tatDB: a database of Ago1-mediated targets of transfer RNA fragments.

tRNA-derived fragments (tRFs) are a class of emerging post-transcriptional regulators of gene expression likely binding to the transcripts of target genes. However, only a few tRFs targets have been experimentally validated, making it hard to extrapolate the functions or binding mechanisms of tRFs. The paucity of resources supporting the identification of the targets of tRFs creates a bottleneck in the fast-developing field. We have previously analyzed chimeric reads in crosslinked Argonaute1-RNA complexes to help infer the guide-target pairs and binding mechanisms of multiple tRFs based on experimental data in human HEK293 cells. To efficiently disseminate these results to the research community, we designed a web-based database tatDB (targets of tRFs DataBase) populated with close to 250 000 experimentally determined guide-target pairs with ∼23 000 tRF isoforms. tatDB has a user-friendly interface with flexible query options/filters allowing one to obtain comprehensive information on given tRFs (or targets). Modes of interactions are supported by secondary structures of potential guide-target hybrids and binding motifs, essential for understanding the targeting mechanisms of tRFs. Further, we illustrate the value of the database on an example of hypothesis-building for a tRFs potentially involved in the lifecycle of the SARS-CoV-2 virus. tatDB is freely accessible at https://grigoriev-lab.camden.rutgers.edu/tatdb.

Large-Scale Computational Discovery of Binding Motifs in tRNA Fragments.

Accumulating evidence has suggested that tRNA-derived fragments (tRFs) could be loaded to Argonaute proteins and function as regulatory small RNAs. However, their mode of action remains largely unknown, and investigations of their binding mechanisms have been limited, revealing little more than microRNA-like seed regions in a handful of tRFs and a few targets. Here, we identified such regions of potential interaction on a larger scale, using in vivo formed hybrids of guides and targets in crosslinked chimeric reads in two orientations. We considered "forward pairs" (with guides located on the 5' ends and targets on the 3' ends of hybrids) and "reverse pairs" (opposite orientation) and compared them as independent sets of biological constructs. We observed intriguing differences between the two chimera orientations, including the paucity of tRNA halves and abundance of polyT-containing targets in forward pairs. We found a total of 197 quality-ranked motifs supported by ∼120,000 tRF-mRNA chimeras, with 103 interacting motifs common in forward and reverse pairs. By analyzing T→C conversions in human and mouse PAR-CLIP datasets, we detected Argonaute crosslinking sites in tRFs, conserved across species. We proposed a novel model connecting the formation of asymmetric pairs in two sets to the potential binding mechanisms of tRFs, involving the identified interaction motifs and crosslinking sites to Argonaute proteins. Our results suggest the way forward for further experimental elucidation of tRF-binding mechanisms.

Computational meta-analysis of ribosomal RNA fragments: potential targets and interaction mechanisms.

The most abundant cellular RNA species, ribosomal RNA (rRNA), appears to be a source of massive amounts of non-randomly generated fragments. We found rRNA fragments (rRFs) in immunoprecipitated Argonaute (Ago-IP) complexes in human and mouse cells and in small RNA sequencing datasets. In human Ago1-IP, guanine-rich rRFs were preferentially cut in single-stranded regions of mature rRNAs between pyrimidines and adenosine, and non-randomly paired with cellular transcripts in crosslinked chimeras. Numerous identical rRFs were found in the cytoplasm and nucleus in mouse Ago2-IP. We report specific interaction motifs enriched in rRF-target pairs. Locations of such motifs on rRFs were compatible with the Ago structural features and patterns of the Ago-RNA crosslinking in both species. Strikingly, many of these motifs may bind to double-stranded regions on target RNAs, suggesting a potential pathway for regulating translation by unwinding mRNAs. Occurring on either end of rRFs and matching intronic, untranslated or coding regions in targets, such interaction sites extend the concept of microRNA seed regions. Targeting both borders of certain short introns, rRFs may be involved in their biogenesis or function, facilitated by Ago. Frequently dismissed as noise, rRFs are poised to greatly enrich the known functional spectrum of small RNA regulation.

