Progress and New Directions in Genetics of Tuberculosis

Report of a National Heart, Lung, and Blood Institute Working Group

Am J Respir Crit Care Med Vol 172. pp 1491–1496, 2005 Internet address www.atsjournals.org

Issar Smith, Carl Nathan, and Hannah H. Peavy

TB Center, The Public Health Research Institute, Newark, New Jersey; Department of Microbiology and Immunology, Weill Medical College of Cornell University, New York, New York; and Division of Lung Diseases, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland

This workshop, sponsored by the National Heart, Lung, and Blood Institute of the National Institutes of Health, was held in Bethesda, Maryland, September 27 and 28, 2004.

Tuberculosis (TB), along with AIDS and malaria, is one of the three major killers among infectious diseases. New approaches to preventing, diagnosing, and curing TB are needed, which depend on a better understanding of Mycobacterium tuberculosis and the host. The National Heart, Lung, and Blood Institute convened a working group to develop recommendations for future TB research, including genetic aspects of the disease. The following areas were identified: (1) animal model research to improve understanding of persistence, reactivation, and granulomatous reactions; (2) preclinical studies aimed at shortening treatment of TB; (3) new resources for manipulating and characterizing the M. tuberculosis genome, proteome chips for more specific diagnoses, and studies of genes that appear to be essential but whose functions are not known; (4) prospective studies associated with clinical trials in populations with or at risk of TB to advance development of diagnostics and prognostics; (5) new quantitative and bioinformatic approaches to study the interaction between M. tuberculosis and the infected host and how this influences the infection process; (6)molecular characterization of M. tuberculosis genome diversity and phylogenetic analysis; (7) coordinated studies of human genome scans; (8) genetic epidemiology studies; (9) activities to foster knowledge dissemination, education, and training; and (10) coordination between the National Institutes of Health, the Gates Foundation, the Global Alliance for Tuberculosis Drug Development, and other organizations

Keywords: bacterial virulence factors; genetics; host immunologic response; Mycobacterium tuberculosis; tuberculosis

Infection can lead to a potentially fatal outcome or an asymptomatic quiescent or latent state, but one in which latent bacteria can be reactivated to cause disease. Immunocompromised individuals and those subject to the ills of poverty are particularly at risk. The frightening public health problem of TB exists despite the widespread use of the Mycobacterium bovis bacillus Calmette-Guerin live attenuated vaccine and several antibiotics. This vaccine is variable in protection and does not prevent reactivation or reinfection (5), and the antibiotics in current use are losing their efficacy because of emerging drug resistance (6, 7). To rationally develop new strategies, we must learn how these bacteria circumvent host defenses to survive in infected human hosts, sometimes for years, and can then be reactivated to cause disease. It is essential to understand why relatively few individuals exposed to Mtb do contract the disease (1), because defining genetic determinants and correlates of host resistance/susceptibility to TB can help in the design of more efficient vaccines. It is equally important to understand why some clinical Mtb strains are more virulent than others, at least in animal models (8), and how Mtb reacts to the host at different stages of infection. Host responses to different Mtb clinical strains are also an important area for study. Successful research in these areas will aid in the design of new therapies.

CLINICAL MANIFESTATIONS OF DISEASE

Most TB infections are initiated by the respiratory route of exposure now that milk products are generally pasteurized, at least in the developed world. One study in 1978, before the AIDS epidemic, showed that 85% of new TB cases were pulmonary (9). Thus, the different forms of the disease that are discussed below usually arise from dissemination of the bacilli from the infected lung. TB in many cases follows a general pattern as described by Wallgren (10), who divided progression and resolution of the disease into four stages. In the first stage, dating from 3 to 8 weeks, after Mtb is inhaled in aerosols and implants in alveoli, the bacteria are disseminated by the lymphatic circulation to regional lymph nodes in the lung, forming the so-called primary or Ghon complex. At this time, conversion to tuberculin reactivity occurs. The second stage, lasting about 3months, is marked by hematogenous circulation of bacteria to many organs, including other parts of the lung, and at this time sometimes fatal disease can occur in the form of tuberculous meningitis or miliary (disseminated) TB.

Pleurisy or inflammation of the pleural surfaces can occur during the third stage, lasting 3 to 7 months, causing severe chest pain, but this stage can be delayed for up to 2 years. It is believed that this condition is caused by either hematogenous dissemination or the release of bacteria into the pleural space from subpleural concentrations of bacteria. The free bacteria or their components are believed to interact with sensitized T lymphocytes that are attracted and then proliferate and release inflammatory cytokines (11). The last stage, resolution of the primary complex, where the disease does not progress, may take up to 3 years. In this stage, longer developing extrapulmonary lesions—for example, those in bones and joints and frequently presenting as chronic back pain—can appear in some individuals. In addition, other organ systems (e.g., genitourinary, nervous, circulatory) can be affected.

