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Accelerated Detection of Mycobacterium tuberculosis Genes Essential for Bacterial Survival in Guinea Pigs, Compared with Mice

  1. S. Moises Hernandez-Abanto1,a,
  2. Qi-Jian Cheng1,
  3. Prabhpreet Singh1,
  4. Lan H. Ly3,
  5. Lee G. Klinkenberg1,
  6. Norman E. Morrison1,
  7. Paul J. Converse1,
  8. Eric Nuermberger1,
  9. Jacques Grosset1,
  10. David N. McMurray3,
  11. Petros C. Karakousis1,
  12. Gyanu Lamichhane1,
  13. William R. Bishai1 and
  14. Sanjay K. Jain1,2,a
  1. 1 Center for Tuberculosis Research, Baltimore, Maryland
  2. 2 Division of Pediatric Infectious Diseases, Johns Hopkins University School of Medicine, Baltimore, Maryland
  3. 3 Department of Microbial and Molecular Pathogenesis, Texas A&M University System Health Science Center, College Station, Texas
  1. Reprints or correspondence: Dr. Sanjay K. Jain, Center for Tuberculosis Research, Johns Hopkins University School of Medicine, 1550 Orleans St., Baltimore, MD 21231 (sjain5{at}jhmi.edu).

  • ↵a S.K.J. and S.M.H.-A. contributed equally to the study.

 
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Abstract

Background. Mouse and guinea pig models have been used to identify Mycobacterium tuberculosis mutants attenuated for survival. However, unlike mice, M. tuberculosis–infected guinea pigs form caseating granulomas, which may simulate human disease more closely.

Methods. We used designer arrays for defined mutant analysis, a high-throughput subtractive competition assay, for genotypically defined M. tuberculosis mutants and compared the survival of the same mutant pools in guinea pig and mouse aerosol models. Selected mutants found to be attenuated in either aerosol model were also analyzed in the mouse hollow-fiber model.

Results. M. tuberculosis mutants representing 74 genes were tested. Eighteen M. tuberculosis mutants were attenuated for survival in either aerosol model, with 70% of selected mutants also attenuated in the mouse hollow-fiber model. The majority of attenuated mutants in the mouse aerosol model were detected only after 90 days of infection. There was a high degree of concordance between the genes identified by the 2 aerosol models, with detection being significantly earlier in the guinea pig (P < .0003).

Conclusions. We identified M. tuberculosis genes required for survival in mammalian lungs. The majority of mouse late-stage survival mutants were detected significantly earlier in the guinea pig, which suggests that differences in tuberculosis-induced lung pathologic changes may account for this accelerated detection.

Tuberculosis is one of the leading infectious causes of morbidity and mortality worldwide, with an estimated 8.8 million new patients acquiring tuberculosis each year [1]. Humans become infected with Mycobacterium tuberculosis via the respiratory route. After successful implantation, M. tuberculosis replicates in the lungs. This initial infection may lead to disease; however, it is frequently controlled by the host immune system, leading to latent infection. Identification of the M. tuberculosis genes required for survival in mammalian lungs may lead to the identification of key targets for drug and vaccine development [2].

Several defective growth phenotypes have been described by studying the in vivo growth patterns of M. tuberculosis mutants in mice [2, 3]. However, these phenotypes have not been characterized extensively in other animal models, particularly the ones that display caseous necrosis—the hallmark of the immune response to M. tuberculosis in humans. High-throughput techniques such as transposon (Tn) site hybridization have been developed, for the subtractive identification of attenuated Tn mutants, on the basis of microarray analysis [4, 5]. Recently, Lamichhane et al. [6] described designer arrays for defined mutant analysis (DeADMAn), a high-throughput, high-sensitivity approach for the subtractive identification of previously archived, genotypically defined M. tuberculosis Himar1 Tn mutants attenuated for survival in mice after intravenous infection. In the present study, we used DeADMAn for the identification of M. tuberculosis genes required for in vivo survival in mammalian lungs after aerosol infection. We also conducted a cross-species analysis by comparing the bacterial growth kinetics of identical pools of M. tuberculosis mutants in guinea pig and mouse aerosol models. Comparing species enabled a direct evaluation of the impact of caseous necrosis, which is present in guinea pig tuberculosis but absent in mouse tuberculosis [7].

