The SMC-family Wadjet complex protects bacteria from plasmid transformation by recognition and cleavage of closed-circular DNA.

Self versus non-self discrimination is a key element of innate and adaptive immunity across life. In bacteria, CRISPR-Cas and restriction-modification systems recognize non-self nucleic acids through their sequence and their methylation state, respectively. Here, we show that the Wadjet defense system recognizes DNA topology to protect its host against plasmid transformation. By combining cryoelectron microscopy with cross-linking mass spectrometry, we show that Wadjet forms a complex similar to the bacterial condensin complex MukBEF, with a novel nuclease subunit similar to a type II DNA topoisomerase. Wadjet specifically cleaves closed-circular DNA in a reaction requiring ATP hydrolysis by the structural maintenance of chromosome (SMC) ATPase subunit JetC, suggesting that the complex could use DNA loop extrusion to sense its substrate's topology, then specifically activate the nuclease subunit JetD to cleave plasmid DNA. Overall, our data reveal how bacteria have co-opted a DNA maintenance machine to specifically recognize and destroy foreign DNAs through topology sensing.

Architecture and self-assembly of the jumbo bacteriophage nuclear shell.

Bacteria encode myriad defences that target the genomes of infecting bacteriophage, including restriction-modification and CRISPR-Cas systems1. In response, one family of large bacteriophages uses a nucleus-like compartment to protect its replicating genomes by excluding host defence factors2-4. However, the principal composition and structure of this compartment remain unknown. Here we find that the bacteriophage nuclear shell assembles primarily from one protein, which we name chimallin (ChmA). Combining cryo-electron tomography of nuclear shells in bacteriophage-infected cells and cryo-electron microscopy of a minimal chimallin compartment in vitro, we show that chimallin self-assembles as a flexible sheet into closed micrometre-scale compartments. The architecture and assembly dynamics of the chimallin shell suggest mechanisms for its nucleation and growth, and its role as a scaffold for phage-encoded factors mediating macromolecular transport, cytoskeletal interactions, and viral maturation.

A phage nucleus-associated RNA-binding protein is required for jumbo phage infection.

Large-genome bacteriophages (jumbo phages) of the proposed family Chimalliviridae assemble a nucleus-like compartment bounded by a protein shell that protects the replicating phage genome from host-encoded restriction enzymes and DNA-targeting CRISPR-Cas nucleases. While the nuclear shell provides broad protection against host nucleases, it necessitates transport of mRNA out of the nucleus-like compartment for translation by host ribosomes, and transport of specific proteins into the nucleus-like compartment to support DNA replication and mRNA transcription. Here, we identify a conserved phage nuclear shell-associated protein that we term Chimallin C (ChmC), which adopts a nucleic acid-binding fold, binds RNA with high affinity in vitro, and binds phage mRNAs in infected cells. ChmC also forms phase-separated condensates with RNA in vitro. Targeted knockdown of ChmC using mRNA-targeting dCas13d results in accumulation of phage-encoded mRNAs in the phage nucleus, reduces phage protein production, and compromises virion assembly. Taken together, our data show that the conserved ChmC protein plays crucial roles in the viral life cycle, potentially by facilitating phage mRNA translocation through the nuclear shell to promote protein production and virion development.

The metabolic fingerprint of chronic hepatitis C progression: Metabolome shifts and cutting-edge diagnostic options.

Hepatitis C virus infection causes chronic diseases such as cirrhosis and hepatocellular carcinoma. Metabolomics research has been shown to be linked to pathophysiologic pathways in liver illnesses. The aim of this study was to investigate the serum metabolic profile of patients with chronic hepatitis C (CHC) infection and to identify underlying mechanisms as well as potential biomarkers associated with the disease. Nuclear magnetic resonance (NMR) was used to evaluate the sera of 83 patients with CHC virus and 52 healthy control volunteers (NMR). Then, multivariate statistical analysis was used to find distinguishing metabolites between the two groups. Sixteen out of 40 metabolites including include 3-HB, betaine, carnitine, creatinine, fucose, glutamine, glycerol, isopropanol, lysine, mannose, methanol, methionine, ornithine, proline, serine, and valine-were shown to be significantly different between the CHC and normal control (NC) groups (variable importance in projection >1 and p < 0.05). All the metabolic perturbations in this disease are associated with pathways of Glycine, serine, and threonine metabolism, glycerolipid metabolism, arginine and proline metabolism, aminoacyl-tRNA biosynthesis, cysteine and methionine metabolism, alanine, aspartate, and glutamate metabolism. Multivariate statistical analysis constructed using these expressed metabolites showed CHC patients can be discriminated from NCs with high sensitivity (90%) and specificity (99%). The metabolomics approach may expand the diagnostic armamentarium for patients with CHC while contributing to a comprehensive understanding of disease mechanisms.

