Introduction Bacterial Outer Membrane Vesicles (OMVs) are naturally produced by all Gram-negative bacteria and are emerging as an attractive vaccine platform. The exploitation of OMVs in cancer immunotherapy was tested by decorating them with cancer epitopes and by following their capacity to elicit protective immune responses, alone or in combination, using cancer mouse models. Furthermore, we tested whether cancer cell-derived exosomes (TEXs) and OMVs can form complexes and whether such complexes, which carry TEX- associated tumor antigens and OMV-associated immune-stimulatory molecules, elicit anti-tumor immune responses in vivo. Results Immunization with OMVs engineered with two B cell epitopes (EGFRvIII and D8-mFAT1) induced tumor growth inhibition after mouse challenge with cell lines expressing the corresponding epitopes. Furthermore, mice immunized with engineered OMVs carrying two cancer epitopes, the EGFRvIII B cell epitope and the M30 CD4+ T cell epitope, were completely protected from EGFRvIIIB16F10 cell line expressing both epitopes, indicating the importance of multi-antigen immunization in cancer immunotherapy. Furthermore TEXs- OMVs immunization induced antibody responses against exosome antigens with a Th1-type profile. Finally, the combination of CT26-derived TEXs and MBP-D8-mFAT1 OMVs elicited synergistic protective activity against mouse challenge with CT26 cell line. Conclusions OMVs decorated with tumor antigens elicit antigen-specific, protective anti- tumor responses in mice. The synergistic protective activity of multiple epitopes simultaneously administered with OMVs, either by direct OMV engineering or by TEX-OMV combination, demonstrates the attractiveness of the OMV platform in cancer immunotherapy.

The SERINC family is a highly conserved group of genes which in the human genome comprises 5 members, encoding five homologous multipass transmembrane proteins. SERINC5, and to a lesser extent SERINC3, are powerful inhibitors of Human immunodeficiency virus 1 (HIV-1). SERINC5, expressed in virus-producing cells, is incorporated into the envelope of newly formed retroviral particles and inhibits an early stage of the virus infection process of the target cell, by preventing the delivery of the retroviral core into the target cell cytoplasm. Nef, an accessory protein of HIV-1, counteracts the antiretroviral activity of SERINC5 by promoting its endocytosis, which results in its removal from the cell surface, preventing its incorporation into retroviral particles. SERINC5 inhibits not only HIV-1, but also other divergent retroviruses, such as Murine leukemia virus (MLV). During my Ph.D. studies I demonstrated that the S2 auxiliary protein from Equine infectious anemia virus (EIAV) functionally resembles Nef and MLV glycoGag and counteracts SERINC5 with a similar mechanism. While the inhibitory effect of SERINC5 has been established on retroviruses, nothing is yet known about its effect on other viruses. Here I describe evidence which indicates the possible inhibitory activity of the SERINC gene family against other RNA viruses.

The mechanism responsible for lipoprotein sorting is conserved among bacterial species. However, the final destination of lipoproteins may vary among species. In some species lipoproteins are surface-exposed while in others they are associated to the outer membrane but facing the periplasm. To elucidate whether the difference in lipoprotein location is intrinsic to lipotrotein sequence/structure or rather is due to specific transport systems present in some bacterial species and absent in others lipoproteins were expressed in different species and their compartmentalization analyzed. The data seem to indicate that the destiny of lipoproteins depends upon specific structural signatures but the recognition of such signatures can be species-specific. Interestingly, lipoproteins can be exploited as chaperones to deliver foreign proteins to the outer membrane compartment.

