Salmonella Membrane Structural Remodeling Increases Resistance to Antimicrobial Peptide LL-37

Abstract

Gram-negative bacteria are protected from their environment by an outer membrane that is primarily composed of lipopolysaccharides (LPSs). Under stress, pathogenic serotypes of Salmonella enterica remodel their LPSs through the PhoPQ two-component regulatory system that increases resistance to both conventional antibiotics and antimicrobial peptides (AMPs). Acquired resistance to AMPs is contrary to the established narrative that AMPs circumvent bacterial resistance by targeting the general chemical properties of membrane lipids. However, the specific mechanisms underlying AMP resistance remain elusive. Here we report a 2-fold increase in bacteriostatic concentrations of human AMP LL-37 for S. enterica with modified LPSs. LPSs with and without chemical modifications were isolated and investigated by Langmuir films coupled with grazing-incidence X-ray diffraction (GIXD) and specular X-ray reflectivity (XR). The initial interactions between LL-37 and LPS bilayers were probed using all-atom molecular dynamics simulations. These simulations suggest that initial association is nonspecific to the type of LPS and governed by hydrogen bonding to the LPS outer carbohydrates. GIXD experiments indicate that the interactions of the peptide with monolayers reduce the number of crystalline domains but greatly increase the typical domain size in both LPS isoforms. Electron densities derived from XR experiments corroborate the bacteriostatic values found in vitro and indicate that peptide intercalation is reduced by LPS modification. We hypothesize that defects at the liquid-ordered boundary facilitate LL-37 intercalation into the outer membrane, whereas PhoPQ-mediated LPS modification protects against this process by having innately increased crystallinity. Since induced ordering has been observed with other AMPs and drugs, LPS modification may represent a general mechanism by which Gram-negative bacteria protect against host innate immunity.

RpoS-regulated SEN1538 gene promotes resistance to stress and influences Salmonella enterica serovar enteritidis virulence

“…The upregulation of phoP in Δ1538 corroborated with a previous report on phoP upregulation in the mutant of ortholog STM14_1829 [19]. We further investigated the expression of PhoP-regulated genes in Δ1538, as these have been previously reported in stress resistance by Salmonella [34,36,37]. Genes encoding Mg 2+ transporters mgtA, mgtB, and mgtC showed 23.73fold, 320.77-fold, and 216.48-fold downregulation in Δ1538 compared to WT, respectively (Figure 2(c)).…”

Lipopolysaccharide Simulations are Sensitive to Phosphate Charge and Ion Parameterization

“…7,8 LPS modification processes, such as the PhoPQ system depicted in Figure 1, reduce this charge through adornment of the lipid A phosphate groups. [9][10][11] In S. enterica, these modifications are activated by a variety of environmental stimuli, such as a low concentration of divalent cations, 12,13 acidic conditions, 14,15 hyperosmotic stress, 16 or antimicrobial peptide presence, 17,18 indicating that modifications may confer a survival advantage in such conditions. Additionally, previous simulations have shown that the presence of aminoarabinose disrupts the cation network, allowing direct inter-lipid hydrogen bonding to instead stabilize the leaflet and potentially reducing the reliance on divalent cations for stability.…”

The Potential of Human Peptide LL-37 as an Antimicrobial and Anti-Biofilm Agent

“…A summary of the resistance mechanisms can be seen in Figure 1. Most commonly, resistance to LL-37 occurs as a result of structural changes to the bacterial membranes [7], including modifications to the cell membrane [20], cell surface charge [9,21], capsule [25], and efflux pumps [26]. Furthermore, resistance may develop through the upregulation and downregulation of specific genes [25,27], and through the resulting alterations to cellular processes and secretions, including metabolism [19], the expression of virulence factors [28], proteases [12,27,29], and outer membrane proteins (OMP) and vesicles [12,27,29].…”