Systems Biology Analysis of Brucella Infected Peyers Patch Reveals Rapid Invasion with Modest Transient Perturbations of the Host Transcriptome

Brucella melitensis causes the most severe and acute symptoms of all Brucella species in human beings and infects hosts primarily through the oral route. The epithelium covering domed villi of jejunal-ileal Peyer’s patches is an important site of entry for several pathogens, including Brucella. Here, we use the calf ligated ileal loop model to study temporal in vivo Brucella-infected host molecular and morphological responses. Our results document Brucella bacteremia occurring within 30 min after intraluminal inoculation of the ileum without histopathologic traces of lesions. Based on a system biology Dynamic Bayesian Network modeling approach (DBN) of microarray data, a very early transient perturbation of the host enteric transcriptome was associated with the initial host response to Brucella contact that is rapidly averted allowing invasion and dissemination. A detailed analysis revealed active expression of Syndecan 2, Integrin alpha L and Integrin beta 2 genes, which may favor initial Brucella adhesion. Also, two intestinal barrier-related pathways (Tight Junction and Trefoil Factors Initiated Mucosal Healing) were significantly repressed in the early stage of infection, suggesting subversion of mucosal epithelial barrier function to facilitate Brucella transepithelial migration. Simultaneously, the strong activation of the innate immune response pathways would suggest that the host mounts an appropriate protective immune response; however, the expression of the two key genes that encode innate immunity anti-Brucella cytokines such as TNF-a and IL12p40 were not significantly changed throughout the study. Furthermore, the defective expression of Toll-Like Receptor Signaling pathways may partially explain the lack of proinflammatory cytokine production and consequently the absence of morphologically detectable inflammation at the site of infection. Cumulatively, our results indicate that the in vivo pathogenesis of the early infectious process of Brucella is primarily accomplished by compromising the mucosal immune barrier and subverting critical immune response mechanisms.The open access fee for this work was funded through the Texas A&M University Open Access to Knowledge (OAK) Fund

Dengue infection atlas - A systems biology approach using high-throughput data integration

Dengue virus (DENV) causes millions of infections around the world and is classified as an emerging pathogen in the USA. Though some of the steps in Dengue virus infection and replication are known, understanding of molecular mechanisms is still elusive. An unpublished microarray gene expression data from Dengue infected HuH7 liver cells at 18 hr and 24 hr p.i. was analyzed to identify differentially expressed genes (DEGs). Transcription of 30 genes was found to be perturbed (26 induced and four down-regulated). Pathway and functional analysis of the DEGs, strongly suggests that the Endoplasmic Reticulum plays an important role in Dengue virus infection. Integration of high-throughput data, DEGs, human proteins interacting with Dengue proteins (DHPINs) and human genes required for Dengue replication (DRNAi), confirms the significance of the Endoplasmic reticulum-associated protein-degradation pathway in DENV infection. DEGs identified in this work did not show extensive overlap with previously reported DEGs. Application of consistent analytical and statistical techniques to the gene expression data from ten DENV infection studies did not substantially improve the amount of overlap between the reported DEGs. Meta-analysis of the combined data using a leave-out dataset cross-validation approach, however, found a total of 1,568 differentially expressed genes. In analyses that include time points early in infection, pathways related to cancer and cell junction are enriched in DEGs, but in analyses in which early time-points are excluded, metabolic and cell signaling related pathways are enriched. This confirms that there is a progressive shift in the effect of the virus on cellular gene expression as infection progresses. Topological profiling, with respect to the human protein-interaction network (HPIN), of DEGs, DRNAi, and DHPINs from DENV shows that DEGs and DHPINs prefer proteins with high degree and betweenness centrality measures but DRNAi do not show a similar trend. However, DENV does not target proteins with high clustering coefficient. DHPINs do not interact with hub proteins (top 5% degree proteins) but rather interact with bottleneck proteins (top 5% betweenness centrality proteins). The combination of these studies substantially extends our understanding of the effects of Dengue virus on host molecular processes

Pathway Score Heat Map for Cell Communication Related Pathways.

<p>This heat map shows the repressed state of activity for the Tight Junction (TJ) and Trefoil Factors (TF) pathway in comparison to the Gap Junction, Integrin-mediated Cell Adhesion and Cell Adhesion Molecules pathways. Note that the TJ and TF pathways have a complex activation pattern. The TJ is tri-phasic in that it is highly repressed in the early stage, becomes moderately activated at 60 and 120 min p.i, and then becomes repressed at 240 min p.i. The TF pathway is bi-phasic in that it is highly repressed in the early stage but becomes moderately activated at 120 and 240 min p.i. The darker red gradients indicate higher Bayesian activation scores (more up-regulated gene expression within the pathway) while the darker green gradients indicate more repressed pathway activity (more down-regulated gene expression).</p

Heatmap of Significantly Perturbed Pathways Scores in Bovine Peyer's Patch Infected with <i>B. melitensis</i>.

<p>Thirty-seven pathways were identified as significantly perturbed (97.5% confidence) from the early stage of infection (15–60 m.p.i.). The darker red gradients indicate higher Bayesian activation scores (more up-regulated gene expression within the pathway) while the darker green gradients indicate more repressed pathway activity (more down-regulated gene expression).</p

Genes Significantly Expressed in the TLRS Pathway (<i>z</i>-score |2.24|).

<p>The table lists significantly perturbed (activated or repressed) genes at any time among the 5 different time points (0.25 h through 4 h p.i.).</p

Graphic Representation of Bovine Genes Differentially Expressed throughout the Experiment.

<p>(DE  =  differentially expressed). Solid bars represent genes up-regulated; open bars represent genes down-regulated.</p

Tight Junction Pathway Bayesian Network Model and Gene Score Heat Map.

<p>(A) Tight Junction pathway Bayesian network representation at 30 min post-inoculation. Gene nodes with orange circles on the network are those defined as mechanistic genes that surpass a threshold Bayesian z-score| >2.24|. The network shows gene nodes with gradient colors representing the level of expression (deeper red for higher up-regulated genes and deeper green for down-regulated). (B) The Bayesian score heat map for the gene expression of <i>Brucella</i> infected host Peyer's patch versus non-infected controls. The heat map is colorized and corresponds to the gene node expression levels. Grey color represents little to no expression difference between <i>Brucella</i>-infected and control loops. The heat map columns are by time post-infection in minutes.</p

Chemokine Gene Score Heat Map by Time Point Post-Inoculation.

<p>The heat map shows a number of both up-regulated and down-regulated genes (15–240 minutes post-inoculation). Note that there were large numbers of down-regulated and non-expressed chemokine genes. Red indicates an activated state while green indicates repression and grey is no expression change from control.</p

Significantly Altered Genes (Bayesian <i>z</i>-score |2.24|) in the Extra-Cellular Membrane (ECM) pathway.

<p>The table lists significantly perturbed (activated or repressed) genes at any time among the 5 different time points (0.25 h through 4 h p.i.).</p