- About this Journal
- Abstracting and Indexing
- Aims and Scope
- Annual Issues
- Article Processing Charges
- Articles in Press
- Author Guidelines
- Bibliographic Information
- Citations to this Journal
- Contact Information
- Editorial Board
- Editorial Workflow
- Free eTOC Alerts
- Publication Ethics
- Reviewers Acknowledgment
- Submit a Manuscript
- Subscription Information
- Table of Contents
Clinical and Developmental Immunology
Volume 2011 (2011), Article ID 814943, 17 pages
Pathogenesis, Immunology, and Diagnosis of Latent Mycobacterium tuberculosis Infection
Department of Microbiology, Faculty of Medicine, Kuwait University, P.O. Box 24923, Safat 13110, Kuwait
Received 6 September 2010; Accepted 28 October 2010
Academic Editor: James Triccas
Copyright © 2011 Suhail Ahmad. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Phagocytosis of tubercle bacilli by antigen-presenting cells in human lung alveoli initiates a complex infection process by Mycobacterium tuberculosis and a potentially protective immune response by the host. M. tuberculosis has devoted a large part of its genome towards functions that allow it to successfully establish latent or progressive infection in the majority of infected individuals. The failure of immune-mediated clearance is due to multiple strategies adopted by M. tuberculosis that blunt the microbicidal mechanisms of infected immune cells and formation of distinct granulomatous lesions that differ in their ability to support or suppress the persistence of viable M. tuberculosis. In this paper, current understanding of various immune processes that lead to the establishment of latent M. tuberculosis infection, bacterial spreading, persistence, reactivation, and waning or elimination of latent infection as well as new diagnostic approaches being used for identification of latently infected individuals for possible control of tuberculosis epidemic are described.
Tuberculosis (TB) has afflicted mankind from the time immemorial. Evidence of spinal disease has been found in Egyptian mummies of several thousand years BC and references to TB are found in ancient Babylonian and Chinese writings. Recent molecular genetic studies have shown that Mycobacterium tuberculosis, the most common cause of TB in humans worldwide, has a progenitor ~3 million years old . Mycobacterium tuberculosis is a member of the M. tuberculosis complex (MTBC) which includes six other closely related species: M. bovis, M. africanum, M. microti, M. pinnipedii, M. caprae, and M. canetti. Although all MTBC members are obligate pathogens and cause TB, they exhibit distinct phenotypic properties and host range. The MTBC members are genetically extremely closely related, the genome of M. tuberculosis shows <0.05% difference with M. bovis, the latter species primarily infects cattle but can also cause TB in other mammals including humans [2, 3].
Tuberculosis is one of the most prevalent infections of human beings and a formidable public health challenge that shows little sign of abating. The disease contributes considerably to illness and death around the world, exacting a heavy toll on the world’s most vulnerable citizens. The current TB epidemic is being sustained and fuelled by two important factors: the human immunodeficiency virus (HIV) infection and its association with active TB disease and increasing resistance of Mycobacterium tuberculosis strains to the most effective (first-line) anti-TB drugs . Other contributing factors include population expansion, poor case detection and cure rates in impoverished countries, active transmission in overcrowded hospitals, prisons, and other public places, migration of individuals from high-incidence countries due to wars or famine, drug abuse, social decay, and homelessness. Active disease patients with sputum smear-positive pulmonary TB are the main source of infection in a community. Primary infection with M. tuberculosis leads to clinical disease in only ~10% of individuals. In the remaining cases, the ensuing immune response arrests further growth of M. tuberculosis. However, the pathogen is completely eradicated in only ~10% people, while the immune response in the remaining ~90% individuals only succeeds in containment of infection as some bacilli escape killing by blunting the microbicidal mechanisms of immune cells (such as phagosome-lysosome fusion, antigen presentation by MHC class I, class II, and CD1 molecules, production of nitric oxide, and other reactive nitrogen intermediates) and remain in nonreplicating (dormant or latent) state in old lesions. The process is termed as latent tuberculosis infection (LTBI), and the dormant bacilli retain the ability to resuscitate and to cause active TB if a disruption of immune response (as in HIV infection) occurs. The World Health Organization (WHO) has estimated that one-third of the total world population is latently infected with M. tuberculosis and 5%–10% of the infected individuals will develop active TB disease during their life time [4, 5]. However, the risk of developing active disease is 5%–15% every year and lifetime risk is ~50% in HIV coinfected individuals [4, 6]. Most of the active disease cases in low TB incidence countries arise from this pool of latently infected individuals.
According to WHO estimates, 9.27 million new active disease cases corresponding to an estimated incidence of 139 per 100,000 population occurred throughout the world in 2007 . Only 5.5 million of 9.27 million cases of TB (new cases and relapse cases) were notified to national tuberculosis programs of various countries, while the rest were based on assessments of effectiveness of surveillance systems. The highest number of TB cases occurred in Asia (55%) followed by Africa (31%). The highest incidence rate (363 per 100,000 population) was recorded for the African region, mainly due to high prevalence of HIV infection. The six most populous countries of Asia (China, India, Indonesia, Pakistan, Bangladesh, and Philippines) accounted for >50% of all TB cases worldwide. An estimated 1.37 million (15%) of incident TB cases in 2007 were coinfected with HIV. Nearly 80% of the HIV-infected TB patients were living in the African region . Globally, 13.7 million total prevalent TB cases were recorded in 2007 corresponding to 206 cases per 100 000 population that resulted in 1.756 million deaths (including 456 000 among TB patients coinfected with HIV) . Nearly 500 000 cases of multidrug-resistant TB (MDR-TB, defined as infection with M. tuberculosis strains resistant at least to the two most important first-line drugs, rifampin and isoniazid) occurred in 2007 . By the end of 2008, extensively drug-resistant TB (XDR-TB; defined as MDR-TB strains additionally resistant to a fluoroquinolone and an injectable agent such as kanamycin, amikacin, viomycin, or capreomycin) has been found in 55 countries and territories of the world . While MDR-TB is difficult and expensive to treat, XDR-TB is virtually an untreatable disease in most of the developing countries .
Population-based studies have shown that some individuals are more at risk of acquiring infection and developing active disease than others. Active transmission also occurs more frequently in small households and crowded places in countries with a high incidence of TB [8, 9]. Molecular epidemiological studies have shown that there are distinct differences in the disease presentation and population demographics in low TB incidence and high TB incidence countries. In several African and Asian countries, where the transmission of M. tuberculosis has been stable or increased in the last few years, the incidence rate is highest among young adults with most cases resulting from recent episodes of infection or reinfection. On the contrary, in low TB incidence countries of Western Europe and North America, a higher proportion of cases occur in older patients or among immigrants from high TB incidence countries [8, 10]. Pulmonary TB accounts for >85% of active TB cases in high TB incidence countries due to higher rates of active transmission, while extrapulmonary TB is also common in low TB incidence countries of the developed world, particularly among HIV-infected individuals and immigrants originating from TB endemic countries [11, 12].
2. Transmission of M. tuberculosis Infection
Tuberculosis is a communicable disease and patients with pulmonary TB are the most important source of infection. Infection is initiated by inhalation of droplet nuclei, which are particles of 1–5 μm in diameter containing M. tuberculosis, expectorated by patients with active pulmonary TB (open TB), typically when the patient coughs. The droplet nuclei, due to their small size, can remain suspended in the air for several minutes to hours. The risk of infection (Figure 1) is dependant on several factors such as the infectiousness of the source case, the closeness of contact, the bacillary load inhaled, and the immune status of the potential host [8–10]. The primary route of infection involves the lungs. Inhaled droplet nuclei avoid the defenses of the bronchi due to their small size and penetrate into the terminal alveoli where they are engulfed by phagocytic immune cells (macrophages and dendritic cells). M. tuberculosis can also infect nonphagocytic cells in the alveolar space including M cells, alveolar endothelial, and type 1 and type 2 epithelial cells (pneumocytes) [13–19]. In the early phase of infection, M. tuberculosis, internalized by phagocytic immune cells, replicates intracellularly, and the bacteria-laden immune cells may cross the alveolar barrier to cause systemic dissemination [14, 15]. The intracellular replication and simultaneous dissemination of the pathogen to the pulmonary lymph nodes and to various other extrapulmonary sites occur prior to the development of the adaptive immune responses. This exemplifies the extraordinary ability of M. tuberculosis to establish a protected niche where they can avoid elimination by the immune system and to persist indefinitely [20, 21].
In the vast majority of the infected individuals, an effective cell-mediated immune response develops 2–8 weeks after infection that stops further multiplication of the tubercle bacilli (Figure 1). The activated T lymphocytes, macrophages, and other immune cells form granulomas that wall off the growing necrotic tissue limiting further replication and spread of the tubercle bacilli. Most of the M. tuberculosis are killed in the caseating granulomas, and disease progression is arrested. However, the pathogen is not completely eradicated in some individuals as M. tuberculosis has evolved effective strategies to evade the immune response resulting in survival and persistence of some bacilli in a nonreplicating state in the host (LTBI) [8, 21, 22]. In support of this hypothesis, M. tuberculosis has been cultured and presence of M. tuberculosis DNA has been demonstrated from lung tissues of individuals who died from other diseases and who did not exhibit any pathological sign of TB disease [23, 24]. Furthermore, a recent report showing transmission of infection from father to son in Denmark in 1961 and reactivation of latent infection in the son more than 30 years later (documented by molecular fingerprinting of their respective M. tuberculosis isolates) has indicated that the surviving bacilli may remain dormant for a long time (lasting up to a lifetime) . A subsequent defect in cell-mediated immunity may result in reactivation of dormant bacilli causing active disease many years after the infection (reactivation TB). The current understanding of the mechanisms leading to the establishment of latent infection and the transition back to active growth in reactivation of latent disease is described below.
3. Entry Mechanisms of M. tuberculosis
Infection with M. tuberculosis starts with phagocytosis of the bacilli by phagocytic antigen-presenting cells in the lung including alveolar macrophages and dendritic cells. The recognition of pathogen-associated molecular patterns (PAMP) by specific pathogen recognition receptors (PRRs) is central to the initiation and coordination of the host innate immune response . M. tuberculosis internalized through different receptors may also have different fate.
The M. tuberculosis or M. tuberculosis components are recognized by host receptors that include Toll-like receptors (TLRs), nucleotide-binding oligomerization domain- (NOD-) like receptors (NLRs), and C-type lectins. The C-type lectins include mannose receptor (CD207), the dendritic cell-specific intercellular adhesion molecule grabbing nonintegrin (DC-SIGN) and Dectin-1 [27, 28]. Other potential receptors include complement receptors, scavenger receptors, surfactant protein A receptors (Sp-A), and cholesterol receptors . Some of these receptors (such as TLRs) are expressed on both, immune cells (such as macrophages, dendritic cells, B cells, and specific types of T cells) and nonimmune cells (like fibroblasts and epithelial cells). The interaction of M. tuberculosis with TLRs initiates an intracellular signaling cascade that culminates in a proinflammatory response (beneficial to the host); however, the bacterium has also evolved strategies that can trigger signals that dampen or modulate the innate immune response (beneficial to the pathogen). Other membrane bound PRRs (CD207, DC-SIGN, and Dectin-1) contribute to the relay of inflammatory signals while cytosolic PRRs (such as Nod-like receptor) modulate host recognition of the pathogen [27, 28].
