Comparative Analyses of Transcriptional Profiles in Mouse Organs Using a Pneumonic Plague Model after Infection with Wild-Type Yersinia pestis CO92 and Its Braun Lipoprotein Mutant

Abstract

We employed Murine GeneChips to delineate the global transcriptional profiles of the livers, lungs, and spleens in a mouse pneumonic plague infection model with wild-type (WT) Y. pestis CO92 and its Braun lipoprotein (Deltalpp) mutant with reduced virulence. These organs showed differential transcriptional responses to infection with WT Y. pestis, but the overall host functional processes affected were similar across all three tissues. Gene expression alterations were found in inflammation, cytokine signaling, and apoptotic cell death-associated genes. Comparison of WT and Deltalpp mutant-infected mice indicated significant overlap in lipopolysaccharide- (LPS-) associated gene expression, but the absence of Lpp perturbed host cell signaling at critical regulatory junctions resulting in altered immune response and possibly host cell apoptosis. We generated a putative signaling pathway including major inflammatory components that could account for the synergistic action of LPS and Lpp and provided the mechanistic basis of attenuation caused by deletion of the lpp gene from Y. pestis in a mouse model of pneumonic plague.

Figure 1

Venn diagram showing the overlap of major functions of genes identified as significantly altered in the liver, spleen, and lung of mice infected with WT Y. pestis CO92. Functions were obtained using Ingenuity software, with genes identified at 12 hours or 48 hours in each tissue type analyzed separately. Fisher's Exact Test was used as the scoring method for determining significance of overrepresented molecular functions and pathways.

Figure 2

Hierarchical clustering of genes determined to be significantly altered in mice at 12 hours and 48 hours in response to WT Y. pestis CO92 infection (a) and in the livers (b), lungs (c), and spleens (d) of mice infected for 48 hours with a Δlpp mutant of Y. pestis CO92, compared to WT Y. pestis. Clustering was performed using Genespring GX 10.0 on normalized and log transformed signal ratios. The three replicate samples representing the three experimental conditions (uninfected animals and mice infected with WT Y. pestis CO92 or its Δlpp mutant) are labeled as control, WT, and mutant, respectively. Note that mice infected for 48 hours with WT Y. pestis CO92 exhibited a collection of altered gene expressions that were common to all three tissues examined (panel a). Most notably, the three replicates representing tissue (liver, lung, or spleen) infected with the mutant clustered together, and mutant-infected samples clustered apart from uninfected controls and mice infected with WT Y. pestis for each tissue examined (panels b–d). The vertical dendrograms indicate relative similarity between samples (columns), while the horizontal dendrograms indicate clusters for genes (rows). Bright red indicates the highest normalized intensity value, bright blue the lowest, and yellow represents median values.

Figure 3

A graphical representation of the changes observed in transcriptional profiles of WT Y. pestis CO92-infected mouse spleens at 48 hours post infection. Genes or gene products are represented as nodes and the biological relationship is represented as a line. All lines are supported by at least one reference from literature, textbook, or from canonical information stored in the Ingenuity Pathways Knowledge Base. The red color indicates transcriptional upregulation, based on microarray results. Those signaling molecules which were not colored (e.g., NF-κB complex) were not transcriptionally altered; however, microarray data suggested they were activated non-transcriptionally.

Figure 4

Putative host signaling pathway induced by Y. pestis bacterial effectors, LPS and Lpp. TLR-2, TLR-4, CD14, and INF-γ were transcriptionally upregulated in host tissues in response to WT Y. pestis infection. Binding of TLR-4 by LPS and TLR-2 by Lpp is inferred from literature and canonical pathway databases (e.g., Biocarta). Myd88 and IRAK, which are TLR adaptor molecules, were upregulated by WT. Y. pestis, resulting in the activation of mitogen-activated protein kinases (MAPKs), three of which (Map3k6, Map3k8, and Map4k5) were transcriptionally upregulated in WT Y. pestis-infected mice based on microarray analysis results. MAPKs are known to phsophorylate and activate nemo-like kinase (Nik), which was upregulated in WT bacteria-infected mice but not in animals infected with the Δlpp mutant. Phosphorylation of Nik is known to cause activation of NF-κB, which was also transcriptionally upregulated in WT Y. pestis-infected mice. Engagement of IFN-γ to its receptor also leads to NF-κB activation via STAT 2 and 3, which were upregulated in WT Y. pestis-infected animals. NF-κB activation results in transcription of proinflammatory cytokines, which were indeed upregulated based on microarray analysis (examples are listed in the diagram). The three MAPKs that were transcriptionally upregulated based on microarray analysis are known activators of c-jun N-terminal kinases (JNK) which leads to activation of Elk-1 and AP-1 transcription factors. AP-1 is composed of c-Jun and Fos subunits, both of which were upregulated in WT infected mice. Leukemia inhibitory factor (Lif) was uniquely upregulated in mice infected with the mutant and thus is likely inhibited in the presence of lpp, as shown. Cyclin D3 (Ccnd3) was uniquely upregulated in the spleen of WT-infected mice and the lung of Δlpp mutant infected mice and leads to increased cell proliferation. Prostaglandin E synthase (Ptges), Bak1, Bcl2l1, and hexokinase 1 (Hk1) are all known to regulate apoptosis. Each of the genes encoding these proteins was differentially expressed in Δlpp mutant-infected animals, compared to those infected with WT Y. pestis. Most likely, Lpp contributes to inhibition of host cell apoptosis and modulates inflammatory responses in coordination with LPS [22].