Reduced Local Growth and Spread but Preserved Pathogenicity of a {Delta}purC Mycobacterium tuberculosis Auxotrophic Mutant in Gamma Interferon Receptor-Deficient Mice after Aerosol Infection

We compared the growth levels and pathogenicities of the Mycobacteriumtuberculosis MT103 clinical strain with those of the ${Delta}$ purC mutantstrain MYC1551, which is auxotrophic for purine in wild-typeand gamma interferon receptor (IFN- ${gamma}$ R)-deficient mice. The ${Delta}$ purCstrain MYC1551 grew initially in both wild-type and IFN- ${gamma}$ R-deficientmice upon aerosol infection, but it grew much less than strainMT103 did. Despite the comparable bacterial burdens of the mice,IFN- ${gamma}$ R-deficient mice succumbed to infection with ${Delta}$ purC strainMYC1551 from necrotic pneumonia within 6 weeks of those infectedwith MT103. In conclusion, the ${Delta}$ purC mutant MYC1551 displaysreduced growth but retains pathogenicity. Therefore, the useof mutant strains of M. tuberculosis as live vaccines may notbe recommended.

INTRODUCTION

Top

Introduction

Tuberculosis (TB) remains a major global public health issue,with a projected mortality of 90 million cases over the next3 decades without any further interventions (7). Vaccinationwith the attenuated strain of Mycobacterium bovis BCG has beeneffective against severe forms of TB in children, but the levelsof protection vary in different geographical locations, andthe strain is not effective against the adult form of TB (3).Considerable efforts have been made to identify specific mycobacterialantigens involved in the protective immune response to TB (8,15). The possibility of using live mutant strains of mycobacteriaas vaccines has also been explored (1, 11, 12, 17). An advantageof using attenuated live vaccines instead of subunit vaccinesis that the former may provide a greater complement of antigens,since live vaccines produce most antigens that are normallyexpressed in vivo. Live attenuated strains of Mycobacteriumtuberculosis should express antigens which the BCG vaccine lacksand which may be important in the immune response to TB (10).Attenuated auxotrophic mutants of bacterial pathogens have alreadyshown potential as live vaccine candidates (5, 19). Further,severely immunodeficient mice have survived BCG auxotrophicinfection, although they succumbed to wild-type (WT) BCG infections(1, 12, 14). It was therefore proposed that attenuated BCG strainscould present potentially safe and useful vaccines against TBinfection (10) and, specifically, that they could be used inpatients infected with human immunodeficiency virus. StrainBCG and M. tuberculosis ${Delta}$ purC and ${Delta}$ leuD mutants that are auxotrophicfor purine have been tested with animal models (1, 12, 14),confirming reduced virulence after intravenous infections inmice.

Here, we determined the pathogenicity of MYC1551, the ${Delta}$ purC mutantfrom clinical strain MT103, with a physiological model of aerosolinfection, aiming to understand whether the mutant strain hasreduced pathogenicity in an immunosuppressed host. In view ofthe critical role that gamma interferon (IFN- ${gamma}$ ) plays in controllingmycobacterial infections (4, 6, 9), we used immunodeficientIFN- ${gamma}$ receptor-deficient (IFN- ${gamma}$ R^–/–) mice (13).

The M. tuberculosis ${Delta}$ purC strain MYC1551 is lethal in IFN- ${gamma}$ R^–/– mice.

Top

The m. tuberculosis {delta}purc...

Since the auxotrophic ${Delta}$ purC strain MYC1551 is attenuated anddoes not grow in in vitro-cultured macrophages (14), we wantedto determine whether this mutant has reduced pathogenicity inan immunodeficient host. Therefore, IFN- ${gamma}$ R^–/– micewere infected with WT MT103 or the ${Delta}$ purC mutant MYC1551 by aerosolexposure (100 CFU per lung) and compared to WT mice. The immunodeficientIFN- ${gamma}$ R^–/– mice infected with MT103 started to losebody weight within 2 weeks (data not shown) and succumbed toinfection between weeks 4 and 6 (Fig. 1A), and those infectedwith the ${Delta}$ purC mutant MYC1551 lost weight within 4 weeks andsurvived slightly longer but succumbed to infection in weeks5 through 7 (Fig. 1B). By contrast, most of the WT mice (8 of10) infected with either MT103 or the ${Delta}$ purC mutant MYC1551 survived(Fig. 1). As MYC1551 has a reduced capacity for in vitro intracellulargrowth in resting macrophages (14), we wanted to determine whetherMYC1551 has a reduced capacity for growth in the host in vivo.

