Integrating High-Content Imaging and Chemical Genetics to Probe Host Cellular Pathways Critical for <em>Yersinia Pestis</em> Infection

<div><p>The molecular machinery that regulates the entry and survival of <em>Yersinia pestis</em> in host macrophages is poorly understood. Here, we report the development of automated high-content imaging assays to quantitate the internalization of virulent <em>Y. pestis</em> CO92 by macrophages and the subsequent activation of host NF-κB. Implementation of these assays in a focused chemical screen identified kinase inhibitors that inhibited both of these processes. Rac-2-ethoxy-3 octadecanamido-1-propylphosphocholine (a protein Kinase C inhibitor), wortmannin (a PI3K inhibitor), and parthenolide (an IκB kinase inhibitor), inhibited pathogen-induced NF-κB activation and reduced bacterial entry and survival within macrophages. Parthenolide inhibited NF-κB activation in response to stimulation with Pam3CSK4 (a TLR2 agonist), <em>E. coli</em> LPS (a TLR4 agonist) or <em>Y. pestis</em> infection, while the PI3K and PKC inhibitors were selective only for <em>Y. pestis</em> infection. Together, our results suggest that phagocytosis is the major stimulus for NF-κB activation in response to <em>Y. pestis</em> infection, and that <em>Y. pestis</em> entry into macrophages may involve the participation of protein kinases such as PI3K and PKC. More importantly, the automated image-based screening platform described here can be applied to the study of other bacteria in general and, in combination with chemical genetic screening, can be used to identify host cell functions facilitating the identification of novel antibacterial therapeutics.</p> </div

Hits identified from the LOPAC¹²⁸⁰ library screening in the phagocytosis assay and their corresponding target class and target selectivity.

*<p>False positive.</p

Chemical Structures.

<p>Structures of select ‘hit compounds’ identified during focused library screening. *For clarity, the structure of Prochlorperazine is shown in the organic form (versus the salts indicated in the ‘Compound Name’).</p

Inhibition of early NF-κB activation by select compounds is dependent on the applied inducer.

<p>Raw264.7 macrophages pretreated for 2 hr with: 1 & 2) DMSO control; 3) rac-2-Ethoxy-3 octadecanamido-1-propylphosphocholine; 4) parthenolide; 5) wortmannin; 6) tyrphostin A9; 7) pifithrin-mu, and then treated for 30 min with inducers (lanes 2–7) (<b>A</b>) Pam3CSK4 (1 µg/ml); (<b>B</b>) LPS (1 µg/ml); and (<b>C</b>) <i>Y</i>. <i>pestis</i> CO92 (10∶1 MOI). The data is the average of six replicates ± standard deviation, and is representative of three independent experiments. (<b>D</b>) Western blot analysis of cell lysates obtained from macrophages pretreated for 2 hr with: 1 & 2) DMSO control; 3) rac-2-Ethoxy-3 octadecanamido-1-propylphosphocholine; 4) parthenolide; 5) wortmannin; 6) tyrphostin A9 and then infected for 30 min with <i>Y. pestis</i> (lanes 2–6). In the left panel, the blots were probed with α pIκBα (Ser 32) antibody to measure IκBα phosphorylation and IκBα antibody to measure IκBα degradation. Transferrin receptor antibody was used as loading control. The right panel is a bar graph of the densitometric scan of the pIκBα and transferrin receptor bands and is depicted as a ratio of the pixel intensities for the two bands.</p

Regulation of <i>Y</i>. <i>pestis</i> infection by select hit compounds.

<p>A bar graph showing the percentage of viable bacteria in RAW264.7 macrophages treated with DMSO control (and normalized to 100% viable bacteria) or 10 µM of the compounds (except Wortmannin which was applied at 20 µM). Bacterial viability was determined at 2 hr following infection. The percentage of viable bacteria is the average ± standard deviation from two replicates of two independent experiments.</p

Quantitative imaging of internalized <i>Y. pestis</i>.

<p>(<b>A</b>) Transmission electron microscopy showing <i>Y. pestis</i> CO92 internalized within RAW264.7 macrophages at 2 hr (left panel; scale bar –500 nm) and 8 hr (right panel; scale bar –2 microns) post infection. Some extracellular bacteria (arrow outside the cell) are also visible at 2 hr post infection. Bacteria confined within the vacuoles of the macrophages are shown by a single arrow inside the cell. N - Nucleus. (<b>B</b>) Immunofluorescent staining of RAW264.7 macrophages that were not infected (left panel) or infected with <i>Y</i>. <i>pestis</i> CO92 following pre-treatment with DMSO control (middle panel) or cytochalasin D (right panel). Green - <i>Y</i>. <i>pestis</i> stained with α-F1 antibody, red - phalloidin, blue – nucleus stained with Hoechst dye. Scale bar = 5 µm. (<b>C</b>) Image segmentation of macrophages infected with <i>Y. pestis</i>. Images acquired in UV channel were segmented to mark the boundaries of the nuclei (top left), cell boundaries were segmented on the images based on a 640 nm laser channel (cytoplasm channel, top right), segmentation of total spots (bacteria) for image fields acquired in the 488 nm channel (bottom left), classification of spot candidates (internalized bacteria) based on the attribute values for contrast and spot-to-cell intensity above the limits set by the input parameters (bottom right). The different colored spots in the bottom panels represent individual spots (bacteria) and were used for better visualization. The non-segmented white spots in the bottom right panel represent extracellular bacteria. Scale bar = 20 µm. (<b>D</b>) Dual staining of GFP expressing <i>Y. pestis</i> (Pgm⁻, Pla⁻) in infected RAW264.7 macrophages to quantitate cell-associated and internalized bacteria. RAW264.7 macrophages were infected for 2 hr with 30∶1 MOI of GFP expressing bacteria. After fixation but without permeabilization of the macrophages, the GFP labeled bacteria were immunostained with αF1 antibody followed by staining with Alexa 568 secondary antibody. Graphical representation of number of bacteria expressing GFP alone or dual staining is shown in the Top panel. Bottom panel is the scatter plot representation of single cell analysis of triplicate wells. The mean relative spot (bacteria) intensity in the red channel is plotted against the green channel for all cells with ≥ one classified spot. (<b>E</b>) Enumerating internalized bacteria. Graphical representations of the four output features (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055167#pone.0055167.s008" target="_blank">Table S1</a>) that were collected from the image analysis of RAW264.7 macrophages that were either not infected or infected with <i>Y</i>. <i>pestis</i> following pretreatment with DMSO control or cytochalasin D. The data represents the average of six replicates ± standard deviation, and is representative of three independent experiments.</p

