Browsing by Subject "Antimicrobial peptides"

Now showing 1 - 5 of 5
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    Biophysical characterization of membrane proteins and antimicrobial peptides by solution and solid-state NMR spectroscopy.
    (2011-03) Verardi, Raffaello
    Membrane proteins and antimicrobial peptides represent two diverse and challenging classes of macromolecules to characterize at the molecular level. They are linked by the interaction with the lipid bilayer of the cell membrane. Within the lipid bilayer, membrane proteins are involved in vital biochemical processes such as ion transport, signal transduction and cell adhesion. Antimicrobial peptides are a broad class of polypeptides produced by all living organisms, representing the first line of defense against bacterial infections. They work by selectively targeting the bacterial membranes and subsequently killing the cell by a variety of mechanisms such as membrane disruption, membrane potential dissipation and enzyme inactivation. Although very important, membrane proteins and antimicrobial peptides are underrepresented in terms of available high-resolution structural information compared to water-soluble proteins and this limits the current understanding of how they work in living cells. In this thesis I summarize my contribution towards the elucidation of the high-resolution structures of the integral membrane protein phospholamban and the mechanism of action of two important antimicrobial peptides (LL37 and distinctin) by a hybrid solution and solid-state nuclear magnetic resonance spectroscopy approach. These results provide new insights and methodologies to study and understand how key membrane proteins and antimicrobial peptides elicit their function.
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    Computer Simulations and Experimental Designs to Study the Mechanisms of Actions of Antimicrobial Peptides
    (2018-10) Lai, Pin-Kuang
    Antimicrobial peptides (AMPs) are promising alternatives to traditional antibiotics, which have a serious resistance crisis. We studied the mechanisms of three different types of AMPs using computational and experimental methods. First, computer simulations are performed to study the AMP microcin J25 (MJ25), a 21-mer peptide with an unusual lasso structure and high activity against Gram-negative bacteria. MJ25 has intracellular targets. The initial step for MJ25 acquisition in bacterial cells is binding to the outer membrane receptor FhuA. Molecular dynamics simulation is implemented to study the binding mechanism of MJ25 to FhuA and to search for important binding residues. The absolute binding free energy calculated from combined free energy perturbation (FEP) and thermodynamic integration (TI) methods agrees well with experimental data. In addition, computational mutation analysis reveals that the His5 is the key residue responsible for MJ25 and FhuA association. We find that the number of hydrogen bonds is essential for MJ25 binding to FhuA. This atomistic, quantitative insight sheds light on the mechanism of action of MJ25, and may pave a path for designing active MJ25 analogues. Second, protegrin-1 (PG-1) is a cationic arginine-rich AMP. It is widely accepted that PG-1 induces membrane disruption by forming pores that lead to cell death. However, the insertion mechanism for these highly cationic peptides into the hydrophobic membrane environment is still poorly understood at the molecular scale. It has previously been determined that the association of arginine guanidinium and lipid phosphate groups results in strong bidentate bonds that stabilize peptide-lipid complexes. It has also been suggested that arginine residues are able to drag phosphate groups as they insert inside the membrane to form a toroidal pore. However, whether bidentate bonds play a significant role in inducing a pore formation remains unclear. To investigate the role of bidentate complexes in PG-1 translocation, we conducted molecular dynamics simulations. Two computational electroporation methods were implemented to examine the translocation process. We found PG-1 could insert into the membrane provided the external electric potential is large enough to first induce a water column or pore within the lipid bilayer membrane. We also found that the highly charged PG-1 is capable in itself of inducing molecular electroporation. Substitution of arginines with charge-equivalent lysines showed a markedly reduced tendency for insertion. This indicates the guanidinium group likely facilitates PG-1 translocation. Potential of mean force calculations suggest that peptide insertion inside the hydrophobic environment of the membrane core is not favored. We found that formation of a water column or pore might be a prerequisite for PG-1 translocation. We also found PG-1 can stabilize the pore after insertion. We suggest that PG-1 could be a pore inducer and stabilizer. This work sheds some light on PG-1 translocation mechanisms at the molecular level. Methods presented in this study may be extended to other arginine-rich antimicrobial and cell-penetrating peptides. Third, oncocin is a proline-rich antimicrobial peptide that inhibits protein synthesis by binding to the bacterial ribosome. In this work, the antimicrobial activity of oncocin was improved by systematic peptide mutagenesis and activity evaluation. We found that a pair of cationic substitutions (P4K and L7K/R) improves the activity by 2-4 fold (p<0.05) against multiple Gram-negative bacteria. An in vitro transcription / translation assay indicated that the increased activity was not because of stronger ribosome binding. Rather a cellular internalization assay revealed a higher internalization rate for the optimized analogs thereby suggesting a mechanism to increase potency. In addition, we found that the optimized peptides’ benefit is dependent upon nutrient depleted media conditions. The molecular design and characterization strategies have broad potential for development of antimicrobial peptides.