The TLR engagement, particularly TLR2 and TLR4, with M. tuberculosis/M. tuberculosis component is an early event in the interaction of the pathogen with host cells and TLR signaling is the main arm of the innate immune response during M. tuberculosis infection [27, 28, 30]. The TLR polymorphisms regulate the innate immune response to mycobacterial lipopeptides and clinical susceptibility to pathogens . Typically, signals generated by the interactions of TLRs with ligands on M. tuberculosis induce the activation of proinflammatory and antimicrobial innate immune response.
The M. tuberculosis cell envelope is composed of a cell wall that is covered with a thick waxy mixture of lipids and polysaccharides and is characterized by a high content of mycolic acids. The most important M. tuberculosis cell surface ligands that interact with TLRs and other receptors include the 19 and 27 kDa lipoproteins, 38 kDa glycolipoprotein, the lipomannan (LM) and mannose-capped lipoarabinomannan (ManLAM) [32–34]. Other ligands include LprA and LprG lipoproteins [35, 36] and, perhaps also, surface-exposed mammalian cell entry (Mce) proteins encoded by mce1 and mce3 operons [37–39]. The interaction of M. tuberculosis ligand(s) with TLRs eventually results in activation of nuclear transcription factor (NF)-κB and production of proinflammatory cytokines such as tumor necrosis factor (TNF)-α, interleukin (IL)-1, IL-12, chemokines, and nitric oxide through either myeloid differentiation primary response protein 88 (MyD88)-dependant or MyD88-independent pathway [27, 34, 40, 41].
Restricting TLR-induced proinflammatory signals is essential to avoid the risk of producing excessive inflammation that could damage host tissues. A family of receptor tyrosine kinases termed Tyro3/Axl/Mer (TAM) provide a negative feedback mechanism to both TLR-mediated and cytokine-driven proinflammatory immune responses . This property has been exploited by M. tuberculosis to its advantage. The 19 kDa lipoprotein of M. tuberculosis is an agonist of the TLR2 and modulates the innate immunity and antigen presenting cell function . Studies have shown that prolonged TLR2 signaling by lipoproteins of M. tuberculosis inhibits major histocompatibility complex (MHC)-II expression and processing of antigens by macrophages [43, 44]. Thus, a subset of infected macrophages with decreased antigen-presenting cell function may be unable to present M. tuberculosis antigens to CD4+ T cells resulting in insufficient activation of effector T cells leading to evasion of immune surveillance and creation of niches where M. tuberculosis survives and persists [27, 32].
The mannose receptors interact with ManLAM present in the cell envelop of M. tuberculosis. The phagocytosis of tubercle bacilli by macrophages through mannose receptor is associated with an anti-inflammatory response as ManLAM inhibits mannose receptor-dependant IL-12 production. This inhibition of macrophage response to M. tuberculosis promotes infection and subsequent survival of M. tuberculosis in macrophages. The ManLAM exerts its effects on phagolysosome maturation by limiting phagosome-lysosome fusion [45, 46].
4. Immune Response of the Host to M. tuberculosis
The alveolar macrophages, after entry of M. tuberculosis, produce inflammatory cytokines and chemokines that serve as a signal for infection. The monocytes, neutrophils, and lymphocytes migrate to the focal site of infection, but they are unable to kill the bacteria efficiently. During this time, the bacilli resist the bactericidal mechanisms of the macrophage (phagolysosome) by preventing phagosome-lysosome fusion, multiply in the phagosome, and cause macrophage necrosis . The released bacilli multiply extracellularly, are phagocytosed by another macrophage that also fails to control the growth of M. tuberculosis, and likewise are destroyed. In the meantime, dendritic cells with engulfed bacilli mature, migrate to the regional lymph node, and prime T cells (both CD4+ and CD8+) against mycobacterial antigens . The specific immune response produces primed T cells which migrate back to the focus of infection, guided by the chemokines produced by the infected cells. The accumulation of macrophages, T cells, and other host cells (dendritic cells, fibroblasts, endothelial cells, and stromal cells) leads to the formation of granuloma at the site of infection .
The granuloma formation walls off tubercle bacilli from the rest of the lung tissue, limits bacterial spread, and provides microenvironment for interactions among macrophages and other cells of the immune system and the cytokines produced by these cells. The CD4+ T cells producing interferon-γ (IFN-γ) recognize infected macrophages presenting antigens from M. tuberculosis and kill them . The infection progression is halted; however, some resistant bacilli capable of surviving under the stressful conditions generated by the host escape killing and enter a state of dormancy and persist by avoiding elimination by the immune system [22, 51, 52]. Recent studies have shown that differences exist in the immunological response mounted by different individuals that lead to the formation of physiologically distinct granulomatous lesions in individuals exposed to M. tuberculosis. Some of these lesions suppress (sterilizing immunity) while others promote the persistence of viable M. tuberculosis in the microenvironment . Low-dose infection of cynomolgus macaques that reproduce the clinical characteristics of human latent TB leads to the formation of at least two types of tuberculous granuloma [54, 55]. Histopathological studies have shown that the classic caseous granuloma are composed of epithelial macrophages, neutrophils, and other immune cells surrounded by fibroblasts. The central caseous necrotic region in this type of granuloma consists of dead macrophages/other cells and is hypoxic with M. tuberculosis residing inside macrophages in the hypoxic center [55, 56]. The other kind of granulomas seen in latent tuberculosis in both humans and cynomolgus macaques are fibrotic lesions, composed almost exclusively of fibroblasts that contain very few macrophages . However, it is not known at present whether M. tuberculosis is located inside macrophages or in the fibrotic area in these lesions.
The microenvironment of the granuloma (hypoxia, low pH, presence of nitric oxide and carbon monoxide, etc.) increases the expression of several M. tuberculosis genes involved in dormancy induction [57–60]. Recent findings of formation of spore-like structures in M. bovis BCG, M. marinum, and M. smegmatis in response to prolonged stationary phase or nutrient starvation suggest that sporulation may be a general mechanism for mycobacterial dormancy [60–62]. The dormant bacilli can inhabit the granuloma during the lifetime of the host, but are able to resuscitate (or germinate) in the event of local immunodepression. The latent infection in a person without overt signs of the disease is indicated by the delayed-type hypersensitivity (DTH) response to purified protein derivative (PPD) prepared from culture filtrates of M. tuberculosis (tuberculin skin test) .
5. Specific Roles of Immune Cells and Cytokines in M. tuberculosis Infection
Studies in animal models and in humans have demonstrated that a wide range of immune components are involved in an effective immune response against M. tuberculosis. These include, beside macrophages and dendritic cells, αβ-T cells (both CD4+ and CD8+), CD1 restricted T cells, γδ-T cells, and cytotoxic T cells, as well as the cytokines produced by these immune cells [22, 63, 64]. The most important among these are CD4+ T cells and the cytokine IFN-γ. Although CD4+ T cells along with CD8+ T cells and the natural killer (NK) cells are the major producers of IFN-γ, studies carried out in CD4+ deficient mice have shown that it is the early production of IFN-γ by CD4+ T cells and subsequent activation of macrophages that determine the outcome of infection [65, 66]. The CD4+ T cells also play other roles in the defense against infection that is independent of IFN-γ production. Depletion of CD4+ T cells was associated with the reactivation of infection in a chronically infected mice and resulted in increasing pathological features and death, even though IFN-γ levels were still high due to a strong response from CD8+ T cells and normal levels of inducible nitric oxide synthase (iNOS) .
The CD4+ T cells carry out several functions that are important to control infection in the granuloma. These include apoptosis of infected macrophages through Fas/Fas ligand interaction, production of other cytokines (such as IL-2 and TNF-α), induction of other immune cells (macrophages or dendritic cells) to produce other immunoregulatory cytokines such as IL-10, IL-12, and IL-15, and activation of macrophages through direct contact via CD40 ligand [63, 66, 68, 69]. The CD4+ T cells also appear to be critical for the cytotoxic function of CD8+ T cells that is mediated by IL-15 [66, 70]. It has also been shown that CD4+ T cells can control the intracellular growth of M. tuberculosis by a nitric oxide-dependent mechanism that is independent of IFN-γ production [66, 71]. Thus, CD4+ T cells, in addition to early production of IFN-γ appear to have several other secondary functions that are critical in the control of M. tuberculosis infection.
The CD8+ T-cells, in addition to producing IFN-γ and other cytokines, may also be cytotoxic for M. tuberculosis-infected macrophages, and thus play an important role in providing immunity to TB. The CD8+ T-cells can directly kill M. tuberculosis via granulysin, and facilitate the control of both the acute as well as chronic infection [66, 72]. The abundant presence of M. tuberculosis-specific CD8+ T cells in latently infected individuals shows that the CD8+ T cells also have a role in the control of latent infection. This is also supported by reactivation of latent infection following depletion of CD8+ T cells in the Cornell model of latent TB .
Studies in primate models of TB have shown that unconventional T cells such as CD1 restricted T cells, and γδ-T cells also contribute to the protection against TB [64, 74, 75]. The CD1 restricted T cells recognize the glycolipids such as LAM that are abundant in the mycobacterial cell wall while γδ-T cells recognize small metabolites containing phosphate (phospholigands) . Although it is well established that mycobacterial antigens in the phagosome of macrophages or dendritic cells are picked up by the MHC class II molecules and presented to CD4+ T cells, studies have shown that the phagosomal membrane is also equipped with the MHC class I processing machinery [76, 77]. Also, CD1 proteins have the capability to present lipid antigens and lipopeptides to T cells, and thus play important roles in the immune response against lipid-rich M. tuberculosis [75, 78, 79]. Further, the vesicles formed due to apoptosis of M. tuberculosis-infected macrophages and containing mycobacterial antigens such as ManLAM, lipoproteins, and so forth are taken up by dendritic cells and presented to the T cells through the MHC class I and CD1 molecules [75, 78, 80].
The IFN-γ is the key cytokine for a protective immune response against M. tuberculosis. Humans and mice defective in IFN-γ or IFN-γ receptor genes are more susceptible to M. tuberculosis infection [63, 66, 81]. The IFN-γ, produced mainly by CD4+, CD8+ T cells, and the NK cells, synergizes with TNF-α and activates macrophages to kill intracellular bacilli. The IFN-γ also augments antigen presentation, leading to recruitment of CD4+ T-cells and/or cytotoxic CD8+ T-cells, which participate in mycobacterial killing and also prevents exhaustion of memory T cells [63, 82]. Furthermore, IFN-γ induces the transcription of more than 200 genes in macrophages including the upregulation of MHC class II expression and the production of antimicrobial effectors such as oxygen radicals and nitric oxide. A major effector mechanism responsible for the antimicrobial activity of IFN-γ in association with TNF-α is the induction of the production of nitric oxide and other reactive nitrogen intermediates (RNI) by macrophages via iNOS [63, 66, 83]. However, some M. tuberculosis factor(s), such as the 19-kDa lipoprotein, have the potential to attenuate the response of macrophages to IFN-γ by blocking the transcription of a subset of IFN-γ-responsive genes (Table 1) [44, 84, 85].
