An atomistic model of STX is developed and it is demonstrated that STX significantly increases lipid packing and acyl chain order of STX–PG bilayer mixtures, and reduces the gel-to-liquid crystalline phase transition temperature, indicating a general stabilizing effect for the fluid phase of PG lipids of varying length.
Abstract
Staphyloxanthin (STX) is a carotenoid synthesized by the human pathogen Staphylococcus aureus. The golden color of this bacterium is due to this carotenoid. STX protects Staphylococcus aureus from oxidative stress by scavenging free radical species. Furthermore, STX has been shown to mechanically strengthen the Staphylococcus aureus membrane and to form microdomains that recruit antibiotic-resistance factors. Thus, inhibition of STX is a promising strategy for intervening against multidrug-resistant strains of this pathogen. However, the molecular mechanisms by which STX regulates the membrane structure and function of Staphylococcus aureus remain unclear. More specifically, the localization of STX within phosphatidylglycerol (PG) bilayers, the primary phospholipid of this bacterium’s membrane, and how this localization drives macroscopic biophysical changes remain unresolved questions. Here, we addressed this issue by integrating molecular dynamics (MD) simulations, X-ray scattering experiments, and fluorescence spectroscopy. We developed an atomistic model of STX, which was validated against X-ray scattering data and which is suitable for all-atom MD simulations. We demonstrate that STX significantly increases lipid packing and acyl chain order of STX–PG bilayer mixtures. In addition, STX self-assembles into clusters, where the long and rigid conjugated triterpenoid chain interdigitates across both leaflets, modifying locally the density of the surrounding PG molecules. These findings provide a molecular explanation to the reduced headgroup spacing and core dynamics observed in fluorescence experiments and are consistent with the formation of structurally-distinct STX-enriched microdomains. Notably, STX reduces the gel-to-liquid crystalline phase transition temperature, indicating a general stabilizing effect for the fluid phase of PG lipids of varying length. Overall, our findings provide molecular insights into how STX enhances membrane mechanical integrity. It will be highly interesting to establish how the membrane remodeling effects observed here connect with STX’s dual roles, acting as an antioxidant and preventing pore formation and other mechanical perturbations induced by antimicrobial molecules.
Staphylococcus aureus (S. aureus) is a prominent foodborne pathogen that poses a continuous threat to global public health and food safety due to its possession of a variety of toxins and its multidrug resistance. Medium-chain fatty acids (MCFAs), notably lauric acid (LA), exhibit strong antimicrobial properties, but their application is heavily constrained by poor water solubility. In this study, optimized LA emulsions stabilized by chitosan (CS) and polyvinyl alcohol (PVA) were evaluated for their antibacterial activity and detailed mode of action against S. aureus ATCC 6538. The antibacterial activities were evaluated by the maximum inhibition zone, with the 20 CS-PVA/DLTA-LA formulation exhibiting stable dispersion and potent antibacterial activity at 1%. The underlying antibacterial mechanisms against S. aureus were specifically focused on cell membranes and peptidoglycan. Therein, the binding of emulsion droplets to the anionic bacterial surface was driven by electrostatic attraction. Membrane degradation was also observed with membrane dysfunctions involving membrane depolarization, increased permeability, and fluidity reduction triggered by their subsequent insertion into the lipid bilayer, which may cause cell dysmetabolism, disintegration, and eventual cell death. Overall, these findings substantiate that LA emulsions disrupt S. aureus by operating potentially multi-targeted effects involving cell membrane disruption and peptidoglycan interference, offering a promising alternative approach warranting further investigation for foodborne pathogen control.
