Role of the methylcitrate cycle in propionate metabolism and detoxification in Mycobacterium smegmatis

Catabolism of odd-chain-length fatty acids yields acetyl-CoA and propionyl-CoA. A common pathway of propionyl-CoA metabolism in micro-organisms is the methylcitrate cycle, which includes the dedicated enzymes methylcitrate synthase (MCS), methylcitrate dehydratase (MCD) and methylisocitrate lyase (MCL). The methylcitrate cycle is essential for propionate metabolism in Mycobacterium tuberculosis. Unusually, M. tuberculosis lacks an MCL orthologue and this activity is provided instead by two isoforms of the glyoxylate cycle enzyme isocitrate lyase (ICL1 and ICL2). These bifunctional (ICL/MCL) enzymes are jointly required for propionate metabolism and for growth and survival in mice. In contrast, the non-pathogenic species Mycobacterium smegmatis encodes a canonical MCL enzyme in addition to ICL1 and ICL2. The M. smegmatis gene encoding MCL (prpB) is clustered with genes encoding MCS (prpC) and MCD (prpD). Here we show that deletion of the M. smegmatis prpDBC locus reduced but did not eliminate MCL activity in cell-free extracts. The residual MCL activity was abolished by deletion of icl1 and icl2 in the DeltaprpDBC background, suggesting that these genes encode bifunctional ICL/MCL enzymes. A DeltaprpB Deltaicl1 Deltaicl2 mutant was unable to grow on propionate or mixtures of propionate and glucose. We hypothesize that incomplete propionyl-CoA metabolism might cause toxic metabolites to accumulate. Consistent with this idea, deletion of prpC and prpD in the DeltaprpB Deltaicl1 Deltaicl2 background paradoxically restored growth on propionate-containing media. These observations suggest that the marked attenuation of ICL1/ICL2-deficient M. tuberculosis in mice could be due to the accumulation of toxic propionyl-CoA metabolites, rather than inability to utilize fatty acids per se.

The Phosphate Specific Transport (Pst) System in Fast- and Slow-Growing Mycobacteria

Cyntia De Piano

Although mycobacteria are known to accumulate polyphosphate (PolyP), a linear polymer of orthophosphate, when subjected to stresses, the exact roles of this molecule are not yet clearly understood. We found that deletion of the pstA gene encoding for a transmembrane domain of the high-affinity phosphate-specific ABC transporter system (Pst system) resulted in accumulation of high amounts of PolyP during growth in phosphate (Pi)-replete conditions in Mycobacterium tuberculosis and Mycobacterium smegmatis. These PolyP stores were gradually used during Pi starvation by the ApstA mutants in order to sustain growth and this allowed the mutant strains to reach a higher cell density than the wild-type (WT) parental strain. Neither the mRNA nor the protein levels of polyphosphate kinase 1 (PPK1) could explain the PolyP accumulation observed in the mutant, suggesting that another regulatory mechanism is involved. The ApstA mutant of M. smegmatis was hypersensitive to several in vitro stresses, including detergent, oxidative stress, and cell wall targeting antibiotics. Deletion of the main enzyme involved in PolyP synthesis, PPK1, in the ApstA mutant background of M. smegmatis completely reversed the PolyP accumulation and partially reversed the stress sensitivity as well as the derepression of PHO regulon genes observed in the ApstA mutant. The mechanism of PolyP accumulation in M. smegmatis was investigated using a genetic approach. We found that this mechanism differs from the (p)ppGpp-mediated PolyP accumulation observed in Escherichia coli, since deletion of the RelA enzyme in a ApstA mutant background did not prevent PolyP accumulation. The mechanism of PolyP degradation during Pi starvation in the M. smegmatis ApstA mutant was also analyzed. Deletion of both polyphosphatase enzymes (PPX1 and PPX2) as well as the polyphosphate kinase 2 (PPK2) enzyme did not have any impact on PolyP degradation. PPK1 is a bifunctional enzyme that can either synthesize or degrade PolyP. Our current hypothesis is that PPK1 might perform the reverse reaction (PolyP degradation) during Pi starvation. The aim of the second part of my project was to make a comparative study of the expression and regulation of the phosphate specific transporter (pst) operon in fast-growing [M. smegmatis] and slow-growing [M. tuberculosis] mycobacteria. To achieve this goal, pst reporter strains of M. smegmatis and M. tuberculosis were constructed by precise insertion of a modified dest_gfp gene (provided by Giulia Manina) encoding a destabilized green fluorescent protein (dest_GFP) into the corresponding pst locus as transcriptional ) fusions. The transcriptional reporter construct [p) was introduced in both the WT and the ApstA mutant backgrounds. The kinetics of induction of the pst-gfp reporters in WT and ApstA mutant strains in response to Pi starvation was analyzed. Population-averaged measurements were made using qRT-PCR and Western blotting. Single-cell measurements using flow cytometry and timelapse fluorescence microscopy were also performed. For the microscopy experiments, the bacteria were cultured in custom-made microfluidic devices that permit rapid switching of the culture medium, e.g., switching between Pi-replete and Pi-depleted media. Because these studies require precise environmental control and resolution of individual and temporal phenotypes, my principal experimental tool was the integrated microfluidics and timelapse fluorescence microscopy systems that have recently been developed in the laboratory. Although there have been many studies published on the population-averaged behavior of bacteria responding to external stresses such as Pi limitation, we are not aware of any studies addressing the individuality of such responses using a real-time single-cell approach. We observed that the kinetics of induction of the Pst operon upon Pi starvation scaled with the growth rate of the bacteria and peak induction occurred within about two generations for both smegmatis and tuberculosis. Interestingly, we found that the basal level of Pst expression and the steady state level reached upon induction by Pi starvation were similar in tuberculosis and M. smegmatis, although the kinetics of induction were much faster in M. smegmatis. These studies contributed to improve the understanding of the regulation and the kinetics of response of the PHO regulon in fast- and slow-growing mycobacteria. The finding that PolyP plays a role in this process could also apply to other organisms where the PHO regulon is regulated in a similar way.