Most humans who are infected with TB do not exhibit progression of the disease. One-third of exposed HIV-negative individuals become infected and, of this number, 3 to 10% develop the disease in their lifetimes, whereas HIV-positive individuals infected with Mtb have a 7 to 10% chance of developing TB each year (12). TB in HIV-positive adults, whether resulting from reactivation or new infection, is frequently pulmonary (13), but extrapulmonary TB is often associated with AIDS and has been accepted as one criterion for an AIDS-defining diagnosis; its frequency among patients with AIDS ranges from 25 to 66% (14). It is believed that the host inflammatory response, especially overproduction of the cytokine tumor necrosis factor alpha (TNF-alpha), plays a major role in tissue damage associated with active TB (15). In the absence of antibiotic treatment or if Mtb resistant to front-line antibiotics is involved in the infection, over 50% of individuals with active TB die (14). This high mortality rate results from widespread destruction of the patient’s lungs.

EVENTS IN THE INFECTIOUS PROCESS

In the human host, Mtb usually enters the alveolar passages in the form of aerosols, where its initial contact is with resident macrophages and dendritic cells (DCs). The bacteria are phagocytosed in a process that is initiated by bacterial contact with macrophage mannose and/or complement receptors (16). On entry into an immunologically naive host macrophage, Mtb initially resides in an endocytic vacuole called the phagosome that does not progress into a later endosome. This avoids the normal endocytic maturation cycle observed with phagocytosed particles (i.e., phagosome–lysosome fusion), in which bacteria encounter a hostile environment that includes acid pH, reactive oxygen intermediates, and lysosomal enzymes. Reactive nitrogen intermediates (RNIs) such as nitric oxide (NO) and its derivatives can arise from the activity of inducible NO synthase (iNOS) in numerous cells, including activated macrophages. RNIs are major elements in antimycobacterial activity in the mouse (17). Although iNOS activity or RNIs are not usually observed in human macrophages from cell lines or macrophages derived in vitro from blood monocytes of healthy humans, alveolar and tissue macrophages from lungs of patients with TB show high levels of functional iNOS (18). In addition, infected lung tissue from patients with TB shows high levels of nitrotyrosine, a product of NOS activity, as well as elevated levels of iNOS activity (19). It is not known how Mtb sometimes avoids the killing activity of macrophages that is observed with other pathogens. However, cell wall components like lipoarabinomannan and secreted proteins like the 19-kD lipoprotein interact with macrophage receptors, and these interactions can modulate the signaling systems used by infected macrophages and DCs to activate the innate immune response (20). For example, the 19-kD protein binds to Toll-like receptor 2, and this interaction results in the repression of the interleukin (IL)-12 and the major histocompatibility complex (MHC) class II presentation pathways (21).

On the basis of much of the above evidence, many researchers favor the possibility that the macrophage has the major role in killing Mtb in the human host (22), and one research goal is to identify macrophage effector molecules. In the mouse, these include but are not limited to RNIs; others, some of which may be under the control of the LRG47 protein (23), remain to be identified. Another hypothesis is that the key cell types responsible for killing Mtb during infections are lymphocytes that produce granulysin and perforin, although granulysin seems to be lacking in the mouse and no clear role for perforin in the control of murine TB has been defined. This line of inquiry tends to focus on the strength on human cytotoxic lymphocyte (CTL) responses and how they may be enhanced by vaccination strategies.

The relative ease of working in tissue culture has provided much useful information on Mtb entrance and trafficking in the macrophage and on other responses of the infected cells, but much less is known about how the bacterium survives and grows during later stages of infection in the lung. Infected macrophages, through their production of chemokines, attract unactivated monocytes, lymphocytes, and neutrophils (24), none of which kill the bacteria efficiently (22). In some infected people, the disease does not progress past this stage and bacteria are ultimately cleared completely. Other individuals form granulomatous focal lesions composed of macrophage-derived giant cells and lymphocytes. This process is generally an effective means for containing the spread of the bacteria. A strong cell-mediated immune (Th1) response is necessary for granuloma formation, and TNF-alpha is especially important in this process (25). In the enclosed granuloma, bacilli-loaded macrophages are killed, and this results in the formation of the caseous center that is surrounded by a cellular zone of fibroblasts, lymphocytes, and blood-derived monocytes (26). Although Mtb bacilli are postulated to be unable to multiply within this caseous tissue, due to its acidic pH, low availability of oxygen, and the presence of toxic fatty acids, some organisms may remain dormant but alive, for decades. The strength of the host cell-mediated immune response determines whether an infection is arrested here or progresses to the next stages. This enclosed infection is referred to as “latent” TB and can persist throughout a person’s life, in an asymptomatic and nontransmissible state. With efficient cellmediated immunity, the infection may be arrested permanently at this point. The granulomas subsequently heal, leaving small fibrous and calcified lesions. However, if an infected person cannot control the initial infection in the lung or if a latently infected person’s immune system becomes weakened by immunosuppressive drugs, HIV infection, malnutrition, diabetes mellitus, aging, or other factors, the center of the granuloma can become liquefied in an unknown manner and then serves as a rich medium in which the now-revived bacteria can replicate very rapidly. At this point, viable Mtb can escape from the granuloma and spread within the lungs (active pulmonary TB). It is at this stage that Mtb can enter alveoli and bronchi of the lung, and the infected individual becomes infectious because the bacilli can be disseminated to others by coughing or expectoration. Mtb can also be spread to other tissues via the lymphatic system and the blood (miliary or extrapulmonary TB). When the disease progresses to this point, intensive antibiotic therapy is essential for survival (26).

