DNase Activity Research Articles

In vivo model of Propionibacterium (Cutibacterium) spp. biofilm in Drosophila melanogaster

ObjectivesAcne vulgaris is a common inflammatory disorder of the pilosebaceous unit and Propionibacterium acnes biofilm-forming ability is believed to be a contributing factor to the disease development. In vivo models mimicking hair follicle environment are lacking. The aim of this study was to develop an in vivo Propionibacterium spp. biofilm model in Drosophila melanogaster (fruit fly). MethodsWe created a sterile line of D. melanogaster able to sustain Propionibacterium spp. biofilms in the gut. In order to mimic the lipid-rich, anaerobic environment of the hair follicle, fruit flies were maintained on lipid-rich diet. Propionibacterium spp. biofilms were visualized by immunofluorescence and scanning electron microscopy. We further tested if the biofilm-dispersal activity of DNase I can be demonstrated in the developed model. ResultsWe have demonstrated the feasibility of our in vivo model for development and study of P. acnes, P. granulosum and P. avidum biofilms. The model is suitable to evaluate dispersal as well as other agents against P. acnes biofilm. ConclusionsWe report a novel in vivo model for studying Propionibacterium spp. biofilms. The model can be suitable for both mechanistic as well as interventional studies.

Secretory abzyme IgA, enzymes and DNA of oral fluid in chronic periodontitis

Catalytic abzyme activity of IgA and corresponding enzymes of oral fluid in patients with chronic periodontitis was studied. The individuals with healthy state of oral cavity were used as controls. Various kinds of proteolytic, oxidoreductase, and DNase activity were investigated; the concentarion of oral fluid DNA was evaluated as well. IgA fractions were isolated from the oral fluids by affinity chromatography on a matrix with polyclonal antibodies against human IgA, Elastase, proteolytic, cathepsin-like and amidase activities were investigated by colorimetric methods. DNase activity was assessed by the rivanol clot test. DNA concentrations in the oral fluid were determined by fluorimetric DNA test with PicoGreen fluorochrome. It has been established that certain kinds of IgA abzyme activities and oral fluid enzymes become substantially elevated following the exacerbations of chronic periodontitis in comparison with healthy controls (p<0.01-0.001). It refers to elastase, peroxidase, cathepsin, and catalase activity of oral cavity. The progression of chronic periodontitis is also characterized by high levels of patients’ DNA contents in oral fluid (p<0.001). For the diagnosis of chronic periodontitis the ROC-analysis of examined laboratory markers found the maximum diagnostic value of oral elastase testing (specificity of the test – 0,97; sensitivity – 0,85).

Characterization of recombinant pumpkin 2S albumin and mutation studies to unravel potential DNA/RNA binding site

The native pumpkin 2S albumin, a multifunctional protein, possess a variety of potential biotechnologically exploitable properties. The present study reports the characterization of recombinant pumpkin 2S albumin (rP2SA) and unraveling of its potential DNA/RNA binding site. The purification and characterization of the rP2SA established that it retains the characteristic α-helical structure and exhibited comparable DNase, RNase, antifungal and anti-proliferative activities as native protein. In vitro studies revealed that rP2SA exhibits potent antiviral activity against chikungunya virus (CHIKV) at a non-toxic concentration with an IC50 of 114.5 μg/mL. In silico studies and site-directed mutagenesis were employed to unravel the potential DNA/RNA binding site. A strong positive charge distribution due to presence of many arginine residues in proximity of helix 5 was identified as a potential site. The two of the arginine residues, conserved in some 2S albumins, were selected for the mutation studies. The mutated forms of recombinant protein (R84A and R91A) showed a drastic reduction in DNase and RNase activities suggesting their presence at binding site and involvement in the nuclease activity. A metal binding site was also identified adjacent to DNA/RNA binding site. The present study demonstrated the structural and functional integrity of the rP2SA and reports potential antiviral activity against CHIKV. Further, potential DNA/RNA binding site was unraveled through mutation studies and bioinformatics analysis.

Occurrence of Virulence and Antimicrobial Resistance Determinants in Vibrio harveyi Isolated from Marine Food Fish Cultured in Korea.