TNF-α, produced by macrophages, dendritic cells, and T-cells, is another cytokine that has a major protective role against M. tuberculosis infection both in mice and humans [104, 105]. Paradoxically, TNF-α also contributes significantly to the development of immunopathology associated with TB . Mice deficient in TNF-α or TNF-α receptors are more susceptible to mycobacterial infections . This cytokine is involved in both immune and immunomodulatory responses and acts in synergy with IFN-γ to enhance the expression of iNOS and the antimycobacterial activity of macrophages [63, 83]. TNF-α also initiates cell migration and formation of microbicidal granulomas while disruption of TNF-α responses leads to overgrowth of the mycobacterial pathogens [63, 66]. The TNF-α produced by the infected macrophages induces the expression of chemokines, such as IL-8, MCP-1, and RANTES which provide signals for migration of immune cells to the sites of M. tuberculosis infection . Both T cell- and macrophage-derived TNF-α are required for sufficient and long-term protection against M. tuberculosis infection . The phenolic glycolipid, a virulence factor in the cell wall of a hypervirulent strain of M. tuberculosis (W-Beijing family) inhibits the release of pro-inflammatory cytokines TNF-α, IL-6 and IL-12 by macrophages .
The importance of IL-12 is also evident from increased susceptibility of mice and humans deficient in IL-12 responses to mycobacterial infections . Individuals with defects in the production of IL-12 or its receptor are highly susceptible to active TB disease . The T-cell-derived cytokines, IFN-γ and TNF-α, are produced abundantly by activated CD4+ T cells under the influence of IL-12, and the role of IFN-γ and TNF-α in activating and augmenting the microbicidal effector functions of phagocytic cells during a protective immune response against M. tuberculosis infection is well established [63, 66, 83].
6. Antigen Presentation Pathways and Their Modulation by M. tuberculosis Components
The tubercle bacilli reside in the phagosome soon after their entry inside alveolar macrophages and dendritic cells. The priming of CD4+ T cells for a protective immune response requires the presentation of M. tuberculosis antigens through MHC class II pathway. The phagosomal membrane is also equipped with the MHC class I processing machinery [74, 77]. Further, mycobacterial glycolipids, lipids, and other phospholigands may also be presented to the T cells [78, 80]. Some M. tuberculosis factors particularly those associated with the cell wall modulate antigen-processing pathways by MHC class I, MHC class II, and CD1 molecules [28, 63, 111]. The ManLAM, trehalose 6,6′-dimycolate (TDM, also known as cord factor), and the 19 kDa lipoprotein downregulate IFN-γ-inducible genes including those involved in antigen presentation by MHC class II machinery (Table 1) [32, 86, 94, 111, 112]. Other mechanisms that modulate antigen presentation include antigen processing and binding of peptides to MHC class II molecules [32, 87]. The 19 kDa lipoprotein also inhibits MHC class I antigen processing via Toll-like receptor signaling . It is probable that continuous attenuation of antigen presentation through multiple mechanisms is advantageous for slowly growing pathogens like M. tuberculosis [111, 112]. Inhibition of antigen presentation by these mechanisms results in persistence of M. tuberculosis inside macrophages .
7. Dampening of Other Macrophage Functions by M. tuberculosis Components
The two major antimycobacterial mechanisms of macrophages include the generation of nitric oxide and other RNI which exert toxic effects on the bacilli and fusion of the phagosomes containing mycobacteria with lysosomes that is bactericidal [83, 92, 113]. The T cell-derived cytokines, mainly IFN-γ and TNF-α, activate macrophages, which then generate nitric oxide and other RNI by iNOS and are mycobactericidal . Direct demonstration of the presence of nitrotyrosine, an RNI derived from tyrosine and peroxynitrite in the lungs of infected mice, has shown that RNI are formed in tuberculous granuloma. Furthermore, inhibition of iNOS activity or disruption of iNOS gene, required for the production of RNI, not only abolished the protective effect of RNI but also led to reactivation of latent infection in mice . Although these studies point towards an essential role for iNOS in the control of both acute and chronic persistent infection, the RNI generated through these mechanisms is not sufficient to eliminate the bacterium completely.
The protective role of RNI in human TB has also been suggested [92, 114]. Studies have shown that M. tuberculosis has evolved several strategies to evade the RNI toxicity. It has been shown that iNOS, a cytoplasmic protein, may be recruited to the phagosomes and this recruitment may be inhibited by M. tuberculosis . The M. tuberculosis gene, alkyl hydroperoxide reductase subunit C (ahpC), detoxifies, in conjunction with some other proteins, the highly reactive peroxynitrite anion (formed by the reaction of nitric oxide with superoxide) . Another potential mechanism for blunting the toxic effects of RNI is the presence of two haemoglobin-like proteins encoded by glbN and glbO in M. tuberculosis. The glbN knockout mutant of M. bovis BCG was highly attenuated, and its growth, in vitro, was also inhibited by nitric oxide under aerobic conditions . Microarray analyses have shown that more than 30 M. tuberculosis genes are induced by RNI and hypoxia [59, 118]. Furthermore, hypoxia and inhibition of respiration by nitric oxide induce a dormancy program in M. tuberculosis that leads to increased survival and persistence of the pathogen in immune cells [59, 119].
8. Phagolysosome Maturation and Its Inhibition by M. tuberculosis Components
The phagocytosis of M. tuberculosis by macrophages is followed by the maturation of phagosomes containing the pathogen through a series of fusion and fission events with several endocytic vesicles that culminate in a phagolysosome [120, 121]. The fission-fusion events remodel the phagosomal membrane, and the recruitment of vacuolar-proton transporting ATPase (vH+-ATPase) lowers the internal pH that allows lysosome-derived acid hydrolases to function efficiently for their microbicidal effect [122, 123]. Furthermore, phagosome maturation is dependant on Ca+2 signaling cascade that begins with phosphorylation of sphingosine to sphingosine 1-phosphate by sphingosine kinase resulting in elevation of cytosolic [Ca+2] inside macrophages due to release of Ca+2 from intracellular stores in the endoplasmic reticulum and continues through Ca+2-calmodulin complex-dependant activation of protein kinase II and phosphatidyl inositol 3-kinase (PI-3K). The cascade culminates in phosphorylation of phosphatidyl inositol to phosphatidyl inositol 3-phosphate (PI-3P) by PI-3K in the phagosome membrane and maturation of phagosome to an acidic bactericidal compartment (phagolysosome) after binding of early endosomal antigen-1 (EEA-1) to PI-3P [89, 124–126].
M. tuberculosis has also evolved several strategies to avoid the destruction by lysosomal enzymes by disrupting the maturation of bacilli-containing phagosomes into phagolysosmes [46, 90, 92, 127, 128]. Exclusion of vH+-ATPase during maturation of phagosomes contributes to the acidification defect that prevents the fusion of phagosomes with lysosomes . Similarly, modulation of Ca+2 signaling cascade, such as SapM-mediated hydrolysis and inactivation of PI-3P, inhibits phagosome maturation leading to enhanced intracellular survival of M. tuberculosis (Table 1) [101, 126–128].
Other M. tuberculosis components such as ManLAM and TDM (Table 1) also affect phagosome maturation by interfering with the tethering and fusion machinery of vesicular transport in mammalian cells and promote persistence of the bacterium inside macrophages [46, 89, 90, 93, 101]. The targets include the soluble N-ethylmaleimide-sensitive factor-attachment protein receptors (SNAREs), the tethering proteins (such as EEA-1), and the Rab family of GTPases [89, 120, 126, 128]. Some of the membrane trafficking processes affected by mycobacterial factors are also affected by HIV during viral budding and this overlap partially contributes towards the synergism observed between AIDS and active TB . Another mechanism by which mycobacteria interfere with phagosome maturation is by retention of the host tryptophan aspartate rich coat protein (TACO) (homolog of coronin-1) on the cytoplasmic side of their phagosomes that likely inhibits the normal process of phagosome-lysosome fusion . The serine/threonine protein kinase G encoded by pknG of M. tuberculosis (Table 1) is implicated as the potential effector of the inhibition of phagosome-lysosome fusion [100, 111].
Another component of the antimicrobial repertoire of macrophages includes lysosomal killing of M. tuberculosis mediated by ubiquitin-derived peptides . The ubiquitination destroys tubercle bacilli by autophagy as a ubiquitin-derived peptide impairs the membrane integrity of M. tuberculosis that allows nitric oxide to kill more efficiently. On the contrary, decreased outer membrane permeability protects M. tuberculosis from killing by ubiquitin-derived peptides .
9. Apoptosis of Infected Macrophages and Its Inhibition by M. tuberculosis Components
The apoptosis of infected macrophages participates in host defense against infection as apoptotic vesicles containing mycobacterial antigens are taken up by dendritic cells for CD8+ T cell activation by phagosome-enclosed antigens [79, 80]. The CD8+ T cells activated by apoptotic vesicles from M. tuberculosis-infected cells produce IFN-γ, which causes uninfected macrophages to produce RNI to effectively kill intracellular M. tuberculosis. Several M. tuberculosis-derived factors are capable of modulating (activating as well as inhibiting) the apoptosis of infected macrophages through differential expression of proapoptotic and antiapoptotic genes . The mycobacterial components modulating apoptosis of macrophages usually target the caspase cascade or the one involving TLRs. The M. tuberculosis components that inhibit apoptosis include cell wall components, ManLAM, virulence-related secretion system encoded by secA2 that transports superoxide dismutase (encoded by sodA) to control reactive oxygen intermediates, and NADH dehydrogenase (encoded by nuoG) (Table 1) [91, 102, 103]. Two secretory proteins of M. tuberculosis encoded by Rv3654c and Rv3655c that inhibit apoptosis of macrophages have also been identified recently . By inhibiting apoptosis of macrophages, M. tuberculosis avoids host defenses and escapes from infected cells by causing necrotic cell death .
10. Escape of M. tuberculosis from Phagosome/Phagolysosome
Although it has been known for quite some time that M. tuberculosis survives in the phagosome by blocking (or slowing down) its maturation into phagolysosome and persists, one of the mechanism by which it escapes from phagosome/phagolysosome to infect other macrophages and other immune/alveolar cells has been elucidated recently. Initial subtractive hybridization-based studies identified a genomic region, termed region of difference 1 (RD1), that was present in all virulent M. tuberculosis and M. bovis strains but was absent in the vaccine strain M. bovis BCG [2, 134, 135]. Subsequently, it was shown that RD1 is crucial for the virulence of M. tuberculosis as it encoded proteins that formed a novel protein secretion system (ESX-1). ESX-1 (type VII secretion system) is involved in the export of several M. tuberculosis proteins including two potent T cell antigens encoded by RD1 itself, the 6-kDa early secreted antigenic target (ESAT-6) (encoded by esxA) and 10-kDa culture filtrate protein (CFP-10) (encoded by esxB) that lack signal sequences for their export [2, 134, 136–140].
