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.

Many bacteria live as biofilms to cope with unfavourable surroundings. Biofilms start from (i) a planktonic stage, (ii) initial adhesion to surfaces and (iii) formation of sessile micro-colonies that secrete extracellular polymeric substance (EPS), leading to bacterial resistance to antibiotics. Antimicrobial peptides (AMPs) are extensively studied with regard to planktonic bacteria but much less so with regard to biofilm formation. In the present study, we investigated how the above three steps are affected by the properties of the AMPs using a series of peptides composed of six lysines and nine leucines, which differ in their sequences and hence their biophysical properties. Treatment with bactericidal peptides at non-inhibitory concentrations resulted in reduced biofilm growth, for some starting from 25 nM which is 0.2 and 0.4% of their minimum inhibitory concentration (MIC 6.3 and 12.5 μM, respectively), continuing in a dose-dependent manner. We suggest that reduced bacterial adhesion to surfaces and decreased biofilm growth are due to the peptide's ability to coat either the biomaterial surface or the bacterium itself. Degradation of established biofilms by bactericidal and non-bactericidal peptides, within 1 h of incubation, occurs by either killing of embedded bacteria or detachment of live ones. In addition to shedding light on the mechanism of biofilm inhibition and degradation, these data may assist in the design of anti-biofilm AMPs.

Despite clear differences in immune system responses and in the prevalence of autoimmune diseases between males and females, there is little understanding of the processes involved. Here, we identified a gene signature of immature-like neutrophils (Immature-Like Neutrophil Signature, ILNS), characterized by the overexpression of genes encoding for several granule-containing proteins, which was found in higher levels (up to 3-fold) in young (20 – 30 years old) but not older (60 – >89 years old) males compared with females. Functional and phenotypic characterization of peripheral blood neutrophils revealed more mature and responsive neutrophils in young females, which also exhibited an elevated capacity in neutrophil extracellular trap (NET) formation at baseline and upon microbial or sterile autoimmune stimuli. Expression levels of the ILNS increased linearly with pregnancy, an immune state of increased susceptibility to and severity of certain infections. Using mass cytometry, we also find increased frequencies of immature forms of neutrophils in the blood of women in late pregnancy. Thus, our findings show novel sex-differences in innate immunity and identify a common neutrophil signature in males and in pregnant women.

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Natural and synthetic membrane active peptides as well as fragments from membrane proteins interact with membranes. In several cases, such interactions cause the insertion of the peptides to the membrane and their assembly within the lipid bilayer. Here we present spectroscopic approaches utilizing NBD and rhodamine fluorescently labeled peptides to measure peptide-membrane interaction and peptide-peptide interaction within the membrane. The usage of the physical properties of NBD and rhodamine in solution and in membranes provides useful information on the interplay between peptides and lipids.