Aerosols optical depth and Ångström exponent over different regions in Garhwal Himalaya, India.

Aerosol optical depth (AOD) and Ångström exponent (AE) are observed to be important parameters in understanding the status of ambient aerosol concentration over a particular location and depend not only upon the local but also on the large-scale dynamics of the atmosphere. The present article analyses the AOD and AE parameters retrieved with Moderate Resolution Imaging Spectrometer (MODIS) and Multi-angle Imaging Spectro-Radiometer (MISR) instruments onboard satellites, for the upper (Chamoli) and foothill (Dehradun) regions of Garhwal Himalaya in Uttarakhand, India, from 2006 to 2015. Aerosol properties are investigated at monthly, seasonal, and annual scales. The monthly mean values of MODIS-derived AOD and AE were observed to be 0.18 (± 0.14) and 1.05 (± 0.43) respectively over the Dehradun region. The seasonal maximums in AOD with MODIS and MISR were observed as 0.23 ± 0.06 and 0.29 ± 0.07 respectively in the pre-monsoon season, and the minimum values (0.099 ± 0.02) were observed in the post-monsoon season, over the Dehradun region. In contrast, in the Chamoli region, the maximum AOD (MODIS) was 0.21 ± 0.06 observed in the monsoon season and the minimum was 0.036 ± 0.007 in the post-monsoon season. Over a decade, the AE for Chamoli and Dehradun was found to vary from 0.07 to 0.17 and from 0.14 to 0.20 respectively. The median AE for Chamoli and Dehradun was found to be 1.49 and 1.47 respectively, marking the dominance of fine mode particles of anthropogenic origin. Observations show the presence of dust and polluted dust resulting from the long-range transport from the west. The comparison of AOD values from the two sensors shows a significant correlation (0.73) with slightly higher values from MISR over the year. The results obtained are important in understanding the climatic implications due to the atmospheric aerosols over the abovementioned Himalayan region of Uttarakhand, India.

Structural, functional and biological insights into the role of Mycobacterium tuberculosis VapBC11 toxin-antitoxin system: targeting a tRNase to tackle mycobacterial adaptation.

Toxin-antitoxin (TA) systems are involved in diverse physiological processes in prokaryotes, but their exact role in Mycobacterium tuberculosis (Mtb) virulence and in vivo stress adaptation has not been extensively studied. Here, we demonstrate that the VapBC11 TA module is essential for Mtb to establish infection in guinea pigs. RNA-sequencing revealed that overexpression of VapC11 toxin results in metabolic slowdown, suggesting that modulation of the growth rate is an essential strategy for in vivo survival. Interestingly, overexpression of VapC11 resulted in the upregulation of chromosomal TA genes, suggesting the existence of highly coordinated crosstalk among TA systems. In this study, we also present the crystal structure of the VapBC11 heterooctameric complex at 1.67 Å resolution. Binding kinetic studies suggest that the binding affinities of toxin-substrate and toxin-antitoxin interactions are comparable. We used a combination of structural studies, molecular docking, mutational analysis and in vitro ribonuclease assays to enhance our understanding of the mode of substrate recognition by the VapC11 toxin. Furthermore, we have also designed peptide-based inhibitors to target VapC11 ribonuclease activity. Taken together, we propose that the structure-guided design of inhibitors against in vivo essential ribonucleases might be a novel strategy to hasten clearance of intracellular Mtb.

