30 research outputs found
An <i>O</i>-Methyltransferase Is Required for Infection of Tick Cells by <i>Anaplasma phagocytophilum</i>
<div><p><i>Anaplasma phagocytophilum</i>, the causative agent of Human Granulocytic Anaplasmosis (HGA), is an obligately intracellular α-proteobacterium that is transmitted by <i>Ixodes</i> spp ticks. However, the pathogen is not transovarially transmitted between tick generations and therefore needs to survive in both a mammalian host and the arthropod vector to complete its life cycle. To adapt to different environments, pathogens rely on differential gene expression as well as the modification of proteins and other molecules. Random transposon mutagenesis of <i>A</i>. <i>phagocytophilum</i> resulted in an insertion within the coding region of an <i>o</i>-methyltransferase (<i>omt</i>) family 3 gene. In wild-type bacteria, expression of <i>omt</i> was up-regulated during binding to tick cells (ISE6) at 2 hr post-inoculation, but nearly absent by 4 hr p.i. Gene disruption reduced bacterial binding to ISE6 cells, and the mutant bacteria that were able to enter the cells were arrested in their replication and development. Analyses of the proteomes of wild-type versus mutant bacteria during binding to ISE6 cells identified Major Surface Protein 4 (Msp4), but also hypothetical protein APH_0406, as the most differentially methylated. Importantly, two glutamic acid residues (the targets of the OMT) were methyl-modified in wild-type Msp4, whereas a single asparagine (not a target of the OMT) was methylated in APH_0406. <i>In vitro</i> methylation assays demonstrated that recombinant OMT specifically methylated Msp4. Towards a greater understanding of the overall structure and catalytic activity of the OMT, we solved the <i>apo</i> (PDB_ID:4OA8), the S-adenosine homocystein-bound (PDB_ID:4OA5), the SAH-Mn<sup>2+</sup> bound (PDB_ID:4PCA), and SAM- Mn<sup>2+</sup> bound (PDB_ID:4PCL) X-ray crystal structures of the enzyme. Here, we characterized a mutation in <i>A</i>. <i>phagocytophilum</i> that affected the ability of the bacteria to productively infect cells from its natural vector. Nevertheless, due to the lack of complementation, we cannot rule out secondary mutations.</p></div
<i>A</i>. <i>phagocytophilum</i> HZ proteins that are differentially abundant in the ΔOMT, according to iTRAQ results.
<p>*Average of peptides used for the quantification of the proteins.</p><p><i>**</i>Ratios <1.0 are less abundant in ΔOMT and ratios >1.0 are more abundant in ΔOMT.</p><p><i>A</i>. <i>phagocytophilum</i> HZ proteins that are differentially abundant in the ΔOMT, according to iTRAQ results.</p
Time course of wild type and ΔOMT <i>A</i>. <i>phagocytophilum</i> development within ISE6 cells
<p>Host cell-free wild-type (ApHZ) and mutant (ApΔOMT) <i>A</i>. <i>phagocytophilum</i> were harvested from HL-60 cells and incubated with ISE6 cells expressing mCherryLifeAct (red, to provide contrast) grown in MatTek chambers for 1 hr at a high MOI (100–300 bacteria/cell) to maximal internalization that permitted observation of phenotypic defects in the ΔOMT which was not possible using a stringent invasion assay. Subsequently, unbound bacteria were washed away and samples were taken at the indicated times post infection. Panels for each sampling time show wild-type bacteria at left, and mutant at right. A) 1 hr, B) 20 hr, C) 44 hr, D) 3 days, E) 4 days, F) 5 days, G) 7 days, and H) 10 days. Bacteria were labeled with FITC (green), cell nuclei were labeled with DAPI (blue), and samples were viewed by confocal microscopy to compare development of bacteria within ISE6 cells. Saturated binding allowed sufficient numbers of ApΔOMT to be internalized (1 hr panels) so that their development could be visually tracked for 10 days. From 44 hr through 10 days, small to very large morulae developed in ApHZ, while the ΔOMT persisted as individual bacteria within the ISE6 cells and never formed morulae. The size bar represents 20 μm.</p
Reduced binding of <i>A</i>. <i>phagocytophilum</i> to ISE6 cells caused by <i>omt</i> mutation or inhibition with AdOx.
<p>A) Mutation of <i>omt</i> (ΔOMT; green bar) in <i>A</i>. <i>phagocytophilum</i> HZ caused a reduction in the number of bacteria adhering to tick cells when compared to the number of wild-type bacteria per tick cell (purple bar). Bacteria purified from HL-60 cells were incubated with ISE6 cells for 30 min at room temperature. Unbound bacteria were removed using vigorous washes and the remaining attached bacteria were counted using immunofluorescence microscopy. The difference in binding was statistically significant (t-value = -4.1011; P = 0.0001). Bars represent the average number of adherent bacteria per cell, and vertical lines indicate the standard error of the mean. B) Effect of AdOx (Adenosine dialdehyde; an inhibitor of SAM-depended methyltransferases) on adherence of <i>A</i>. <i>phagocytophilum</i> HZ wild-type to ISE6 cells in comparison to the effect of mutation of the <i>omt</i> gene in ΔOMT bacteria. Wild-type <i>A</i>. <i>phagocytophilum</i> were incubated with different concentrations of AdOx for 1 hr while ΔOMT was held in medium alone, and then mixed with ISE6 cells. Bars represent the average number of bacteria bound to ISE6 cells in each treatment, and bars with the same letter are not significantly different, whereas different letters indicate a significant difference (P<0.001). The standard error of the mean from four replicates is shown as vertical lines.</p
<i>In vitro</i> methylation of recombinant <i>A</i>. <i>phagocytophilum</i> proteins by rOMT and catalytic effect of metal ions.
