Localization and Quantification of Post-Translational Modifications of Proteins Using Electron Activated Dissociation Fragmentation on a Fast-Acquisition Time-of-Flight Mass Spectrometer.

Protein post-translational modifications (PTMs) are crucial and dynamic players in a large variety of cellular processes and signaling. Proteomic technologies have emerged as the method of choice to profile PTMs. However, these analyses remain challenging due to potential low PTM stoichiometry, the presence of multiple PTMs per proteolytic peptide, PTM site localization of isobaric peptides, and neutral losses. Collision-induced dissociation (CID) is commonly used to characterize PTMs, but the application of collision energy can lead to neutral losses and incomplete peptide sequencing for labile PTM groups. In this study, we assessed the performance of an alternative fragmentation, electron activated dissociation (EAD), to characterize, site localize, and quantify peptides with labile modifications in comparison to CID, both operated on a recently introduced fast-scanning quadrupole-time-of-flight (QqTOF) mass spectrometer. We analyzed biologically relevant phosphorylated, succinylated, malonylated, and acetylated synthetic peptides using targeted parallel reaction monitoring (PRM or MRMHR) assays. We report that electron-based fragmentation preserves the malonyl group from neutral losses. The novel tunable EAD kinetic energy maintained labile modification integrity and provided better peptide sequence coverage with strong PTM-site localization fragment ions. Activation of a novel trap-and-release technology significantly improves the duty cycle and provided significant MS/MS sensitivity gains by an average of 6-11-fold for EAD analyses. Evaluation of the quantitative EAD PRM workflows revealed high reproducibility with coefficients of variation of ∼2-7%, as well as very good linearity and quantification accuracy. This novel workflow combining EAD and trap-and-release technology provides high sensitivity, alternative fragmentation information to achieve confident PTM characterization and quantification.

Vancomycin-Arginine Conjugate Inhibits Growth of Carbapenem-Resistant E. coli and Targets Cell-Wall Synthesis.

The emergence of multi-drug-resistant Gram-negative bacteria, including carbapenem-resistant Enterobacteriaceae, is a major health problem that necessitates the development of new antibiotics. Vancomycin inhibits cell-wall synthesis in Gram-positive bacteria but is generally ineffective against Gram-negative bacteria and is unable to penetrate the outer membrane barrier. In an effort to determine whether vancomycin and other antibiotics effective against Gram-positive bacteria could, through modification, be rendered effective against Gram-negative bacteria, we discovered that the covalent attachment of a single arginine to vancomycin yielded conjugates with order-of-magnitude improvements in activity against Gram-negative bacteria, including pathogenic E. coli. The vancomycin-arginine conjugate (V-R) exhibited efficacy against actively growing bacteria, induced the loss of rod cellular morphology, and resulted in the intracellular accumulation of peptidoglycan precursors, all consistent with cell-wall synthesis disruption as its mechanism of action. Membrane permeabilization studies demonstrated an enhanced outer membrane permeability of V-R as compared with vancomycin. The conjugate exhibited no mammalian cell toxicity or hemolytic activity in MTT and hemolysis assays. Our study introduces a new vancomycin derivative effective against Gram-negative bacteria and underscores the broader potential of generating new antibiotics through combined mode-of-action and synthesis-informed design studies.

A Dual-Function Antibiotic-Transporter Conjugate Exhibits Superior Activity in Sterilizing MRSA Biofilms and Killing Persister Cells.

New strategies are urgently needed to target MRSA, a major global health problem and the leading cause of mortality from antibiotic-resistant infections in many countries. Here, we report a general approach to this problem exemplified by the design and synthesis of a vancomycin-d-octaarginine conjugate (V-r8) and investigation of its efficacy in addressing antibiotic-insensitive bacterial populations. V-r8 eradicated MRSA biofilm and persister cells in vitro, outperforming vancomycin by orders of magnitude. It also eliminated 97% of biofilm-associated MRSA in a murine wound infection model and displayed no acute dermal toxicity. This new dual-function conjugate displays enhanced cellular accumulation and membrane perturbation as compared to vancomycin. Based on its rapid and potent activity against biofilm and persister cells, V-r8 is a promising agent against clinical MRSA infections.

Phosphoethanolamine cellulose enhances curli-mediated adhesion of uropathogenic Escherichia coli to bladder epithelial cells.

Uropathogenic Escherichia coli (UPEC) are the major causative agents of urinary tract infections, employing numerous molecular strategies to contribute to adhesion, colonization, and persistence in the bladder niche. Identifying strategies to prevent adhesion and colonization is a promising approach to inhibit bacterial pathogenesis and to help preserve the efficacy of available antibiotics. This approach requires an improved understanding of the molecular determinants of adhesion to the bladder urothelium. We designed experiments using a custom-built live cell monolayer rheometer (LCMR) to quantitatively measure individual and combined contributions of bacterial cell surface structures [type 1 pili, curli, and phosphoethanolamine (pEtN) cellulose] to bladder cell adhesion. Using the UPEC strain UTI89, isogenic mutants, and controlled conditions for the differential production of cell surface structures, we discovered that curli can promote stronger adhesive interactions with bladder cells than type 1 pili. Moreover, the coproduction of curli and pEtN cellulose enhanced adhesion. The LCMR enables the evaluation of adhesion under high-shear conditions to reveal this role for pEtN cellulose which escaped detection using conventional tissue culture adhesion assays. Together with complementary biochemical experiments, the results support a model wherein cellulose serves a mortar-like function to promote curli association with and around the bacterial cell surface, resulting in increased bacterial adhesion strength at the bladder cell surface.