Macrophage infectivity potentiator protein, a peptidyl prolyl cis-trans isomerase, essential for Coxiella burnetii growth and pathogenesis.

Coxiella burnetii is a Gram-negative intracellular pathogen that causes the debilitating disease Q fever, which affects both animals and humans. The only available human vaccine, Q-Vax, is effective but has a high risk of severe adverse reactions, limiting its use as a countermeasure to contain outbreaks. Therefore, it is essential to identify new drug targets to treat this infection. Macrophage infectivity potentiator (Mip) proteins catalyse the folding of proline-containing proteins through their peptidyl prolyl cis-trans isomerase (PPIase) activity and have been shown to play an important role in the virulence of several pathogenic bacteria. To date the role of the Mip protein in C. burnetii pathogenesis has not been investigated. This study demonstrates that CbMip is likely to be an essential protein in C. burnetii. The pipecolic acid derived compounds, SF235 and AN296, which have shown utility in targeting other Mip proteins from pathogenic bacteria, demonstrate inhibitory activities against CbMip. These compounds were found to significantly inhibit intracellular replication of C. burnetii in both HeLa and THP-1 cells. Furthermore, SF235 and AN296 were also found to exhibit antibiotic properties against both the virulent (Phase I) and avirulent (Phase II) forms of C. burnetii Nine Mile Strain in axenic culture. Comparative proteomics, in the presence of AN296, revealed alterations in stress responses with H2O2 sensitivity assays validating that Mip inhibition increases the sensitivity of C. burnetii to oxidative stress. In addition, SF235 and AN296 were effective in vivo and significantly improved the survival of Galleria mellonella infected with C. burnetii. These results suggest that unlike in other bacteria, Mip in C. burnetii is required for replication and that the development of more potent inhibitors against CbMip is warranted and offer potential as novel therapeutics against this pathogen.

N-acetyl-ß-hexosaminidase activity is important for chitooligosaccharide metabolism and biofilm formation in Burkholderia pseudomallei.

Burkholderia pseudomallei is a saprophytic Gram-negative bacillus that can cause the disease melioidosis. Although B. pseudomallei is a recognised member of terrestrial soil microbiomes, little is known about its contribution to the saprophytic degradation of polysaccharides within its niche. For example, while chitin is predicted to be abundant within terrestrial soils the chitinolytic capacity of B. pseudomallei is yet to be defined. This study identifies and characterises a putative glycoside hydrolase, bpsl0500, which is expressed by B. pseudomallei K96243. Recombinant BPSL0500 was found to exhibit activity against substrate analogues and GlcNAc disaccharides relevant to chitinolytic N-acetyl-ß-d-hexosaminidases. In B. pseudomallei, bpsl0500 was found to be essential for both N-acetyl-ß-d-hexosaminidase activity and chitooligosaccharide metabolism. Furthermore, bpsl0500 was also observed to significantly affect biofilm deposition. These observations led to the identification of BPSL0500 activity against model disaccharide linkages that are present in biofilm exopolysaccharides, a feature that has not yet been described for chitinolytic enzymes. The results in this study indicate that chitinolytic N-acetyl-ß-d-hexosaminidases like bpsl0500 may facilitate biofilm disruption as well as chitin assimilation, providing dual functionality for saprophytic bacteria such as B. pseudomallei within the competitive soil microbiome.

Identification of active main metabolites of anti-infective inhibitors of the macrophage infectivity potentiator protein by liquid chromatography using mass detection.

Due to increasing antibiotic resistance, the development of anti-infectives with new mechanisms of action is crucial. Virulence factors such as the "macrophage infectivity potentiator" (Mip) protein, which catalyzes the folding of proline-containing proteins by means of their cis-trans isomerase (PPIase) activity, have come into focus as a potential new target. Since the inhibition of Mip by small molecules has been shown to lead to reduced virulence and survival in vitro, especially of Gram-negative bacteria such as Burkholderia pseudomallei (Bp), Neisseria meningitidis (Nm), and Neisseria gonorrhoeae (Ng), or Coxiella burnetii (Cb), among many others, a library of Mip inhibitors was developed. As drug metabolism has a significant impact on the overall therapeutic outcome, this report describes the biotransformation of the most potent Mip inhibitors. Therefore, the anti-infectives were treated using human liver microsomes in vitro. Liquid chromatography with tandem mass spectrometry (LC/MS-MS) methods were applied to identify the metabolites and quantify the metabolic degradation of the hit compounds. Active metabolites, N-oxides, were found, leading to new opportunities for further drug development.