EPFL2013

Quantitative Mass Spectrometry Reveals Plasticity of Metabolic Networks in Mycobacterium smegmatis

Florence Armand, Diego Chiappe, Tarun Chopra, Romain Hamelin, John McKinney, Marc Moniatte

Mycobacterium tuberculosis has a remarkable ability to persist within the human host as a clinically inapparent or chronically active infection. Fatty acids are thought to be an important carbon source used by the bacteria during long term infection. Catabolism of fatty acids requires reprogramming of metabolic networks, and enzymes central to this reprogramming have been targeted for drug discovery. Mycobacterium smegmatis, a nonpathogenic relative of M. tuberculosis, is often used as a model system because of the similarity of basic cellular processes in these two species. Here, we take a quantitative proteomics-based approach to achieve a global view of how the M. smegmatis metabolic network adjusts to utilization of fatty acids as a carbon source. Two-dimensional liquid chromatography and mass spectrometry of isotopically labeled proteins identified a total of 3,067 proteins with high confidence. This number corresponds to 44% of the predicted M. smegmatis proteome and includes most of the predicted metabolic enzymes. Compared with glucose-grown cells, 162 proteins showed differential abundance in acetate- or propionate-grown cells. Among these, acetate-grown cells showed a higher abundance of proteins that could constitute a functional glycerate pathway. Gene inactivation experiments confirmed that both the glyoxylate shunt and the glycerate pathway are operational in M. smegmatis. In addition to proteins with annotated functions, we demonstrate carbon source-dependent differential abundance of proteins that have not been functionally characterized. These proteins might play as-yet-unidentified roles in mycobacterial carbon metabolism. This study reveals several novel features of carbon assimilation in M. smegmatis, which suggests significant functional plasticity of metabolic networks in this organism.

Amer Soc Biochemistry Molecular Biology Inc2014

Synthesis and antimycobacterial evaluation of benzofurobenzopyran analogues

Stewart Cole

We recently reported that 3,3-dimethyl-3H-benzofuro[3,2,f][1]-benzopyran and its hydrogenated analogue are selective in vitro inhibitors of mycobacterial growth. However, their lack of in vivo activity on a murine model of Mycobacterium tuberculosis infection due to their poor bioavailability led to a structure-activity relationship investigation. We wish to report here the preparation of some structural analogues along with their biological effect on the growth of Mycobacterium smegmatis, M. tuberculosis, as well as on VERO cells for the most active compound.