Vibrio harveyi is a significant cause of infection in both marine animals and humans. It has been reported frequently in seafood-borne infections worldwide. This study was conducted to determine the potential health impact of the V. harveyi isolated from marine food fish cultured in Korea concerning their virulence and antimicrobial resistance. A total of 49 V. harveyi samples were isolated by biochemical tests and multiplex PCR. Phenotypic detection of virulence factors resulted DNase activity (81.63%), hemolysis (α = 75.51% and β = 12.25), gelatinase activity (71.43%), protease production (71.43%), phospholipase activity (65.31%), and lipase production (34.69%). Virulence genes, including VPI, tlh, tdh, toxR, VAC, and ctxAB, were detected in 57.14%, 44.90%, 36.73%, 22.45%, 12.24%, and 8.16% of the isolates, respectively. Resistance to ampicillin (77.55%), oxacillin (69.39%), nalidixic acid (53.06%), amoxicillin (46.94%), oxytetracycline (46.94%), colistin sulfate (34.69%), fosfomycin (34.69%), chloramphenicol (32.65%), streptomycin (32.65%), cephalothin (28.57%), oxytetracycline (26.53%), ceftriaxone (20.41%), erythromycin (14.29%), and cefoxitin (12.24%) was detected in disc diffusion assay. Most of the isolates were classified as multidrug resistant as they scored multiple antimicrobial resistance index ≥0.2. Furthermore, antimicrobial resistance genes tetB, qnrA, intI1 (Class 1 integron integrase), aac(6')-Ib, blaSHV, blaCTX-M, strA-strB, tetA, aphAI-IAB, qnrC, qnrS, and blaTEM were found in 81.63%, 67.35%, 61.22%, 46.94%, 44.90%, 44.90%, 36.73%, 18.37%, 10.20%, 10.20%, 8.16% and 6.12% of the isolates, respectively. In conclusion, the development of antimicrobial resistance among V. harveyi will ultimately reduce the efficacy of antimicrobials used for treating and can favor the development of more virulent V. harveyi strains.

DNase activity in human seminal plasma and follicular fluid and its inhibition by follicular fluid chelating agents

Research questionWhat is the mechanism by which human follicular fluid inhibits seminal plasma DNase activity? DesignHuman genomic DNA was incubated with human follicular fluid and seminal plasma (reaction mixture) under different experimental conditions; increasing volumes of human follicular fluid; proteinase K digested or heat inactivated human follicular fluid; and the addition of Ca2+ or Mg2+ to the reaction mixture. ResultsIncreasing volume of human follicular fluid resulted in a dose-dependent inhibition of seminal plasma DNase activity. Inhibition was not caused by proteins in the human follicular fluid as digestion with proteinase K or heat inactivation of human follicular fluid failed to abolish its inhibitory effect. Addition of divalent cations resulted in a reversion of the inhibitory effect, providing evidence that human follicular fluid inhibition of seminal plasma DNase activity seems to be mediated by a compound with chelating activity. Furthermore, incubation of genomic DNA with human follicular fluid in the presence of divalent cations served to elicit the existence of DNase activity. ConclusionsHuman follicular fluid seems to contain a molecule or molecules with chelating capacity that inhibits DNase activity of both follicular fluid and seminal plasma. Our findings provide new insight to understanding sperm preservation and the physiology of fertilization biology.

Genomic insights on DNase production in Streptococcus agalactiae ST17 and ST19 strains.

Streptococcus agalactiae evasion from the human defense mechanisms has been linked to the production of DNases. These were proposed to contribute to the hypervirulence of S. agalactiae ST17/capsular-type III strains, mostly associated with neonatal meningitis. We performed a comparative genomic analysis between ST17 and ST19 human strains with different cell tropism and distinct DNase production phenotypes. All S. agalactiae ST17 strains, with the exception of 2211-04, were found to display DNase activity, while the opposite scenario was observed for ST19, where 1203-05 was the only DNase(+) strain. The analysis of the genetic variability of the seven genes putatively encoding secreted DNases in S. agalactiae revealed an exclusive amino acid change in the predicted signal peptide of GBS0661 (NucA) of the ST17 DNase(-), and an exclusive amino acid change alteration in GBS0609 of the ST19 DNase(+) strain. Further core-genome analysis identified some specificities (SNVs or indels) differentiating the DNase(-) ST17 2211-04 and the DNase(+) ST19 1203-05 from the remaining strains of each ST. The pan-genomic analysis evidenced an intact phage without homology in S. agalactiae and a transposon homologous to TnGBS2.3 in ST17 DNase(-) 2211-04; the transposon was also found in one ST17 DNase(+) strain, yet with a different site of insertion. A group of nine accessory genes were identified among all ST17 DNase(+) strains, including the Eco47II family restriction endonuclease and the C-5 cytosine-specific DNA methylase. None of these loci was found in any DNase(-) strain, which may suggest that these proteins might contribute to the lack of DNase activity. In summary, we provide novel insights on the genetic diversity between DNase(+) and DNase(-) strains, and identified genetic traits, namely specific mutations affecting predicted DNases (NucA and GBS0609) and differences in the accessory genome, that need further investigation as they may justify distinct DNase-related virulence phenotypes in S. agalactiae.