The importance of ESX-1 secreted proteins in virulence of M. tuberculosis has been shown by deletion of RD1 or disruption of ESAT-6 from M. tuberculosis genome that resulted in reduced virulence (spreading) both, in cultured macrophages and in mice [140, 141]. Furthermore, the introduction of RD1 genes in M. bovis BCG resulted in altered colonial morphology, increased virulence in severely combined immune deficient mice including the formation of granuloma, and longer persistence in immunocompetent mice . In M. tuberculosis, ESAT-6 complexes with CFP-10 in 1 : 1 ratio before its export outside the cell but can dissociate from its partner (CFP-10) at lower pH. Individually, ESAT-6, but not CFP-10, can cause disruption of artificial membranes as well as cytolysis [143–145]. ESAT-6 alone has also been shown to associate strongly with liposomes containing dimyristoylphosphatidylcholine and cholesterol (constituents of mammalian cell membranes) and causing destabilization and lysis of liposomes [95, 96, 144].
The studies carried out by de Jonge et al.  have shown that ESAT-6:CFP-10 complex secreted by live M. tuberculosis inside phagosome splits apart when tubercle bacilli are stressed following acidification of phagosome, and ESAT-6 inserts itself into lipid bilayer, causing lysis and escape of M. tuberculosis from phagosome. Further studies have shown that ESAT-6 also induces apoptosis of macrophages via the extrinsic (caspase-dependent) pathway by formation of pores in cell membrane  and contributes (or helps) in the translocation of M. tuberculosis from the phagolysosomes to the cytoplasm in myeloid cells . More recently, ESAT-6 has also been shown to cause cytolysis of type 1 and type 2 alveolar epithelial cells. This ESAT-6-mediated cytolysis was shown to help in the dissemination of M. tuberculosis through alveolar wall . The ESAT-6 homolog from Mycobacterium marinum, the bacterium that causes tuberculous granuloma in zebrafish, has also been demonstrated to cause lysis of red blood cells and macrophages by forming pores in their membranes [147, 148]. The presence of a capsular layer containing high amounts of proteins that are secreted via the ESX-1 secretion system including ESAT-6 has also been demonstrated in pathogenic mycobacteria recently . Furthermore, ESX-1-associated proteins in the capsular layer enhanced the interaction of mycobacteria with macrophages and also dampened proinflammatory cytokine response of macrophage. These studies have established the role of ESX-1 secretion system and ESAT-6 protein of M. tuberculosis in facilitating macrophage infection and subsequent bacterial escape to infect other nearby cells (Table 1).
11. Persistence and Reactivation of Latent TB Infection
The hallmark of M. tuberculosis infection in humans is the inability of an otherwise effective immune response to completely eliminate the pathogen. The tubercle bacilli have evolved multiple strategies to manipulate infected host cells in order to evade or modify the ensuing immune response so as to avoid elimination and thus persist in the host. As described above, several M. tuberculosis factors, ManLAM and 19-kDa lipoprotein notable among them, have been identified that modulate antigen presentation pathways and either blunt the microbicidal functions of macrophages and other immune cells (such as RNI) or prevent their maturation (phagolysosome) (Table 1).
Two experimental strategies have been employed to identify other M. tuberculosis factors, which promote persistence of the pathogen in immune cells including macrophages. One approach involves cloning of M. tuberculosis genes in nonpathogenic mycobacteria and studying their increased survival in macrophages or other mammalian cells while the other approach uses knockout mutants of M. tuberculosis for selected genes for persistence of the pathogen in macrophages and other immune cells. Several additional M. tuberculosis factors promoting persistence or increased survival in mammalian cells have been identified. Some of these factors include phospholipases encoded by plcA, plcB, plcC, and plcD , the two PhoP and PhoQ regulatory proteins , phosphate-binding proteins PstS1 and PstS2 , and proteins encoded by mce operons . Thus, M. tuberculosis has devoted a large part of its genome towards functions that promote its intracellular survival in mammalian cells including macrophages.
Reactivation of latent infection requires latent M. tuberculosis cells to exit dormancy. Several factors can trigger the development of active disease from the reactivation of remote infection, and this typically involve the weakening of the immune system. HIV infection is the most important single risk factor for progression to active disease in adults as it causes depletion of CD4+ T cells and functional abnormalities of CD4+ and CD8+ T-cells which play an important role in providing protection against active TB disease [4, 6]. Likewise, M. tuberculosis infection accelerates the progression of asymptomatic HIV infection to acquired immunodeficiency syndrome (AIDS) and eventually to death. This is recognized in the current AIDS case definition as pulmonary or extrapulmonary TB in HIV-infected patient is sufficient for the diagnosis of AIDS. Old age, malnutrition, and medical conditions that compromise the immune system such as poorly controlled diabetes mellitus, renal failure, and therapy with immunosuppressive drugs are other factors that lead to immunodepression and reactivation of a dormant infection [6, 8, 152, 153]. The reactivation TB can occur in any organ system in which the tubercle bacilli were seeded during the primary infection; however, in immunocompetent individuals, the reactivation usually occurs in the upper lobes, where higher oxygen pressure supports good bacillary growth. The lytic transglycosylases known as resuscitation promoting factors (RPFs) and an endopeptidase (RipA) of M. tuberculosis have recently been recognized as vital components for revival from latency [154–156].
12. Current Dynamic Model of Latent Infection
The LTBI has traditionally been defined as infection with M. tuberculosis in foci within granuloma that remain in nonreplicating state but retain their ability to come out of latency and cause active TB if and when a disruption of the immune response occurs . However, recent experimental data supports a dynamic model of LTBI where endogenous reactivation as well as damage response occurs constantly in immunocompetent individuals . The model suggests that during infection, M. tuberculosis grows well inside phagosomes; however, some bacilli released from necrotic macrophages in extracellular milieu in developing granulomas stop replicating. The arrest of bacterial growth occurs even before an effective immune response has fully been developed due to hypoxic and acidic environment (conditions that mimic stationary bacterial cultures) in the extracellular milieu and release of bactericidal enzymes from dead macrophages and neutrophils. The actively growing bacillary population is eventually killed due to the development of an effective immune response; however, nonreplicating bacilli resist killing and survive [158, 159].
The model further suggests that some macrophages (foamy macrophages) also emerge during the chronic inflammatory process, as they have phagocytosed the cellular debris rich in fatty acids and cholesterol derived from cellular membranes [160, 161]. The foamy macrophages also phagocytose extracellular nonreplicating lipid-rich M. tuberculosis; however, the bacilli do not grow in the intracellular environment of activated macrophages but are also not killed due to the nonreplicating state of the bacilli . The nonreplicating bacilli-laden foamy macrophages drain from lung granuloma towards the bronchial tree and return to a different region of lung parenchyma due to aerosols generated by inspired air and begin the infection process at this new location once again [157, 160, 161, 163]. In this dynamic process, reinfection in the upper lobe may have the chance to cause cavitary lesion. This is aided by higher oxygen pressure in the upper lobes that can support rapid extracellular bacillary growth resulting in bacillary concentration that can not be controlled by the optimum immune response by the host. The subsequent much stronger inflammatory response leads to tissue destruction, liquefaction, and extracellular bacillary growth which amplifies the response further and causes cavitation [157, 158].
The dynamic infection process leading to active disease in the upper lobes has some parallels with immune reconstitution inflammatory syndrome and the active TB disease that occurs in HIV-infected patients. The presence of bacilli is tolerated by the HIV-infected patients with low CD4+ cell counts as the host is unable to mount an inflammatory response needed to control their growth. However, the sudden increase in CD4+ T cells in AIDS patients receiving highly active antiretroviral treatment causes an aggressive granulamatous response and active TB disease [164, 165]. The possibility of slow clearance of latent infection proposed by the dynamic infection model has also been supported by a recent study from Norway comprising a population of individuals exposed to a minimal risk of active transmission of infection. Cohort analysis of data from National Tuberculosis Registry to calculate rates and changes in rates of active TB disease, among patients previously exposed to M. tuberculosis, has shown that the rate of reactivated TB has progressively decreased over time . The study further suggested that the number of individuals with latent infection could be reduced in half in approximately 9 years in populations in which new infections are effectively prevented. The dynamic infection model also explains how therapy for a relatively short time (9 months) with a single drug (isoniazid), active only against actively dividing bacilli , is highly effective for a latent infection that can possibly remain dormant for the entire lifetime of the host. As isoniazid will prevent episodes of reinfection by bacilli resuscitated from dormancy, slow drainage and destruction of nonreplicating bacilli in the stomach will eventually lead to clearance of latent infection [157, 166].
13. Diagnosis of Latent M. tuberculosis Infection
The persons infected with M. tuberculosis may be identified by tuberculin skin test six to eight weeks after exposure to the bacilli. The test is based on a delayed-type hypersensitivity (DTH) response to a complex cocktail of M. tuberculosis antigens, known as purified protein derivative (PPD). The induration of more than 5 mm, recorded 48 to 72 hours after injection of PPD, is considered as positive. Surveys conducted with PPD skin test suggest that nearly a third of the world’s and half of Asia’s population is infected with M. tuberculosis . Skin test reaction over 20 mm is usually due to active disease; however, a negative skin test in an active TB patient may also result from anergy or incorrect administration of the test. The tuberculin skin test lacks sensitivity and specificity as it can not differentiate between infection with M. tuberculosis and sensitization with other environmental mycobacteria [5, 8]. Also, BCG vaccination may cause false-positive reactions, but these generally last only a few years after vaccination and are in the moderate range (5 to 10 mm).
More sensitive and specific tests such as cell-mediated immunity-based interferon-gamma (IFN-γ) release assays (IGRAs) have also been developed that detect T cell responses after stimulation by two M. tuberculosis-specific antigens, early secreted antigenic target-6 (ESAT-6) and culture filtrate protein-10 (CFP-10) [168–173]. The IGRAs have excellent specificity as the antigens (ESAT-6 and CFP-10) used in these assays are encoded by genes deleted in the vaccine strain M. bovis BCG and majority of environmental mycobacteria of clinical relevance [134, 135, 170]. Another variation of conventional IGRAs has also been developed by using flow cytometry . Although flow cytometric approach has an advantage over conventional IGRAs as a smaller blood volume (<1 ml) is required for testing, the assay has limited utility in much of the developing world due to the requirement of technical expertise and the high cost of flow cytometers. The detection of significant levels of antibodies to some M. tuberculosis-specific proteins has also been noted in latently infected individuals as well as in patients with active TB disease but not in healthy subjects [175–178]. However, antibody-based tests have not been used so far for the detection of LTBI.
