Alcohol use is a leading cause of chronic liver disease worldwide, and changes in the microbiome associated with alcohol use contribute to patients' risk for liver disease progression. Less is known about the effects of alcohol use on the intestinal viral microbiome (virome) and interactions between bacteriophages and their target bacteria. We studied changes in the intestinal virome of 62 clinically well-characterized patients with alcohol use disorder (AUD) during active alcohol use and after 2 weeks of alcohol abstinence, by extracting virus-like particles and performing metagenomic sequencing. We observed decreased abundance of Propionibacterium, Lactobacillus, and Leuconostoc phages in patients with active AUD when compared with controls, whereas after 2 weeks of alcohol abstinence, patients with AUD demonstrated an increase in the abundance of Propionibacterium, Lactobacillus, and Leuconostoc phages. The intestinal virome signature was also significantly different in patients with AUD with progressive liver disease, with increased abundance of phages targeting Enterobacteria and Lactococcus species phages compared with patients with AUD with nonprogressive liver disease. By performing moderation analyses, we found that progressive liver disease is associated with changes in interactions between some bacteriophages and their respective target bacteria. In summary, active alcohol use and alcohol-associated progressive liver disease are associated with changes in the fecal virome, some of which are partially reversible after a short period of abstinence. Progression of alcohol-associated liver disease is associated with changes in bacteriophage-bacteria interactions.

Platelet and fibrin clots occlude blood vessels in hemostasis and thrombosis. Here we report a noncanonical mechanism for vascular occlusion based on neutrophil extracellular traps (NETs), DNA fibers released by neutrophils during inflammation. We investigated which host factors control NETs in vivo and found that two deoxyribonucleases (DNases), DNase1 and DNase1-like 3, degraded NETs in circulation during sterile neutrophilia and septicemia. In the absence of both DNases, intravascular NETs formed clots that obstructed blood vessels and caused organ damage. Vascular occlusions in patients with severe bacterial infections were associated with a defect to degrade NETs ex vivo and the formation of intravascular NET clots. DNase1 and DNase1-like 3 are independently expressed and thus provide dual host protection against deleterious effects of intravascular NETs.

Jellyfish venoms are rich sources of toxins designed to capture prey or deter predators, but they can also elicit harmful effects in humans. In this study, an integrated transcriptomic and proteomic approach was used to identify putative toxins and their potential role in the venom of the scyphozoan jellyfish Chrysaora fuscescens. A de novo tentacle transcriptome, containing more than 23,000 contigs, was constructed and used in proteomic analysis of C. fuscescens venom to identify potential toxins. From a total of 163 proteins identified in the venom proteome, 27 were classified as putative toxins and grouped into six protein families: proteinases, venom allergens, C-type lectins, pore-forming toxins, glycoside hydrolases and enzyme inhibitors. Other putative toxins identified in the transcriptome, but not the proteome, included additional proteinases as well as lipases and deoxyribonucleases. Sequence analysis also revealed the presence of ShKT domains in two putative venom proteins from the proteome and an additional 15 from the transcriptome, suggesting potential ion channel blockade or modulatory activities. Comparison of these potential toxins to those from other cnidarians provided insight into their possible roles in C. fuscescens venom and an overview of the diversity of potential toxin families in cnidarian venoms.

Deoxyribonucleases (DNases) cleave extracellular DNA (ecDNA) and are under intense research as interventions for diseases associated with high ecDNA, such as acute live injury. DNase I treatment decreases morbidity and mortality in this animal model. Endogenous DNase activity has high interindividual variability. In this study, we tested the hypothesis that high endogenous DNase activity is beneficial in an animal model of acute liver failure. DNase activity was measured in the plasma of adult male mice taken before i.p. injection of thioacetamide to induce acute liver failure. The survival of mice was monitored for 48 h. Mice were retrospectively divided into two groups based on the median DNase activity assessed using the gel-based single-radial enzyme diffusion assay. In acute liver failure, mice with a higher baseline DNase activity had lower mortality after 48 h (by 25%). Different protection of ecDNA against nucleases by vesicles or DNA-binding proteins could play a role and should be further evaluated. Similarly, the role of endogenous DNase activity should be analyzed in other disease models associated with high ecDNA.