System-Wide Analysis Unravels the Differential Regulation and In Vivo Essentiality of Virulence-Associated Proteins B and C Toxin-Antitoxin Systems of Mycobacterium tuberculosis.

Toxin-antitoxin (TA) systems are bicistronic genetic modules that are ubiquitously present in bacterial genomes. The Mycobacterium tuberculosis genome encodes 90 putative TA systems, and these are considered to be associated with maintenance of bacterial genomic stability or bacterial survival under unfavorable environmental conditions. The majority of these in M. tuberculosis have been annotated as belonging to the virulence-associated protein B and C (VapBC) family. However, their precise role in bacterial physiology has not been elucidated. Here, we functionally characterized VapC toxins from M. tuberculosis and show that overexpression of some homologs inhibits growth of Mycobacterium bovis bacillus Calmette-Guérin in a bacteriostatic manner. Expression profiling of messenger RNA revealed that these VapC toxins were differentially induced upon exposure of M. tuberculosis to stress conditions. We also unraveled that transcriptional cross-activation exists between TA systems in M. tuberculosis. This study provides the first evidence for the essentiality of VapBC3 and VapBC4 systems in M. tuberculosis virulence.

Calcimycin mediates mycobacterial killing by inducing intracellular calcium-regulated autophagy in a P2RX7 dependent manner.

Phenotypic screening led to the identification of calcimycin as a potent inhibitor of Mycobacterium bovis BCG (M. bovis BCG) growth in vitro and in THP-1 cells. In the present study, we aim to decipher the mechanism of antimycobacterial activity of calcimycin. We noticed that treatment with calcimycin led to up-regulation of different autophagy markers like Beclin-1, autophagy-related gene (Atg) 7, Atg 3 and enhanced microtubule-associated protein 1A/1B-light chain 3-I (LC3-I) to LC3-II conversion in macrophages. This calcimycin-mediated killing of intracellular M. smegmatis and M. bovis BCG was abrogated in the presence of 3-methyladenine (3-MA). We also demonstrate that calcimycin binding with purinergic receptor P2X7 (P2RX7) led to increase in intracellular calcium level that regulates the extracellular release of ATP. ATP was able to regulate calcimycin-induced autophagy through P2RX7 in an autocrine fashion. Blocking of either P2RX7 expression by 1-[N,O-bis(5-Isoquinolinesulfonyl)-N-methyl-l-tyrosyl]-4-phenylpiperazine (KN-62) or reducing intracellular calcium levels by 1,2-Bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra (acetoxy-methyl) ester (BAPTA-AM) abrogated the antimycobacterial activity of calcimycin. Taken together, these results showed that calcimycin exerts its antimycobacterial effect by regulating intracellular calcium-dependent ATP release that induces autophagy in a P2RX7 dependent manner.

Architecture and infection-sensing mechanism of the bacterial PARIS defense system.

Bacteria and the viruses that infect them (bacteriophages or phages) are engaged in an evolutionary arms race that has resulted in the development of hundreds of bacterial defense systems and myriad phage-encoded counterdefenses1-5. While the mechanisms of many bacterial defense systems are known1, how these systems avoid toxicity outside infection yet activate quickly upon sensing phage infection is less well understood. Here, we show that the bacterial Phage Anti-Restriction-Induced System (PARIS) operates as a toxin-antitoxin system, in which the antitoxin AriA sequesters and inactivates the toxin AriB until triggered by the T7 phage counterdefense protein Ocr. Using cryoelectron microscopy (cryoEM), we show that AriA is structurally similar to dimeric SMC-family ATPases but assembles into a distinctive homohexameric complex through two distinct oligomerization interfaces. In the absence of infection, the AriA hexamer binds up to three monomers of AriB, maintaining them in an inactive state. Ocr binding to the AriA-AriB complex triggers rearrangement of the AriA hexamer, releasing AriB and allowing it to dimerize and activate. AriB is a toprim/OLD-family nuclease whose activation arrests cell growth and inhibits phage propagation by globally inhibiting protein translation. Collectively, our findings reveal the intricate molecular mechanisms of a bacterial defense system that evolved in response to a phage counterdefense protein, and highlight how an SMC-family ATPase has been adapted as a bacterial infection sensor.

