Campylobacter (C.) jejuni and C. coli are common zoonotic bacteria in pigs, which typically act as asymptomatic carriers. However, the effects of Campylobacter colonisation on the porcine intestinal microbiota and metabolome remain poorly understood. This study investigated microbiome and metabolome alterations associated with co-colonisation by C. jejuni and C. coli in the different intestinal segments of pigs. Thirty-two weaned piglets were assigned to a control group and a group inoculated with C. coli ST5777/CT828 and C. jejuni ST122/CT206. Four weeks post inoculation, jejunal and caecal contents were analysed for Campylobacter counts, metabolite profiles and microbial composition. All animals remained clinically healthy. Both Campylobacter species colonised the jejunum and caecum, with higher C. coli counts in the caecum. Campylobacter-colonised pigs showed significantly altered metabolite profiles, including reduced cysteine and urea and increased glycine in the jejunum, as well as elevated 3-hydroxybutyrate levels in the caecum. In contrast, short-chain fatty acid concentrations in the caecum were unaffected by infection. Microbiota analysis revealed a significant reduction in caecal alpha diversity, whereas jejunal diversity remained unchanged. Infected pigs exhibited increased relative abundances of Lactobacillaceae and Bifidobacteriaceae and a decreased abundance of Pseudomonadota, including Enterobacteriaceae. In conclusion, Campylobacter co-colonisation induces distinct microbiome and metabolome alterations in pigs despite the absence of clinical disease. These findings highlight complex host-microbiota-pathogen interactions that may be relevant for future Campylobacter control strategies in pig production.

Abstract Vibrio natriegens is a halophilic, Gram-negative marine bacterium that is increasingly used in metabolic engineering applications due to its fast growth rate. In sparse minimal medium the organism has a doubling time of 25 minutes, which is about twice as fast as Escherichia coli under similar conditions. Given that its protein density is similarly constrained to that of E. coli , this necessitates that its metabolic enzymes are able to catalyze flux at a higher rate to sustain its metabolism. In this work, we measure the apparent turnover numbers of metabolically active enzymes in V. natriegens under a variety of growth conditions. The apparent turnover numbers of V. natriegens enzymes were measured in vivo by conducting coupled quantitative proteomics and 13 C metabolic flux analysis experiments under seven different carbon source conditions in sparse minimal medium. A high quality genome-scale metabolic model was constructed and curated using additional experimental data. This model was extended with enzyme constraints, and subsequently used to find kinetic parameters that minimize the difference between model predictions and experimental observations. This model guided data fusion approach enabled the estimation of 357 apparent turnover numbers for metabolically active enzymes in V. natriegens . Our results reveal that the metabolic enzymes of V. natriegens are in median 14-fold faster than those of E. coli under similar conditions. Moreover, we show that machine learning generated turnover number estimates substantially underestimate the kinetics of V. natriegens . Our turnover number estimates were used to parameterize multiple condition dependent enzyme constrained flux balance analysis models of V. natriegens , which improved their predictive accuracy compared to the machine learning parameterisation. The combined experimental-computational approach employed here sheds light on the mechanism V. natriegens uses to accelerate its growth. This approach can also be extended to other bacteria, increasing the availability of in vivo measured enzyme turnover numbers, and improving the predictive accuracy of enzyme constrained metabolic models of other microbes.

Despite the extensive research that has been conducted into the metabolism of Gram-positive bacteria, relatively little is known about the adaptations of Streptococcus dysgalactiae subsp. equisimilis (SDSE) during biofilm maturation. This study uses targeted metabolomics and MALDI MS imaging to investigate time-dependent changes in the metabolome composition. Key metabolites of energy metabolism were quantified in planktonic and biofilm bacteria. A distinct decline in the abundance of metabolites from all analyzed metabolic pathways was observed in sessile SDSE. Furthermore, we observed a broad decrease of nutrient utilization in biofilm-associated bacteria. Additionally, we examined the impact of deleting streptokinase on SDSE metabolism. Especially, concentrations of glycolysis intermediates and adenosine phosphates exhibited significant alterations when comparing the S118 strain with its mutant. The absence of the ska gene led to drastic changes in both energy and structural metabolism. These findings provide new insight into the metabolic adaptations of SDSE and represent an initial step toward understanding its role in pathogenesis.