Peipei Ma, Run-run Zhang, Chen Li et al.· Foods· 0 citations
Phenol-soluble modulin α1 (PSMα1) is a cytolytic peptide secreted by Staphylococcus aureus that contributes to host-cell damage and biofilm stability, yet the relationship between its assembly behavior and function remains incompletely understood. Here, we combine cellular assays, molecular spectroscopy, and high-resolution structural approaches to elucidate how environmental conditions govern PSMα1 activity and supramolecular organization. Live-cell imaging and cytotoxicity assays show that PSMα1 accumulates at the plasma membrane of human cells prior to membrane permeabilization, linking membrane association to cytotoxic outcomes. This process is strongly attenuated by epigallocatechin gallate (EGCG). Cryogenic electron microscopy (cryo-EM) reveals two polymorphic canonical amyloid fibril architectures that share a conserved hydrophobic core and protofilament interface. In parallel, we identify pH as a key determinant of PSMα1 assembly pathways, driving a bifurcation between cross-β amyloid fibrils at extreme acidic and alkaline conditions and heterogeneous, long-lived, thermally stable α-helical nanotubular assemblies at acidic, near-neutral, and slightly alkaline conditions, which act as transient intermediates under highly acidic conditions. Together, these findings demonstrate that PSMα1 is not a single amyloid structure but a condition-dependent structural system in which environmental cues dictate assembly, membrane interaction, and cytotoxic function. This work provides a framework for understanding how polymorphic assembly of bacterial virulence peptides interfaces with host-cell interactions and suggests new avenues for targeting PSM-mediated pathogenicity. Statement of significance Staphylococcus aureus causes severe infections and uses the peptide PSMα1 to damage host cells and strengthen protective biofilms. Like many disease-associated proteins, PSMα1 self-assembles into amyloid fibrils, though their role in virulence remains unclear. We show that PSMα1 does not adopt a single architecture. Instead, environmental changes, such as those at infection sites, drive the peptide into distinct assemblies, including cross-β amyloid fibrils and unexpectedly stable nanotubes with α-helical features. Live-cell imaging shows PSMα1 accumulates at the plasma membrane before cell death, and that epigallocatechin gallate reduces membrane association and toxicity. These findings show that bacterial virulence can be regulated through environmentally controlled transitions between protein assemblies, identifying membrane accumulation as a promising anti-virulence target.
Sambhasan Banerjee, V. Skoryk, Bader Rayan et al.· bioRxiv· 0 citations
This study aims to explore the synergistic bactericidal interaction of carvacrol (CAR) combined with ε-polylysine (ε-PL) against Staphylococcus aureus and its underlying mechanism. The combination of CAR (12.50 µg mL−1) and ε-PL (7.66 µg mL−1) exhibited synergistic bactericidal and antibiofilm activities, primarily attributed to combined destruction of the bacterial cell membrane, as evidenced by increased membrane permeability, time-dependent depolarization, and reduced cell surface hydrophobicity after 3 h of treatment. Membrane damage facilitated intracellular accumulation of CAR, which rapidly triggered energy depletion and early metabolic disturbances. Using metabolomics, the combination after 0.5 h was found to primarily perturb three key pathways, including tryptophan metabolism, cofactor biosynthesis (thiamine, biotin, nicotinamide), and nucleotide metabolism. These early metabolic changes, along with ATP decline, occurred prior to full membrane depolarization, oxidative stress, and pH imbalance at 3 h. Ultimately, the synergy caused irreversible energy depletion, membrane disruption, and severe oxidative damage. Furthermore, the combination reduced S. aureus counts by approximately 1.0 log10 CFU g−1 in raw beef under refrigeration, supporting its practical potential. This study for the first time constructs a complete temporal mechanism chain from initial membrane damage and rapid metabolic disorder to downstream cellular dysfunction for the CAR-ε-PL synergy, providing a novel theoretical basis for developing natural antimicrobial strategies in food preservation.
Xiangyu Zang, Chen Du, Shuai Wan et al.· RSC Advances· 0 citations
Therapeutic interventions for bacterial infections are increasingly challenging due to the growing threat of antibiotic resistance, particularly from Methicillin-resistant Staphylococcus aureus (MRSA), which poses significant risks to global health. Generally, MRSA has a high mortality rate and can lead to severe illnesses such as sepsis and pneumonia, mainly due to the lack of effective treatments and growing drug resistance. To this end, the present study introduces azobenzene-conjugated photo-responsive polypeptide nanofibers (PNFs) that mimic host-defense peptides to combat antibiotic-resistant bacterial pathogens. We show that incorporating an azobenzene moiety into the PNF enables light-mediated control via photoinduced conformational changes. The polypeptide structure incorporates these azobenzene units, synthesized via ring-opening polymerization (ROP) of N-carbobenzoxy-l-lysine N-carboxyanhydride (Cbz-l-Lys NCA) with NAP-AZO as the initiator. Moreover, we show that conformational changes in the PNF affect its interactions with bacterial membranes. While the azobenzene moieties provide light-regulated activity, the amino groups from lysine enhance electrostatic membrane disruption. This has resulted in effective membrane interaction of PNFs, facilitating the generation of reactive oxygen species (ROS) against MRSA, which are crucial for bacterial death or growth inhibition. Overall, the present study suggests that photo-responsive polypeptide PNFs represent a promising and versatile therapeutic strategy for combating antibiotic-resistant bacterial infections, owing to their dual functional capabilities.
