Mycobacterium tuberculosis is the world's most deadly bacterial pathogen. In 2015, the World Health Organization (WHO) reported
that more than 9 million people were newly diagnosed with the disease, and that 1.6 million people died from Tuberculosis (TB). The
recent emergence of multi-drug resistant (MDR) strains of M. tuberculosis requires patients diagnosed with MDR-TB to be treated with second-line, toxic drugs for 20 months. Even so, almost half of MDR-TB patients died last year (190,000 of 480,000 patients). The need for new drugs to treat TB is a clear global priority.
Recently, it was shown that M. tuberculosis lacks a complete TCA cycle; specifically that the gene annotated as alpha-ketoglutarate
decarboxylase does not possess that activity. It was suggested instead that alpha-ketoglutarate was converted to L-glutamate and then
to gamma-aminobutyric acid (GABA), and finally to succinate semialdehyde by glutamate decarboxylase (Gad) and GABA
transaminase, respectively. Orthologues of each of these enzymes have been identified in the genome of M. tuberculosis, but none
have been expressed or functionally characterized. The only of the "GABA shunt" enzymes to have been enzymatically characterized
is succinate semialdehyde dehydrogenase, which generates succinate, the TCA cycle intermediate, and NADH.
All L-glutamate decarboxylases characterized so far contain the organic cofactor pyridoxal phosphate (PLP) to which the substrate
covalently binds and is subsequently chemically converted to product. We propose to heterologously express M. tuberculosis Gad,
purify the protein to homogeneity, determine the chemical state of bound PLP spectroscopically and spectrofluorimetrically, assess the
pH-dependent enzymatic activity and its oligomeric state.
This knowledge will provide a platform for the later examination of potential inhibitors of the enzyme as a "druggable target" in M.
tuberculosis.
During the infectious process, bacterial pathogens are continuously exposed to hostile environments. Evolution and natural selection has favored those possessing protective mechanisms to cope with such life-threatening conditions. Among them, the fluctuations in pH are one of the most frequent and severe stresses encountered during the colonization of the human host. Others include exposure to reactive oxygen and nitrogen species (Lund et al., 2014).
In this respect, the human pathogen Mycobacterium tuberculosis is exposed to all of these stresses and many acidic environments during its intracellular life-cycle. The most representative is the macrophage phagosome and phagolysosome, whose pH values range from a mildly acidic value of pH 6.0 to a more acidic pH of 4.5-5.0, depending on the activation state of macrophage. Phagosome acidity can provide a critical cue for adaptation to the host niche. However, how the bacilli can survive and replicate inside the hostile environment of the phagosome is still poorly understood.
To date, except for the production of ammonia by urease, none of the best suited protective mechanisms for acid resistance, reported in Gram-positive and Gram-negative bacterial pathogens, has been found to play a role in M. tuberculosis acid resistance (Vandal et al., 2009). Thus, discovering how the organism adapts within the acidic environment of the phagosome may guide the development of new drugs.
The glutamate-dependent acid resistance system (GDAR system), the most powerful acid resistance mechanism reported in E. coli, consists of an L-glutamate decarboxylase, GadB, and the cognate antiporter, GadC. GadB catalyzes the irreversible decarboxylation of
L-glutamate to produce GABA, which is then exported out of the cell by GadC, which exchanges one molecule of L-glutamate for one molecule of GABA (ratio 1:1) and in the process consumes one intracellular proton (De Biase and Pennacchietti, 2012).
L-glutamate is the most abundant intracellular metabolite in E. coli as it is the predominant amino group donor in many essential biosynthetic pathways (Bennet et al., 2009). This, along with the consumption of cytoplasmic protons during the decarboxylation reaction, and the GABA-mediated export of positive charges, make the GDAR the most efficient system to counteract strong extracellular acidity (Lund et al., 2014).
The only role for L-glutamate decarboxylase that has been suggested so far in M. tuberculosis is as a key enzyme in the GABA "shunt" pathway, which, along with GABA transaminase and succinate semialdehyde dehydrogenase, restores the otherwise "incomplete" TCA cycle of this pathogenic bacterium due to the lack of a functional alpha-ketoglutarate decarboxylase. A similar situation has also
been reported in Listeria monocytogenes, a well-known Gram-positive pathogen responsible for listeriosis, a disease with a mortality rate up to 30%, in which the GABA-shunt plays a role in survival under acidic conditions but also as filling the gap of the TCA cycle.
Indeed, like in M. tuberculosis, the GABA "shunt" is thought to compensate for the "incomplete" TCA cycle in Listeria as genomic evidences suggest that this bacterium lacks both the alpha-ketoglutarate dehydrogenase and succinyl-CoA synthetase (Feehily et al., 2013).
Interestingly, the alignment of the presumed M. tuberculosis GadB sequence with that E. coli GadB (48% overall identity), shows a perfect conservation of all the residues known as "GAD signature". Together, these residues make up a biological fingerprint of all L-glutamate decarboxylases known in prokaryotes since they represent residues associated with binding of the cofactor (PLP), the
decarboxylation of the substrate and the structural changes associated with a reduction in pH.
With these issues in mind, I propose to investigate the pH-dependent activity of the expressed and purified M. tuberculosis GadB, with particular emphasis on the enzymatic activity and spectroscopic properties at low pH. The results I obtain will clarify if this enzyme plays a potential role in acid resistance in this pathogen and serves as a novel virulence factor. Together with its role in central carbon metabolism as a constituent of the GABA "shunt", completing the TCA cycle, it may represent an attractive and compelling target for inhibitor design due to its implication in two important pathways in M. tuberculosis (Rhee et al., 2011).
References
Bennet, et. al, Nature Chemical Biology 5 (2009) 593-599
De Biase and Pennacchietti, Molecular Microbiology 86 (2012) 770-786
Feehily, et. al, Applied and Environmental Microbiology 79 (2013) 74-80
Lund, et. al, FEMS Microbiol Rev 38 (2014) 1091-1125
Rhee, et. al, Microbs and Metabolism 19 (2011) 307-314
Vandal, et. al, J. Bacteriol 191 (2009) 4714-4721