Macrophages are immune cells, capable to remodel the extracellular matrix, which can harbor extracellular DNA incorporated into neutrophil extracellular traps (NETs). To study the breakdown of NETs we studied the capability of macrophage subsets to degrade these structures in vitro and in vivo in a murine thrombosis model. Furthermore, we analyzed human abdominal aortic aneurysm samples in support of our in vitro and in vivo results. Approach and Results: Macrophages were seeded onto blood clots or isolated NETs and polarized. All macrophages were capable to degrade NETs. For initial breakdown, macrophages relied on extracellular deoxyribonucleases. Proinflammatory polarization enhanced NET degradation. The boost in degradation was because of increased macropinocytosis, as inhibition by imipramine diminished their NET breakdown. Inhibition of macropinocytosis in a murine thrombosis model led to increased NET burden and reduced thrombus resolution in vivo. When analyzing abdominal aortic aneurysm samples, macrophage density furthermore corresponded negatively with the amount of local NETs in the intraluminal thrombi as well as in the vessel wall, as increased macrophage density was associated with a reduction in NET burden.

Group A Streptococcus (GAS) secretes deoxyribonucleases and evades neutrophil extracellular killing by degrading neutrophil extracellular traps (NETs). However, limited information is currently available on the interaction between GAS and NETs in the pathogenicity of GAS pharyngitis. In this study, we modified a mouse model of GAS pharyngitis and revealed an essential role for DNase in this model. After intranasal infection, the nasal mucosa was markedly damaged near the nasal cavity, at which GAS was surrounded by neutrophils. When neutrophils were depleted from mice, GAS colonization and damage to the nasal mucosa were significantly decreased. Furthermore, mice infected with deoxyribonuclease knockout GAS mutants (∆spd, ∆endA, and ∆sdaD2) survived significantly better than those infected with wild-type GAS. In addition, the supernatants of digested NETs enhanced GAS-induced cell death in vitro. Collectively, these results indicate that NET degradation products may contribute to the establishment of pharyngeal infection caused by GAS.

Post-transcriptional control of mitochondrial gene expression, including the processing and generation of mature transcripts as well as their degradation, is a key regulatory step in gene expression in human mitochondria. Consequently, identification of the proteins responsible for RNA processing and degradation in this organelle is of great importance. The metallo-β-lactamase (MBL) is a candidate protein family that includes ribo- and deoxyribonucleases. In this study, we discovered a function for LACTB2, an orphan MBL protein found in mammalian mitochondria. Solving its crystal structure revealed almost perfect alignment of the MBL domain with CPSF73, as well as to other ribonucleases of the MBL superfamily. Recombinant human LACTB2 displayed robust endoribonuclease activity on ssRNA with a preference for cleavage after purine-pyrimidine sequences. Mutational analysis identified an extended RNA-binding site. Knockdown of LACTB2 in cultured cells caused a moderate but significant accumulation of many mitochondrial transcripts, and its overexpression led to the opposite effect. Furthermore, manipulation of LACTB2 expression resulted in cellular morphological deformation and cell death. Together, this study discovered that LACTB2 is an endoribonuclease that is involved in the turnover of mitochondrial RNA, and is essential for mitochondrial function in human cells.

The confluence of wireless technology and biosensors offers the possibility to detect and manage medical conditions outside of clinical settings. Wound infections represent a major clinical challenge in which timely detection is critical for effective interventions, but this is currently hindered by the lack of a monitoring technology that can interface with wounds, detect pathogenic bacteria, and wirelessly transmit data. Here, we report a flexible, wireless, and battery-free sensor that provides smartphone-based detection of wound infection using a bacteria-responsive DNA hydrogel. The engineered DNA hydrogels respond selectively to deoxyribonucleases associated with pathogenic bacteria through tunable dielectric changes, which can be wirelessly detected using near-field communication. In a mouse acute wound model, we demonstrate that the wireless sensor can detect physiologically relevant amounts of Staphylococcus aureus even before visible manifestation of infection. These results demonstrate strategies for continuous infection monitoring, which may facilitate improved management of surgical or chronic wounds.

