We report the synthesis, characterization, activity, and biocompatibility of a novel series of antimicrobial peptide-polymer conjugates. Using parent peptide aurein 2.2, we designed a peptide array (∼100 peptides) with single and multiple W and R mutations and identified antimicrobial peptides (AMPs) with potent activity against Staphylococcus aureus (S. aureus). These novel AMPs were conjugated to hyperbranched polyglycerols (HPGs) of different molecular weights and number of peptides to improve their antimicrobial activity and toxicity. The cell and blood compatibility studies of these conjugates demonstrated better properties than those of the AMP alone. However, conjugates showed lower antimicrobial activity in comparison to that of peptides, as determined from minimal inhibition concentrations (MICs) against S. aureus, but considerably better than that of the available polymer-AMP conjugates in the literature. In addition to measuring MICs and characterizing the biocompatibility, circular dichroism spectroscopy was used to investigate the interaction of the novel conjugates with model bacterial biomembranes. Moreover, the novel conjugates were exposed to trypsin to evaluate their stability. It was found that the conjugates resist proteolysis in comparison with unprotected peptides. The peptide conjugates were active in serum and whole blood. Overall, the results show that combining a highly active AMP and low-molecular-weight HPG yields bioconjugates with excellent biocompatibility, MICs below 100 μg/mL, and proteolytic stability, which could potentially improve its utility for in vivo applications.
Clostridium thermocellum is actively being developed as a microbial platform to produce biofuels and chemicals from renewable plant biomass. An attractive feature of this bacterium is its ability to efficiently degrade lignocellulose using surfacedisplayed cellulosomes, large multi-protein complexes that house different types of cellulase enzymes. Clostridium thermocellum tailors the enzyme composition of its cellulosome using nine membrane-embedded anti-σ factors (RsgI1-9), which are thought to sense different types of extracellular carbohydrates and then elicit distinct gene expression programs via cytoplasmic σ factors. Here we show that the RsgI9 anti-σ factor interacts with cellulose via a C-terminal bi-domain unit. A 2.0 Å crystal structure reveals that the unit is constructed from S1C peptidase and NTF2-like protein domains that contain a potential binding site for cellulose. Small-angle X-ray scattering experiments of the intact ectodomain indicate that it adopts a bi-lobed, elongated conformation. In the structure, a conserved RsgI extracellular (CRE) domain is connected to the bi-domain via a proline-rich linker, which is expected to project the carbohydrate-binding unit $160 Å from the cell surface. The CRE and prolinerich elements are conserved in several other C. thermocellum anti-σ factors, suggesting that they will also form extended structures that sense carbohydrates.
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