It is demonstrated for the first time that amyloid formation, similar to MV production, is regulated by environmental stresses, and a potential role for this complex in the environmental adaptation of S. aureus is suggested.
Abstract
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.
The 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
ABSTRACT Staphylococcus aureus is a leading cause of biofilm-associated infections, in which communities of bacterial cells are encased in an extracellular matrix composed of polysaccharides, proteins, and extracellular DNA (eDNA) that protect bacteria from host immune defense and antibiotics. Despite their importance, the mechanisms by which matrix components are released from bacterial cells and incorporated into the biofilm matrix remain poorly understood. Using a drip-flow biofilm system, we showed that membrane vesicles (MVs) were associated with the biofilm matrix formed by S. aureus clinical isolate MN8. Proteomic analysis of biofilm matrix proteins and purified MVs showed that biofilm-derived MVs carried cytoplasmic, membrane, and extracellular proteins that closely resembled the protein composition of the biofilm matrix, but differed significantly from MVs produced by planktonic cultures. Biofilm-derived MVs carried significantly higher levels of DNA than MVs from planktonic cultures, and MV-associated DNA was sensitive to DNase treatment, suggesting that eDNA is primarily associated with the MV surface. Although strain MN8 is known to form polysaccharide-dependent biofilms, exogenously added DNase or proteinase K significantly impaired biofilm formation and integrity. Importantly, supplementation with biofilm-derived MVs, but not MVs from planktonic cultures, significantly restored biofilm formation in enzyme-treated static cultures. Together, these findings provide evidence that S. aureus MVs are generated within biofilms, and that these MVs serve as an important resource of matrix components and contribute to biofilm formation. IMPORTANCE Extracellular membrane vesicles (MVs) are important mediators of intercellular communication and have been implicated in the bacterial physiology and pathogenesis. MVs in fungi and gram-negative bacteria mediate key biofilm processes, such as formation and structural maintenance. However, MV production and function in biofilm formation in gram-positive bacteria have remained largely unexplored. Here, we report for the first time the purification and characterization of MVs derived from Staphylococcus aureus biofilms. Our studies demonstrate that S. aureus MVs are important components of the biofilm matrix that contribute to biofilm formation by serving as carriers of key matrix components. This work advances our limited understanding of MVs in gram-positive bacteria and reveals a previously unrecognized mechanism contributing to S. aureus biofilm formation. Extracellular membrane vesicles (MVs) are important mediators of intercellular communication and have been implicated in the bacterial physiology and pathogenesis. MVs in fungi and gram-negative bacteria mediate key biofilm processes, such as formation and structural maintenance. However, MV production and function in biofilm formation in gram-positive bacteria have remained largely unexplored. Here, we report for the first time the purification and characterization of MVs derived from Staphylococcus aureus biofilms. Our studies demonstrate that S. aureus MVs are important components of the biofilm matrix that contribute to biofilm formation by serving as carriers of key matrix components. This work advances our limited understanding of MVs in gram-positive bacteria and reveals a previously unrecognized mechanism contributing to S. aureus biofilm formation.
Jinger Lei, Misaki Foster, Emery L. Ng et al.· Journal of Bacteriology· 0 citations
ABSTRACT Quorum sensing (QS) plays a central role in the adaptive biology of Streptococcus mutans, yet the extent to which competence-stimulating peptide (CSP) signaling intersects with membrane vesicle production and function remained unclear. In this study, CSP activation markedly increased membrane vesicle output during the stationary phase and drove extensive changes in vesicle protein composition. Proteomic analysis revealed broad remodeling of vesicle cargo, including shifts in subcellular origin, and enrichment of multiple functional protein classes. Notably, CSP-induced vesicles carried the persistence-associated peptide Pep299, suggesting a link between QS-dependent cargo loading and antibiotic survival. To examine the functional consequences of this remodeling, S. mutans cells were pre-exposed to vesicles prior to antibiotic challenge. Vesicles derived from CSP-induced cultures promoted persister formation in a concentration-dependent manner, whereas vesicles from non-induced cultures exhibited minimal activity. Wild-type vesicles increased persistence, and vesicles from Pep299-overexpressing cells further enhanced this phenotype at lower vesicle concentrations. In contrast, vesicles derived from a Δ299 mutant failed to promote persistence, demonstrating that Pep299 is a key determinant of this activity. At higher vesicle abundance, the responses of wild-type and Pep299-enriched vesicles converged, consistent with endogenous CSP-dependent Pep299 loading. Fusion assays using R18 dequenching showed that vesicles from wild-type, Pep299-overexpressing, and Δ299 strains fused with comparable efficiency, indicating that differences in persistence arise from cargo composition rather than altered delivery efficiency. Together, these findings reveal that CSP QS coordinates biogenesis and selective cargo remodeling in S. mutans and identify Pep299-containing vesicles as a quorum-regulated mechanism promoting antibiotic persistence. IMPORTANCE Quorum sensing enables bacterial populations to coordinate adaptive behaviors, yet its influence on membrane vesicle biology is not fully understood. This study shows that competence-stimulating peptide (CSP) signaling in Streptococcus mutans not only increases vesicle production but also remodels vesicle protein cargo, including enrichment of the persistence-associated peptide Pep299. CSP-induced vesicles enhance the formation of persister cells through a cargo-dependent mechanism rather than through changes in vesicle–cell fusion. These findings uncover a previously unrecognized connection between quorum sensing and vesicle-mediated persistence and reveal a strategy through which S. mutans modulates community behavior and resilience within the oral biofilm environment. Quorum sensing enables bacterial populations to coordinate adaptive behaviors, yet its influence on membrane vesicle biology is not fully understood. This study shows that competence-stimulating peptide (CSP) signaling in Streptococcus mutans not only increases vesicle production but also remodels vesicle protein cargo, including enrichment of the persistence-associated peptide Pep299. CSP-induced vesicles enhance the formation of persister cells through a cargo-dependent mechanism rather than through changes in vesicle–cell fusion. These findings uncover a previously unrecognized connection between quorum sensing and vesicle-mediated persistence and reveal a strategy through which S. mutans modulates community behavior and resilience within the oral biofilm environment.
