Cationic antimicrobial peptides (CAMPs) serve as the first line of defense of the innate immune system against invading microbial pathogens. Gram-positive bacteria can resist CAMPs by modifying their anionic teichoic acids (TAs) with D-alanine, but the exact mechanism of resistance is not fully understood. Here, we utilized various functional and biophysical approaches to investigate the interactions of the human pathogen Group B Streptococcus (GBS) with a series of CAMPs having different properties. The data reveal that: (i) D-alanylation of lipoteichoic acids (LTAs) enhance GBS resistance only to a subset of CAMPs and there is a direct correlation between resistance and CAMPs length and charge density; (ii) resistance due to reduced anionic charge of LTAs is not attributed to decreased amounts of bound peptides to the bacteria; and (iii) D-alanylation most probably alters the conformation of LTAs which results in increasing the cell wall density, as seen by Transmission Electron Microscopy, and reduces the penetration of CAMPs through the cell wall. Furthermore, Atomic Force Microscopy reveals increased surface rigidity of the cell wall of the wild-type GBS strain to more than 20-fold that of the dltA mutant. We propose that D-alanylation of LTAs confers protection against linear CAMPs mainly by decreasing the flexibility and permeability of the cell wall, rather than by reducing the electrostatic interactions of the peptide with the cell surface. Overall, our findings uncover an important protective role of the cell wall against CAMPs and extend our understanding of mechanisms of bacterial resistance.

The slow kinetics of G protein‐activated K+ (GIRK) channels expressed in Xenopus oocytes were studied in single‐channel, inside‐out membrane patches. Channels formed by GIRK1 plus GIRK4 subunits, which are known to form the cardiac acetylcholine (ACh)‐activated GIRK channel (KACh), were activated by a near‐saturating dose of G protein βγ subunits (Gβγ; 20 nM). The kinetic parameters of the expressed GIRK1/4 channels were similar to those of cardiac KACh. GIRK1/4 channels differed significantly from channels formed by GIRK1 with the endogenous oocyte subunit GIRK5 (GIRK1/5) in some of their kinetic parameters and in a 3‐fold lower open probability, Po. The unexpectedly low Po (0.025) of GIRK1/4 was due to the presence of closures of hundreds of milliseconds; the channel spent ∼90 % of the time in the long closed states. GIRK1∼4 channels displayed a clear modal behaviour: on a time scale of tens of seconds, the Gβγ‐activated channels cycled between a low‐Po mode (Po of about 0.0034) and a bursting mode characterized by an ∼30‐fold higher Po and a different set of kinetic constants (and, therefore, a different set of channel conformations). The available evidence indicates that the slow modal transitions are not driven by binding and unbinding of Gβγ. The GTPγS‐activated Gαi1 subunit, previously shown to inhibit GIRK channels, substantially increased the time spent in closed states and apparently shifted the channel to a mode similar, but not identical, to the low‐Po mode. This is the first demonstration of slow modal transitions in GIRK channels. The detailed description of the slow gating kinetics of GIRK1∼4 may help in future analysis of mechanisms of GIRK gating.

The increasing number of resistant bacteria is a major threat worldwide, leading to the search for new antibiotic agents. One of the leading strategies is the use of antimicrobial peptides (AMPs), cationic and hydrophobic innate immune defense peptides. A major target of AMPs is the bacterial membrane. Notably, accumulating data suggest that AMPs can activate the twocomponent systems (TCSs) of Gram-negative bacteria. These include PhoP-PhoQ (PhoPQ) and PmrA-PmrB (PmrAB), responsible for remodeling of the bacterial cell surface. To better understand this mechanism, we utilized bacteria deficient either in one system alone or in both and biophysical tools including fluorescence spectroscopy, single-cell atomic force microscopy, electron microscopy, and mass spectrometry (

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