SARS-CoV-2 vaccine ChAdOx1 nCoV-19 (AstraZeneca) causes a thromboembolic complication termed vaccine-induced immune thrombotic thrombocytopenia (VITT). Using biophysical techniques, mouse models, and ...analysis of VITT patient samples, we identified determinants of this vaccine-induced adverse reaction. Super-resolution microscopy visualized vaccine components forming antigenic complexes with platelet factor 4 (PF4) on platelet surfaces to which anti-PF4 antibodies obtained from VITT patients bound. PF4/vaccine complex formation was charge-driven and increased by addition of DNA. Proteomics identified substantial amounts of virus production-derived T-REx HEK293 proteins in the ethylenediaminetetraacetic acid (EDTA)-containing vaccine. Injected vaccine increased vascular leakage in mice, leading to systemic dissemination of vaccine components known to stimulate immune responses. Together, PF4/vaccine complex formation and the vaccine-stimulated proinflammatory milieu trigger a pronounced B-cell response that results in the formation of high-avidity anti-PF4 antibodies in VITT patients. The resulting high-titer anti-PF4 antibodies potently activated platelets in the presence of PF4 or DNA and polyphosphate polyanions. Anti-PF4 VITT patient antibodies also stimulated neutrophils to release neutrophil extracellular traps (NETs) in a platelet PF4-dependent manner. Biomarkers of procoagulant NETs were elevated in VITT patient serum, and NETs were visualized in abundance by immunohistochemistry in cerebral vein thrombi obtained from VITT patients. Together, vaccine-induced PF4/adenovirus aggregates and proinflammatory reactions stimulate pathologic anti-PF4 antibody production that drives thrombosis in VITT. The data support a 2-step mechanism underlying VITT that resembles the pathogenesis of (autoimmune) heparin-induced thrombocytopenia.
The stringent response is initiated by rapid (p)ppGpp synthesis, which leads to a profound reprogramming of gene expression in most bacteria. The stringent phenotype seems to be species specific and ...may be mediated by fundamentally different molecular mechanisms. In Staphylococcus aureus, (p)ppGpp synthesis upon amino acid deprivation is achieved through the synthase domain of the bifunctional enzyme RSH (RelA/SpoT homolog). In several firmicutes, a direct link between stringent response and the CodY regulon was proposed. Wild-type strain HG001, rsh(Syn), codY and rsh(Syn), codY double mutants were analyzed by transcriptome analysis to delineate different consequences of RSH-dependent (p)ppGpp synthesis after induction of the stringent response by amino-acid deprivation. Under these conditions genes coding for major components of the protein synthesis machinery and nucleotide metabolism were down-regulated only in rsh positive strains. Genes which became activated upon (p)ppGpp induction are mostly regulated indirectly via de-repression of the GTP-responsive repressor CodY. Only seven genes, including those coding for the cytotoxic phenol-soluble modulins (PSMs), were found to be up-regulated via RSH independently of CodY. qtRT-PCR analyses of hallmark genes of the stringent response indicate that an RSH activating stringent condition is induced after uptake of S. aureus in human polymorphonuclear neutrophils (PMNs). The RSH activity in turn is crucial for intracellular expression of psms. Accordingly, rsh(Syn) and rsh(Syn), codY mutants were less able to survive after phagocytosis similar to psm mutants. Intraphagosomal induction of psmα1-4 and/or psmβ1,2 could complement the survival of the rsh(Syn) mutant. Thus, an active RSH synthase is required for intracellular psm expression which contributes to survival after phagocytosis.