D. Dufour, C. Leiva-Sabadini, S. Aguayo et al.· Journal of Bacteriology· 0 citations
Bacterial extracellular vesicles (BEVs) are nano-sized lipid bilayer particles secreted by bacteria, capable of carrying various proteins, lipids, nucleic acids, and pathogen-associated molecular patterns (PAMPs). The biosynthetic pathway of BEVs determines their load components, physicochemical properties, and different biological activities. Increasing evidence indicates that BEVs play an important role in mediating host immune responses and the dynamic regulation of bacterial biofilms. Additionally, BEVs may serve as a molecular bridge between the two. BEVs derived from pathogens can trigger pro-inflammatory cascades, assist bacteria in immune evasion, and further accelerate the maturation of biofilms, forming a vicious cycle of persistent infection and inflammatory damage. In contrast, BEVs derived from probiotics can maintain host immune homeostasis and exert direct anti-biofilm and synergistic antibacterial effects, thereby breaking the pathological cycle. However, significant methodological research bottlenecks have greatly hindered the comparability and clinical translation of BEVs research. This article systematically summarizes the classification of BEVs and their biosynthetic mechanisms, compares the differential effects of BEVs from pathogenic bacteria and probiotic bacteria on immunity, clarifies the dual regulatory role of BEVs throughout the life cycle of biofilms, and highlights the bridging function of BEVs in the immune–biofilm interaction. Additionally, this article also discusses the current development of BEVs in clinical translation applications, such as vaccine development, antibiotic delivery, and mucosal inflammation intervention, and outlines the key industrial and clinical challenges faced in the future development of BEVs-based therapeutic approaches.
Qingyu Zhang, Bei-Lei Zhang, Mohd Shafiq Aazmi et al.· Biomolecules· 0 citations
Increased intravascular coagulation is a molecular hallmark of sepsis and we showed that extracellular vesicles (EVs) from human neutrophils challenged with bacteria activate the coagulation cascade. Discovery of the EV biogenesis pathway may lead to the ability to interfere with EV production and reduce coagulation.
To investigate the mechanism of EV biogenesis, we performed proteomics on three EV subsets: those produced spontaneously, in response to fMLF, and after staphylococcal challenge. After protein abundances were normalized, we compared fold changes between subsets. We validated the proteomic results with immunoblotting. The role of nSmase was evaluated using GW4869, an nSmase inhibitor.
Although the production of some neutrophil EV subsets relies on membrane curvature caused by neutral sphingomyelinase (nSmase), the production of EVs in response to staphylococcal challenge was nSmase-independent. Annexins and proteins involved in the formation of the membrane attack complex (C5b-9) were enriched in EVs produced after staphylococcal challenge.
Based on these results, we propose that complement-mediated bystander activation produces membrane damage, which initiates Annexin-mediated repair and EV production. To test this, we opsonized bacteria with C9-sufficient or deficient serum, and measured production of EVs by monitoring Flotillin-1. In the absence of C9, less Flotillin-1 was observed compared to the control. These data suggest a role for complement in EV production.
The Frost Fund in the Cal Poly Bailey College of Science and Mathematics
Innate Immune Responses and Host Defense: Cellular Mechanisms (INC)
Justin W Grapentine, Gabriella Richardson, Oliver Smith et al.· Journal of Immunology· 0 citations
Dopamine was taken up by the bacteria and was associated with enhanced pathogenic phenotypes, including increased proliferation, toxin production, biofilm formation, motility, adhesion, and multidrug resistance and deletion of saeS markedly attenuated these dopamine-associated effects.
Ben Xu, Ziyue Wang, Jinmei Chai et al.· BMC Microbiology· 0 citations
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