View larger version (14K):
[in this window]
[in a new window]
FIG. 1. The M. tuberculosis ${Delta}$ purC mutant MYC1551 causes rapid lethality in IFN- ${gamma}$ R^–/– mice. WT mice and IFN- ${gamma}$ R^–/– mice were exposed to aerogenic infections (100 CFU) with the WT MT103 (A) and ${Delta}$ purC mutant MYC1551 (B) strains. Each group contained 10 mice. The values on the y axes indicate percent survival.

Reduced bacterial burdens in M. tuberculosis ${Delta}$ purC strain MYC1551-infected mice.

Top

Reduced bacterial burdens in...

Organs of infected mice were assessed for mycobacterial growthat 4 weeks of infection. Mycobacterial loads of MT103 in thelungs, spleens, and livers of IFN- ${gamma}$ R^–/– mice werehigher (for lungs and livers, P < 0.05; for spleens, P <0.001) than those in WT mice (Fig. 2). The ${Delta}$ purC mutant MYC1551grew much less in vivo upon aerosol infection, but some growthwas preserved, unlike after intravenous infection (17). Thebacterial counts in these lungs, spleens, and livers were significantlylower than the bacterial counts in those with MT103 infections(P < 0.01) (Fig. 2).

View larger version (16K):
[in this window]
[in a new window]
FIG. 2. MYC1551 mutant mycobacteria have reduced growth in vivo. The bacterial burdens in the lung, spleen, and liver were determined 4 weeks after aerosol infection of WT (A) and IFN- ${gamma}$ R^–/– (B) mice infected with the WT MT103 (open bars) and the ${Delta}$ purC mutant MYC1551 (filled bars). Bars represent mean CFU values (n = 5), with error bars indicating standard deviations. The data are representative of two independent experiments.

The bacterial loads in lungs and especially in spleens and liversof IFN- ${gamma}$ R^–/– mice infected with the ${Delta}$ purC mutant MYC1551were significantly higher than those in the organs of WT mice(lungs, P < 0.05; spleens and livers, P < 0.01) (Fig.2). Indeed, MYC1551 CFU counts in organs were 3 (in lungs) and10 (in spleens and livers) times higher in IFN- ${gamma}$ R^–/–mice, which suggests that there is residual growth, not an absenceof growth as reported previously (17), which is IFN- ${gamma}$ sensitive.Therefore, the data suggest that the mutant mycobacteria havea reduced capacity for growth in normal and IFN- ${gamma}$ R^–/–hosts, and we wanted to determine whether the inflammatory changesmight be reduced.

The M. tuberculosis ${Delta}$ purC mutant MYC1551 causes acute pneumonia in IFN- ${gamma}$ R^–/– mice.

Top

The m. tuberculosis {delta}purc...

The inflammatory and granuloma responses were investigated bylight microscopy at 4 and 8 weeks after aerosol infection. InMT103-infected WT mice, typical lung granulomas consisting ofactivated macrophages and abundant lymphocytes were found at4 weeks after infection, a response that increased slightlyat 8 weeks (Fig. 3A). MT103 infections in IFN- ${gamma}$ R^–/–mice caused necrotic pneumonia with abundant extracellular growthof mycobacteria in the absence of proper granuloma formation(Fig. 3A). In addition, there were many small granulomas inthe liver (data not shown).

View larger version (162K):
[in this window]
[in a new window]
FIG. 3. Diminished inflammatory and granuloma responses in the lungs of WT mice compared to IFN- ${gamma}$ R^–/– mice upon infection with the WT MT103 (A) and the mutant MYC1551 (B). Shown are images of confluent inflammation and necrosis in IFN- ${gamma}$ R^–/– mice infected with the MT103 and MYC1551 strains. These images are representative micrographs of the lungs from WT mice at 4 and 8 weeks and IFN- ${gamma}$ R^–/– mice at 4 weeks after infection. Sections were stained with hematoxylin and eosin and are representative of five mice each. Magnification, x80.