Phosphorylation regulates tau's phase separation behavior and interactions with chromatin.

Tau is a microtubule-associated protein often found in neurofibrillary tangles (NFTs) in the brains of patients with Alzheimer's disease. Beyond this context, mounting evidence suggests that tau localizes into the nucleus, where it may play a role in DNA protection and heterochromatin regulation. The molecular mechanisms behind these observations are currently unclear. Using in vitro biophysical experiments, here we demonstrate that tau can undergo liquid-liquid phase separation (LLPS) with DNA, mononucleosomes, and reconstituted nucleosome arrays under low salt conditions. Low concentrations of tau promote chromatin compaction and protect DNA from digestion. While the material state of samples at physiological salt is dominated by chromatin oligomerization, tau can still associate strongly and reversibly with nucleosome arrays. These properties are driven by tau's strong interactions with linker and nucleosomal DNA. In addition, tau co-localizes into droplets formed by nucleosome arrays and phosphorylated HP1α, a key heterochromatin constituent thought to function through an LLPS mechanism. Importantly, LLPS and chromatin interactions are disrupted by aberrant tau hyperphosphorylation. These biophysical properties suggest that tau may directly impact DNA and chromatin accessibility and that loss of these interactions could contribute to the aberrant nuclear effects seen in tau pathology.

Loop extrusion-mediated plasmid DNA cleavage by the bacterial SMC Wadjet complex.

Structural maintenance of chromosomes (SMC) protein complexes play pivotal roles in genome organization and maintenance across all domains of life. In prokaryotes, SMC family Wadjet complexes structurally resemble the widespread MukBEF genome-organizing complexes but serve a defensive role by inhibiting plasmid transformation. We previously showed that Wadjet specifically cleaves circular DNA; however, the molecular mechanism underlying DNA substrate recognition remains unclear. Here, we use in vitro single-molecule imaging to directly visualize DNA loop extrusion and plasmid cleavage by Wadjet. We find that Wadjet is a symmetric DNA loop extruder that simultaneously reels in DNA from both sides of a growing loop and that this activity requires a dimeric JetABC supercomplex containing two dimers of the JetC motor subunit. On surface-anchored plasmid DNAs, Wadjet extrudes the full length of a 44 kilobase pair plasmid, stalls, and then cleaves DNA. Our findings reveal the role of loop extrusion in the specific recognition and elimination of plasmids by Wadjet, and establish loop extrusion as an evolutionarily conserved mechanism among SMC complexes across kingdoms of life.

Phospho-KNL-1 recognition by a TPR domain targets the BUB-1-BUB-3 complex to C. elegans kinetochores.

During mitosis, the Bub1-Bub3 complex concentrates at kinetochores, the microtubule-coupling interfaces on chromosomes, where it contributes to spindle checkpoint activation, kinetochore-spindle microtubule interactions, and protection of centromeric cohesion. Bub1 has a conserved N-terminal tetratricopeptide (TPR) domain followed by a binding motif for its conserved interactor Bub3. The current model for Bub1-Bub3 localization to kinetochores is that Bub3, along with its bound motif from Bub1, recognizes phosphorylated "MELT" motifs in the kinetochore scaffold protein Knl1. Motivated by the greater phenotypic severity of BUB-1 versus BUB-3 loss in C. elegans, we show that the BUB-1 TPR domain directly recognizes a distinct class of phosphorylated motifs in KNL-1 and that this interaction is essential for BUB-1-BUB-3 localization and function. BUB-3 recognition of phospho-MELT motifs additively contributes to drive super-stoichiometric accumulation of BUB-1-BUB-3 on its KNL-1 scaffold during mitotic entry. Bub1's TPR domain interacts with Knl1 in other species, suggesting that collaboration of TPR-dependent and Bub3-dependent interfaces in Bub1-Bub3 localization and functions may be conserved.

Phospho-KNL-1 recognition by a TPR domain targets the BUB-1-BUB-3 complex to C. elegans kinetochores.

