Generation of markerless and multiple-gene knockout in Glaesserella parasuis based on natural transformation and Flp recombinase.

Glaesserella parasuis is an important bacterial pathogen that affects the swine industry worldwide. Research on the pathogenic mechanism and genetically engineered vaccine remains undeveloped because an effective markerless and multiple-gene knockout system is unavailable for G. parasuis yet. To establish a markerless knockout, deleted allelic genes with kanamycin resistance (KanR) cassettes were introduced into the genome of G. parasuis by using natural transformation with suicide plasmids. Then, the KanR cassette was excised with a thermosensitive plasmid pGF conferring a constitutive Flp expression. To realize the markerless and multiple-gene knockout, plasmid pGAF was constructed by placing the Flp gene under the control of an arabinose-inducible promoter. Firstly, pGAF was introduced into G. parasuis by electroporation, and the marked mutants were produced following natural transformation. Finally, the KanR cassette was excised from the genome by the inducible expression of Flp upon arabinose action. Based on the natural transformation and the inducible expression of Flp, the markerless single-gene knockout mutants of ΔhsdR, ΔneuA2, ΔespP2, Δapd, and ΔnanH were constructed. In addition, a five-gene knockout mutant of ΔhsdRΔneuA2ΔespP2ΔapdΔnanH was generated by successive natural transformation with five suicide plasmids. Taken together, a markerless and multiple-gene deletion system was established for G. parasuis in the present study for the first time. This system is simple, efficient, and easy to manipulate for G. parasuis; thus, our technique will substantially aid the understanding of the etiology, pathogenesis, and genetic engineering of G. parasuis and other bacteria that can be naturally transformed in laboratory conditions. KEY POINTS: • Flp recombinase excised the KanR gene flanked by FRT sites in Glaesserella parasuis. • The regulatory expression of Flp enabled a multiple-gene knockout forG. parasuis. • The technique will promote the understanding of Glässer's disease pathogens.

A corpus-driven study of lexicalization models of English intransitive verbs.

This bottom-up study applied a corpus-driven approach to extract the major lexicalization models of English intransitive verbs (EIVs) through an analysis of their meanings under the Ideal Motion Event Category inspired by Talmy. A 710-photo specialized multimodal corpus of EIVs was constructed for this purpose. Data analysis showed that the major lexicalization models of EIVs include [Motion + Patient], [Motion + Manner], [Motion + Path], [Motion + Result], [Motion + Location], and [Motion + Purpose]. In-depth analysis of these models identified three major possible reasons why EIVs cannot originally take direct objects: the incorporation of [Patient], the inheritance of the intransitive feature, and the internalization of the actions expressed by EIVs. By comparing Chinese learners' most misused EIVs with their corresponding Chinese verbs, the current study provides empirical data to illustrate why transitive misusages of EIVs might occur among Chinese learners. The findings of this study will help English learners and users better apply EIVs.

Glaesserella parasuis autotransporters EspP1 and EspP2 are novel IgA-specific proteases.

Background: Glaesserella parasuis causes Glässer's disease, which is associated with severe polyarthritis, fibrinous polyserositis and meningitis, and leads to significant economic losses to the swine industry worldwide. IgA is one of the most important humoral immune factors present on mucosal surfaces, and it plays a crucial role in neutralizing and removing pathogens. G. parasuis is able to colonize the mucosal membrane of respiratory tract without being eliminated. Nevertheless, the immune evasion mechanism of G. parasuis in thwarting IgA remains unclear. Aims: The object of this study is to characterize the IgA degradation activity of Mac-1-containing autotransporter EspP1 and EspP2 from G. parasuis. Methods: The swine IgA was purified and incubated with EspP1 and EspP2 respectively. Western blotting was used to detect the cleavage of swine IgA. Generation of EspP1 and EspP2 mutant protein were used to explore the putative active sites of EspPs. LC-MS/MS based N/C-terminal sequencing was performed to measure the cleavage sites in swine IgA. Result: Our results show that G. parasuis EspP1 and EspP2 cleave swine IgA in a dose- and time- dependent manner. G. parasuis lose the IgA protease activity after simultaneously delete espP1 and espP2 indicating that EspP1 and EspP2 are the only two IgA proteases in G. parasuis. The IgA protease activity of EspP1 and EspP2 is affected by the putative active sites which contain Cys47, His172 and Asp194/195. Swine IgA is cleaved within Cα1 and Cα3 domains upon incubation with EspPs. Moreover, EspPs can degrade neither IgG nor IgM while G. parasuis possess the ability to degrade IgM unexpectedly. It suggests that G. parasuis can secrete other proteases to cleave IgM which have never been reported. Conclusion: We report for the first time that both EspP1 and EspP2 are novel IgA-specific proteases and cleave swine IgA within the Cα1 and Cα3 domains. These findings provide a theoretical basis for the EspPs-induced immune evasion.