Abstract
Mycobacterium tuberculosis is an intracellular pathogen. Within macrophages, M. tuberculosis thrives in a specialized membrane-bound vacuole, the phagosome, whose pH is slightly acidic, and where access to nutrients is limited. Understanding how the bacillus extracts and incorporates nutrients from its host may help develop novel strategies to combat tuberculosis. Here we show that M. tuberculosis employs the asparagine transporter AnsP2 and the secreted asparaginase AnsA to assimilate nitrogen and resist acid stress through asparagine hydrolysis and ammonia release. While the role of AnsP2 is partially spared by yet to be identified transporter(s), that of AnsA is crucial in both phagosome acidification arrest and intracellular replication, as an M. tuberculosis mutant lacking this asparaginase is ultimately attenuated in macrophages and in mice. Our study provides yet another example of the intimate link between physiology and virulence in the tubercle bacillus, and identifies a novel pathway to be targeted for therapeutic purposes. © 2014 Gouzy et al.
Figures
![Figure 4. Requirement of AnsP2 and AnsA for M. tuberculosis resistance to acid in host macrophages. (A,B) Exogenous asparagine accumulates in the mycobacterial phagosome. (A) Images from a representative infected cell showing the locations of M. tuberculosis (13C% map, middle) and 15N-asparagine uptake (15N/14N ratio map, right), as derived from secondary ion mass spectrometry (SIMS) analysis of M. tuberculosis H37Rv-infected mouse bone marrow-derived macrophages (BMMs). 13C-labeled bacteria were used to infect BMMs at a multiplicity of infection of 10 bacteria per cell. At 20 h post-infection, infected cells were pulsed for 4 h with 5 mM 15N1 (amine)-asparagine, and 13C and 15N isotope proportions were analyzed. (B) Quantification of 15N isotope enrichment in surface areas chosen in the intracellular 13C-labeled bacteria; ‘‘background’’ indicates the level of enrichment measured in the host cell cytoplasm. For more details about the technique, see [30]. (C) IFNc- and LPS-activated BMMs were infected with M. tuberculosis wild type (H37Rv), the ansA-KO mutant or its complemented strain (Compl.) at a multiplicity of infection (MOI) of 0.1 bacterium/cell for 4 h at 37uC. Cells were washed and further incubated with fresh medium for 0, 2 or 5 days. At the indicated time-points, cells lysates were plated for CFU scoring. (D) Confocal microscopy analysis of activated BMMs infected for 1 h with Alexa Fluor 488-labeled M. tuberculosis wild type (H37Rv), the ansA-KO mutant or its complemented strain (Compl.) (green), and stained with LysoTracker Red DND-99 (red) and DAPI (blue) to visualize nuclei. Bar represents 10 mm. Arrowheads point to example phagosomes considered positive for LysoTracker staining. (E) Quantification of LysoTracker-positive phagosomes in samples prepared as in (h) 2 or 4 h after infection. Colocalisation events were recorded in <300 phagosomes observed in <10 different fields. (F) Phagosomal pH measured by flow cytometry in activated BMMs infected with M. tuberculosis wild type (H37Rv), the ansA-KO mutant or its complemented counterpart (Compl.). (G) Cells were pre-incubated with 100 nM bafilomycin A1 for 1 h, infected as in (C) and bafilomycin A1 was removed after 24 h. All data are representative of at least three independent experiments. In (C), (E) and (G), data represent mean6s.d. of triplicate samples, and were analyzed using the Student’s t test. *, P,0.05; **, P,0.01; ***, P,0.001; ****, P,0.0001. doi:10.1371/journal.ppat.1003928.g004](https://s3-eu-west-1.amazonaws.com/com.mendeley.prod.article-extracted-content/images/3ae2a95b-e1d6-31ab-8820-3821189e9bd6/thumbnail-93aff2cf-4278-4d95-9489-74ed0ab1bd1c-3.png)