The successful sustainable cultivation of the well-known medicinal plant sundew on rewetted peatlands not only leads to the preservation of natural populations, but also provides a basis for the sustainable pharmaceutical use of the plant. The bioactive compounds of sundew, flavonoids and naphthoquinones, show biofilm-inhibiting properties against multidrug-resistant, ESBL-producing E. coli strains and open up new therapeutic possibilities. This study investigates the molecular mechanisms of these compounds in biofilm inhibition through proteomic analyses. Specific fractions of flavonoids and naphthoquinones, as well as individual substances like 7-methyljuglone and 2″-O-galloylhyperoside, are analyzed. Results show that naphthoquinones appear to act via central regulatory proteins such as OmpR and alter the stress response while flavonoids likely affect biofilm formation by creating an iron-poor environment through iron complexation and additionally influence polyamine balance, reducing intracellular spermidine levels. Further investigations including assays for iron complexation and analysis of polyamines confirmed the proteomic data. Safety evaluations through cytotoxicity tests in 3D cell cultures and the Galleria mellonella in vivo model confirm the safety of the extracts used. These findings highlight sundew as a promising candidate for new phytopharmaceuticals.

Biofilms of pathogenic bacteria are responsible for persistent infections in humans, therefore investigations of biofilm formation and treatment strategies are required. The gram-negative enterobacterium Escherichia (E.) coli is the most common pathogen causing chronic or recurring urinary tract infections. Metabolomics approaches targeted the bacterium to investigate specific metabolic patterns of biofilms and regulatory influences on biofilm formation. In this study, we aimed to investigate the metabolome of biofilms formed by the multidrug-resistant extended-spectrum beta-lactamase-producing (ESBL) E. coli PBIO729. For this purpose, a protocol for fast sampling of the macrocolony biofilms and efficient extraction of metabolites was optimized. Validation of an LC-MS/MS method confirmed its usability for the analysis of nucleotides and other phosphorylated metabolites. A GC-MS approach was used to monitor nutrient uptake from the medium in addition to the analysis of amino acid content and metabolites of glycolysis and TCA cycle in E. coli biofilms.

Background Streptococcus dysgalactiae subspecies equisimilis (SDSE) is increasingly recognized as an emerging cause of invasive diseases including necrotizing soft tissue infections (NSTIs). In contrast to the closely related Streptococcus pyogenes, SDSE infections mainly affect older and comorbid patients. Biofilm formation has been demonstrated in soft tissue biopsies of S. pyogenes NSTI cases. Results Here, we show that bacterial aggregations indicative of biofilms are also present in SDSE NSTI. Although streptokinase (Ska) activity and biofilm formation did not correlate in a diverse set of clinical SDSE isolates, addition of exogenous Ska at an early time point prevented biofilm formation for selected strains. Deletion of ska in SDSE S118 strain resulted in increased biofilm forming capacity. Ska-deficient mutant strain was characterized by a higher metabolic activity and consequent metabolome profiling of biofilms identified higher deposition of a wide range of metabolites as compared to the wild-type. Conclusions Our results argue that Ska suppresses biofilm formation in SDSE independent of its original plasminogen converting activity. However, the impact of biofilms and its consequences for patient outcomes in streptococcal NSTIs remain to be elucidated.