Two commercial IGRAs, whole blood, ELISA-based QuantiFERON (QFN)-TB Gold assay (Cellestis Ltd., Carnegie, Australia) and peripheral blood mononuclear cell (PBMC) and enzyme-linked immunospot (ELISPOT) technology-based T SPOT-TB (Oxford Immunotec, Oxford, UK) test have also been developed and approved by Food and Drug Administration (FDA) for detecting LTBI. The tests were initially based on stimulation of T lymphocytes with ESAT-6 and CFP-10 proteins and measurement of IFN-γ production (QFN-TB Gold) or detection of T-cells themselves (T SPOT-TB). These tests have undergone further improvement. The newer version of the QFN-TB Gold is called QuantiFERON-TB-Gold-In-Tube (QFT-G-IT) (Cellestis Ltd., Carnegie, Australia) that uses ESAT-6 and CFP-10 and TB7.7 (corresponding to Rv2654 ) peptides as antigens. The newer version of T-Spot-TB also uses peptides of ESAT-6 and CFP-10 instead of whole ESAT-6 and CFP-10 proteins as antigens (Oxford Immunotec, Oxford, UK). The performance of both QFT-G-IT and T-Spot-TB tests have recently been evaluated extensively, and several systematic reviews are available for a more detailed description [173, 179–182]. Although IGRAs can not distinguish between LTBI and active TB disease in immunocompetent adults , in high-risk individuals with immunosuppressive conditions and children, IGRAs may help in the diagnosis of active disease as adjunctive diagnostic tests, particularly if specimens from the suspected site of infection (such as bronchoalveolar lavage, cerebrospinal fluid) rather than blood is used [183, 184]. While the results of IGRAs exhibit better correlation with surrogate measures of exposure to M. tuberculosis in low TB incidence countries; however, their performance is suboptimal in countries with a high TB incidence [173, 180–182, 185].
In low TB incidence countries of North America and Western Europe, the majority of active TB disease cases occur in foreign-born persons. Previous studies have shown that majority of active disease cases in immigrants/expatriates originating from TB endemic countries occur as a result of reactivation of previously acquired infection mostly within two years of their migration [8, 12, 171, 172]. Application of IGRAs to identify latently infected individuals and their treatment for LTBI has greatly helped in lowering the incidence of TB in rich, advanced countries [171, 172, 182, 186, 187]. Some other low-intermediate TB incidence countries which contain large expatriate populations originating from TB endemic countries [188–192] are also evolving similar strategies for controlling TB [12, 193].
With nearly 9 million new active TB cases and 2 million deaths occurring every year, TB remains a major infectious disease of global proportion. Active disease patients with sputum smear-positive pulmonary TB are the main source of infection. Primary infection with M. tuberculosis leads to clinical disease in ~10% of individuals. In the remaining cases, the ensuing immune response arrests further growth of M. tuberculosis. However, the pathogen is eradicated completely in ~10% people while the immune response in the remaining ~90% individuals only succeeds in containment of infection as some bacilli escape killing by blunting the microbicidal mechanisms of immune cells and remain in nonreplicating (dormant or latent) state in old lesions. The dormant bacilli retain their ability to induce reactivation and to cause active TB if a disruption of immune response occurs. While active transmission is a significant contributor of active disease cases in high TB burden countries, most cases in low TB incidence countries arise from this pool of latently infected individuals. The positive tuberculin skin test or more recent and specific T cell-based IGRAs in a person without overt signs of the disease indicates LTBI. Two commercial IGRAs, QFT-G-IT and T-Spot-TB, are also available. Application of IGRAs to identify latently infected individuals and their treatment for LTBI has greatly helped in lowering the incidence of TB in rich, advanced countries. Similar approaches also hold great promise for other countries with low-intermediate rates of TB incidence.
S. Ahmad was supported by Kuwait University Research Administration Grant MI 05/00.
- M. G. Cristina, S. Brisse, R. Brosch et al., “Ancient origin and gene mosaicism of the progenitor of Mycobacterium tuberculosis,” PLoS Pathogens, vol. 1, no. 1, article e5, 2005.
- S. T. Cole, R. Brosch, J. Parkhill et al., “Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence,” Nature, vol. 393, no. 6685, pp. 537–544, 1998.
- T. Garnier, K. Eiglmeier, J.-C. Camus et al., “The complete genome sequence of Mycobacterium bovis,” Proceedings of the National Academy of Sciences of the United States of America, vol. 100, no. 13, pp. 7877–7882, 2003.
- World Health Organization, “Global tuberculosis control: surveillance, planning and financing,” WHO/HTM/TB/2009.411, WHO, Geneva, Switzerland, 2009.
- C. Dye, S. Scheele, P. Dolin, V. Pathania, and M. C. Raviglione, “Consensus statement. Global burden of tuberculosis: estimated incidence, prevalence, and mortality by country. WHO Global Surveillance and Monitoring Project,” JAMA, vol. 282, no. 7, pp. 677–686, 1999.
- C. D. Wells, J. P. Cegielski, L. J. Nelson et al., “HIV infection and multidrug-resistant tuberculosis—the perfect storm,” Journal of Infectious Diseases, vol. 196, no. 1, pp. S86–S107, 2007.
- S. Ahmad and E. Mokaddas, “Recent advances in the diagnosis and treatment of multidrug-resistant tuberculosis,” Respiratory Medicine, vol. 103, no. 12, pp. 1777–1790, 2009.
- T. R. Frieden, T. R. Sterling, S. S. Munsiff, C. J. Watt, and C. Dye, “Tuberculosis,” The Lancet, vol. 362, no. 9387, pp. 887–899, 2003.
- P. C. Hill, R. H. Brookes, A. Fox et al., “Large-scale evaluation of enzyme-linked immunospot assay and skin test for diagnosis of Mycobacterium tuberculosis infection against a gradient of exposure in The Gambia,” Clinical Infectious Diseases, vol. 38, no. 7, pp. 966–973, 2004.
- B. Mathema, N. Kurepina, D. Fallows, and B. N. Kreiswirth, “Lessons from molecular epidemiology and comparative genomics,” Seminars in Respiratory and Critical Care Medicine, vol. 29, no. 5, pp. 467–480, 2008.
- M. P. Golden and H. R. Vikram, “Extrapulmonary tuberculosis: an overview,” American Family Physician, vol. 72, no. 9, pp. 1761–1768, 2005.
- E. Mokaddas, S. Ahmad, and I. Samir, “Secular trends in susceptibility patterns of Mycobacterium tuberculosis isolates in Kuwait, 1996-2005,” International Journal of Tuberculosis and Lung Disease, vol. 12, no. 3, pp. 319–325, 2008.
- L. E. Bermudez and J. Goodman, “Mycobacterium tuberculosis invades and replicates within type II alveolar cells,” Infection and Immunity, vol. 64, no. 4, pp. 1400–1406, 1996.
- R. Teitelbaum, W. Schubert, L. Gunther et al., “The M cell as a portal of entry to the lung for the bacterial pathogen Mycobacterium tuberculosis,” Immunity, vol. 10, no. 6, pp. 641–650, 1999.
- L. E. Bermudez, F. J. Sangari, P. Kolonoski, M. Petrofsky, and J. Goodman, “The efficiency of the translocation of Mycobacterium tuberculosis across a bilayer of epithelial and endothelial cells as a model of the alveolar wall is a consequence of transport within mononuclear phagocytes and invasion of alveolar epithelial cells,” Infection and Immunity, vol. 70, no. 1, pp. 140–146, 2002.
- L. Danelishvili, J. McGarvey, Y.-J. Li, and L. E. Bermudez, “Mycobacterium tuberculosis infection causes different levels of apoptosis and necrosis in human macrophages and alveolar epithelial cells,” Cellular Microbiology, vol. 5, no. 9, pp. 649–660, 2003.
- B. E. García-Pérez, R. Mondragón-Flores, and J. Luna-Herrera, “Internalization of Mycobacterium tuberculosis by macropinocytosis in non-phagocytic cells,” Microbial Pathogenesis, vol. 35, no. 2, pp. 49–55, 2003.
- P. K. Mehta, R. K. Karls, E. H. White, E. W. Ades, and F. D. Quinn, “Entry and intracellular replication of Mycobacterium tuberculosis in cultured human microvascular endothelial cells,” Microbial Pathogenesis, vol. 41, no. 2-3, pp. 119–124, 2006.
- A. G. Kinhikar, I. Verma, D. Chandra et al., “Potential role for ESAT6 in dissemination of M. tuberculosis via human lung epithelial cells,” Molecular Microbiology, vol. 75, no. 1, pp. 92–106, 2010.
- A. A. Chackerian, J. M. Alt, T. V. Perera, C. C. Dascher, and S. M. Behar, “Dissemination of Mycobacterium tuberculosis is influenced by host factors and precedes the initiation of T-cell immunity,” Infection and Immunity, vol. 70, no. 8, pp. 4501–4509, 2002.
- S. M. Hingley-Wilson, V. K. Sambandamurthy, and W. R. Jacobs Jr., “Survival perspectives from the world's most successful pathogen, Mycobacterium tuberculosis,” Nature Immunology, vol. 4, no. 10, pp. 949–955, 2003.
- J. M. Tufariello, J. Chan, and J. L. Flynn, “Latent tuberculosis: mechanisms of host and bacillus that contribute to persistent infection,” The Lancet Infectious Diseases, vol. 3, no. 9, pp. 578–590, 2003.
- E. L. Opie and J. D. Anderson, “Tubercle bacilli in latent tuberculosis lesions and in lung tissue without tuberculosis lesions,” Archives of Pathology and Laboratory Medicine, vol. 4, no. 1, pp. 1–21, 1927.
- R. Hernández-Pando, M. Jeyanathan, G. Mengistu et al., “Persistence of DNA from Mycobacterium tuberculosis in superficially normal lung tissue during latent infection,” The Lancet, vol. 356, pp. 2133–2138, 2000.
- T. Lillebaek, A. Dirksen, I. Baess, B. Strunge, V. Ø. Thomsen, and Å. B. Andersen, “Molecular evidence of endogenous reactivation of Mycobacterium tuberculosis after 33 years of latent infection,” Journal of Infectious Diseases, vol. 185, no. 3, pp. 401–404, 2002.
- S. Akira, S. Uematsu, and O. Takeuchi, “Pathogen recognition and innate immunity,” Cell, vol. 124, no. 4, pp. 783–801, 2006.
- E.-K. Jo, “Mycobacterial interaction with innate receptors: TLRs, C-type lectins, and NLRs,” Current Opinion in Infectious Diseases, vol. 21, no. 3, pp. 279–286, 2008.
- C. V. Harding and W. H. Boom, “Regulation of antigen presentation by Mycobacterium tuberculosis: a role for Toll-like receptors,” Nature Reviews Microbiology, vol. 8, no. 4, pp. 296–307, 2010.
- S. H. El-Etr and J. D. Cirillo, “Entry mechanisms of mycobacteria,” Front Biosci, vol. 6, pp. D737–747, 2001.
- C.-S. Yang, J.-S. Lee, C.-H. Song et al., “Protein kinase C zeta plays an essential role for Mycobacterium tuberculosis-induced extracellular signal-regulated kinase 1/2 activation in monocytes/macrophages via Toll-like receptor 2,” Cellular Microbiology, vol. 9, no. 2, pp. 382–396, 2007.
