Pathophysiological Implications of Cell Envelope Structure in Mycobacterium tuberculosis and Related Taxa

Mycobacterium tuberculosis has a cell envelope incorporating a peptidoglycan-linked arabinogalactan esterified by long-chain mycolic acids. A range of “free” lipids are associated with the “bound” mycolic acids, producing an effective envelope outer membrane. The distribution of these lipids is discontinuous among mycobacteria and such lipids have proven potential for biomarker use in tracing the evolution of tuberculosis. A plausible evolutionary scenario involves progression from an environmental organism, such as Mycobacterium kansasii, through intermediate “smooth” tubercle bacilli, labelled “Mycobacterium canettii”; cell envelope lipid composition possibly correlates with such a progression. M. kansasii and “M. canettii” have characteristic lipooligosaccharides, associated with motility and biofilms, and glycosyl phenolphthiocerol dimycocerosates (“phenolic glycolipids”). Both these lipid classes are absent in modern M. tuberculosis sensu stricto, though simplified phenolic glycolipids remain in certain current biotypes. Dimycocerosates of the phthiocerol family are restricted to smaller phthiodiolone diesters in M. kansasii. Diacyl and pentaacyl trehaloses are present in “M. canettii” and M. tuberculosis, where they are accompanied by related sulfated acyl trehaloses. In comparison with environmental mycobacteria, subtle modifications in mycolic acid structures in “M. canettii” and M. tuberculosis are notable. The probability of essential tuberculosis evolution taking place in Pleistocene megafauna, rather than Homo sapiens, is reemphasised

Biochemical and structural characterization of mycobacterial aspartyl-tRNA synthetase AspS, a promising TB drug target.

The human pathogen Mycobacterium tuberculosis is the causative agent of pulmonary tuberculosis (TB), a disease with high worldwide mortality rates. Current treatment programs are under significant threat from multi-drug and extensively-drug resistant strains of M. tuberculosis, and it is essential to identify new inhibitors and their targets. We generated spontaneous resistant mutants in Mycobacterium bovis BCG in the presence of 10× the minimum inhibitory concentration (MIC) of compound 1, a previously identified potent inhibitor of mycobacterial growth in culture. Whole genome sequencing of two resistant mutants revealed in one case a single nucleotide polymorphism in the gene aspS at 535GAC>535AAC (D179N), while in the second mutant a single nucleotide polymorphism was identified upstream of the aspS promoter region. We probed whole cell target engagement by overexpressing either M. bovis BCG aspS or Mycobacterium smegmatis aspS, which resulted in a ten-fold and greater than ten-fold increase, respectively, of the MIC against compound 1. To analyse the impact of inhibitor 1 on M. tuberculosis AspS (Mt-AspS) activity we over-expressed, purified and characterised the kinetics of this enzyme using a robust tRNA-independent assay adapted to a high-throughput screening format. Finally, to aid hit-to-lead optimization, the crystal structure of apo M. smegmatis AspS was determined to a resolution of 2.4 Å

Mycolic acids: deciphering and targeting the A

Mycolic acids are unique long chain fatty acids found in the lipid‐rich cell walls of mycobacteria including the tubercle bacillus M ycobacterium tuberculosis. Essential for viability and virulence, enzymes involved in the biosynthesis of mycolic acids represent novel targets for drug development. This is particularly relevant to the impact on global health given the rise of multidrug resistant and extensively drug resistant strains of M . tuberculosis. In this review, we discuss recent advances in our understanding of how mycolic acid are synthesised, especially the potential role of specialised fatty acid synthase complexes. Also, we examine the role of a recently reported mycolic acid transporter MmpL3 with reference to several reports of the targeting of this transporter by diverse compounds with anti‐M . tuberculosis activity. Additionally, we consider recent findings that place mycolic acid biosynthesis in the context of the cell biology of the bacterium, viz its localisation and co‐ordination with the bacterial cytoskeleton, and its role beyond maintaining cell envelope integrity

Structures of different LOS subclasses from <i>M</i>. <i>kansasii</i>

<p>(A) LOS-I-III (n = 1–3), (B) LOS-IV-VII (n = 2–4), (C) LOS-VII; R1, R2 and R3 represents the acyl chain attached to tetra glucose core of LOS. (D) Schematic showing predicted domains and topology of <i>MKAN27435</i>. The amino and carboxy terminals are indicated as ‘N’ and ‘C’ respectively, and numbers represent predicted borders of domains which are depicted as grey areas. The regions corresponding to the predicted transmembrane domains are indicated with black bars.</p

MALDI-TOF mass spectroscopy analysis of purified LOS’s from <i>M</i>.<i>kansasii</i> WT and Δ<i>MKAN27435</i> strains in positive ion mode.

