases., (© 2024 Federation of American Societies for Experimental Biology.)

Objectives: The rapid dissemination of the mcr-1 gene via plasmid-mediated transfer has raised concerns regarding the efficacy of colistin as a last-resort treatment for multidrug-resistant Gram-negative bacterial infections. Current mcr-1 gene detection methods mainly focus on cultured bacteria, which is a complex and time-consuming process requiring skilled personnel, making it unsuitable for field analysis., Methods: A rapid detection technique combining recombinase polymerase amplification with a lateral flow dipstick targeting uncultured clinical samples was developed., Results: This new method targeting the mcr-1 gene region (23 232-23 642 bp, no. KP347127.1) achieved a low detection limit of 10 copies/μL. The whole process was carried out with high specificity and was completed within 20 min. The evaluation assay was conducted using 45 human faecal samples; 16 strains yielded a 98% accuracy, closely matching antimicrobial susceptibility outcomes., Conclusions: The novel method integrates nucleic acid extraction, isothermal amplification, and a test assay, suggesting the potential for timely colistin resistance surveillance in frontline disease control and healthcare settings, supporting future prevention and clinical standardization efforts., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Background: Hereditary spastic paraplegia 81 is a recently identified, rare autosomal recessive disease, caused by biallelic pathogenic variants in the SELENOI gene, with only two families reported to date. The features documented in the two previous affected families include sensorineural deafness, blindness, cleft palate, delayed motor development, regression of motor skills, impaired intellectual development, poor speech and language acquisition, spasticity, hyperreflexia, white matter abnormalities and cerebral and cerebellar atrophy., Methods: In the present study, we performed exome sequencing analysis in a single family with two affected siblings to identify the genetic cause of complicated hereditary spastic paraplegia. The results were further confirmed by Sanger sequencing, cDNA analysis and 3D protein modelling., Results: Exome sequencing identified a homozygous, synonymous variant in the SELENOI gene (NM&#95;033505.4:c.126G>A:p.(Lys42Lys)) in both of the siblings. Sanger sequencing confirmed the heterozygous status in both parents consistent with the autosomal recessive inheritance. This variant has been found to disrupt normal splicing and lead to skipping of exon 2, causing in-frame deletion of SELENOI N-terminal 23 amino acids [NM&#95;033505.4:c.57&#95;126del:p.(Tyr20&#95;Lys42del)] and further leading to structural changes in the protein., Conclusions: We report a novel homozygous synonymous variant in the SELENOI gene causing abnormal splicing in two patients affected with hereditary spastic paraplegia 81. This report further expands the phenotypic and genotypic spectrum of hereditary spastic paraplegia 81., (© 2023 John Wiley & Sons Ltd.)

Objectives: Colistin-resistant Gram-negative pathogens have become a serious worldwide medical problem. This study was designed to reveal the effects of an intrinsic phosphoethanolamine transferase from Acinetobacter modestus on Enterobacterales., Methods: A strain of colistin-resistant A. modestus was isolated from a sample of nasal secretions taken in 2019 from a hospitalised pet cat in Japan. The whole genome was sequenced by next generation sequencing, and transformants of Escherichia coli, Klebsiella pneumoniae, and Enterobacter cloacae harbouring the phosphoethanolamine transferase-encoding gene from A. modestus were constructed. Lipid A modification in E. coli transformants was analysed using electrospray ionization mass spectrometry., Results: Sequencing of the entire genome revealed that the isolate harboured a phosphoethanolamine transferase-encoding gene, eptA&#95;AM, on its chromosome. Transformants of E. coli, K. pneumoniae, and E. cloacae harbouring both the promoter and eptA&#95;AM gene from A. modestus had 32-fold, 8-fold, and 4-fold higher minimum inhibitory concentrations (MICs) for colistin, respectively, than transformants harbouring a control vector. The genetic environment surrounding eptA&#95;AM in A. modestus was similar to that surrounding eptA&#95;AM in Acinetobacter junii and Acinetobacter venetianus. Electrospray ionization mass spectrometry analysis revealed that EptA&#95;AM modified lipid A in Enterobacterales., Conclusion: This is the first report to describe the isolation of an A. modestus strain in Japan and show that its intrinsic phosphoethanolamine transferase, EptA&#95;AM, contributes to colistin resistance in Enterobacterales and A. modestus., Competing Interests: Competing interests None declared., (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

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., (Copyright © 2022 The Author(s). Published by Elsevier Ltd.. All rights reserved.)