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.

Broad-spectrum in vitro activity of macrophage infectivity potentiator inhibitors against Gram-negative bacteria and Leishmania major.

BACKGROUND: The macrophage infectivity potentiator (Mip) protein, which belongs to the immunophilin superfamily, is a peptidyl-prolyl cis/trans isomerase (PPIase) enzyme. Mip has been shown to be important for virulence in a wide range of pathogenic microorganisms. It has previously been demonstrated that small-molecule compounds designed to target Mip from the Gram-negative bacterium Burkholderia pseudomallei bind at the site of enzymatic activity of the protein, inhibiting the in vitro activity of Mip. OBJECTIVES: In this study, co-crystallography experiments with recombinant B. pseudomallei Mip (BpMip) protein and Mip inhibitors, biochemical analysis and computational modelling were used to predict the efficacy of lead compounds for broad-spectrum activity against other pathogens. METHODS: Binding activity of three lead compounds targeting BpMip was verified using surface plasmon resonance spectroscopy. The determination of crystal structures of BpMip in complex with these compounds, together with molecular modelling and in vitro assays, was used to determine whether the compounds have broad-spectrum antimicrobial activity against pathogens. RESULTS: Of the three lead small-molecule compounds, two were effective in inhibiting the PPIase activity of Mip proteins from Neisseria meningitidis, Klebsiella pneumoniae and Leishmania major. The compounds also reduced the intracellular burden of these pathogens using in vitro cell infection assays. CONCLUSIONS: These results indicate that Mip is a novel antivirulence target that can be inhibited using small-molecule compounds that prove to be promising broad-spectrum drug candidates in vitro. Further optimization of compounds is required for in vivo evaluation and future clinical applications.

Peptidyl-Prolyl Isomerase ppiB Is Essential for Proteome Homeostasis and Virulence in Burkholderia pseudomallei.

Burkholderia pseudomallei is the causative agent of melioidosis, a disease endemic to Southeast Asia and northern Australia. Mortality rates in these areas are high even with antimicrobial treatment, and there are few options for effective therapy. Therefore, there is a need to identify antibacterial targets for the development of novel treatments. Cyclophilins are a family of highly conserved enzymes important in multiple cellular processes. Cyclophilins catalyze the cis-trans isomerization of xaa-proline bonds, a rate-limiting step in protein folding which has been shown to be important for bacterial virulence. B. pseudomallei carries a putative cyclophilin B gene, ppiB, the role of which was investigated. A B. pseudomalleiΔppiB (BpsΔppiB) mutant strain demonstrates impaired biofilm formation and reduced motility. Macrophage invasion and survival assays showed that although the BpsΔppiB strain retained the ability to infect macrophages, it had reduced survival and lacked the ability to spread cell to cell, indicating ppiB is essential for B. pseudomallei virulence. This is reflected in the BALB/c mouse infection model, demonstrating the requirement of ppiB for in vivo disease dissemination and progression. Proteomic analysis demonstrates that the loss of PpiB leads to pleiotropic effects, supporting the role of PpiB in maintaining proteome homeostasis. The loss of PpiB leads to decreased abundance of multiple virulence determinants, including flagellar machinery and alterations in type VI secretion system proteins. In addition, the loss of ppiB leads to increased sensitivity toward multiple antibiotics, including meropenem and doxycycline, highlighting ppiB inhibition as a promising antivirulence target to both treat B. pseudomallei infections and increase antibiotic efficacy.

Virulence of the Melioidosis Pathogen Burkholderia pseudomallei Requires the Oxidoreductase Membrane Protein DsbB.

