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… Show more
“…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)).…”
Section: Sen1538 Promotes Resistance To Stressorssupporting
Salmonella enterica serovar Enteritidis (S. Enteritidis; wild type (WT)) is a major cause of foodborne illness globally. The ability of this pathogen to survive stress inside and outside the host, such as encountering antimicrobial peptides and heat stress, determines the efficiency of enteric infection. These stressors concertedly trigger virulence factors encoded on Salmonella pathogenicity islands (SPIs). Although RpoS is a well-known central transcriptional stress and virulence regulator, functional information regarding the genes of the regulon is currently limited. Here, we identified SEN1538 as a conserved RpoS-regulated gene belonging to the KGG protein superfamily. We further assessed its role in pathogenic stress responses and virulence. When SEN1538 was deleted (Δ1538), the pathogen showed reduced survival during antimicrobial peptide introduction and heat stress at 55°C compared to WT. The mutant displayed 70% reduced invasion in the HCT116 colon epithelial cell line, 5-fold attenuated phagocytic survival in RAW264.7 cells, and downregulation of several SPI-1 and SPI-2 genes encoding the three secretion system apparatus and effector proteins. Δ1538 also showed decreased virulence compared to WT, demonstrated by its reduced bacterial counts in the feces, mLN, spleen, and cecum of C57BL/6 mice. Comparative transcriptomic analysis of Δ1538 against WT revealed 111 differentially regulated genes, 103 of which were downregulated (fold change ≤ −1.5, P < 0.05). The majority of these genes were in clusters for metabolism, transporters, and pathogenesis, driving pathogenic stress responses and virulence. SEN1538 is, therefore, an important virulence determinant contributing to the resilience of S. Enteritidis to stress factors during infection.
“…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)).…”
Section: Sen1538 Promotes Resistance To Stressorssupporting
Salmonella enterica serovar Enteritidis (S. Enteritidis; wild type (WT)) is a major cause of foodborne illness globally. The ability of this pathogen to survive stress inside and outside the host, such as encountering antimicrobial peptides and heat stress, determines the efficiency of enteric infection. These stressors concertedly trigger virulence factors encoded on Salmonella pathogenicity islands (SPIs). Although RpoS is a well-known central transcriptional stress and virulence regulator, functional information regarding the genes of the regulon is currently limited. Here, we identified SEN1538 as a conserved RpoS-regulated gene belonging to the KGG protein superfamily. We further assessed its role in pathogenic stress responses and virulence. When SEN1538 was deleted (Δ1538), the pathogen showed reduced survival during antimicrobial peptide introduction and heat stress at 55°C compared to WT. The mutant displayed 70% reduced invasion in the HCT116 colon epithelial cell line, 5-fold attenuated phagocytic survival in RAW264.7 cells, and downregulation of several SPI-1 and SPI-2 genes encoding the three secretion system apparatus and effector proteins. Δ1538 also showed decreased virulence compared to WT, demonstrated by its reduced bacterial counts in the feces, mLN, spleen, and cecum of C57BL/6 mice. Comparative transcriptomic analysis of Δ1538 against WT revealed 111 differentially regulated genes, 103 of which were downregulated (fold change ≤ −1.5, P < 0.05). The majority of these genes were in clusters for metabolism, transporters, and pathogenesis, driving pathogenic stress responses and virulence. SEN1538 is, therefore, an important virulence determinant contributing to the resilience of S. Enteritidis to stress factors during infection.
“…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 high proportion of lipopolysaccharide (LPS) molecules in the outer membrane of Gram-negative bacteria make it a highly effective barrier to small molecules, antibiotic drugs, and other antimicrobial agents. Given this vital role in protecting bacteria from potentially hostile environments, simulations of LPS bilayers and outer membrane systems represent a critical tool for understanding the mechanisms of bacterial resistance and the development of new antibiotic compounds that circumvent these defenses. The basis of these simulations are parameterizations of LPS, which have been developed for all major molecular dynamics force fields. However, these parameterizations differ in both the protonation state of LPS as well as how LPS membranes behave in the presence of various ion species. To address these discrepancies and understand the effects of phosphate charge on bilayer properties, simulations were performed for multiple distinct LPS chemotypes with different ion parameterizations in both protonated or deprotonated lipid A states. These simulations show that bilayer properties, such as the area per lipid and inter-lipid hydrogen bonding, are highly influenced by the choice of phosphate group charges, cation type, and ion parameterization, with protonated LPS and monovalent cations with modified nonbonded parameters providing the best match to experiments. Additionally, alchemical free energy simulations were performed to determine theoretical pK a values for LPS, and subsequently validated by 31 P solid-state NMR experiments. Results from these complementary computational and experimental studies demonstrate that the protonated state dominates at physiological pH, contrary to the deprotonated form modeled by many LPS force fields.In all, these results highlight the sensitivity of LPS simulations to phosphate charge and ion parameters, while offering recommendations for how existing models should be updated for consistency between force fields as well as to best match experiments.
“…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].…”
The rise in antimicrobial resistant bacteria threatens the current methods utilized to treat bacterial infections. The development of novel therapeutic agents is crucial in avoiding a post-antibiotic era and the associated deaths from antibiotic resistant pathogens. The human antimicrobial peptide LL-37 has been considered as a potential alternative to conventional antibiotics as it displays broad spectrum antibacterial and anti-biofilm activities as well as immunomodulatory functions. While LL-37 has shown promising results, it has yet to receive regulatory approval as a peptide antibiotic. Despite the strong antimicrobial properties, LL-37 has several limitations including high cost, lower activity in physiological environments, susceptibility to proteolytic degradation, and high toxicity to human cells. This review will discuss the challenges associated with making LL-37 into a viable antibiotic treatment option, with a focus on antimicrobial resistance and cross-resistance as well as adaptive responses to sub-inhibitory concentrations of the peptide. The possible methods to overcome these challenges, including immobilization techniques, LL-37 delivery systems, the development of LL-37 derivatives, and synergistic combinations will also be considered. Herein, we describe how combination therapy and structural modifications to the sequence, helicity, hydrophobicity, charge, and configuration of LL-37 could optimize the antimicrobial and anti-biofilm activities of LL-37 for future clinical use.
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