Streptococcus pneumoniae is the most common causative agent of community-acquired pneumonia worldwide. A key pathogenic mechanism that exacerbates severity of disease is the disruption of the alveolar-capillary barrier. However, the specific virulence mechanisms responsible for this in the human lung are not yet fully understood. In this study, we infected living human lung tissue with Strep. pneumoniae and observed a significant degradation of the central junctional proteins occludin and vascular endothelial cadherin, indicating barrier disruption. Surprisingly, neither pneumolysin, bacterial hydrogen peroxide nor pro-inflammatory activation were sufficient to cause this junctional degradation. Instead, pneumococcal infection led to a significant decrease of pH (similar to 6), resulting in the acidification of the alveolar microenvironment, which was linked to junctional degradation. Stabilising the pH at physiological levels during infection reversed this effect, even in a therapeutic-like approach. Further analysis of bacterial metabolites and RNA sequencing revealed that sugar consumption and subsequent lactate production were the major factors contributing to bacterially induced alveolar acidification, which also hindered the release of critical immune factors. Our findings highlight bacterial metabolite-induced acidification as an independent virulence mechanism for barrier disruption and inflammatory dysregulation in pneumonia. Thus, our data suggest that strictly monitoring and buffering alveolar pH during infections caused by fermentative bacteria could serve as an adjunctive therapeutic strategy for sustaining barrier integrity and immune response.

Marine Bacteroidetes that degrade polysaccharides contribute to carbon cycling in the ocean. Organic matter, including glycans from terrestrial plants, might enter the oceans through rivers. Whether marine bacteria degrade structurally related glycans from diverse sources including terrestrial plants and marine algae was previously unknown. We show that the marine bacterium Flavimarina sp. Hel_I_48 encodes two polysaccharide utilization loci (PULs) which degrade xylans from terrestrial plants and marine algae. Biochemical experiments revealed activity and specificity of the encoded xylanases and associated enzymes of these PULs. Proteomics indicated that these genomic regions respond to glucuronoxylans and arabinoxylans. Substrate specificities of key enzymes suggest dedicated metabolic pathways for xylan utilization. Some of the xylanases were active on different xylans with the conserved β-1,4-linked xylose main chain. Enzyme activity was consistent with growth curves showing Flavimarina sp. Hel_I_48 uses structurally different xylans. The observed abundance of related xylan-degrading enzyme repertoires in genomes of other marine Bacteroidetes indicates similar activities are common in the ocean. The here presented data show that certain marine bacteria are genetically and biochemically variable enough to access parts of structurally diverse xylans from terrestrial plants as well as from marine algal sources.

Staphylococcus aureus is a major pathogen, which has to defend against reactive oxygen and electrophilic species encountered during infections. Activated macrophages produce the immunometabolite itaconate as potent electrophile and antimicrobial upon pathogen infection. In this work, we used transcriptomics, metabolomics and shotgun redox proteomics to investigate the specific stress responses, metabolic changes and redox modifications caused by sublethal concentrations of itaconic acid in S. aureus. In the RNA-seq transcriptome, itaconic acid caused the induction of the GlnR, KdpDE, CidR, SigB, GraRS, PerR, CtsR and HrcA regulons and the urease-encoding operon, revealing an acid and oxidative stress response and impaired proteostasis. Neutralization using external urea as ammonium source improved the growth and decreased the expression of the glutamine synthetase-controlling GlnR regulon, indicating that S. aureus experienced ammonium starvation upon itaconic acid stress. In the extracellular metabolome, the amounts of acetate and formate were decreased, while secretion of pyruvate and the neutral product acetoin were strongly enhanced to avoid intracellular acidification. Exposure to itaconic acid affected the amino acid uptake and metabolism as revealed by the strong intracellular accumulation of lysine, threonine, histidine, aspartate, alanine, valine, leucine, isoleucine, cysteine and methionine. In the proteome, itaconic acid caused widespread S-bacillithiolation and S-itaconation of redox-sensitive antioxidant and metabolic enzymes, ribosomal proteins and translation factors in S. aureus, supporting its oxidative and electrophilic mode of action in S. aureus. In phenotype analyses, the catalase KatA, the low molecular weight thiol bacillithiol and the urease provided protection against itaconic acid-induced oxidative and acid stress in S. aureus. Altogether, our results revealed that under physiological infection conditions, such as in the acidic phagolysome, itaconic acid is a highly effective antimicrobial against multi-resistant S. aureus isolates, which acts as weak acid causing an acid, oxidative and electrophilic stress response, leading to S-bacillithiolation and itaconation.