Horizontal gene transfer (HGT) allows rapid exchanges of large genetic elements and is known to play an important role in bacterial evolution and adaptation. Conjugative transfer of genomic islands (GIs) has recently been reported in the opportunistic pathogen Pseudomonas aeruginosa. PAPI-1, one of the largest pathogenicity islands of P. aeruginosa, encodes several putative virulence genes and a major regulator of biofilm formation and antibiotic-resistant traits and was found to be horizontally transferable into strains lacking it. The conjugation of PAPI-1 island transfer is mediated by type IV pilus, which is encoded by ten genes located in PAPI-1. Nevertheless, the acquisition mechanism of PAPI-1 is currently not well understood. The first part of this thesis was aimed at identifying the receptor for conjugative transfer on the bacterial cell surface. Based on previous knowledge on bacterial conjugation, we designed and performed a series of mating experiments and analyzed transfer efficiency between PAPI-1 donor and recipient strains. Our data showed that A-band lipopolysaccharide (LPS) is required to initiate PAPI-1 transfer, supporting the idea that this structure acts as a receptor for conjugative type IV pilus in recipient strains. These results were verified by PAPI-1 transfer inhibition experiments with outer membrane (OM) or LPS preparations. The addition of a low amount of OM or LPS derived from strains producing A-band decreased PAPI-1 transfer efficiency by 80% compared to controls. In the second part, we demonstrated that P. aeruginosa strains which already acquired a copy of PAPI-1 almost completely lost the ability to receive additional copies of the island. Combination of strains with or without PAPI-1 were mated in-pair to investigate the redundancy in PAPI-1 transfer. The surface exclusion of PAPI-1 was characterized by investigating the effects of the addition of OM and LPS derived from strains with or without PAPI-1. In addition, LPS of the different strains were analyzed by western blot using antibodies directed against different parts of the molecule and by testing the in-vitro binding capacity of LPS to pilin protein. All experiments indicated that the strains carrying PAPI-1 produced much less A-band LPS compared to those lacking the island and lost the ability to bind to conjugative pilin. Finally, the screening of a series of mutants highlighted a role for two PAPI-1 genes in an entry exclusion activity, possibly through PAPI-1 island destabilization. This study contributes with a step forward in the understanding of the acquisition of genomic islands in P. aeruginosa, which may be generalized to other gram-negative bacteria and may lead to the future development of new strategies to limit the spread of virulence or resistance functions in populations of pathogenic bacteria.

In the 2007 report of World Health Organization (WHO) it was reported that in 2005 a great proportion of 1.8 million people died because of food and drinking water contamination (Velusamy et al., 2010). Fresh food product such as, fruits and vegetables carry a natural non-pathogenic epiphytic micro-flora, but during the food chain: harvest, transportation and further processing and handling the produce can be contaminated with pathogens from human or animal sources (Anon, 2002). While conventional methods used to evaluate pasteurization efficiency are based on cultivation in vitro, it has been ascertained that, under environmental stress conditions (e.g. nutrient limitation, pressure, temperature), a number of pathogens enter in a so-called Viable But Not Cultivable (VBNC) state, becoming eventually more resistant to stress and thus escaping to detection by cultivation methods. Improving health risk assessment associated with the increasing consumption of minimally processed fresh food products is a crucial need. To reach this objective, in the first part of my PhD project I set up and validated cultivation-independent bacterial viability assays, propidium monoazide quantitative PCR (PMA-qPCR) and flow cytometry (FCM), to monitor bacterial populations in food after Supercritical Carbon Dioxide (SC-CO2) treatment, that is one of the most promising non-thermal pasteurization technology in the age of the increasing demand for “ready-to-eat” and minimally-processed food products. The efficiency of SC-CO2 treatment was evaluated on bacterial liquid cultures, on bacteria spiked both on a synthetic solid substrate (LB agar) and on some fresh food products, including carrots, coconut and dry cured ham. The results indicated that the treatment is more efficient on bacteria spiked on LB agar, and that bacterial inactivation is accompanied by a reduction of their biovolume. Total