Drug screening approach against mycobacterial fatty acyl-AMP ligase FAAL32 renews the interest of the salicylanilide pharmacophore in the fight against tuberculosis.

Tuberculosis (TB) remains a global health crisis, further exacerbated by the slow pace of new treatment options, and the emergence of extreme and total drug resistance to existing drugs. The challenge to developing new antibacterial compounds with activity against Mycobacterium tuberculosis (Mtb), the causative agent of TB, is in part due to unique features of this pathogen, especially the composition and structure of its complex cell envelope. Therefore, targeting enzymes involved in cell envelope synthesis has been of major interest for anti-TB drug discovery. FAAL32 is a fatty acyl-AMP ligase involved in the biosynthesis of the cell wall mycolic acids, and a potential target for drug discovery. To rapidly advance research in this area, we initiated a drug repurposing campaign and screened a collection of 1280 approved human or veterinary drugs (Prestwick Chemical Library) using a biochemical assay that reads out FAAL32 inhibition. These efforts led to the discovery of salicylanilide closantel, and some of its derivatives as inhibitors with potent in vitro activity against M. tuberculosis. These results suggest that salicylanilide represents a potentially promising pharmacophore for the conception of novel anti-tubercular candidates targeting FAAL32 that would open new targeting opportunities. Moreover, this work illustrates the value of drug repurposing campaigns to discover new leads in challenging drug discovery fields.

Elionurus tristis Essential Oil: GC-MS Analysis and Antioxidant and Antituberculosis Activities.

Essential oil was obtained in a yield 1.1%, w/w, by steam distillation of Elionurus tristis leaves from Madagascar. The chemical composition was analyzed qualitatively and quantitatively by GC-MS and GC-FID, respectively. To the best of our knowledge, this is the first chemical analysis of this essential oil. Seventy-three compounds were identified, corresponding to 94.9% of the total essential oil. The principal compounds were sesquiterpenes and the more represented were ß-gudjunene (18.4%), neoclovene (15.8%) and nootkatone (10.4%). Through a comparative study, we observed a large variability between the components of E. tristis essential oil and those from others species of the same genus. Evaluation of the antioxidant (ABTS and DPPH assays) and anti- tuberculosis activities of the essential oil showed weak antioxidant potency but an interesting anti-tuberculosis activity with a MIC of 32 mg/L. This activity prompted us to evaluate individually the major components for the treatment of tuberculosis.

Current knowledge on mycolic acids in Corynebacterium glutamicum and their relevance for biotechnological processes.

Corynebacterium glutamicum is the world's largest producer of glutamate and lysine. Industrial glutamate overproduction is induced by empirical processes, such as biotin limitation, supplementation with specific surfactants or addition of sublethal concentration of certain antibiotics to the culture media. Although Gram-positive bacteria, C. glutamicum and related bacterial species and genera contain, in addition to the plasma membrane, an outer permeability membrane similar to that of Gram-negative microorganisms. As the amino acids have to cross both membranes, their integrity, composition and fluidity influence the export process. While the precise mechanism of the export of the amino acids by C. glutamicum is not fully understood, the excretion of amino acids through the inner membrane involved at least a major export system mechanosensitive channel MscS family (MscCG) encoded by NCgl1221. As the various industrial treatments have been shown to affect the lipid content of the bacterial cell, it is strongly believed that defects in the hallmark of the outer membrane, 2-alkyl, 3-hydroxylated long-chain fatty acids (mycolic acids), could be key factors in the glutamate overproduction. This review aims at giving an overview of the current knowledge on mycolic acids structure, biosynthesis and transfer in C. glutamicum and their relevance for amino acid biotechnological production.

A monoacylglycerol lipase from Mycobacterium smegmatis Involved in bacterial cell interaction.

MSMEG_0220 from Mycobacterium smegmatis, the ortholog of the Rv0183 gene from M. tuberculosis, recently identified and characterized as encoding a monoacylglycerol lipase, was cloned and expressed in Escherichia coli. The recombinant protein (rMSMEG_0220), which exhibits 68% amino acid sequence identity with Rv0183, showed the same substrate specificity and similar patterns of pH-dependent activity and stability as the M. tuberculosis enzyme. rMSMEG_0220 was found to hydrolyze long-chain monoacylglycerol with a specific activity of 143 +/- 6 U mg(-1). Like Rv0183 in M. tuberculosis, MSMEG_0220 was found to be located in the cell wall. To assess the in vivo role of the homologous proteins, an MSMEG_0220 disrupted mutant of M. smegmatis (MsDelta0220) was produced. An intriguing change in the colony morphology and in the cell interaction, which were partly restored in the complemented mutant containing either an active (ComMsDelta0220) or an inactive (ComMsDelta0220S111A) enzyme, was observed. Growth studies performed in media supplemented with monoolein showed that the ability of both MsDelta0220 and ComMsDelta0220S111A to grow in the presence of this lipid was impaired. Moreover, studies of the antimicrobial susceptibility of the MsDelta0220 strain showed that this mutant is more sensitive to rifampin and more resistant to isoniazid than the wild-type strain, pointing to a critical structural role of this enzyme in mycobacterial physiology, in addition to its function in the hydrolysis of exogenous lipids.

The Mycobacterium tuberculosis PhoPR two-component system regulates genes essential for virulence and complex lipid biosynthesis.

Two-component signal transduction systems (2-CS) play an important role in bacterial pathogenesis. In the work presented here, we have studied the effects of a mutation in the Mycobacterium tuberculosis (Mtb) PhoPR 2-CS on the pathogenicity, physiology and global gene expression of this bacterial pathogen. Disruption of PhoPR causes a marked attenuation of growth in macrophages and mice and prevents growth in low-Mg2+ media. The inability to grow in THP-1 macrophages can be partially overcome by the addition of excess Mg2+ during infection. Global transcription assays demonstrate PhoP is a positive transcriptional regulator of several genes, but do not support the hypothesis that the Mtb PhoPR system is sensing Mg2+ starvation, as is the case with the Salmonella typhimurium PhoPQ 2-CS. The genes that were positively regulated include those found in the pks2 and the msl3 gene clusters that encode enzymes for the biosynthesis of sulphatides and diacyltrehalose and polyacyltrehalose respectively. Complementary biochemical studies, in agreement with recent results from another group, indicate that these complex lipids are also absent from the phoP mutant, and the lack of these components in its cell envelope may indirectly cause the mutant's high-Mg2+ growth requirement. The experiments reported here provide functional evidence for the PhoPR 2-CS involvement in Mtb pathogenesis, and they suggest that a major reason for the attenuation observed in the phoP mutant is the absence of certain complex lipids that are known to be important for virulence.