Opposing effects of PhoPQ and PmrAB on the properties of Salmonella enterica serovar Typhimurium: implications on resistance to antimicrobial peptides

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 two-component 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 (Moskowitz, S. M.; Antimicrob. Agents Chemother. 2012, 56, 1019-1030; Cheng, H. Y.; J. Biomed. Sci. 2010, 17, 60). Our data suggested that the two systems have opposing effects on the properties of Salmonella enterica. The knockout of PhoPQ made the bacteria more susceptible to AMPs by making the surface less rigid, more polarized, and permeable with a slightly more negatively charged cell wall. In addition, the periplasmic space is thinner. In contrast, the knockout of PmrAB did not affect its susceptibility, while it made the bacterial outer layer very rigid, less polarized, and less permeable than the other two mutants, with a negatively charged cell wall similar to the WT. Overall, the data suggest that the coexistence of systems with opposing effects on the biophysical properties of the bacteria contribute to their membrane flexibility, which, on the one hand, is important to accommodate changing environments and, on the other hand, may inhibit the development of meaningful resistance to AMPs

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

International audienceCationic 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 resistanc

Investigation of GBS cell wall properties using atomic force microscopy.

<p>The root mean square (RMS) of surface roughness calculated from topography images (A) and the average surface rigidity calculated from DMT modulus images (B) are shown. Data are means ± SD of n≥8 cells.</p

Investigation of GBS cell wall structure using transmission electron microscopy.

<p>High resolution images of the cell wall of WT (A), <i>dltA</i> mutant (B), and <i>dltA</i> complemented (C) strains, as revealed by using the freeze substitution method. The inner region of the cell wall is pointed by arrow. Bars for the main picture and for the whole cell inserted picture represent 50 nm and 200 nm, respectively.</p

Investigation of GBS morphology using atomic force microscopy.

<p>Deflection (A–D) and topography (E–H) images of GBS surface morphology. Representative images of non-treated WT (A and E), <i>dltA</i> mutant (B and F), and LL37-treated (10 µM) WT (C and G) and <i>dltA</i> mutant (D and H).</p

Quantitative binding assay.

<p>The binding of cytochrome C (A) and NBD-labeled CAMPs (B) to WT (black) and <i>dltA</i> (gray) mutant strains was studied. Data are means ± SD of triplicate measurements from three independent experiments and are presented as the percentage of the maximum signal (peptide with no bacteria) ± SD.</p

Peptide designation and properties.

a<p>Underlined and bold amino acids are D-enantiomers. All linear peptides are amidated in their C-terminus.</p>b<p>Calculated by dividing the net charge by the total number of amino acids.</p>c<p>The peptides were eluted in 40 min using a linear gradient of acetonitrile (AcN) from 30 to 70% v/v in water containing TFA (0.05% v/v) on a C4 reverse analytical column.</p

Susceptibility of GBS strains to perforation by CAMPs.

<p>Peptide-dependent influx of the vital dye SYTOX green following 30 min exposure of GBS to magainin 2 (A), LL-37 (B), 5D-K₆L₉ (C) and K₅L₇ (D). Solid lines represent the WT strain whereas dashed lines represent the <i>dltA</i> muatnt. All readings were normalized by subtracting the basal fluorescence of the dye. Data are means ± SD of triplicate measurements.</p