22 research outputs found
Alignment of Clostridial Group NEAT domains.
<p>The alignment was generated by ClustalX. The canonical 3<sub>10</sub>-helix sequence, SXXXXY, present in 33 of the 51 NEAT domains in this group, is highlighted in blue. The locations of two highly conserved phenylalanine residues are boxed in green. The methionine residue predicted to interact with heme (<a><b>Figure 6</b></a>) is highlighted in red and boxed in orange. The <i>C. botulinum</i> protein4, which was chosen for structural modeling analysis, is highlighted in magenta text.</p
Phylogenetic tree demonstrating the relationship of the 343 identified putative NEAT domains.
<p>Each unique NEAT domain is included and each genus is indicated by a different color. Branch lengths are not representative phylogenetic distance; numbers on branches are bootstrap values obtained from 100 replicates. Colored arrowheads point to NEAT domains that have been previously characterized. The black arches indicate NEAT Groups discussed chosen for analysis. The green and magenta asterisks indicate Pp-p4 and Cb-p4, which are described in detail in the text.</p
A Product of Heme Catabolism Modulates Bacterial Function and Survival
<div><p>Bilirubin is the terminal metabolite in heme catabolism in mammals. After deposition into bile, bilirubin is released in large quantities into the mammalian gastrointestinal (GI) tract. We hypothesized that intestinal bilirubin may modulate the function of enteric bacteria. To test this hypothesis, we investigated the effect of bilirubin on two enteric pathogens; enterohemorrhagic <i>E. coli</i> (EHEC), a Gram-negative that causes life-threatening intestinal infections, and <i>E. faecalis</i>, a Gram-positive human commensal bacterium known to be an opportunistic pathogen with broad-spectrum antibiotic resistance. We demonstrate that bilirubin can protect EHEC from exogenous and host-generated reactive oxygen species (ROS) through the absorption of free radicals. In contrast, <i>E. faecalis</i> was highly susceptible to bilirubin, which causes significant membrane disruption and uncoupling of respiratory metabolism in this bacterium. Interestingly, similar results were observed for other Gram-positive bacteria, including <i>B. cereus</i> and <i>S. aureus</i>. A model is proposed whereby bilirubin places distinct selective pressure on enteric bacteria, with Gram-negative bacteria being protected from ROS (positive outcome) and Gram-positive bacteria being susceptible to membrane disruption (negative outcome). This work suggests bilirubin has differential but biologically relevant effects on bacteria and justifies additional efforts to determine the role of this neglected waste catabolite in disease processes, including animal models.</p></div
Bilirubin protects EHEC from killing by J774A.1 macrophages.
<p>EHEC (86-24) was cultured with solvent (white), bilirubin (orange, 250 µM), biliverdin (green, 250 µM) or α-tocopherol (blue, 250 µM), in minimal media for 6 hours before addition to macrophages (J774A.1) at an MOI of approximately 3. (A) The amount of EHEC exposed to the macrophages is compared to the amount internalized by macrophages after 30 minutes. (B) The percentage of internalized EHEC over 2 hours was monitored for bacteria cultured with solvent (white diamonds, NaOH), bilirubin (orange squares), biliverdin (green circles) and α-tocopherol (blue triangles). Error bars represent ± one standard deviation, n = 3. Data are representative of a single experiment repeated three times with similar results and the (*) denotes a significant (P≤0.05) difference while (**) denotes a non-significant difference (P>0.05) between treated samples and solvent-treated samples.</p
<i>E. faecalis</i> metabolism decreases after exposure to bilirubin.
<p>(A) <i>E. coli</i> (86-24), <i>E. faecalis</i>, <i>B. cereus</i>, and <i>S. aureus</i> were supplemented with resazurin and incubated with heme (50 µM), biliverdin (500 µM), bilirubin (50 µM), and bilirubin ditaurate (500 µM) for either 30 minutes or 2 hours. Unreduced resazurin was monitored by absorbance at 600 nm. (B) <i>E. faecalis</i> cultures were supplemented with resazurin and solvent (NaOH, Sol.), bilirubin (100 µM, BR), biliverdin (100 µM, BV), or TTF (1 mM, a known inhibitor of succinate dehydrogenase) while incubated in 1× PBS with 0.5% sucrose for 30 minutes at 37°C. (C) Similar to panel B, <i>E. faecalis</i> cultures were supplemented with resazurin and either superoxide dismutase (SOD, 1000 U/mL) or heat-inactivated superoxide dismutase (SODi, 1000 U/mL). (D) <i>E. faecalis</i> supplemented with resazurin and diluted into 1× PBS with 0.5% sucrose (+Suc.) or without sucrose (−Suc.) and incubated for 30 minutes at 37°C. (E) <i>E. faecalis</i> supplemented with resazurin and increasing amounts of bilirubin (1, 5, 10, 20, 30, 50, and 100 µM) in similar conditions as panels B and C. Error bars represent ± one standard deviation, n = 3, and the (*) denotes a significant (P≤0.05) difference while (**) denotes a non-significant difference (P>0.05) between treated samples and solvent-treated samples.</p
Structure of the <i>B. anthracis</i> IsdX1 NEAT domain.