Campylobacter (C.) spp. represent one of the most important causes for food-borne bacterial pathogen in humans worldwide. The aim of this study was to investigate metabolic requirements of two Campylobacter strains of different species based on substrate utilisation (in vitro). Based on these results, a correlation between the colonisation and the available substrates in different intestinal sections was recorded using an animal model. Campylobacter coli (ST-5777) and C. jejuni (ST-122) were used to inoculate 16 pigs, respectively, and one group of 16 pigs was used as control. The strains differed significantly in substrate utilisation -C. coli was able to metabolise various substrates (acetate, asparagine, serine, fucose, and propionate), while C. jejuni only utilised serine. Metabolomic analysis of intestinal content from different gut sections showed the presence of all previ-ously tested metabolites, except for fucose. A significantly larger amount of glucose was found in the jejunum of those pigs infected with C. coli, while neither strain utilised it in vitro. The analysis of the intestinal contents revealed a very low proportion of Campylobacterales in the total microbiome, suggesting that the small per-centage of the inoculated Campylobacter strains in the gut microflora of the animals is too low to cause differences between the control and infected groups in the composition of the metabolome. Nevertheless, knowledge of specific nutritional requirements of the pathogens combined with proof of different metabolites in the intestinal segments may provide clues about the site of colonisation in the host and improve our understanding of this zoonotic germ.

Vaccine-induced immune thrombotic thrombocytopenia (VITT; synonym, thrombosis with thrombocytopenia syndrome, is associated with high-titer immunoglobulin G antibodies directed against platelet factor 4 (PF4). These antibodies activate platelets via platelet FcγIIa receptors, with platelet activation greatly enhanced by PF4. Here we summarize the current concepts in the pathogenesis of VITT. We first address parallels between heparin-induced thrombocytopenia and VITT, and provide recent findings on binding of PF4 to adenovirus particles and non-assembled adenovirus proteins in the 2 adenovirus vector-based COVID-19 vaccines, ChAdOx1 nCoV-19 and Ad26.COV2.S. Further, we discuss the potential role of vaccine constituents such as glycosaminoglycans, EDTA, polysorbate 80, human cell-line proteins and nucleotides as potential binding partners of PF4. The immune response towards PF4 in VITT is likely triggered by a proinflammatory milieu. Human cell-line proteins, non-assembled virus proteins, and potentially EDTA may contribute to the proinflammatory state. The transient nature of the immune response towards PF4 in VITT makes it likely that-as in heparin-induced thrombocytopenia -marginal zone B cells are key for antibody production. Once high-titer anti-PF4 antibodies have been formed 5 to 20 days after vaccination, they activate platelets and granulocytes. Activated granulocytes undergo NETosis and the released DNA also forms complexes with PF4, which fuels the Fcγ receptor-dependent cell activation process, ultimately leading to massive thrombin generation. Finally, we summarize our initial observations indicating that VITT-like antibodies might also be present in rare patients with recurrent venous and arterial thrombotic complications, independent of vaccination.

