The Host Response to a Clinical MDR Mycobacterial Strain Cultured in a Detergent-Free Environment: A Global Transcriptomics Approach

Abstract

During Mycobacterium tuberculosis (M.tb) infection, the initial interactions between the pathogen and the host cell determines internalization and innate immune response events. It is established that detergents such as Tween alter the mycobacterial cell wall and solubilize various lipids and proteins. The implication of this is significant since induced changes on the cell wall affect macrophage uptake and the immune response to M.tb. Importantly, during transmission between hosts, aerosolized M.tb enters the host in its native form, i.e. in a detergent-free environment, thus in vitro and in vivo studies should mimic this as closely as possible. To this end, we have optimized a procedure for growing and processing detergent-free M.tb and assessed the response of murine macrophages (BMDM) infected with multi drug-resistant M.tb (R179 Beijing 220 clinical isolate) using RNAseq. We compared the effects of the host response to M.tb cultured under standard laboratory conditions (Tween 80 containing medium -R179T), or in detergent-free medium (R179NT). RNAseq comparisons reveal 2651 differentially expressed genes in BMDMs infected with R179T M.tb vs. BMDMs infected with R179NT M.tb. A range of differentially expressed genes involved in BMDM receptor interaction with M.tb (Mrc1, Ifngr1, Tlr9, Fpr1 and Itgax) and pro-inflammatory cytokines/chemokines (Il6, Il1b, Tnf, Ccl5 and Cxcl14) were selected for analysis through qPCR. BMDMs infected with R179NT stimulate a robust inflammatory response. Interestingly, R179NT M.tb induce transcription of Fpr1, a receptor which detects bacterial formyl peptides and initiates a myriad of immune responses. Additionally we show that the host components Cxcl14, with an unknown role in M.tb infection, and Tlr9, an emerging role player, are only stimulated by infection with R179NT M.tb. Taken together, our results suggest that the host response differs significantly in response to Tween 80 cultured M.tb and should therefore not be used in infection experiments.

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.

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.