Separation of the Cell Envelope for Gram-negative Bacteria into Inner and Outer Membrane Fractions with Technical Adjustments for &lt;em&gt;Acinetobacter baumannii&lt;/em&gt;

Cian, Melina B.; Giordano, Nicole; Mettlach, Joshua A.; Minor, Keaton E.; Dalebroux, Zachary D.

doi:10.3791/60517

Cited by 14 publications

(20 citation statements)

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“…Lastly, we devised an assay to detect defects in anterograde transport that monitors outer membrane vesicle (OMV) formation with a pulse-chase in lieu of the membrane separations and LC-MS/MS assay used by Kamischke et al At this time, Kamischke et al is the only publication reporting the successful separation of A. baumannii inner and outer membrane fractions, which we ultimately found to be unreproducible ( Kamischke et al, 2019 ). Both our lab and the Dalebroux lab have found the methodology to be insufficient as reported here and in JOVE ( Cian et al, 2020 ). As we were unable to reliably separate membranes of A. baumannii, we utilized the OMV pulse-chase to circumvent these caveats.…”

Section: Introductionmentioning

confidence: 69%

The Mla pathway in Acinetobacter baumannii has no demonstrable role in anterograde lipid transport

Powers

Simpson

Trent

2020

eLife

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The asymmetric outer membrane (OM) of Gram-negative bacteria functions as a selective permeability barrier to the environment. Perturbations to OM lipid asymmetry sensitize the cell to antibiotics. As such, mechanisms involved in lipid asymmetry are fundamental to our understanding of OM lipid homeostasis. One such mechanism, the Maintenance of lipid asymmetry (Mla) pathway has been proposed to extract mislocalized glycerophospholipids from the outer leaflet of the OM and return them to the inner membrane (IM). Work on this pathway in Acinetobacter baumannii support conflicting models for the directionality of the Mla system being retrograde (OM to IM) or anterograde (IM to OM). Here we show conclusively that A. baumannii mla mutants exhibit no defects in anterograde transport. Furthermore, we identify an allele of the GTPase obgE that is synthetically sick in the absence of Mla; providing another link between cell envelope homeostasis and stringent response.

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Section: Introductionmentioning

confidence: 69%

The Mla pathway in Acinetobacter baumannii has no demonstrable role in anterograde lipid transport

Powers

Simpson

Trent

2020

eLife

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“…Therefore, the GPL composition of the wild-type and mlaF -deficient strains was measured. Total membrane and OM fractions were collected from logarithmic phase bacteria ( Figure S4A ) and GPL was quantified for each fraction by liquid chromatography-tandem mass spectrometry (LC-MS/MS) ( Cian et al, 2020 ; Dalebroux et al, 2014 , 2015 ; Osborn et al, 1972 ). There was no significant difference in the total GPL in the strains ( Figure S4B ).…”

Section: Resultsmentioning

confidence: 99%

“…To quantify differences in LOS abundance, LOS molecules were extracted from bacterial suspensions that had been normalized to cell number by optical density and colony-forming units ( Figure S4E ). Using a hot phenol-chloroform extraction method, gradient-gel electrophoresis, and Pro-Q Emerald 300 staining ( Figures 4C , S4F , and S4G ), LOS levels were quantified for multiple biological and technical replicates from mid-log cultures ( Cian et al, 2020 ). Relative to the wild-type strain, the LOS levels of the Δ mlaF mutant were significantly decreased, whereas the LOS levels of the suppressor mutant were significantly increased ( Figure 4C ).…”

Section: Resultsmentioning

confidence: 99%

“…Membranes were isolated, fractionated, and characterized similarly to previously described methods ( Cian et al 2020 ). Overnight cultures were grown in 50 mL LB and diluted 1:100 into 1 L LB in a 2-L flask and incubated at 37°C with shaking until OD 600 of 0.6 to 0.8.…”

