TARGETING THE LIPOPOLYSACCHARIDE TRANSPORT TO DEVELOP NOVEL ANTIMICROBIAL DRUGS

The emergence of multidrug-resistant strains of Gram-negative pathogens that rapidly spread in the clinic is of great concern, since the range of antibiotics still effective against these organisms is limited and will continue to diminish. Therefore, the identification of novel and unexplored drug targets is an urgent need. Gram-negative bacteria possess an outer membrane (OM) with highly selective permeability properties due to its asymmetric structure with LPS in the outer leaflet and phospholipids in the inner leaflet. Dissecting the biogenesis of the OM and gaining insights into the multiprotein machineries that assemble this structure is vital if we want to succeed in developing novel antibiotic compounds that can target these machineries. This thesis focuses on the machinery that transports lipopolysaccharide (LPS) to the cell surface: the LPS transport (Lpt) machinery. In Escherichia coli, the Lpt system is composed of seven essential proteins spanning the cell envelope: the ABC transporter LptB2FGC powers the LPS extraction from the inner membrane (IM) and its transport along the periplasmic bridge, comprising LptA, to the OM LptDE translocon, which assembles LPS on the cell surface. Due to its vital role in cell physiology, the Lpt system represents a good target for the development of antibiotics with an innovative mechanism of action. Encouragingly, two promising inhibitors of this machinery have been discovered: murepavadin, which is currently in preclinical development, and thanatin. The research project of this thesis focuses on two main topics: elucidating the mechanism behind thanatin’s antibacterial activity, and the characterization of a mutant six-component Lpt machinery that is functional without LptC. Thanatin is a host-defence antimicrobial peptide recently shown to cause defects in membrane assembly and to bind to the N-terminal β-strand of LptA in vitro (Vetterli et al., 2018). Since this region is involved in both LptA dimerization and interaction with LptC, we implemented the Bacterial Adenylate Cyclase Two-Hybrid (BACTH) system to detect these interactions in the periplasm and probe which is the target of thanatin. With this technique, we found that thanatin targets both interactions and has a stronger inhibitory effect on the LptC-LptA interaction (Moura et al., 2020: https://doi.org/10.3389/fmicb.2020.00909). Further demonstrating a direct effect upon the LPS transport, we observed in thanatin-treated cells the degradation of LptA and the accumulation of LPS decorated with colanic acid (Moura et al., 2020), both of which have been previously reported to be indicative of LPS transport defects (Sperandeo et al., 2008, 2011). We further explored how thanatin affects the integrity of the cell envelope and observed that it induces promoters regulated by envelope-specific stress response systems (unpublished data). Although all seven Lpt proteins have been shown to be essential, viable mutants lacking LptC but carrying suppressor mutations at the residue R212 in the periplasmic domain of LptF were isolated by our group (Benedet et al., 2016). Interestingly, LptC was recently proposed to have a regulatory role on the LptB2FGC transporter by modulating its ATPase activity (Owens et al., 2019; Li et al., 2019), thus adding to the mystery of how the suppressor mutants can survive without LptC. In the second part of the project, we elucidated how the cell can bypass the presence of LptC and its regulatory role in the machinery by performing a biochemical characterization of the most representative suppressor mutant (manuscript ready for submission). Moreover, by analysing the interaction networks around the residue R212 of LptF, we also formulated a putative mechanism adopted by the Lpt transporter to regulate LPS transfer from LptB2FGC to LptA.