Inhibition of the <i>in vitro</i> MDH activity by phosphorylation.

<p>The MDH activity was assayed by measuring the decrease in NADH absorbance at 340 nm. The assay was carried out before (■) or after (●) the phosphorylation of MDH by the PknD at a room temperature (20°C), or in the absence of MDH but in the presence of PknD (▲). Results are the means ± s.e.m. of n experiments performed in triplicates.</p

The active form of the <i>Mtb</i> MDH consists in a dimer.

<p>A: Native polyacrylamide gel (8%) was stained for MDH activity as described under Materials and Methods. B: Autoradiography of the gel. Lane 1: 2 μg of non-phosphorylated MDH; Lane 2: 2 μg of MDH phosphorylated by 0.25 μg of PknD; Lane 3: 0.25 μg of autophosphorylated PknD.</p

Phosphorylation of mycobacterial proteins by PknD.

<p>After separation by chromatography of the proteins from a homogenate from <i>M</i>. <i>bovis</i> BCG 1173P2, some fractions were pooled and incubated in the presence of (γ-³³P)- ATP and of PKnD. Proteins were separated by SDS-PAGE and the phosphoproteins were visualized by autoradiography. Lanes M : standards of various molecular weights ; 1 : chromatographic fractions 42–44; 2 : chromatographic fractions 45–47; 3 : chromatographic fractions 48–50; 4: chromatographic fractions 66–69; 5 : chromatographic fractions 71–75; 6: no fraction from the chromatography. After transfer to a PVDF membrane, the bands 1 to 5 were sequenced using the Edman degradation method.</p

SDS-PAGE of the His₆-MDH stained by Coomassie-Blue R-250.

<p>His₆-MDH was overproduced as a soluble protein and purified on a Ni²⁺-immobilized HiTrap column. Lanes 1 (30 μg) and 2 (30 μg) show <i>E</i>. <i>coli</i> soluble extract before and after its loading on the HiTrap column, respectively. Lane 3 (1.5 μg) shows the purified His₆-MDH. The purified His₆-MDH was also observed by Western blot using a mAb anti-Xpress against His₆-tag epitope (lane 4).</p

Phosphorylation of the <i>Mtb</i> MDH by <i>Mtb</i> STPKs during various bacterial growth conditions.

<p>The immunoprecipitated MDH was separated on SDS-PAGE and transferred onto nitrocellulose membranes for Western blot analyses using antibodies recognizing the MDH (anti-MDH) or phosphothreonine residue (anti-pThr). A: Bacteria cultured until early, middle and late exponential phases, (corresponding to OD_600nm 0.3, 0.6 or 0.9, respectively) were further cultured for 3 hours in phosphate-repleted medium or bacteria cultured until late exponential phase (corresponding to OD_600nm 0.9) were further cultured in phosphate limited medium. B: Bacteria cultured until OD_600nm 0.6 in Dubos normal medium (DM) or under deprivation of oxygen (DM-O₂).</p

Kinetic parameters of the purified recombinant <i>Mtb</i> MDH.

<p>Kinetic parameters of the purified recombinant <i>Mtb</i> MDH.</p

Determination of the major proteins phosphorylated by PknD in a homogenate from <i>M</i>. <i>bovis</i>.

<p>Various fractions of the chromatography of a homogenate from <i>M</i>. <i>bovis</i> were incubated with PknD and the phosphorylated proteins were separated by SDS-PAGE. Five bands (numbered on the gels) were extracted and sequenced using the Edman degradation. They were identified by comparison with the genome of the H37Rv strain.</p><p>Determination of the major proteins phosphorylated by PknD in a homogenate from <i>M</i>. <i>bovis</i>.</p

Repartition of the clusters according to the links identified between the patients.

<p>Clusters are represented by unique identification numbers. Next to the cluster number, the number of patients for who an epidemiological link was detected is indicated (i.e. 2/3: two of the three patients included in the cluster present this link). f: familial link; o: same geographic origin; p: geographic proximity. Clusters in orange contain more than 3 patients and cluster in grey more than 6. The purple boxes indicate the number of clusters belonging to each category (or combination of categories) of links. The blue boxes indicate clusters comprising exclusively Belgian-born patients. The green boxes indicate clusters with strains presenting the 776000000000171 spoligotype (S-family).</p

Characteristics of <i>Mycobacterium tuberculosis</i> complex strains isolated from tuberculosis patients living in Brussels and two subgroups; no-clustered and clustered strains, 2010–2013.

<p>Characteristics of <i>Mycobacterium tuberculosis</i> complex strains isolated from tuberculosis patients living in Brussels and two subgroups; no-clustered and clustered strains, 2010–2013.</p

Molecular epidemiology of <i>Mycobacterium tuberculosis</i> complex in Brussels, 2010–2013

<div><p>The tuberculosis (TB) incidence rate in Brussels-Capital Region is 3-fold higher than in Belgium as a whole. Eight years after the realization of initial prospective population-based molecular epidemiology investigations in this Region, a similar study over the period 2010–2013 was conducted. TB strains isolated from 945 patients were submitted to genotyping by standardized 24-locus-MIRU-VNTR typing and spoligotyping. The phylogenetic analysis showed that the LAM (16.7%) and Haarlem (15.7%) branches are the two most prevalent TB lineages circulating in Brussels. Analysis of the MDR subgroup showed an association with Beijing strains (39.9%) and patients native of Eastern Europe (40.7%). Genotyping detected 113 clusters involving 321 patients, giving a recent transmission index of 22.9%. Molecular-guided epidemiological investigations and routine surveillance activities revealed family transmission or social contact for patients distributed over 34 clusters. Most of the patients were foreign-born (75.7%). However, cluster analysis revealed only limited trans-national transmission. Comparison with the previous study shows a stable epidemiological situation except for the mean age difference between Belgian-born and foreign-born patients which has disappeared. This study confirms that molecular epidemiology has become an important determinant for TB control programs. However, sufficient financial means need to be available to perform all required epidemiological investigations.</p></div