Abstract
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.
Highlights
The innate immune system of almost all living organisms produce cationic antimicrobial peptides (CAMPs) to protect against bacterial invaders
The investigated Group B Streptococcus (GBS) strains were previously characterized revealing that 20.8% of the glycerophosphate residues of the lipoteichoic acids (LTAs) of the WT strain NEM316 were substituted with D-alanyl esters whereas insertional inactivation of dltA caused complete absence of D-alanine [21]
The dltA mutant was highly susceptible to colistin compared to the WT strain and resistance was recovered at an intermediate level in a complemented strain where D-alanine incorporation was partially restored to 12.8%, confirming that there is a positive correlation between D-alanine content and resistance [22]
Summary
The innate immune system of almost all living organisms produce cationic antimicrobial peptides (CAMPs) to protect against bacterial invaders. Teichoic acids are negatively charged poly-glycerophosphate (Gro-P) chains that can be either covalently linked to PGN (i.e. wall teichoic acids or WTAs) or anchored to the cytoplasmic membrane (i.e. lipoteichoic acids or LTAs) [9]. The anionic property of teichoic acids confers a global negative charge, which is thought to contribute to the preferential accumulation of CAMPs on the bacterial cell surface [10]. These peptides traverse the PGN barrier, reach the anionic phospholipid of the cytoplasmic membrane, and perturb it via several mechanisms, depending on the peptide used [11,12,13]
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