A Novel Tail-Associated O91-Specific Polysaccharide Depolymerase from a Podophage Reveals Lytic Efficacy of Shiga Toxin-Producing Escherichia coli.

Shiga toxin-producing Escherichia coli (STEC) strains are important zoonotic foodborne pathogens, causing diarrhea, hemorrhagic colitis, and life-threatening hemolytic uremic syndrome (HUS) in humans. However, antibiotic treatment of STEC infection is associated with an increased risk of HUS. Therefore, there is an urgent need for early and effective therapeutic strategies. Here, we isolated lytic T7-like STEC phage PHB19 and identified a novel O91-specific polysaccharide depolymerase (Dep6) in the C terminus of the PHB19 tailspike protein. Dep6 exhibited strong hydrolase activity across wide ranges of pH (pH 4 to 8) and temperature (20 to 60°C) and degraded polysaccharides on the surface of STEC strain HB10. In addition, both Dep6 and PHB19 degraded biofilms formed by STEC strain HB10. In a mouse STEC infection model, delayed Dep6 treatment (3 h postinfection) resulted in only 33% survival, compared with 83% survival when mice were treated simultaneously with infection. In comparison, pretreatment with Dep6 led to 100% survival compared with that of the control group. Surprisingly, a single PHB19 treatment resulted in 100% survival in all three treatment protocols. Moreover, a significant reduction in the levels of proinflammatory cytokines was observed at 24 h postinfection in Dep6- or PHB19-treated mice. These results demonstrated that Dep6 or PHB19 might be used as a potential therapeutic agent to prevent STEC infection.IMPORTANCE Shiga toxin-producing Escherichia coli (STEC) is an important foodborne pathogen worldwide. The Shiga-like toxin causes diarrhea, hemorrhagic colitis, and life-threatening hemolytic uremic syndrome (HUS) in humans. Although antibiotic therapy is still used for STEC infections, this approach may increase the risk of HUS. Phages or phage-derived depolymerases have been used to treat bacterial infections in animals and humans, as in the case of the "San Diego patient" treated with a phage cocktail. Here, we showed that phage PHB19 and its O91-specific polysaccharide depolymerase Dep6 degraded STEC biofilms and stripped the lipopolysaccharide (LPS) from STEC strain HB10, which was subsequently killed by serum complement in vitro In a mouse model, PHB19 and Dep6 protected against STEC infection and caused a significant reduction in the levels of proinflammatory cytokines. This study reports the use of an O91-specific polysaccharide depolymerase for the treatment of STEC infection in mice.

Age-Related Argonaute Loading of Ribosomal RNA Fragments.

BACKGROUND: Accumulating evidence points to the functional roles of rRNA derived Fragments (rRFs), often considered degradation byproducts. Small RNAs, including miRNAs and tRNA-derived Fragments (tRFs), have been implicated in the aging process and we considered rRFs in this context. OBJECTIVE: We performed a computational analysis of Argonaute-loaded rRFs in Drosophila melanogaster to study rRF changes with age. We determined rRF abundance in Ago1 and Ago2 at 3 and 30 days to identify Ago1-guided and Ago2-guided fragments. We searched for putative seed sequences in rRFs based on frequent matches of sliding k-mer windows to the conserved regions of 12 Drosophila genomes. We predicted putative targets (containing matches to seeds identified in four rRFs) and studied their functional enrichments using Gene Ontology. RESULTS: We identified precise cleavage sites of distinct rRF isoforms from both nuclear and mitochondrial rRNAs. The most prominent rRF isoforms were enriched in Ago2 at 3 days and that loading strongly decreased with age. For less abundant rRFs, loading of Ago2-guided rRFs generally increased in Ago2, whereas Ago1-guided rRFs revealed diverse patterns. The distribution of seed matches in targets suggested that rRFs may bind to various conserved regions of many genes, possibly via miRNA-like seed-based mechanisms. CONCLUSION: Our observations suggest that rRFs may be functional molecules, with age-dependent Argonaute loading, comparable to that of miRNAs and tRFs. The putative rRF targets showed significant enrichment in developmental processes and biological regulation, similar to tRFs and consistent with a possible involvement of these newly identified small RNAs in the Drosophila aging.