Section: Methodsmentioning

confidence: 99%

“…An adjusted sucrose gradient was prepared as follows: 2 mL of 73% sucrose, 4 mL of 45% sucrose, the sample, and the void filled in by 20% sucrose. Forty-five percent sucrose was used in place of the 53% described in previous protocols to allow for the separation of OM; this strain of Acinetobacter the OM was not sufficiently dense to flow through the 53% sucrose ( Cian et al, 2020 ). The sucrose gradients were then centrifuged overnight at 288,000 x g using a SW-41 swinging bucket rotor in a Beckman ultracentrifuge.…”

Section: Methodsmentioning

confidence: 99%

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Modulating Isoprenoid Biosynthesis Increases Lipooligosaccharides and Restores Acinetobacter baumannii Resistance to Host and Antibiotic Stress

et al. 2020

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SUMMARY Acinetobacter baumannii is a leading cause of ventilator-associated pneumonia and a critical threat due to multidrug resistance. The A. baumannii outer membrane is an asymmetric lipid bilayer composed of inner leaflet glycerophospholipids and outer leaflet lipooligosaccharides. Deleting mlaF of the maintenance of lipid asymmetry (Mla) system causes A. baumannii to become more susceptible to pulmonary surfactants and antibiotics and decreases bacterial survival in the lungs of mice. Spontaneous suppressor mutants isolated from infected mice contain an IS Aba11 insertion upstream of the ispB initiation codon, an essential isoprenoid biosynthesis gene. The insertion restores antimicrobial resistance and virulence to Δ mlaF . The suppressor strain increases lipooligosaccharides, suggesting that the mechanism involves balancing the glycerophospholipids/lipooligosaccharides ratio on the bacterial surface. An identical insertion exists in an extensively drug-resistant A. baumannii isolate, demonstrating its clinical relevance. These data show that the stresses bacteria encounter during infection select for genomic rearrangements that increase resistance to antimicrobials.

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Biosynthetic neoantigen displayed on bacteria derived vesicles elicit systemic antitumour immunity

Meng

Zhang

et al. 2022

J of Extracellular Vesicle

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Neoantigens derived from mutant proteins in tumour cells could elicit potent personalized anti‐tumour immunity. Nevertheless, the layout of vaccine vehicle and synthesis of neoantigen are pivotal for stimulating robust response. The power of synthetic biology enables genetic programming bacteria to produce therapeutic agents under contol of the gene circuits. Herein, we genetically engineered bacteria to synthesize fusion neoantigens, and prepared bacteria derived vesicles (BDVs) presenting the neoantigens (BDVs‐Neo) as personalized therapeutic vaccine to drive systemic antitumour response. BDVs‐Neo and granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) were inoculated subcutaneously within hydrogel (Gel), whereas sustaining release of BDVs‐Lipopolysaccharide (LPS) and GM‐CSF recruited the dendritic cells (DCs). Virtually, Gel‐BDVs‐Neo combined with the programmed cell death protein 1 (PD‐1) antibody intensively enhanced proliferation and activation of tumour‐infiltrated T cells, as well as memory T cell clone expansion. Consequently, BDVs‐Neo combining with checkpoint blockade therapy effectively prevented tumour relapse and metastasis.

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Separation of the Cell Envelope for Gram-negative Bacteria into Inner and Outer Membrane Fractions with Technical Adjustments for <em>Acinetobacter baumannii</em>

Cited by 14 publications

References 0 publications

The Mla pathway in Acinetobacter baumannii has no demonstrable role in anterograde lipid transport

The Mla pathway in Acinetobacter baumannii has no demonstrable role in anterograde lipid transport

Modulating Isoprenoid Biosynthesis Increases Lipooligosaccharides and Restores Acinetobacter baumannii Resistance to Host and Antibiotic Stress

Biosynthetic neoantigen displayed on bacteria derived vesicles elicit systemic antitumour immunity

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