![Figure 5. In vitro expression and localization of recombinant AnsA-His6 in M. tuberculosis wild-type and ESX-1, ESX-5 or SecA2 mutant strains. (A) Asparaginase activity in the supernatant of M. tuberculosis ansA-KO mutant and its wild-type and complemented counterparts. Fifty mL of cultures (OD600<0.5) were concentrated 50 times. The concentrates were incubated with 15N1 (amine)-asparagine at 37uC; at the indicated time points, the reaction mixtures were mixed with an equal volume of acetonitrile:methanol:water (2:2:1) and analyzed by MS for the presence of 15N-aspartate. (B) Expression and secretion of AnsA-His6 in M. tuberculosis wild-type and DESX-1 [63], DESX-5 [62], DSecA2 (Bottai et al. unpublished data) mutants. Fifteen mg of total cell lysates or culture filtrate proteins from the different mycobacterial strains were subjected to SDS-PAGE and tested in Western blotting by using a mouse anti-His6 monoclonal antibody. As control, samples were tested with the anti-ESAT-6 and anti-SodA monoclonal antibodies. Preparations were also tested with the anti-GroEL2 antibody, which was used for lysis control. As expected, cell lysates and cell culture filtrates from all M. tuberculosis strains transformed with the empty vector p-VV16 were negative when tested with the anti-His6 monoclonal antibody (data not shown). (C) Quantification of the relative expression of AnsA-HIS6 in the culture filtrate, as compared to in the cell pellet, in the WT and ESX-5-KO strains. Data represent mean6s.d. of triplicate samples, and were analyzed using the Student’s t test. ****, P,0.0001. (D) Ni-NTA-Nanogold detection of AnsA-HIS6 by electron microscopy in ultrathin sections of M. tuberculosis-infected BMMs. Bar indicates 0.5 mm; arrowheads indicate gold particles in the phagosomal lumen. doi:10.1371/journal.ppat.1003928.g005](https://s3-eu-west-1.amazonaws.com/com.mendeley.prod.article-extracted-content/images/3ae2a95b-e1d6-31ab-8820-3821189e9bd6/thumbnail-5521b8dd-124d-4166-bd67-a07a93511aa7-4.png)
![Figure 6. Schematic representation of the role of asparagine catabolism in nitrogen incorporation, resistance to acid and intracellular survival. Within macrophages, asparagine enters the M. tuberculosis phagosome through an unknown mechanism. Asparagine is captured by M. tuberculosis through AnsP2 and one or more other yet to be identified transporter(s), and hydrolyzed by cytosolic AnsA resulting in nitrogen assimilation into glutamine and glutamate, and release of ammonia. AnsA is secreted in the lumen of the phagosome through, at least in part, an ESX-5-dependent mechanism. AnsA can also hydrolyze asparagine in the lumen of the phagosome, resulting in the production of aspartate and ammonia. Aspartate is imported by AnsP1 [30] for nitrogen assimilation. In the phagosomal lumen, ammonia reacts with protons transported by the V-ATPase to form ammonium ions allowing phagosomal pH buffering. doi:10.1371/journal.ppat.1003928.g006](https://s3-eu-west-1.amazonaws.com/com.mendeley.prod.article-extracted-content/images/3ae2a95b-e1d6-31ab-8820-3821189e9bd6/thumbnail-5b437cd0-fad8-4ede-971e-e36d160e7d06-5.png)
Register to see more suggestions
Mendeley helps you to discover research relevant for your work.
Cite
CITATION STYLE
Gouzy, A., Larrouy-Maumus, G., Bottai, D., Levillain, F., Dumas, A., Wallach, J. B., … Neyrolles, O. (2014). Mycobacterium tuberculosis Exploits Asparagine to Assimilate Nitrogen and Resist Acid Stress during Infection. PLoS Pathogens, 10(2). https://doi.org/10.1371/journal.ppat.1003928
Readers over time
Readers' Seniority
Readers' Discipline