Problems and trends in the development of dairy livestock in Russia

Staphylococci are widespread in the environment, which is facilitated by the duration of their survival on various objects. In this regard, rapid and accurate identification of staphylococcal species is in demand, with the aim to assess their pathogenic properties and prescribe adequate drug therapy. This year, 48 staphylococcal cultures were isolated from the udder secretions of cows (sick with mastitis) from six regional agricultural enterprises and tested for a number of biochemical properties. By the minimum number of tests, including catalase activity, mannitol fermentation and plasma coagulation, isolated cultures were differentiated into coagulase-positive and coagulase-negative ones. According to the research results for S. aureus, the three above-mentioned tests are sufficiently informative to classify them as pathogenic staphylococci. S. intermedius strains are catalase- and coagulase-positive, and with respect to mannitol fermentation, the reaction can be either positive or negative, which requires additional research. In addition to the tests listed above, the presence of hemolytic and DNAse activity was confirmed in strains of pathogenic staphylococci. The use of the reagents set of “Multimicrotests for biochemical identification of staphylococci (“MMT S”)” makes it possible to determine the species belonging of coagulase-negative staphylococci. Determination of biochemical properties of staphylococci isolated during bacteriological analysis makes it possible to identify them and assess their pathogenic properties.

DNase and Gelatinase Activities Of β-Hemolysin Aeromonas hydrophila Isolated from Catfish (Clarias batrachus)

Aeromonas hydrophila infection can cause mass mortality fish and result economic losses for farmers. These is bacterium secretes substances or compounds that support its virulence by producing toxins and extracellular proteins in the form of enzymes. This study aimed to determine the activity of β-hemolysin toxin, DNase enzyme and gelatinase enzyme of A. hydrophila isolated from catfish (Clarias batrachus) in Sidoarjo. This research was carried out in November 2020 in Dukuh Tengah Village, Buduran District, Wedoro Village, Waru District, Wadung Asri Village, Waru District and Keboan Sikep Village, Gedangan District. The sampling technique was carried out by researchers by following activities in the field and visiting correspondents directly in the catfish cultivator ponds, then the catfish samples were carried out by biochemical tests and toxin and enzyme activity tests to obtain the information and data needed. The results showed that A. hydrophila bacteria in catfish produced positive β-hemolysin toxin, DNase enzyme and gelatinase enzyme. The conclusion of the research was that 10 isolates of A. hydrophila bacteria isolated from catfish (Clarias batrachus) in Sidoarjo showed the activity of β hemolysin toxin, DNase enzyme and gelatinase enzyme.

The core Cas1 protein of CRISPR-Cas I-B in Leptospira shows metal-tunable nuclease activity.

Leptospira interrogans serovar Copenhageni strain Fiocruz L1-130 is the causative agent of leptospirosis in animals and humans. This organism carries a functional cas1 gene classified under CRISPR-Cas I-B. In this study, using various nuclease assays and bioinformatics analysis, we report that the recombinant Cas1 (LinCas1) possesses metal-ion dependent DNase activity, which is inhibited upon substitution or chelation of metal-ion and/or interaction with recombinant Cas2 (LinCas2) of L. interrogans. Model of LinCas1 structure shows a shorter N-terminal domain unlike other Cas1 orthologs reported to date. The C-terminal domain of LinCas1 contains conserved divalent-metal binding residues (Glu108, His176, and Glu191) and the mutation of these residues leads to abolition in DNase activity. Immunoassay using anti-LinCas2 demonstrates that LinCas1 interacts with LinCas2 and attains a saturation point. Moreover, the nuclease activity of the LinCas1-Cas2 mixture on ds-DNA displayed a reduction in activity compared to the pure core LinCas proteins under in vitro condition. The DNase activity for LinCas1 is consistent with a role for this protein in the recognition/cleavage of foreign DNA and integration of foreign DNA as spacer into the CRISPR array.