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    Engineering Probiotic Bacteria for Use as Antibiotic Alternatives
    (2018-02) Forkus, Brittany Anne
    Decades of overuse of antibiotics has led to the emergence of resistant infections across the globe. Healthcare professionals are running out of viable options, as clinical isolates have begun resisting treatment to even last resort therapies. The emergence of these ‘superbugs’, coupled with the lack of new drugs in the discovery pipeline, has led to the possibility of a ‘post-antibiotic’ era. With the primary driving force for resistance development being the overuse of antibiotics, technologies are being sought to limit their injudicious application within the clinical and agricultural sectors. For decades, antimicrobial peptides (AMPs) have been proposed as a promising contender in the fight against microbial resistance. AMPs are small peptides that are produced natively from organisms across all domains of life as a first line of defense against microbial challenge. However, despite their therapeutic potential, AMPs have widely failed in translational success due to delivery and synthesis challenges. In this work, we propose engineering probiotic bacteria as AMP-delivery vehicles to overcome the inherent transport barriers of AMP-therapy. We focus on developing engineered probiotics to target resident pathogens of the gastrointestinal tract. The success of this technology could aid in the resistance crisis by unlocking the antibiotic power of many otherwise unusable peptide antibiotics. We have developed several derivatives of the probiotic strain, E.coli Nissle 1917 (EcN), which are capable of eliciting antibiotic activity against clinical and foodborne pathogens. The foundation of this work lays in the reorganization of AMP biosynthetic gene clusters for functional utility. We describe our development of the engineered probiotic, EcN(J25), which led to the first in vivo success of AMP-producing probiotics. Treatment with EcN(J25) was capable of reducing Salmonella carriage in pre-harvest poultry by 97% just 14-days post-treatment. In a similar workflow, we then focused on the development of EcN(C7) for use in decolonizing multidrug resistant E.coli in human carriers. Along the way we studied mechanisms of resistance, applied bioinformatics techniques, and developed novel synthetic biology tools for use in future engineered bacteria. The work within describes many of the challenges and potential of engineered probiotics, laying a foundation for future work in the field.
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    Multiscale models of antimicrobial peptides.
    (2010-12) Bolintineanu, Dan
    Antimicrobial peptides (AMPs) are small proteins that constitute a first line of defense against invading pathogens in the innate immune systems of countless plant and animal species. Their mechanism of action relies to a large extent on selectively disrupting the cell membranes of bacteria, which makes them promising therapeutic agents in the fight against infectious pathogens, including antibiotic-resistant bacterial strains. However, AMPs also exhibit toxicity towards mammalian cells, which presents a significant bottleneck in the development of AMPs for antibiotic treatment in humans. Thus, before the full potential of AMPs as therapeutic agents can be unlocked, their fundamental mechanism of action must be understood in order to identify targets for mutation that can improve activity and ameliorate toxic effects. To this end, we carry out computer simulations and modeling studies in order to understand the interactions of protegrin, a particularly potent and well-studied AMP, with lipid bilayers that mimic the compositions of bacterial and mammalian cell membranes. In particular, we attempt to connect molecular-level thermodynamic information to biologically relevant membrane association equilibria; we elucidate the ion transport characteristics of protegrin pores, and show how the atomistic structure leads to experimentally observed conductance behavior; we provide multi-scale models that can quantitatively link the structure of protegrin and protegrin pores to the leakage of potassium ions from bacterial cells, which appears to be an essential element in the bactericidal mechanism of action of protegrin; finally, we investigate the structure of protegrin pores using molecular dynamics simulations with atomistic resolution. Our work reveals this link for the first time, and provides a united, quantitative connection between molecular-level structural and thermodynamic information and mesoscopic, measurable quantities. Our modeling tools can be easily extended to other AMPs, and protein-membrane systems in general.
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    Seasonal benefits of a natural propolis envelope to honey bee immunity and colony health
    (Company of Biologists, 2015) Borba, Renata S.; Klyczek, Karen K.; Mogen, Kim L.; Spivak, Marla
    Honey bees, as social insects, rely on collective behavioral defenses that produce a colony-level immune phenotype, or social immunity, which in turn impacts the immune response of individuals. One behavioral defense is the collection and deposition of antimicrobial plant resins, or propolis, in the nest. We tested the effect of a naturally constructed propolis envelope within standard beekeeping equipment on the pathogen and parasite load of large field colonies, and on immune system activity, virus and storage protein levels of individual bees over the course of a year. The main effect of the propolis envelope was a decreased and more uniform baseline expression of immune genes in bees during summer and autumn months each year, compared with the immune activity in bees with no propolis envelope in the colony. The most important function of the propolis envelope may be to modulate costly immune system activity. As no differences were found in levels of bacteria, pathogens and parasites between the treatment groups, the propolis envelope may act directly on the immune system, reducing the bees’ need to activate the physiologically costly production of humoral immune responses. Colonies with a natural propolis envelope had increased colony strength and vitellogenin levels after surviving the winter in one of the two years of the study, despite the fact that the biological activity of the propolis diminished over the winter. A natural propolis envelope acts as an important antimicrobial layer enshrouding the colony, benefiting individual immunity and ultimately colony health.