The naturally antibiotic-resistant bacterium Burkholderia pseudomallei is the causative agent of melioidosis, a disease with stubbornly high mortality and a complex, protracted treatment regimen. The worldwide incidence of melioidosis is likely grossly underreported, though it is known to be highly endemic in northern Australia and Southeast Asia. Bacterial disulfide bond (DSB) proteins catalyze the oxidative folding and isomerization of disulfide bonds in substrate proteins. In the present study, we demonstrate that B. pseudomallei membrane protein disulfide bond protein B (BpsDsbB) forms a functional redox relay with the previously characterized virulence mediator B. pseudomallei disulfide bond protein A (BpsDsbA). Genomic analysis of diverse B. pseudomallei clinical isolates demonstrated that dsbB is a highly conserved core gene. Critically, we show that DsbB is required for virulence in B. pseudomallei A panel of B. pseudomalleidsbB deletion strains (K96243, 576, MSHR2511, MSHR0305b, and MSHR5858) were phenotypically diverse according to the results of in vitro assays that assess hallmarks of virulence. Irrespective of their in vitro virulence phenotypes, two deletion strains were attenuated in a BALB/c mouse model of infection. A crystal structure of a DsbB-derived peptide complexed with BpsDsbA provides the first molecular characterization of their interaction. This work contributes to our broader understanding of DSB redox biology and will support the design of antimicrobial drugs active against this important family of bacterial virulence targets.

Burkholderia pseudomallei sequencing identifies genomic clades with distinct recombination, accessory, and epigenetic profiles.

Burkholderia pseudomallei (Bp) is the causative agent of the infectious disease melioidosis. To investigate population diversity, recombination, and horizontal gene transfer in closely related Bp isolates, we performed whole-genome sequencing (WGS) on 106 clinical, animal, and environmental strains from a restricted Asian locale. Whole-genome phylogenies resolved multiple genomic clades of Bp, largely congruent with multilocus sequence typing (MLST). We discovered widespread recombination in the Bp core genome, involving hundreds of regions associated with multiple haplotypes. Highly recombinant regions exhibited functional enrichments that may contribute to virulence. We observed clade-specific patterns of recombination and accessory gene exchange, and provide evidence that this is likely due to ongoing recombination between clade members. Reciprocally, interclade exchanges were rarely observed, suggesting mechanisms restricting gene flow between clades. Interrogation of accessory elements revealed that each clade harbored a distinct complement of restriction-modification (RM) systems, predicted to cause clade-specific patterns of DNA methylation. Using methylome sequencing, we confirmed that representative strains from separate clades indeed exhibit distinct methylation profiles. Finally, using an E. coli system, we demonstrate that Bp RM systems can inhibit uptake of non-self DNA. Our data suggest that RM systems borne on mobile elements, besides preventing foreign DNA invasion, may also contribute to limiting exchanges of genetic material between individuals of the same species. Genomic clades may thus represent functional units of genetic isolation in Bp, modulating intraspecies genetic diversity.

Characterization of secreted sphingosine-1-phosphate lyases required for virulence and intracellular survival of Burkholderia pseudomallei.

Sphingosine-1-phosphate (S1P), a bioactive sphingolipid metabolite, plays a critical role in the orchestration of immune responses. S1P levels within the mammalian host are tightly regulated, in part through the activity of S1P lyase (S1PL) which catalyses its irreversible degradation. Herein, we describe the identification and characterization of secreted S1PL orthologues encoded by the facultative intracellular bacteria Burkholderia pseudomallei and Burkholderia thailandensis. These bacterial orthologues exhibited S1PL enzymatic activity, functionally complemented an S1PL-deficient yeast strain and conferred resistance to the antimicrobial sphingolipid D-erythro-sphingosine. We report that secretion of these bacterial S1PLs is pH-dependent, and is observed during intracellular infection. S1PL-deficient mutants displayed impaired intracellular replication in murine macrophages (associated with an inability to evade the maturing phagosome) and were significantly attenuated in murine and larval infection models. Furthermore, treatment of Burkholderia-infected macrophages with either S1P or a selective agonist of S1P receptor 1 enhanced bacterial colocalisation with LAMP-1 and reduced their intracellular survival. In summary, our studies confirm bacterial-encoded S1PL as a critical virulence determinant of B. pseudomallei and B. thailandensis, further highlighting the pivotal role of S1P in host-pathogen interactions. In addition, our data suggest that S1P pathway modulators have potential for the treatment of intracellular infection.

