Insights in the maturation of pathogenic bacteria vaccine candidates using mass spectrometry based approaches

The study of the maturation process that occurs to a protein is of pivotal importance for the understanding of its function. This is true also in the vaccine field but in this case is also important to evaluate if inappropriate protein conformation and maturation play roles in the impairment of the functional immunogenicity of protein vaccines. Mass spectrometry (MS) is the method of choice for the study of the maturation process since each modification that occurs during the maturation will lead to a change in the mass of the entire protein. Therefore the aim of my thesis is the development of mass spectrometry-based approaches to study the maturation of proteins and the application of these methods to proteic vaccine candidates. The thesis is divided in two main parts. In the first part, I focused my attention on the study of the maturation of different vaccine candidates using native mass spectrometry. The analyses in this case have been performed using recombinant proteins produced in E. coli. In the second part I applied different MS strategies for the identification of unknown PTMs on pathogenic bacteria surface proteins since modified surface proteins are now considered for vaccine candidate selection.

Purple bacteria for the biotechnological valorisation of agro-industrial streams

Waste management worldwide has received increasing attention from global policies in recent years. In particular, agro-industrial streams represent a global concern due to the huge volumes generated and a high number of residues, which produce an environmental and economic impact on the ecosystem. The use of biotechnological approaches to treat these streams could allow the production of desirable by-products to be reinjected into the production cycle through sustainable processes. Purple phototrophic bacteria (PPB) are targeted as microorganisms capable to reduce the pressure of agro-industrial streams on environmental issues, due to their metabolic versatility (autotrophic and/or heterotrophic growth under different conditions). This Ph.D. research project aims to assess the effectiveness of PPB cultivation for industrial streams valorisation in the applications of biogas desulfurization and microbial protein production. For these purposes, the first part of the present work is dedicated to the cultivation of purple sulfur bacteria (PSB) for biogas streams upgrading, cleaning biogas from sulfur compounds (H2S), and producing elemental sulfur (S0), potentially suitable as a slow-release fertilizer. The second part of the thesis, instead, sees the application of purple non-sulfur bacteria (PNSB) on streams rich in organics, such as molasses, generating biomass with high content of proteins and pigments, useful as supplements in animal feed. The assessment of the main metabolic mechanisms involved in the two processes is evaluated at a laboratory scale using flasks and a photobioreactor, to define the consumption of substrates and the accumulation of products both in the autotrophic (on biogas) and in heterotrophic grow (on molasses). In conclusion, the effectiveness of processes employing PPB for a sustainable valorisation of several agro-industrial streams has been proved promising, using actual residues, and coupling their treatments with the production of added-value by-products.