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Materials and Methods

M. tuberculosis Himar1 Tn mutants and media. Random insertion mutagenesis of M. tuberculosis CDC 1551 strain was performed in our laboratory using the Himar1 Tn as part of a comprehensive insertional mutagenesis study. Tn insertion sites were identified by sequencing the insertion junction as described elsewhere [8, 9]. Each mutant was grown separately at 37°C in Dubos broth base (Becton Dickinson) supplemented with Dubos medium albumin (Becton Dickinson), 5% glycerol, 0.01% sodium pyruvate, and 20 µg/mL kanamycin. All mutants used in the study, except for the positive and negative controls, were selected randomly. Two pools (A and B) containing 30 and 50 mutants, respectively, were prepared by combining a pure culture of each mutant grown to an OD600 of 0.8. Only mutants whose in vitro growth was similar to that of the wild type were used for the present study. Mutants that harbored a Tn in the region upstream of the terminal 100 bp (or, if the gene was <500 bp in length, within the 5′ 80% of the gene) were considered for screening, to decrease the likelihood of selecting distal mutations that might not disrupt gene-product function. In addition, 7 M. tuberculosis Tn mutants that harbored a Tn in the terminal 100 bp (or, if the gene was <500 bp in length, within the 3′ 20% of the gene) were also screened. M. tuberculosis Tn mutants JHU2583c-322 and JHU2675c-564 were included as positive and negative controls in each pool, respectively, because previous studies have shown that JHU2583c-322 (a Tn mutant in gene MT2660) is attenuated for survival in mice, whereas JHU2675c-564 (a Tn mutant in gene MT2749) is fully virulent [6]. Furthermore, M. tuberculosis Tn mutants JHU0842–1196 and JHU3833–375 (Tn mutants in genes MT0864 and MT3941, respectively) were present in both pools A and B. All mutants used in the study, including the controls, were subsequently found to have a $#0394;sigF background [10].

Guinea pig infection. Hartley strain guinea pigs weighing 250–300 g (Charles River Laboratories) were infected via the aerosol route using the latest version of the Madison Aerosol Chamber from the University of Wisconsin Engineering Shops [11]. Five guinea pigs per group were killed at days 1, 21, and 42 (pool A) and days 1, 21, 49, and 63 (pool B) after infection, and the survival of each mutant in the lungs was analyzed. Both lungs in their entirety were homogenized in PBS, and a significant proportion of the entire homogenate was plated on Middlebrook 7H10 solid medium (Becton Dickinson). For day 1 colony-forming unit counts, both lungs in their entirety were homogenized, and half of the homogenate was plated as above. Colony-forming unit counts were obtained by multiplying these results by a factor of 2. All plates were incubated at 37°Cfor at least 3 weeks before the colonies were counted or used for DNA preparation.

Mouse infection. Female BALB/c mice, 5–6 weeks old (Charles River Laboratories), were infected via the aerosol route using the Glas-Col inhalation exposure system (Glas-Col). Three mice per group were killed at days 1, 21, 49, 96, 147, and 360 after infection for both pools A and B, and the survival of each mutant in the lungs was analyzed. Both lungs in their entirety were homogenized in PBS and plated on Dubos broth base (Becton Dickinson) supplemented with Dubos medium albumin (Becton Dickinson), 1.5% agar, 5% glycerol, 0.01% sodium pyruvate, and 20 µg/mL kanamycin. For day 1 colony-forming unit counts, both lungs in their entirety were homogenized, and all of the homogenate was plated as described above. All plates were incubated at 37°C for at least 3 weeks before the colonies were counted or used for DNA preparation.

