Bacterial contact-dependent growth inhibition (CDI) is mediated by the CdiB/CdiA family of two-partner secretion proteins. CDI systems deploy a variety of distinct toxins, which are contained within ...the polymorphic C-terminal region (CdiA-CT) of CdiA proteins. Several CdiA-CTs are nucleases, suggesting that the toxins are transported into the target cell cytoplasm to interact with their substrates. To analyze CdiA transfer to target bacteria, we used the CDI system of uropathogenic Escherichia coli 536 (UPEC536) as a model. Antibodies recognizing the amino- and carboxyl-termini of CdiA(UPEC536) were used to visualize transfer of CdiA from CDI(UPEC536+) inhibitor cells to target cells using fluorescence microscopy. The results indicate that the entire CdiA(UPEC536) protein is deposited onto the surface of target bacteria. CdiA(UPEC536) transfer to bamA101 mutants is reduced, consistent with low expression of the CDI receptor BamA on these cells. Notably, our results indicate that the C-terminal CdiA-CT toxin region of CdiA(UPEC536) is translocated into target cells, but the N-terminal region remains at the cell surface based on protease sensitivity. These results suggest that the CdiA-CT toxin domain is cleaved from CdiA(UPEC536) prior to translocation. Delivery of a heterologous Dickeya dadantii CdiA-CT toxin, which has DNase activity, was also visualized. Following incubation with CDI(+) inhibitor cells targets became anucleate, showing that the D.dadantii CdiA-CT was delivered intracellularly. Together, these results demonstrate that diverse CDI toxins are efficiently translocated across target cell envelopes.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The legume symbiont Sinorhizobium meliloti is chemoattracted to compounds exuded by germinating seeds of its host alfalfa. This response is mainly mediated by the S. meliloti chemoreceptor McpU. McpU ...also has a prominent contribution in sensing a synthetic amino acid (aa) mixture mimicking the amounts and composition observed in seed exudate. Here, we used the hydrogel capillary assay to quantify chemotactic responses of S. meliloti to individual aa exuded by germinating alfalfa seeds and to define the role of McpU in this behavior. S. meliloti exhibited positive chemotaxis responses to all proteinogenic aa, except for aspartate, and to citrulline, cystine, gamma-aminobutyric acid, and ornithine. Wild-type responses were diverse in intensity, while a strain lacking mcpU displayed strongly diminished responses. Differential scanning fluorimetry demonstrated interaction of the purified periplasmic region of McpU (McpU-PR) with the aa, except glutamate and aspartate. We additionally tested organic acids and sugars, but there were no significant interactions with the McpU ligand-binding domain, except for citrate. Using ligand displacement, we confirmed the interaction of McpU-PR with aa representing strong and weak attractants. Our results show that S. meliloti McpU is a broad-range aa receptor mediating differential responses to individual attractants, which does not bind negatively charged aa.
Adhesive hydrogels have been recently proposed as a potential option to seal and treat gastric perforation (GP) which causes high mortality despite advancements in surgical treatments. However, to be ...effective, the hydrogels must have sufficient tissue adhesiveness, tough mechanical property, tunable biodegradability and ideally are easy to apply and form. Herein, we report an adhesive and resilient hydrogel for the sealing and treatment of gastric perforation. The hydrogel consists of a bioactive, transglutaminase (TG)-crosslinked gelatin network and a dynamic, borate-crosslinked poly-N-Tris(hydroxymethyl)methylacrylamide (PTH) network. The hydrogel can be formed in situ, facilitating easy delivery to the GP and allowing for precise sealing of the defects. In vivo experiments, using a perforated stomach mouse model, shows that the adhesive hydrogel plug effectively seals GP defects and promotes gastric mucosa regeneration. Overall, this hydrogel represents a promising biomaterial for GP treatment.
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•The interpenetrate double network hydrogel plug can be formed in situ with strong adhesiveness and toughness.•The hydrogel plug could adhere to the GP site and withstand the peristaltic movement of the stomach.•The hydrogel plug can effectively treated the GP and promoted mucosa regeneration in a mouse model.
