RESUMO
Bacteria that occupy an intracellular niche can evade extracellular host immune responses and antimicrobial molecules. In addition to classic intracellular pathogens, other bacteria including uropathogenic Escherichia coli (UPEC) can adopt both extracellular and intracellular lifestyles. UPEC intracellular survival and replication complicates treatment, as many therapeutic molecules do not effectively reach all components of the infection cycle. In this study, we explored cell-penetrating antimicrobial peptides from distinct structural classes as alternative molecules for targeting bacteria. We identified two ß-hairpin peptides from the horseshoe crab, tachyplesin I and polyphemusin I, with broad antimicrobial activity toward a panel of pathogenic and non-pathogenic bacteria in planktonic form. Peptide analogs [I11A]tachyplesin I and [I11S]tachyplesin I maintained activity toward bacteria, but were less toxic to mammalian cells than native tachyplesin I. This important increase in therapeutic window allowed treatment with higher concentrations of [I11A]tachyplesin I and [I11S]tachyplesin I, to significantly reduce intramacrophage survival of UPEC in an in vitro infection model. Mechanistic studies using bacterial cells, model membranes and cell membrane extracts, suggest that tachyplesin I and polyphemusin I peptides kill UPEC by selectively binding and disrupting bacterial cell membranes. Moreover, treatment of UPEC with sublethal peptide concentrations increased zinc toxicity and enhanced innate macrophage antimicrobial pathways. In summary, our combined data show that cell-penetrating peptides are attractive alternatives to traditional small molecule antibiotics for treating UPEC infection, and that optimization of native peptide sequences can deliver effective antimicrobials for targeting bacteria in extracellular and intracellular environments.
Assuntos
Antibacterianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/farmacologia , Peptídeos Antimicrobianos/farmacologia , Bactérias/efeitos dos fármacos , Infecções Bacterianas/tratamento farmacológico , Proteínas de Ligação a DNA/farmacologia , Peptídeos Cíclicos/farmacologia , Animais , Células da Medula Óssea , Membrana Celular/efeitos dos fármacos , Células Cultivadas , Eritrócitos , Caranguejos Ferradura/metabolismo , Humanos , Camundongos Endogâmicos C57BL , Cultura Primária de CélulasRESUMO
Rapid clearance by renal filtration is a major impediment to the translation of small bioactive biologics into drugs. To extend serum t1/2, a commonly used approach is to attach drug leads to the G-related albumin-binding domain (ABD) to bind albumin and evade clearance. Despite the success of this approach in extending half-lives of a wide range of biologics, it is unclear whether the existing constructs are optimized for binding and size; any improvements along these lines could lead to improved drugs. Characterization of the biophysics of binding of an ABD to albumin in solution could shed light on this question. Here, we examine the binding of an ABD to human serum albumin using isothermal titration calorimetry and assess the structural integrity of the ABD using CD, NMR, and molecular dynamics. A structure-activity analysis of truncations of the ABD suggests that downsized variants could replace the full-length domain. Reducing size could have the benefit of reducing potential immunogenicity problems. We further showed that one of these variants could be used to design a bifunctional molecule with affinity for albumin and a serum protein involved in cholesterol metabolism, PCSK9, demonstrating the potential utility of these fragments in the design of cholesterol-lowering drugs. Future work could extend these in vitro binding studies to other ABD variants to develop therapeutics. Our study presents new understanding of the solution structural and binding properties of ABDs, which has implications for the design of next-generation long-lasting therapeutics.
Assuntos
Portadores de Fármacos/química , Simulação de Acoplamento Molecular , Albumina Sérica Humana/química , Humanos , Domínios Proteicos , Albumina Sérica Humana/genética , Relação Estrutura-AtividadeRESUMO
Melioidosis is a neglected tropical disease caused by the Gram-negative soil bacterium Burkholderia pseudomallei. Current antibiotic regimens used to treat melioidosis are prolonged and expensive, and often ineffective because of intrinsic and acquired antimicrobial resistance. Efforts to develop new treatments for melioidosis are limited by the risks associated with handling pathogenic B. pseudomallei, which restricts research to facilities with biosafety level three containment. Closely related nonpathogenic Burkholderia can be investigated under less stringent biosafety level two containment, and we hypothesized that they could be used as model organisms for developing therapies that would also be effective against B. pseudomallei. We used microbroth dilution assays to compare drug susceptibility profiles of three B. pseudomallei strains and five nonpathogenic Burkholderia strains. Burkholderia humptydooensis, Burkholderia thailandensis, and Burkholderia territorii had similar susceptibility profiles to pathogenic B. pseudomallei that support their potential as safer in vitro models for developing new melioidosis therapies.