<p>All molecular ions are [M+Na]⁺ and species indicated with an asterisk (*) correspond to <i>m/z</i> values with an intact acyl group.</p

Autoradiograph of 2-D TLC analysis of polar lipids extracted from <i>M</i>. <i>kansasii</i> WT, Δ<i>MKAN27435</i>, Δ<i>MKAN27435</i>-C strains.

<p>Arrows indicate the different LOS species. I and II indicate system E dimension 1 and 2 respectively. Dimension I: Chloroform: Methanol: H₂O (60:30:6); Dimension II: Chloroform: Acetic acid: Methanol: H₂O (40:25:3:6).</p

MALDI-TOF mass spectroscopy analyses tabulated to list LOS species with corresponding <i>m/z</i> values in <i>M</i>. <i>kansasii</i> WT and Δ<i>MKAN27435</i> strains.

<p>All molecular ions are [M+Na]⁺and an asterisk (*) indicates species with an intact acyl group. Presence (+) or absence (-) of each species is indicated for each strain.</p><p>MALDI-TOF mass spectroscopy analyses tabulated to list LOS species with corresponding <i>m/z</i> values in <i>M</i>. <i>kansasii</i> WT and Δ<i>MKAN27435</i> strains.</p

The role of hydrophobicity in tuberculosis evolution and pathogenicity

The evolution of tubercle bacilli parallels a route from environmental Mycobacterium kansasii, through intermediate "Mycobacterium canettii", to the modern Mycobacterium tuberculosis complex. Cell envelope outer membrane lipids change systematically from hydrophilic lipooligosaccharides and phenolic glycolipids to hydrophobic phthiocerol dimycocerosates, di- and pentaacyl trehaloses and sulfoglycolipids. Such lipid changes point to a hydrophobic phenotype for M. tuberculosis sensu stricto. Using Congo Red staining and hexadecane-aqueous buffer partitioning, the hydrophobicity of rough morphology M. tuberculosis and Mycobacterium bovis strains was greater than smooth "M. canettii" and M. kansasii. Killed mycobacteria maintained differential hydrophobicity but defatted cells were similar, indicating that outer membrane lipids govern overall hydrophobicity. A rough M. tuberculosis H37Rv ΔpapA1 sulfoglycolipid-deficient mutant had significantly diminished Congo Red uptake though hexadecane-aqueous buffer partitioning was similar to H37Rv. An M. kansasii, ΔMKAN27435 partially lipooligosaccharide-deficient mutant absorbed marginally more Congo Red dye than the parent strain but was comparable in partition experiments. In evolving from ancestral mycobacteria, related to "M. canettii" and M. kansasii, modern M. tuberculosis probably became more hydrophobic by increasing the proportion of less polar lipids in the outer membrane. Importantly, such a change would enhance the capability for aerosol transmission, affecting virulence and pathogenicity

Substrate dependence of Mt-AspS activity.

<p>Michaelis-Menten curves were fitted for <b>A</b>) varying ADPCP as substrate. <b>B</b>) <b>L</b>-Asp as substrate at fixed saturating concentrations of ADPCP and PP<i>_i.</i><b>C</b>) PP<i>_i</i> as substrate at fixed concentrations of ADPCP and <b>L</b>-Asp. <b>D</b>) ADPNP as substrate. <b>E</b>) <b>L</b>-Asp as substrate at fixed concentrations of ADPNP and PP<i>_i</i>. <b>F</b>) ATP dependence of hexokinase/glucose-6-phosphate dehydrogenase activity. The initial velocity data (dA/min) were plotted against the substrate concentration. Each assay was done in triplicate and expressed as mean ± standard error of mean.</p

Comparison of the expression levels of the transcript produced by wild type <i>M. bovis</i> BCG, <i>aspS</i> mutant 1 and promoter up mutant 2 by RT-PCR.

<p>RT-PCR was performed with RT (upper panel) and without RT (lower panel). The RT-PCR cycling conditions were programmed for 25, 30, 40 and 50 cycles, respectively, to get sufficient yields of the 200 bp amplified product and analysed on a 2% agarose gel following staining.</p