Structural evidence for dimerization-regulated activation of an integral membrane phospholipase

Snijder, H.J.; Ubarretxena-Belandia, Iban; Blaauw, Mieke; Kalk, K.H.; Verheij, Hubertus M.; Egmond, Maarten R.; Dekker, Niek; Dijkstra, Bauke W.

doi:10.1038/44890

Cited by 139 publications

(20 citation statements)

References 35 publications

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“…Effects of PldA and PagP on the yejM1163-mediated suppression. Elevated LPE levels in the yejM1163 background could be caused by the action of two OM enzymes, PldA (35) and PagP (36), which generate LPE either by PE hydrolysis (PldA) or during the transfer of a palmitate chain from PE to hexa-acylated LPS (PagP). These enzymes are normally dormant but are activated in response to the perturbation of the OM structure (e.g., lipid asymmetry) due to the mislocalization of PL to the outer leaflet of the OM (37)(38)(39).…”

Section: What Causes Om Permeability Increase In the Yejm1163 Mutant?mentioning

confidence: 99%

Overcoming Iron Deficiency of an Escherichia coli tonB Mutant by Increasing Outer Membrane Permeability

Qiu

Misra

2019

J Bacteriol

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The intake of certain nutrients, including ferric ion, is facilitated by the outer membrane-localized transporters. Due to ferric insolubility at physiological pH, Escherichia coli secretes a chelator, enterobactin, outside the cell and then transports back the enterobactin-ferric complex via an outer membrane receptor protein, FepA, whose activity is dependent on the proton motive force energy transduced by the TonB-ExbBD complex of the inner membrane. Consequently, ΔtonB mutant cells grow poorly on a medium low in iron. Prolonged incubation of ΔtonB cells on low-iron medium yields faster-growing colonies that acquired suppressor mutations in the yejM (pbgA) gene, which codes for a putative inner-to-outer membrane cardiolipin transporter. Further characterization of suppressors revealed that they display hypersusceptibility to vancomycin, a large hydrophilic antibiotic normally precluded from entering E. coli cells, and leak periplasmic proteins into the culture supernatant, indicating a compromised outer membrane permeability barrier. All phenotypes were reversed by supplying the wild-type copy of yejM on a plasmid, suggesting that yejM mutations are solely responsible for the observed phenotypes. The deletion of all known cardiolipin synthase genes (clsABC) did not produce the phenotypes similar to mutations in the yejM gene, suggesting that the absence of cardiolipin from the outer membrane per se is not responsible for increased outer membrane permeability. Elevated lysophosphatidylethanolamine levels and the synthetic growth phenotype without pldA indicated that defective lipid homeostasis in the yejM mutant compromises outer membrane lipid asymmetry and permeability barrier to allow enterobactin intake, and that YejM has additional roles other than transporting cardiolipin. IMPORTANCE The work presented here describes a positive genetic selection strategy for isolating mutations that destabilize the outer membrane permeability barrier of E. coli. Given the importance of the outer membrane in restricting the entry of antibiotics, characterization of the genes and their products that affect outer membrane integrity will enhance the understanding of bacterial membranes and the development of strategies to bypass the outer membrane barrier for improved drug efficacy.

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Section: What Causes Om Permeability Increase In the Yejm1163 Mutant?mentioning

confidence: 99%

Overcoming Iron Deficiency of an Escherichia coli tonB Mutant by Increasing Outer Membrane Permeability

Qiu

Misra

2019

J Bacteriol

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“…Three lipid asymmetry-maintaining systems, primarily studied in Escherichia coli , are the phospholipase A PldA [4], the LPS palmitoyltransferase PagP [5], and the m aintenance of l ipid a symmetry (Mla) [6] systems. PagP and PldA both remove phospholipids from the outer membrane by destroying the phospholipid.…”

Section: Introductionmentioning

confidence: 99%

Neisseria gonorrhoeae MlaA influences gonococcal virulence and membrane vesicle production

et al. 2019

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The six-component maintenance of lipid asymmetry (Mla) system is responsible for retrograde transport of phospholipids, ensuring the barrier function of the Gram-negative cell envelope. Located within the outer membrane, MlaA (VacJ) acts as a channel to shuttle phospholipids from the outer leaflet. We identified Neisseria gonorrhoeae MlaA (ngo2121) during high-throughput proteomic mining for potential therapeutic targets against this medically important human pathogen. Our follow-up phenotypic microarrays revealed that lack of MlaA results in a complex sensitivity phenome. Herein we focused on MlaA function in cell envelope biogenesis and pathogenesis. We demonstrate the existence of two MlaA classes among 21 bacterial species, characterized by the presence or lack of a lipoprotein signal peptide. Purified truncated N. gonorrhoeae MlaA elicited antibodies that cross-reacted with a panel of different Neisseria. Little is known about MlaA expression; we provide the first evidence that MlaA levels increase in stationary phase and under anaerobiosis but decrease during iron starvation. Lack of MlaA resulted in higher cell counts during conditions mimicking different host niches; however, it also significantly decreased colony size. Antimicrobial peptides such as polymyxin B exacerbated the size difference while human defensin was detrimental to mutant viability. Consistent with the proposed role of MlaA in vesicle biogenesis, the ΔmlaA mutant released 1.7-fold more membrane vesicles. Comparative proteomics of cell envelopes and native membrane vesicles derived from ΔmlaA and wild type bacteria revealed enrichment of TadA–which recodes proteins through mRNA editing–as well as increased levels of adhesins and virulence factors. MlaA-deficient gonococci significantly outcompeted (up to 16-fold) wild-type bacteria in the murine lower genital tract, suggesting the growth advantage or increased expression of virulence factors afforded by inactivation of mlaA is advantageous in vivo. Based on these results, we propose N. gonorrhoeae restricts MlaA levels to modulate cell envelope homeostasis and fine-tune virulence.