Little is known about the mechanism(s) of TB reactivation. Some experiments on “persistence” or “latency” have been performed using chronic mouse infection models in which bacterial numbers can be maintained in a steady state, in the absence of disease. Reactivation of bacterial growth and histopathology in these models can be induced by the administration of the iNOS inhibitors N-iminoethyl-L-lysine (27) and aminoguanidine (28). These results and other data strongly suggest that cell-mediated immunity (i.e., IFN-gamma induction of iNOS and Th1 cytokines) plays amajor role in limiting bacterial growth in infected animals. It is still not certain whether the bacteria in the chronic disease models are actually viable but nongrowing, which would reflect a true latent state, or whether they are growing and dying at the same rate. An early observation showed that Mtb in chronically infected mice is partially susceptible to isoniazid (29), a drug that only is effective against growing Mtb (30), providing evidence for both explanations—that is, noncycling persistence and balanced growth and death. Recent experiments now strongly suggest that the bacteria in the chronic disease models are actually viable but nongrowing (31). Moreover, mouse models of acute and chronic TB are different from the human disease in many regards. Other animal models of TB, such as guinea pig, rabbit, and, more recently, monkey infections, are closer in some respects to their human counterpart (32, 33). This is especially true for the formation and development of pulmonary granulomas in human TB infections that show caseation and cavitation (34). Another issue with the mouse “ latency and reactivation” experiments is that iNOS plays a major role in these processes. Although iNOS is expressed in tuberculous lesions in humans, as discussed above, there is no direct evidence for a protective role for RNIs in human TB and the role of these compounds in human TB is unclear.

Although we know that strong cell-mediated immune responses are necessary for protection and that CD4+ T cells are needed to end the lymphohematogenous stage of early infection in animal models and humans, we still lack critical knowledge regarding the mechanism(s) that govern the induction and maintenance of latent TB infection in humans. Investigations involving persons who have had active versus latent TB infection suggest that latency might be associated with stronger CD8+ CTL responses (35). In addition, Mtb-specific CD8+ T cells preferentially recognize and lyse heavily infected antigen-presenting cells, suggesting a role for these lymphocytes in immunosurveillance (36). This hypothesis is consistent with experiments in mice in which suppression of CD8+ T cells with anti-CD8 antisera hastens reactivation of TB in a latency model of infection (37). The antigen-presentation pathways involved in inducing CD8+ CTL responses in humans are being actively studied. Acurrently discussed possibility is that either Mtb or its antigens escape from the phagosome into the cytosol and are presented via proteasome–MHC class I pathways (38). A related hypothesis proposes apoptosis-associated cross-priming in which mycobacterial antigens in the cytosol of the infected macrophages are
packaged into apoptotic blebs. These structures subsequently fuse with DCs, the primary antigen-presenting cells to naive T cells, so that the antigen contents of the blebs become cytosolic in the DCs and can be presented efficiently (39, 40). Thus, differences in the apoptosis of monocytes and macrophages during initial infection with Mtb might determine how well microbial antigens enter into apoptosis-associated cross-priming pathways and might thereby influence whether active or latent disease develops. Also, a role for TNF-alpha in controlling later stages of human TB infection and latency has been postulated, as elevated levels of this cytokine are found in chronically infected human lung tissue (36), and the use of anti–TNF-alpha agents like infliximab to treat rheumatoid arthritis and Crohn’s disease is associated with the reactivation of latent TB (41).

Currently, there is little information on how Mtb responds to the environment of the lung. There is biochemical evidence that the intermediary metabolism of Mtb is different during the course of mouse infections than that observed in bacteria growing in broth culture because fatty acids seem to be a preferred carbon source for Mtb in vivo (42–44), but the significance of these apparent changes in metabolism for acute or chronic infection is not known. Recent experiments have shown that RNIs induce a unique pattern of gene expression in Mtb (45), and specific Mtb genes show different patterns of expression in wildtype as compared with IFN-gamma knockout mice (46). This is consistent with Mtb in lungs responding to the onset of cell-mediated immunity.

Recent methodologic advances in Mtb genetics, such as more efficient allelic replacement and transposon mutagenesis (47, 48), as well as the DNA sequencing and annotation of the Mtb H37Rv genome (49) and those of related mycobacteria that have been or are currently being completed by the Sanger Center–Pasteur Institute consortium and by the Institute for Genomic Research (50) have led to a burst of new information on the genetics of Mtb virulence. The demonstration that large-sequence polymorphisms are unique events that generate robust phylogeographic bacterial population structures provides a rational framework for understanding the consequences of genetic variation among clinical isolates of Mtb (51). Recent scientific advances have demonstrated that over 200 genes and the proteins they encode are necessary for Mtb virulence (52, 53). These genetic and molecular approaches have allowed researchers to analyze the genes responsible for differences in virulence that are observed in clinical Mtb strains (8) and to characterize the proteins they encode as being responsible for these differences in ability to cause disease (54). New National Institutes of Health (NIH) initiatives to fund a comprehensive mutant analysis of the Mtb genome are now underway and approximately 900 transposon mutants have been obtained (NO1 30036, awarded to W. Bishai, Jr.). At the same time, some progress has been made in discovering genetic loci in mice that determine susceptibility/resistance to Mtb infection (55–58). Likewise, specific human population cohorts are being analyzed to elucidate the reasons for high susceptibility to TB (59–62). In some cases, correlations have been made with specific human genetic loci (63, 64). A new project to use whole genome scans with multiple single nucleotide poymorphisms (SNPs) to study 1,000 patients with TB and 1,000 control subjects is now underway and should provide valuable information on human genetic loci (A.V. Hill, personal communication, 2005). Thus, genetic tools are now in place for integrating rigorous population genetic analysis of both the pathogen and the host.