An integrated computational-experimental approach reveals Yersinia pestis genes essential across a narrow or a broad range of environmental conditions.

BACKGROUND: The World Health Organization has categorized plague as a re-emerging disease and the potential for Yersinia pestis to also be used as a bioweapon makes the identification of new drug targets against this pathogen a priority. Environmental temperature is a key signal which regulates virulence of the bacterium. The bacterium normally grows outside the human host at 28 °C. Therefore, understanding the mechanisms that the bacterium used to adapt to a mammalian host at 37 °C is central to the development of vaccines or drugs for the prevention or treatment of human disease. RESULTS: Using a library of over 1 million Y. pestis CO92 random mutants and transposon-directed insertion site sequencing, we identified 530 essential genes when the bacteria were cultured at 28 °C. When the library of mutants was subsequently cultured at 37 °C we identified 19 genes that were essential at 37 °C but not at 28 °C, including genes which encode proteins that play a role in enabling functioning of the type III secretion and in DNA replication and maintenance. Using genome-scale metabolic network reconstruction we showed that growth conditions profoundly influence the physiology of the bacterium, and by combining computational and experimental approaches we were able to identify 54 genes that are essential under a broad range of conditions. CONCLUSIONS: Using an integrated computational-experimental approach we identify genes which are required for growth at 37 °C and under a broad range of environments may be the best targets for the development of new interventions to prevent or treat plague in humans.

A miniaturized peptidyl-prolyl isomerase enzyme assay.

Prolyl-peptidyl isomerases (PPIases) are enzymes that are found in all living organisms. They form an essential part of the cellular protein folding homeostasis machinery. PPIases are associated with many important human diseases, e.g. cardiovascular disease, cancer and Alzheimer's. The development of novel PPIase inhibitors has been limited by the lack of a rapid, laboratory-based assay for these enzymes, as their substrates and products are challenging to distinguish. A well described continuous assay, coupled with the hydrolysis of a peptide by chymotrypsin is highly effective, but comparatively slow. To address this, we developed an improved version of the traditional assay using a temperature controlled plate reader. This assay allows semi-automated medium throughput assays in an academic laboratory for 84 samples per day. The assay shows lower errors, with an average Z' of 0.72. We further developed the assay using a fluorogenic peptide-based FRET probe. This provides an extremely sensitive PPIase assay using substrate at 200 nM, which approaches single turnover conditions. The fluorescent probe achieves an excellent quenching efficiency of 98.6%, and initial experiments showed acceptable Z' of 0.31 and 0.30 for cyclophilin A and hFKBP12 respectively. The assays provide an improved toolset for the quantitative, biochemical analysis of PPIases.

Survival protein A is essential for virulence in Yersinia pestis.

Plague is a highly pathogenic disease caused by the bacterium Yersinia pestis. There is currently no vaccine available for prophylaxis and antibiotic resistant strains have been isolated, thus there is a need for the development of new countermeasures to treat this disease. Survival protein A (SurA) is a chaperone that has been linked to virulence in several species of bacteria, including the close relative Yersinia pseudotuberculosis. In this study, we aimed to evaluate the role of SurA in virulence of the highly pathogenic Y. pestis by creating an unmarked surA deletion mutant. The Y. pestis ΔsurA mutant was found to be more susceptible to membrane perturbing agents and was completely avirulent in a mouse infection model when delivered up to 2.1 × 10(5) CFU by the subcutaneous route. This provides strong evidence that SurA would make a promising antimicrobial target.

Development, synthesis and structure-activity-relationships of inhibitors of the macrophage infectivity potentiator (Mip) proteins of Legionella pneumophila and Burkholderia pseudomallei.

