• Mass Spectrometry based structural analysis and systems immunoproteomics strategies deduce specifics of host-pathogen interactions

      Khan, Mohd M.; Goodlett, David Robinson, 1960- (2018)
      The innate immune system is the first line of defense against pathogens. Pattern recognition receptors (PRRs), such as the Toll-like receptors (TLRs) sense and sample pathogen-associated molecular patterns (PAMPs). On the host myeloid cell surface, the proinflammatory Gram-negative bacterial outer membrane component lipopolysaccharide (LPS, also known as endotoxin) activates the innate immune system via TLR4. Intracellularly, LPS is detected by the noncanonical inflammasome through caspase4/5/11. In the present work, mass spectrometry (MS)-based top-down structural analysis of LPS uncovered major determinants of molecular pathogenesis, and MS-based systems immunoproteomics elucidated specific features of the immune response against endotoxin. We used targeted proteomics to profile the host response to the pathogens Escherichia coli, Staphylococcus aureus, and Burkholderia cenocepacia, and we discovered significant temporal changes in the macrophage secretome. Additionally, we identified global changes in protein secretion in TLR4- and caspase11- stimulated macrophages. Finally, we observed bacterial proteomic rewiring within the biofilm forms of Burkholderia, possibly explaining the observed lowering in sensitivity to antibiotics.
    • Mechanisms of endotoxin-induced endothelial monolayer disruption: Roles of tyrosine phosphorylation, caspase activation, and the actin-associated adherens junctions

      Bannerman, Douglas Donald; Goldblum, Simeon E. (1999)
      Endothelial cell injury and/or dysfunction contributes to a variety of complications associated with Gram-negative septicemia including systemic vascular collapse, disseminated intravascular coagulation, and vascular leak syndromes. Endotoxin or bacterial lipopolysaccharide, a component of the outer membrane of Gram-negative bacteria, directly provokes endothelial injury in vitro and in vivo. Specifically, endotoxin in the presence of serum induces F-actin depolymerization, opening of the paracellular pathway, and increased endothelial monolayer permeability to macromolecules. We have identified lipid A as the bioactive moiety of bacterial lipopolysaccharide responsible for inducing an array of endothelial cell responses including increased protein tyrosine phosphorylation, actin depolymerization, increased monolayer permeability, and apoptosis. We have also found that the influence of endotoxin on endothelial cell actin organization and barrier function is mediated, in part, through a signaling pathway that is dependent on tyrosine phosphorylation events. Protein tyrosine kinase inhibition, which blocks endotoxin-induced tyrosine phosphorylation of the focal adhesion protein paxillin, protects against downstream endothelial responses including actin depolymerization, intercellular gap formation, and loss of barrier function. Further, we have studied the effect that caspase-mediated cleavage of adherens junction proteins has on mediating endotoxin-induced changes in cell-cell and cell-matrix adhesion. Endotoxin-provoked increments in transendothelial albumin flux and endothelial cell detachment occur at doses and times which are compatible with endotoxin-induced caspase activation and apoptosis. Proteins associated with the zonula adherens and focal adhesions, which mediate cell-cell and cell-matrix adhesion, respectively, are targets of caspase proteolysis. Cleavage of focal adhesion kinase leads to its dissociation from paxillin, a substrate for focal adhesion kinase tyrosine phosphorylation. The time-dependent cleavage of focal adhesion kinase and its dissociation from paxillin parallels a decrease in the phosphotyrosine content of paxillin. Caspase inhibition blocks focal adhesion kinase cleavage, decrements in paxillin phosphotyrosine content, and endothelial cell detachment, but fails to protect against endotoxin-induced endothelial barrier dysfunction. Protein tyrosine kinase inhibition, however, fails to block proteolysis but does protect against increased monolayer permeability. These findings suggest a bifurcation in the pathways through which endotoxin influences cell-cell adhesion and opening of the paracellular pathway versus endothelial cell adhesion to and detachment from the underlying extracellular matrix.
    • Modulation of microbial growth and antimicrobial activity of aminoglycosides by oxygen tensions in gram-negative bacteria

      Park, Matthew Kihoon; Marzella, Louis (1991)
      Oxygen tensions alter microbial growth, antimicrobial activity, and host responses to infections. In particular, hyperoxia is bactericidal for microorganisms with deficient antioxidant defenses and influences the activity of several classes of antimicrobial agents. I hypothesized that hyperoxia can augment the bacteriostatic and bactericidal effects of protein synthesis inhibitors by increasing the generation of reactive oxygen species. To test this hypothesis, I have characterized the interactions between hyperoxia and aminoglycosides on the growth of gram-negative bacteria. Growth inhibition was quantitated by determining the postantibiotic effect (PAE; the period of bacterial growth suppression that follows a brief exposure to an antibiotic) under normoxic and hyperoxic conditions. I found that hyperoxia (100% O{dollar}\sb2{dollar}, 101.3 kPa) enhanced the PAE of tobramycin against P. aeruginosa. The delay in growth recovery was associated with reduced rates of protein synthesis. Hyperbaric oxygen (100% O{dollar}\sb2{dollar}, 274.5 kPa) further suppressed the growth recovery of P. aeruginosa. I next examined the effects of hyperoxia on bacterial growth and killing by aminoglycosides in the family Enterobacteriaceae. While hyperoxia did not influence bacterial growth, hyperoxia markedly influenced the bactericidal activity of aminoglycosides in a strain-specific manner. This finding extends the range of oxygen tensions that are known to influence the bactericidal activity of tobramycin. Finally, I investigated the role of reactive oxygen species in the hyperoxic enhancement of the tobramycin-induced PAE in P. aeruginosa. Hyperoxia is known to increase the intracellular flux of reactive oxygen species. I found that hyperoxia induced antioxidant defenses and that tobramycin blocked this induction. However, antioxidants did not decrease the susceptibility of tobramycin-exposed P. aeruginosa to hyperoxia. I conclude that hyperoxia influences the bacteriostatic and bactericidal activities of aminoglycosides against gram-negative bacteria. These findings have potential implications for the treatment of infections in patients exposed to high fractional inspired oxygen concentrations.