FUTURE DIRECTIONS

The National Heart, Lung, and Blood Institute Working Group identified the following areas as important for future research. It is important to stress that the goals of the workshop did not include vaccine development, clinical management, diagnostics, or drug development. This did not reflect a lack of interest. On the contrary, these issues have such high priority that they are being addressed by other working groups and funding mechanisms.

1. Animal model research to illuminate stages of human TB, such as persistence and reactivation, and to improve understanding of specific granulomatous reactions, such as caseation and cavitation, including the following:

• Conditions in which mice, guinea pigs, rabbits, and monkeys can be used to model each stage and manifestation of human disease (e.g., to gain a better understanding of the granuloma)
• A better characterized and more uniform set of experiments to be used to delineate TB pathogenesis in diverse experimental animal models. This will be helpful in comparing results from different laboratories.
• Metabolic characterization of host compartments (e.g., granulomas and cavities) in animal models with respect to PO2, carbon sources, and other factors relevant to Mtb’s metabolism
• Studies to provide a better understanding of host susceptibility to TB, including the genetic and nongenetic aspects (e.g., environmental factors, such as nutritional state of host, age, etc.)
• Better understanding of host responses to Mtb infection at the molecular, cellular, and whole animal level. As an example, studies of suppressive/regulatory pathways of immunity and their genetic control in lungs and central lymphoid organs and the host signaling pathways that are affected by Mtb infection.
• New technologies to study the above issues

2. Preclinical studies to identify chemotherapeutic approaches that may shorten treatment times for active TB. Specific areas are as follows:

• Development and use of target-specific whole cell assay systems, including human cells and cell–cell interactions, suitable for functional or chemical genomics including screening for chemical inhibitors. For these studies, hypomorphs and hypermorphs, which are defined as strains expressing each gene at subnormal or supranormal levels, will be valuable tools for assessing the specificity of chemical inhibitors of the gene products, according to the increased susceptibility or resistance of the mutant strain compared with the wildtype. Also, global gene expression profiling, secondary promoter–reporter approaches to identify inhibitors of gene products of primary interest via the response of genes whose transcription is affected secondarily by disruption of the primary gene.
• Resources for chemical biology, including support to set up chemistry core facilities and high-throughput screening facilities and to purchase diversity collections and focused, secondary chemical libraries
• Support for multidisciplinary groups whose expertise includes microbiology, immunology, genetics, biochemistry, structural biology, chemistry, medicinal chemistry and/or pharmacology, and quantitative or systems approaches to biology

3. New resources for manipulating and characterizing the Mtb genome, including a comprehensive program to inactivate every Mtb open reading frame. This will complement existing work in progress. The production of bacterial hypomorphic and hypermorphic alleleles for each Mtb gene is a valuable goal as noted above. Advanced proteomic techniques to produce complete proteome chips that could be used for earlier and more specific diagnoses and monitoring of prophylactic and therapeutic interventions. Specific areas are as follows:

• Regulated promoters for titratable, in vivo–active conditional gene activation and inactivation
• Phage bank and distribution resource for constructs to disrupt all Mtb genes in different Mtb clinical and laboratory strains
• Phage bank and distribution resource for a panel of hypomorphic and hypermorphic alleles of Mtb genes. Phage bank and distribution resource for unmarked biological constructs for multiple deletions.
• Studies of the functions of bacterial genes that appear to be essential for virulence but whose function is unknown, as this will lead to an improved understanding of Mtb biology in the context of the infectious process
• New and improved methods of target validation in vivo, including humans (e.g., Mtb gene expression analysis at a global level in vivo at low bacterial numbers). These studies will be very important for a better understanding of latency, persistence, and reactivation.

4. Prospective studies associated with vaccine or other clinical trials in populations with or at risk of TB to advance the development of diagnostics and prognostics, including the following:

• Serology at a pan-proteomic level—for example, to allow tests of specific antibodies that might help reveal what proteins are expressed by Mtb at what stage in which hosts, and to see if reactivation can be detected earlier via the host antibody response than can be achieved by conventional methods such as chest X-rays and sputum analysis
• Assessment of subjects’ medical status with respect to diabetes, atherosclerosis, hypertension, cardiovascular disease, obesity, and “metabolic syndrome” as possible risk factors for reactivation and predictors for response to vaccination
• Studies to identify sterilization of infection versus latent infection
• Studies to determine who should be treated for latent infection. The recently suggested promise of DNA vaccines for the prevention of reactivation and reinfection of Mtb during chemotherapy (65) illustrates an important connection with this area of development.
• Studies to predict reactivation and prevent transmission
• Identification of genetic and immune markers for human protection and susceptibility (to better understand why only 10% of infected individuals contract TB)
• Rapid spot funding to investigate any of the above, as well as mechanistic questions, in the framework of clinical studies, where the new issues arise after the clinical study was designed

