Antibacterial T6SS effectors with a VRR-Nuc domain induce target cell death via DNA Double-Strand Breaks

The T6SS (Type VI secretion System) secretes antibacterial effectors into target competitors. Salmonella spp. encode five phylogenetically distinct T6SSs. Here we characterize the function of the SPI-22 T6SS of S. bongori, showing that it has antibacterial activity. We identify a group of antibacterial T6SS effectors (TseV1-4) containing an N-terminal PAAR-like domain and a C-terminal VRR-Nuc domain encoded next to cognate immunity proteins that contain the DUF3396 domain (TsiV1-4). TseV2 and TseV3 are toxic when expressed in Escherichia coli and bacterial competition assays confirm that TseV2 and TseV3 are secreted by the SPI-22 T6SS. Phylogenetic analysis reveals that TseV1-4 are evolutionarily related to enzymes involved in DNA repair. TseV2 and TseV3 maintained the ability to bind DNA, but instead cause specific DNA double-strand breaks and induce the SOS response in target cells. The crystal structure of the TseV3:TsiV3 complex reveals that the immunity protein likely blocks the effector interaction with the DNA substrate. These results expand our knowledge on the function of Salmonella pathogenicity islands, the evolution of toxins used in biological conflicts, and the endogenous mechanism regulating the activity of these toxins.

A dynamic antibacterial T6SS in Pantoea agglomerans pv. betae delivers a lysozyme-like effector to antagonize competitors

ABSTRACTThe type VI secretion system (T6SS) is deployed by numerous Gram-negative bacteria to deliver toxic effectors into neighboring cells. The genome of Pantoea agglomerans pv. betae (Pab) phytopathogenic bacteria contains a gene cluster (T6SS1) predicted to encode a complete T6SS. Using secretion and competition assays, we found that T6SS1 in Pab is a functional antibacterial system that allows this pathogen to outcompete rival plant-associated bacteria found in its natural environment. Computational analysis of the T6SS1 gene cluster revealed that antibacterial effector and immunity proteins are encoded within three dynamic genomic islands that harbor arrays of orphan immunity genes or toxin and immunity cassettes. Functional analysis demonstrated that the specialized antibacterial effector VgrG contains a C-terminal catalytically active glucosaminidase domain that is used to degrade prey peptidoglycan. Moreover, we confirmed that a bicistronic unit at the end of the T6SS1 cluster encodes a novel antibacterial T6SS effector and immunity pair. Together, these results demonstrate that Pab T6SS1 is an antibacterial system delivering a lysozyme-like effector to eliminate competitors, and indicate that this bacterium contains novel T6SS effectors.Significance StatementIn this work, we describe the identification of a Pantoea agglomerans T6SS as an antibacterial determinant used by this phytopathogen to outcompete bacterial rivals. Furthermore, we provide an in-depth analysis of the T6SS gene cluster and the putative effector and immunity genes that comprise it, and we propose explanations for its dynamic evolution and effector diversification in Pantoea strains. Lastly, we experimentally validate two predicted effector and immunity pairs, and we demonstrate that one is a potent lysozyme-like toxin.

Inhibition of the DOT1L/MPL/JAK2 Signaling Axis Selectively Targets TET2 Mutation-Driven Clonal Hematopoiesis