Respiratory tract infections are a worldwide health problem for humans and animals. Different cell types produce lipid mediators in response to infections, which consist of eicosanoids like hydroxyeicosatetraenoic acids (HETEs) or oxylipins like hydroxydocosahexaenoic acids (HDHAs). Both substance classes possess immunomodulatory functions. However, little is known about their role in respiratory infections. Here, we aimed to analyze the lipid mediator imprint of different organs of C57BL/6J mice after intranasal mono-infections with Streptococcus pneumoniae (pneumococcus), Staphylococcus aureus or Influenza A virus (IAV) as wells as pneumococcal-IAV co-infection. C57BL/6J mice were infected with different pathogens and lungs, spleen, and plasma were collected. Lipid mediators were analyzed using HPLC-MS/MS. In addition, spatial-distribution of sphingosine 1-phosphate (S1P) and ceramide 1-phosphates (C1P) in tissue samples was examined using MALDI-MS-Imaging. The presence of bacterial pathogens in the lung was confirmed via immunofluorescence staining. We found IAV specific changes for different HDHAs and HETEs in mouse lungs as well as enhanced levels of 20-HETE in severe S. aureus infection. Moreover, MALDI-MS-Imaging analysis showed an accumulation of C1P and a decrease of S1P during co-infection in lung and spleen. Long chain C1P was enriched in the red and not in the white pulp of the spleen. Lipid mediator analysis showed that host synthesis of bioactive lipids is in part specific for a certain pathogen, in particular for IAV infection. Furthermore, MS-Imaging displayed great potential to study infections and revealed changes of S1P and C1P in lungs and spleen of co-infected animals, which was not described before.

Tafazzin-an acyltransferase-is involved in cardiolipin (CL) remodeling. CL is associated with mitochondrial function, structure and more recently with cell proliferation. Various tafazzin isoforms exist in humans. The role of these isoforms in cardiolipin remodeling is unknown. Aim of this study was to investigate if specific isoforms like Δ5 can restore the wild type phenotype with respect to CL composition, cellular proliferation and gene expression profile. In addition, we aimed to determine the molecular mechanism by which tafazzin can modulate gene expression by applying promoter analysis and (Ingenuity Pathway Analyis) IPA to genes regulated by TAZ-deficiency. Expression of Δ5 and rat full length TAZ in C6-TAZ- cells could fully restore CL composition and-as proven for Δ5-this is naturally associated with restoration of mitochondrial respiration. A similar restoration of CL-composition could not be observed after re-expression of an enzymatically dead full-length rat TAZ (H69L; TAZMut). Re-expression of only rat full length TAZ could restore proliferation rate. Surprisingly, the Δ5 variant failed to restore wild-type proliferation. Further, as expected, re-expression of the TAZMut variant completely failed to reverse the gene expression changes, whereas re-expression of the TAZ-FL variant largely did so and the Δ5 variant to somewhat less extent. Very likely TAZ-deficiency provokes substantial long-lasting changes in cellular lipid metabolism which contribute to changes in proliferation and gene expression, and are not or only very slowly reversible.

Formaldehyde is a toxic metabolite that is formed in large quantities during bacterial utilization of the methoxy sugar 6‐ O ‐methyl‐ d ‐galactose, an abundant monosaccharide in the red algal polysaccharide porphyran. Marine bacteria capable of metabolizing porphyran must therefore possess suitable detoxification systems for formaldehyde. We demonstrate here that detoxification of formaldehyde in the marine Flavobacterium Zobellia galactanivorans proceeds via the ribulose monophosphate pathway. Simultaneously, we show that the genes encoding the key enzymes of this pathway are important for maintaining high formaldehyde resistance. Additionally, these genes are upregulated in the presence of porphyran, allowing us to connect porphyran degradation to the detoxification of formed formaldehyde.

The global turkey industry is confronted with emerging challenges regarding health and welfare. Performance and disease resilience are directly linked to gut health. A clear definition of a healthy gut is a prerequisite to developing new strategies for improved gut health and, thus, general health, welfare and productivity. To date, detailed knowledge about gut health characteristics, especially during the critical fattening period, is still lacking for turkeys. Therefore, the goal of this study was to describe the morphology, microbiota, and metabolome along the intestinal tract of clinically healthy Salmonella- and Campylobacter-free commercial turkey hens throughout the fattening period from 7 to 10 wk posthatch, and obtain information on the stability of the investigated values over time. Feed changes were avoided directly preceding and during the investigation period. Investigation methods included histomorphometric measurement of intestinal villi and crypts, Illumina-sequencing for microbiota analysis, and proton nuclear magnetic resonance spectroscopy for metabolite identification and quantification. Overall, the study demonstrated a high repeatability across all 3 experiments and gut section differences observed coincided with their functions. It was demonstrated that gut maturation, defined by gut microbiota stability, is reached earlier in the ceca than any other intestinal section where morphological changes are ongoing throughout the fattening period. Therefore, the present study provides valuable information necessary to advise future studies on the development and implementation of measures to support gut maturation and establish a protective microbiota in commercial turkeys.