Microarray and real-time polymerase chain reaction (PCR) analysis. For each time point, the colonies from the plates were scraped and pooled, and genomic DNA was prepared using standard methods [12]. Two custom microarray sets (1 for each pool) were printed on poly-L-lysine–coated glass slides. Sixty-base oligonucleotides corresponding to the sequence at the junction of each Tn insertion were spotted 4 times in tandem on the microarray. The genomic DNA was digested with AluI, the adapter was ligated, and junctions of Tn insertion sites were selectively PCR amplified using Tn-specific primers and were labeled with Cy3 and Cy5 monoffluorescent dyes (Amersham Pharmacia) as described elsewhere [6]. Probes prepared from the input pool (1 day after infection) and output pools were cohybridized to custom microarrays and scanned by using a Genepix Axon 4000B device (Axon Instruments). Microarrays were performed at least in duplicate for each time point. The data were analyzed by use of a custom developed program [6]. Mutants with the input:output pool ratio significantly greater than or equal to the average of the negative and positive controls at a given time point were considered to be attenuated.

Real-time PCR analysis was performed to confirm and quantify the microarray results for mutants found to be attenuated by DeADMAn. Mutant-specific primer sets using Tn and gene-specific primers were designed to amplify 150–200-bp DNA fragments. These primer sets were tested using conventional PCR. All successful primer sets were used for mutant-specific real-time PCR using the iCycler IQ system (version 3.1.7050; Bio-Rad). The cycle threshold for genomic DNA from the input pool (1 day after infection) was compared with that from the output pool. For mutants not found to be attenuated at the time point indicated by microarray analysis, real-time PCR was performed at all subsequent time points available for that pool. Real-time PCR was performed at least in triplicate for each mutant.

Mouse hollow-fiber infection. Pool B culture containing 50 mutants was encapsulated into polyvinylidene ffluoride hollow fibers and implanted subcutaneously into 6–8-week-old SKH1 hairless mice (Charles River Laboratories), as described elsewhere [13]. Four mice (each of which contained 2 hollow fibers) were killed at days 1 and 56 after infection. The hollow-fiber contents were recovered and plated on Middlebrook 7H10 solid medium (Becton Dickinson). For each time point, the colonies from the plates were scraped and pooled, and genomic DNA was prepared using standard methods [12]. Real-time PCR analysis was performed for all pool B mutants found to be attenuated for survival in the guinea pig and mouse aerosol models. The cycle threshold for genomic DNA from the input pool (1 day after infection) was compared with that from the output pool (56 days after infection). Real-time PCR was performed at least in triplicate for each mutant.

Statistical analysis. Kaplan-Meier survival curve analysis was done using Prism 4 software (version 4.01; GraphPad Software).

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Results

M. tuberculosis Tn mutants tested and attenuated for survival in the guinea pig and mouse aerosol models. A total of 80 M. tuberculosis Tn mutants were used to infect both the guinea pig and mouse via the aerosol route (table 1). After aerosol infection, day 1 log10 colony-forming units were 3.48 ± 0.06 and 3.40 ± 0.28 for pools A and B, respectively, for mouse lungs and 2.07± 0.17 and 2.29± 0.07 for pools A and B, respectively, for guinea pig lungs.

Table 1.
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Table 1.

Mycobacterium tuberculosis transposon (Tn) mutants (n = 76) tested in the guinea pig and mouse aerosol models.

DeADMAn screening on day 1 (input pool) confirmed the lung implantation of each mutant in both pools and models used. As expected, the positive and negative controls were found to be attenuated for survival and fully virulent, respectively, in both pools and models. Similarly, M. tuberculosis Tn mutants JHU0842-1196 and JHU3833-375 were found to yield similar results in both pools and models. Thirty-four M. tuberculosis Tn mutants were found to be attenuated for survival in either the guinea pig or mouse lung. Of these, 26 were found to be attenuated for survival in the guinea pig model, whereas 29 were attenuated in the mouse model (figure 1A). There was a high degree of agreement between the mutants found to be attenuated for survival in the guinea pig lung, compared with mouse lung (κ coefficient, 0.63; agreement, 0.83). To confirm and quantify the DeADMAn results, real-time PCR was performed for the 34 M. tuberculosis Tn mutants attenuated for survival. Primer sets for 5 mutants (JHU1021-27, JHU1127c10, JHU2202c-74, JHU3447a-419, and JHU3352c-22) were unsuccessful in amplifying a specific PCR product and were removed from further analysis. Eighteen mutants were found to be attenuated for survival in either the guinea pig or mouse aerosol models. Of these, 15 were found to be attenuated for survival in the guinea pig, whereas 13 were attenuated in the mouse (table 2). Again, there was a high degree of agreement between the mutants found to be attenuated for survival in the guinea pig lung, compared with mouse lung (κ coefficient, 0.64; agreement, 0.89), which suggests a high degree of concordance for M. tuberculosis genetic requirements for growth between the 2 models (figure 1B).