Clonal Hematopoiesis of Indeterminate Potential (CHIP) refers to the clonal expansion of hematopoietic stem and progenitor cells (HSPCs) carrying mutations associated with hematologic malignancies in individuals without evidence of a blood disorder. CHIP carriers are at a 10-fold higher risk of developing blood cancers relative to non-carriers. The time interval from acquisition of CHIP to overt neoplasia can span many years. This finding suggests a potential window of opportunity to intervene to prevent or delay malignant transformation; however, this goal is not yet possible due to the lack of known effective interventions. One of the most commonly mutated genes in CHIP is TET2, which encodes an epigenetic modifier involved in DNA demethylation. Tet2 knockout mouse models have an expansion of HSPCs and are at increased risk of development of myeloid malignancies, thus establishing TET2 mutation as a bona fide driver of pre-leukemia. To identify drugs that selectively target Tet2-deficient HSPCs, we generated isogenic murine cell lines by overexpressing HOXB4 in Sca-1 + HSPCs derived from a Tet2 knockout (Tet2-/-) mouse and a Tet2 wildtype (Tet2 +/+) littermate. HOXB4 overexpression has previously been shown to expand and immortalize HSPCs indefinitely without causing leukemogenesis. Using this isogenic cell line system termed HPC HOXB4, we conducted a drug screen of 37 small-molecule inhibitors of epigenetic signaling to find compounds that selectively reduced the proportion of Tet2-/- to Tet2+/+ HPC HOXB4 cells in competition assays. Through this screen, we discovered that DOT1L inhibitors, including SGC0946 and pinometostat, preferentially reduced the competitive advantage of Tet2 -/- HPC HOXB4 cells. Importantly, SGC0946 treatment also reduced the competitive advantage of unmodified Tet2 -/- murine HSPCs over wildtype cells in short-term competition assays. Mechanistic studies revealed that DOT1L inhibition induced higher levels of apoptosis in Tet2 -/- than in Tet2+/+ HPC HOXB4 cells and promoted the differentiation of Tet2-/- HPC HOXB4 cells. Genetic knockdown of Dot1l expression using shRNAs phenocopied the effects of pharmacologic DOT1L inhibition. DOT1L is a histone methyltransferase that catalyzes histone H3K79 methylation. Unexpectedly, we found that global H3K79me2 and H3K79me3 levels were higher in Tet2 -/- than in Tet2+/+ HPC HOXB4 cells. SGC0946 treatment effectively removed the H3K79me2/3 mark in Tet2 -/- cells. Next, we conducted RNA-seq analysis to gain insights into the transcriptomic changes in Tet2 -/- HPC HOXB4 after DOT1L inhibition. We found that expression of Mpl, which encodes the thrombopoietin receptor (TPOR), was over 20-fold higher in Tet2 -/- HPC HOXB4 than in wildtype cells at baseline and was strongly decreased after SGC0946 treatment. In contrast, SGC0946 had no effect on Mpl expression in Tet2 +/+ HPC HOXB4 cells. Mpl expression was also elevated in unmodified Tet2 -/- HSPCs compared with Tet2+/+ cells. Given that TPO signaling promotes HSC self-renewal and proliferation, we hypothesized that the effects of Dot1L inhibition are mediated through suppression of Mpl. Consistent with our hypothesis, enforced expression of Mpl in Tet2 -/- HPC HOXB4 cells completely rescued the effects of DOT1L inhibition, and downregulation of Mpl expression using shRNAs phenocopied the effects of DOT1L inhibition on Tet2 -/- HPC HOXB4 cells. Based on the findings above, we hypothesized that inhibition of TPOR signaling would selectively target Tet2 -/- over Tet2+/+ HSPCs. Given that Janus Kinase 2 (JAK2) is required to transduce signals downstream of the TPOR, we studied the effects of two potent JAK2 inhibitors, fedratinib and AZD1480, along with a JAK1/2 inhibitor, ruxolitinib, on Tet2 -/- and Tet2+/+ HPC HOXB4 cells in competition assays. In line with our hypothesis, all three compounds reduced the competitive advantage of Tet2 -/- HPC HOXB4 cells. Ruxolitinib also reduced the competitive advantage of unmodified Tet2 -/- HPSCs cells over wildtype cells in a dose-dependent manner. In summary, our data demonstrate that 1) the TPOR signaling pathway is upregulated in TET2-mutated HSPCs through epigenetic dysregulation by DOT1L, and 2) inhibition of DOT1L or the MPL/JAK2 signaling axis selectively targets TET2-mutated HSPCs. Our findings have important implications in the development of pharmacologic interventions against TET2-mutation driven CHIP. Disclosures Chan: AbbVie: Research Funding; BMS: Research Funding.

The origin and impeded dissemination of the DNA phosphorothioation system in prokaryotes

Phosphorothioate (PT) modification by the dnd gene cluster is the first identified DNA backbone modification and constitute an epigenetic system with multiple functions, including antioxidant ability, restriction modification, and virus resistance. Despite these advantages for hosting dnd systems, they are surprisingly distributed sporadically among contemporary prokaryotic genomes. To address this ecological paradox, we systematically investigate the occurrence and phylogeny of dnd systems, and they are suggested to have originated in ancient Cyanobacteria after the Great Oxygenation Event. Interestingly, the occurrence of dnd systems and prophages is significantly negatively correlated. Further, we experimentally confirm that PT modification activates the filamentous phage SW1 by altering the binding affinity of repressor and the transcription level of its encoding gene. Competition assays, concurrent epigenomic and transcriptomic sequencing subsequently show that PT modification affects the expression of a variety of metabolic genes, which reduces the competitive fitness of the marine bacterium Shewanella piezotolerans WP3. Our findings strongly suggest that a series of negative effects on microorganisms caused by dnd systems limit horizontal gene transfer, thus leading to their sporadic distribution. Overall, our study reveals putative evolutionary scenario of the dnd system and provides novel insights into the physiological and ecological influences of PT modification.

Overdominant and partially dominant mutations drive short-term adaptation in diploid yeast

Identification of adaptive targets in experimental evolution typically relies on extensive replication and allele reconstructions. An alternative approach is to directly assay all mutations in an evolved clone by generating pools of segregants that contain random combinations of the evolved mutations. Here, we apply this method to 6 clones isolated from 4 diploid populations that were clonally evolved for 2,000 generations in rich glucose medium. Each clone contains ~20-25 mutations relative to the ancestor. We derived intermediate genotypes between the founder and the evolved clones by bulk mating sporulated cultures of each evolved clone to a barcoded haploid version of the founder. We competed the barcoded segregants en masse and quantified the fitness of each barcode. We estimated average fitness effects of evolved mutations using barcode fitness and whole genome sequencing for a subset of segregants or time-course whole population whole genome sequencing. In contrast to our previous work in haploid populations, we find that diploids carry fewer evolved mutations with a detectable fitness effect (6%), contributing a modest fitness advantage (up to 5.4%). In agreement with theoretical expectations, reconstruction experiments show that all adaptive mutations manifest some degree of dominance over the ancestral allele, and most are overdominant. Competition assays under conditions that deviated from the evolutionary environment show that adaptive mutations are often pleiotropic.