TfCa, a promiscuous carboxylesterase from Thermobifida fusca, was found to hydrolyze polyethylene terephthalate (PET) degradation intermediates such as bis(2-hydroxyethyl) terephthalate (BHET) and mono-(2-hydroxyethyl)-terephthalate (MHET). In this study, we elucidated the structures of TfCa in its apo form, as well as in complex with a PET monomer analogue and with BHET. The structure-function relationship of TfCa was investigated by comparing its hydrolytic activity on various ortho- and para-phthalate esters of different lengths. Structure-guided rational engineering of amino acid residues in the substrate-binding pocket resulted in the TfCa variant I69W/V376A (WA), which showed 2.6-fold and 3.3-fold higher hydrolytic activity on MHET and BHET, respectively, than the wild-type enzyme. TfCa or its WA variant was mixed with a mesophilic PET depolymerizing enzyme variant [Ideonella sakaiensis PETase (IsPETase) PM] to degrade PET substrates of various crystallinity. The dual enzyme system with the wild-type TfCa or its WA variant produced up to 11-fold and 14-fold more terephthalate (TPA) than the single IsPETase PM, respectively. In comparison to the recently published chimeric fusion protein of IsPETase and MHETase, our system requires 10% IsPETase and one-fourth of the reaction time to yield the same amount of TPA under similar PET degradation conditions. Our simple dual enzyme system reveals further advantages in terms of cost-effectiveness and catalytic efficiency since it does not require time-consuming and expensive cross-linking and immobilization approaches.

ABSTRACT To be a successful pathogen, Staphylococcus aureus has to adapt its metabolism to the typically oxygen- and glucose-limited environment of the host. Under fermenting conditions and in the presence of glucose, S. aureus uses glycolysis to generate ATP via substrate-level phosphorylation and mainly lactic acid fermentation to maintain the redox balance by reoxidation of NADH equivalents. However, it is less clear how S. aureus proceeds under anoxic conditions and glucose limitation, likely representing the bona fide situation in the host. Using a combination of proteomic, transcriptional, and metabolomic analyses, we show that in the absence of an abundant glycolysis substrate, the available carbon source pyruvate is converted to acetyl coenzyme A (AcCoA) in a pyruvate formate-lyase (PflB)-dependent reaction to produce ATP and acetate. This process critically depends on derepression of the catabolite control protein A (CcpA), leading to upregulation of pflB transcription. Under these conditions, ethanol production is repressed to prevent wasteful consumption of AcCoA. In addition, our global and quantitative characterization of the metabolic switch prioritizing acetate over lactate fermentation when glucose is absent illustrates examples of carbon source-dependent control of colonization and pathogenicity factors. IMPORTANCE Under infection conditions, S. aureus needs to ensure survival when energy production via oxidative phosphorylation is not possible, e.g., either due to the lack of terminal electron acceptors or by the inactivation of components of the respiratory chain. Under these conditions, S. aureus can switch to mixed-acid fermentation to sustain ATP production by substrate level phosphorylation. The drop in the cellular NAD + /NADH ratio is sensed by the repressor Rex, resulting in derepression of fermentation genes. Here, we show that expression of fermentation pathways is further controlled by CcpA in response to the availability of glucose to ensure optimal resource utilization under growth-limiting conditions. We provide evidence for carbon source-dependent control of colonization and virulence factors. These findings add another level to the regulatory network controlling mixed-acid fermentation in S. aureus and provide additional evidence for the lifestyle-modulating effect of carbon sources available to S. aureus .