<p>The ribbon structure of the heme-bound form of IsdX1 is shown. NEAT domains share a conserved β-barrel fold including a 3<sub>10</sub>-helix (green) that conventionally begins with a serine and ends with a tyrosine, and eight β-strands (teal). The two tyrosine residues (Tyr-109 and Tyr-113) within the heme-binding sequence YXXXY are indicated in purple. Tyr-109 non-covalently binds to the iron atom within the heme molecule at a predicted distance of 2.2 Å. Tyr-113 H-bonds with Tyr-109 as indicated by the dotted black line (2.4 Å). Ser-24 (green) H-bonds with the buried propionate group of the heme porphyrin, increasing binding affinity (2.6 Å). PDB code: 3SIK; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104794#pone.0104794-Ekworomadu1" target="_blank">[36]</a>.</p
Fold reduction in viability of bacteria exposed to bilirubin.
<p>Mid-log phase bacteria were exposed to 200 µM bilirubin and plated to determine CFUs. Fold reduction was calculated by the quantity of bacteria unexposed to bilirubin divided by the quantity present when exposed to bilirubin, n = 3.</p
Molecular and Evolutionary Analysis of NEAr-Iron Transporter (NEAT) Domains
<div><p>Iron is essential for bacterial survival, being required for numerous biological processes. <u>NEA</u>r-iron <u>T</u>ransporter (NEAT) domains have been studied in pathogenic Gram-positive bacteria to understand how their proteins obtain heme as an iron source during infection. While a 2002 study initially discovered and annotated the NEAT domain encoded by the genomes of several Gram-positive bacteria, there remains a scarcity of information regarding the conservation and distribution of NEAT domains throughout the bacterial kingdom, and whether these domains are restricted to pathogenic bacteria. This study aims to expand upon initial bioinformatics analysis of predicted NEAT domains, by exploring their evolution and conserved function. This information was used to identify new candidate domains in both pathogenic and nonpathogenic organisms. We also searched metagenomic datasets, specifically sequence from the Human Microbiome Project. Here, we report a comprehensive phylogenetic analysis of 343 NEAT domains, encoded by Gram-positive bacteria, mostly within the phylum Firmicutes, with the exception of <i>Eggerthella</i> sp. (Actinobacteria) and an unclassified Mollicutes bacterium (Tenericutes). No new NEAT sequences were identified in the HMP dataset. We detected specific groups of NEAT domains based on phylogeny of protein sequences, including a cluster of novel clostridial NEAT domains. We also identified environmental and soil organisms that encode putative NEAT proteins. Biochemical analysis of heme binding by a NEAT domain from a protein encoded by the soil-dwelling organism <i>Paenibacillus polymyxa</i> demonstrated that the domain is homologous in function to NEAT domains encoded by pathogenic bacteria. Together, this study provides the first global bioinformatics analysis and phylogenetic evidence that NEAT domains have a strong conservation of function, despite group-specific differences at the amino acid level. These findings will provide information useful for future projects concerning the structure and function of NEAT domains, particularly in pathogens where they have yet to be studied.</p></div
Amino acid sequence of Pp-IsdC and heme binding analysis.
<p><b><i>A</i></b>- Amino acid sequence of full-length Pp-IsdC. The signal cleavage site is indicated by the arrowhead, and the NEAT domain (Pp-IsdC<sub>N</sub>) is highlighted in grey. The 3<sub>10</sub>-helix is boxed in black, and the two tyrosine residues within the heme-binding signature sequence are bolded. <b><i>B</i></b>- Spectral analysis of purified Pp-IsdC shows the presence of a Soret band at 404 nm, indicating that the protein can bind heme. The red spectra, magnified ×5, indicates Q bands at 500 and 630 nm, demonstrating that the heme bound by Pp-IsdC is oxidized (Fe<sup>3+</sup>) and is in a high-spin coordination <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104794#pone.0104794-Berry1" target="_blank">[67]</a>. <b><i>Inset:</i></b> SDS-PAGE of purified Pp-IsdC<sub>N</sub> at 14 kDa.</p
The effect of bile on EHEC growth in the presence of ROS.
<p>(A, Left) Wideband absorbance (420–580 nm) of EHEC (EDL933) cultures supplemented with plumbagin (0, 25, 50, and 75 µM) was monitored while cultures were grown at 37°C with shaking. (A, Right) The time to mid-log phase of each culture was calculated from the growth curves. (B) EHEC (EDL933) cultures supplemented with (grey bars) or without (white bars) plumbagin (50 µM) and/or BSA (2, 20, and 200 uM BSA) and/or ox bile (50, 100, 500, 1000 ug/mL ox bile). (C) EHEC (86-24) cultures were supplemented with plumbagin (50 µM) (grey bars) or without plumbagin (white bars) and either ox, rabbit (Rb), or human (Hu) bile (1 and 10 mg/mL ox bile; 0.5 and 5.0% rabbit and human bile). Error bars represent ± one standard deviation, n = 3, and (*) denotes a significant (P≤0.05) difference between treated samples and solvent-treated samples.</p