Secretion of extracellular vesicles (EVs), a process common to eukaryotes, archae, and bacteria, represents a secretory pathway that allows cell-free intercellular communication. Microbial EVs ...package diverse proteins and influence the host-pathogen interaction, but the mechanisms underlying EV production in Gram-positive bacteria are poorly understood. Here we show that EVs purified from community-associated methicillin-resistant Staphylococcus aureus package cytosolic, surface, and secreted proteins, including cytolysins. Staphylococcal alpha-type phenol-soluble modulins promote EV biogenesis by disrupting the cytoplasmic membrane; whereas, peptidoglycan cross-linking and autolysin activity modulate EV production by altering the permeability of the cell wall. We demonstrate that EVs purified from a S. aureus mutant that is genetically engineered to express detoxified cytolysins are immunogenic in mice, elicit cytolysin-neutralizing antibodies, and protect the animals in a lethal sepsis model. Our study reveals mechanisms underlying S. aureus EV production and highlights the usefulness of EVs as a S. aureus vaccine platform.
Release of extracellular vesicles (EVs) is a common feature among eukaryotes, archaea, and bacteria. However, the biogenesis and downstream biological effects of EVs released from gram-positive ...bacteria remain poorly characterized. Here, we report that EVs purified from a community-associated methicillin-resistant Staphylococcus aureus strain were internalized into human macrophages in vitro and that this process was blocked by inhibition of the dynamin-dependent endocytic pathway. Human macrophages responded to S. aureus EVs by TLR2 signaling and activation of NLRP3 inflammasomes through K⁺ efflux, leading to the recruitment of ASC and activation of caspase-1. Cleavage of pro– interleukin (IL)-1β, pro-IL-18, and gasdermin-D by activated caspase-1 resulted in the cellular release of the mature cytokines IL-1β and IL-18 and induction of pyroptosis. Consistent with this result, a dose-dependent cytokine response was detected in the extracellular fluids of mice challenged intraperitoneally with S. aureus EVs. Pore-forming toxins associated with S. aureus EVs were critical for NLRP3-dependent caspase-1 activation of human macrophages, but not for TLR2 signaling. In contrast, EV-associated lipoproteins not onlymediated TLR2 signaling to initiate the priming step of NLRP3 activation but also modulated EV biogenesis and the toxin content of EVs, resulting in alterations in IL-1β, IL-18, and caspase-1 activity. Collectively, our study describes mechanisms by which S. aureus EVs induce inflammasome activation and reveals an unexpected role of staphylococcal lipoproteins in EV biogenesis. EVs may serve as a novel secretory pathway for S. aureus to transport protected cargo in a concentrated form to host cells during infections to modulate cellular functions.
Staphylococcus aureus generates extracellular membrane vesicles (MVs) during both in vitro culture and in vivo infection.S. aureus MVs are generated by either blebbing of the cytoplasmic membrane or ...explosive cell lysis.Proteins and nucleic acids encapsulated within S. aureus MVs are protected from degradation or neutralization by host factors.S. aureus MVs exhibit cytotoxicity to a variety of host cell types and induce both host innate and adaptive immune responses in animal infection models.MVs exploit different endocytic pathways for the delivery of their cargo into host cells. Deciphering the intracellular fate of S. aureus MVs after cellular entry may deepen our limited understanding of how MVs impact the host during staphylococcal infections.
Staphylococcus aureus is an important bacterial pathogen that causes a wide variety of human diseases in community and hospital settings. S. aureus employs a diverse array of virulence factors, both surface-associated and secreted, to promote colonization, infection, and immune evasion. Over the past decade, a growing body of research has shown that S. aureus generates extracellular membrane vesicles (MVs) that package a variety of bacterial components, many of which are virulence factors. In this review, we summarize recent advances in our understanding of S. aureus MVs and highlight their biogenesis, cargo, and potential role in the pathogenesis of staphylococcal infections. Lastly, we present some emerging questions in the field.
Staphylococcus aureus is an important bacterial pathogen that causes a wide variety of human diseases in community and hospital settings. S. aureus employs a diverse array of virulence factors, both surface-associated and secreted, to promote colonization, infection, and immune evasion. Over the past decade, a growing body of research has shown that S. aureus generates extracellular membrane vesicles (MVs) that package a variety of bacterial components, many of which are virulence factors. In this review, we summarize recent advances in our understanding of S. aureus MVs and highlight their biogenesis, cargo, and potential role in the pathogenesis of staphylococcal infections. Lastly, we present some emerging questions in the field.
