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Scholarly Works (5 results)

  • Thesis
  • Peer Reviewed
UC San Diego Electronic Theses and Dissertations (2016)

Multidrug-resistant Gram-negative (MDRGN) bacterial pathogens pose a significant threat to public health in the US and worldwide. The lack of therapeutic options for MDRGN bacterial infection have forced clinicians to use older, once rarely used antibiotics that are associated with significant toxicity. An increasing number of immunocompromised individuals, including the elderly, premature neonates, surgical patients, organ transplant recipients, and those receiving cancer chemotherapy, represent an expanding reservoir for MDRGN bacteria in the hospital setting. Research to understand the molecular host-pathogen interactions involved in MDRGN bacterial infections and reveal novel therapeutic targets are imperative if we are to prevent the advent of a post-antibiotic era.

For this PhD dissertation project, I began by searching for genes in the MDRGN bacterial pathogen Acinetobacter baumannii that are important in resisting the bactericidal action of the human cathelicidin antimicrobial peptide, LL-37, a critical front-line effector of host innate defense. A transposon insertion in vacJ, a component of the mla system conserved in all Gram-negative species, proved important in mediating resistance to LL-37, detergents, whole blood, complement, and neutrophils. A. baumannii lacking vacJ demonstrated reduced virulence compared to wild-type parent strain in a murine pneumonia model. To expand the general significance of this work, I examined the virulence capacity of multiple mla mutants in Pseudomonas aeruginosa, a leading MDRGN opportunistic human pathogen. P. aeruginosa lacking an mla system exhibited increased sensitivity to LL-37 and its murine homolog, as well as membrane active pharmaceuticals, serum, and whole blood. A murine pneumonia model revealed reduced bacterial burden and mortality when infected with bacteria lacking vacJ. Finally, I explored how a common antibiotic, azithromycin, traditionally considered ineffective against MDRGN bacteria in standard testing assays, gained entry to its ribosomal target when endogenous antimicrobial peptides modify the outer membrane, allowing it to provide clear efficacy in an in vivo model of A. baumannii pneumonia. Altogether, this dissertation furthered our understanding on the importance outer membrane integrity for Gram-negative bacteria to resist host immune clearance. This research points to a possible therapeutic target in the mla pathway to control MDRGN infections by sensitizing them to the host innate immune system.

    Cover page: New Approaches to Sensitize Multidrug-Resistant Gram-Negative Bacterial Pathogens to Host Innate Immune Clearance
    • Article
    • Peer Reviewed
    UC San Diego Previously Published Works (2019)
    To maintain the lipid asymmetry of the cell envelope in Gram-negative bacteria, the MlaC protein serves as a lipid transfer factor and delivers phospholipids from the outer to the inner membrane. A strategy of antibiotic discovery is to design a proper compound that can tightly bind to the MlaC protein and inhibit the MlaC function. In this study, we performed virtual screening on multiple MlaC structures obtained from molecular dynamics simulations to identify potential MlaC binders. Our results suggested that clorobiocin is a compound that could bind to the MlaC protein. Through the comparison of the bound geometry between clorobiocin and novobiocin, we pointed out that the methyl-pyrrole group of the noviose sugar in clorobiocin forms hydrophobic interactions with amino acids in the phospholipid binding pocket, which allows the compound to bind deep in the active site. This also explains why clorobiocin shows a tighter binding affinity than novobiocin. Our study highlights a practical path of antibiotic development against Gram-negative bacteria.
      • Article
      • Peer Reviewed
      UC San Diego Previously Published Works (2016)
      The composition of the outer membrane in Gram-negative bacteria is asymmetric, with the lipopolysaccharides found in the outer leaflet and phospholipids in the inner leaflet. The MlaC protein transfers phospholipids from the outer to inner membrane to maintain such lipid asymmetry in the Mla pathway. In this work, we have performed molecular dynamics simulations on apo and phospholipid-bound systems to study the dynamical properties of MlaC. Our simulations show that the phospholipid forms hydrophobic interactions with the protein. Residues surrounding the entrance of the binding site exhibit correlated motions to control the site opening and closing. Lipid binding leads to increase of the binding pocket volume and precludes entry of the water molecules. However, in the absence of the phospholipid, water molecules can freely move in and out of the binding site when the pocket is open. Dehydration occurs when the pocket closes. This study provides dynamic information of the MlaC protein and may facilitate the design of antibiotics against the Mla pathway of Gram-negative bacteria.
        • Article
        • Peer Reviewed
        UC San Diego Previously Published Works (2017)
        Pseudomonas aeruginosa is an important opportunistic pathogen that has become a serious problem due to increased rates of antibiotic resistance. Due to this along with a dearth in novel antibiotic development, especially against Gram-negative pathogens, new therapeutic strategies are needed to prevent a post-antibiotic era. Here, we describe the importance of the vacJ/Mla pathway in resisting bactericidal actions of the host innate immune response. P. aeruginosa tn5 transposon mutants in genes from the VacJ/Mla pathway showed increased susceptibility to killing by the host cathelicidin antimicrobial peptide, LL-37, when compared to the wild-type parent strain. The P. aeruginosa vacJ - mutant demonstrated increased membrane permeability upon damage as well as sensitivity to killing in the presence of the detergent sodium dodecyl sulfate and the divalent cation chelator EDTA. When exposed to human whole blood and serum complement, the vacJ - mutant was killed more rapidly when compared to the wild-type parent strain and complemented mutant. Finally, in an in vivo mouse lung infection model, infection with the vacJ - mutant resulted in reduced mortality, lower bacterial burden, and reduced lung damage when compared to the wild-type strain. This study highlights the potential in therapeutically targeting the VacJ/Mla pathway in sensitizing P. aeruginosa to killing by the host innate immune response.