- T. R. Hawn, E. A. Misch, S. J. Dunstan et al., “A common human TLR1 polymorphism regulates the innate immune response to lipopeptides,” European Journal of Immunology, vol. 37, no. 8, pp. 2280–2289, 2007.
- E. H. Noss, R. K. Pai, T. J. Sellati et al., “Toll-like receptor 2-dependent inhibition of macrophage class II MHC expression and antigen processing by 19-kDa lipoprotein of Mycobacterium tuberculosis,” The Journal of Immunology, vol. 167, no. 2, pp. 910–918, 2001.
- S.-B. Jung, C.-S. Yang, J.-S. Lee et al., “The mycobacterial 38-kilodalton glycolipoprotein antigen activates the mitogen-activated protein kinase pathway and release of proinflammatory cytokines through Toll-like receptors 2 and 4 in human monocytes,” Infection and Immunity, vol. 74, no. 5, pp. 2686–2696, 2006.
- E.-K. Jo, C.-S. Yang, C. H. Choi, and C. V. Harding, “Intracellular signalling cascades regulating innate immune responses to mycobacteria: branching out from Toll-like receptors,” Cellular Microbiology, vol. 9, no. 5, pp. 1087–1098, 2007.
- A. J. Gehring, K. M. Dobos, J. T. Belisle, C. V. Harding, and W. H. Boom, “Mycobacterium tuberculosis LprG (Rv1411c): a novel TLR-2 ligand that inhibits human macrophage class II MHC antigen processing,” The Journal of Immunology, vol. 173, no. 4, pp. 2660–2668, 2004.
- N. D. Pecora, A. J. Gehring, D. H. Canaday, W. H. Boom, and C. V. Harding, “Mycobacterium tuberculosis LprA is a lipoprotein agonist of TLR2 that regulates innate immunity and APC function,” The Journal of Immunology, vol. 177, no. 1, pp. 422–429, 2006.
- S. Chitale, S. Ehrt, I. Kawamura et al., “Recombinant Mycobacterium tuberculosis protein associated with mammalian cell entry,” Cellular Microbiology, vol. 3, no. 4, pp. 247–254, 2001.
- S. Ahmad, S. El-Shazly, A. S. Mustafa, and R. Al-Attiyah, “The six mammalian cell entry proteins (Mce3A-F) encoded by the mce3 operon are expressed during in vitro growth of Mycobacterium tuberculosis,” Scandinavian Journal of Immunology, vol. 62, no. 1, pp. 16–24, 2005.
- S. El-Shazly, S. Ahmad, A. S. Mustafa, R. Al-Attiyah, and D. Krajci, “Internalization by HeLa cells of latex beads coated with mammalian cell entry (Mce) proteins encoded by the mce3 operon of Mycobacterium tuberculosis,” Journal of Medical Microbiology, vol. 56, no. 9, pp. 1145–1151, 2007.
- M. Yamamoto, S. Sato, H. Hemmi et al., “Role of adaptor TRIF in the MyD88-independent toll-like receptor signaling pathway,” Science, vol. 301, no. 5633, pp. 640–643, 2003.
- Y. Xu, C. Jagannath, X.-D. Liu, A. Sharafkhaneh, K. E. Kolodziejska, and N. T. Eissa, “Toll-like receptor 4 Is a sensor for autophagy associated with innate immunity,” Immunity, vol. 27, no. 1, pp. 135–144, 2007.
- C. V. Rothlin, S. Ghosh, E. I. Zuniga, M. B. A. Oldstone, and G. Lemke, “TAM receptors are pleiotropic inhibitors of the innate immune Response,” Cell, vol. 131, no. 6, pp. 1124–1136, 2007.
- S. A. Fulton, S. M. Reba, R. K. Pai et al., “Inhibition of major histocompatibility complex II expression and antigen processing in murine alveolar macrophages by mycobacterium bovis BCG and the 19-kilodalton mycobacterial lipoprotein,” Infection and Immunity, vol. 72, no. 4, pp. 2101–2110, 2004.
- R. K. Pai, M. E. Pennini, A. A. R. Tobian, D. H. Canaday, W. H. Boom, and C. V. Harding, “Prolonged toll-like receptor signaling by Mycobacterium tuberculosis and its 19-kilodalton lipoprotein inhibits gamma interferon-induced regulation of selected genes in macrophages,” Infection and Immunity, vol. 72, no. 11, pp. 6603–6614, 2004.
- J. Nigou, C. Zelle-Rieser, M. Gilleron, M. Thurnher, and G. Puzo, “Mannosylated lipoarabinomannans inhibit IL-12 production by human dendritic cells: evidence for a negative signal delivered through the mannose receptor,” The Journal of Immunology, vol. 166, no. 12, pp. 7477–7485, 2001.
- P. B. Kang, A. K. Azad, J. B. Torrelles et al., “The human macrophage mannose receptor directs Mycobacterium tuberculosis lipoarabinomannan-mediated phagosome biogenesis,” Journal of Experimental Medicine, vol. 202, no. 7, pp. 987–999, 2005.
- M. Chen, H. Gan, and H. G. Remold, “A mechanism of virulence: virulent Mycobacterium tuberculosis strain H37Rv, but not attenuated H37Ra, causes significant mitochondrial inner membrane disruption in macrophages leading to necrosis,” The Journal of Immunology, vol. 176, no. 6, pp. 3707–3716, 2006.
- K. A. Bodnar, N. V. Serbina, and J. L. Flynn, “Fate of Mycobacterium tuberculosis within murine dendritic cells,” Infection and Immunity, vol. 69, no. 2, pp. 800–809, 2001.
- M. Gonzalez-Juarrero, O. C. Turner, J. Turner, P. Marietta, J. V. Brooks, and I. M. Orme, “Temporal and spatial arrangement of lymphocytes within lung granulomas induced by aerosol infection with Mycobacterium tuberculosis,” Infection and Immunity, vol. 69, no. 3, pp. 1722–1728, 2001.
- A. J. Wolf, L. Desvignes, B. Linas et al., “Initiation of the adaptive immune response to Mycobacterium tuberculosis depends on antigen production in the local lymph node, not the lungs,” Journal of Experimental Medicine, vol. 205, no. 1, pp. 105–115, 2008.
- J. L. Flynn and J. Chan, “Immune evasion by Mycobacterium tuberculosis: living with the enemy,” Current Opinion in Immunology, vol. 15, no. 4, pp. 450–455, 2003.
- J. L. Flynn and J. Chan, “What's good for the host is good for the bug,” Trends in Microbiology, vol. 13, no. 3, pp. 98–102, 2005.
- D. B. Young, H. P. Gideon, and R. J. Wilkinson, “Eliminating latent tuberculosis,” Trends in Microbiology, vol. 17, no. 5, pp. 183–188, 2009.
- P. L. Lin, M. Rodgers, L. Smith et al., “Quantitative comparison of active and latent tuberculosis in the cynomolgus macaque model,” Infection and Immunity, vol. 77, no. 10, pp. 4631–4642, 2009.
- C. E. Barry III, H. I. Boshoff, V. Dartois et al., “The spectrum of latent tuberculosis: rethinking the biology and intervention strategies,” Nature Reviews Microbiology, vol. 7, no. 12, pp. 845–855, 2009.
- L. E. Via, P. L. Lin, S. M. Ray et al., “Tuberculous granulomas are hypoxic in guinea pigs, rabbits, and nonhuman primates,” Infection and Immunity, vol. 76, no. 6, pp. 2333–2340, 2008.
- T. Ulrichs and S. H. E. Kaufmann, “New insights into the function of granulomas in human tuberculosis,” Journal of Pathology, vol. 208, no. 2, pp. 261–269, 2006.
- A. Kumar, J. S. Deshane, D. K. Crossman et al., “Heme oxygenase-1-derived carbon monoxide induces the Mycobacterium tuberculosis dormancy regulon,” The Journal of Biological Chemistry, vol. 283, no. 26, pp. 18032–18039, 2008.
- T. R. Rustad, A. M. Sherrid, K. J. Minch, and D. R. Sherman, “Hypoxia: a window into Mycobacterium tuberculosis latency,” Cellular Microbiology, vol. 11, no. 8, pp. 1151–1159, 2009.
- M. U. Shiloh and P. A. DiGiuseppe Champion, “To catch a killer. What can mycobacterial models teach us about Mycobacterium tuberculosis pathogenesis?” Current Opinion in Microbiology, vol. 13, no. 1, pp. 86–92, 2010.
- J. Ghosh, P. Larsson, B. Singh et al., “Sporulation in mycobacteria,” Proceedings of the National Academy of Sciences of the United States of America, vol. 106, no. 26, pp. 10781–10786, 2009.
- A. M. Anuchin, A. L. Mulyukin, N. E. Suzina, V. I. Duda, G. I. El-Registan, and A. S. Kaprelyants, “Dormant forms of Mycobacterium smegmatis with distinct morphology,” Microbiology, vol. 155, no. 4, pp. 1071–1079, 2009.
- J. Chan and J. Flynn, “The immunological aspects of latency in tuberculosis,” Clinical Immunology, vol. 110, no. 1, pp. 2–12, 2004.
- S. Beetz, D. Wesch, L. Marischen, S. Welte, H.-H. Oberg, and D. Kabelitz, “Innate immune functions of human γδ T cells,” Immunobiology, vol. 213, no. 3-4, pp. 173–182, 2008.
- A. M. Caruso, N. Serbina, E. Klein, K. Triebold, B. R. Bloom, and J. L. Flynn, “Mice deficient in CD4 T cells have only transiently diminished levels of IFN-γ, yet succumb to tuberculosis,” The Journal of Immunology, vol. 162, no. 9, pp. 5407–5416, 1999.
- A. M. Cooper, “Cell-mediated immune responses in tuberculosis,” Annual Review of Immunology, vol. 27, pp. 393–422, 2009.
- C. A. Scanga, V. P. Mohan, K. Yu et al., “Depletion of T cells causes reactivation of murine persistent tuberculosis despite continued expression of interferon γ and nitric oxide synthase 2,” Journal of Experimental Medicine, vol. 192, no. 3, pp. 347–358, 2000.
- M. Cella, D. Scheidegger, K. Palmer-Lehmann, P. Lane, A. Lanzavecchia, and G. Alber, “Ligation of CD40 on dendritic cells triggers production of high levels of interleukin-12 and enhances T cell stimulatory capacity: T-T help via APC activation,” Journal of Experimental Medicine, vol. 184, no. 2, pp. 747–752, 1996.
- M. Oddo, T. Renno, A. Attinger, T. Bakker, H. R. MacDonald, and P. R. A. Meylan, “Fas ligand-induced apoptosis of infected human macrophages reduces the viability of intracellular Mycobacterium tuberculosis,” The Journal of Immunology, vol. 160, no. 11, pp. 5448–5454, 1998.
- N. V. Serbina, V. Lazarevic, and J. L. Flynn, “ T cells are required for the development of cytotoxic T cells during Mycobacterium tuberculosis infection,” The Journal of Immunology, vol. 167, no. 12, pp. 6991–7000, 2001.
