pH Induced Conformational Transitions in the Transforming Growth Factor β-Induced Protein (TGFβIp) Associated Corneal Dystrophy Mutants

Most stromal corneal dystrophies are associated with aggregation and deposition of the mutated transforming growth factor-β induced protein (TGFβIp). The 4th_FAS1 domain of TGFβIp harbors ~80% of the mutations that forms amyloidogenic and non-amyloidogenic aggregates. To understand the mechanism of aggregation and the differences between the amyloidogenic and non-amyloidogenic phenotypes, we expressed the 4th_FAS1 domains of TGFβIp carrying the mutations R555W (non-amyloidogenic) and H572R (amyloidogenic) along with the wild-type (WT). R555W was more susceptible to acidic pH compared to H572R and displayed varying chemical stabilities with decreasing pH. Thermal denaturation studies at acidic pH showed that while WT did not undergo any conformational transition, the mutants exhibited a clear pH-dependent irreversible conversion from αβ conformation to β-sheet oligomers. The β-oligomers of both mutants were stable at physiological temperature and pH. Electron microscopy and dynamic light scattering studies showed that β-oligomers of H572R were larger compared to R555W. The β-oligomers of both mutants were cytotoxic to primary human corneal stromal fibroblast (pHCSF) cells. The β-oligomers of both mutants exhibit variations in their morphologies, sizes, thermal and chemical stabilities, aggregation patterns and cytotoxicities

Insight into membrane selectivity of linear and branched polyethylenimines and their potential as biocides for advanced wound dressings

We report here structure-property relationship between linear and branched polyethylene imines by examining their antimicrobial activities against wide range of pathogens. Both the polymers target the cytoplasmic membrane of bacteria and yeasts, eliciting rapid microbicidal properties. Using multiscale molecular dynamic simulations, we showed that, in both fully or partially protonated forms LPEI discriminates between mammalian and bacterial model membranes whereas BPEI lacks selectivity for both the model membranes. Simulation results suggest that LPEI forms weak complex with the zwitterionic lipids whereas the side chain amino groups of BPEI sequester the zwitterionic lipids by forming tight complex. Consistent with these observations, label-free cell impedance measurements, cell viability assays and high content analysis indicate that BPEI is cytotoxic to human epithelial and fibroblasts cells. Crosslinking of BPEI onto electrospun gelatin mats attenuate the cytotoxicity for fibroblasts while retaining the antimicrobial activity against Gram-positive and yeasts strains. PEI crosslinked gelatin mats elicit bactericidal activity by contact-mediated killing and durable to leaching for 7days. The potent antimicrobial activity combined with enhanced selectivity of the crosslinked ES gelatin mats would expand the arsenel of biocides in the management of superficial skin infections. The contact-mediated microbicidal properties may avert antimicrobial resistance and expand the diversity of applications to prevent microbial contamination.NRF (Natl Research Foundation, S’pore)MOE (Min. of Education, S’pore)NMRC (Natl Medical Research Council, S’pore)Accepted versio

Branched Peptide, B2088, Disrupts the Supramolecular Organization of Lipopolysaccharides and Sensitizes the Gram-negative Bacteria

Dissecting the complexities of branched peptide-lipopolysaccharides (LPS) interactions provide rationale for the development of non-cytotoxic antibiotic adjuvants. Using various biophysical methods, we show that the branched peptide, B2088, binds to lipid A and disrupts the supramolecular organization of LPS. The disruption of outer membrane in an intact bacterium was demonstrated by fluorescence spectroscopy and checkerboard assays, the latter confirming strong to moderate synergism between B2088 and various classes of antibiotics. The potency of synergistic combinations of B2088 and antibiotics was further established by time-kill kinetics, mammalian cell culture infections model and in vivo model of bacterial keratitis. Importantly, B2088 did not show any cytotoxicity to corneal epithelial cells for at least 96 h continuous exposure or hemolytic activity even at 20 mg/ml. Peptide congeners containing norvaline, phenylalanine and tyrosine (instead of valine in B2088) displayed better synergism compared to other substitutions. We propose that high affinity and subsequent disruption of the supramolecular assembly of LPS by the branched peptides are vital for the development of non-cytotoxic antibiotic adjuvants that can enhance the accessibility of conventional antibiotics to the intracellular targets, decrease the antibiotic consumption and holds promise in averting antibiotic resistance.NRF (Natl Research Foundation, S’pore)ASTAR (Agency for Sci., Tech. and Research, S’pore)NMRC (Natl Medical Research Council, S’pore)Published versio