Morphotype of the KdpF-overexpressing strain.

<p><b>A.</b> Single <i>M. bovis</i> BCG colonies were grown on cord-reading agar and visualized after 3 weeks. Magnification is 16x for the main figures and 63x for the insets. <b>B.</b> SEM micrographs of single <i>M. bovis</i> BCG colonies grown in 7H10 agar plates at 6000× magnification. The inset micrographs are at 13740x and 12000× magnification for <i>M. bovis</i> BCG pMV261 and <i>M. bovis</i> BCG overexpressing <i>kdpF,</i> respectively.</p

Effect of <i>kdpF</i> overexpression on the expression of the <i>kdp</i> operons inside macrophages.

<p>RNA was extracted from <i>M. bovis</i> BCG overexpressing <i>kdpF</i> grown Sauton’s liquid medium or after 6 days infection of BMDM. Quantitative RT-PCR was used to study the expression of the <i>kdp</i> operon genes relative to the one of the <i>sigA</i> gene. Data are means ± SD calculated from three independent biological samples analyzed in triplicate. Statistical significance was performed using a generalized mixed effects model.</p

Growth of <i>M. bovis</i> BCG overexpressing <i>kdpF</i> in murine and human primary macrophages.

<p><b>A</b>. Kinetic of growth of <i>M. bovis</i> BCG overexpressing <i>kdpF</i> in murine BMDM over a 8-day period. <b>B.</b> Bacterial number upon infection of HMDM infected with <i>M. bovis</i> BCG overexpressing <i>kdpF</i> at day 6 post-infection. The means ± SD calculated from three independent experiments (each performed in triplicate) are shown. Asterisks indicate statistical significance using a generalized mixed effects model (*** P<0.001).</p

Genetic organization of the <i>kdp</i> operons in various bacteria and overexpression of the <i>kdpF</i> gene in <i>M. bovis</i><b> BCG.</b>

<p><b>A.</b> Transcriptional organization of the <i>kdp</i> operon in <i>E. coli</i> and various mycobacteria. An intergenic region of 192 bp separates the <i>kdpDE</i> operon from the <i>kdpFABC</i> operon in <i>M. tuberculosis</i> and <i>M. bovis</i> BCG. Arrows indicate the direction of transcription. <b>B.</b> Expression of <i>kdpF</i> transcripts relatively to those of s<i>igA</i> gene was measured by qRT-PCR from <i>M. bovis</i> BCG strains grown in Sauton’s medium carrying the pMV261 vector or p<i>kdpF</i> plasmid. Results are expressed as means ± SD from three independent experiments (each performed in triplicate).</p

<i>In vivo</i> interaction of KdpF with MmpL7 variants using the BACTH system.

<p><b>A</b>. Predicted topology of MmpL7 using the topological analysis server TMHMM (<a href="http://www.cbs.dtu.dk/services/TMHMM-2.0/" target="_blank">http://www.cbs.dtu.dk/services/TMHMM-2.0/</a>). The TM regions are indicated by cylinders and are numbered while the two non-TM loops are indicated as Domain 1 and Domain 2. <b>B.</b> Interaction between KdpF-T25 and MmpL7-T18 constructs. <i>E.coli</i> BTH101 were co-transformed with <i>kdpF</i>-T25 plasmid and different constructs derived from pUT18 plasmid encoding MmpL7 full-length protein or portions of MmpL7 (TM 2–6, TM 8–12, cytoplasmic Domain 2). The portion coding for MmpL7 TM 8–12 was also cloned into pUT18c vector to obtain a reverse membrane orientation. The bars represent β-galactosidase activity expressed in Miller units ± SD of three independent experiments performed in triplicate.</p

<i>In vivo</i> interaction of KdpF with KdpD and effect of <i>kdpF</i> overexpression on the regulation of <i>kdp</i> genes by K⁺.