During mitosis, the Bub1-Bub3 complex concentrates at kinetochores, the microtubule-coupling interfaces on chromosomes, where it contributes to spindle checkpoint activation, kinetochore-spindle microtubule interactions, and protection of centromeric cohesion. Bub1 has a conserved N-terminal tetratricopeptide repeat (TPR) domain followed by a binding motif for its conserved interactor Bub3. The current model for Bub1-Bub3 localization to kinetochores is that Bub3, along with its bound motif from Bub1, recognizes phosphorylated "MELT" motifs in the kinetochore scaffold protein Knl1. Motivated by the greater phenotypic severity of BUB-1 versus BUB-3 loss in C. elegans, we show that the BUB-1 TPR domain directly recognizes a distinct class of phosphorylated motifs in KNL-1 and that this interaction is essential for BUB-1-BUB-3 localization and function. BUB-3 recognition of phospho-MELT motifs additively contributes to drive super-stoichiometric accumulation of BUB-1-BUB-3 on its KNL-1 scaffold during mitotic entry. Bub1's TPR domain interacts with Knl1 in other species, suggesting that collaboration of TPR-dependent and Bub3-dependent interfaces in Bub1-Bub3 localization and functions may be conserved.

Eight versus twelve weeks of sofosbuvir-velpatasvir in treatment-naïve non-cirrhotic patients with chronic hepatitis C virus infection: Study protocol for a multicentric, open labelled, randomized, non-inferiority trial (RESOLVE trial).

BACKGROUND: Hepatitis C virus (HCV) is a common cause of liver cirrhosis and hepatocellular carcinoma. Globally, nearly 71 million people have chronic HCV infection, and approximately 399,000 dies annually. In patients without cirrhosis, HCV infection is treated with 12 weeks of sofosbuvir/velpatasvir combination. Results from available small, single-centre observational studies suggest that the sofosbuvir/velpatasvir combination given for 8 weeks may be as effective as the standard 12 weeks of treatment. We propose to compare the treatment response of 12 weeks versus 8 weeks of sofosbuvir/velpatasvir in non-cirrhotic people with chronic HCV infection. METHODS: This multicentric, randomized, open-label, non-inferiority trial will include 880 (2 arms x 440) treatment naïve, viraemic (HCV RNA >10,000 IU/mL), non-cirrhotic adults (age >18 years) with chronic hepatitis C. People who are at high-risk for HCV reinfection such as haemophiliacs, people who inject drugs, those on maintenance hemodialysis or having HIV will be excluded. The presence or absence of cirrhosis will be determined with a combination of history, examination, ultrasound, liver stiffness measured with transient elastography, APRI, FIB-4, and esophagogastroduodenoscopy. Participants will be randomized to receive either 8- or 12-week sofosbuvir/velpatasvir treatment. A blood specimen will be collected before starting the treatment (to determine the HCV genotype), after 4 weeks of treatment (for early virological response), and at 12 weeks after treatment discontinuation for SVR12. DISCUSSION: The study will provide data on the efficacy of 8 weeks of treatment as compared to the standard of care (12 weeks) in non-cirrhotic patients with chronic HCV infection. Treatment for a shorter duration may improve treatment compliance, reduce the cost of treatment, and ease the treatment implementation from a public health perspective. TRIAL REGISTRATION: Registered with Clinical Trial Registry of India (http://ctri.nic.in) Registration No. CTRI/2022/03/041368 [Registered on: 24/03/2022]-Trial Registered Prospectively.

Structural insights into DarT toxin neutralization by cognate DarG antitoxin: ssDNA mimicry by DarG C-terminal domain keeps the DarT toxin inhibited.