5. New quantitative and bioinformatics approaches to study Mtb–host interactions and their effect on infection outcome, including the following:

• Combined transcriptome analyses of Mtb and infected macrophages and granulomas, using DNA arrays and other technologies (e.g., laser-capture methodology), as well as the development of post-transcriptome analyses. These approaches could help in the identification of new regulatory networks that may help explain pathogenic mechanisms and lead to new therapies.
• Bioinformatic modeling to understand data obtained with DNA arrays and other global searches and its use to predict outcomes

6. Characterization and phylogenetic analysis of Mtb genome diversity to provide novel approaches to understanding gene function and host–pathogen interactions. Specific areas are as follows:

• Complete sequencing of five additional strains representative of geographically distributed phylogeny will add considerable insight to sequences obtained to date.
• Development of high-throughput SNP typing based on five whole genomes and segment sequencing of 100 more strains will be an invaluable tool for large-scale comparative genomic studies.
• Compilation of a minimum panel of strains that are quantitatively representative of genetic diversity among clinical isolates (aside from drug resistance) as an aid to testing newdiagnostics, vaccines, and chemotherapeutics
• Integration of these improved tools for bacterial population analysis of Mtb with ongoing studies of human genetic variability have the potential to fundamentally alter our understanding of the relationship between host and pathogens.

7. Coordinated studies of human genome scans to allow multiple research groups to cooperate to obtain large numbers of samples that would be used for surveying
diverse human populations for susceptibility to TB

8. Genetic epidemiology studies to predict how susceptibility and resistance genes in populations affect the overall prevalence of disease and trends for epidemics

9. Activities to foster knowledge dissemination and education, including the following:

• Workshops for standardization of definitions, reagents, assays, protocols, and mouse strains for vaccine development, and other preclinical and clinical studies
• Sponsorship of meetings to review new technical opportunities, to plan or promote awareness of clinical trials, to coordinate human genome scans
• Training programs in microbial physiology
• Technical transfer to endemic areas
• Future meetings on topics such as latency—what is known, how to model it, how to use human data, limitations, possible drugs, and so forth

10. Coordination between private and public funding agencies should be fostered. However, the product development efforts that use coordinated and focused management structures that are being funded by the NIH, Bill and Melinda Gates Foundation, and others should complement and not compete with existing discovery-based research. The translational activities of organizations such as the Global Alliance for Tuberculosis Drug Development, Aeras Tuberculosis Vaccine Foundation, and the Foundation for Innovative New Diagnostics provide an unprecedented opportunity for synergy between basic and applied research. Coordination would include advisory resources to help guide individual investigators or consortia along a path from target validation to hit identification to lead development to drug development to clinical trials, with each of the relevant agencies contributing expertise at the appropriate point and cooperating to hand off a given project to other agencies as appropriate.

Conflict of Interest Statement:

None of the authors have a financial relationship with a commercial entity that has an interest in the subject of this manuscript.

Acknowledgment:

The authors thank the participants of the workshop who provided the important ideas embodied in this report. These participants included: co-chairs Carl Nathan (New York, NY) and Issar Smith (Newark, NJ); Alexander S. Apt (Moscow, Russia); Samuel M. Behar (Boston, MA); John Chan (Bronx, NY); Zheng Chen (Chicago, IL); Paul Converse (Baltimore, MD); Jeffrey Cox (San Francisco, CA); Ken Duncan (Seattle, WA); Sabine Ehrt (New York, NY); Philip L. Felgner (Irvine, CA); JoAnne Flynn (Pittsburgh, PA); Li M. Fu (Anaheim, CA); Dorothy B. Gail (Bethesda, MD); Anne E. Goldfeld (Boston, MA); James E. Graham (Louisville, KY); Liana Harvath (Bethesda, MD); Sandra Colombini Hatch (Bethesda, MD); Robert L. Hunter (Houston, TX); Gail G. Jacobs (Bethesda, MD); William R. Jacobs, Jr. (Bronx, NY); Chinnaswamy Jagannath (Houston, TX); Gilla Kaplan (Newark, NJ); Douglas S. Kernodle (Nashville, TN); Denise Kirschner (Ann Arbor, MI); Hardy Kornfeld (Worcester, MA); Igor Kramnik (Boston, MA); Mike Kurilla (Bethesda, MD); Yukari C. Manabe (Baltimore, MD); Robin Mason (Bethesda, MD); John Mittler (Seattle, WA); Hannah H. Peavy (Bethesda, MD); Richard Pine (Newark, NJ); Mary Reichler (Atlanta, GA); Jesse Roman (Atlanta, GA); Eric J. Rubin (Boston, MA); James C. Sacchettini (College Station, TX); Erwin Schurr (Montreal, Canada); William K. Scott (Durham, NC); David Sherman (Seattle, WA); Christine Sizemore (Bethesda, MD); Peter Small (Seattle, WA); Andrew A. Vernon (Atlanta, GA); Zhenhua Yang (Ann Arbor, MI); Thomas Zahrt (Milwaukee, WI).