Hypercholesterolemia Impairs Clearance of Neutrophil Extracellular Traps and Promotes Inflammation and Atherosclerotic Plaque Progression.

Objective: Hypercholesterolemia-induced NETosis and accumulation of neutrophil extracellular traps (NETs) in the atherosclerotic lesion exacerbates inflammation and is causally implicated in plaque progression. We investigated whether hypercholesterolemia additionally impairs the clearance of NETs mediated by endonucleases such as DNase1 and DNase1L3 and its implication in advanced atherosclerotic plaque progression. Approach and Results: Using a mouse model, we demonstrate that an experimental increase in the systemic level of NETs leads to a rapid increase in serum DNase activity, which is critical for the prompt clearance of NETs and achieving inflammation resolution. Importantly, hypercholesterolemic mice demonstrate an impairment in this critical NET-induced DNase response with consequent delay in the clearance of NETs and defective inflammation resolution. Administration of tauroursodeoxycholic acid, a chemical chaperone that relieves endoplasmic reticulum stress, rescued the hypercholesterolemia-induced impairment in the NET-induced DNase response suggesting a causal role for endoplasmic reticulum stress in this phenomenon. Correction of the defective DNase response with exogenous supplementation of DNase1 in Apoe-/- mice with advanced atherosclerosis resulted in a decrease in plaque NET content and significant plaque remodeling with decreased area of plaque necrosis and increased collagen content. From a translational standpoint, we demonstrate that humans with hypercholesterolemia have elevated systemic extracellular DNA levels and decreased plasma DNase activity. Conclusions: These data suggest that hypercholesterolemia impairs the NET-induced DNase response resulting in defective clearance and accumulation of NETs in the atherosclerotic plaque. Therefore, strategies aimed at rescuing this defect could be of potential therapeutic benefit in promoting inflammation resolution and atherosclerotic plaque stabilization.

Crystal structure and characterization of nucleoside diphosphate kinase from Vibrio cholerae

Nucleoside diphosphate kinases (NDK) are ubiquitous enzymes that catalyse the transfer of the γ phosphate from nucleoside triphosphates (NTPs) to nucleoside diphosphate (NDPs), to maintain appropriate NTP levels in cells. NDKs are associated with signal transduction, cell development, proliferation, differentiation, tumor metastasis, apoptosis and motility. The critical role of NDK in bacterial virulence renders it a potential drug target. The present manuscript reports crystal structure and functional characterization of Vibrio cholerae NDK (VNDK). The 16 kDa VNDK was crystallized in a solution containing 30% PEG 4000, 100 mM Tris-HCl pH 8.5 and 200 mM sodium acetate in orthorhombic space group P212121 with unit cell parameters a = 48.37, b = 71.21, c = 89.14 Å, α = β = γ = 90° with 2 molecules in asymmetric unit. The crystal structure was solved by molecular replacement and refined to crystallographic Rfactor and Rfree values of 22.8% and 25.8% respectively. VNDK exists as both dimer and tetramer in solution as confirmed by size exclusion chromatography, glutaraldehyde crosslinking and small angle X-ray scattering while the crystal structure appears to be a dimer. The biophysical characterization states that VNDK has kinase and DNase activity with maximum stability at pH 8–9 and temperature up to 40 °C. VNDK shows elevated thermolability as compared to other NDK and shows preferential binding with GTP rationalized using computational studies.

Culprit site extracellular DNA and microvascular obstruction in ST-elevation myocardial infarction.

