D-Alanylation of Lipoteichoic Acids Confers Resistance to Cationic Peptides in Group B Streptococcus by Increasing the Cell Wall Density

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.

Compound-gene interaction mapping reveals distinct roles forStaphylococcus aureusteichoic acids

“…The D-alanine deficient cells had defects in cell wall ultrastructure and displayed increased lysis, showing that the D-alanine modification on LTAs contributes to membrane integrity. It has been proposed that D-alanylation influences LTA conformations, changing the density and rigidity of the cell wall (26). The presence of positively charged D-alanine esters on TAs may also negatively regulate hydrolytic enzymes, and removal of WTAs is also known to result in dysregulated autolysin activity (6,8,10,35).…”

“…This step seemed necessary because some of the knockouts, including ΔdltA, ΔgraR, and ΔvraFG, are known to have increased susceptibility to cationic antimicrobial peptides (CAMPs) and certain other antibiotics with cell envelope targets (7,8,25). Increased susceptibility is proposed to occur because the cell surface lacks the D-alanine modifications that normally repel positively charged toxins at the envelope interface (26). Although tunicamycin is not charged at physiological pH, we wanted to rule out increased access to MraY as an explanation for the lethal phenotypes we observed.…”

The action mechanism of daptomycin

“…The alanylation of these surface-exposed structures results in an increase in the net positive charge of the cell surface, which may affect daptomycin's ability to function in the same manner as PG lysylation. Moreover, teichoic acid D-alanylation reduces penetration of the cell wall by cationic antimicrobial peptides, 107 a mechanism that also appears plausible with daptomycin.…”

Anti-virulence Strategies to Target Bacterial Infections

“…by addition of amino-arabinose or heptoses) and inhibition of several enzymes of LPS biosynthesis have been identified as treatment strategies (Desroy et al 2013;Escaich 2010). Bacterial resistance to CAMPs is linked to an increase in Dalanylation of the lipoteichonic acids in the cell wall of staphylococci and streptococci (Peschel et al 1999;Saar-Dover et al 2012). Furthermore, inhibitors of D-alanylation enzymes have been identified, some of which were also shown to reduce bacteremia (Escaich 2010;Santa Maria et al 2014).…”