• Establishing a Lipid Model of Host-Pathogen Interaction Using Multimodal Mass Spectrometry Imaging in a Francisella Infection

      Scott, Alison June; Ernst, Robert K.; 0000-0001-6969-4707 (2015)
      Host membranes are intimately involved in the immune response to any infection, including formation of lipid docking sites for proteins, organization of immune signaling complexes in lipid rafts, and maintaining a reservoir of fatty acids that contribute to acute inflammation. Francisella species maintain several host immune evasion strategies, one of which involves induction of the immunomodulatory lipid prostaglandin E2 (PGE2). The source of PGE2 is arachidonic acid (AA), a structural component of membrane phospholipids. Mass spectrometry imaging (MSI) was used to map and characterize both host- and pathogen-borne lipids using Francisella infected spleens in a murine model. Here, we identified and mapped the unique bacterial molecule, lipid A within infected mouse spleens by MALDI-MSI, confirming the in vivo structure (m/z 1665.1) in a mammalian infection. Francisella lipid A mapped primarily to the red pulp of the spleen, with signal first appearing between 24 and 36 hours post-infection, corresponding to the onset of bacteremia. Numerous changes in host lipid levels were correlated with progression of the infection. A phosphatidylinositol species, 1-stearoyl, 2-arachidonyl phosphatidylinositol (SAPI) was identified in the periphery of the splenic white pulp, suggesting a cell-specific origin. SAPI abundance peaks at 24 hours and is depleted in the timepoints preceding lethality (48 to 60 hours). In vitro reports demonstrate that SAPI is the earliest source of AA in activated macrophages. We have subsequently linked importation of SAPI into the spleen by monocytic infiltrates, which increases the total SAPI load. Additionally, accumulation of cholesterol was observed by SIMS-Imaging in the infected spleens and may be another indicator of immune infiltration. These data highlight a role for newly immigrant cells in contributing to the pool of total inflammatory lipids. Here, MSI is presented as a new approach to studying lipid-level host-pathogen interactions, facilitating targeted and untargeted discovery.
    • Immune Responses to a Francisella Lipid A Mutant: Characterization and Therapeutic Potential

      Powell, Daniel Alford; Ernst, Robert K. (2012)
      Francisella tularensis tularensis (Ft) is an intracellular Gram-negative bacterium and the causative agent of the severe human disease tularemia with potential for use as a bioweapon. Francisella lipid A, normally the biologically active component of lipopolysaccharide (LPS) has extremely low endotoxic activity. A Francisella tularensis novicida (Fn) lipid A biosynthesis mutant was generated that lacked the 4'-phosphatase enzyme (LpxF). Analysis of lipid A isolated from this mutant strain, compared to WT Fn showed retention of the phosphate moiety at the 4' position and the N-linked fatty acid at the 3' position on the diglucosamine backbone. This mutant was previously shown in our laboratory to be avirulent and confer protective immunity to a lethal WT Fn challenge. Further work has been carried out to elucidate the mechanisms of this protective immunity. The role of B cells was examined using μMT<super>-/-</super> mice, which lack functional B cells. While all mice survived the initial inoculation, all of the μMT<super>-/-</super> mice succumbed to the lethal challenge. This complete lack of protection shows that B cells are absolutely required for the protective response generated by the lpxF-null mutant. While development of an effective vaccine to Francisella remains a priority, we decided to address the therapeutic potential of serum obtained from immunization with the lpxF-null mutant in the possibility of a Francisella outbreak. Mice were infected with Fn and then were treated with serum either from naïve or immune mice. Mice receiving immune serum were completely rescued out to 36 hours post-infection, and were partially rescued at 48 hours, whereas mice receiving naïve serum started succumbing to infection at 60 hours post-infection, indicating the serum has therapeutic potential even late in an infection. The identity of the proteins recognized by this protective serum was further investigated. Our work not only identified known Francisella immunogens but revealed proteins previously unknown to be antigenic. Serum from LVS lpxF-null vaccinated-mice showed a similar protective capacity when given as a therapeutic. Current work is being carried out to generate monoclonal antibodies to these identified proteins and assay their ability to be used in the event of a Francisella outbreak.
    • Modifications to lipid A in Acinetobacter baumannii: Potential Biomarkers for Rapid Diagnosis of Colistin Resistance

      Casella, Leila Guerrero; Ernst, Robert K. (2013)
      One of the major challenges in the management of A. baumannii infections is the rapid dissemination of multidrug resistant (MDR) strains and diminishing antibiotic choices available to treat this troublesome pathogen. In this context, physicians are turning to the potentially nephrotoxic cationic antimicrobial peptide, Colistin (polymyxin E) to treat patients infected with A. baumannii MDR strains. The emergence of A. baumannii Colistin-resistant clinical isolates has raised concerns among physicians as this antibiotic is used as a salvage therapy. More alarming, is the identification of Colistin-heteroresistant subpopulations in susceptible A. baumannii clinical isolates that may lead to the development of complete resistance to Colistin. Studies assessing reliability of current susceptibility test methods have suggested poor concordance and revealed a further problem: unreliable detection of heteroresistant subpopulations. Thus, rapid and reliable susceptibility testing methods are critical to deliver effective antimicrobial therapies and minimize the risk of failed treatments resulting in adverse clinical outcomes. Mass-spectrometry (MS) assays have proven to be an important tool to identify bacterial species. The MS-based identification method (Bruker Biotyper) is based on proteomic profiling using MALDI-TOF (Matrix Assisted Laser Desorption-Ionization Time-of-Flight), but the inability to distinguish antibiotic resistance profiles represents a significant limitation. Lipid profiles, especially modifications to lipid A, the membrane anchor of lipopolysaccharide can be directly linked to antimicrobial peptide resistance patterns. Using this MS approach to analyze modifications to lipid A has enabled us to determine unique diagnostic biomarkers to rapid identify Colistin-sensitivity or -resistance profiles in laboratory-adapted A. baumannii, including characterization of heterosensitivity. This work was further extended to analyze lipid A from patient samples to validate the clinical utility of this biomarker. The detection of a novel, modified lipid A with ethanolamine at the 4'position and a novel hexosamine addition. Subsequent LC/MS analysis identified the positively charged hexosamine as galactosamine attached at the 1 position of lipid A. Modification of lipid A with these positively charged constituents was concordant with antibiotic susceptibility profiles. Together, these findings clearly demonstrate the feasibility of this type of analysis as a diagnostic test for rapid determination of antimicrobial susceptibility profiles in clinical settings.