- S. C. Cowley and K. L. Elkins, “ T Cells Mediate IFN-γ-Independent Control of Mycobacterium tuberculosis Infection Both In Vitro and In Vivo,” The Journal of Immunology, vol. 171, no. 9, pp. 4689–4699, 2003.
- J. E. Grotzke and D. M. Lewinsohn, “Role of T lymphocytes in control of Mycobacterium tuberculosis infection,” Microbes and Infection, vol. 7, no. 4, pp. 776–788, 2005.
- L. A. H. Van Pinxteren, J. P. Cassidy, B. H. C. Smedegaard, E. M. Agger, and P. Andersen, “Control of latent Mycobacterium tuberculosis infection is dependent on CD8 T cells,” European Journal of Immunology, vol. 30, no. 12, pp. 3689–3698, 2000.
- S. H. E. Kaufmann, “New issues in tuberculosis,” Annals of the Rheumatic Diseases, vol. 63, no. 2, pp. ii50–ii56, 2004.
- D. C. Barral and M. B. Brenner, “CD1 antigen presentation: how it works,” Nature Reviews Immunology, vol. 7, no. 12, pp. 929–941, 2007.
- P. Guermonprez, L. Saveanu, M. Kleijmeer, J. Davoust, P. Van Endert, and S. Amigorena, “ER-phagosome fusion defines an MHC class I cross-presentation compartment in dendritic cells,” Nature, vol. 425, no. 6956, pp. 397–402, 2003.
- M. Houde, S. Bertholet, E. Gagnon et al., “Phagosomes are competent organelles for antigen cross-presentation,” Nature, vol. 425, no. 6956, pp. 402–406, 2003.
- D. B. Moody, D. C. Young, T.-Y. Cheng et al., “T cell activation by lipopeptide antigens,” Science, vol. 303, no. 5657, pp. 527–531, 2004.
- F. Winau, S. Weber, S. Sad et al., “Apoptotic vesicles crossprime CD8 T cells and protect against tuberculosis,” Immunity, vol. 24, no. 1, pp. 105–117, 2006.
- U. E. Schaible, F. Winau, P. A. Sieling et al., “Apoptosis facilitates antigen presentation to T lymphocytes through MHC-I and CD1 in tuberculosis,” Nature Medicine, vol. 9, no. 8, pp. 1039–1046, 2003.
- J. L. Flynn, J. Chan, K. J. Triebold, D. K. Dalton, T. A. Stewart, and B. R. Bloom, “An essential role for interferon γ in resistance to Mycobacterium tuberculosis infection,” Journal of Experimental Medicine, vol. 178, no. 6, pp. 2249–2254, 1993.
- M. S. Russell, R. Dudani, L. Krishnan, and S. Sad, “IFN- expressed by T cells regulates the persistence of antigen presentation by limiting the survival of dendritic cells,” The Journal of Immunology, vol. 183, no. 12, pp. 7710–7718, 2009.
- C. A. Scanga, V. P. Mohan, K. Tanaka, D. Alland, J. L. Flynn, and J. Chan, “The inducible nitric oxide synthase locus confers protection against aerogenic challenge of both clinical and laboratory strains of Mycobacterium tuberculosis in mice,” Infection and Immunity, vol. 69, no. 12, pp. 7711–7717, 2001.
- A. J. Gehring, R. E. Rojas, D. H. Canaday, D. L. Lakey, C. V. Harding, and W. H. Boom, “The Mycobacterium tuberculosis 19-kilodalton lipoprotein inhibits gamma interferon-regulated HLA-DR and FcγR1 on human macrophages through toll-like receptor 2,” Infection and Immunity, vol. 71, no. 8, pp. 4487–4497, 2003.
- R. K. Pai, M. Convery, T. A. Hamilton, W. Henry Boom, and C. V. Harding, “Inhibition of IFN-γ-induced class II transactivator expression by a 19-kDa lipoprotein from Mycobacterium tuberculosis: a potential mechanism for immune evasion,” The Journal of Immunology, vol. 171, no. 1, pp. 175–184, 2003.
- M. E. Pennini, R. K. Pai, D. C. Schultz, W. H. Boom, and C. V. Harding, “Mycobacterium tuberculosis 19-kDa lipoprotein inhibits IFN-γ-induced chromatin remodeling of MHC2TA by TLR2 and MAPK signaling,” The Journal of Immunology, vol. 176, no. 7, pp. 4323–4330, 2006.
- M. Torres, L. Ramachandra, R. E. Rojas et al., “Role of phagosomes and major histocompatibility complex class II (MHC-II) compartment in MHC-II antigen processing of Mycobacterium tuberculosis in human macrophages,” Infection and Immunity, vol. 74, no. 3, pp. 1621–1630, 2006.
- A. A. R. Tobian, N. S. Potter, L. Ramachandra et al., “Alternate class I MHC antigen processing is inhibited by Toll-like receptor signaling pathogen-associated molecular patterns: Mycobacterium tuberculosis 19-kDa lipoprotein, CpG DNA, and lipopolysaccharide,” The Journal of Immunology, vol. 171, no. 3, pp. 1413–1422, 2003.
- R. A. Fratti, J. Chua, and V. Deretic, “Induction of p38 mitogen-activated protein Kinase reduces early endosome autoantigen 1 (EEA1) recruitment to phagosomal membranes,” The Journal of Biological Chemistry, vol. 278, no. 47, pp. 46961–46967, 2003.
- V. Briken, S. A. Porcelli, G. S. Besra, and L. Kremer, “Mycobacterial lipoarabinomannan and related lipoglycans: from biogenesis to modulation of the immune response,” Molecular Microbiology, vol. 53, no. 2, pp. 391–403, 2004.
- M. Rojas, L. F. Garcia, J. Nigou, G. Puzo, and M. Olivier, “Mannosylated lipoarabinomannan antagonizes Mycobacterium tuberculosis- induced macrophage apoptosis by altering CA+2-dependent cell signaling,” Journal of Infectious Diseases, vol. 182, no. 1, pp. 240–251, 2000.
- D. M. E. Bowdish, K. Sakamoto, M.-J. Kim et al., “MARCO, TLR2, and CD14 are required for macrophage cytokine responses to mycobacterial trehalose dimycolate and Mycobacterium tuberculosis,” PLoS Pathogens, vol. 5, no. 6, article e1000474, pp. 1–14, 2009.
- S. Axelrod, H. Oschkinat, J. Enders et al., “Delay of phagosome maturation by a mycobacterial lipid is reversed by nitric oxide,” Cellular Microbiology, vol. 10, no. 7, pp. 1530–1545, 2008.
- C. Kan-Sutton, C. Jagannath, and R. L. Hunter Jr., “Trehalose 6,6′-dimycolate on the surface of Mycobacterium tuberculosis modulates surface marker expression for antigen presentation and costimulation in murine macrophages,” Microbes and Infection, vol. 11, no. 1, pp. 40–48, 2009.
- L.-Y. Gao, S. Guo, B. McLaughlin, H. Morisaki, J. N. Engel, and E. J. Brown, “A mycobacterial virulence gene cluster extending RD1 is required for cytolysis, bacterial spreading and ESAT-6 secretion,” Molecular Microbiology, vol. 53, no. 6, pp. 1677–1693, 2004.
- M. I. de Jonge, G. Pehau-Arnaudet, M. M. Fretz et al., “ESAT-6 from Mycobacterium tuberculosis dissociates from its putative chaperone CFP-10 under acidic conditions and exhibits membrane-lysing activity,” Journal of Bacteriology, vol. 189, no. 16, pp. 6028–6034, 2007.
- N. van der Wel, D. Hava, D. Houben et al., “M. tuberculosis and M. leprae translocate from the phagolysosome to the cytosol in myeloid cells,” Cell, vol. 129, no. 7, pp. 1287–1298, 2007.
- M. Sani, E. N.G. Houben, J. Geurtsen et al., “Direct visualization by Cryo-EM of the mycobacterial capsular layer: a labile structure containing ESX-1-secreted proteins,” PLoS Pathogens, vol. 6, no. 3, article e1000794, pp. 1–10, 2010.
- R. Jayachandran, V. Sundaramurthy, B. Combaluzier et al., “Survival of mycobacteria in macrophages is mediated by coronin 1-dependent activation of calcineurin,” Cell, vol. 130, no. 1, pp. 37–50, 2007.
- A. Walburger, A. Koul, G. Ferrari et al., “Protein kinase G from pathogenic mycobacteria promotes survival within macrophages,” Science, vol. 304, no. 5678, pp. 1800–1804, 2004.
- I. Vergne, J. Chua, H.-H. Lee, M. Lucas, J. Belisle, and V. Deretic, “Mechanism of phagolysosome biogenesis block by viable Mycobacterium tuberculosis,” Proceedings of the National Academy of Sciences of the United States of America, vol. 102, no. 11, pp. 4033–4038, 2005.
- J. Hinchey, S. Lee, B. Y. Jeon et al., “Enhanced priming of adaptive immunity by a proapoptotic mutant of Mycobacterium tuberculosis,” The Journal of Clinical Investigation, vol. 117, no. 8, pp. 2279–2288, 2007.
- K. Velmurugan, B. Chen, J. L. Miller et al., “Mycobacterium tuberculosis nuoG is a virulence gene that inhibits apoptosis of infected host cells,” PLoS Pathogens, vol. 3, no. 7, article e110, 2007.
- A. G. D. Bean, D. R. Roach, H. Briscoe et al., “Structural deficiencies in granuloma formation in TNF gene-targeted mice underlie the heightened susceptibility to aerosol Mycobacterium tuberculosis infection, which is not compensated for by lymphotoxin,” The Journal of Immunology, vol. 162, no. 6, pp. 3504–3511, 1999.
- J. Keane, “TNF-blocking agents and tuberculosis: new drugs illuminate an old topic,” Rheumatology, vol. 44, no. 6, pp. 714–720, 2005.
- H. M. S. Algood, J. Chan, and J. L. Flynn, “Chemokines and tuberculosis,” Cytokine and Growth Factor Reviews, vol. 14, no. 6, pp. 467–477, 2003.
- B. M. Saunders, S. Tran, S. Ruuls, J. D. Sedgwick, H. Britton, and B. J. Warwick, “Transmembrane TNF is sufficient to initiate cell migration and granuloma formation and provide acute, but not long-term, control of Mycobacterium tuberculosis infection,” The Journal of Immunology, vol. 174, no. 8, pp. 4852–4859, 2005.
- M. B. Reed, P. Domenech, C. Manca et al., “A glycolipid of hypervirulent tuberculosis strains that inhibits the innate immune response,” Nature, vol. 430, no. 7004, pp. 84–87, 2004.
- E. Jouanguy, R. Doffinger, S. Dupuis, A. Pallier, F. Altare, and J.-L. Casanova, “IL-12 and IFN- in host defense against mycobacteria and salmonella in mice and men,” Current Opinion in Immunology, vol. 11, no. 3, pp. 346–351, 1999.