Table 2.
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Table 2.

Mycobacterium tuberculosis transposon (Tn) mutants found to be attenuated for survival in the guinea pig and mouse aerosol models.

Table 3.
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Table 3.

Functional classification of the genes represented by the Mycobacterium tuberculosis transposon (Tn) mutants.

Figure 1.
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Figure 1.

Results after microarray and real-time polymerase chain reaction (PCR) analysis. A, Thirty-four Mycobacterium tuberculosis transposon (Tn) mutants, attenuated for survival in either the guinea pig or mouse aerosol models after microarray analysis. There was a high degree of agreement between the mutants found to be attenuated for survival in guinea pig lung, compared with mouse lung (κ coefficient, 0.63; agreement, 0.83). B, Eighteen M. tuberculosis Tn mutants, attenuated for survival in either the guinea pig or mouse aerosol models after real-time PCR analysis. Again, there was a high degree of agreement between the mutants found to be attenuated for survival in guinea pig lung, compared with mouse lung (κ coefficient, 0.64; agreement, 0.89). JHU2583c-322 (positive control), JHU2675c-564 (negative control), JHU0842-1196, and JHU3833-375 were present in both pools A and B and yielded the same results in both pools and both models.

Mutants for 9 of 13 genes attenuated for survival in the mouse were detected only after 90 days of infection. Furthermore, as expected, 6 of 7 Tn mutants with Tn insertions in the distal portion of the genes were not found to be attenuated in either the guinea pig or mouse aerosol models. However, mutant JHU1742a-175 was found to be attenuated in both the guinea pig and mouse models. This mutant harbors the Tn insertion just distal (80.5%) to the 80% cutoff used in the study, and it is therefore likely that the Tn insertion was successful in inactivating this gene.

Mouse hollow-fiber infection. As proof of principle, selected mutants found to be attenuated for survival in either the guinea pig or mouse aerosol models were also analyzed using real-time PCR in the mouse hollow-fiber model [13]. In this novel in vivo model, granulomatous lesions develop around encapsulated bacilli in semidiffusible hollow fibers implanted subcutaneously into mice. In this microenvironment, the organisms demonstrated an altered physiologic state characterized by stationary-state colony-forming unit counts and decreased metabolic activity. Seven of 10 mutants tested in this model were found to be attenuated by day 56 after infection (table 2), which suggests that these genes may be important for the extracellular survival of M. tuberculosis within granulomatous lesions.

Functional classification of genes represented by the M. tuberculosis Tn mutants. Table 3 summarizes the functional classification of genes represented by the M. tuberculosis Tn mutants. Mutants in 74 unique genes spanning 11 M. tuberculosis genetic functional classes were tested [14, 15]. Forty-one percent were conserved hypotheticals, 16% were involved with cell wall/cell processes, 16% were involved with intermediary metabolism/respiration, and the remaining 27% distributed among other functional classes. Forty-two percent (5/12) of genes involved with cell wall/cell processes, 25% (3/12) of the genes involved with intermediary metabolism/respiration, and 20% (6/30) of conserved hypotheticals were found to be attenuated for survival. Thirty-three percent (3/9) of the genes identified for late-stage survival (after 90 days) in the mouse lung were involved with cell wall/cell processes.

Time to detection of attenuation for the survival of M. tuberculosis Tn mutants. Of the 13 M. tuberculosis Tn mutants found to be attenuated in the mouse aerosol model, less than one-third (4/13) were detected by day 49. We hypothesized that the guinea pig aerosol model would be immunologically different from the mouse model because of the presence of caseous necrosis [7]. Indeed, 87% (13/15) of Tn mutants found to be attenuated in the guinea pig aerosol model were detected by day 49. Figure 2 shows the comparison of Kaplan-Meier survival curves for the attenuated Tn mutants. Median survival times of Tn mutants in guinea pig and mouse aerosol models were 49 and 360 days, respectively (P < .0001 , log-rank test). When this comparison was made for the 10 Tn mutants attenuated in both the guinea pig and mouse aerosol models, median mutant survival times were still significantly shorter in the guinea pig (P = .0003, log-rank test). These data show that the guinea pig model detects attenuation for the survival of M. tuberculosis mutants earlier than the mouse model.