REFERENCES

1. Dye C, Scheele S, Dolin P, Pathania V, Raviglione MC. Consensus statement. Global burden of tuberculosis: estimated incidence, prevalence, and mortality by country. WHO Global Surveillance and Monitoring Project. JAMA 1999;282:677–686.
2. Tobin MJ. Tuberculosis, lung infections, interstitial lung disease, and journalology in AJRCCM 2002. Am J Respir Crit Care Med 2003;167: 345–355.
3. Tobin MJ. Tuberculosis, lung infections, interstitial lung disease, social issues and journalology in AJRCCM 2003. Am J Respir Crit Care Med 2004;169:288–300.
4. Nemery B, Yew WW, Albert R, Brun-Buisson C, Macnee WF, Martinez JD, Angus C, Abraham E. Tuberculosis, nontuberculous lung infection, pleural disorders, pulmonary function, respiratory muscles, occupational lung disease, pulmonary infections, and social issues in AJRCCM in 2004. Am J Respir Crit Care Med 2005;171:554–562.
5. Colditz, GA, Brewer,TF, Berkey CS, Wilson ME, Burdick E, Fineberg HV, Mosteller F. Efficacy ofBCGvaccine in the prevention of tuberculosis: meta-analysis of the published literature. JAMA 1994;271:698– 702.
6. Smith CV, Sharma V, Sacchettini JC. TB drug discovery: addressing issues of persistence and resistance. Tuberculosis 2004;84:45–55.
7. O’Brien RJ, Nunn PP. The need for new drugs against tuberculosis: obstacles, opportunities, and next steps. Am J Respir Crit Care Med 2001;163:1055–1058.
8. Manca C, Tsenova L, Bergtold A, Freeman S, Tovey M, Musser JM, Barry CE, Freedman VH, Kaplan G. Virulence of a Mycobacterium tuberculosis clinical isolate in mice is determined by failure to induce Th1 type immunity and is associated with induction of IFN-alpha/beta. Proc Natl Acad Sci USA 2001;98:5752–5757.
9. Hopewell PC. Overview of clinical tuberculosis. In: Bloom BR, editor. Tuberculosis: pathogenesis, protection, and control. Washington, DC: American Society for Microbiology; 2000. pp. 25–46.
10. Wallgren A. The time table of tuberculosis. Tubercle 1948;29:245–251.
11. Kamholz SL. Pleural tuberculosis. In: Rom WN, Garay S, editors. Tuberculosis. Boston: Little, Brown and Co; 1996. pp 483–491.
12. Paolo WF, Nosanchuk JD. Tuberculosis in New York City: recent lessons and a look ahead. Lancet Infect Dis 2004;4:287–293.
13. Garay S. Tuberculosis and the human immunodeficiency virus infection. In: Rom WN, Garay S, editors. Tuberculosis. Boston: Little, Brown and Co; 1996. pp. 443–465.
14. Kaye K, Frieden T. Tuberculosis control: the relevance of classic principles in an era of acquired immunodeficiency syndrome and multidrug resistance. Epidemiol Rev 1996;18:52–63.
15. Bekker LG, Moreira AL, Bergtold A, Freeman S, Ryffel B, Kaplan G. Immunopathologic effects of tumor necrosis factor alpha in murine mycobacterial infection are dose dependent. Infect Immun 2000;68: 6954–6961.
16. Ernst JD. Macrophage receptors for Mycobacterium tuberculosis. Infect Immun 1998;66:1277–1281.
17. Chan J, Tanaka K, Carroll D, Flynn J, Bloom BR. Effects of nitric oxide synthase inhibitors on murine infection with Mycobacterium tuberculosis. Infect Immun 1995;63:736–740.
18. Nicholson S, Bonecini-Almeida Mda G, Lapa e Silva JR, Nathan C, Xie QW, Mumford R, Weidner JR, Calaycay J, Geng J, Boechat N, et al. Inducible nitric oxide synthase in pulmonary alveolar macrophages in patients with active pulmonary tuberculosis. J Exp Med 1996;183:2293–2302.
19. Choi HS, Rai PR, Chu HW, Cool C, Chan ED. Analysis of nitric oxide synthase and nitrotyrosine expression in human pulmonary tuberculosis. Am J Respir Crit Care Med 2002;166:178–186.
20. Wieland CW, Knapp S, Florquin S, de Vos AF, Takeda K, Akira S, Golenbock DT, Verbon A, van der Poll T. Non-mannose-capped lipoarabinomannan induces lung inflammation via toll-like receptor 2. Am J Respir Crit Care Med 2004;170:1367–1374.
21. Noss EH, Pai RK, Sellati TJ, Radolf JD, Belisle J, Golenbock DT, Boom WH, Harding CV. Toll-like receptor 2-dependent inhibition of macrophage class II MHC expression and antigen processing by 19-kDa lipoprotein of Mycobacterium tuberculosis. J Immunol 2001; 167:910–918.
22. Fenton MJ, Vermeulen MW. Immunopathology of tuberculosis: roles of macrophages and monocytes. Infect Immun 1996;64:683–690.
23. MacMicking JD, Taylor GA, McKinney JD. Immune control of tuberculosis by IFN-gamma-inducible LRG-47. Science 2003;302:654–659.
