Quantitative RT-PCR validation of RNA-sequencing results.

<p>(A) Quantitative RT-PCR analysis of the mRNA expression of genes differentially expressed after treatment with different levels of H₂O₂. <i>M</i>. <i>smegmatis</i> cultures were treated with 2 mM or 7 mM H₂O₂ for 30 min before extraction of RNA for qRT-PCR. The data represent 3 independent experiments. (B) Fold changes of selected genes differentially expressed genes after treatment with 0.2 mM and 7 mM H₂O₂ obtained by the RNA-sequencing.</p

RNA-sequencing mapping statistics.

<p>RNA-sequencing mapping statistics.</p

Connected network of the enriched differentially expressed genes following exposure to 0.2 mM H₂O₂.

<p>(A) Connected network of enriched differentially expressed genes involved in fatty acid metabolism (RM018 and RM020). (B) Partial fatty acid metabolism in <i>M</i>. <i>smegmatis</i>. Genes expressed differentially after 0.2 mM H₂O₂ treatment assigned to RM018 and RM020 are marked in red.</p

Overview of the differential expression profiles in response to 0.2 mM H₂O₂ in <i>M</i>. <i>smegmatis</i>.

<p>(A) Enrichment analysis. The differently colored bars indicate the gene number for the enrichment of the annotations. (B) Interaction network of the differentially expressed genes of <i>M</i>. <i>smegmatis</i> induced by 0.2 mM H₂O₂ using STRING (9.1) at confidence scores ≥ 0.4. The network is enriched among the 634 differentially expressed genes and 111 interactions were observed (p value = 0).</p

Distinct Responses of <i>Mycobacterium smegmatis</i> to Exposure to Low and High Levels of Hydrogen Peroxide

<div><p>Hydrogen peroxide (H₂O₂) is a natural oxidant produced by aerobic organisms and gives rise to oxidative damage, including DNA mutations, protein inactivation and lipid damage. The genus <i>Mycobacterium</i> utilizes redox sensors and H₂O₂ scavenging enzymes for the detoxification of H₂O₂. To date, the precise response to oxidative stress has not been fully elucidated. Here, we compared the effects of different levels of H₂O₂ on transcription in <i>M</i>. <i>smegmatis</i> using RNA-sequencing. A 0.2 mM H₂O₂ treatment had little effect on the growth and viability of <i>M</i>. <i>smegmatis</i> whereas 7 mM H₂O₂ was lethal. Analysis of global transcription showed that 0.2 mM H₂O₂ induced relatively few changes in gene expression, whereas a large proportion of the mycobacterial genome was found to be differentially expressed after treatment with 7 mM H₂O₂. Genes differentially expressed following treatment with 0.2 mM H₂O₂ included those coding for proteins involved in glycolysis-gluconeogenesis and fatty acid metabolism pathways, and expression of most genes encoding ribosomal proteins was lower following treatment with 7 mM H₂O₂. Our analysis shows that <i>M</i>. <i>smegmatis</i> utilizes the sigma factor MSMEG_5214 in response to 0.2 mM H₂O₂, and the RpoE1 sigma factors MSMEG_0573 and MSMEG_0574 in response to 7 mM H₂O₂. In addition, different transcriptional regulators responded to different levels of H₂O₂: MSMEG_1919 was induced by 0.2 mM H₂O₂, while high-level induction of DevR occurred in response to 7 mM H₂O₂. We detected the induction of different detoxifying enzymes, including genes encoding KatG, AhpD, TrxB and Trx, at different levels of H₂O₂ and the detoxifying enzymes were expressed at different levels of H₂O₂. In conclusion, our study reveals the changes in transcription that are induced in response to different levels of H₂O₂ in <i>M</i>. <i>smegmatis</i>.</p></div

Fold changes of genes differentially expressed after treatment with 0.2 mM and 7 mM H₂O₂ (treated vs untreated).

<p>Fold changes of genes differentially expressed after treatment with 0.2 mM and 7 mM H₂O₂ (treated vs untreated).</p

Connected network of the enriched differentially expressed genes following exposure to 0.2 mM H₂O₂.

<p>(A) Connected network of enriched differentially expressed genes involved in fatty acid metabolism (RM018 and RM020). (B) Partial fatty acid metabolism in <i>M</i>. <i>smegmatis</i>. Genes expressed differentially after 0.2 mM H₂O₂ treatment assigned to RM018 and RM020 are marked in red.</p

Gene ontology analysis of 345 differentially expressed proteins between <i>M</i>. <i>smegmatis</i> mc²155 and the copper resistant strain.

<p>(A) Classification based on protein functions; (B) classification based on the proteins-associated biological processes.</p

Growth curve and the susceptibility of <i>M</i>. <i>smegmatis</i> to copper and isoniazid.

<p>(a) The growth curve of <i>M</i>. <i>smegmatis</i> mc²155 and the copper resistant strain mc²155-Cu were measured in 7H9 media. Experiments were performed in triplicate. Squares, mc2155-Cu strains; circle, mc2155 strain; (b) The bacterial growth on 7H10 plates for <i>M</i>. <i>smegmatis</i> mc²155 and mc²155-Cu that were treated with CuSO₄ at different concentrations for 3 days, respectively. The panels show serial dilution (1:10) of mc²155 and mc²155-Cu. Diluted M. smegmatis cultures were spotted onto solid 7H10 media in the presence of CuSO₄ ranged from 0 to 500 μM. Images were taken after 3 days incubation at 37°C. Images stand for 3 independent experiments.; and (c) The bacterial survival rate for <i>M</i>. <i>smegmatis</i> mc²155 and mc²155-Cu that were treated with 0.1 mg/ml isoniazid.***<i>p</i><0.001; n = 3.</p

Proteomic Analysis of Drug-Resistant Mycobacteria: Co-Evolution of Copper and INH Resistance

<div><p>Tuberculosis, caused by the pathogen <i>Mycobacterium tuberculosis</i>, is a worldwide public health threat. <i>Mycobacterium tuberculosis</i> is capable of resisting various stresses in host cells, including high levels of ROS and copper ions. To better understand the resistance mechanisms of mycobacteria to copper, we generated a copper-resistant strain of <i>Mycobacterium smegmatis</i>, mc²155-Cu from the selection of copper sulfate treated-bacteria. The mc²155-Cu strain has a 5-fold higher resistance to copper sulfate and a 2-fold higher resistance to isoniazid (INH) than its parental strain mc²155, respectively. Quantitative proteomics was carried out to find differentially expressed proteins between mc²155 and mc²155-Cu. Among 345 differentially expressed proteins, copper-translocating P-type ATPase was up-regulated, while all other ABC transporters were down-regulated in mc²155-Cu, suggesting copper-translocating P-type ATPase plays a crucial role in copper resistance. Results also indicated that the down-regulation of metabolic enzymes and decreases in cellular NAD, FAD, mycothiol, and glutamine levels in mc²155-Cu were responsible for its slowing growth rate as compared to mc²155. Down-regulation of KatG2 expression in both protein and mRNA levels indicates the co-evolution of copper and INH resistance in copper resistance bacteria, and provides new evidence to understanding of the molecular mechanisms of survival of <i>mycobacteria</i> under stress conditions.</p></div