Figure 2.
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Figure 2.

Kaplan-Meier survival comparison for attenuated mutants. A, Survival-curve analysis for 18 Mycobacterium tuberculosis transposon (Tn) mutants attenuated either in the guinea pig or mouse aerosol model. Median survival times of Tn mutants in the guinea pig model were significantly shorter than those in the mouse model (P < .0001, log-rank test). B, Survival-curve analysis for 10 M. tuberculosis Tn mutants attenuated in both the guinea pig and mouse models. Median survival times of Tn mutants in the guinea pig model were significantly less than those in the mouse model (P = .0003, log-rank test). These data show that the guinea pig model detects attenuation for survival earlier than the mouse aerosol model.

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Discussion

M. tuberculosis is an extensively host-adapted pathogen that has developed strategies to survive intracellularly (in macrophages and nonprofessional phagocytic cells), disseminate outside of the lungs, and resist both innate and adaptive immune mechanisms, the latter of which is required in humans for the development of caseating granulomas. At least 4 classes of M. tuberculosis survival defects have been described through the study of M. tuberculosis mutants in mice; these include defective growth in vivo attenuation (giv), severe growth in vivo (sgiv), persistence (per), and pathology (pat) or immunopathology (imp) phenotypes [2, 16]. However, mutants displaying these phenotypes have not been extensively characterized in animal models, such as the guinea pig, that form caseating necrosis as part of their adaptive immune response to M. tuberculosis. Previous studies have shown that the M. tuberculosis ΔsigC mutant was found to have a giv phenotype in guinea pigs [17] but an imp phenotype in mice [18]. Recently, Converse et al. have shown that the M. tuberculosis ΔdosR mutant has a giv phenotype in guinea pigs. In mice, however, this same mutant has a giv phenotype at 4–8 weeks but no significant growth defect by 6 months after infection (P. J. Converse, P. C. Karakousis, S. S. Allen, et al., unpublished data). These data suggest different bacterial survival kinetics for M. tuberculosis mutants in guinea pig and mouse models. Therefore, in the present study, we compared the kinetics of bacterial survival in mammalian lungs of identical pools of M. tuberculosis mutants in 2 aerosol models, to directly evaluate the impact of caseous necrosis seen in guinea pig tuberculosis [7] but not in mouse tuberculosis. Furthermore, mutants for select genes required for survival in mammalian lungs were also evaluated in the hollow-fiber dormancy mouse granuloma model.

DeADMAn is a high-throughput, subtractive identification screening tool for the competitive survival of defined mutant pools. This approach has been shown to be highly sensitive for up to 100 mutants/pool [6]. In the present study, we used smaller pools of up to 50 mutants, because only a limited number of bacilli can be implanted in the lungs by the aerosol route. It should be noted that DeADMAn uses pooled mutant infections, which may confound results because of complementation of defective mutants by extracellular factors secreted by other mutants in the same pool. Furthermore, because DeADMAn is an indirect measure of colony-forming units, it is not useful for the identification of imp/pat mutants. All mutants used in the present study have a $#0394;sigF background [10]. However, because mutants were normalized against controls with the same $#0394;sigF background, a Tn-disrupted gene rather than a sigF deletion is likely responsible for the observed phenotype. Nonetheless, potential interaction of the sigF deletion with the Tn-disrupted gene cannot be completely ruled out. Finally, the M. tuberculosis CDC 1551 strain was used in the study, and certain animal models have shown that this strain is less virulent than the H37Rv strain [19]. However, we believe that these 2 strains are highly comparable. Furthermore, because comparisons were made against controls with the same background, the Tn-disrupted gene rather than the background strain is responsible for the observed phenotype. Nonetheless, it is possible that a similar analysis in the H37Rv background may result in a somewhat different list of mutants attenuated for survival than found in the present study.

