Mycobacterium tuberculosis carrying a rifampicin drug resistance mutation reprograms macrophage metabolism through cell wall lipid changes

Abstract

Tuberculosis is a significant global health threat, with one-third of the world's population infected with its causative agent Mycobacterium tuberculosis (Mtb). The emergence of multidrug-resistant (MDR) Mtb that is resistant to the frontline anti-tubercular drugs rifampicin and isoniazid forces treatment with toxic second-line drugs. Currently, ~4% of new and ~21% of previously treated tuberculosis cases are either rifampicin-drug-resistant or MDR Mtb infections¹. The specific molecular host-pathogen interactions mediating the rapid worldwide spread of MDR Mtb strains remain poorly understood. W-Beijing Mtb strains are highly prevalent throughout the world and associated with increased drug resistance². In the early 1990s, closely related MDR W-Beijing Mtb strains (W strains) were identified in large institutional outbreaks in New York City and caused high mortality rates³. The production of interleukin-1β (IL-1β) by macrophages coincides with the shift towards aerobic glycolysis, a metabolic process that mediates protection against drug-susceptible Mtb⁴. Here, using a collection of MDR W-Mtb strains, we demonstrate that the overexpression of Mtb cell wall lipids, phthiocerol dimycocerosates, bypasses the interleukin 1 receptor, type I (IL-1R1) signalling pathway, instead driving the induction of interferon-β (IFN-β) to reprogram macrophage metabolism. Importantly, Mtb carrying a drug resistance-conferring single nucleotide polymorphism in rpoB (H445Y)⁵ can modulate host macrophage metabolic reprogramming. These findings transform our mechanistic understanding of how emerging MDR Mtb strains may acquire drug resistance single nucleotide polymorphisms, thereby altering Mtb surface lipid expression and modulating host macrophage metabolic reprogramming.

Figure 1.. IL-1R1 is dispensable for protective immunity against MDR Mtb strain, W_7642.

B6 (n=10) and gene deficient mice (Myd88^−/− HN878 n=3, W_7642 n=6; Ifngr^−/− HN878 n=5, W_7642 n=6; Tnfr1^−/− n=4; Nos2^−/− n=5; Il1r1^−/− HN878 n=9, W_7642 n=5; Ifnar^−/− n=5; Il10^−/− n=5) were aerosol infected with 100 CFU Mtb HN878 or W_7642. Lung bacterial burden was determined on 30 dpi (a) or at defined time points (b, B6 n=5, Il1r1^−/− n=9 except W_7642 D15, D20 Il1r1^−/− n=5). HN878-infected Il1r1^−/− mice were sacrificed on 30 dpi due to severe TB disease (b). On 30 dpi, formalin-fixed, paraffin embedded (FFPE) lung sections from B6 and Il1r1^−/− mice were stained with H&E and inflammatory area was measured. Micrographs are representative images - 5x magnification (c, HN878 B6 n=9, HN878 Il1r1^−/− n=8, W_7642 n=7). Total number of lung neutrophils, monocytes, and recruited macrophages were determined on 30 dpi using flow cytometry (d, B6 n=5, Il1r1^−/− n=4, UI B6 n=4). Cytokine and chemokine protein levels in lung homogenates were measured in B6 and Il1r1^−/− mice at 30 dpi (e, n=5). UI-uninfected. (a) 1-way ANOVA with Tukey’s post-test, (b,d,e) 2-way ANOVA with Bonferroni post-test, (c) two tailed Student’s t-test. The data points represent the mean (±SD) of values. *p≤0.05, **p≤0.01, ***p≤0.001, ns-not significant (p>0.05).

Figure 2.. MDR Mtb W_7642 infection induces the type I IFN pathway and distinctive host macrophage metabolism.

B6 and Il1r1^−/− macrophages were infected with HN878 or W_7642 (MOI1). RNA was extracted on 6 dpi and RNA sequencing was performed. The expression of genes in the glycolysis pathway in HN878- or W_7642-infected B6 macrophages over uninfected macrophages is shown (a, n=3, except W_7642 B6 n=2). The expression of genes in the oxidative phosphorylation and type I IFN pathways in W_7642- over HN878-infected B6 macrophages is shown (b, n=3, except W_7642 B6 n=2). The heat map of marker mRNAs from the annotated pathways are shown from individual samples within the different groups (c). Cytokine protein, lactate (d-e) and nitrite levels (f) were measured in supernatants, and intracellular Mtb CFU (g) was determined on 6 dpi (n=4–6). Infected macrophages were treated with IFNAR blocking antibody or isotype (25 μg/ml) at −1 and 3 dpi. IL-1β (n=5) and lactate levels (n=4) were measured in supernatants (h), and intracellular Mtb CFU (i, n=5) was determined on 6 dpi. B6 macrophages (n=5) were co-infected with HN878 and W_7642 (3 HN878:1 W_7642, 1 HN878:1 W_7642, total MOI1) for 6 dpi. IL-1β, lactate, and IFN-β levels were measured in supernatants (j). B6 macrophages were infected with HN878 or W_7642 while in glucose or galactose (25mM each)-containing media. IL-1β (n=6), lactate (n=4), and IFN-β levels (n=4) (k), and intracellular Mtb CFU (l, n=6) were determined 3 dpi. UN-untreated, UI-uninfected, nd-not detectable. (a-b) Gene set enrichment analysis was done using an FGSEA R package as in methods, (d-g,j,k) 1-way ANOVA with Tukey’s post-test, (h,i) 2-way ANOVA with Bonferroni post-test. The data points represent the mean (±SD) of values. *p≤0.05, **p≤0.01, ***p≤0.001, ****p≤0.0001, ns-not significant (p>0.05).

