Emerging antibiotic resistance in Gram-negative pathogens

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PROJECT SUMMARY Burkholderia multivorans is a successful pathogen and a member of the B. cepacia complex (Bcc) that causes pneumonia in immunocompromised individuals with underlying lung diseases, such as cystic fibrosis (CF) and chronic granulomatous disease (CGD). Bcc consists of a group of 17 closely related Gram-negative bacteria with extreme genetic capacity and metabolic diversity. Several Bcc members can trigger chronic airway infections in CF patients and have emerged as opportunistic pulmonary pathogens. B. multivorans and B. cenocepacia are the two most commonly isolated species, which are threats for outbreaks. Bcc infections in CF patients are associated with enhanced morbidity and mortality. They also have the capacity to cause rapid clinical deterioration with septicemia that leads to death. Several outbreaks of B. multivorans causing severe morbidity and mortality in both CF and non-CF patients have occurred. Bcc pathogens are intrinsically resistant to a broad range of antimicrobials, including b-lactams, fluoroquinolones, aminoglycosides, polymyxins and cationic peptides, creating a major challenge to the treatment of Bcc pulmonary infections. Hopanoids play a predominant role in supporting outer membrane stability and barrier function in B. multivorans, thus participating in the resistance to polymyxin B and colistin. Hopanoids are pentacyclic triterpenoid lipids that are capable of inserting in bacterial membranes and contributing to their stability and stiffness. Hopanoids help membranes withstand damaging stress conditions, including high temperature, low pH and the presence of antibiotics. Importantly, hopanoid production plays an important role in the physiology and pathogenesis of B. cenocepacia. In spite of the importance of hopanoids in bacteria, the mechanism of intracellular hopanoid trafficking has not been explored. We propose to target the B. multivorans HpnN (hopanoid biosynthesis-associated resistance- nodulation-cell division (RND)) transporter, which is essential for cell wall remodeling in this Gram-negative bacterium. Our working hypothesis is that HpnN plays a major role in the intrinsic antimicrobial resistance of B. multivorans by shuttling hopanoids from the cytoplasmic membrane to outer membrane, strengthening the cell wall. The process of intracellular hopanoid trafficking may also require the participation of the periplasmic lipophilic protein HpnM. We will elucidate the molecular mechanisms of multidrug resistance in B. multivorans mediated by HpnN and HpnM. We will define crystal structures of B. multivorans HpnN both in the absence and presence of hopanoids. Based on the structural information, we will identify important residues for hopanoid recognition and transport. Our preliminary data strongly suggest that HpnN shuttles hopanoid molecules from the outer leaflet of the inner membrane to the outer membrane. Simulations have shown the exact pathway through HpnN for diploptene, indicating how this hopanoid molecule is exported through the channel formed by the HpnN transporter. We will ascertain the role of HpnM in hopanoid trafficking. We will also apply phage display methodology to identify novel peptides that strongly interact with HpnN or HpnM, inhibiting their function to transport hopanoids. We hypothesize that we will be able to produce unique inhibitors that render B. multivorans susceptible to antibiotics. Peptides that inhibit the function of HpnN will be used to co-crystallize with this transporter. The structures will allow us to understand the mechanism of inhibition. In addition, Galleria mellonella and mouse models of infection will be used to test the efficacy of these peptide-based inhibitors. These peptides would not inhibit the growth of Burkholderia cells in the absence of antibiotics. However, they can render bacteria susceptible to antibiotics and act as “antibiotic adjuvants” for the treatment of infections. If successful, our strategy could be transferred to other bacterial pathogens, which would provide an added mechanism to treat infections.