Eighteen M. tuberculosis genes were identified in the present study to be required for survival in mammalian lungs. These include MT1847 (Rv1798) and MT3648 (Rv3544c), which have been previously described for in vivo survival in mouse lungs and spleens, respectively [5, 6], and MT3594 (otsA, Rv3490), whose deletion mutant has been shown to be attenuated in mouse lung and spleen after intravenous infection [20]. MT1847 (Rv1798), MT3236 (nuoD, Rv3148), and MT3594 (otsA, Rv3490) have been found to be up-regulated during nutrient starvation, which suggests their role in mycobacterial responses to the host environment [21]. Of the genes identified in the present study, 9 were required for in vivo survival in the mouse lung for >90 days. We believe that these genes are required for late-stage survival and represent key virulence factors required for survival in maturing granulomatous lesions. These late-stage survival genes include MT1102 (Rv1072) and MT2050 (Rv1994c), which have been found to be up-regulated during nutrient starvation and oxidative stress [21, 22]. MT1102 (Rv1072) and MT2050 (Rv1994c) are both SigH-dependent genes [16, 23], which suggests their role in adaptive responses to the host immune system. Similarly, MT0361 (Rv0346c) and MT2174 (Rv2114) were found to be up-regulated under different stress conditions, including the anaerobic/micro-aerophilic environment [24] and nutrient starvation [21], which suggests their possible role in survival against the host immune response. MT1698 (pks10, Rv1660) is required for phthiocerol dimycocerosate synthesis, and mutants deficient in this gene are attenuated for survival in the mouse lung [25]. In addition, it is also up-regulated during nutrient starvation [21]. MT3978 (Rv3864) has been previously described to be required for in vivo survival in the mouse spleen [5] and has also been found to be up-regulated during nutrient starvation [21]. Further studies are under way to validate our screen and to characterize these mutants to better understand their function(s).

Unlike mice, M. tuberculosis disease in guinea pigs leads to well-formed caseous granulomas [7]. It was therefore hypothesized that mouse late-stage survival mutants might lack key virulence pathways required for survival in maturing granulomatous lesions and that the presence of caseous necrosis in guinea pig granulomas might accelerate their detection. In other words, well-formed granulomas in guinea pigs might impose more intense, albeit similar, stressors, compared with the poorly formed granulomas observed in mice, and lead to accelerated detection of these late-stage survival mutants. Indeed, as hypothesized, the guinea pig model was found to detect attenuation for survival of the majority of the Tn mutants earlier than the mouse lung model. Furthermore, 10 mutants required for survival in mammalian lungs were evaluated in the hollow-fiber mouse granuloma model, and 7 were found to be attenuated for survival in this model, which suggests that the genes represented by those mutants are important for M. tuberculosis survival within granulomatous lesions. Our hypothesis is further strengthened by the fact that several of the genes detected earliest in the guinea pig model, compared with the mouse model (MT0361 [Rv0346c], MT1102 [Rv1072], MT2050 [Rv1994c], and MT3978 [Rv3864]), are likely to be involved with responses to stressors generated by the host immune response, such as granulomas [16, 21, 22, 23, 24].

In summary, using a competition survival assay, we have identified genes required for bacterial survival in mammalian lungs after aerosol infection. There was a high degree of agreement between the genes identified by each of the models used. Long-term mouse infection showed that certain late-stage survival bacterial mutants survived for >90 days but later were out-competed by other mutants in the pools between days 150 and 360. Majority of these mouse late-stage survival mutants were identified in <65 days in the guinea pig model. Mouse late-stage survival mutants may lack key virulence pathways required for survival in maturing granulomatous lesions, and the presence of caseous necrosis in guinea pig granulomas may accelerate their detection. Further studies are under way to characterize these mutants and to better understand their function(s) in well-formed granulomas.

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Acknowledgments

We appreciate the assistance of Nacer Lounis in the study.

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Footnotes

  • Potential conflicts of interest: none reported.

  • Financial support: National Institutes of Health (grants and contracts AI36973, AI43846, AI37856, and N01 AI30036).

  • Received November 2, 2006.
  • Accepted December 21, 2006.
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  1. J Infect Dis. (2007) 195 (11): 1634-1642. doi: 10.1086/517526
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