Inferring targeting modes of Argonaute-loaded tRNA fragments.

Transfer RNA fragments (tRFs) are an emerging class of small RNA molecules derived from mature or precursor tRNAs. They are found across a wide range of organisms and tissues, in small RNA fraction or loaded to Argonaute in numbers comparable to microRNAs. Their functions and mechanisms of action are largely unknown, and results obtained on individual tRFs are often hard to generalize. Here we predicted binding mechanisms and specific target interaction sites of 26 human Argonaute-loaded tRFs of different types using large-scale meta-analyses of available experimental data. Strikingly, our findings matched all interaction sites detected in a recent experimental screen, confirming the validity of our computational approach. Such sites are primarily located on the 5' end of tRFs and often involve additional binding along the tRF length, similar to microRNAs. Indicative of multiple layers of regulation, diverse regulatory non-coding RNAs comprised a third of the tRF targets, with the rest being protein-coding transcripts. In the latter, coding sequence and 3' UTRs were the likely primary target regions, although we observed interactions of tRFs with 5' UTRs. Another novel phenomenon we report, a large number of putative interactions between tRFs and introns, is compatible with the roles of Argonaute in the nucleus. Further, observed tRF-intron binding modes suggest a mechanism of interaction of tRFs with Argonaute-dependent introns, and we predict here >20 candidate introns of this type. Taken together, these results present tRFs as regulatory molecules with a rich functional spectrum.

Conjugation of DM1 to anti-CD30 antibody has potential antitumor activity in CD30-positive hematological malignancies with lower systemic toxicity.

An anti-CD30 antibody-drug conjugate incorporating the antimitotic agent DM1 and a stable SMCC linker, anti-CD30-MCC-DM1, was generated as a new antitumor drug candidate for CD30-positive hematological malignancies. Here, the in vitro and in vivo pharmacologic activities of anti-CD30-MCC-DM1 (also known as F0002-ADC) were evaluated and compared with ADCETRIS (brentuximab vedotin). Pharmacokinetics (PK) and the safety profiles in cynomolgus monkeys were assessed. Anti-CD30-MCC-DM1 was effective in in vitro cell death assays using CD30-positive lymphoma cell lines. We studied the properties of anti-CD30-MCC-DM1, including binding, internalization, drug release and actions. Unlike ADCETRIS, anti-CD30-MCC-DM1 did not cause a bystander effect in this study. In vivo, anti-CD30-MCC-DM1 was found to be capable of inducing tumor regression in subcutaneous inoculation of Karpas 299 (anaplastic large cell lymphoma), HH (cutaneous T-cell lymphoma) and L428 (Hodgkin's disease) cell models. The half-lives of 4 mg/kg and 12 mg/kg anti-CD30-MCC-DM1 were about 5 days in cynomolgus monkeys, and the tolerated dose was 30 mg/kg in non-human primates, supporting the tolerance of anti-CD30-MCC-DM1 in humans. These results suggest that anti-CD30-MCC-DM1 presents efficacy, safety and PK profiles that support its use as a valuable treatment for CD30-positive hematological malignancies.

Multi-targeting therapeutic mechanisms of the Chinese herbal medicine QHD in the treatment of non-alcoholic fatty liver disease.