The bacteria Burkholderia pseudomallei and Legionella pneumophila cause severe diseases like melioidosis and Legionnaire's disease with high mortality rates despite antibiotic treatment. Due to increasing antibiotic resistances against these and other Gram-negative bacteria, alternative therapeutical strategies are in urgent demand. As a virulence factor, the macrophage infectivity potentiator (Mip) protein constitutes an attractive target. The Mip proteins of B. pseudomallei and L. pneumophila exhibit peptidyl-prolyl cis/trans isomerase (PPIase) activity and belong to the PPIase superfamily. In previous studies, the pipecolic acid moiety proved to be a valuable scaffold for inhibiting this PPIase activity. Thus, a library of pipecolic acid derivatives was established guided by structural information and computational analyses of the binding site and possible binding modes. Stability and toxicity considerations were taken into account in iterative extensions of the library. Synthesis and evaluation of the compounds in PPIase assays resulted in highly active inhibitors. The activities can be interpreted in terms of a common binding mode obtained by docking calculations.

Evaluating the role of phage-shock protein A in Burkholderia pseudomallei.

The phage-shock protein (Psp) response is an extracytoplasmic response system that is vital for maintenance of the cytoplasmic membrane when the cell encounters stressful conditions. The paradigm of the Psp response has been established in Escherichia coli. The response has been shown to be important for survival during the stationary phase, maintenance of the proton motive force across membranes and implicated in virulence. In this study, we identified a putative PspA homologue in Burkholderia pseudomallei, annotated as BPSL2105. Similar to the induction of PspA in E. coli, the expression of B. pseudomallei BPSL2105 was induced by heat shock. Deletion of BPSL2105 resulted in a survival defect in the late stationary phase coincident with dramatic changes in the pH of the culture medium. The B. pseudomallei BPSL2105 deletion mutant also displayed reduced survival in macrophage infection - the first indication that the Psp response plays a role during intracellular pathogenesis in this species. The purified protein formed large oligomeric structures similar to those observed for the PspA protein of E. coli, and PspA homologues in Bacillus, cyanobacteria and higher plants, providing further evidence to support the identification of BPSL2105 as a PspA-like protein in B. pseudomallei.

A structural biology approach enables the development of antimicrobials targeting bacterial immunophilins.

Macrophage infectivity potentiators (Mips) are immunophilin proteins and essential virulence factors for a range of pathogenic organisms. We applied a structural biology approach to characterize a Mip from Burkholderia pseudomallei (BpML1), the causative agent of melioidosis. Crystal structure and nuclear magnetic resonance analyses of BpML1 in complex with known macrocyclics and other derivatives led to the identification of a key chemical scaffold. This scaffold possesses inhibitory potency for BpML1 without the immunosuppressive components of related macrocyclic agents. Biophysical characterization of a compound series with this scaffold allowed binding site specificity in solution and potency determinations for rank ordering the set. The best compounds in this series possessed a low-micromolar affinity for BpML1, bound at the site of enzymatic activity, and inhibited a panel of homologous Mip proteins from other pathogenic bacteria, without demonstrating toxicity in human macrophages. Importantly, the in vitro activity of BpML1 was reduced by these compounds, leading to decreased macrophage infectivity and intracellular growth of Burkholderia pseudomallei. These compounds offer the potential for activity against a new class of antimicrobial targets and present the utility of a structure-based approach for novel antimicrobial drug discovery.

The serine protease inhibitor Ecotin is required for full virulence of Burkholderia pseudomallei.

Burkholderia pseudomallei is a Gram negative soil saprophyte that causes the disease melioidosis where clinical symptoms can vary from localised infection to pneumonia and septic shock. Ecotin is a potent periplasmic serine protease inhibitor first identified in Escherichia coli. Ecotin, although present in only a small subset of genera, can inhibit a broad range of serine proteases including those typically associated with the innate immune system such as neutrophil elastase and cathepsin G. An Ecotin orthologue identified in B. pseudomallei was recombinantly expressed and found to inhibit elastase. To study the role of Ecotin in B. pseudomallei virulence an in-frame unmarked deletion mutant was created. Infection of a murine macrophages-like cell line revealed Ecotin was necessary for the early stages of colonisation allowing replication following cell entry. Attenuation of the Δeco mutant strain in the murine model of melioidosis further supported Ecotin as a virulence factor of B. pseudomallei.

High Affinity Inhibitors of the Macrophage Infectivity Potentiator Protein from Trypanosoma cruzi, Burkholderia pseudomallei, and Legionella pneumophila─A Comparison.

