Project Portugal 2030

Summary

Challenges: Antibiotic resistance is exponentially becoming a serious global health threat associated with increased morbidity, mortality, prolonged hospitalisation and healthcare costs [19]. Many Gram-Positive (G+) bacteria are among the most common multidrug-resistant pathogens associated with this issue [20]. Traditionally, strategies to fight microbial infections rely on the disruption of the pathogen growth cycle and consequently the organism destruction. Although highly effective, these approaches result in extensive impact on the microbe population, rapidly selecting for resistant subpopulations [21]. Rather than focusing on therapeutics that affect bacterial viability, an alternative approach is to target essential aspects for infection, such as virulence mechanisms required to invade the host and cause damage and disease [22-24]. This approach has several potential advantages such as expanding the repertoire and specificity of bacterial targets, exerting less selective pressure and preserving the host microbiota. To succeed with these novel anti-virulence strategies, new specific conserved virulence factors need to be discovered and very well characterised in order to better design directed therapeutics. We identified the wall teichoic acids (WTA) glycosyltransferase RmlT as involved in the virulence and resistance to antimicrobials of Listeria monocytogenes (Lm), a major G+ foodborne pathogen [6-8]. Being not essential for bacterial growth, RmlT thus appears as a promising target for next-generation anti-virulence/resistance strategies. Facing the mentioned challenges, the overall objective of our project is to enhance the understanding of bacterial WTA glycosylation, pathogenesis and antibiotic resistance mechanisms, while contributing to the development of innovative approaches to combat antibiotic-resistant infections. Three specific objectives were established: 1 - Elucidate the enzymatic mechanism of RmlT: - Determine the 3D structure of RmlT in a ternary complex with TDP-rhamnose and synthetic WTA, using X-ray crystallography. - Identify key residues involved in substrate recognition and catalysis through site-directed mutagenesis and functional assays. - Discover the transition state structure of RmlT reaction. 2 - Investigate the behaviour of Lm WTA glycosylations during infection: - Characterise Lm WTA glycosylation profiles in the course of mouse infection. - Evaluate the impact of WTA compositions in different phases of Lm infection. 3 - Develop RmlT inhibitors for anti-virulence strategies: - Conduct high-throughput screening of synthetic compound libraries using the optimised RmlT biochemical assay to identify potential inhibitors. - Perform structure-based virtual screening using ternary complex experimental structures and biophysical data to identify druggable sites for inhibitor design. - Validate hit compounds through in vitro potency and efficiency studies and in vivo pharmacokinetics and toxicity. This proposal thus seeks to decipher the fundamental mechanisms of Lm virulence/resistance to develop a novel approach based on drugs inhibiting specific sugar decorations, simultaneously diminishing bacterial virulence, increasing susceptibility to host innate defenses and potentiating the action of antibiotics. RmlT presenting marked similarities with WTA glycosyltransferases from Staphylococci or Enterococci, our aim is to go beyond Lm to propose new antimicrobial solutions against major G+ human pathogens.