bacterial inactivation on food products was reached for both Escherichia coli and Listeria monocytogenes, satisfying both the US and European requirements (CFSAN/FSIS, 2003; European Commission, 2005). Salmonella enterica was instead more resistant to treatment, suggesting future experiments consisting in the application of a combination between SC-CO2 and other techniques alternative to heat pasteurization, such as ultrasounds or Pulsed Electrical Field. FCM and PMA-qPCR data showed that a fraction of bacterial cells not detectable by plate counts maintained the integrity of their membrane (at least 102 cells/g for each bacterial species) suggested that the cells entered in a VBNC state. Comprehensively, the FCM assay showed the best performance as a bacterial viability test method, permitting to evaluate with high sensitivity the efficiency of treatment, to discriminate subpopulations of cells with different level of membrane permeabilization, and to identify variations in biovolume and alterations of the cellular surface. The method could be applied, with some adjustments, to any field where determining microbial viability status is of importance, including food, environment or in the clinic. Permeabilization of the cell membrane has been proposed to be the first event leading to cell inactivation or death after SC-CO2 treatment (Garcia-Gonzalez et al., 2007; Spilimbergo et al., 2009).The Permeabilization of membrane induced by SC-CO2 was also observed in Salmonella enterica (Kim et al., 2009a; Tamburini et al., 2013) and in Saccharomyces cerevisiae (Spilimbergo et al., 2010). Whether SC-CO2 has a direct effect on the bacterial membrane or permeabilization is a consequence of cell death remains an open question. In the second part of the Thesis to increase knowledge on the mechanism of bacterial inactivation mediated by SC-CO2 lipidomic profiles (HPLC-IT-ESI-MS), bacterial depolarization/permeabilization analysis (FCM) and gene expression studies of enzymes involved in phospholipids biosynthesis were performed on E. coli K12 MG1665. The data indicated that after 15 min of SC-CO2 treatment most of bacterial cells lost their membrane potential (95%) and membrane integrity (81% of permeabilized and 18% of partially-permeabilized cells). Bacterial permeabilization was associated to a 20% decrease of cellular biovolume and to a strong reduction (more than 50%) of all Phosphatidylglycerol (PG) membrane species, but without altering their average unsaturation index (1.30 ±0.02) and the average acyl chain on the glycerol backbone (33.30 ±0.03). The process acts more efficiently on PG than on PE (Phosphatidylethanolamine) head group phospholipids. Bacteria responded to treatment up-regulating the expression level of PssA gene, involved in PEs synthesis, since PssA activity is regulated by mole fraction of PGs and Cardiolin in the membrane. However still remains to understand why only PG species have been found to strongly decrease during the treatments. Further studies would be necessary, including phospholipid biosynthesis mutant analysis.

Plant cell division cycle is a highly complex and tightly controlled process which is regulated by both cell cycle genes conserved across kingdoms and plant-specific regulators. TCP family genes are plant-specific transcription factors reported in recent years that function in many aspects of plant growth, and broadly control the morphology of different organs via positive or negative regulating target genes expression. The initial work in our study was to identify the transcriptional factors regulating miRNA168a gene expression. A TCP family gene AtTCP7 was isolated as a putative regulator of Arabidopsis miR168a from yeast one-hybrid screening. Transgenic plants over-expressing AtTCP7 gene in Arabidopsis thaliana showed a decrease of mature miRNA168a accumulation, while later crossing analysis co-expressing 35S::AtTCP7 and pMiR168a::GUS showed a minor affection on reporter gene GUS expression, it indicated that the AtTCP7 may not directly interact with miR168a promoter in vivo. In-depth phenotypic characterization of the transgenic lines displayed a variety of growth defects and developmental delay, including shorter roots, delayed flowering and reduced fertility. Analyses at different developmental stages demonstrated that over-expression of AtTCP7 differentially affected cell size and DNA content in different organs by altering cell cycle genes expression and by restricting the interphase transition from G1 to S phase. Further expression analyses on over-expressing lines uncovered a complex network of genetic interactions between the members of the TCP family, highlighting the functional interaction especially between TCP7 and TCP23.

