Profiling of Intracellular Metabolites: An Approach to Understanding the Characteristic Physiology of <i>Mycobacterium leprae</i>

<div><p><i>Mycobacterium leprae</i> is the causative agent of leprosy and also known to possess unique features such as inability to proliferate <i>in vitro</i>. Among the cellular components of <i>M</i>. <i>leprae</i>, various glycolipids present on the cell envelope are well characterized and some of them are identified to be pathogenic factors responsible for intracellular survival in host cells, while other intracellular metabolites, assumed to be associated with basic physiological feature, remain largely unknown. In the present study, to elucidate the comprehensive profile of intracellular metabolites, we performed the capillary electrophoresis-mass spectrometry (CE-MS) analysis on <i>M</i>. <i>leprae</i> and compared to that of <i>M</i>. <i>bovis</i> BCG. Interestingly, comparison of these two profiles showed that, in <i>M</i>. <i>leprae</i>, amino acids and their derivatives are significantly accumulated, but most of intermediates related to central carbon metabolism markedly decreased, implying that <i>M</i>. <i>leprae</i> possess unique metabolic features. The present study is the first report demonstrating the unique profiles of <i>M</i>. <i>leprae</i> metabolites and these insights might contribute to understanding undefined metabolism of <i>M</i>. <i>leprae</i> as well as pathogenic characteristics related to the manifestation of the disease.</p></div

Comparative profiling of metabolites detected from <i>M</i>. <i>leprae</i> and <i>M</i>. <i>bovis</i> BCG.

<p>A) Principal component analysis (PCA) on relative peak area of metabolites detected from three independent groups of <i>M</i>. <i>leprae</i> and <i>M</i>. <i>bovis</i> BCG. Total relative peak area for analysis was derived from 193 metabolites of <i>M</i>. <i>leprae</i> and 137 metabolites of <i>M</i>. <i>bovis</i> BCG. B) Venn diagram of identified metabolites. C) Proportion of the classified metabolites based on their putative functions in each metabolism. Each of mycobacterial metabolites was derived from 2.5x10¹⁰ cells.</p

Functional classification of the metabolites having over 10-fold difference in the mean ratio of relative peak areas [<i>M</i>. <i>leprae</i> (n = 3)/<i>M</i>. <i>bovis</i> BCG (n = 3)].

<p>Functional classification of the metabolites having over 10-fold difference in the mean ratio of relative peak areas [<i>M</i>. <i>leprae</i> (n = 3)/<i>M</i>. <i>bovis</i> BCG (n = 3)].</p

Functional classification of the metabolites having less than 0.1-fold difference in the mean ratio of relative peak areas [<i>M</i>. <i>leprae</i> (n = 3)/<i>M</i>. <i>bovis</i> BCG (n = 3)].

<p>Functional classification of the metabolites having less than 0.1-fold difference in the mean ratio of relative peak areas [<i>M</i>. <i>leprae</i> (n = 3)/<i>M</i>. <i>bovis</i> BCG (n = 3)].</p

Schematic representation for retrieving the <i>M</i>. <i>leprae</i>-derived metabolites identified by CE-MS analysis.

<p>In each of common 48 metabolites, the mean of triplicate values (relative peak areas) of uninfected nude mice was subtracted from all triplicate value of <i>M</i>. <i>leprae</i>-infected nude mice and resultant 35 metabolites were retrieved as abundantly present in <i>M</i>. <i>leprae</i>-infected nude mice.</p

Quantitative comparison of 84 metabolites common to <i>M</i>. <i>leprae</i> and <i>M</i>. <i>bovis</i> BCG.

<p>Relative peak areas are expressed as means± S.D. of triplicate. These values were retrieved by CE-MS analysis performing on methanol extract from 2.5x10¹⁰ cells of each mycobacteria collected by filtration. As for those of <i>M</i>. <i>leprae</i>, nude mice-derived values were subtracted. Red circle and green square represent the mean value of each metabolite detected from <i>M</i>. <i>leprae</i> and <i>M</i>. <i>bovis</i> BCG, respectively. 84 metabolites are arranged in descending order of their mean ratio (<i>M</i>. <i>leprae</i>/<i>M</i>. <i>bovis</i> BCG). Of these metabolites, their mean ratio showing over 10-fold and less than 0.1-fold are indicated by arrows.</p

Mutations conferring resistance in <i>Mycobacterium leprae</i> are detected by the GenoType LepraeDR DNA strip test.

<p>Lane 1 is a negative control (only the CC band). Lanes 2 to 11 showed various profiles for resistant strains: lane 2, <i>rpoB</i> mutation S456L with wild type <i>gyrA</i> and <i>folP1</i> alleles; lane 3, wild type <i>rpoB</i> and <i>gyrA</i> alleles with a <i>folP1</i> mutation to be defined; lane 4, <i>rpoB</i> mutation S456L with a wild type <i>gyrA</i> allele but a mutation in <i>folP1</i>; lane 5, <i>rpoB</i> mutation (Q438V) with wild type <i>gyrA</i> and <i>folP1</i> alleles; lane 6, wild type <i>rpoB</i> and <i>gyrA</i> alleles with a P55L mutation in <i>folP1</i>; lane 7, <i>rpoB</i> mutation S456L with a A91V <i>gyrA</i> mutation and a P55L mutation in <i>folP</i>; lane 8 and lane 9, wild type <i>rpoB</i> and <i>gyrA</i> alleles with a P55L mutation in <i>folP1</i> ; lane 10 and lane 11, <i>rpoB</i> mutation S456L with wild type <i>gyrA</i> and <i>folP1</i> alleles. The numbering system used is that of the <i>Mycobacterium leprae</i> genome strain NT (n°NC 002677).</p

<i>Mycobacterium leprae</i> susceptible strains showed a wild type profile in the GenoType LepraeDR test.

<p>Lane 1 to 16 (except lane 8) showed wild type profiles for susceptible <i>M. leprae</i> strains. Lane 8 showed a multiresistant profile with mutations in <i>rpoB</i>, <i>gyrA</i> and <i>folP1</i> genes. Lanes 17 and 18 showed result of negative controls.</p

Concordance of results for the DNA strip test (GenoType LepraeDR) and the susceptibility phenotypic and genotypic pattern of antibiotic resistance for the <i>M. leprae</i> strains studied.

*<p>For strains growing in vivo and yielding interpretable susceptibility results. Tests were stopped for dapsone due to new regulation for antibiotic animal feeding. Tests for ofloxacin were restricted to patient with previous treatment by fluoroquinolones.</p>**<p>including two strains with a mutation at codon 447: Ser447Cys for one strain and a silent mutation for the second strain (see text for details).</p

Probes and primers used in the GenoType Leprae DR test for molecular detection of antileprosy resistance.

<p>na, non applicable.</p>*<p>numbering system used in the <i>M. leprae</i> genome of strain NT (sequence NC 002677 in GenBank).</p