Extracellular chromatin and deoxyribonuclease (DNase) have been identified as important players of thrombosis, inflammation, and homeostasis in a murine model. We previously demonstrated that activated neutrophils release neutrophil extracellular traps (NETs) at the culprit site in ST-elevation myocardial infarction (STEMI), which significantly contribute to extracellular chromatin burden, and are associated with larger infarcts. To understand the correlation between neutrophil activation, extracellular chromatin, and infarct size (IS), we investigated these parameters in a porcine myocardial infarction model, and at different time points and sites in a prospective STEMI trial with cardiac magnetic resonance (CMR) endpoints. In a prospective STEMI trial (NCT01777750), 101 STEMI patients were included and blood samples were obtained from first medical contact until 6 months after primary percutaneous coronary intervention (pPCI) including direct sampling from the culprit site. CMR was performed 4 ± 2 days and 6 months after pPCI. Neutrophil counts, markers of extracellular chromatin, and inflammation were measured. Double-stranded deoxyribonucleic acid (dsDNA), citrullinated histone 3, nucleosomes, myeloperoxidase, neutrophil elastase, and interleukin (IL)-6 were significantly increased, while DNase activity was significantly decreased at the culprit site in STEMI patients. High neutrophil counts and dsDNA levels at the culprit site correlated with high microvascular obstruction (MVO) and low ejection fraction (EF). High DNase activity at the culprit site correlated with low MVO and high EF. In correspondence, dsDNA correlated with IS in the porcine myocardial infarction model. In porcine infarcts, neutrophils and extracellular chromatin were detected in congested small arteries corresponding with MVO. Markers of neutrophil activation, extracellular chromatin, DNase activity and CMR measurements correlated with markers of systemic inflammation C-reactive protein and IL-6 in patients. NETs and extracellular chromatin are important determinants of MVO in STEMI. Rapid degradation of extracellular chromatin by DNases appears to be crucial for microvascular patency and outcome.

Unusual finding of the human-adapted hypervirulent serotype III/ST17 clone in a historical bovine Group B Streptococcus isolate from Brazil.

Group B Streptococcus (GBS) is a leading cause of human neonatal infections and bovine mastitis. We report here the unusual finding of the human-adapted hypervirulent serotype III/ST17 clone in a bovine GBS isolated in 1987 in Brazil. This isolate shared several phenotypic and genotypic characteristics with serotype III/ST17 strains obtained from human sources, including PFGE pattern, pilus genes, lactose fermentation, DNase activity, and antimicrobial susceptibility profile, highlighting the importance of continued tracking of GBS in the One Health scope. The study brings new evidence for the potential interspecies transmission and sheds new light into evolution aspects of the pathogen Group B Streptococcus (GBS) by reporting the occurrence of an ancient bovine GBS isolate belonging to a variant currently known to be exclusively found in human hosts.

Mechanistic insights into the R-loop formation and cleavage in CRISPR-Cas12i1

Cas12i is a newly identified member of the functionally diverse type V CRISPR-Cas effectors. Although Cas12i has the potential to serve as genome-editing tool, its structural and functional characteristics need to be investigated in more detail before effective application. Here we report the crystal structures of the Cas12i1 R-loop complexes before and after target DNA cleavage to elucidate the mechanisms underlying target DNA duplex unwinding, R-loop formation and cis cleavage. The structure of the R-loop complex after target DNA cleavage also provides information regarding trans cleavage. Besides, we report a crystal structure of the Cas12i1 binary complex interacting with a pseudo target oligonucleotide, which mimics target interrogation. Upon target DNA duplex binding, the Cas12i1 PAM-interacting cleft undergoes a remarkable open-to-closed adjustment. Notably, a zipper motif in the Helical-I domain facilitates unzipping of the target DNA duplex. Formation of the 19-bp crRNA-target DNA strand heteroduplex in the R-loop complexes triggers a conformational rearrangement and unleashes the DNase activity. This study provides valuable insights for developing Cas12i1 into a reliable genome-editing tool.

The CRISPR-associated Cas4 protein from Leptospira interrogans demonstrate versatile nuclease activity.