        Key messages

        • The Mla pathway regulates outer membrane dynamics in human pathogen Pseudomonas aeruginosa (PA). • Disruption of Mla pathway gene vacJ sensitizes PA to host cathelicidin antimicrobial peptide LL-37. • Loss of vacJ expression renders PA more sensitive to human whole blood and serum killing. • Loss of vacJ expression reduces PA survival and virulence in a murine lung infection model. • The Mla pathway merits exploration as a pharmacologic target to sensitize PA to host innate immunity.
          Cover page: The Mla pathway is critical for Pseudomonas aeruginosa resistance to outer membrane permeabilization and host innate immune clearance
          • Article
          • Peer Reviewed
          UC San Diego Previously Published Works (2017)
          We asked whether beta-lactamase inhibitors (BLIs) increased the activity of daptomycin (DAP) against methicillin-resistant Staphylococcus aureus (MRSA), the peptide antibiotic colistin (COL) against the emerging Gram-negative nosocomial pathogen Acinetobacter baumannii, and the human host defense peptide cathelicidin LL37 against either pathogen. DAP and LL37 kill curves were performed with or without BLIs against MRSA, vancomycin-intermediate S. aureus (VISA), and heterogeneous VISA (hVISA). COL and LL37 kill curves were performed against A. baumannii Boron-dipyrromethene (BODIPY)-labeled DAP binding to MRSA grown with the BLI tazobactam (TAZ) was assessed microscopically. The combination of COL plus TAZ was studied in a murine model of A. baumannii pneumonia. TAZ alone lacked in vitro activity against MRSA or A. baumannii The addition of TAZ to DAP resulted in a 2- to 5-log10 reduction in recoverable MRSA CFU at 24 h compared to the recoverable CFU with DAP alone. TAZ plus COL showed synergy by kill curves for 4 of 5 strains of A. baumannii tested. Growth with 20 mg/liter TAZ resulted in 2- to 2.5-fold increases in the intensity of BODIPY-DAP binding to MRSA and hVISA strains. TAZ significantly increased the killing of MRSA and A. baumannii by LL37 in vitro TAZ increased the activity of COL in a murine model of A. baumannii pneumonia. Classical BLIs demonstrate synergy with peptide antibiotics. Since BLIs have scant antimicrobial activity on their own and are thus not expected to increase selective pressure toward antibiotic resistance, their use in combination with peptide antibiotics warrants further study.
            Cover page: Classical β-Lactamase Inhibitors Potentiate the Activity of Daptomycin against Methicillin-Resistant Staphylococcus aureus and Colistin against Acinetobacter baumannii
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