Figure 3.. Identification of unique SNPs in rpoB in MDR Mtb strains that alter macrophage reprogramming.

WGS data was used to generate a SNP maximum-likelihood tree to determine the phylogenetic relationship between the Mtb strains. Reads were mapped onto the sequence of Mtb H37Rv (AL123456) (a). The WGS of the Mtb strains was used to generate a SNP density map. Loci where W12_1811 and W_7642 have different non-synonymous SNPs are indicated on the outer track and with labels (red labels indicate 2+ such loci per gene). On all tracks, darker shades indicate higher SNP rates. 1 tick = 100kb, total length = 4.4mbp, GC content = 65.6%. “Lolliplot” representation of rpoB (RVBD_0667) shows the positions of non-synonymous (orange) and synonymous (black) SNPs and the corresponding amino acid change (b). B6 and Il1r1^−/− mice were aerosol infected with 100 CFU of Mtb strains and lung bacterial burden was determined on 30 dpi (W12_1811 n=15, W12_15183 and W12_3474 n=10, W_7642 n=5) (c). B6 and Il1r1^−/− macrophages were infected with Mtb strains (MOI1) and IL-1β (d, n=5), lactate (d, n=4), and IFN-β (e, n=5), levels were measured in supernatants, and intracellular CFU (f, n=5) was determined by after 6 dpi. B6 macrophages were treated with hkMtb strains (20 μg/ml each) for 48 hours and ECAR was measured in treated cells (UN n=10, W12_1811 n=5, W12_15183 and W12_3474 n=6) (g). UN-untreated, UI-uninfected. (c-g) 1-way ANOVA with Tukey’s post-test. The data points represent the mean (±SD) of values. *p≤0.05, **p≤0.01, ***p≤0.001, ****p≤0.0001, ns-not significant (p>0.05).

Figure 4.. W-Beijing Mtb strains carrying the rpoB-H445Y SNP overexpress PDIM and bypass the IL-1R1 signaling pathway for protective immunity.

B6 and Il1r1^−/− macrophages were infected with HN878 or three independently isolated HN878 rpoB-S450L or HN878 rpoB-H445Y mutants (MOI1) for 6 days (n=4). IL-1β, lactate, and IFN-β levels were measured in supernatants (a) and intracellular CFU was determined (b). B6 and Il1r1^−/− mice (n=5) were aerosol infected with 100 CFU Mtb HN878 rpoB-S450L or HN878 rpoB-H445Y. Lung bacterial burden was determined on 30 dpi (c). The LC/MS reconstructed ion chromatogram (RIC, m/z 1330–1450) of PDIM (d) and the internal standard TAG (e) from HN878 and W_7642 are overlayed (the two earlier elution peaks of the TAG chromatogram are from endogenous Mtb 18:1/16:0/26-TAG (second isotope) and 18:0/16:0/26:0-TAG). The amounts of 20:0/20:0/20:0-TAG internal standard in the two samples are nearly equal. The LC/MS RIC (m/z 1330–1450) of PDIM (f) and the internal standard TAG (g) from rpoB-H445Y and rpoB-S450L are overlayed. The 20:0/20:0/20:0-TAG internal standard in the rpoB-S450L sample is about 1.5 times of the amount in rpoB-H445Y. Trace data is representative of at least two replicates. PDIM was isolated from each Mtb strain and coated onto polystyrene beads. B6 macrophages (n=4, except rpoB-H445Y and rpoB-S450L PDIM n=8) were treated with PDIM coated beads (200:1) from different Mtb strains or control HSA coated beads (200:1) alone or in combination with HN878 infection (MOI1). IL-1β, lactate and IFN-β protein levels were determined in 6 dpi supernatants (h). UN-untreated, UI-uninfected, nd-not detectable. (a-c) 2-way ANOVA with Bonferroni post-test, (h) 1-way ANOVA with Tukey’s post-test. The data points represent the mean (±SD) of values. *p≤0.05, ***p≤0.001, ns-not significant (p>0.05).