Engineering Chirally Blind Protein Pseudocapsids into Antibacterial Persisters

Abstract

Antimicrobial resistance stimulates the search for antimicrobial forms that may be less subject to acquired resistance. Here we report a conceptual design of protein pseudocapsids exhibiting a broad spectrum of antimicrobial activities. Unlike conventional antibiotics, these agents are effective against phenotypic bacterial variants, while clearing “superbugs” in vivo without toxicity. The design adopts an icosahedral architecture that is polymorphic in size, but not in shape, and that is available in both l and d epimeric forms. Using a combination of nanoscale and single-cell imaging we demonstrate that such pseudocapsids inflict rapid and irreparable damage to bacterial cells. In phospholipid membranes they rapidly convert into nanopores, which remain confined to the binding positions of individual pseudocapsids. This mechanism ensures precisely delivered influxes of high antimicrobial doses, rendering the design a versatile platform for engineering structurally diverse and functionally persistent antimicrobial agents.

Revealing Sources of Variation for Reproducible Imaging of Protein Assemblies by Electron Microscopy

“…Consistent with observations by others [14][15][16][17], variations in staining were due to the accumulation of stain in the interior of the spheroids, which contrasts with solid nanoparticles assembled through condensation with nucleic acids [18]. Model 1 comprises of two layers of β-pleated networks arranging into a double-wall shell [8]. This may explain the alternation of stain patterns observed in individual spheroids, suggesting a double wall structure ( Figure 1C).…”

“…This study employs two main types of self-assembled protein structures. One type, spherical capsid-like shells, is exemplified by model 1, a virus-like capsid assembled from a re-engineered antimicrobial domain of human breast milk protein lactoferrin [8], and model 2, the capsids of the MS2 bacteriophage. The other type is represented by protein filaments of different lengths, which are exemplified in models 3 and 4 [9,10,12].…”

“…Model 1 comprises of two layers of β-pleated networks arranging into a double-wall shell [8]. This may explain the alternation of stain patterns observed in individual spheroids, suggesting a double wall structure ( Figure 1C).…”

“…Models 1, 3, and 4 were assembled from synthetic polypeptide constructs prepared as reported elsewhere [8][9][10]. Briefly, monomer units were synthesized on a Liberty Blue microwave peptide synthesizer (CEM Corp., Matthews, NC, USA) on a Rink amide 4-methylbenzhydrylamine resin (model 1) and a Fmoc-Gln(Trt)-Wang resin (models 3 and 4) using 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU) and diisopropylethylamine as coupling reagents and conventional solid-phase Fmoc/tBu protocols.…”

“…Briefly, monomer units were synthesized on a Liberty Blue microwave peptide synthesizer (CEM Corp., Matthews, NC, USA) on a Rink amide 4-methylbenzhydrylamine resin (model 1) and a Fmoc-Gln(Trt)-Wang resin (models 3 and 4) using 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU) and diisopropylethylamine as coupling reagents and conventional solid-phase Fmoc/tBu protocols. Fmoc-Lys(Mtt)-OH was used to enable orthogonal conjugation for model 1 [2] and Fmoc-Lys(Alloc)-OH was used to label model 4 with Alexa Fluor 488 [8]. After cleavage from the resin and deprotection, the synthesized constructs were purified by reversed-phase high performance liquid chromatography (RP-HPLC) and were then lyophilized and had their identities confirmed by matrix-assisted laser desorption/ionization time of flight (MALDI-ToF) mass spectrometry.…”