Clonally derived bacterial populations exhibit significant genotypic and phenotypic diversity that contribute to fitness in rapidly changing environments. Here, we show that serial passage of ...Salmonella enterica serovar Typhimurium LT2 (StLT2) in broth, or within a mouse host, results in selection of an evolved population that inhibits the growth of ancestral cells by direct contact. Cells within each evolved population gain the ability to express and deploy a cryptic "orphan" toxin encoded within the rearrangement hotspot (rhs) locus. The Rhs orphan toxin is encoded by a gene fragment located downstream of the "main" rhs gene in the ancestral strain StLT2. The Rhs orphan coding sequence is linked to an immunity gene, which encodes an immunity protein that specifically blocks Rhs orphan toxin activity. Expression of the Rhs orphan immunity protein protects ancestral cells from the evolved lineages, indicating that orphan toxin activity is responsible for the observed growth inhibition. Because the Rhs orphan toxin is encoded by a fragmented reading frame, it lacks translation initiation and protein export signals. We provide evidence that evolved cells undergo recombination between the main rhs gene and the rhs orphan toxin gene fragment, yielding a fusion that enables expression and delivery of the orphan toxin. In this manner, rhs locus rearrangement provides a selective advantage to a subpopulation of cells. These observations suggest that rhs genes play important roles in intra-species competition and bacterial evolution.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Bacterial contact-dependent growth inhibition (CDI) is mediated by the CdiB/CdiA family of two-partner secretion proteins. CdiA effector proteins are exported onto the surface of CDI(+) inhibitor ...cells, where they interact with susceptible bacteria and deliver effectors/toxins derived from their C-terminal regions (CdiA-CT). CDI(+) cells also produce an immunity protein that binds the CdiA-CT and blocks its activity to prevent autoinhibition. Here, we show that the CdiA-CT from uropathogenic Escherichia coli strain 536 (UPEC536) is a latent tRNase that requires activation by the biosynthetic enzyme CysK (O-acetylserine sulfhydrylase A). UPEC536 CdiA-CT exhibits no nuclease activity in vitro, but cleaves within transfer RNA (tRNA) anti-codon loops when purified CysK is added. CysK and CdiA-CT form a stable complex, and their binding interaction appears to mimic that of the CysK/CysE cysteine synthase complex. CdiA-CT activation is also required for growth inhibition. Synthesis of CdiA-CT in E. coli cysK(+) cells arrests cell growth, whereas the growth of ΔcysK mutants is unaffected by the toxin. Moreover, E. coli ΔcysK cells are completely resistant to inhibitor cells expressing UPEC536 CdiA, indicating that CysK is required to activate the tRNase during CDI. Thus, CysK acts as a permissive factor for CDI, providing a potential mechanism to modulate growth inhibition in target cells.
The Neutron star Interior Composition Explorer observed several rotation-powered millisecond pulsars (MSPs) to search for or confirm the presence of X-ray pulsations. When broad and sine-like, these ...pulsations may indicate thermal emission from hot polar caps at the magnetic poles on the neutron star surface. We report confident detections (≥4.7 after background filtering) of X-ray pulsations for five of the seven pulsars in our target sample: PSR J0614−3329, PSR J0636+5129, PSR J0751+1807, PSR J1012+5307, and PSR J2241−5236, while PSR J1552+5437 and PSR J1744−1134 remain undetected. Of those, only PSR J0751+1807 and PSR J1012+5307 had pulsations previously detected at the 1.7 and almost 3 confidence levels, respectively, in XMM-Newton data. All detected sources exhibit broad sine-like pulses, which are indicative of surface thermal radiation. As such, these MSPs are promising targets for future X-ray observations aimed at constraining the neutron star mass-radius relation and the dense matter equation of state using detailed pulse profile modeling. Furthermore, we find that three of the detected MSPs exhibit a significant phase offset between their X-ray and radio pulses.