The dominant molecular species contributing to the surface-enhanced Raman spectroscopy (SERS) spectra of bacteria excited at 785 nm are the metabolites of purine degradation: adenine, hypoxanthine, ...xanthine, guanine, uric acid, and adenosine monophosphate. These molecules result from the starvation response of the bacterial cells in pure water washes following enrichment from nutrient-rich environments. Vibrational shifts due to isotopic labeling, bacterial SERS spectral fitting, SERS and mass spectrometry analysis of bacterial supernatant, SERS spectra of defined bacterial mutants, and the enzymatic substrate dependence of SERS spectra are used to identify these molecular components. The absence or presence of different degradation/salvage enzymes in the known purine metabolism pathways of these organisms plays a central role in determining the bacterial specificity of these purine-base SERS signatures. These results provide the biochemical basis for the development of SERS as a rapid bacterial diagnostic and illustrate how SERS can be applied more generally for metabolic profiling as a probe of cellular activity.
Graphical Abstract
Bacterial typing by metabolites released under stress
The microbial secretome modulates how the organism interacts with its environment. Included in the Staphylococcus aureus secretome are extracellular membrane vesicles (MVs) that consist of ...cytoplasmic and membrane proteins, as well as exoproteins, some cell wall-associated proteins, and glycopolymers. The extent to which MVs contribute to the diverse composition of the secretome is not understood. We performed a proteomic analysis of MVs purified from the S. aureus strain MRSA252 along with a similar analysis of the whole secretome (culture supernatant) before and after depletion of MVs. The MRSA252 secretome was comprised of 1,001 proteins, of which 667 were also present in MVs. Cell membrane-associated proteins and lipoteichoic acid in the culture supernatant were highly associated with MVs, followed by cytoplasmic and extracellular proteins. Few cell wall-associated proteins were contained in MVs, and capsular polysaccharides were found both in the secretome and MVs. When MVs were removed from the culture supernatant by ultracentrifugation, 54 of the secretome proteins were significantly depleted in abundance. Proteins packaged in MVs were characterized by an isoelectric point that was significantly higher than that of proteins excluded from MVs. Our data indicate that the generation of S. aureus MVs is a mechanism by which lipoteichoic acid, cytoplasmic, and cell membrane-associated proteins are released into the secretome.
The secretome of Staphylococcus aureus includes soluble molecules and nano-sized extracellular membrane vesicles (MVs). The protein composition of both the secretome and MVs includes cytoplasmic and membrane proteins, as well as exoproteins, some cell wall-associated proteins, and glycopolymers. How the MV cargo differs from the protein composition of the secretome has not yet been addressed. Although the compositions of the secretome and MVs were strikingly similar, we identified 54 proteins that were specifically packaged in MVs. Proteins highly associated with MVs were characterized by their abundance in the secretome, an association with the bacterial membrane, and a basic isoelectric point. This study deepens our limited understanding about the contribution of MVs to the secretome of S. aureus.
Worldwide, nearly 800,000 individuals die by suicide each year; however, longitudinal prediction of suicide attempts remains a major challenge within the field of psychiatry. The objective of the ...present research was to develop and evaluate an evidence-based suicide attempt risk checklist i.e., the Durham Risk Score (DRS) to aid clinicians in the identification of individuals at risk for attempting suicide in the future. Three prospective cohort studies, including a population-based study from the United States i.e., the National Epidemiologic Survey on Alcohol and Related Conditions (NESARC) study as well as 2 smaller US veteran cohorts i.e., the Assessing and Reducing Post-Deployment Violence Risk (REHAB) and the Veterans After-Discharge Longitudinal Registry (VALOR) studies, were used to develop and validate the DRS. From a total sample size of 35,654 participants, 17,630 participants were selected to develop the checklist, whereas the remaining participants (N = 18,024) were used to validate it. The main outcome measure was future suicide attempts (i.e., actual suicide attempts that occurred after the baseline assessment during the 1- to 3-year follow-up period). Measure development began with a review of the extant literature to identify potential variables that had substantial empirical support as longitudinal predictors of suicide attempts and deaths. Next, receiver operating characteristic (ROC) curve analysis was utilized to identify variables from the literature review that uniquely contributed to the longitudinal prediction of suicide attempts in the development cohorts. We observed that the DRS was a robust prospective predictor of future suicide attempts in both the combined development (area under the curve AUC = 0.91) and validation (AUC = 0.92) cohorts. A concentration of risk analysis found that across all 35,654 participants, 82% of prospective suicide attempts occurred among individuals in the top 15% of DRS scores, whereas 27% occurred in the top 1%. The DRS also performed well among important subgroups, including women (AUC = 0.91), men (AUC = 0.93), Black (AUC = 0.92), White (AUC = 0.93), Hispanic (AUC = 0.89), veterans (AUC = 0.91), lower-income individuals (AUC = 0.90), younger adults (AUC = 0.88), and lesbian, gay, bisexual, transgender, and queer or questioning (LGBTQ) individuals (AUC = 0.88). The primary limitation of the present study was its its reliance on secondary data analyses to develop and validate the risk score. In this study, we observed that the DRS was a strong predictor of future suicide attempts in both the combined development (AUC = 0.91) and validation (AUC = 0.92) cohorts. It also demonstrated good utility in many important subgroups, including women, men, Black, White, Hispanic, veterans, lower-income individuals, younger adults, and LGBTQ individuals. We further observed that 82% of prospective suicide attempts occurred among individuals in the top 15% of DRS scores, whereas 27% occurred in the top 1%. Taken together, these findings suggest that the DRS represents a significant advancement in suicide risk prediction over traditional clinical assessment approaches. While more work is needed to independently validate the DRS in prospective studies and to identify the optimal methods to assess the constructs used to calculate the score, our findings suggest that the DRS is a promising new tool that has the potential to significantly enhance clinicians' ability to identify individuals at risk for attempting suicide in the future.
