Bacterial outer membrane vesicles (OMVs) represent an interesting vaccine platform for their built-in adjuvanticity and simplicity of production process. Moreover, OMVs can be decorated with foreign antigens using different synthetic biology approaches. However, the optimal OMV engineering strategy, which should guarantee the OMV compartmentalization of most heterologous antigens in quantities high enough to elicit protective immune responses, remains to be validated. In this work we exploited the lipoprotein transport pathway to engineer OMVs with foreign proteins. Using 5 Staphylococcus aureus protective antigens expressed in Escherichia coli as fusions to a lipoprotein leader sequence, we demonstrated that all 5 antigens accumulated in the vesicular compartment at a concentration ranging from 5 to 20% of total OMV proteins, suggesting that antigen lipidation could be a universal approach for OMV manipulation. Engineered OMVs elicited high, saturating antigen-specific antibody titers when administered to mice in quantities as low as 0.2 μg/dose. Moreover, the expression of lipidated antigens in E. coli BL21(DE3)ΔompAΔmsbBΔpagP was shown to affect the lipopolysaccharide structure, with the result that the TLR4 agonist activity of OMVs was markedly reduced. These results, together with the potent protective activity of engineered OMVs observed in mice challenged with S. aureus Newman strain, makes the 5-combo-OMVs a promising vaccine candidate to be tested in clinics.

Over the last years the zebrafish imposed itself as a powerful model to study skeletal diseases, but a limit to its use is the poor characterization of collagen type I, the most abundant protein in bone and skin. In tetrapods collagen type I is a trimer mainly composed of two α1 chains and one α2 chain, encoded by COL1A1 and COL1A2 genes, respectively. In contrast, in zebrafish three type I collagen genes exist, col1a1a, col1a1b and col1a2 coding for α1(I), α3(I) and α2(I) chains. During embryonic and larval development the three collagen type I genes showed a similar spatio-temporal expression pattern, indicating their co-regulation and interdependence at these stages. In both embryonic and adult tissues, the presence of the three α(I) chains was demonstrated, although in embryos α1(I) was present in two distinct glycosylated states, suggesting a developmental-specific collagen composition. Even though in adult bone, skin and scales equal amounts of α1(I), α3(I) and α2(I) chains are present, the presented data suggest a tissue-specific stoichiometry and/or post-translational modification status for collagen type I. In conclusion, this data will be useful to properly interpret results and insights gained from zebrafish models of skeletal diseases.

In conclusion, the combination of proteomic data with system biology platforms allowed us to amplify the information obtained processing the results and indicated the principal pathways involved. These information can point to potential biomarkers and new therapeutic targets opening the way for further analysis.

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Because of their potent adjuvanticity, ease of manipulation and simplicity of production Gram‐negative Outer Membrane Vesicles OMVs have the potential to become a highly effective vaccine platform. However, some optimization is required, including the reduction of the number of endogenous proteins, the increase of the loading capacity with respect to heterologous antigens, the enhancement of productivity in terms of number of vesicles per culture volume. In this work we describe the use of Synthetic Biology to create Escherichia coli BL21(DE3)Δ60, a strain releasing OMVs (OMVsΔ60) deprived of 59 endogenous proteins. The strain produces large quantities of vesicles (> 40 mg/L under laboratory conditions), which can accommodate recombinant proteins to a level ranging from 5% to 30% of total OMV proteins. Moreover, also thanks to the absence of immune responses toward the inactivated endogenous proteins, OMVsΔ60 decorated with heterologous antigens/epitopes elicit elevated antigens/epitopes‐specific antibody titers and high frequencies of epitope‐specific IFN‐γ‐producing CD8+ T cells. Altogether, we believe that E. coli BL21(DE3)Δ60 have the potential to become a workhorse factory for novel OMV‐based vaccines.

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Phenotypic variability in the presence of an identical molecular defect is a recurrent feature in heritable disorders and it was also reported in osteogenesis imperfecta (OI). OI is a prototype for skeletal dysplasias mainly caused by mutations in the two genes coding for type I collagen. No definitive cure is available for this disorder, but the understanding of molecular basis in OI phenotypic modulation will have a pivotal role in identifying possible targets to develop novel drug therapy. We used a functional proteomic approach to address the study of phenotypic variability using the skin of the OI murine model Brtl. Brtl mice reproduce the molecular defect, dominant transmission and phenotypic variability of human OI patients. In the presence of a Gly349Cys substitution in α1(I)-collagen Brtl mice can have a lethal or a moderately severe outcome. Differential expression of chaperones, proteasomal subunits, metabolic enzymes, and proteins related to cellular fate demonstrated that a different ability to adapt to cellular stress distinguished mutant from wild-type mice and mutant lethal from surviving mutant animals. Interestingly, class discovery analysis identified clusters of differentially expressed proteins associated with a specific outcome, and functional analysis contributed to a deeper investigation into biochemical and cellular pathways affected by the disease. This article is part of a Special Issue entitled: Translational Proteomics.

The human follicular fluid (HFF) contains molecules and proteins that may affect follicle growth, oocyte maturation and competence acquiring. Despite the numerous studies, an integrated broad overview on biomolecular and patho/ physiological processes that are proved or supposed to take place in HFF during folliculogenesis and oocyte development is still missing. In this review we report, for the first time, all the proteins unambiguously detected in HFF and, applying DAVID (Database for Annotation, Visualization and Integrated Discovery) and MetaCore bioinformatic resources, we shed new lights on their functional correlation, delineating protein patterns and pathways with reasonable potentialities for oocyte quality estimation in in vitro fertilisation (IVF) programs. Performing a rigorous PubMed search, we redacted a list of 617 unique proteins unambiguously-annotated as HFF components. Their functional processing suggested the occurrence in HFF of a tight and highly dynamic functional-network, which is balanced by specific effectors, primarily involved in extracellular matrix degradation and remodelling, inflammation and coagulation. Metalloproteinases, thrombin and vitamin-Dreceptor/retinoid-X-receptor-alpha resulted as the main key factors in the nets and their differential activity may be indicative of ovarian health and oocyte quality. Despite future accurate clinical investigations are absolutely needed, the present analysis may provide a starting point for more accurate oocyte quality estimation and for defining personalised therapies in reproductive medicine.