Beneficial effects of the Chinese herbal medicine Qushi Huayu Decoction (QHD) were observed with non-alcoholic fatty liver disease (NAFLD) patients and animal models. The impact of QHD or its active components (geniposide and chlorogenic acid, GC) on NAFLD liver transcriptome and gut microbiota was examined with NAFLD rats. Increased expression for genes required for glutathione production and decreased expression for genes required for lipid synthesis was observed in NAFLD livers treated with QHD and GC. GC treatment decreased serum LPS, which could be explained by reduced mucosal damage in the colon of GC-treated rats. Further, our data suggest an increased abundance of Treg-inducing bacteria that stimulated the Treg activity in GC treated colon, which in turn down-regulated inflammatory signals, improved gut barrier function and consequently reduced hepatic exposure to microbial products. Our study suggests that QHD simultaneously enhanced the hepatic anti-oxidative mechanism, decreased hepatic lipid synthesis, and promoted the regulatory T cell inducing microbiota in the gut.

A controllable bacterial lysis system to enhance biological safety of live attenuated Vibrio anguillarum vaccine.

Bacterial strains used as backbone for the generation of vaccine prototypes should exhibit an adequate and stable safety profile. Given the fact that live attenuated vaccines often contain some potential risks in virulence recovery and spread infections, new approaches are greatly needed to improve their biological safety. Here, a critically iron-regulated promoter PviuA was screened from Vibrio anguillarum, which was demonstrated to respond to iron-limitation signal both in vitro and in vivo. By using PviuA as a regulatory switch to control the expression of phage P22 lysis cassette 13-19-15, a novel in vivo inducible bacterial lysis system was established in V. anguillarum. This system was proved to be activated by iron-limitation signals and then effectively lyse V. anguillarum both in vitro and in vivo. Further, this controllable bacterial lysis system, after being transformed into a live attenuated V. anguillarum vaccine strain MVAV6203, was confirmed to significantly improve biological safety of the live attenuated vaccine without impairing its immune protection efficacy.

Cell-penetrating peptides mediated protein cross-membrane delivery and its use in bacterial vector vaccine.

It is an attractive strategy to develop a recombinant bacterial vector vaccine by expressing exogenous protective antigen to induce the immune response, and the main concern is how to enhance the cellular internalization of antigen produced by bacterial vector. Cell-penetrating peptides (CPPs) are short cationic/amphipathic peptides which facilitate cellular uptake of various molecular cargoes and therefore have great potentials in vector vaccine design. In this work, eleven different CPPs were fused to the C-terminus of EGFP respectively, and the resultant EGFP-CPP fusion proteins were expressed and purified to assay their cross-membrane transport in macrophage J774 A.1 cells. Among the tested CPPs, TAT showed an excellent capability to deliver the cargo protein EGFP into cytoplasm. In order to establish an efficient antigen delivery system in Escherichia coli, the EGFP-TAT synthesis circuit was combined with an in vivo inducible lysis circuit PviuA-E in E. coli to form an integrated antigen delivery system, the resultant E. coli was proved to be able to lyse upon the induction of a mimic in vivo signal and thus release intracellular EGFP-TAT intensively, which were assumed to undergo a more efficient intracellular delivery by CPP to evoke protective immune responses. Based on the established antigen delivery system, the protective antigen gene flgD from an invasive intracellular fish pathogen Edwardsiella tarda EIB202, was applied to establish an E. coli recombinant vector vaccine. This E. coli vector vaccine presented superior immune protection (RPS = 63%) under the challenge with E. tarda EIB202, suggesting that the novel antigen delivery system had great potential in bacterial vector vaccine applications.

Identification of an iron acquisition machinery in Flavobacterium columnare.

Flavobacterium columnare, a fastidious Gram-negative pathogen and the causative agent of columnaris disease, is one of the most harmful pathogens in the freshwater fish-farming industry. Nevertheless the virulence mechanisms of F. columnare are not well understood. Bacterial iron uptake from the host during infection is an important mechanism of virulence. Here we identified and analyzed part of the iron uptake machinery of F. columnare. Under iron-limited conditions during in vitro growth, synthesis of an outer membrane protein of ~86 kDa was upregulated. This protein was identified as a TonB-dependent ferrichrome-iron receptor precursor (FhuA). Synthesis of siderophores in F. columnare was corroborated by chrome azurol S assays. A putative ferric uptake regulator (Fur) protein was also identified in the F. columnare genome. Structural analysis of the F. columnare Fur protein revealed that it was similar to Fur proteins involved in iron uptake regulation of other bacteria. Furthermore, Salmonella enterica serovar Typhimurium (S. Typhimurium) Δfur mutants were partially complemented by the F. columnare fur gene. We conclude that a siderophore-mediated iron uptake system exists in F. columnare, and fur from F. columnare could partially complement S. Typhimurium Δfur mutant.