24. van Crevel R, Ottenhoff TH, van der Meer JW. Innate immunity to Mycobacterium tuberculosis. Clin Microbiol Rev 2002;15:294–309.
25. Algood HM, Lin PL, Yankura D, Jones A, Chan J, Flynn JL. TNF influences chemokine expression of macrophages in vitro and that of CD11b+ cells in vivo during Mycobacterium tuberculosis infection.J Immunol 2004;172:6846–6857.
26. Dannenberg AM, Rook JA. 1994. Pathogenesis of pulmonary tuberculosis: an interplay of tissue-damaging and macrophage-activating immune responses. Dual mechanisms that control bacillary multiplication. In: Bloom BR, editor. Tuberculosis. Pathogenesis, protection and control. Washington, DC: ASM Press; 2004. 459–483.
27. MacMicking JD. North R, LaCourse R, Mudgett, J, Shah S, Nathan C. Identification of nitric oxide synthase as a protective locus against tuberculosis. Proc Natl Acad Sci USA 1997;94:5243–5248.
28. Flynn JL, Scanga CA, Tanaka KE, Chan J. Effects of aminoguanidine on latent murine tuberculosis. J Immunol 1998;160:1796–1803.
29. Rees JRW, Hart PDA. Analysis of the host-parasite equilibrium in chronic murine tuberculosis by total and viable bacilary counts. Br J Exp Pathol 1961;42:83–88.
30. Wayne LG, Sramek HA. Metronidazole is bactericidal to dormant cells of Mycobacterium tuberculosis. Antimicrob Agents Chemother 1994;38: 2054–2058.
31. Munoz-Elias EJ, Timm J, Botha T, Chan WT, Gomez JE, McKinney JD.Replication dynamics of Mycobacterium tuberculosis in chronically infected mice. Infect Immun 2005;73:546–551.
32. Capuano SV, Croix DA, Pawar S, Zinovik A, Myers A, Lin PL, Bissel S, Fuhrman C, Klein E, Flynn JL. Experimental Mycobacterium tuberculosis infection of cynomolgus macaques closely resembles the various manifestations of human M. tuberculosis infection. Infect Immun 2003; 71:5831–5844.
33. Shen Y, Zhou D, Qiu L, Lai X, Simon M, Shen L, Kou Z, Wang Q, Jiang L, Estep J, et al. Adaptive immune response of Vgamma2Vdelta2+ T cells during mycobacterial infections. Science 2002;295:2255–2258.
34. Converse PJ, Dannenberg AM, Estep JE, Sugisaki K, Abe Y, Schofield BH, Pitt ML. Cavitary tuberculosis produced in rabbits by aerosolized virulent tubercle bacilli. Infect Immun 1996;64:4776–4787.
35. Pathan AA, Wilkinson KA,Wilkinson RJ, Latif M, McShane H, Pasvol G, Hill AV, Lalvani A. High frequencies of circulating IFN-gamma secreting CD8 cytotoxic T cells specific for a novel MHC class Irestricted Mycobacterium tuberculosis epitope in M. tuberculosisinfected subjects without disease. Eur J Immunol 2000;30:2713–2721.
36. Lewinsohn DA, Heinzel AS, Gardner JM, Zhu, L, Alderson MR, Lewinsohn DM. Mycobacterium tuberculosis-specific CD8+ T cells preferentially recognize heavily infected cells. Am J Respir Crit Care Med 2003;168:1346–1352.
37. van Pinxteren LA, Cassidy JP, Smedegaard BH, Agger EM, Andersen P. Control of latent Mycobacterium tuberculosis infection is dependent on CD8+ T cells. Eur J Immunol 2000;30:3689–3698.
38. Teitelbaum R, CammerM, Maitland ML, Freitag NE, Condeelis J, Bloom BR. Mycobacterial infection of macrophages results in membranepermeable phagosomes. Proc Natl Acad Sci USA1999;96:15190–15195.
39. Albert ML, Sauter B, Bhardwaj N. Dendritic cells acquire antigen from apoptotic cells and induce class I-restricted CTLs. Nature 1998;392: 86–89.
40. Schaible UE, Winau F, Sieling PA, Fischer K. K, Collins HL, Hagens K, Modlin RL, Brinkmann, V, Kaufmann SH. Apoptosis facilitates antigen presentation to T lymphocytes through MHC-I and CD1 in tuberculosis. Nat Med 2003;9:1039–1046.
41. Keane J,Gershon S,Wise RP,Mirabile-LevensE,Kasznica J, Schwieterman WD, Siegel JN, Braun MM. Tuberculosis associated with infliximab, a tumor necrosis factor alpha-neutralizing agent. N Engl J Med 2001;345:1098–1104.
42. Segal W, Bloch H. Biochemical differentiation of Mycobacterium tuberculosis grown in vivo and in vitro. J Bacteriol 1956;72:132–141.
43. Segal W, Bloch H. Pathogenic and immunogenic differentiation of Mycobacterium tuberculosis grown in vivo and in vitro. Am Rev Tubercl Pulm Dis 1957;75:495–500.
44. McKinney JD, Bentrup KHZ, Munoz-Elias EJ,Miczak A, Chen B, Chan WT, Swenson D, Sacchettini JC, Jacobs WR, Russell DG. Persistence of Mycobacterium tuberculosis in macrophages and mice requires the glyoxalate shunt enzyme isocitrate lyase. Science 2000;406:735–738.
