Design, expression, and characterization of FimH antigen as single recombinant protein or exposed on nanoparticles

Uropathogenic Escherichia coli (UPEC) accounts for approximately 85% of all urinary tract infections (UTIs), causing a global economic burden. E. coli is one of the pathogens mentioned in the ESKAPEE list drafted by OMS, meaning that the increasing antibiotic resistance acquired by UPEC is and will be a serious health problem in the future. Amongst the immunogenic antigens exposed on the surface of UPEC, FimH represent a potential target for vaccine development, since it is involved in the early stages of infection. As already demonstrated, immunizations with FimH elicit functional antibodies that prevent UPEC infections even though the number of doses required to elicit a strong immune response is not optimal. In this work, we aimed to stabilize FimH as a soluble recombinant antigen exploiting the donor strand complementation mechanism by generating different chimeric constructs constituted by FimH and FimG donor strand. To explore the potential of self-assembling nanoparticles to display FimH through genetic fusion, different constructs have been computationally designed and produced. In this work a structure-based design, using available crystal structures of FimH and three different NPs was performed to generate different constructs with optimized properties. Despite the different conditions tested, all the constructs designed (single antigen or chimeric NPs), resulted to be un-soluble proteins in E. coli. To overcome this issue a mammalian expression system has been tested. Soluble antigen expression was achieved for all constructs tested in the culture supernatants. Three novel chimeric NPs have been characterized by transmission electron microscopy (TEM) confirming the presence of correctly assembled NPs displaying UPEC antigen. In vivo study has shown a higher immunogenicity of the E. coli antigen when displayed on NPs surface compared to the single recombinant antigen. The antibodies elicited by chimeric NPs showed a higher functionality in the inhibition of bacterial adhesion.

Phenotypic and molecular characterization of ethylene biosynthesis in apples

Ethylene plays an important role in apple fruit development. Its biosynthesis is catalyzed by two enzymes ACS and ACO. The first is considered to catalyzes the rate-limiting step of ethylene production and in apple two different alleles (MdACS1-1 and MdACS1-2) of this gene have been identified. The presence in the promoter region of MdACS1-2 allele of a SINE insertion is considered to be responsible for a low transcription level and a pronounced reduction in ethylene production in apple cultivar homozygous for this allele. However, the specific expression of each MdACS1 allele has never been reported as well as any in vivo analysis of its 5’-flanking region. With the present study we addressed these issues by developing a set of qPCR allele specific primers for MdACS1 and by a functional characterization of the MdACS1 promoters by transient expression analysis. qPCR analysis on different apple tissues and stages of development demonstrated that MdACS1-2 allele is never express and that MdACS1-1 allele is ripening-related and expresses predominantly but not exclusively in apple fruit. To test MdACS1 promoter in fruit the only protocol available in literature for transient transformation of apple fruit was evaluated and optimized. Twenty chimeric promoter::reporter constructs were generated and analyzed by Agrobacterium-transient transformation. The in vivo analysis allowed to identify an enhancer-like region of 261 bp in MdACS1 promoter and a region of 57 bp in MdACS1-2 responsible, also if not alone, in the inactivation of the MdACS1-2 allele. Through the assessment of ethylene production in a segregating progeny derived from the cross between Fuji and Mondial Gala (homozygous for MdACS1-2 allele) we demonstrated that at least two other genes may be involved in apple ethylene production. An hypothesis that could explain the difference between Fuji and Mondial Gala have been proposed.