Genes involved in nitrogen metabolism in the slow growing mycobacterium <i>M. bovis</i> BCG.

<p>Ammonia is assimilated in the production of L-glutamine and L-glutamate. Together, L-glutamine, L-glutamate, and L-aspartate act as precursors or nitrogen donors to most other nitrogenous compounds in the mycobacterium. The map was constructed from the combined PATRIC pathways for <i>M. bovis</i> BCG <i>str</i>. Pasteur 1743P2 nitrogen metabolism and alanine, aspartate and glutamate metabolism [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084452#B3" target="_blank">3</a>]. Genes were assigned to the EC numbers by PATRIC and/or Refseq and/or Legacy BRC.</p

Growth of <i>Δgdh</i> in 7H9 with different nitrogen sources.

<p>Growth of wt BCG, the <i>Δgdh</i> mutant and the <i>Δgdh</i> complemented strain in (A) standard 7H9 containing approximately 4 mM ammonium sulphate (AS, (NH₄)₂SO₄) and 3 mM L-Glu, (B) ‑N7H9 (nitrogen-depleted 7H9) + 3 mM L-Glu, (C) 7H9 + 30 mM L-Glu, (D) 7H9 + 30 mM L-Asn, (E) -N7H9 + 30 mM L-Asn, (F) 7H9 + 3 mM L-Asn, (G) -N7H9 + 3 mM L-Asn, or (H) 7H9 + 30 mM L-Asp. Growth of the <i>Δgdh</i> mutant in (I) ‑N7H9 + 3 mM L-Glu supplemented with increasing concentrations of AS. Cultures for cfu/ml determinations were inoculated to OD₆₀₀ = 0.0005 (cfu/ml of approximately 10⁵). Log₁₀(cfu/ml) of <i>Δgdh</i> cultured in -N7H9 + 3 mM Glu was different from log₁₀(cfu/ml) of <i>Δgdh</i> cultured in -N7H9 + 3 mM Glu supplemented with 1 mM AS at day 9 and 14 (p < 0.01). Log₁₀(cfu/ml) of <i>Δgdh</i> cultured in 7H9 + 30 mM L-Asn was different from log₁₀(cfu/ml) of <i>Δgdh</i> cultured in -N7H9 + 3 mM Glu supplemented with 1 mM AS at day 6, 9 and 14 (p < 0.01). (J) Growth of the <i>Δgdh</i> mutant cultured in ‑N7H9 + 3 mM L-Glu for three weeks when sub-cultured in fresh 7H9 or –N7H9 + 3 mM L-Glu. Aliquots of three week old <i>Δgdh</i> mutant ‑N7H9 + 3 mM L-Glu cultures were washed once with ‑N7H9 and used to inoculate fresh 7H9 or ‑N7H9 + 3 mM L-Glu to an OD₆₀₀ = 0.020. (K) Determination of growth of single colonies obtained from three week old <i>Δgdh</i> mutant ‑N7H9 + 3 mM L-Glu cultures in fresh –N7H9 + 3 mM L-Glu. A1 and A2 were obtained from the first growth curve experiment, B1 and B2 from the second and C1-C3 from the third. Mean OD measurements with standard deviations presented in panels A-H and J and mean log₁₀(cfu/ml) with standard errors presented in panel I were calculated with growth curve data obtained from three independent experiments performed for each condition tested. In some instances error bars were smaller than the symbols used to depict the means. AS, ammonium sulphate.</p

Replacement of the <i>gltBD</i> operon with a hygromycin cassette and gene deletion of <i>gdh</i>.

<p>A) Comparison of the wild-type <i>M. bovis</i> BCG and mutant <i>gltBD</i> regions. B) Southern blot analysis of wild-type <i>M. bovis</i> BCG (lane 1) and <i>ΔgltBD::hyg</i> (lane 2) with a probe that hybridises upstream of <i>gltB</i>. Southern blot analysis of wild-type <i>M. bovis</i> BCG (lane 3) and <i>ΔgltBD::hyg</i> (lane 4) with a probe that hybridises downstream of <i>gltD</i>. C) Comparison of the wild-type <i>M. bovis</i> BCG and mutant <i>gdh</i> regions. The GDH domain region is the sequence in <i>gdh</i> which aligned with a 98% query coverage (66% identity, 80% positives) in a blastp to the GDH domain sequence of the previously characterised Streptomyces <i>clavuligerus</i> L-180 GDH [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084452#B36" target="_blank">36</a>]. The NruI fragment spanning the GDH domain is deleted in the <i>Δgdh</i> chromosome. The probe which is complementary to fragments both upstream and downstream of the GDH domain does not hybridise across its full length with wild-type <i>M. bovis</i> BCG DNA, but does with <i>Δgdh</i> mutant DNA, as indicated in the figure. D) Southern blot analysis of wild-type <i>M. bovis</i> BCG (lane 1) and <i>Δgdh</i> (lane 2). <i>S</i>, SphI; <i>hyg</i>^<i>R</i>, hygromycin resitance cassette; <i>K</i>, KpnI; <i>N</i>, NruI; U, upstream; D, downstream.</p

Growth of <i>M. bovis</i> BCG in standard 7H9, 7H9 lacking nitrogen sources (‑N7H9) and 7H9 containing alanine as sole nitrogen source (‑N7H9 + 3 mM L-Ala).

<p>Mean OD measurements with standard deviations were calculated with growth curve data obtained from three independent experiments performed for each condition tested.</p

Growth of <i>ΔgltBD</i> in 7H9 with different nitrogen sources.