Viruses, and particularly bacteriophages, are key players in many microbial ecosystems and can profoundly influence the human microbiome and its impact on human health. While the bacterial and archaeal fraction of the human microbiome can now be profiled at an unprecedented resolution via cultivation-free metagenomics, viral metagenomics is still extremely challenging. The lack of universal viral genetic markers limits the de-novo discovery of viral entities, and the low number of available viral reference genomes from cultivation studies does not cover well the phage diversity in human microbiome samples. Viral-like particle (VLP) purification has been proposed as a set of experimental tools to concentrate viruses in samples prior to sequencing, but it remains unclear how efficient and reproducible such tools are in practice. In this thesis we aim to address some of these challenges and better exploit the potential of viral metagenomics in the context of the human microbiome. First, we performed and studied the performance of VLP procedures on freshwater and sediment samples. We found that bacteria can still be abundant at the end of the filtration process, thus lowering the efficiency of the enrichment. Analyzing samples with a low enrichment may lead to inconsistent conclusions, as the residual bacterial contamination might misdirect the computational analysis. To better quantify the extent of non-viral contamination in VLP sequencing, we designed ViromeQC, a novel open-source tool able to assess and rank viromes by their viral purity directly from the raw reads. In ViromeQC, rRNA genes and bacterial single-copy proteins are used as a proxy to estimate non-viral contamination. With the ViromeQC, we conducted the largest meta-analysis on the degree of enrichment of thousands of viral metagenomes, and concluded that the vast majority of them are three-fold less enriched than a standard metagenome. ViromeQC was then used to select the human gut viromes that had the highest enrichment as a starting point for a novel reference-free pipeline for the discovery of previously uncharacterized viral entities. The approach included metagenomic assembly of the enriched viromes as well as extensive mining of many thousands of assembled metagenomes, and led to a catalog of 162,876 sequences of highly-trusted viral origin. Most of these predicted viral sequences had no match against any known virus in RefSeq even though some of them showed a prevalence in gut metagenomes of up to 70%. Our analyses and publicly available tools and resources are helping to uncover the still hidden virome diversity and improve the support for current and future investigations of the human virome.

SERINC5 is a restriction factor for retroviruses, antagonized by Nef of primate lentiviruses, by glycoGag of Moloney Murine Leukaemia Virus (MoMLV) and by S2 of Equine Infectious Anaemia virus (EIAV). In addition, SERINC5 sensitizes HIV-1 to neutralizing antibodies (nAbs) targeting the MPER in gp41. However, since the identification of SERINC5 as an inhibitor of retrovirus infectivity, many features of the host factor await clarification, notably the molecular mechanisms of restriction and viral counteraction. Furthermore, SERINC5 cellular role beyond restriction is still obscure. This thesis explores multiple aspects of the mutual antagonism governing the SERINC5 interplay with retroviruses. We first describe a contribution towards the determination of the structure of SERINC5 and the identification of the determinants crucial for antiviral activity, virus sensitization to neutralization and counteraction by retroviruses. By performing a structure-based mutagenesis screening, we identified SERINC5 ECL3, ECL5 and the interface between subdomains as regions essential for inhibition of HIV-1 infectivity and virus sensitization to 4E10 and 2F5 nAbs. The simultaneous impairment of both SERINC5 antiviral effects indicates that they are mechanistically related and support the hypothesis of a SERINC5-mediated impairment of the envelope glycoproteins. We included a comparative analysis of the antiviral activity of human SERINC paralogs and their sensitivity to retroviral counteraction. It has been previously established that SERINC3 inhibits HIV-1 infectivity less potently than SERINC5, while SERINC2 has no antiviral effects. We report here that similarly to SERINC3, SERINC1 is endowed with a modest antiviral activity; in contrast, SERINC4 severely inhibits HIV-1 infectivity, despite being poorly expressed. Irrespectively of their antiretroviral potency, all SERINC proteins are incorporated into virus particles. Interestingly, we observed that virion-associated SERINC2 is specifically cleaved by the viral protease, but proteolysis does not explain the lack of antiretroviral effects. Furthermore, SERINC5 and SERINC2 have different glycomic profiles, but diverse post-translational modification is irrelevant for their opposite activity against HIV-1. In addition, we reported that human SERINCs are differently targeted by retroviral counteracting factors, with SERINC5 being the paralog most efficiently downregulated, while SERINC1 being completely resistant. A cysteines cluster within ICL4 emerged as the major determinant of SERINC5 responsiveness to different nef alleles, while it proved irrelevant for internalization by MoMLV glycoGag and EIAV S2, indicating that diverse retroviral counteractors likely target the host factor differently. Though SERINC5 ICL4 harbours multiple motifs governing SERINC5 sensitivity to antagonization, insertion of this loop within SERINC2 was not enough to transfer susceptibility to Nef activity, suggesting that the overall conformation of the protein is essential for downregulation by Nef. Importantly, the cysteine stretch within ICL4 is palmitoylated, suggesting that this modification may be important for counteraction by the lentiviral factor. SERINC5 and CD4 downregulation by Nef are functionally related, as they both require AP-2 mediated endocytosis. However, regions in Nef selectively governing SERINC5 internalization are unknown. We reported here that Phe90 within Nef αA-helix genetically uncouples the activities on SERINC5 and CD4, being selectively involved in SERINC5 downregulation. In parallel, we explored SERINC5 antagonization by different glycoGag alleles and observed that the ability to target the host factor is not conserved across divergent γ-retroviruses. Finally, we observed that HIV-1 may evade SERINC5 restriction by direct cell-to-cell infection, suggesting that the host factor may have a broader role in retroviral spreading, requiring the evolution and the conservation of active viral counteraction. To this end, we preliminary investigated a positive contribution of SERINC5 to intracellular signalling.