Sangita Rajwar, C. Kumar, Shreya Ghosh et al.· ACS Applied Bio Materials· 0 citations
ABSTRACT Staphylococcus aureus is one of the most frequently co-isolated pathogens in polymicrobial infections, where interspecies interactions contribute to enhanced virulence, persistence, and antimicrobial tolerance. Nutrient availability plays a central role in these interactions as microorganisms compete for resources to sustain essential cellular processes. For instance, branched-chain amino acids (BCAAs) are critical for protein synthesis, and valine synthesis pathway precursors are essential for energy production. In S. aureus, BCAAs are also the precursors for branched-chain fatty acids (BCFAs), the dominant fatty acids in the S. aureus membrane. We previously identified a second pathway that uses branched-chain carboxylic acids (BCCAs) and the high-affinity acyl-CoA synthetase MbcS to catalyze the formation of primers for BCFA synthesis. However, the physiological role of this pathway and the conditions triggering its activation remain unclear. Here, we show that mbcS is restricted to S. aureus and closely related human-associated staphylococci. Phylogenetic analyses suggest that MbcS arose from a refunctionalization event and represents a non-orthologous replacement for the phosphotransbutyrylase (Ptb) and butyrate kinase (Buk) enzymes. Consistent with this model, Ptb and Buk from Staphylococcus pseudintermedius catalyze the formation of branched-chain acyl-CoAs from BCCAs, but only at high substrate concentrations. We further show that mbcS expression is upregulated in a CodY-deficient strain, implicating this pathway in BCAA-limited conditions. In support, we show that MbcS is required for optimal fitness during intra-species competition. Together, our findings support a model in which the MbcS-dependent pathway enables S. aureus to scavenge BCFA precursors under nutrient-limited conditions, providing a competitive advantage in polymicrobial environments. IMPORTANCE Staphylococcus aureus is a major contributor to polymicrobial infections, where competition for nutrients can influence bacterial physiology and survival. A deeper understanding of how S. aureus adapts to nutrient limitation is therefore essential to explain its success as a human pathogen. In S. aureus, the acyl-CoA synthetase MbcS supports branched-chain fatty acid (BCFA) synthesis from branched-chain amino acid (BCAA)-derived carboxylic acids and aldehydes, which are released into the environment as by-products of bacterial metabolism. Herein, we provide evidence that S. aureus acquired the acyl-CoA synthetase MbcS as an adaptive trait. This metabolic innovation allows this bacterium to maintain membrane homeostasis under nutrient limitation and compete against neighboring bacteria. Our findings highlight an adaptive strategy that may contribute to the persistence of S. aureus in polymicrobial infections. Staphylococcus aureus is a major contributor to polymicrobial infections, where competition for nutrients can influence bacterial physiology and survival. A deeper understanding of how S. aureus adapts to nutrient limitation is therefore essential to explain its success as a human pathogen. In S. aureus, the acyl-CoA synthetase MbcS supports branched-chain fatty acid (BCFA) synthesis from branched-chain amino acid (BCAA)-derived carboxylic acids and aldehydes, which are released into the environment as by-products of bacterial metabolism. Herein, we provide evidence that S. aureus acquired the acyl-CoA synthetase MbcS as an adaptive trait. This metabolic innovation allows this bacterium to maintain membrane homeostasis under nutrient limitation and compete against neighboring bacteria. Our findings highlight an adaptive strategy that may contribute to the persistence of S. aureus in polymicrobial infections.
Marcelle C. dos Santos Ferreira, T. Stephens, Shaun R. Brinsmade· Journal of Bacteriology· 0 citations
Staphylococcus aureus releases extracellular membrane vesicles (MVs) that exert diverse biological functions depending on their cargo. Moreover, MV cargos can be reprogrammed in response to different growth conditions. In this study, we aimed to investigate whether the protein cargo in MVs can reflect specific environmental stresses, including iron depletion and antibiotic exposure, which S. aureus may encounter during infection. Unexpectedly, we found that extensive fibrillar amyloids were co-purified with S. aureus MVs when iron was depleted from the medium. This phenotype was also observed in bacteria grown in normal medium, albeit to a much lesser extent, and displayed distinct characteristics under antibiotic-treated conditions. To further elucidate the relationship between this phenotype and environmental stresses, we compared the proteomes of the MV-amyloid complexes derived from S. aureus cultured under the aforementioned conditions. Our results revealed stress-specific proteomic alterations in the MV-amyloid complex, as demonstrated by functional enrichment analyses of differentially expressed proteins. In summary, our findings demonstrate for the first time that amyloid formation, similar to MV production, is regulated by environmental stresses. The stress-specific proteomic changes of the MV-amyloid complex suggest a potential role for this complex in the environmental adaptation of S. aureus.
Zhuang Zhu, Jinxi Yue, Yalan Feng et al.· Microbial Pathogenesis· 0 citations