Bacterial chitinase with phytopathogen control capacity from suppressive soil revealed by functional metagenomics.

Plant disease caused by fungal pathogens results in vast crop damage globally. Microbial communities of soil that is suppressive to fungal crop disease provide a source for the identification of novel enzymes functioning as bioshields against plant pathogens. In this study, we targeted chitin-degrading enzymes of the uncultured bacterial community through a functional metagenomics approach, using a fosmid library of a suppressive soil metagenome. We identified a novel bacterial chitinase, Chi18H8, with antifungal activity against several important crop pathogens. Sequence analyses show that the chi18H8 gene encodes a 425-amino acid protein of 46 kDa with an N-terminal signal peptide, a catalytic domain with the conserved active site F175DGIDIDWE183, and a chitinase insertion domain. Chi18H8 was expressed (pGEX-6P-3 vector) in Escherichia coli and purified. Enzyme characterization shows that Chi18H8 has a prevalent chitobiosidase activity with a maximum activity at 35 °C at pH lower than 6, suggesting a role as exochitinase on native chitin. To our knowledge, Chi18H8 is the first chitinase isolated from a metagenome library obtained in pure form and which has the potential to be used as a candidate agent for controlling fungal crop diseases. Furthermore, Chi18H8 may also answer to the demand for novel chitin-degrading enzymes for a broad range of other industrial processes and medical purposes.

Two PDZ binding motifs within NS5 have roles in Tick-borne encephalitis virus replication.

The flavivirus genus includes important human neurotropic pathogens like Tick-borne encephalitis virus (TBEV) and West-Nile virus (WNV). Flavivirus replication occurs at replication complexes, where the NS5 protein provides both RNA cap methyltransferase and RNA-dependent RNA polymerase activities. TBEVNS5 contains two PDZ binding motifs (PBMs) important for specific targeting of human PDZ proteins including Scribble, an association important for viral down regulation of cellular defense systems and neurite outgrowth. To determine whether the PBMs of TBEVNS5 affects virus replication we constructed a DNA based sub-genomic TBEV replicon expressing firefly luciferase. The PBMs within NS5 were mutated individually and in concert and the replicons were assayed in cell culture. Our results show that the replication rate was impaired in all mutants, which indicates that PDZ dependent host interactions influence TBEV replication. We also find that the C-terminal PBMs present in TBEVNS5 and WNVNS5 are targeting various human PDZ domain proteins. TBEVNS5 has affinity to Zonula occludens-2 (ZO-2), GIAP C-terminus interacting protein (GIPC), calcium/calmodulin-dependent serine protein kinase (CASK), glutamate receptor interacting protein 2, (GRIP2) and Interleukin 16 (IL-16). A different pattern was observed for WNVNS5 as it associate with a broader repertoire of putative host PDZ proteins.

First dating of a recombination event in mammalian tick-borne flaviviruses.

The mammalian tick-borne flavivirus group (MTBFG) contains viruses associated with important human and animal diseases such as encephalitis and hemorrhagic fever. In contrast to mosquito-borne flaviviruses where recombination events are frequent, the evolutionary dynamic within the MTBFG was believed to be essentially clonal. This assumption was challenged with the recent report of several homologous recombinations within the Tick-borne encephalitis virus (TBEV). We performed a thorough analysis of publicly available genomes in this group and found no compelling evidence for the previously identified recombinations. However, our results show for the first time that demonstrable recombination (i.e., with large statistical support and strong phylogenetic evidences) has occurred in the MTBFG, more specifically within the Louping ill virus lineage. Putative parents, recombinant strains and breakpoints were further tested for statistical significance using phylogenetic methods. We investigated the time of divergence between the recombinant and parental strains in a Bayesian framework. The recombination was estimated to have occurred during a window of 282 to 76 years before the present. By unravelling the temporal setting of the event, we adduce hypotheses about the ecological conditions that could account for the observed recombination.

Sequencing of a tick-borne encephalitis virus from Ixodes ricinus reveals a thermosensitive RNA switch significant for virus propagation in ectothermic arthropods.

Tick-borne encephalitis virus (TBEV) is a flavivirus with major impact on global health. The geographical TBEV distribution is expanding, thus making it pivotal to further characterize the natural virus populations. In this study, we completed the earlier partial sequencing of a TBEV pulled out of a pool of RNA extracted from 115 ticks collected on Torö in the Stockholm archipelago. The total RNA was sufficient for all sequencing of a TBEV genome (Torö-2003), without conventional enrichment procedures such as cell culturing or suckling mice amplification. To our knowledge, this is the first time that the genome of TBEV has been sequenced directly from an arthropod reservoir. The Torö-2003 sequence has been characterized and compared with other TBE viruses. In silico analyses of secondary RNA structures formed by the two untranslated regions revealed a temperature-sensitive structural shift between a closed replicative form and an open AUG accessible form, analogous to a recently described bacterial thermoswitch. Additionally, novel phylogenetic conserved structures were identified in the variable part of the 3'-untranslated region, and their sequence and structure similarity when compared with earlier identified structures suggests an enhancing function on virus replication and translation. We propose that the thermo-switch mechanism may explain the low TBEV prevalence often observed in environmentally sampled ticks. Finally, we were able to detect variations that help in the understanding of virus adaptations to varied environmental temperatures and mammalian hosts through a comparative approach that compares RNA folding dynamics between strains with different mammalian cell passage histories.

Rac1 and Scribble are targets for the arrest of neurite outgrowth by TBE virus NS5.

Tick-borne encephalitis virus (TBEV) causes extensive CNS disease in humans known as TBE, however, relatively little is known of the molecular mechanisms for its progress. Here, we now show that TBEV produces defects in neuronal development of PC12 cells through a function of the viral NS5 protein. The methyltransferase domain of NS5 is critical and sufficient for restriction of nerve growth factor induced neurite outgrowth. This effect is reversed by expression of NS5 mutants unable to bind Scribble and unexpectedly, in Scribble depleted cells with binding-competent NS5. Furthermore, we also demonstrate that the Rho GTPase Rac1 and the guanine nucleotide-exchange factor, betaPIX are outcompeted by NS5 for binding to Scribble, linking to effects on neurite outgrowth by TBEV. Together, these findings provide the first experimental evidence that Rac1 and betaPIX are indirect targets of NS5 acting through the multifunctional polarity protein Scribble to oppose neuronal differentiation. In conclusion, our results offer a potential mechanism by which TBEV alters neuronal circuitry and opens new avenues for therapeutic interventions.