Type II restriction endonucleases (REases) are deoxyribonucleases that cleave DNA sequences with remarkable specificity. Type II REases are highly divergent in sequence as well as in topology, i.e. the connectivity of secondary structure elements. A widely held assumption is that a structural core of five beta-strands flanked by two alpha-helices is common to these enzymes. We introduce a systematic procedure to enumerate secondary structure elements in an unambiguous and reproducible way, and use it to analyze the currently available X-ray structures of Type II REases. Based on this analysis, we propose an alternative definition of the core, which we term the alphabetaalpha-core. The alphabetaalpha-core includes the most frequently observed secondary structure elements and is not a sandwich, as it consists of a five-strand beta-sheet and two alpha-helices on the same face of the beta-sheet. We use the alphabetaalpha-core connectivity as a basis for grouping the Type II REases into distinct structural classes. In these new structural classes, the connectivity correlates with the angles between the secondary structure elements and with the cleavage patterns of the REases. We show that there exists a substructure of the alphabetaalpha-core, namely a common conserved core, ccc, defined here as one alpha-helix and four beta-strands common to all Type II REase of known structure.

Extracellular deoxyribonucleases (DNases) contribute to the spread of pathogenic bacteria through the evasion from host innate immunity. Our main objective was to evaluate the production of extracellular DNases by human and bovine Streptococcus agalactiae clinical strains and perform a correlation of genetic lineages and DNase activity with capsular type, genetic determinants, clinical origin (colonization and infection), and host (human or bovine). DNase activity was evaluated by qualitative and quantitative assays for a collection of 406 human (n = 285) and bovine (n = 121) strains. All (121/121) bovine were isolated from mastitis and revealed to be DNase (+), indicating a putative pathogenic role in this clinical scenario. From the human S. agalactiae strains, 86% (245/285) showed DNase activity, among which all strains belonging to capsular types, namely, Ia, Ib, III-2, and IV. All CC17 strains (n = 58) and 56/96 (58.3%) of the CC19 displayed DNase activity. DNase (-) strains belonged to the CC19 group. However, the subcharacterization of CC19 S. agalactiae strains through multiple-locus variable number tandem repeat analysis (MLVA), antibiotic resistance, mobile elements, and surface proteins did not provide any distinction among DNase producers and non-producers. The production of DNases by all human CC17 strains, about two-fifths of human CC19, and all bovine strains, suggest an important contribution of DNases to hypervirulence.

Streptococcus gordonii and Streptococcus sanguinis are pioneer colonizers of dental plaque and important agents of bacterial infective endocarditis (IE). To gain a greater understanding of these two closely related species, we performed comparative analyses on 14 new S. gordonii and 5 S. sanguinis strains using various bioinformatics approaches. We revealed S. gordonii and S. sanguinis harbor open pan-genomes and share generally high sequence homology and number of core genes including virulence genes. However, we observed subtle differences in genomic islands and prophages between the species. Comparative pathogenomics analysis identified S. sanguinis strains have genes encoding IgA proteases, mitogenic factor deoxyribonucleases, nickel/cobalt uptake and cobalamin biosynthesis. On the contrary, genomic islands of S. gordonii strains contain additional copies of comCDE quorum-sensing system components involved in genetic competence. Two distinct polysaccharide locus architectures were identified, one of which was exclusively present in S. gordonii strains. The first evidence of genes encoding the CylA and CylB system by the α-haemolytic S. gordonii is presented. This study provides new insights into the genetic distinctions between S. gordonii and S. sanguinis, which yields understanding of tooth surfaces colonization and contributions to dental plaque formation, as well as their potential roles in the pathogenesis of IE.

