Genomic and functional insights into the interactions between vaginal Lactobacillus strains and pathogens

The vaginal microbiota of healthy pre-menopausal women is typically dominated by one Lactobacillus species among L. crispatus, L. gasseri, L. jensenii and L. iners. Thanks to a series of antimicrobial activities, strains belonging to these species represent the first barrier against infections and maintain niche homeostasis. On the other hands, the increase abundance in pathogen species is associated with the onset of numerous diseases, leading also to an increase risk of other infections acquisition. The deciphering of factors which influence Lactobacillus survival, as well as the interactions between lactobacilli-pathogens and pathogens-pathogens represent an important topic of study for improving woman health and investigating effective probiotic strategies. Here, we investigated environmental factors and genetic traits that lead to the dominance of either L. crispatus or L. gasseri in the vaginal niche and the possible applications of liposomes loaded with L. gasseri biosurfactants for the treatment and prevention of Staphylococcus aureus biofilm infections. Furthermore, considering the increasing relevance acquired by bacterial extracellular vesicles (EVs) we analysed the role of EVs derived from vaginal lactobacilli and pathogens on both bacterial growth and HIV-1 infections. As a result, we reported for the first time i) common and species-specific genotypic and phenotypic features of L. crispatus and L. gasseri ii) significant antibiofilm activity of liposomes loading vaginal Lactobacillus biosurfactants against multi-drug resistant S. aureus strains iii) absence of growth regulation mediated by EVs derived from lactobacilli on pathogen cultures and vice versa iv) anti-HIV-1 activity of protein derived from L. gasseri EVs and unexpected antiviral effect of pathogen-derived EVs on HIV-1 infections in vitro. In conclusion, this PhD thesis explored characteristics and possible applications of vaginal lactobacilli for the human health, as well as promising antiviral effects of both lactobacilli and pathogen derived EVs.

A genome editing approach to the study of Parvovirus B19

Parvovirus B19 (B19V) is a ssDNA virus, with a 5596 nt long genome encapsidated within an icosahedral capsid with a diameter of 22 nm. Viral proteins are subdivided into structural and non-structural: the main non-structural one is the NS1, while the 2 structural proteins VP1 and VP2 assemble originating the capsid shell. B19V tropism is mainly limited to erythroid progenitor cells (EPCs), however, virus can be detected in several districts persisting in tissues possibly lifelong. The virus can induce anemia and erythroid aplasia. Therapeutic strategies are only symptomatic, so the search for antivirals is strongly active, with screenings showing the activity in vitro of different compounds like hydroxyurea, cidofovir and brincidofovir. In the first project, a functional minigenome of B19V was developed, able to express only the NS1 protein. This minigenome proved able to replicate and express the NS1 at levels comparable to unmodified clones. Furthermore, the ability of this minigenome to complement the function of NS1-deficient genomes was demonstrated, thus providing a proof-of-concept of B19V genome editing possibility and, at the same time, a useful tool to study the NS1 protein also as an antiviral target. In the second project I addressed the interplay between B19V and the cellular restriction factor APOBEC3B (A3B), a cytidine deaminase acting on ssDNA, whose footprint on B19V genome was proved by a bioinformatic sequence analysis performed by the hosting lab. To understand whether A3B still exerts activity and a potential antiviral effect on B19V, the UT7/EpoS1 cells were transduced with lentiviral vectors to silence A3B expression, then used as a model to study viral behavior. No significant role of A3B on B19V was demonstrated, in agreement with the hypothesis of viral adaptation to this cellular restriction factor; anyway, virus ability to alter A3B expression would deserve further investigations.