Mycobacterium tuberculosis nitrogen assimilation and host colonization require aspartate

Abstract

Here we identify the amino acid transporter AnsP1 as the unique aspartate importer in the human pathogen Mycobacterium tuberculosis. Metabolomic analysis of a mutant with an inactive AnsP1 revealed that the transporter is essential for M. tuberculosis to assimilate nitrogen from aspartate. Virulence of the AnsP1 mutant is impaired in vivo, revealing that aspartate is a primary nitrogen source required for host colonization by the tuberculosis bacillus.

Figure 1. AnsP1 is essential for M. tuberculosis growth on aspartate as sole nitrogen source

(a,b) Growth of M. tuberculosis H37Rv, the ansP1-KO mutant and the ansP1-KO complemented (Compl.) strains in various conditions. Growth was measured by monitoring turbidity; data represent mean ±s.d. of triplicate samples and are representative of at least three independent experiments. (a) Bacteria were grown in minimal medium containing 5 mM asparagine (Asn), aspartate (Asp), glutamine (Gln) or glutamate (Glu), as sole nitrogen sources. (b) Bacteria were grown in minimal medium containing 50 mM aspartate (Asp) only, 50 mM aspartate and 15 mM ammonium (Asp+NH_4⁺), or 50 mM aspartate and 10 g/L glycerol (Asp+Gro), as nitrogen or carbon sources. (c) ¹⁴C-aspartate uptake assay with M. tuberculosis H37Rv, the ansP1-KO mutant and its complemented strains (Compl.). Bacteria previously grown in 7H9 with 5 mM aspartate, were harvested and resuspended in an uptake buffer containing a mix of ¹⁴C-radiolabeled and non-labeled aspartate to obtain a final concentration of 20 μM aspartate. Bacteria were incubated at 37°C and samples were removed; bacteria-associated ¹⁴C radioactivity was quantified at the indicated time points. Data are expressed as the number of disintegrations per minute (DPM) per total protein concentration (¹⁴C-Asp (DPM).μg protein⁻¹), represent mean ±s.d. of triplicate samples, are representative of three independent experiments, and were analyzed using the Student’s t test; *, P<0.05; **, P<0.01.

Figure 2. AnsP1 is essential for Mycobacterium tuberculosis nitrogen assimilation from aspartate

(a) Nitrogen incorporation pathways from aspartate into various N-containing metabolites resulting from transamination (dotted line) and/or transformation (plain lines) reactions. (b) Frequency of ¹⁵N-glutamine and ¹⁵N-glutamate detected in the presence of ¹⁵N-aspartate in either M. tuberculosis wild type (H37Rv), the ansP1-KO mutant, or its complemented strain (Compl.). Data represent mean±s.d. of triplicate samples and are representative of at least two independent experiments. *, no signal detected. Any signal below 5-10% corresponds to background noise. (c) ¹³C-labeled bacteria were used to infect mouse macrophages. After pulsing the cells with ¹⁵N-aspartate, ¹³C and ¹⁵N isotope compositions were analyzed by NanoSIMS. Images display a representative infected cell. Left panel is the as recorded ¹²C¹⁴N⁻ image showing the histological aspect of the cell (scale bar represents 5 μm). The central panel represents the ¹³C atomic fraction map (in %) of the corresponding area. The right panel shows the ¹⁵N/¹⁴N ratio image indicating the ¹⁵N-aspartate uptake. For enhanced visibility, the ratio was multiplied by 1×10⁴. The ¹⁵N/¹⁴N ratio at natural ¹⁵N abundance appears blue. (d) Quantification of ¹⁵N enrichment (compared to the resin) in surface areas chosen in the cell cytoplasm (n=5), and intracellular ¹³C labeled bacteria (n=43). Data represent mean ±s.d. and were analyzed using the Student’s t test. The ¹⁵N-enriched phagosomes, arbitrarily defined as those vacuoles with a ¹⁵N enrichment above mean+3s.d. of that observed in the host cell cytoplasm, represent 34.9% of all phagosomes.