Extracellular Polymeric Substance Protects Some Cells in an Escherichia coli Biofilm from the Biomechanical Consequences of Treatment with Magainin 2.

Bacterial biofilms have long been recognized as a source of persistent infections and industrial contamination with their intransigence generally attributed to their protective layer of extracellular polymeric substances (EPS). EPS, consisting of secreted nucleic acids, proteins, and polysaccharides, make it difficult to fully eliminate biofilms by conventional chemical or physical means. Since most bacteria are capable of forming biofilms, understanding how biofilms respond to new antibiotic compounds and components of the immune system has important ramifications. Antimicrobial peptides (AMPs) are both potential novel antibiotic compounds and part of the immune response in many different organisms. Here, we use atomic force microscopy to investigate the biomechanical changes that occur in individual cells when a biofilm is exposed to the AMP magainin 2 (MAG2), which acts by permeabilizing bacterial membranes. While MAG2 is able to prevent biofilm initiation, cells in an established biofilm can withstand exposure to high concentrations of MAG2. Treated cells in the biofilm are classified into two distinct populations after treatment: one population of cells is indistinguishable from untreated cells, maintaining cellular turgor pressure and a smooth outer surface, and the second population of cells are softer than untreated cells and have a rough outer surface after treatment. Notably, the latter population is similar to planktonic cells treated with MAG2. The EPS likely reduces the local MAG2 concentration around the stiffer cells since once the EPS was enzymatically removed, all cells became softer and had rough outer surfaces. Thus, while MAG2 appears to have the same mechanism of action in biofilm cells as in planktonic ones, MAG2 cannot eradicate a biofilm unless coupled with the removal of the EPS.

The phosphorylation state of both hERG and KvLQT1 mediates protein-protein interactions between these complementary cardiac potassium channel alpha subunits.

KvLQT1 and hERG are the α-subunits of the voltage-gated K+ channels which carry the cardiac repolarizing currents IKs and IKr, respectively. These currents function in vivo with some redundancy to maintain appropriate action potential durations (APDs) in cardiomyocytes. As such, protein-protein interactions between hERG and KvLQT1 may be important in normal cardiac electrophysiology, as well as in arrhythmia and sudden cardiac death. Previous phenomenological observations of functional, mutual downregulation between these complementary repolarizing currents in transgenic rabbit models and human cell culture motivate our investigations into protein-protein interactions between hERG and KvLQT1. Previous data suggest that a dynamic, physical interaction between hERG and KvLQT1 modulates the respective currents. However, the mechanism by which hERG-KvLQT1 interactions are regulated is still poorly understood. Phosphorylation is proposed to play a role since modifying the phosphorylation state of each protein has been shown to alter channel kinetics, and both hERG and KvLQT1 are targets of the Ser/Thr protein kinase PKA, activated by elevated intracellular cAMP. In this work, quantitative apFRET analyses of phosphonull and phosphomimetic hERG and KvLQT1 mutants indicate that unphosphorylated hERG does not interact with KvLQT1, suggesting that hERG phosphorylation is important for wild-type proteins to interact. For proteins already potentially interacting, phosphorylation of KvLQT1 appears to be the driving factor abrogating hERG-KvLQT1 interaction. This work increases our knowledge about hERG-KvLQT1 interactions, which may contribute to the efforts to elucidate mechanisms that underlie many types of arrhythmias, and also further characterizes novel protein-protein interactions between two distinct potassium channel families.

Hybrids made from antimicrobial peptides with different mechanisms of action show enhanced membrane permeabilization.