Colonization of the human nose by Staphylococcus aureus in one-third of the population represents a major risk factor for invasive infections. The basis for adaptation of S. aureus to this specific ...habitat and reasons for the human predisposition to become colonized have remained largely unknown. Human nasal secretions were analyzed by metabolomics and found to contain potential nutrients in rather low amounts. No significant differences were found between S. aureus carriers and non-carriers, indicating that carriage is not associated with individual differences in nutrient supply. A synthetic nasal medium (SNM3) was composed based on the metabolomics data that permits consistent growth of S. aureus isolates. Key genes were expressed in SNM3 in a similar way as in the human nose, indicating that SNM3 represents a suitable surrogate environment for in vitro simulation studies. While the majority of S. aureus strains grew well in SNM3, most of the tested coagulase-negative staphylococci (CoNS) had major problems to multiply in SNM3 supporting the notion that CoNS are less well adapted to the nose and colonize preferentially the human skin. Global gene expression analysis revealed that, during growth in SNM3, S. aureus depends heavily on de novo synthesis of methionine. Accordingly, the methionine-biosynthesis enzyme cysteine-γ-synthase (MetI) was indispensable for growth in SNM3, and the MetI inhibitor DL-propargylglycine inhibited S. aureus growth in SNM3 but not in the presence of methionine. Of note, metI was strongly up-regulated by S. aureus in human noses, and metI mutants were strongly abrogated in their capacity to colonize the noses of cotton rats. These findings indicate that the methionine biosynthetic pathway may include promising antimicrobial targets that have previously remained unrecognized. Hence, exploring the environmental conditions facultative pathogens are exposed to during colonization can be useful for understanding niche adaptation and identifying targets for new antimicrobial strategies.
During infection processes, Staphylococcus aureus is able to survive within the host and to invade tissues and cells. For studying the interaction between the pathogenic bacterium and the host cell, ...the bacterial growth behaviour and its metabolic adaptation to the host cell environment provides first basic information. In the present study, we therefore cultivated S. aureus COL and HG001 in the eukaryotic cell culture medium RPMI 1640 and analyzed the extracellular metabolic uptake and secretion patterns of both commonly used laboratory strains. Extracellular accumulation of D-isoleucine was detected starting during exponential growth of COL and HG001 in RPMI medium. This non-canonical D-amino acid is known to play a regulatory role in adaptation processes. Moreover, individual uptake of glucose, accumulation of acetate, further overflow metabolites, and intermediates of the branched-chain amino acid metabolism constitute unique metabolic footprints. Altogether these time-resolved footprint analyses give first metabolic insights into staphylococcal growth behaviour in a culture medium used for infection related studies.
Social arthropods such as termites, ants, and bees are among others the most successful animal groups on earth. However, social arthropods face an elevated risk of infections due to the dense colony ...structure, which facilitates pathogen transmission. An interesting hypothesis is that social arthropods are protected by chemical compounds produced by the arthropods themselves, microbial symbionts, or plants they associate with.
Stegodyphus dumicola
is an African social spider species, inhabiting communal silk nests. Because of the complex three-dimensional structure of the spider nest antimicrobial volatile organic compounds (VOCs) are a promising protection against pathogens, because of their ability to diffuse through air-filled pores. We analyzed the volatilomes of
S. dumicola
, their nests, and capture webs in three locations in Namibia and assessed their antimicrobial potential. Volatilomes were collected using polydimethylsiloxane (PDMS) tubes and analyzed using GC/Q-TOF. We showed the presence of 199 VOCs and tentatively identified 53 VOCs. More than 40% of the tentatively identified VOCs are known for their antimicrobial activity. Here, six VOCs were confirmed by analyzing pure compounds namely acetophenone, 1,3-benzothiazole, 1-decanal, 2-decanone, 1-tetradecene, and docosane and for five of these compounds the antimicrobial activity were proven. The nest and web volatilomes had many VOCs in common, whereas the spider volatilomes were more differentiated. Clear differences were identified between the volatilomes from the different sampling sites which is likely justified by differences in the microbiomes of the spiders and nests, the plants, and the different climatic conditions. The results indicate the potential relevance of the volatilomes for the ecological success of
S. dumicola
.
The potential benefit of soy isoflavones in breast cancer chemoprevention, as suggested by epidemiological studies, has aroused the interest of numerous scientists for over twenty years. Although ...intensive work has been done in this field, the preclinical results continue to be controversial and the molecular mechanisms are far from being fully understood. The antiproliferative effect of soy isoflavones has been commonly linked to the estrogen receptor interaction, but there is growing evidence that other pathways are influenced as well. Among these, the regulation of apoptosis, cell proliferation and survival, inhibition of angiogenesis and metastasis or antioxidant properties have been recently explored using various isoflavone doses and various breast cancer cells. In this review, we offer a comprehensive perspective on the molecular mechanisms of isoflavones observed in in vitro studies, emphasizing each time the dose-effect relationship and estrogen receptor status of the cells. Furthermore, we present future research directions in this field which could provide a better understanding of the inner molecular mechanisms of soy isoflavones in breast cancer.
