Outer membrane biogenensis in Gram negative bacteria as a target for innovative antibacterial drugs

Despite the significant and continuous advances in the treatment of infectious diseases in this “antibiotic age”, pathogenic microorganisms still pose a threat to human and animal health worldwide. In addition to disease agents whose control has never been fully reached, new opportunistic pathogens have emerged and old well-known pathogens, that were thought to be defeated, have re-emerged. The onset of multidrug resistant bacterial strains further aggravates this scenario, and continuously demands novel antibiotic molecules. It is thus evident that investigations on the molecular mechanisms that underlay bacterial infections and host interactions should be strongly addressed to identify new molecular targets in bacteria. The currently available proteomics and genomics tools, coupled to structure-based drug discovery, provide a most productive approach to this aim; together, such tools build the most promising and rational strategy currently available to face evolution of the microbial world, and to prevent a dramatic drift towards a “pre-antibiotic age”. Cell surface in bacterial pathogens is the first site of host interaction, and is a major target for the antibacterial activity of the host. Among microbial components, lipopolysaccharide (LPS), an essential structure in the outer membrane (OM) of Gram-negative bacteria, is a key component that is sensed by the host, representing a potent stimulant of the innate immune response. Lipid A (endotoxin) is the toxic portion of LPS and is the crucial moiety in the interaction with the TLR4-MD2 receptor on the surface of immune cells. Excessive response to LPS, caused by circulating microbial antigens in the blood of affected patients, results in severe sepsis, a rapidly progressing inflammatory disease with up to 29% mortality. As LPS biogenesis is a key pathway essential for cell survival and pathogenicity, focusing on the molecular bases of infectious diseases as a target for innovative therapeutic strategies is a central issue for human health protection. In this context, the Project here presented aims to study the functional/molecular role of key proteins implicated in LPS biogenesis, and to design/synthesize novel lead compounds inhibiting the LPS biogenetic pathway. The specific objectives of the project are addressed by two main approaches described below: a) Structure-function studies of key proteins in the LPS biogenetic pathway Using this approach, in addition to the cellular, mutational and functional studies, the 3D-structures of key proteins implicated in the LPS biogenetic pathway (KdsD, LptA and LptC) will be solved and structure-function studies will be performed as a basis for rational drug design. These studies will be carried out using both Escherichia coli, a Gram-negative model organism and a pathogen implicated in severe gastrointestinal and extraintestinal diseases, and Pseudomonas aeruginosa, an opportunistic pathogen that causes a wide variety of infections in compromised patients, given that intrinsic and acquired resistance of the pathogen to most conventional drugs makes the treatment of such infections very difficult. b) Rational drug design and synthesis of novel antibacterial leads This aim will be pursued through the design and testing of novel, specific inhibitors, based on structural and functional studies of the target proteins known to play key roles in LPS biogenesis. Inhibitory molecules will be synthesized, screened for their antibacterial activities and tested for their specific effects against LPS biogenesis, using an assay we developed while studying the role of LptA. Additionally, the synthesized inhibitors will be further used in 3D-structure studies by analyzing protein inhibitor complexes; such stage will represent an optimization cycle, whereby inhibitor recognition principles will drive