Ixodid ticks represent vectors and reservoirs for a broad range of zoonotic pathogens. Collected ticks from field studies are therefore usually stored in ethanol, which in higher concentrations effectively inactivates most of the known tick-borne pathogens. Although commonly practiced as gold standard for inactivation, hardly any scientific data demonstrate that ethanol sufficiently penetrates the comparatively thick cuticula of ticks. Therefore, Amblyomma hebraeum tick pools were stored for 21 days in ethanol (96%). Afterwards, the ethanol was removed and the ticks were homogenized. Quantitative 1H-NMR spectroscopic analysis was applied to determine the residual concentration of ethanol inside the ticks. 1H-NMR spectroscopic analysis revealed that ethanol constituted 28.3–42.6 mg of the total weight of three ticks in the pools (89.9–121.5 mg). In addition, the low-pathogenic Hazara orthonairovirus (HAZV) was used as a cell culture model for this study. The virus was exposed to ethanol concentrations between 0 and 60% and incubated under various temperature conditions for four time periods. Afterwards, the residual virus infectivity was determined by titration. Following ethanol exposure, HAZV did not grow in cells after 9 h of exposure to an ethanol concentration of 25%. These results demonstrate an extremely low ethanol resistance of the virus, which was generally in line with previously reported ethanol inactivation data for Crimean-Congo hemorrhagic fever orthonairovirus (CCHFV). After prolonged storage and impregnation, comparable ethanol concentrations are achieved in the ticks, indicating the suitability of this inactivation method also for Bunyaviruses in ticks. At the very least, a massive virus inactivation can be assumed. Definitive proof of virus inactivation would require a bioassay of ethanol-treated infected ticks under appropriate biosafety conditions.

Background SARS-CoV-2 vaccine ChAdOx1 nCov-19 rarely causes vaccine-induced immune thrombotic thrombocytopenia (VITT) that—like autoimmune heparin-induced thrombocytopenia—is mediated by platelet-activating anti-platelet factor 4 (PF4) antibodies. Methods We investigated vaccine, PF4, and VITT patient-derived anti-PF4 antibody interactions using dynamic light scattering, 3D-super-resolution microscopy, and electron microscopy. Mass spectrometry was used to analyze vaccine composition. We investigated the mechanism for early post-vaccine inflammatory reactions as potential co-stimulant for anti-PF4 immune response. Finally, we evaluated VITT antibodies for inducing release of procoagulant DNA-containing neutrophil extracellular traps (NETs), and measured DNase activity in VITT patient serum. Results Biophysical analyses showed formation of complexes between PF4 and vaccine constituents, including virus proteins that were recognized by VITT antibodies. EDTA, a vaccine constituent, increased microvascular leakage in mice allowing for circulation of virus- and virus-producing cell culture-derived proteins. Antibodies in normal sera cross-reacted with human proteins in the vaccine and likely contribute to commonly observed acute ChAdOx1 nCov-19 post-vaccination inflammatory reactions. Polyphosphates and DNA enhanced PF4-dependent platelet activation by VITT antibodies. In the presence of platelets, PF4 enhanced VITT antibody-driven procoagulant NETs formation, while DNase activity was reduced in VITT sera, with granulocyte-rich cerebral vein thrombosis observed in a VITT patient. Conclusions ChAdOx1 nCoV-19 vaccine constituents (i) form antigenic complexes with PF4, (ii) EDTA increases microvascular permeability, and (iii) vaccine components cause acute inflammatory reactions. Antigen formation in a proinflammatory milieu offers an explanation for anti-PF4 antibody production. High-titer anti-PF4 antibodies activate platelets and induce neutrophil activation and NETs formation, fueling the VITT prothrombotic response.