RadA, a MSCRAMM Adhesin of the Dominant Symbiote Ruminococcus gnavus E1, Binds Human Immunoglobulins and Intestinal Mucins

Adhesion to the digestive mucosa is considered a key factor for bacterial persistence within the gut. In this study, we show that Ruminococcus gnavus E1 can express the radA gene, which encodes an adhesin of the MSCRAMMs family, only when it colonizes the gut. The RadA N-terminal region contains an all-β bacterial Ig-like domain known to interact with collagens. We observed that it preferentially binds human immunoglobulins (IgA and IgG) and intestinal mucins. Using deglycosylated substrates, we also showed that the RadA N-terminal region recognizes two different types of motifs, the protein backbone of human IgG and the glycan structure of mucins. Finally, competition assays with lectins and free monosaccharides identified Galactose and N-Acetyl-Galactosamine motifs as specific targets for the binding of RadA to mucins and the surface of human epithelial cells.

Structure of Venezuelan equine encephalitis virus in complex with the LDLRAD3 receptor

LDLRAD3 is a recently defined attachment and entry receptor for Venezuelan equine encephalitis virus (VEEV)1, a New World alphavirus that causes severe neurological disease in humans. Here we present near-atomic-resolution cryo-electron microscopy reconstructions of VEEV virus-like particles alone and in a complex with the ectodomains of LDLRAD3. Domain 1 of LDLRAD3 is a low-density lipoprotein receptor type-A module that binds to VEEV by wedging into a cleft created by two adjacent E2–E1 heterodimers in one trimeric spike, and engages domains A and B of E2 and the fusion loop in E1. Atomic modelling of this interface is supported by mutagenesis and anti-VEEV antibody binding competition assays. Notably, VEEV engages LDLRAD3 in a manner that is similar to the way that arthritogenic alphaviruses bind to the structurally unrelated MXRA8 receptor, but with a much smaller interface. These studies further elucidate the structural basis of alphavirus–receptor interactions, which could inform the development of therapies to mitigate infection and disease against multiple members of this family.

A chemical probe based on the PreQ1 metabolite enables transcriptome-wide mapping of binding sites

The role of metabolite-responsive riboswitches in regulating gene expression in bacteria is well known and makes them useful systems for the study of RNA-small molecule interactions. Here, we study the PreQ1 riboswitch system, assessing sixteen diverse PreQ1-derived probes for their ability to selectively modify the class-I PreQ1 riboswitch aptamer covalently. For the most active probe (11), a diazirine-based photocrosslinking analog of PreQ1, X-ray crystallography and gel-based competition assays demonstrated the mode of binding of the ligand to the aptamer, and functional assays demonstrated that the probe retains activity against the full riboswitch. Transcriptome-wide mapping using Chem-CLIP revealed a highly selective interaction between the bacterial aptamer and the probe. In addition, a small number of RNA targets in endogenous human transcripts were found to bind specifically to 11, providing evidence for candidate PreQ1 aptamers in human RNA. This work demonstrates a stark influence of linker chemistry and structure on the ability of molecules to crosslink RNA, reveals that the PreQ1 aptamer/ligand pair are broadly useful for chemical biology applications, and provides insights into how PreQ1, which is similar in structure to guanine, interacts with human RNAs.

PixR, a novel activator of conjugative transfer of IncX4 resistance plasmids, mitigates the fitness cost of mcr-1 carriage in Escherichia coli

The emergence of the plasmid-borne colistin resistance gene mcr-1 threats public health. IncX4-type plasmids are one of the most epidemiologically successful vehicles for spreading mcr-1 worldwide. Since MCR-1 is known for imposing a fitness cost to its host bacterium, the successful spread of mcr-1-bearing plasmids might be linked to high conjugation frequency, which would enhance the maintenance of the plasmid in the host without antibiotic selection. However, the mechanism of IncX4 plasmids conjugation remains unclear. In this study, we used high-density transposon mutagenesis to identify factors required for IncX4 plasmid transfer and 18 genes were identified, including five with annotations unrelated to conjugation. The Cappable-seq and RNA-seq analysis confirmed that a novel transcriptional regulator gene, pixR, directly regulates the transfer of IncX4 plasmids by binding the promoter of 13 essential transfer genes to increase their transcription. Plasmid invasion and co-culture competition assays revealed that pixR is essential for the spread and persistence of mcr-1>-bearing IncX4 plasmids in bacterial populations, and effective conjugation is crucial for alleviating the fitness cost exerted by mcr-1 carriage. The existence of the IncX4-specific pixR gene increases plasmid transmissibility while promoting the invasion and persistence of mcr-1-bearing plasmids in bacterial populations, which helps explain their global prevalence.