Asd-based balanced-lethal system in attenuated Edwardsiella tarda to express a heterologous antigen for a multivalent bacterial vaccine.

Edwardsiella tarda is an enteric Gram-negative invasive intracellular pathogen, which causes enteric septicemia in fish. It could be potentially used to develop a recombinant attenuated E. tarda vaccine for the aquaculture industry. Because live vaccine strains can potentially be released into the environment upon vaccination, medical and environmental safety issues must be considered. Deletion of the asdB gene in E. tarda resulted in a diaminopimelic acid (DAP)-dependent mutant. The wild type asdB gene was inserted in place of the antibiotic-resistance gene in the plasmid, and the resultant non-antibiotic resistant vector was transformed into the attenuated and DAP-dependent E. tarda vaccine strain (WEDΔasdB) to obtain a balanced-lethal system for heterologous antigen expression. The balanced-lethal expression system was further optimized by comparing plasmid replicons with different Shine-Dalgarno sequences and start codons for the asdB gene. Utilizing the optimized balanced-lethal expression system, the protective antigen gene gapA34 from the fish pathogen Aeromonas hydrophila LSA34 was expressed in the attenuated E. tarda to generate the multivalent vaccine candidate WEDΔasdB/pUTta4DGap. This vaccine was shown to evoke an effective immune response against both E. tarda and A. hydrophila LSA34 by vaccinating turbot via a simple immersion route. This multivalent E. tarda vector vaccine has great potential for broad applications in aquaculture.

Development of a Fur-dependent and tightly regulated expression system in Escherichia coli for toxic protein synthesis.

BACKGROUND: There is a continuous demanding for tightly regulated prokaryotic expression systems, which allow functional synthesis of toxic proteins in Escherichia coli for bioscience or biotechnology application. However, most of the current promoter options either are tightly repressed only with low protein production levels, or produce substantial protein but lacking of the necessary repression to avoid mutations initiated by leaky expression in the absence of inducer. The aim of this study was to develop a tightly regulated, relatively high-efficient expression vector in E. coli based on the principle of iron uptake system. RESULTS: By using GFP as reporter, PfhuA with the highest relative fluorescence units, but leaky expression, was screened from 23 iron-regulated promoter candidates. PfhuA was repressed by ferric uptake regulator (Fur)-Fe2+ complex binding to Fur box locating at the promoter sequence. Otherwise, PfhuA was activated without Fur-Fe2+ binding in the absence of iron. In order to improve the tightness of PfhuA regulation for toxic gene expression, Fur box in promoter sequence and fur expression were refined through five different approaches. Eventually, through substituting E. coli consensus Fur box for original one of PfhuA, the induction ratio of modified PfhuA (named PfhuA1) was improved from 3 to 101. Under the control of PfhuA1, strong toxic gene E was successfully expressed in high, middle, low copy-number vectors, and other two toxic proteins, Gef and MazF were functionally synthesized without E. coli death before induction. CONCLUSIONS: The features of easy control, tight regulation and relatively high efficiency were combined in the newly engineered PfhuA1. Under this promoter, the toxic genes E, gef and mazF were functionally expressed in E. coli induced by iron chelator in a tightly controllable way. This study provides a tightly regulated expression system that might enable the stable cloning, and functional synthesis of toxic proteins for their function study, bacterial programmed cell death in biological containment system and bacterial vector vaccine development.

A novel in vivo inducible expression system in Edwardsiella tarda for potential application in bacterial polyvalence vaccine.

