Alveolar Macrophages Infected with Ames or Sterne Strain of <i>Bacillus anthracis</i> Elicit Differential Molecular Expression Patterns

<div><p>Alveolar macrophages (AMs) phagocytose <i>Bacillus anthracis</i> following inhalation and induce the production of pro-inflammatory cytokines and chemokines to mediate the activation of innate immunity. Ames, the virulent strain of <i>B. anthracis</i>, contains two plasmids that encode the antiphagocytic poly-γ-d-glutamic acid capsule and the lethal toxin. The attenuated Sterne strain of <i>B. anthracis</i>, which lacks the plasmid encoding capsule, is widely adapted as a vaccine strain. Although differences in the outcome of infection with the two strains may have originated from the presence or absence of an anti-phagocytic capsule, the disease pathogenesis following infection will be manifested via the host responses, which is not well understood. To gain understanding of the host responses at cellular level, a microarray analysis was performed using primary rhesus macaque AMs infected with either Ames or Sterne spores. Notably, 528 human orthologs were identified to be differentially expressed in AMs infected with either strain of the <i>B. anthracis.</i> Meta-analyses revealed genes differentially expressed in response to <i>B. anthracis</i> infection were also induced upon infections with multiple pathogens such as <i>Francisella Novicida</i> or <i>Staphylococcus aureus</i>. This suggests the existence of a common molecular signature in response to pathogen infections. Importantly, the microarray and protein expression data for certain cytokines, chemokines and host factors provide further insights on how cellular processes such as innate immune sensing pathways, anti-apoptosis versus apoptosis may be differentially modulated in response to the virulent or vaccine strain of <i>B. anthracis</i>. The reported differences may account for the marked difference in pathogenicity between these two strains.</p></div

Overlap between the genes identified from current study and studies referenced.

<p>Explanation of the column headings in table are as follows: Pathogens: name of the pathogen used in the referenced microarray studies; # of overlapping genes: the number of genes that are overlapping between the current study and the referenced studies; Cells used in the study: the type of cells used in the referenced microarray studies; Selected Overlapping Genes: selected gene symbols that are overlapping between the two studies; P-value: the P-value of the overlap; PMID or GEO ID: the reference of the published microarray studies.</p

AMs infected with Ames or Sterne spores show differential pro-inflammatory cytokine/chemokine protein expression patterns.

<p>AMs were infected with either Ames or Sterne spores at an MOI of 10. Supernatants were collected at indicated time points and cytokine/chemokines levels were quantified. The experiment was performed at least three times and data for three rhesus macaque donors are shown. Scatter plots are presented as mean ± Standard Deviation. Experiments for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087201#pone-0087201-g003" target="_blank">Figure 3</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087201#pone.0087201.s001" target="_blank">Figure S1</a> were performed concurrently and shared the same controls. P-value is calculated using a paired Student’s t-test. *represents p-value <0.05 and **represents p-value <0.01.</p

Microarray analyses identified 528 human orthologs which were differentially expressed between Ames and Sterne infected AMs.

<p>AMs obtained from five rhesus macaque donors were infected with Ames or Sterne spores at an MOI of 10 for indicated time points. Total mRNAs were purified and hybridized to rhesus macaque cDNA microarrays. “Ames vs Sterne” bar depicts the ratio of gene expressions (in logarithmic scale) between Ames-infected AMs to Sterne-infected counterparts. “Ames” and “Sterne” bar depict fold change of a gene expression (in logarithmic scale) by normalizing Ames or Sterne treated AMs with the 0 h time point. The level of fold changes are colored coded, where red stands for high values (>1) and blue for low fold changes (<1). To highlight statistically significant changes for “Ames vs. Sterne” for the ease of visualization, we decreased the contrast by three folds for the portion of the heat map, where differential expression is not significant.</p

Gene ontology (GO) analysis.

<p>Statistically significant overrepresentation of selected functional classes and protein families based upon gene ontology (GO) analysis.</p

Time-dependent kinetic difference in the induction of COX-2 and PGE₂ expression (A) AMs were infected with either Ames or Sterne spores for 90 min, 4 h or 18 h.

<p>Cells were lysed and the mRNA was purified and quantified by real time PCR. Fold expression was calculated by normalizing to time 0. (B) AMs were infected with Ames or Sterne spores at an MOI of 10. The amount of PGE₂ was quantified by ELISA. Data shown in (A) and (B) are representative of n = 3 experiments. Scatter plots are presented as mean ± Standard deviation. P-value is calculated using a paired two tailed Student’s t-test. *represent p-value <0.05.</p

A Schematic diagram of observed differential modulation of host responses to Ames and Sterne spores of <i>B. anthracis</i>.

<p>The diagram is based on the observed differences in the cytokine/chemokine and host responses in AMs infected with either Ames or Sterne spores. TFs: Transcription factors.</p

L'Écho : grand quotidien d'information du Centre Ouest

14 février 19441944/02/14 (A73,N726).Appartient à l’ensemble documentaire : PoitouCh

L'Écho : grand quotidien d'information du Centre Ouest

25 décembre 19241924/12/25 (A53).Appartient à l’ensemble documentaire : PoitouCh

siRNA Screen Identifies Trafficking Host Factors that Modulate Alphavirus Infection

<div><p>Little is known about the repertoire of cellular factors involved in the replication of pathogenic alphaviruses. To uncover molecular regulators of alphavirus infection, and to identify candidate drug targets, we performed a high-content imaging-based siRNA screen. We revealed an actin-remodeling pathway involving Rac1, PIP5K1- α, and Arp3, as essential for infection by pathogenic alphaviruses. Infection causes cellular actin rearrangements into large bundles of actin filaments termed actin foci. Actin foci are generated late in infection concomitantly with alphavirus envelope (E2) expression and are dependent on the activities of Rac1 and Arp3. E2 associates with actin in alphavirus-infected cells and co-localizes with Rac1–PIP5K1-α along actin filaments in the context of actin foci. Finally, Rac1, Arp3, and actin polymerization inhibitors interfere with E2 trafficking from the trans-Golgi network to the cell surface, suggesting a plausible model in which transport of E2 to the cell surface is mediated via Rac1- and Arp3-dependent actin remodeling.</p></div