The Cas4 protein is one of the core CRISPR-associated (Cas) proteins implicated in the adaptation module in many variants of the CRISPR-Cas system in prokaryotes against the invading genetic elements. Cas4 is recognized as a DNA exonuclease that contains a RecB nuclease domain and a Fe-S cluster-binding module. In Leptospira interrogans serovar Copenhageni strain Fiocruz L1-130, the cas4 gene is functionally transcribed as an active component of the CRISPR-Cas I-B system. Investigation of nuclease activity of Cas4 (LinCas4) of the L. interrogans illustrated divalent-metal cofactor (Mn2+ or Mg2+) dependent endonuclease activity on the DNA substrate. In agreement, mutation of the selective metal interacting residues (Asp74 and Glu87) curtails the DNA cleavage activity in LinCas4. Computational modeling shows metal-ion interacting residues (Asp74 and Glu87) in the LinCas4 to be a part of the RecB motifs II and III, the same as other Cas4 orthologs. The mutation of a potential DNA interacting residue in the LinCas4 (LinCas4Y132A) or one of the four cysteine residues (LinCas4C18A) involved in coordinating the 4Fe-4S cluster did not perturb its DNase activity. Iron chelation assay of the purified LinCas4 demonstrated it in the apostate conformation. Reconstitution of the Fe-S cluster in the LinCas4 under in vitro condition displayed its coordination with four iron atoms per LinCas4 monomer and was confirmed by the UV and CD spectroscopy studies.

Comparison of ultrafiltration and iron chloride flocculation in the preparation of aquatic viromes from contrasting sample types.

Viral metagenomes (viromes) are a valuable untargeted tool for studying viral diversity and the central roles viruses play in host disease, ecology, and evolution. Establishing effective methods to concentrate and purify viral genomes prior to sequencing is essential for high quality viromes. Using virus spike-and-recovery experiments, we stepwise compared two common approaches for virus concentration, ultrafiltration and iron chloride flocculation, across diverse matrices: wastewater influent, wastewater secondary effluent, river water, and seawater. Viral DNA was purified by removing cellular DNA via chloroform cell lysis, filtration, and enzymatic degradation of extra-viral DNA. We found that viral genomes were concentrated 1-2 orders of magnitude more with ultrafiltration than iron chloride flocculation for all matrices and resulted in higher quality DNA suitable for amplification-free and long-read sequencing. Given its widespread use and utility as an inexpensive field method for virome sampling, we nonetheless sought to optimize iron flocculation. We found viruses were best concentrated in seawater with five-fold higher iron concentrations than the standard used, inhibition of DNase activity reduced purification effectiveness, and five-fold more iron was needed to flocculate viruses from freshwater than seawater—critical knowledge for those seeking to apply this broadly used method to freshwater virome samples. Overall, our results demonstrated that ultrafiltration and purification performed better than iron chloride flocculation and purification in the tested matrices. Given that the method performance depended on the solids content and salinity of the samples, we suggest spike-and-recovery experiments be applied when concentrating and purifying sample types that diverge from those tested here.

Optimization of gamma-aminobutyric acid production by Lactobacillus brevis PML1 in dairy sludge-based culture medium through response surface methodology.

Gamma‐aminobutyric acid (GABA) is a pharmaceutical, bioactive amino acid that can produce by some species of Lactic Acid Bacteria (LAB). For the first time, we evaluated the production of GABA by Lactobacillus brevis PML1 in the medium that contain the contaminant food bio‐product like dairy sludge and soybean meal. GABA production was analyzed by chromatography (TLC, HPLC) and the features of fermented extract which contains this amino acid were evaluated. The results of Response Surface Methodology (RSM) of Central Composite Design (CCD) at p < .05 showed 300 ppm of GABA production in optimal treatment including 14.77% dairy sludge powder, 6.27% soybean meal, and 0.49% ammonium sulfate (32°C for 120 hr fermentation). The results of fermented extract also showed the acceptable antimicrobial, antioxidant, and toxicity (against cancer cell) properties. Also, L. brevis PML1has not shown any hemolytic or DNase activity which confirm its safety aspects. According to the results, this new culture can be used as a cheap substrate to biological production of GABA, by L. brevis PML1 in various food and pharmaceutical formulations.

Defective NET clearance contributes to sustained FXII activation in COVID-19-associated pulmonary thrombo-inflammation.

