Construction and Phenotypic Characterization of an Auxotrophic Mutant of Mycobacterium tuberculosis Defective in l-Arginine Biosynthesis

The vector pARG7S1 was constructed by cloning the hsp60-sacB cassette (where the promoter of the Bacillus subtilis sacB gene [20] was replaced by that of the M. bovis BCG hsp60 gene) in pARG7, which contains a ΔargF::hyg allele and a lacZ marker for identification of single crossovers (17). Allelic exchange was carried out essentially as described by Parish and Stoker (18). M. tuberculosis H37Rv (ATCC 27294) was electroporated with 5 μg of UV-pretreated pARG7S1 (9) and plated on Middlebrook 7H10 agar containing hygromycin (Hyg; 50 μg/ml) and 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-Gal; 50 μg/ml). Plating of a blue, single-crossover recombinant colony on medium containing Hyg, X-Gal, sucrose (7, 18), and l-arginine (0.24 mM) produced 184 sucrose-resistant colonies, one of which remained white upon repatching on fresh, l-arginine-supplemented indicator plates.

The growth rate of the mutant strain was compared to that of the wild type by inoculating 30 ml of Middlebrook 7H9 broth into stirred or rolled cultures containing 0, 0.24, or 0.96 mM l-arginine at a bacterial density of 10⁶ CFU/ml. CFU were enumerated by plating duplicate samples of at least 3 serial dilutions on supplemented 7H10 agar. Substrate utilization experiments were performed by streaking equivalent amounts of logarithmic-phase cultures of the wild-type and the argF mutant strains onto 7H10 plates containing l-ornithine, l-citrulline, or argininosuccinate at concentrations of 1 to 10 mM.

Median survival times (MSTs) and growth kinetics in SCID and DBA/2 mice infected with M. tuberculosis strains were assessed as previously described (23).

Allelic exchange with the ΔargF::hyg allele was achieved by two-step mutagenesis (18) using the vector pARG7S1. As predicted from earlier work (17), a large number of colonies recovered from the sucrose counterselection step had to be screened before one white, Hyg-resistant colony was obtained, which was suggestive of a double-crossover gene replacement event. Restriction sites used to confirm the mutant genotype are shown for the wild-type and mutant alleles in Fig. 1A along with the Southern blot analysis (Fig. 1B). The 749-bp fragment observed in the wild type and partial merodiploids (Fig. 1B, lanes 2 and 3) was lost in the knockout mutant (lane 4) and replaced by two cross-hybridizing fragments (456 and 1,886 bp) by the introduction of a new NotI site from the hyg gene.

To demonstrate its auxotrophic phenotype, the mutant strain was grown in axenic culture alongside wild-type H37Rv in Middlebrook 7H9 medium supplemented with 0, 0.24, or 0.96 mM l-arginine (Fig. 2A). In the absence of this supplement, the mutant strain showed a steady decline from 10⁶ CFU/ml to an undetectable level after 14 days in culture. In the presence of 0.24 mM l-arginine, the growth rate of the mutant strain was significantly lower than that of its parental wild type and the maximum cell density achieved was approximately 10-fold lower. However, the mutant strain grew at a rate approaching that of the wild type and achieved a comparable bacterial density in stationary phase in the presence of 0.96 mM l-arginine, although it did display a longer lag phase, reaching its peak of growth at 15 days compared to 10 days for the wild type. By comparison, the wild-type strain displayed little difference in growth rate or maximum cell density in the presence of 0.24 mM or 0.96 mM l-arginine compared to that of the l-arginine-free control (Fig. 2B). The high concentration of supplement required to restore optimal growth rates in the mutant suggests that the normal demand for l-arginine in M. tuberculosis is high.

In a further set of experiments in vitro, argF mutant bacteria were found to survive within bone marrow-derived macrophages cultured as described by Smith et al. (23) in medium containing 0.4 mM l-arginine (Dulbecco’s modified Eagle’s medium) and to elicit the secretion of NO^•, tumor necrosis factor α, interleukin-10, and interleukin-12 to the same extent as wild-type bacteria (data not shown).

