14 research outputs found

    Unique Structural Modifications Are Present in the Lipopolysaccharide from Colistin-Resistant Strains of \u3ci\u3eAcinetobacter baumannii\u3c/i\u3e

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    Acinetobacter baumannii is a nosocomial opportunistic pathogen that can cause severe infections, including hospital-acquired pneumonia, wound infections, and sepsis. Multidrug-resistant (MDR) strains are prevalent, further complicating patient treatment. Due to the increase in MDR strains, the cationic antimicrobial peptide colistin has been used to treat A. baumannii infections. Colistin-resistant strains of A. baumannii with alterations to the lipid A component of lipopolysaccharide (LPS) have been reported; specifically, the lipid A structure was shown to be hepta-acylated with a phosphoethanolamine (pEtN) modification present on one of the terminal phosphate residues. Using a tandem mass spectrometry platform, we provide definitive evidence that the lipid A isolated from colistin-resistant A. baumannii MAC204 LPS contains a novel structure corresponding to a diphosphoryl hepta-acylated lipid A structure with both pEtN and galactosamine (GalN) modifications. To correlate our structural studies with clinically relevant samples, we characterized colistin-susceptible and -resistant isolates obtained from patients. These results demonstrated that the clinical colistin-resistant isolate had the same pEtN and GalN modifications as those seen in the laboratory-adapted A. baumannii strain MAC204. In summary, this work has shown complete structure characterization including the accurate assignment of acylation, phosphorylation, and glycosylation of lipid A from A. baumannii, which are important for resistance to colistin

    The Crystal Structure of Zn(II)-Free Treponema pallidum TroA, a Periplasmic Metal-Binding Protein, Reveals a Closed Conformation

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    We previously demonstrated that Treponema pallidum TroA is a periplasmic metal-binding protein (MBP) with a distinctive alpha-helical backbone. To better understand the mechanisms of metal binding and release by TroA, we determined the crystal structure of the apoprotein at a resolution of 2.5 Å and compared it to that of the Zn(II)-bound form (Protein Data Bank accession code 1toa). apo-TroA shows a conformation even more closed than that of its Zn(II)-bound counterpart due to a 4° tilt of the C-terminal domain (residues 190 through 308) about an axis parallel to the poorly flexible backbone helix. This domain tilting pushes two loops (residues 248 through 253 and 277 through 286) towards the metal-binding site by more than 1 Å, resulting in an unfavorable interaction of I251 with D66. To avoid this contact, D66 shifts towards H68, one of the four Zn(II)-coordinating residues. The approach of this negative charge coincides with the flipping of the imidazole side chain of H68, resulting in the formation of a new hydrogen bond. The conformational change of H68, along with a slight rearrangement of D279, a C-terminal domain Zn(II)-coordinating residue, distorts the metal-binding site geometry, presumably causing the release of the bound metal ion. Ligand binding and release by TroA, and presumably by other members of the MBP cluster, differs from the “Venus flytrap” mechanism utilized by bacterial nonmetal solute-binding receptors

    Cellular responsiveness to <i>Ft</i> is conferred principally by TLR2.

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    <p>(A) HEK 293 cells stably transfected to express individual members of the mouse TLR family were incubated with a whole cell lysate of <i>Ft</i> LVS or a TLR-specific agonist as a control. The extent of cellular responsiveness after 24 h is presented in the form of relative luciferase units. *<i>P</i><0.05. (B) Similar results were obtained using HEK 293 cells transfected with human TLR homologues. *<i>P</i><0.05. Control agonists used in (A) and (B) include 1 µg/ml Pam<sub>3</sub>Cys (TLR2), 1 µg/ml poly(I∶C) (TLR3), 100 ng/ml flagellin (TLR5), 100 ng/ml LPS (TLR4), and 100 µM loxoribine (TLR7/8). HEK 293 cells stably transfected with mouse (C) and human (D) TLR9 were incubated with either 20 or 200 µg/ml of genomic DNA purified from <i>Ft</i> LVS or unmethylated CpG DNA as a positive control. Cellular responses measured after 24 h are presented in the form of relative luciferase units. **<i>P</i><0.01. (E) Bone marrow-derived macrophages (BMDMs) (2.5×10<sup>5</sup> cells/well) from wild-type (C57BL/6) and TLR9<sup>−/−</sup> mice were incubated with 8 µg/ml of genomic DNA purified from <i>Ft</i> LVS either in the absence or presence of DOTAP. The levels of cytokines released after 24 h were determined by Cytometric Bead Array (CBA) or ELISA. Results represent the mean ± SEM from three independent experiments. *** <i>P</i><0.001. (All results shown were subjected to One-way ANOVA with Bonferroni's Post-test).</p

    Discordant Results Obtained with <em>Francisella tularensis</em> during In Vitro and In Vivo Immunological Studies Are Attributable to Compromised Bacterial Structural Integrity

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    <div><p><i>Francisella tularensis</i> (<i>Ft</i>) is a highly infectious intracellular pathogen and the causative agent of tularemia. Because <i>Ft</i> can be dispersed via small droplet-aerosols and has a very low infectious dose it is characterized as a category A Select Agent of biological warfare. Respiratory infection with the attenuated Live Vaccine Strain (LVS) and the highly virulent SchuS4 strain of <i>Ft</i> engenders intense peribronchiolar and perivascular inflammation, but fails to elicit select pro-inflammatory mediators (<i>e.g.</i>, TNF, IL-1β, IL-6, IL-12, and IFN-γ) within the first ∼72 h. This <i>in vivo</i> finding is discordant with the principally T<sub>H</sub>1-oriented response to <i>Ft</i> frequently observed in cell-based studies wherein the aforementioned cytokines are produced. An often overlooked confounding factor in the interpretation of experimental results is the influence of environmental cues on the bacterium's capacity to elicit certain host responses. Herein, we reveal that adaptation of <i>Ft</i> to its mammalian host imparts an inability to elicit select pro-inflammatory mediators throughout the course of infection. Furthermore, <i>in vitro</i> findings that non-host adapted <i>Ft</i> elicits such a response from host cells reflect aberrant recognition of the DNA of structurally-compromised bacteria by AIM2-dependent and -independent host cell cytosolic DNA sensors. Growth of <i>Ft</i> in Muller-Hinton Broth or on Muller-Hinton-based chocolate agar plates or genetic mutation of <i>Ft</i> was found to compromise the structural integrity of the bacterium thus rendering it capable of aberrantly eliciting pro-inflammatory mediators (<i>e.g.</i>, TNF, IL-1β, IL-6, IL-12, and IFN-γ). Our studies highlight the profound impact of different growth conditions on host cell response to infection and demonstrate that not all <i>in vitro</i>-derived findings may be relevant to tularemia pathogenesis in the mammalian host. Rational development of a vaccine and immunotherapeutics can only proceed from a foundation of knowledge based upon <i>in vitro</i> findings that recapitulate those observed during natural infection.</p> </div
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