Combining two known antimicrobial peptides (AMPs) into a hybrid peptide is one promising avenue in the design of agents with increased antibacterial activity. However, very few previous studies have considered the effect of creating a hybrid from one AMP that permeabilizes membranes and another AMP that acts intracellularly after translocating across the membrane. Moreover, very few studies have systematically evaluated the order of parent peptides or the presence of linkers in the design of hybrid AMPs. Here, we use a combination of antibacterial measurements, cellular assays and semi-quantitative confocal microscopy to characterize the activity and mechanism for a library of sixteen hybrid peptides. These hybrids consist of permutations of two primarily membrane translocating peptides, buforin II and DesHDAP1, and two primarily membrane permeabilizing peptides, magainin 2 and parasin. For all hybrids, the permeabilizing peptide appeared to dominate the mechanism, with hybrids primarily killing bacteria through membrane permeabilization. We also observed increased hybrid activity when the permeabilizing parent peptide was placed at the N-terminus. Activity data also highlighted the potential value of considering AMP cocktails in addition to hybrid peptides. Together, these observations will guide future design efforts aiming to design more active hybrid AMPs.

Production and Visualization of Bacterial Spheroplasts and Protoplasts to Characterize Antimicrobial Peptide Localization.

The use of confocal microscopy as a method to assess peptide localization patterns within bacteria is commonly inhibited by the resolution limits of conventional light microscopes. As the resolution for a given microscope cannot be easily enhanced, we present protocols to transform the small rod-shaped gram-negative Escherichia coli (E. coli) and gram-positive Bacillus megaterium (B. megaterium) into larger, easily imaged spherical forms called spheroplasts or protoplasts. This transformation allows observers to rapidly and clearly determine whether peptides lodge themselves into the bacterial membrane (i.e., membrane localizing) or cross the membrane to enter the cell (i.e., translocating). With this approach, we also present a systematic method to characterize peptides as membrane localizing or translocating. While this method can be used for a variety of membrane-active peptides and bacterial strains, we demonstrate the utility of this protocol by observing the interaction of Buforin II P11A (BF2 P11A), an antimicrobial peptide (AMP), with E. coli spheroplasts and B. megaterium protoplasts.

Bacterial Spheroplasts as a Model for Visualizing Membrane Translocation of Antimicrobial Peptides.

Studies attempting to characterize the membrane translocation of antimicrobial and cell-penetrating peptides are frequently limited by the resolution of conventional light microscopy. This study shows that spheroplasts provide a valuable approach to overcome these limits. Spheroplasts produce less ambiguous images and allow for more systematic analyses of localization. Data collected with spheroplasts are consistent with studies using normal bacterial cells and imply that a particular peptide may not always follow the same mechanism of action.

Disparate Response to Methotrexate in Stem Versus Non-Stem Cells.

Methotrexate (MTX) is a commonly used chemotherapeutic agent that kills cancer cells by binding dihydrofolate reductase (DHFR) as a competitive inhibitor. Due to its non-selectivity, MTX also impairs normal (non-cancerous) cell function and causes long-term damage to healthy tissue. These consequences have been investigated extensively in bone-derived cells due to their sensitivity to the drug. While DHFR likely plays a role in normal cell response to MTX, research in this area is limited. Moreover, how MTX sensitivity differs among cell types responsible for maintaining connective tissues is unknown. The goal of this study was to investigate the role of DHFR and subsequent nucleotide synthesis in normal cell response to MTX. We also sought to compare adverse effects of MTX among normal cell types to identify sensitive populations and resistant cell sources for regenerative procedures targeting patients undergoing chemotherapy. DHFR overexpression or exogenous amino acid + nucleoside delivery rescued normal cells from adverse MTX effects. Conversely, DHFR knockdown impaired MTX-treated adipose-derived stem cell (ASC) osteogenesis. Proliferation of ASCs and bone marrow stem cells was more resistant to MTX than that of terminally differentiated osteoblasts. However, stem cells became susceptible to the drug after beginning differentiation. These results suggest that the ability of stem cells to survive and to maintain their surrounding tissues likely depends on whether they are in a "stem" state when exposed to MTX. Therapeutic strategies that delay the differentiation of stem cells until clearance of the drug may produce more favorable outcomes in the long-term health of treated tissues.