<p>Growth of wt-BCG, the <i>ΔgltBD</i> mutant and the <i>ΔgltBD</i> complemented strain in (A) standard 7H9 containing approximately 4 mM ammonium sulphate (AS, (NH₄)₂SO₄) and 3 mM L-Glu, (B) 7H9 + 10 mM L-Glutamate, or (C) ‑N7H9 (nitrogen-depleted 7H9) + 4 mM AS. Growth of the <i>ΔgltBD</i> mutant in (D) ‑N7H9 + 4 mM AS supplemented with increasing concentrations of glutamate. Cultures for cfu/ml determinations were inoculated to OD₆₀₀ = 0.0005 (cfu/ml of approximately 10⁵). Log₁₀(cfu/ml) of <i>ΔgltBD</i> cultured in -N7H9 + 4 mM AS supplemented with 10 mM L-Glu was different from log₁₀(cfu/ml) of <i>ΔgltBD</i> cultured in -N7H9 + 4 mM AS at every time point after and including 3 days (P < 0.001). Log₁₀(cfu/ml) of <i>ΔgltBD</i> cultured in -N7H9 + 4 mM AS supplemented with 3 mM L-Glu was different from log₁₀(cfu/ml) of <i>ΔgltBD</i> cultured in -N7H9 + 4 mM AS at 3 days (p < 0.01) and every following time point (p < 0.001). Log₁₀(cfu/ml) of <i>ΔgltBD</i> cultured in -N7H9 + 4 mM AS supplemented with 0.3 mM L-Glu was different from log₁₀(cfu/ml) of <i>ΔgltBD</i> cultured in -N7H9 + 4 mM AS at 6 days (p < 0.01) and every following time point (p < 0.001). Growth of wt-BCG, the <i>ΔgltBD</i> mutant and the <i>ΔgltBD</i> complement strain in (E) –N7H9 + 3 mM L-Glu, (F) –N7H9 + 3 mM L-Asn, (G) ‑N7H9 + 3 mM L-Gln, (H) –N7H9 + 3 mM L-Asp, (I) unmodified –N7H9, or (J) 7H9 + 30 mM AS. Mean OD measurements with standard deviations presented in panels A-C and E-J and mean log₁₀(cfu/ml) with standard errors presented in panel D were calculated with growth curve data obtained from three independent experiments performed for each condition tested. In some instances error bars were smaller than the symbols used to depict the means. AS, ammonium sulphate.</p

Top Canonical Pathways activated in BMDMs after infection with detergent-free <i>M</i>.<i>tb</i>.

<p>Top Canonical Pathways activated in BMDMs after infection with detergent-free <i>M</i>.<i>tb</i>.</p

qPCR based validation of selected differentially expressed host cytokine and chemokine genes.

<p><b>A.</b> Relative expression (fold change) of cytokines and chemokines in BMDMs infected with R179T and R179NT <i>M</i>.<i>tb</i>. The means and standard error of three independent experiments are shown, * indicates significance p < 0.05 vs. R179T. <b>B.</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).</p

Top 20 up- and downregulated transcripts in BMDMs infected with R179NT <i>M.tb</i> vs. R179T <i>M.tb</i>.

<p>Top 20 up- and downregulated transcripts in BMDMs infected with R179NT <i>M.tb</i> vs. R179T <i>M.tb</i>.</p

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

<div><p>During <i>Mycobacterium tuberculosis</i> (<i>M</i>.<i>tb)</i> 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 <i>M</i>.<i>tb</i>. Importantly, during transmission between hosts, aerosolized <i>M</i>.<i>tb</i> enters the host in its native form, i.e. in a detergent-free environment, thus <i>in vitro</i> and <i>in vivo</i> studies should mimic this as closely as possible. To this end, we have optimized a procedure for growing and processing detergent-free <i>M</i>.<i>tb</i> and assessed the response of murine macrophages (BMDM) infected with multi drug-resistant <i>M</i>.<i>tb</i> (R179 Beijing 220 clinical isolate) using RNAseq. We compared the effects of the host response to <i>M</i>.<i>tb</i> 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 <i>M</i>.<i>tb</i> vs. BMDMs infected with R179NT <i>M</i>.<i>tb</i>. A range of differentially expressed genes involved in BMDM receptor interaction with <i>M</i>.<i>tb</i> (<i>Mrc1</i>, <i>Ifngr1</i>, <i>Tlr9</i>, <i>Fpr1</i> and <i>Itgax</i>) and pro-inflammatory cytokines/chemokines (<i>Il6</i>, <i>Il1b</i>, <i>Tnf</i>, <i>Ccl5</i> and <i>Cxcl14</i>) were selected for analysis through qPCR. BMDMs infected with R179NT stimulate a robust inflammatory response. Interestingly, R179NT <i>M</i>.<i>tb</i> induce transcription of <i>Fpr1</i>, a receptor which detects bacterial formyl peptides and initiates a myriad of immune responses. Additionally we show that the host components <i>Cxcl14</i>, with an unknown role in <i>M</i>.<i>tb</i> infection, and Tlr9, an emerging role player, are only stimulated by infection with R179NT <i>M</i>.<i>tb</i>. Taken together, our results suggest that the host response differs significantly in response to Tween 80 cultured <i>M</i>.<i>tb</i> and should therefore not be used in infection experiments.</p></div

Differential expression of host gene transcripts in R179T versus R179NT infected BMDMs.

<p>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 <i>M</i>.<i>tb</i> exhibit a differential infection profile. C—control/uninfected BMDMs, RT—R179 Tween 80 cultured <i>M</i>.<i>tb</i>, RNT—R179 non-Tween 80 (detergent free) cultured <i>M</i>.<i>tb</i>.</p