<p>A recombinant version of the complete OMT was produced in <i>E</i>. <i>coli</i> Rosetta 2(DE3) pLysS and purified by column affinity chromatography. The eluted rOMT was visualized by Coomassie blue staining after electrophoresis in a 4–16% gel for 1 hr. The expected size of rOMT is indicated on the left; the left lane contains lysate from uninduced <i>E</i>. <i>coli</i> cells carrying the plasmid encoding OMT. The right lane contains rOMT His-tag purified from <i>E</i>. <i>coli</i> carrying the plasmid encoding OMT, following induction with IPTG. B) Enzymatic activity of the rOMT was determined in a methylation assay that measured fluorescence resulting from production of resorufin in each sample. Average fluorescence from three replicates of each sample was plotted against assay time indicated on the X-axis. The blue dotted line represents the reaction using Msp4 as the substrate, which produced a curve expected from an enzymatic reaction. The reactions with APH_0406 (black line), TypA (dotted red line), and p44-16b (dotted gray line) as substrates produced only a minimal increase in fluorescence that was not significant. Green dotted line: negative control. C) Enzymatic activity under different concentrations of additional manganese (as MnCl<sub>2</sub>). (Red = 16 mM, Dark Blue = 8 mM, Green = 2 mM, Purple = 0.5 mM, Brown = 0 mM, Light Blue = Negative control) was tested to determine if Mn<sup>2+</sup> (included at a concentration of 10 mM in the assay kit) was a limiting factor for the OMT. Higher concentrations of MnCl<sub>2</sub> resulted in a proportional increase in velocity of the reaction, indicating that Mn<sup>2+</sup> was a co-factor and required at concentrations greater than 10 mM for optimal enzyme activity. Reactions with 16 mM additional MnCl<sub>2</sub> (17 mM total Mn<sup>2+</sup>) were completed in 90 min with fluorescence levels 25 times higher than with only 10 mM. The line graphs represent the averages from 3 replicates. Standard deviations for substrate testing ranged from 5%–10%, whereas standard deviations for Mn<sup>2+</sup> assays were <5% at all concentrations used.</p
Effects of the mutation on the growth of <i>A</i>. <i>phagocytophilum</i> in tick cell culture.
<p>Growth curves representing the replication of ΔOMT (solid blue line) and wild-type (dashed red line) bacteria in ISE6 (top) or HL-60 cells (bottom). ΔOMT and wild-type bacteria were purified from HL-60 cells and inoculated into ISE6 or HL-60 cultures. The number of bacteria was estimated by determining the copy number of the <i>msp5</i> gene. Each data point represents the average number of bacteria from triplicate samples, and vertical bars indicate the standard deviation. Statistical differences were evaluated by repeated measures ANOVA. ΔOMT bacteria were not able to replicate in ISE6 cells (top) and had decreased already by day 3, which was significantly different from the replication of wild-type bacteria in ISE6 cells (P = 0.008). This is in contrast to ΔOMT growth within HL-60 cells (bottom), in which there was no significant difference between mutant and wild-type bacteria numbers (P = 0.504).</p
Expression of the OMT during infection of ISE6 cells.
<p>qRT_PCR to track the expression of the <i>omt</i> gene in wild-type <i>A</i>. <i>phagocytophilum</i> during adhesion and invasion of ISE6 cells in comparison to <i>omt</i> gene transcripts detected in bacteria interacting with HL-60 cells. Wild-type bacteria were purified from HL-60 cells and inoculated onto cell layers of ISE6 cells or mixed with suspended HL-60 cells. RNA was purified at the indicated times post inoculation, and qRT-PCR was performed. The bars represent the average of the fold change normalized to <i>msp5</i> (blue) or to <i>rpoB</i> (red), and vertical lines represent the standard error of the mean. Up-regulation of <i>omt</i> transcription was seen as early as 30 min, reaching 34-fold change at 2 hr. At 4 hr there was no detectable difference in bacterial gene expression between the two host cell types (0.97 fold change).</p
Pathways that are altered in ΔOMT during binding to and internalization into ISE6 cells.
<p>Pathways that are altered in ΔOMT during binding to and internalization into ISE6 cells.</p
<i>A</i>. <i>phagocytophilum</i> proteins with reduced peptide methylation in the ΔOMT mutant identified by iTRAQ.
<p>* Number of occurrences of each methylated and non-methylated peptide.</p><p>* *Ratios <1.0 are less abundant in ΔOMT and ratios >1.0 are more abundant in ΔOMT.</p><p><i>A</i>. <i>phagocytophilum</i> proteins with reduced peptide methylation in the ΔOMT mutant identified by iTRAQ.</p
Catalytic site of AnphA.01233.a Bound to SAM and Mn<sup>+2</sup>.
<p>Cross-eyed stereo view of the catalytic site of AnphA.01233.a when bound to both SAM and Mn<sup>+2</sup> in stick representation. Carbons are colored yellow, nitrogens blue, oxygens red, sulfurs orange. Coordinating waters and the manganese ion are shown as red and purple spheres, respectively. Carbons from E177 from the neighboring asymmetric unit are colored green for clarity. A) In chain A E177 interacts directly with the Mn<sup>+2</sup>. B) However, in chain B interaction with the Mn<sup>+2</sup> is mediated by water molecules.</p