DESENVOLVIMENTO DE BIOTERAPÊUTICOS PARA CONTROLAR A COLONIZAÇÃO E A DOENÇA PNEUMOCÓCICA

The project aims to address the persistent challenge of preventing and controlling S. pneumoniae infections, which continue to pose a significant burden globally. In particular, the association of pneumococci with respiratory viruses, notably influenza, in secondary bacterial infections exacerbates disease severity and mortality rates. Current therapeutic options have limitations: vaccines are too narrow and antibiotics are too broad. As pneumococcal disease is always preceded by colonization, our central idea is to interfere at this critical stage. We propose the development of two products aimed at precisely targeting S. pneumoniae colonization: - MISSMI, an engineered live biotherapeutic derived from a commensal strain of S. mitis; - OPTMI, a cocktail of engineered bacteriocins derived from S. mitis with improved resistance to proteases. While biotherapeutics are an active area of research, their targeted application for controlling S. pneumoniae colonization represents a promising and innovative approach that, to the best of our knowledge, was not previously attempted. Our previous project, STOPneumo, funded by FCT and successfully concluded, laid the groundwork for MISSMI-OPTMI. We identified seven promising streptococcal strains with some anti-pneumococcal activity, but none had all desired characteristics. Specifically, strain F-ad emerged as a promising candidate for MISSMI development due to its favorable traits: isolation from a healthy individual's upper respiratory tract, amenability to genetic manipulation, and effective asymptomatic colonization of mouse respiratory tract. Moreover, our characterization of seven commensal strains revealed over 50 bacteriocin-coding genes. Deletion of the bacteriocin-encoding regions (a single locus often encodes for multiple bacteriocins), led to loss of anti-pneumococcal activity, implicating them in this process. Based on the above, we have: designed a robust strategy for the development of the current proposal, optimized protocols, consolidated collaborations with colleagues of complementary expertise, and attracted talented PhD students to contribute to the project. The specific objectives of the project are: - synthesize and evaluate the inhibitory activity of 50 promising bacteriocins against S. pneumoniae (TASK 1); - construct strain MISSMI by integrating the most potent bacteriocins selected in TASK 1, along with their dedicated transporter and immunity proteins, into strain F-ad, strategically minimizing lateral gene transfer and resistance development (TASK 2); - design and engineer 30 protease-resistant anti-pneumococcal bacteriocins using predictive deep learning models (TASK 3); - express and purify ten optimized protease-resistant bacteriocins (TASK 4); - determine the optimal dose and treatment regimen of MISSMI and engineered bacteriocins for S. pneumoniae colonization control (TASK 5); - assess the cytotoxicity of MISSMI and OPTMI (TASK 6); - evaluate the efficacy of MISSMI and OPTMI in preventing S. pneumoniae colonization and secondary pneumococcal pneumonia using a murine model (TASK 7); - investigate the potential development of S. pneumoniae bypass mutants and identify cellular targets of MISSMI and OPTMI (TASK 8); - explore potential synergistic effects of OPTMI with commonly used antibiotics to resensitize antibiotic-resistant S. pneumoniae (TASK 9); - ensure effective project management and promotion and dissemination initiatives (TASKS 10 and 11).