The infection of WT mice with the M. tuberculosis ${Delta}$ purC mutantMYC1551 induced an increase in peribronchial cellular infiltration,with the formation of small granulomas, between weeks 4 and8 (Fig. 3B). By contrast, MYC1551 caused extensive pneumoniain IFN- ${gamma}$ R^–/– mice at 4 weeks, with diffuse mononuclearcellular infiltration in the absence of granuloma and necrosis,which was lethal. Therefore, the MYC1551 mutant strain retainedpathogenicity, as observed in the immunodeficient IFN- ${gamma}$ R^–/–mice, even with lower organ bacterial burdens.

Reduced iNOS expression in the absence of IFN- ${gamma}$ R signaling.

Top

Reduced inos expression in...

Mycobacterial infection causes activation of inducible nitricoxidase (iNOS) in infected macrophages. Activated macrophagescan be detected in organs by staining them for their iNOS immunoreactivity.At 4 weeks, WT mice infected with the MT103 strain, or to alesser extent, the ${Delta}$ purC mutant MYC1551, displayed iNOS immunoreactivityin the lungs (Fig. 4A), as well as in the liver and spleen (datanot shown). iNOS expression was reduced in IFN- ${gamma}$ R^–/–mice infected with either strain (Fig. 4B). Nitrotyrosine, whichis the end product of the iNOS reaction, was detected in thelungs of WT mice, and the level detected was very similar tothat obtained from iNOS staining of these tissues (data notshown). As expected, in IFN- ${gamma}$ R^–/– mice, nitrotyrosineimmunoreactivity was diminished, concomitant with reduced iNOSexpression. Therefore, the ${Delta}$ purC mutant MYC1551 has an attenuatedcapacity to induce iNOS expression. In the absence of IFN- ${gamma}$ Rsignaling, reduced activation of macrophages occurred and noproper granulomas were formed.

View larger version (162K):
[in this window]
[in a new window]
FIG. 4. Mice infected with the mutant MYC1551 display reduced activation of pulmonary macrophages compared to mice infected with the WT MT103. Expression levels of iNOS in the lungs of WT and IFN- ${gamma}$ R^–/– mice at 4 weeks after infection were determined as described previously (8, 18). WT and IFN- ${gamma}$ R^–/– mice were infected with the MT103 (A) and MYC1551 (B) strains, and representative immunohistochemistry sections from five mice each are shown. Magnification, x80.

In conclusion, we demonstrated here that the M. tuberculosis ${Delta}$ purC mutant MYC1551 displayed reduced growth in WT mice buthad 0.5- to 2-log-higher growth levels in the lungs and in thelivers and spleens, respectively, of IFN- ${gamma}$ R^–/– mice.Although the organ CFU counts of the ${Delta}$ purC mutant MYC1551 wereabout 2 logs lower than those of the parent strain in IFN- ${gamma}$ R^–/–mice, the mutant mycobacteria were still pathogenic in the immunodeficienthost. The heightened sensitivity of IFN- ${gamma}$ ^–/– or IFN- ${gamma}$ R^–/–mice to mycobacteria is well documented (4, 6, 9). Therefore,the use of such an immunodeficient model to test reduced pathogenicitiesof mycobacterial mutants is justified. Our data indicate thatthe ${Delta}$ purC MYC1551 auxotrophic mutant displays a growth disadvantagein vivo but causes death in IFN- ${gamma}$ R^–/– mice. Thesefindings are relevant for the extrapolation of recombinant vaccinestrategies for the use of live vaccines in immunodepressed patients.

ACKNOWLEDGMENTS

Grants from The Wellcome Trust, NRF, and MRC of South Africasupported this work.

FOOTNOTES

* Corresponding author. Mailing address: CNRS, 3B rue de la Ferollerie, F-45071 Orléans, France. Phone: 0033 238 25 78 60. Fax: 0033 238 25 79 79. E-mail: bryffel{at}cnrs-orleans.fr.

Editor: F. C. Fang

REFERENCES

Top