Recombinant bacterial vector vaccine is an attractive vaccination strategy to induce the immune response to a carried protective antigen, and the main concern of bacterial vector vaccine is to establish a stable antigen expression system in vector bacteria. Edwardsiella tarda is an important facultative intracellular pathogen of both animals and humans, and its attenuated derivates are excellent bacterial vectors for use in recombinant vaccine design. In this study, we design an in vivo inducible expression system in E. tarda and establish potential recombinant E. tarda vector vaccines. With wild type strain E. tarda EIB202 as a vector, 53 different bacteria-originated promoters were examined for iron-responsive transcription in vitro, and the promoters P(dps) and P(yncE) showed high transcription activity. The transcription profiles in vivo of two promoters were further assayed, and P(dps) revealed an enhanced in vivo inducible transcription in macrophage, larvae and adult zebra fish. The gapA34 gene, encoding the protective antigen GAPDH from the fish pathogen Aeromonas hydrophila LSA34, was introduced into the P(dps)-based protein expression system, and transformed into attenuated E. tarda strains. The resultant recombinant vector vaccine WED/pUTDgap was evaluated in turbot (Scophtalmus maximus). Over 60% of the vaccinated fish survived under the challenge with A. hydrophila LSA34 and E. tarda EIB202, suggesting that the P(dps)-based antigen delivery system had great potential in bacterial vector vaccine application.

Iron-regulated lysis of recombinant Escherichia coli in host releases protective antigen and confers biological containment.

The use of a recombinant bacterial vector vaccine is an attractive vaccination strategy to induce an immune response to a carried protective antigen. The superiorities of live bacterial vectors include mimicry of a natural infection, intrinsic adjuvant properties, and the potential for administration by mucosal routes. Escherichia coli is a simple and efficient vector system for production of exogenous proteins. In addition, many strains are nonpathogenic and avirulent, making it a good candidate for use in recombinant vaccine design. In this study, we screened 23 different iron-regulated promoters in an E. coli BL21(DE3) vector and found one, P(viuB), with characteristics suitable for our use. We fused P(viuB) with lysis gene E, establishing an in vivo inducible lysis circuit. The resulting in vivo lysis circuit was introduced into a strain also carrying an IPTG (isopropyl-ß-d-thiogalactopyranoside)-inducible P(T7)-controlled protein synthesis circuit, forming a novel E. coli-based protein delivery system. The recombinant E. coli produced a large amount of antigen in vitro and could deliver the antigen into zebrafish after vaccination via injection. The strain subsequently lysed in response to the iron-limiting signal in vivo, implementing antigen release and biological containment. The gapA gene, encoding the protective antigen GAPDH (glyceraldehyde-3-phosphate dehydrogenase) from the fish pathogen Aeromonas hydrophila LSA34, was introduced into the E. coli-based protein delivery system, and the resultant recombinant vector vaccine was evaluated in turbot (Scophtalmus maximus). Over 80% of the vaccinated fish survived challenge with A. hydrophila LSA34, suggesting that the E. coli-based antigen delivery system has great potential in bacterial vector vaccine applications.

Involvement of LuxS in the regulation of motility and flagella biogenesis in Vibrio alginolyticus.

The fish pathogen Vibrio alginolyticus contains two unique flagellar systems. The LuxS quorum sensing system is reported to regulate the expression of virulence factors in a wide variety of pathogenic bacteria. Our previous work demonstrated that inactive luxS led to decreased virulence in V. alginolyticus. In this study, LuxS-dependent regulation of motility and flagella biogenesis, the potential virulence factors in V. alginolyticus, were further investigated. A luxS-deleted mutant showed deficiency in motility and flagella formation, and an intact luxS complemented the defect. Since motility is flagella dependent, V. alginolyticus flagella biogenesis related genes, including the flagella regulator genes flaK and lafK and the sub-hierarchical flagellar genes fliS and lafA, were cloned and identified. Based on quantitative real-time reverse transcription-PCR, differential expression of these genes was confirmed in wild-type and luxS mutants. Our results indicate that LuxS plays an important role in the regulation of motility and flagella biogenesis in V. alginolyticus.