Abstract Microorganisms preserve their metabolic function against a wide range of external perturbations including biotic or abiotic factors by utilizing cellular adaptations to maintain cell ...homeostasis. Functional genomics aims to detect such adaptive alterations on the level of transcriptome, proteome and metabolome to understand system wide changes and to identify interactions between the different levels of biochemical organization. Microbial metabolomics measures metabolites, the direct biochemical response to the environment, and is pivotal to the understanding of the variability and dynamics of bacterial cell metabolism. Metabolomics can measure many different types of compounds including primary metabolites, secondary metabolites, second messengers, quorum sensing compounds and others, which all contribute to the complex bacterial response to an environmental change. Recent data confirmed that many metabolic processes in pathogenic bacteria are linked to virulence and invasive capabilities. Deciphering bacterial metabolism in response to specific environmental conditions and in specific genetic backgrounds will help map the complex network between the metabolome and the other “-omes”. Here, we will review a selection of case studies for the pathogenic Gram-positive bacterium Staphylococcus aureus and summarize the current state of metabolomics literature covering staphylococci metabolism under different physiological states.
Ongoing resistance developments against antibiotics that also affect last-resort antibiotics require novel antibacterial compounds. Strategies to discover such novel structures have been dimerization ...or hybridization of known antibacterial agents. We found novel antibacterial agents by dimerization of indols and hybridization with carbazoles. They were obtained in a simple one-pot reaction as bisindole tetrahydrocarbazoles. Further oxidation led to bisindole carbazoles with varied substitutions of both the indole and the carbazole scaffold. Both the tetrahydrocarbazoles and the carbazoles have been evaluated in various
strains, including MRSA strains. Those 5-cyano substituted derivatives showed best activities as determined by MIC values. The tetrahydrocarbazoles partly exceed the activity of the carbazole compounds and thus the activity of the used standard antibiotics. Thus, promising lead compounds could be identified for further studies.
The peptidoglycan layer is responsible for maintaining bacterial cell shape and permitting cell division. Cell wall growth is facilitated by peptidoglycan synthases and hydrolases and is potentially ...modulated by components of the central carbon metabolism. In Bacillus subtilis, UgtP synthesises the glucolipid precursor for lipoteichoic acid and has been suggested to function as a metabolic sensor governing cell size. Here we show that ugtP mutant cells have increased levels of cell wall precursors and changes in their peptidoglycan that suggest elevated DL-endopeptidase activity. The additional deletion of lytE, encoding a DL-endopeptidase important for cell elongation, in the ugtP mutant background produced cells with severe shape defects. Interestingly, the ugtP lytE mutant recovered normal rod-shape by acquiring mutations that decreased the expression of the peptidoglycan synthase PBP1. Together our results suggest that cells lacking ugtP must re-adjust the balance between peptidoglycan synthesis and hydrolysis to maintain proper cell morphology.
We are currently facing an antimicrobial resistance crisis, which means that a lot of bacterial pathogens have developed resistance to common antibiotics. Hence, novel and innovative solutions are ...urgently needed to combat resistant human pathogens. A new source of antimicrobial compounds could be bacterial volatiles. Volatiles are ubiquitous produced, chemically divers and playing essential roles in intra- and interspecies interactions like communication and antimicrobial defense. In the last years, an increasing number of studies showed bioactivities of bacterial volatiles, including antibacterial, antifungal and anti-oomycete activities, indicating bacterial volatiles as an exciting source for novel antimicrobial compounds. In this review we introduce the chemical diversity of bacterial volatiles, their antimicrobial activities and methods for testing this activity. Concluding, we discuss the possibility of using antimicrobial volatiles to antagonize the antimicrobial resistance crisis.
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