In the M. tuberculosis genome (3), argF is located within the argCJBDFRGH gene cluster (Fig. 3A). To investigate the effect of the ΔargF::hyg mutation on the function of downstream genes, substrate utilization by the argF mutant strain was analyzed by monitoring its growth when streaked on medium containing l-ornithine, l-citrulline, argininosuccinate, or l-arginine (Fig. 3B). Since disruption of argF would abolish ornithine carbamoyltransferase activity, the failure of the mutant strain to grow on medium containing 1 to 10 mM l-ornithine was as expected. However, the failure of 1 to 10 mM l-citrulline to support its growth indicated that the argF mutant was also defective in argG function. This observation suggests that the ΔargF::hyg mutation exerted polar effects on argR and argG, which is consistent with the operonic arrangement of the argCJBDFRG cluster (3). In contrast, the mutant bacteria grew as well as wild-type bacteria with 10 mM argininosuccinate supplement (data not shown), suggesting that the argH gene is expressed. However, the growth supported by 1 mM argininosuccinate was markedly poorer than that observed with 0.24 mM l-arginine, suggesting that the uptake of argininosuccinate may be relatively inefficient.

To assess the effect of the ΔargF::hyg mutation on bacterial virulence in the absence of specific immunity, SCID mice were infected with either wild-type H37Rv or the argF mutant strain. SCID mice are highly susceptible to M. tuberculosis infection (16), and as previously observed (23), they succumbed to infection approximately 29 days after infection with wild-type bacteria (Fig. 4A). Mice infected with the mutant strain survived significantly longer, with an MST of 83 days (P < 0.0004), suggesting that this strain is less virulent than its wild-type parent. However, the l-arginine auxotroph of M. tuberculosis is significantly more virulent than the corresponding proline and tryptophan auxotrophic mutants, with the former displaying an MST of 130 days and 80% of the experimental group of the latter surviving to more than 300 days (23). These observations presumably reflect differences in the availability of arginine compared with proline and tryptophan to tubercle bacilli in vivo. The concentration of l-arginine has been reported to be 0.1 to 0.3 mM in the plasma of various animal species (2, 25). Although these levels are sufficient to allow the mutant to grow in SCID mice, the attenuation observed in this model suggests that access of the mutant to l-arginine is restricted in the animal.

To assess whether l-arginine auxotrophy affected the growth rate of M. tuberculosis in an immunocompetent host, DBA/2 mice were infected with either H37Rv or the argF mutant strain. Mice infected with H37Rv died with an MST of 95.5 days (Fig. 4B), whereas those infected with the mutant strain survived significantly longer (P > 0.0006). The increased survival of mice infected with the mutant strain correlated with the very low numbers of bacteria recoverable from the organs of immunocompetent mice (Fig. 5). Whereas bacterial growth of the wild-type strain rose 10-fold in the lung over the course of infection and the bacillary load remained above 5 logs in both the liver and spleen, the mutant was attenuated in DBA/2 mice, with bacillary loads in the liver, spleen, and lung of infected mice being at or below the limit of detection at time points up to 60 days. In accordance with this, analysis of histological responses in the tissues revealed little evidence of inflammatory responses in mutant-strain-infected mice (data not shown). There was evidence of low numbers of bacteria in the spleen at day 60, and by 99 days of infection, some bacterial outgrowth of the mutant strain was seen in the livers of infected mice. The rapid decline in the numbers of argF mutant bacteria in DBA/2 mice after high-dose intravenous infection suggests that sufficient l-arginine is unavailable during initial stages of infection in this mouse strain, but the growth observed in the liver at later time points suggests that l-arginine availability may be altered at later stages of infection. However, the survival data suggest that the mutant numbers were nonetheless maintained at extremely low levels.

The data presented herein suggest that l-arginine auxotrophy does confer on M. tuberculosis some of the properties sought for a rationally attenuated vaccine candidate. This study has also raised interesting questions regarding the supply of l-arginine in vivo and the role of the immune system in the control of this auxotroph. In the context of macrophage function, l-arginine is unique among amino acids as it is the substrate for nitric oxide synthase, the inducible isoform of which plays a central role in the antimicrobial activity of activated macrophages against intracellular parasites (15). The l-arginine auxotroph of M. tuberculosis could thus provide a useful tool for investigating the interplay between mycobacterial infection, l-arginine uptake and distribution between host and pathogen, and NO^• production in macrophages (21).