In the DarTG toxin-antitoxin system, the DarT toxin ADP-ribosylates single-stranded DNA (ssDNA), which stalls DNA replication and plays a crucial role in controlling bacterial growth and bacteriophage infection. This toxic activity is reversed by the N-terminal macrodomain of the cognate antitoxin DarG. DarG also binds DarT, but the role of these interactions in DarT neutralization is unknown. Here, we report that the C-terminal domain of DarG (DarG toxin-binding domain [DarGTBD]) interacts with DarT to form a 1:1 stoichiometric heterodimeric complex. We determined the 2.2 Å resolution crystal structure of the Mycobacterium tuberculosis DarT-DarGTBD complex. The comparative structural analysis reveals that DarGTBD interacts with DarT at the DarT/ssDNA interaction interface, thus sterically occluding substrate ssDNA binding and consequently inactivating toxin by direct protein-protein interactions. Our data support a unique two-layered DarT toxin neutralization mechanism of DarG, which is important in keeping the toxin molecules in check under normal growth conditions.

Bacterial Shedu immune nucleases share a common enzymatic core regulated by diverse sensor domains.

Prokaryotes encode diverse anti-bacteriophage immune systems, including the single-protein Shedu nuclease. Here we reveal the structural basis for activation of Bacillus cereus Shedu. In the inactive homotetramer, a key catalytic residue in Shedu's nuclease domain is sequestered away from the catalytic site. Activation involves a conformational change that completes the active site and promotes assembly of a homo-octamer for coordinated double-strand DNA cleavage. Removal of Shedu's N-terminal domain ectopically activates the enzyme, suggesting that this domain allosterically inhibits Shedu in the absence of infection. Bioinformatic analysis of nearly 8,000 Shedu homologs reveals remarkable diversity in their N-terminal regulatory domains: we identify 79 domain families falling into eight functional classes, including diverse nucleic acid binding, enzymatic, and other domains. Together, these data reveal Shedu as a broad family of immune nucleases with a common nuclease core regulated by diverse N-terminal domains that likely respond to a range of infection-related signals.

Phosphorylation regulates tau's phase separation behavior and interactions with chromatin.

Tau is a microtubule-associated protein often found in neurofibrillary tangles (NFTs) in the brains of patients with Alzheimer's disease (AD). Beyond this context, mounting evidence suggests that tau localizes into the nucleus, where it may play a role in DNA protection and heterochromatin regulation. Models of tau depletion or pathology show loss of genetically silent heterochromatin, aberrant expression of heterochromatic genes, and transposable element activation. The molecular mechanisms behind these observations are currently unclear. Using in vitro biophysical experiments, here we demonstrate that tau can undergo liquid-liquid phase separation (LLPS) with DNA, mononucleosomes, and reconstituted nucleosome arrays under low salt conditions. Low concentrations of tau promote chromatin compaction and protect DNA from digestion. While the material state of samples at physiological salt is dominated by chromatin oligomerization, tau can still associate strongly and reversibly with nucleosome arrays. These properties are driven by tau's strong interactions with linker and nucleosomal DNA, while magic angle spinning (MAS) solid-state NMR experiments show that tau binding does not drastically alter nucleosome structure and dynamics. In addition, tau co-localizes into droplets formed by nucleosome arrays and phosphorylated HP1α, a key heterochromatin constituent thought to function through an LLPS mechanism. Importantly, LLPS and chromatin interactions are disrupted by aberrant tau hyperphosphorylation. These biophysical properties suggest that tau may directly impact DNA and chromatin accessibility and that loss of these interactions could contribute to the aberrant nuclear effects seen in tau pathology.

Identification of the bacteriophage nucleus protein interaction network.

In the arms race between bacteria and bacteriophages (phages), some large-genome jumbo phages have evolved a protein shell that encloses their replicating genome to protect it against host immune factors. By segregating the genome from the host cytoplasm, however, the 'phage nucleus' introduces the need to specifically translocate messenger RNA and proteins through the nuclear shell and to dock capsids on the shell for genome packaging. Here, we use proximity labeling and localization mapping to systematically identify proteins associated with the major nuclear shell protein chimallin (ChmA) and other distinctive structures assembled by these phages. We identify six uncharacterized nuclear-shell-associated proteins, one of which directly interacts with self-assembled ChmA. The structure and protein-protein interaction network of this protein, which we term ChmB, suggest that it forms pores in the ChmA lattice that serve as docking sites for capsid genome packaging and may also participate in messenger RNA and/or protein translocation.