In the light of increasing drug resistance in Staphylococcus aureus, bacteriophage endolysins peptidoglycan hydrolases (PGHs) have been suggested as promising antimicrobial agents. The aim of this ...study was to determine the antimicrobial activity of nine enzymes representing unique homology groups within a diverse class of staphylococcal PGHs.
PGHs were recombinantly expressed, purified and tested for staphylolytic activity in multiple in vitro assays (zymogram, turbidity reduction assay and plate lysis) and against a comprehensive set of strains (S. aureus and CoNS). PGH cut sites in the staphylococcal peptidoglycan were determined by biochemical assays (Park-Johnson and Ghuysen procedures) and MS analysis. The enzymes were tested for their ability to eradicate static S. aureus biofilms and compared for their efficacy against systemic MRSA infection in a mouse model.
Despite similar modular architectures and unexpectedly conserved cleavage sites in the peptidoglycan (conferred by evolutionarily divergent catalytic domains), the enzymes displayed varying degrees of in vitro lytic activity against numerous staphylococcal strains, including cell surface mutants and drug-resistant strains, and proved effective against static biofilms. In a mouse model of systemic MRSA infection, six PGHs provided 100% protection from death, with animals being free of clinical signs at the end of the experiment.
Our results corroborate the high potential of PGHs for treatment of S. aureus infections and reveal unique antimicrobial and biochemical properties of the different enzymes, suggesting a high diversity of potential applications despite highly conserved peptidoglycan target sites.
The joint evaluated fission and fusion nuclear data library 3.3 is described. New evaluations for neutron-induced interactions with the major actinides
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, Cr, Cu, Zr, Cd, Hf, W, Au, Pb and Bi are presented. It includes new fission yields, prompt fission neutron spectra and average number of neutrons per fission. In addition, new data for radioactive decay, thermal neutron scattering, gamma-ray emission, neutron activation, delayed neutrons and displacement damage are presented. JEFF-3.3 was complemented by files from the TENDL project. The libraries for photon, proton, deuteron, triton, helion and alpha-particle induced reactions are from TENDL-2017. The demands for uncertainty quantification in modeling led to many new covariance data for the evaluations. A comparison between results from model calculations using the JEFF-3.3 library and those from benchmark experiments for criticality, delayed neutron yields, shielding and decay heat, reveals that JEFF-3.3 performes very well for a wide range of nuclear technology applications, in particular nuclear energy.
We describe the landscape of genomic alterations in cutaneous melanomas through DNA, RNA, and protein-based analysis of 333 primary and/or metastatic melanomas from 331 patients. We establish a ...framework for genomic classification into one of four subtypes based on the pattern of the most prevalent significantly mutated genes: mutant BRAF, mutant RAS, mutant NF1, and Triple-WT (wild-type). Integrative analysis reveals enrichment of KIT mutations and focal amplifications and complex structural rearrangements as a feature of the Triple-WT subtype. We found no significant outcome correlation with genomic classification, but samples assigned a transcriptomic subclass enriched for immune gene expression associated with lymphocyte infiltrate on pathology review and high LCK protein expression, a T cell marker, were associated with improved patient survival. This clinicopathological and multi-dimensional analysis suggests that the prognosis of melanoma patients with regional metastases is influenced by tumor stroma immunobiology, offering insights to further personalize therapeutic decision-making.
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•Represents the largest integrative analysis of cutaneous melanoma (331 patients)•Establishes a framework for melanoma genomic classification: BRAF, RAS, NF1, and Triple-WT•Identifies additional subtypes that may benefit from MAPK- and RTK-targeted therapies•Multi-dimensional analyses identify immune signatures associated with improved survival
An integrative analysis of cutaneous melanomas establishes a framework for genomic classification into four subtypes that can guide clinical decision-making for targeted therapies. A subset of each of the genomic classes expresses considerable immune infiltration markers that are associated with improved survival, with potential implications for immunotherapy.
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