<p><b>A</b>. The interaction between KdpF and KdpD was assayed using the BACTH system by transforming <i>E. coli</i> BTH101 cells with KdpF-T25 and KdpD-T18 plasmids. Liquid β-galactosidase assays were performed from three independent experiments. Error bars represent standard deviations. <b>B.</b> K⁺-regulated expression of <i>kdp</i> operon. <i>M. bovis</i> BCG carrying the pMV261 vector or the p<i>kdpF</i> constructs were grown in Sauton’s medium or in K⁺-depleted Sauton’s medium. The levels of <i>kdpA, kdpB, kdpD</i> and <i>kdpE</i> transcripts relative to those of the <i>sigA</i> gene were measured by qRT-PCR. Results are expressed as means ± SD from three independent experiments (each performed in triplicate). Asterisks indicate statistical significance using a generalized mixed effects model (** P<0.01, *** P<0.001).</p

Cytokines response in T-SPOT supernatants.

<p><b>A-</b> The expression of IFN-γ in ESAT-6 stimulated T cells was quantified by the T-SPOT assay. Results are expressed by numbers of secreted cells (SC) per 250 000 PBMC for 36 patients with ATB (red circles) and 115 patients with LTBI (green squares). The median is shown in each group. The p value is calculated by the Mann-Whitney U test. ** corresponds to p≤0.01. <b>B-</b> The ROC curve displays sensitivity vs specificity for IFN-γ in differentiating the ATB group from the LTBI group. The AUC and a threshold at 100 SC/250000 PBMC are indicated in the graph. <b>C-E-G-I-K-M-</b> IL-4, IL-12, TNF-α, GM-CSF, Eotaxin and IFN-α indexes (cytokine/IFN-γ *100), ATB are represented by red circles and LTBI by green squares. The median indexes are shown in each group of each panel. p values are calculated by the Mann-Whitney U test. ** corresponds to p≤0.01, *** to p≤0.001 and **** to p≤0.0001. <b>D-F-H-J-L-N-</b> Biparametric graphs between the numbers of IFN-γ immunospots in response to ESAT-6 stimulation and the level of cytokines (pg/ml) in the corresponding supernatants. ATB are represented by red circles and LTBI by green squares. Defined risk groups are circled.</p

Identification of cytokine profiles associated with ATB.

<p><b>A.</b> Unsupervised hierarchical clustering was performed according to the similarities in cytokine expression profiles which were visualised using the indicated color scale. Cytokine concentrations were indicated using a color scale that ranges from green (low) through black to red (high). The dendrogram above the heat map illustrated degrees of relatedness between the expression profiles evident within the various patients. Subjects with ATB (n = 35; black boxes on the strip above the heat-map), with high IFN-γ and low IL-12, TNF-α, GM-CSF, Eotaxin and IFN-γ secretions tended to cluster together (cluster I on the dendogram), while subjects with LTBI (n = 125; grey boxes) with IFN-γ and high IL-12, TNF-α, GM-CSF, Eotaxin and IFN-γ secretions also tended to cluster together (cluster II). The dendrogram on the right-hand side of the heat map indicates relationships between the expression profiles of the analysed cytokines across all the patients assessed in each study. <b>B.</b> Partial least squares analysis model of all 7 cytokines classified individuals with 61% overall accuracy for classification and 55% accuracy for cross-validation (red circles, ATB; green squares, LTBI). Latent Variable 1 (LV1), linear combination of the variables included in the model, LV2, orthogonal to the first combination. 95% of each population was circled. <b>C.</b> Latent variable 1 best separated individuals who are ATB from those LTBI. Cytokine loadings indicate multivariate cytokines associated with ATB.</p

Clinical characteristic of 150 subjects included in this study.

<p>Clinical characteristic of 150 subjects included in this study.</p

Design of crude odd ratios by univariate analysis depending on cytokine response from ESAT-6 stimulated PBMC.

<p>Design of crude odd ratios by univariate analysis depending on cytokine response from ESAT-6 stimulated PBMC.</p