BackgroundCoagulopathy and inflammation are hallmarks of Coronavirus disease 2019 (COVID-19) and are associated with increased mortality. Clinical and experimental data have revealed a role for neutrophil extracellular traps (NETs) in COVID-19 disease. The mechanisms that drive thrombo-inflammation in COVID-19 are poorly understood.MethodsWe performed proteomic analysis and immunostaining of postmortem lung tissues from COVID-19 patients and patients with other lung pathologies. We further compared coagulation factor XII (FXII) and DNase activities in plasma samples from COVID-19 patients and healthy control donors and determined NET-induced FXII activation using a chromogenic substrate assay.FindingsFXII expression and activity were increased in the lung parenchyma, within the pulmonary vasculature and in fibrin-rich alveolar spaces of postmortem lung tissues from COVID-19 patients. In agreement with this, plasmaaac acafajföeFXII activation (FXIIa) was increased in samples from COVID-19 patients. Furthermore, FXIIa colocalized with NETs in COVID-19 lung tissue indicating that NETs accumulation leads to FXII contact activation in COVID-19. We further showed that an accumulation of NETs is partially due to impaired NET clearance by extracellular DNases as DNase substitution improved NET dissolution and reduced FXII activation in vitro.InterpretationCollectively, our study supports that the NET/FXII axis contributes to the pathogenic chain of procoagulant and proinflammatory responses in COVID-19. Targeting both NETs and FXIIa may offer a potential novel therapeutic strategy.FundingThis study was supported by the European Union (840189), the Werner Otto Medical Foundation Hamburg (8/95) and the German Research Foundation (FR4239/1-1, A11/SFB877, B08/SFB841 and P06/KFO306).

Acid‐Mediated Dissociation of Haemophilus ducreyi Cytolethal Distending Toxin Subunits Upon Cell Entry

Haemophilus ducreyi is a Gram-negative bacterium responsible for the sexually transmitted disease chancroid. It releases a heterotrimeric cytolethal distending toxin (CDT) that contains a catalytic subunit (CdtB) which has a DNAse activity and two binding components (CdtA and CdtC) responsible for entry into the cell. Our hypothesis predicts that the endocytosed holotoxin disassembles through two steps, where CdtA is dissociated from CdtB/CdtC subunits in the late endosomes due to the low pH of this compartment. Afterward, CdtC is released in the endoplasmic reticulum. This allows CdtB to enter the nucleus, causing DNA damage and cell cycle arrest. To study the mechanism of CdtA dissociation, each Cdt subunit was expressed in bacteria transformed with Cdt expression plasmids. Subunit purification was confirmed using SDS-PAGE with Coomassie stain. Afterward, circular dichroism studies tested the structural stabilities of each Cdt subunit at extracellular pH (7.4), early endosome pH (6.3), and late endosome pH (5.2). These structural studies showed that CdtB and CdtC do not undergo significant secondary structure changes at acidic pH. Acid-induces changes were observed with the CdtA subunit, causing its unfolding, aggregation and precipitation at pH 5.2. Additional studies assembled the holotoxin from the individual subunits and introduced it to different pH levels to evaluate the association and dissociation of the subunits using size exclusion chromatography. The results showed that the active holotoxin disassembles at a pH of 5.2, suggesting that the toxin dissociates in the acidified endosomal compartments due to the unfolding of CdtA. Knowledge of the CDT dissociation mechanism could be used to find treatments for chancroid that would prevent toxin function by blocking its disassembly.

The corruption of innate immunity by Streptococcus anginosus group

Abstract Streptococcus anginosus group (SAG), formerly known as Streptococcus milleri, consists of three distinct streptococcal species: Streptococcus anginosus, Streptococcus constellatus, and Streptococcus intermedius. SAG was considered as commensal bacteria of colon, oral cavity and vagina. Recent observation reported those bacteria as potent pathogens found in brain or liver abscess. Mechanism of SAG pathogenesis is still unknown, despite the strong set of clinical data. In our study, we determined the virulence of different SAG clinical isolates by examining the activity of hemolysins, DNAses and proteases. By using Dictyostelium discoideum as model for screening SAG virulence we selected a few high and low virulence strains out of 41 SAG isolates. Finally, we established and characterized the infection process of selected SAG strains by wax worm Galleria mellonella. Therefore, we aimed to characterize how those bacteria can interact with human innate immunity. In our experiments, using both human serum and whole blood we demonstrated, that these bacteria are resistant to killing by non-specific mechanisms of the immune response. Our studies have demonstrated that SAG have numerous virulence factors which differ among examined strains, and which allow bacteria to effectively colonize the human body and avoid elimination by the immune system. Supported by National Science Centre, Poland 2018/29/B/NZ6/00624