RNAseq reveals hypervirulence-specific host responses to M. tuberculosis infection

Abstract

The distinguishing factors that characterize the host response to infection with virulent Mycobacterium tuberculosis (M.tb) are largely confounding. We present an infection study with 2 genetically closely related M.tb strains that have vastly different pathogenic characteristics. The early host response to infection with these detergent-free cultured strains was analyzed through RNAseq in an attempt to provide information on the subtleties which may ultimately contribute to the virulent phenotype. Murine bone marrow derived macrophages (BMDMs) were infected with either a hyper- (R5527) or hypovirulent (R1507) Beijing M. tuberculosis clinical isolate. RNAseq revealed 69 differentially expressed host genes in BMDMs during comparison of these 2 transcriptomes. Pathway analysis revealed activation of the stress-induced and growth inhibitory Gadd45 signaling pathway in hypervirulent infected BMDMs. Upstream regulators of interferon activation such as and IRF3 and IRF7 were predicted to be upregulated in hypovirulent-infected BMDMs. Additional analysis of the host immune response through ELISA and qPCR included the use of human THP-1 macrophages where a robust proinflammatory response was observed after infection with the hypervirulent strain. RNAseq revealed 2 early-response genes (ier3 and saa3) and 2 host-defense genes (oasl1 and slpi) that were significantly upregulated by the hypervirulent strain. The role of these genes under M.tb infection conditions are largely unknown but here we provide validation of their presence with use of qPCR and Western blot. Further analysis into their biological role during infection with virulent M.tb is required.

Keywords: RNAseq; host-response; infection; mycobacterium tuberculosis; virulence.

Figure 1.

Global transcriptome profile of BMDMs infected with either a hyper-(R5527) or hypovirulent (R1507) M.tb. (A). Percentage uptake of Hypovirulent and hypervirulent M.tb 4 hr post-infection in BMDM (MOI = 1). (B). Principal component analysis of uninfected BMDMs (Green, C1-C3), BMDMs infected with hypervirulent M.tb (Blue, R55-1-R55-3) and hypovirulent M.tb (Red, R50-1-R50-3) which are independently clustered. (C). Heatmap visualization of differentially expressed transcripts as analyzed by RNA-seq. Transcripts with significant fold changes, based on both fold change and FDR adjusted P-value threshold, are shown in the heat map. Gene names are indicated to the right of the heat map and bacterial growth conditions are shown at the top. Red = upregulation, green = downregulation. Dendrogram indicates sample clustering. Differentially expressed genes defined as having an FC > 2 .0 and FDR < 0 .05 in both the common and tagwise dispersion estimate analysis. Analysis was conducted on 3 biological replicates (C1, 2, 3, R55-1, 2, 3 and R150-1, 2, 3).

Figure 2.

qPCR based validation and corresponding secreted proteins of differentially expressed cytokines and chemokines in BMDM after M.tb infection. A. Relative mRNA expression (fold change) of various cytokines and chemokines induced by BMDMs following infection with hypo- and hypervirulent M.tb as analyzed through qPCR (n = 3). B. Corresponding heatmap as generated by RNAseq under the same infection conditions (Red-Upregulation, Green-downregulation). C and D. Corresponding secreted cytokines and chemokines in BMDMs under the same infection conditions as measured by ELISA (n = 6). The means and standard error of a minimum of 3 independent experiments are shown, * indicates significance p < 0.05. Legend corresponds to all 3 graphs (A, C and D).

Figure 3.

qPCR based validation and corresponding secreted proteins of differentially expressed cytokines and chemokines in THP-1 macrophages after infection with hypo- and hypervirulent M.tb. A. Relative expression (fold change) of various cytokines and chemokines induced by THP-1 macrophages following infection with hypo- and hypervirulent M.tb as analyzed through qPCR (n = 3). B. Corresponding secreted cytokines and chemokines in THP-1s under the same infection conditions measure by ELISA (n = 6). The means and standard error of a minimum of 3 independent experiments are shown, *indicates significance p < 0.05. Legend corresponds to all graphs (A and B).

Figure 4.

Virulence-specific gene expression confirmed through qPCR and Western blot in BMDM and THP-1 macrophages infected with hypo- and hypervirulent M.tb. A. Relative mRNA expression (fold change) of selected differentially expressed genes induced by BMDMs following infection with hypervirulent M.tb after 12 h of infection as analyzed through qPCR (n = 4). B. Corresponding heatmap as generated by RNAseq under the same infection conditions in BMDMs (Red-Upregulation, Green-downregulation), Rep = Replicate. C. Relative mRNA expression (fold change) of the same virulence-specific genes induced by THP-1s following infection with hypervirulent M.tb after 12 h of infection as analyzed through qPCR (n = 4). D. Western blot of corresponding proteins expressed by BMDMs and THP-1s under the same infection conditions, GAPDH was used as a loading control (n = 4), Uninf. = Uninfected BMDM and THP-1, Hypov.= Hypovirulent infection, Hyperv. = Hypervirulent infection. *p < 0.05 indicates significance, a minimum of 3 biological replicates were performed for each experiment.