Bacteria have developed mechanisms to communicate and compete with one another in diverse environments. A new form of intercellular communication, contact-dependent growth inhibition (CDI), was ...discovered recently in Escherichia coli. CDI is mediated by the CdiB/CdiA two-partner secretion (TPS) system. CdiB facilitates secretion of the CdiA 'exoprotein' onto the cell surface. An additional small immunity protein (CdiI) protects CDI+ cells from autoinhibition. The mechanisms by which CDI blocks cell growth and by which CdiI counteracts this growth arrest are unknown. Moreover, the existence of CDI activity in other bacteria has not been explored. Here we show that the CDI growth inhibitory activity resides within the carboxy-terminal region of CdiA (CdiA-CT), and that CdiI binds and inactivates cognate CdiA-CT, but not heterologous CdiA-CT. Bioinformatic and experimental analyses show that multiple bacterial species encode functional CDI systems with high sequence variability in the CdiA-CT and CdiI coding regions. CdiA-CT heterogeneity implies that a range of toxic activities are used during CDI. Indeed, CdiA-CTs from uropathogenic E. coli and the plant pathogen Dickeya dadantii have different nuclease activities, each providing a distinct mechanism of growth inhibition. Finally, we show that bacteria lacking the CdiA-CT and CdiI coding regions are unable to compete with isogenic wild-type CDI+ cells both in laboratory media and on a eukaryotic host. Taken together, these results suggest that CDI systems constitute an intricate immunity network with an important function in bacterial competition.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Countries continue to debate the need for decontamination of cold-chain food packaging to reduce possible severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) fomite transmission among ...frontline workers. While laboratory-based studies demonstrate persistence of SARS-CoV-2 on surfaces, the likelihood of fomite-mediated transmission under real-life conditions is uncertain. Using a quantitative microbial risk assessment model of a frozen food packaging facility, we simulated 1) SARS-CoV-2 fomite-mediated infection risks following worker exposure to contaminated plastic packaging; and 2) reductions in these risks from masking, handwashing, and vaccination. In a frozen food facility without interventions, SARS-CoV-2 infection risk to a susceptible worker from contact with contaminated packaging was 1.5 × 10−3 per 1h-period (5th – 95th percentile: 9.2 × 10−6, 1.2 × 10−2). Standard food industry infection control interventions, handwashing and masking, reduced risk (99.4%) to 8.5 × 10−6 risk per 1h-period (5th – 95th percentile: 2.8 × 10−8, 6.6 × 10−5). Vaccination of the susceptible worker (two doses Pfizer/Moderna, vaccine effectiveness: 86–99%) with handwashing and masking reduced risk to 5.2 × 10−7 risk per 1h-period (5th – 95th percentile: 1.8 × 10−9, 5.4 × 10−6). Simulating increased transmissibility of current and future variants (Delta, Omicron), (2-, 10-fold viral shedding) among a fully vaccinated workforce, handwashing and masking continued to mitigate risk (1.4 × 10−6 - 8.8 × 10−6 risk per 1h-period). Additional decontamination of frozen food plastic packaging reduced infection risks to 1.2 × 10−8 risk per 1h-period (5th – 95th percentile: 1.9 × 10−11, 9.5 × 10−8). Given that standard infection control interventions reduced risks well below 1 × 10−4 (World Health Organization water quality risk thresholds), additional packaging decontamination suggest no marginal benefit in risk reduction. Consequences of this decontamination may include increased chemical exposures to workers, food quality and hazard risks to consumers, and unnecessary added costs to governments and the global food industry.
•SARS-CoV-2 infection risk from contact with frozen food packaging is very low.•Standard food worker handwashing and masking practices reduce risk by 99.4%.•Vaccination, handwashing, and masks mitigate risks from more transmissible variants.•Additional packaging decontamination results in minimal risk reduction to workers.
•We generated Affimers against platelet GPVI and mapped their binding sites, revealing functional regions regulating ligand binding.•A dimeric epitope was identified on GPVI for Affimer D18, which ...specifically binds GPVI dimer through a 1:1 interaction.
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Glycoprotein VI (GPVI) plays a key role in collagen-induced platelet aggregation. Affimers are engineered binding protein alternatives to antibodies. We screened and characterized GPVI-binding Affimers as novel tools to probe GPVI function. Among the positive clones, M17, D22, and D18 bound GPVI with the highest affinities (dissociation constant (KD) in the nanomolar range). These Affimers inhibited GPVI-collagen-related peptide (CRP)-XL/collagen interactions, CRP-XL/collagen-induced platelet aggregation, and D22 also inhibited in vitro thrombus formation on a collagen surface under flow. D18 bound GPVI dimer but not monomer. GPVI binding was increased for D18 but not M17/D22 upon platelet activation by CRP-XL and adenosine 5′-diphosphate. D22 but not M17/D18 displaced nanobody 2 (Nb2) binding to GPVI, indicating similar epitopes for D22 with Nb2 but not for M17/D18. Mapping of binding sites revealed that D22 binds a site that overlaps with Nb2 on the D1 domain, whereas M17 targets a site on the D2 domain, overlapping in part with the glenzocimab binding site, a humanized GPVI antibody fragment antigen-binding fragment. D18 targets a new region on the D2 domain. We found that D18 is a stable noncovalent dimer and forms a stable complex with dimeric GPVI with 1:1 stoichiometry. Taken together, our data demonstrate that Affimers modulate GPVI-ligand interactions and bind different sites on GPVI D1/D2 domains. D18 is dimer-specific and could be used as a tool to detect GPVI dimerization or clustering in platelets. A dimeric epitope regulating ligand binding was identified on the GPVI D2 domain, which could be used for the development of novel bivalent antithrombotic agents selectively targeting GPVI dimer on platelets.
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