Since Chagas disease, melioidosis, and Legionnaires' disease are all potentially life-threatening infections, there is an urgent need for new treatment strategies. All causative agents, Trypanosoma cruzi, Burkholderia pseudomallei, and Legionella pneumophila, express a virulence factor, the macrophage infectivity potentiator (MIP) protein, emerging as a promising new therapeutic target. Inhibition of MIP proteins having a peptidyl-prolyl isomerase activity leads to reduced viability, proliferation, and cell invasion. The affinity of a series of pipecolic acid-type MIP inhibitors was evaluated against all MIPs using a fluorescence polarization assay. The analysis of structure-activity relationships led to highly active inhibitors of MIPs of all pathogens, characterized by a one-digit nanomolar affinity for the MIPs and a very effective inhibition of their peptidyl-prolyl isomerase activity. Docking studies, molecular dynamics simulations, and quantum mechanical calculations suggest an extended σ-hole of the meta-halogenated phenyl sulfonamide to be responsible for the high affinity.

Inhibition of macrophage infectivity potentiator in Burkholderia pseudomallei suppresses pro-inflammatory responses in murine macrophages.

Introduction: Melioidosis, caused by the Gram-negative bacterium Burkholderia pseudomallei, is a disease endemic in many tropical countries globally. Clinical presentation is highly variable, ranging from asymptomatic to fatal septicemia, and thus the outcome of infection can depend on the host immune responses. The aims of this study were to firstly, characterize the macrophage immune response to B. pseudomallei and secondly, to determine whether the immune response was modified in the presence of novel inhibitors targeting the virulence factor, the macrophage infectivity potentiator (Mip) protein. We hypothesized that inhibition of Mip in B. pseudomallei would disarm the bacteria and result in a host beneficial immune response. Methods: Murine macrophage J774A.1 cells were infected with B. pseudomallei K96243 in the presence of small-molecule inhibitors targeting the Mip protein. RNA-sequencing was performed on infected cells four hours post-infection. Secreted cytokines and lactose dehydrogenase were measured in cell culture supernatants 24 hours post-infection. Viable, intracellular B. pseudomallei in macrophages were also enumerated 24 hours post-infection. Results: Global transcriptional profiling of macrophages infected with B. pseudomallei by RNA-seq demonstrated upregulation of immune-associated genes, in particular a significant enrichment of genes in the TNF signaling pathway. Treatment of B. pseudomallei-infected macrophages with the Mip inhibitor, AN_CH_37 resulted in a 5.3-fold reduction of il1b when compared to cells treated with DMSO, which the inhibitors were solubilized in. A statistically significant reduction in IL-1ß levels in culture supernatants was seen 24 hours post-infection with AN_CH_37, as well as other pro-inflammatory cytokines, namely IL-6 and TNF-α. Treatment with AN_CH_37 also reduced the survival of B. pseudomallei in macrophages after 24 hours which was accompanied by a significant reduction in B. pseudomallei-induced cytotoxicity as determined by lactate dehydrogenase release. Discussion: These data highlight the potential to utilize Mip inhibitors in reducing potentially harmful pro-inflammatory responses resulting from B. pseudomallei infection in macrophages. This could be of significance since overstimulation of pro-inflammatory responses can result in immunopathology, tissue damage and septic shock.

Phosphoethanolamine Transferases as Drug Discovery Targets for Therapeutic Treatment of Multi-Drug Resistant Pathogenic Gram-Negative Bacteria.

Antibiotic resistance caused by multidrug-resistant (MDR) bacteria is a major challenge to global public health. Polymyxins are increasingly being used as last-in-line antibiotics to treat MDR Gram-negative bacterial infections, but resistance development renders them ineffective for empirical therapy. The main mechanism that bacteria use to defend against polymyxins is to modify the lipid A headgroups of the outer membrane by adding phosphoethanolamine (PEA) moieties. In addition to lipid A modifying PEA transferases, Gram-negative bacteria possess PEA transferases that decorate proteins and glycans. This review provides a comprehensive overview of the function, structure, and mechanism of action of PEA transferases identified in pathogenic Gram-negative bacteria. It also summarizes the current drug development progress targeting this enzyme family, which could reverse antibiotic resistance to polymyxins to restore their utility in empiric therapy.