45. Ohno H, Zhu G, Mohan VP, Chu D, Kohno S, Jacobs WR, Chan J. The effects of reactive nitrogen intermediates on gene expression in Mycobacterium tuberculosis. Cell Microbiol 2003;5:637–648.
46. Shi L, Jung YJ, Tyagi S, Gennaro ML, North RJ. Expression of Th1- mediated immunity in mouse lungs induces a Mycobacterium tuberculosis transcription pattern characteristic of nonreplicating persistence. Proc Natl Acad Sci USA 2003;100:241–246.
47. Pelicic V, Reyrat JM, Gicquel B. Genetic advances for studying Mycobacterium tuberculosis pathogenicity. Mol Microbiol 1998;28:413–420.
48. Glickman MS, Jacobs WR. Microbial pathogenesis of Mycobacterium tuberculosis: dawn of a discipline. Cell 2001;104:477–485.
49. Cole ST, Brosch R, Parkhill J, Garnier T, Churcher C, Harris D, Gordon SV, Eigenmeir K, Gas S, Barry C III. Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence. Nature 1998;393:537–544.
50. Fleischmann RD, Alland D, Eisen JA, Carpenter L, White O, Peterson J, DeBoyR,Dodson R, Gwinn M, Haft D. Whole-genome comparison of Mycobacterium tuberculosis clinical and laboratory strains. J Bacteriol 2002;184:5479–5490.
51. Hirsh AE, Tsolaki AG, DeRiemer K, Feldman MW, Small PM. Stable association between strains of Mycobacterium tuberculosis and their human host populations. Proc Natl Acad Sci USA 2004;101:4871–4876.
52. Sassetti C, Rubin EJ. Genomic analyses of microbial virulence. Curr Opin Microbiol 2002;5:27–32.
53. Smith I. Mycobacterium tuberculosis pathogenesis and molecular determinants of virulence. Clin Microbiol Rev 2003;16:463–496.
54. Domenech P, Reed MB, Dowd CS, Manca C, Kaplan G, Barry CE. The role of MmpL8 in sulfatide biogenesis and virulence of Mycobacterium tuberculosis. J Biol Chem 2004;279:21257–21265.
55. Kramnik I, Dietrich WF, Demant P, Bloom BR. Genetic control of resistance to experimental infection with virulent Mycobacterium tuberculosis. Proc Natl Acad Sci USA 2000;97:8560–8565.
56. Sanchez F, Radaeva TV, Nikonenko BV, Persson AS, Sengul S. Schalling, Schurr ME, Apt AS, Lavebratt C. 2003. Multigenic control of disease severity after virulent Mycobacterium tuberculosis infection in mice. Infect Immun 2003;71:126–131.
57. Kamath AB, Alt J,Debbabi H, Taylor C, Behar SM. The major histocompatibility complex haplotype affects T-cell recognition of mycobacterial antigens but not resistance to Mycobacterium tuberculosis in C3H mice. Infect Immun 2004;72:6790–6798.
58. Pan H, Yan BS, Rojas M, Shebzukhov YV, Zhou H, Kobzik L, Higgins DE, Daly MJ, Bloom BR, Kramnik I. Ipr1 gene mediates innate immunity to tuberculosis. Nature 2005;434:767–772.
59. Sousa AO, Salem JI, Lee FK, Vercosa MC, Cruaud P, Bloom BR, Lagrange PH, David HL. An epidemic of tuberculosis with a high rate of tuberculin anergy among a population previously unexposed to tuberculosis, the Yanomami Indians of the Brazilian Amazon. Proc Natl Acad Sci USA 1997;94:13227–13232.
60. Bellamy R, Beyers N, McAdam KP, Ruwende C, Gie R, Samaai P, Bester D, Meyer M, Corrah T, Collin M, et al. Genetic susceptibility to tuberculosis in Africans: a genome-wide scan. Proc Natl Acad Sci USA 2000;97:8005–8009.
61. Segal S, Hill AV. Genetic susceptibility to infectious disease. Trends Microbiol 2003;11:445–448.
62. Zhang W, Shao L, Weng X, Hu Z, Jin A, Chen S, Pang M, Chen ZW. Variants of the natural resistance-associated macrophage protein 1 gene (NRAMP1) are associated with severe forms of pulmonary tuberculosis.Clin Infect Dis 2005;40:1232–1236.
63. Cervino A. C, Lakiss S, Sow O, Bellamy R, Beyers N, Hoal-van Helden E, van Helden P, McAdam KP, Hill AV. Fine mapping of a putative tuberculosis-susceptibility locus on chromosome 15q11–13 in African families. Hum Mol Genet 2002;11:1599–1603.
64. Bellamy R, Ruwende C, Corrah T, McAdam KP, Whittle HC, Hill AV. Variations in the NRAMP1 gene and susceptibility to tuberculosis in West Africans. N Engl J Med 1998;338:640–644.
65. Ha SJ, Jeon BY, Youn JI, Kim SC, Cho SN, Sung YC. Protective effect of DNA vaccine during chemotherapy on reactivation and reinfection of Mycobacterium tuberculosis. Gene Ther 2005;12:634–638.

Correspondence and requests for reprints should be addressed to:

Issar Smith, M.D.
TB Center
The Public Health Research Institute
225 Warren Street
Newark, NJ 07103
E-mail: smitty@phri.org