TatD enzymes are evolutionarily conserved deoxyribonucleases associated with DNA repair, apoptosis, development, and parasite virulence. Three TatD paralogs exist in humans, but their nuclease functions are unknown. Here, we describe the nuclease activities of two of the three human TatD paralogs, TATDN1 and TATDN3, which represent two phylogenetically distinct clades based on unique active site motifs. We found that in addition to 3'-5' exonuclease activity associated with other TatD proteins, both TATDN1 and TATDN3 exhibited apurinic/apyrimidinic (AP) endonuclease activity. The AP endonuclease activity was observed only in double-stranded DNA, whereas exonuclease activity was operative primarily in single-stranded DNA. Both nuclease activities were observed in the presence of Mg2+ or Mn2+, and we identified several divalent metal cofactors that inhibited exonuclease and supported AP endonuclease activity. Biochemical analysis and a crystal structure of TATDN1 bound to 2'-deoxyadenosine 5'-monophosphate in the active site are consistent with two-metal ion catalysis, and we identify several residues that differentiate nuclease activities in the two proteins. In addition, we show that the three Escherichia coli TatD paralogs are also AP endonucleases, indicating that this activity is conserved across evolution. Together, these results indicate that TatD enzymes constitute a family of ancient AP endonucleases.

Extracellular DNA (ecDNA) is DNA outside of cells, which is a result of various mechanisms. EcDNA is believed to be a cause of various pathogeneses as well as their potential biomarker. EcDNA is believed to also be part of small extracellular vesicles (sEVs) from cell cultures. If ecDNA is present in sEVs in plasma, their membrane may protect it from degradation by deoxyribonucleases. Moreover, sEVs play a role in the intercellular communication, and they can therefore transfer ecDNA between cells. The aim of this study was to investigate the presence of ecDNA in sEVs isolated from fresh human plasma by the ultracentrifugation and density gradient, which serves to exclude the co-isolation of non-sEVs compartments. The novelty of the current study is the investigation of the localization and subcellular origin of the ecDNA associated with sEVs in plasma, as well as the estimation of the approximate concentration. The cup-shaped sEVs were confirmed by transmission electron microscopy. The highest concentration of particles was in the size of 123 nm. The presence of the sEVs markers CD9 and TSG101 was confirmed by western blot. It was found that 60-75% of DNA is on the surface of sEVs, but a part of the DNA is localized inside the sEVs. Moreover, both nuclear and mitochondrial DNA were present in plasma EVs. Further studies should focus on the potential harmful autoimmune effect of DNA carried by plasma EVs or specifically sEVs.

The cytolethal distending toxin (CDT), Haemophilus ducreyi, is one of the bacterial toxins that have recently been considered for targeted therapies, especially in cancer therapies. CDT is an A-B2 exotoxin. Its catalytic subunit (CdtB) is capable of inducing DNA double strand breaks, cell cycle arrest and apoptosis in host eukaryotic cells. The sequence alignment indicates that the CdtB is structurally homologyr to phosphatases and deoxyribonucleases I (DNase I). Recently, it has been found that CdtB toxicity is mainly related to its nuclease activity. The immunogenicity of CDT can reduce its effectiveness in targeted therapies. However, the toxin can be very useful if its immunogenicity is significantly reduced. Detecting hotspot ectopic residues by computational servers and then mutating them to eliminate B-cell epitopes is a promising approach to reduce the immunogenicity of foreign protein-based therapeutics. By the mentioned method, in this study, we try to reduce the immunogenicity of the CdtB- protein sequence. This study initially screened residue of the CdtB is B-cell epitopes both linearly and conformationally. By overlapping the B-cell epitopes with the excluded conserve residues, and active and enzymatic sites, four residues were allowed to be mutated. There were two mutein options that show reduced antigenicity probability. Option one was N19F, G74I, and S161F with a VaxiJen score of 0.45 and the immune epitope database (IEDB) score of 1.80, and option two was N19F, G74I, and S161W with a VaxiJen score of 0.45 and IEDB score of 1.88. The 3D structure of the proposed sequences was evaluated and refined. The structural stability of native and mutant proteins was accessed through molecular dynamic simulation. The results showed that the mutations in the mutants caused no considerable changes in their structural stability. However, mutant 1 reveals more thermodynamic stability during the simulation. The applied approaches in this study can be used as rough guidelines for finding hot spot immunogen regions in the therapeutic proteins. Our results provide a new version of CdtB that, due to reduced immunogenicity and increased stability, can be used in toxin-based drugs such as immunotoxins.