NF-κB screening assay results.

<p> (<b>A</b>) Variance accounted for by each of the principal components (PCs). (<b>B–D</b>) Scatter plots showing PC1 and PC2 (<b>B</b>), PC1 and PC3 (<b>C</b>), and PC2 and PC3 (<b>D</b>) for compounds tested (black) and DMSO controls (green). Input variables are represented by red vectors: a = number of cells Z-score, b = nucleus size Z-score, c = intensity contrast Z-score, d = normalized nucleus intensity Z-score, e = intensity ratio Z-score, f = intensity difference Z-score, g = nucleus intensity Z-score, h = cytoplasm intensity Z-score, and i = normalized cytoplasm intensity Z-score. (<b>E</b>) Mahalanobis distance in PC1-3 space from each well to the medoid DMSO control well. Compounds with Mahalanobis distance >15 and cell number Z score ≥ −3 were considered hits. (<b>F</b>) A pie chart showing the number of hits (19) and cytotoxic compounds (38) from a total of 1280 compounds screened. (<b>G</b>) A pie chart showing the distribution of the identified hits based on their target classes. (<b>H</b>) Dose response curves of select inhibitors of the NF-κB translocation assay. 1 - rac-2-Ethoxy-3 octadecanamido-1-propylphosphocholine; 2– parthenolide; 3– wortmannin; 4– pifithrin-mu; 5– tyrphostin A9. The data is the average of two replicates ± standard deviation, and is representative of three independent experiments.</p

Phagocytosis screening assay results.

<p>(<b>A</b>) Variance accounted for by each of the principal components (PCs). (<b>B</b>) A biplot showing PC1 and PC2 for compounds (black) and DMSO controls (green). Input variables are represented by red vectors: a = number of cells Z-score, b = number of spots Z-score, c = spots per cell Z-score, and d = background-corrected spot signal per cell area Z-score. (<b>C</b>) Mahalanobis distance in PC1–PC2 space from each well to the medoid DMSO treated and infected control well. Compounds with Mahalanobis distance >15 and cell number Z score ≥ −3 were considered as hits. (<b>D</b>) A pie chart showing the number of hits (16) and cytotoxic compounds (26) from a total of 1280 compounds screened. (<b>E</b>) A pie chart showing the distribution of the identified hits based on their target classes. (<b>F</b>) Dose response curves of select inhibitors from the phagocytosis assay. The percent inhibition is the average ± standard deviation from duplicate wells of three independent experiments.</p

Rapid Real-Time PCR Assays for Detection of Klebsiella pneumoniae with the rmpA or magA Genes Associated with the Hypermucoviscosity Phenotype: Screening of Nonhuman Primates

The relationship of mucoviscosity-associated (magA) and/or regulator of mucoid phenotype (rmpA) genes to the Klebsiella pneumoniae hypermucoviscosity (HMV) phenotype has been reported. We previously demonstrated that rmpA+ K. pneumoniae can cause serious disease in African green monkeys and isolated rmpA+ and magA+ HMV K. pneumoniae from other species of non-human primates. To rapidly screen African green monkeys/non-human primates for these infections, we developed three real-time PCR assays. The first was K. pneumoniae-specific, targeting the khe gene, while the others targeted rmpA and magA. Primer Express 2 was used with the three K. pneumoniae genes to generate sequence-specific TaqMan/TaqMan-Minor Groove Binder assays. Oral/rectal swabs and necropsy samples were collected; swabs were used for routine culture and DNA extraction. K. pneumoniae colonies were identified on the Vitek 2 with DNA tested using the K. pneumoniae-specific assays. Testing of 45 African green monkeys resulted in 19 khe+ samples from 14 animals with none positive for either rmpA or magA. Of these 19 khe+ samples, five were culture-positive, but none were HMV “string test”-positive. Subsequent testing of 307 non-human primates resulted in 64 HMV K. pneumoniae isolates of which 42 were rmpA+ and 15 were magA+. Non-human primate testing at the U.S. Army Medical Research Institute of Infectious Diseases demonstrated the ability to screen both live and necropsied animals for K. pneumoniae by culture and real-time PCR to determine HMV genotype