The human gut microbiota plays a crucial role in the functioning of the gastrointestinal tract and its alteration can lead to gastrointestinal abnormalities and inflammation. Additionally, the gut microbiota modulates central nervous system (CNS) activities affecting several aspect of host physiology. Motivated by the increasing evidences of the role of the gut microbiota in the complex set of interactions connecting the gut and the CNS, known as gut-brain axis, in this Ph.D. thesis we asked whether the gastrointestinal abnormalities and inflammation commonly associated with neurological disorders such as Rett syndrome (RTT) and Autism could be related to alterations of the bacterial and fungal intestinal microbiota. First, since only few reports have explored the fungal component of the gut microbiota in health and disease, we characterized the gut mycobiota in a cohort of healthy individuals, in order to reduce the gap of knowledge concerning factors influencing the intestinal microbial communities. Next, we compared the gut microbiota of three cohorts of healthy, RTT and autistic subjects to investigate if these neurological disorders harbour alterations of the gut microbiota. Culture-based and metataxonomics analysis of the faecal fungal populations of healthy volunteers revealed that the gut mycobiota differs in function of individuals’ life stage in a gender-related fashion. Different fungal species were isolated showing phenotypic adaptation to the intestinal environment. High frequency of azoles resistance was also found, with potential clinical significance. It was further observed that autistic subjects are characterized by a reduced incidence of Bacteroidetes and that Collinsella, Corynebacterium, Dorea and Lactobacillus were the taxa predominating in the gut microbiota of autistic subjects. Constipation has been associated with different bacterial patterns in autistic and neurotypical subjects, with constipated autistic individuals characterized by higher levels of Escherichia/Shigella and Clostridium cluster XVIII than constipated neurotypical subjects. RTT is a neurological disorder caused by loss-of-function mutations of MeCP2 and it is commonly associated with gastrointestinal dysfunctions and constipation. We showed that RTT subjects harbour bacterial and fungal microbiota altered from those of healthy controls, with a reduced microbial richness and dominated by Bifidobacterium, different Clostridia and Candida. The alterations of the gut microbiota observed did not depend on the constipation status of RTT subjects while this microbiota produced altered SCFAs profiles potentially contributing to the constipation itself. Phenotypical and immunological characterizations of faecal fungal isolates from RTT subjects showed Candida parapsilosis as the most abundant species isolated in RTT, genetically unrelated to healthy controls’ isolates and with elevated resistance to azoles. Furthermore these isolates induced high levels of IL-10 suggesting increased tolerance and persistence within the host. Finally, the importance of multiple sequence alignment (MSA) accuracy in microbiome research was investigated comparing three implementations of the widely used NAST algorithm. By now, different implementations of NAST have been developed but no one tested the performances and the accuracy of the MSAs generated with these implementations. We showed that micca, a new bioinformatics pipeline for metataxonomics data improves the quality of NAST alignments by using a fast and memory efficient reimplementation of the NAST algorithm.