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“…S1g), using a 240 to 205 nm scan, shows minimum mean residue molecular ellipticity [Θ] (MRW) around 217 nm and a crossover at 210 nm, indicating refolded OmpLA contains mainly of β-structure, similar to EcOmpLA 11,12,13 . Protocol described here (Methods) yields more refolded OmpLA than previously published methods 14,15 . Refolded OmpLA was crystallized in various conditions using MemGold 1 and 2.…”

Section: Resultsmentioning

confidence: 99%

Crystal structure of calcium bound outer membrane phospholipase A (OmpLA) fromSalmonella typhiandin silicoanti-microbial screening

Perumal

Raina

et al. 2020

Preprint

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24Antimicrobial resistance is widespread in Salmonella infections that affect millions 25 worldwide. Salmonella typhi and other Gram-negative bacterial pathogens encode an outer 26 membrane phospholipase A (OmpLA), crucial for their membrane integrity. Further, OmpLA 27 is implicated in pathogen internalization, haemolysis, acid tolerance, virulence and sustained 28 infection in human hosts. OmpLA is an attractive drug target for developing novel anti-29 microbials that attenuate virulence, as the abrogation of OmpLA encoding pldA gene causes 30 loss of virulence. Here, we present the crystal structure of Salmonella typhi OmpLA in 31 dimeric calcium bound activated state at 2.95 Å. Structure analysis suggests that OmpLA is a 32 potential druggable target. Further, we have identified and shortlisted small molecules that 33 bind at the dimer interface using structure based in silico screening, docking and molecular 34 dynamics. While it requires further experimental validation, anti-microbial discovery 35 targeting OmpLA from gram-negative pathogens offers an advantage as OmpLA is required 36 for virulence. 37 38 Keywords: Outer membrane phospholipase A (OmpLA), OmpLA, Salmonella typhi, crystal 39 structure, antibiotic resistance, antimicrobial design 40 41 42 43 44 65 strongly suggests OmpLA is not essential for growth but is a major virulence factor and 66 hence a potential drug target. Interestingly, bacterial OmpLA shows no sequence or structural 67 homology with soluble phospholipases in human, indicative of its usefulness as a unique drug 68 target. 69 4 70 Results and discussion 71 Structure determination of S. typhi OmpLA 72S. typhi OmpLA (StOmpLA), cloned without the signal sequence ( Fig. S1a, b), was 73 overexpressed in E. coli as inclusion bodies. Scouting urea concentration for unfolding 74 OmpLA shows sharp rise in absorption at OD280 at 4 M which stabilizes at 6 M ( Fig. S1c, d). 75Large-scale unfolding was done using 8 M urea, and refolding was achieved in presence of 76 0.3% Polyoxyethylene (9) lauryl ether (C12E9). Refolded OmpLA was further purified using 77 ion-exchange and size exclusion chromatography ( Fig. S1e, f) and concentrated to 14 mg/ml. 78Then the protein was detergent exchanged from C12E9 to β-octyl-glucoside (β-OG) using an 79 ion-exchange column, concentrated to 14 mg/ml, flash frozen in liquid nitrogen and stored at 80 -80 ºC before crystallization. Total yield of refolded and detergent exchanged OmpLA was 81 15 mg/gm of purified inclusion bodies. Circular dichroism (CD) data (Fig. S1g), using a 240 82 to 205 nm scan, shows minimum mean residue molecular ellipticity [Ɵ] (MRW) around 217 83 nm and a crossover at 210 nm, indicating refolded OmpLA contains mainly of β-structure, 84 similar to EcOmpLA 11,12,13 . Protocol described here (Methods) yields more refolded OmpLA 85 than previously published methods 14,15 . Refolded OmpLA was crystallized in various 86 conditions using MemGold 1 and 2. Diffraction quality crystals (Fig. S1h) were obtained in a 87 condition containing 0.1 ...

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Structural evidence for dimerization-regulated activation of an integral membrane phospholipase

Cited by 139 publications

References 35 publications

Overcoming Iron Deficiency of an Escherichia coli tonB Mutant by Increasing Outer Membrane Permeability

Overcoming Iron Deficiency of an Escherichia coli tonB Mutant by Increasing Outer Membrane Permeability

Neisseria gonorrhoeae MlaA influences gonococcal virulence and membrane vesicle production

Crystal structure of calcium bound outer membrane phospholipase A (OmpLA) fromSalmonella typhiandin silicoanti-microbial screening

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