Fig 1.
Differential expression of host gene transcripts in R179T versus R179NT infected BMDMs.
Heatmap visualization of differentially expressed transcripts as analyzed by RNA-seq where R179T and R179NT were compared to uninfected BMDMs. Transcripts with significant fold changes, based on both fold change and FDR adjusted P-value threshold, are shown in the heat map. The level of expression of each gene, in each sample, relative to the mean level of expression of that gene across all of the samples, is represented by using a red–green color scale as shown in the key with a range of − 1.2 to + 2.09 on a log (10) scale. 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. Analysis was conducted on three biological replicates (C1, 2, 3, RT1, 2, 3 and RNT1, 2, 3). BMDMs infected with detergent-free M.tb exhibit a differential infection profile. C—control/uninfected BMDMs, RT—R179 Tween 80 cultured M.tb, RNT—R179 non-Tween 80 (detergent free) cultured M.tb.
Table 1.
Top 20 up- and downregulated transcripts in BMDMs infected with R179NT M.tb vs. R179T M.tb.
Table 2.
Top Canonical Pathways activated in BMDMs after infection with detergent-free M.tb.
Table 3.
Top Upstream regulators activated in BMDMs after infection with detergent-free M.tb.
Fig 2.
qPCR based validation of selected differentially expressed host receptor genes.
A. Relative expression (fold change) of downregulated receptors in BMDMs infected with R179T and R179NT M.tb. B. Relative expression (fold change) of upregulated receptors BMDMs infected with R179T and R179NT M.tb. The means and standard error of three independent experiments are shown, * indicates significance p < 0.05. Legend corresponds to both graphs (A and B).C. Corresponding heatmap visualization of differentially expressed transcripts as analyzed by RNA-seq. The level of expression of each gene, in each sample, relative to the mean level of expression of that gene across all of the samples, is represented by using a red–green color scale as shown in the key with a range of − 1.2 to + 2.09 on a log(10) scale. Red = upregulation, green = downregulation (FC >2.0 and FDR <0.05).
Fig 3.
qPCR based validation of selected differentially expressed host cytokine and chemokine genes.
A. Relative expression (fold change) of cytokines and chemokines in BMDMs infected with R179T and R179NT M.tb. The means and standard error of three independent experiments are shown, * indicates significance p < 0.05 vs. R179T. B. Corresponding heatmap visualization of differentially expressed transcripts as analyzed by RNA-seq The level of expression of each gene, in each sample, relative to the mean level of expression of that gene across all of the samples, is represented by using a red–green color scale as shown in the key with a range of − 1.2 to + 2.09 on a log(10) scale, Red = upregulation, green = downregulation (FC >2.0 and FDR <0.05).
Fig 4.
The host response to infection with detergent-free cultured M.tb.
Using the canonical pathway from IPA depicting the ‘Role of Pattern Recognition Receptors in Recognition of Bacteria and Viruses’, expression values from RNAseq data was overlaid where red molecules indicate upregulation and green molecules indicate downregulation. Uncoloured molecules indicate no expression. Tlr1, 2 and 6 are stimulated by M.tb infection, whereas Tlr5 and 9 expression is downregulated. We included Fpr1, as it is highly stimulated upon infection with R179NT M.tb. Under infection conditions, it indirectly activates a number of proinflammatory cytokines as well as ERK1/2 and JNK. Cxcl14 was also included in the pathway as it was observed to be downregulated by M.tb infection. It has anti-microbial properties and was observed that infection by other pathogens decreases its expression. Solid lines indicate direct activation, broken lines indicate indirect activation.