Background. Infections caused by Pseudomonas aeruginosa are the main cause of morbidity and mortality in Cystic Fibrosis (CF) patients and occur via primary colonisation of the airway followed by the accumulation of pathoadaptive mutations in the bacterial genome which increase fitness in the lung environment and result in chronicization. A better understanding of i) the evolutionary dynamics occurring during chronic airway infections in CF patients and ii) the genetic adaptation of strains to the CF lung environment, might give further clues for preventive measures or novel therapies to control CF infections in the future. In this work, we obtained genomic sequences of 40 P. aeruginosa isolates from a single CF patient collected over an eight-year period (2007-2014) and analysed the population in terms of clonality of the isolates, phylogenetic relationships, and presence of polymorphisms and variants between the strains. Population structure and microevolution. In silico Multilocus Sequence Typing (MLST) analysis revealed a characteristic single clonal population dominated by a previously characterized sequence type (ST390) and a small number of new, closely related ST variants (ST1863, ST1864, ST1923). EBURST analysis of the sequence types revealed that all members of this population belong to the same clonal lineage and likely evolved from a single ancestral colonizing strain. Furthermore, the phylogenetic analysis based on SNPs also divided the population into two subpopulations derived from the evolution of the first infecting strain. The annotation of SNPs allowed us to identify mutations with moderate or high impact. Genes with high impact variants encoded respiratory nitrate reductase subunit gamma nail, polyprotein signal peptidase lspA, the ABC transporter-binding protein aaltP, the copper resistance protein A precursor pcoAin, and four hypothetical proteins. The evolution of strains in the CF airway is characterized by the loss of many virulence traits, including motility and protease secretion, along with the acquisition of multidrug resistance. Functional phenotypic assays of the collection, including motility and secretion of proteases, showed a decrease over time in the persistent isolates. We also determined the antibiotic susceptibility profile of the collection; while early isolates were found to be susceptible to almost all these antibiotics, resistant phenotypes dramatically increased over time in the population. Functional studies on specific strains. To identify additional functional variations related to pathoadaptive mutations occurring in the course of chronic infection in CF, we then selected three isolates for further characterization: one early CF isolate (TNCF_23 isolated in 2007); one clonal late CF isolate (TNCF_175 isolated in 2014); one clinical isolate (VrPa97) from a non-CF patient belonging to the same sequence type (ST390) as the former isolates. With this approach, we aimed to identify additional phenotypic and functional variations between isolates with a very homogeneous genomic background, in an attempt to find out new pathoadaptive mutations occurring in the course of chronic infection in CF. Specifically, the following traits were investigated: killing of C. elegans and G. mellonella (in vivo virulence); immunomodulatory properties (IL-8 ELISA assay); competitive growth in Artificial Sputum Medium (ASM); functionality of Type Six Secretion System (T6SS). Despite their close genetic relatedness, considerable variations were observed between the three isolates, among which the late isolate TNCF_175 showed several alterations 7 putatively resulting from the adaptation process to the CF lung. TNCF_175 presented a mutation in tssK3, part of H3-T6SS; this mutation (C958T) was therefore introduced in the reference strains PAO1 and PA14, and mutated strains were subsequently complemented; killing rate on C. elegans and growth rate in ASM in mutant and complemented strains were evaluated. Conclusions. A rare feature of this strain collection is the consistent number of clonal isolates obtained from a single patient over a rather long period of 8 eight years, thus providing a model to look at microevolutionary trends within a highly homogenous bacterial population, and avoiding potential biases due to the host genetic background and clinical history. In spite of the close genomic relatedness of all isolates, a surprisingly high diversity was observed for the majority of tested phenotypes. Investigating the competitive ability of early versus late strains we propose a role for T6SS in the adaptation process to the CF lung environment. Our data suggest that once persistence has been established, a strain no longer requires its T6SS, allowing loss of function mutations to occur. Conversely, acute and early CF strains still carry a number of virulence factors, including T6SS that potentially provide an advantage in outcompeting other microorganisms in the initial stage of CF infection.