- E. G. R. Lichtenauer-Kaligis, T. de Boer, F. A. W. Verreck et al., “Severe Mycobacterium bovis BCG infections in a large series of novel IL-12 receptor β1 deficient patients and evidence for the existence of partial IL-12 receptor β1 deficiency,” European Journal of Immunology, vol. 33, no. 1, pp. 59–69, 2003.
- A. Baena and S. A. Porcelli, “Evasion and subversion of antigen presentation by Mycobacterium tuberculosis,” Tissue Antigens, vol. 74, no. 3, pp. 189–204, 2009.
- S. T. Chang, J. J. Linderman, and D. E. Kirschner, “Multiple mechanisms allow Mycobacterium tuberculosis to continuously inhibit MHC class II-mediated antigen presentation by macrophages,” Proceedings of the National Academy of Sciences of the United States of America, vol. 102, no. 12, pp. 4530–4535, 2005.
- J. Chan, Y. Xing, R. S. Magliozzo, and B. R. Bloom, “Killing of virulent Mycobacterium tuberculosis by reactive nitrogen intermediates produced by activated murine macrophages,” Journal of Experimental Medicine, vol. 175, no. 4, pp. 1111–1122, 1992.
- H.-S. Choi, P. R. Rai, H. W. Chu, C. Cool, and E. D. Chan, “Analysis of nitric oxide synthase and nitrotyrosine expression in human pulmonary tuberculosis,” American Journal of Respiratory and Critical Care Medicine, vol. 166, no. 2, pp. 178–186, 2002.
- B. H. Miller, R. A. Fratti, J. F. Poschet et al., “Mycobacteria inhibit nitric oxide synthase recruitment to phagosomes during macrophage infection,” Infection and Immunity, vol. 72, no. 5, pp. 2872–2878, 2004.
- R. Bryk, C. D. Lima, H. Erdjument-Bromage, P. Tempst, and C. Nathan, “Metabolic enzymes of mycobacteria linked to antioxidant defense by a thioredoxin-like protein,” Science, vol. 295, no. 5557, pp. 1073–1077, 2002.
- H. Ouellet, Y. Ouellet, C. Richard et al., “Truncated hemoglobin HbN protects Mycobacterium bovis from nitric oxide,” Proceedings of the National Academy of Sciences of the United States of America, vol. 99, no. 9, pp. 5902–5907, 2002.
- H. Ohno, G. Zhu, V. P. Mohan et al., “The effects of reactive nitrogen intermediates on gene expression in Mycobacterium tuberculosis,” Cellular Microbiology, vol. 5, no. 9, pp. 637–648, 2003.
- M. I. Voskuil, D. Schnappinger, K. C. Visconti et al., “Inhibition of respiration by nitric oxide induces a Mycobacterium tuberculosis dormancy program,” Journal of Experimental Medicine, vol. 198, no. 5, pp. 705–713, 2003.
- O. V. Vieira, R. J. Botelho, and S. Grinstein, “Phagosome maturation: aging gracefully,” Biochemical Journal, vol. 366, no. 3, pp. 689–704, 2002.
- K. Rhode, R. M. Yates, G. E. Purdy, and D. G. Russell, “Mycobactrium tuberculosis and the environment within the phagosome,” Immunological Reviews, vol. 219, pp. 37–54, 2007.
- S. Sturgill-Koszycki, P. H. Schlesinger, P. Chakraborty et al., “Lack of acidification in Mycobacterium phagosomes produced by exclusion vesicular proton-ATPase,” Science, vol. 263, no. 5147, pp. 678–681, 1994.
- R. M. Yates, A. Hermetter, and D. G. Russell, “The kinetics of phagosome maturation as a function of phagosome/lysosome fusion and acquisition of hydrolytic activity,” Traffic, vol. 6, no. 5, pp. 413–420, 2005.
- D. L. Clemens and M. A. Horwitz, “Characterization of the Mycobacterium tuberculosis phagosome and evidence that phagosomal maturation is inhibited,” Journal of Experimental Medicine, vol. 181, no. 1, pp. 257–270, 1995.
- J. Chua and V. Deretic, “Mycobacterium tuberculosis reprograms waves of phosphatidylinositol 3-phosphate on phagosomal organelles,” The Journal of Biological Chemistry, vol. 279, no. 35, pp. 36982–36992, 2004.
- D. J. Kusner, “Mechanisms of mycobacterial persistence in tuberculosis,” Clinical Immunology, vol. 114, no. 3, pp. 239–247, 2005.
- Z. A. Malik, C. R. Thompson, S. Hashimi, B. Porter, S. S. Iyer, and D. J. Kusner, “Cutting edge: Mycobacterium tuberculosis blocks signaling and phagosome maturation in human macrophages via specific inhibition of sphingosine kinase,” The Journal of Immunology, vol. 170, no. 6, pp. 2811–2815, 2003.
- G. B. Kyei, I. Vergne, J. Chua et al., “Rab14 is critical for maintenance of Mycobacterium tuberculosis phagosome maturation arrest,” The EMBO Journal, vol. 25, no. 22, pp. 5250–5259, 2006.
- V. Deretic, I. Vergne, J. Chua et al., “Endosomal membrane traffic: convergence point targeted by Mycobacterium tuberculosis and HIV,” Cellular Microbiology, vol. 6, no. 11, pp. 999–1009, 2004.
- S. Alonso, K. Pethe, D. G. Russell, and G. E. Purdy, “Lysosomal killing of Mycobacterium mediated by ubiquitin-derived peptides is enhanced by autophagy,” Proceedings of the National Academy of Sciences of the United States of America, vol. 104, no. 14, pp. 6031–6036, 2007.
- G. E. Purdy, M. Niederweis, and D. G. Russell, “Decreased outer membrane permeability protects mycobacteria from killing by ubiquitin-derived peptides,” Molecular Microbiology, vol. 73, no. 5, pp. 844–857, 2009.
- L. Danelishvili, Y. Yamazaki, J. Selker, and L. E. Bermudez, “Secreted Mycobacterium tuberculosis Rv3654c and Rv3655c proteins participate in the suppression of macrophage apoptosis,” PloS ONE, vol. 5, no. 5, article e10474, 2010.
- H. Gan, J. Lee, F. Ren, M. Chen, H. Kornfeld, and H. G. Remold, “Mycobacterium tuberculosis blocks crosslinking of annexin-1 and apoptotic envelope formation on infected macrophages to maintain virulence,” Nature Immunology, vol. 9, no. 10, pp. 1189–1197, 2008.
- G. G. Mahairas, P. J. Sabo, M. J. Hickey, D. C. Singh, and C. K. Stover, “Molecular analysis of genetic differences between Mycobacterium bovis BCG and virulent M. bovis,” Journal of Bacteriology, vol. 178, no. 5, pp. 1274–1282, 1996.
- M. A. Behr, M. A. Wilson, W. P. Gill et al., “Comparative genomics of BCG vaccines by whole-genome DNA microarray,” Science, vol. 284, no. 5419, pp. 1520–1523, 1999.
- M. Harboe, T. Oettinger, H. G. Wiker, I. Rosenkrands, and P. Andersen, “Evidence for occurrence of the ESAT-6 protein in Mycobacterium tuberculosis and virulent Mycobacterium bovis and for its absence in Mycobacterium bovis BCG,” Infection and Immunity, vol. 64, no. 1, pp. 16–22, 1996.
- F.-X. Berthet, P. B. Rasmussen, I. Rosenkrands, P. Andersen, and B. Gicquel, “A Mycobacterium tuberculosis operon encoding ESAT-6 and a novel low-molecular-mass culture filtrate protein (CFP-10),” Microbiology, vol. 144, no. 11, pp. 3195–3203, 1998.
- R. L. V. Skjøt, T. Oettinger, I. Rosenkrands et al., “Comparative evaluation of low-molecular-mass proteins from Mycobacterium tuberculosis identifies members of the ESAT-6 family as immunodominant T-cell antigens,” Infection and Immunity, vol. 68, no. 1, pp. 214–220, 2000.
- A. S. Mustafa, “Development of new vaccines and diagnostic reagents against tuberculosis,” Molecular Immunology, vol. 39, no. 1-2, pp. 113–119, 2002.
- K. M. Guinn, M. J. Hickey, S. K. Mathur et al., “Individual RD1—region genes are required for export of ESAT-6/CFP-10 and for virulence of Mycobacterium tuberculosis,” Molecular Microbiology, vol. 51, no. 2, pp. 359–370, 2004.
- K. N. Lewis, R. Liao, K. M. Guinn et al., “Deletion of RD1 from Mycobacterium tuberculosis mimics bacille Calmette-Guérin attenuation,” Journal of Infectious Diseases, vol. 187, no. 1, pp. 117–123, 2003.
- A. S. Pym, P. Brodin, L. Majlessi et al., “Recombinant BCG exporting ESAT-6 confers enhanced protection against tuberculosis,” Nature Medicine, vol. 9, no. 5, pp. 533–539, 2003.
- P. S. Renshaw, P. Panagiotidou, A. Whelan et al., “Conclusive evidence that the major T-cell antigens of the Mycobacterium tuberculosis complex ESAT-6 and CFP-10 form a tight, 1:1 complex and characterization of the structural properties of ESAT-6, CFP-10, and the ESAT-6·CFP-10 complex. Implications for pathogenesis and virulence,” The Journal of Biological Chemistry, vol. 277, no. 24, pp. 21598–21603, 2002.
- T. Hsu, S. M. Hingley-Wilson, B. Chen et al., “The primary mechanism of attenuation of bacillus Calmette-Guérin is a loss of secreted lytic function required for invasion of lung interstitial tissue,” Proceedings of the National Academy of Sciences of the United States of America, vol. 100, no. 21, pp. 12420–12425, 2003.
- P. Brodin, I. Rosenkrands, P. Andersen, S. T. Cole, and R. Brosch, “ESAT-6 proteins: protective antigens and virulence factors?” Trends in Microbiology, vol. 12, no. 11, pp. 500–508, 2004.
- S. C. Derrick and S. L. Morris, “The ESAT6 protein of Mycobacterium tuberculosis induces apoptosis of macrophages by activating caspase expression,” Cellular Microbiology, vol. 9, no. 6, pp. 1547–1555, 2007.
- J. Smith, J. Manoranjan, M. Pan et al., “Evidence for pore formation in host cell membranes by ESX-1-secreted ESAT-6 and its role in Mycobacterium marinum escape from the vacuole,” Infection and Immunity, vol. 76, no. 12, pp. 5478–5487, 2008.
- H. E. Volkman, T. C. Pozos, J. Zheng, J. M. Davis, J. F. Rawls, and L. Ramakrishnan, “Tuberculous granuloma induction via interaction of a bacterial secreted protein with host epithelium,” Science, vol. 327, no. 5964, pp. 466–469, 2010.
- C. Raynaud, C. Guilhot, J. Rauzier et al., “Phospholipases C are involved in the virulence of Mycobacterium tuberculosis,” Molecular Microbiology, vol. 45, no. 1, pp. 203–217, 2002.
- P. Peirs, P. Lefèvre, S. Boarbi et al., “Mycobacterium tuberculosis with disruption in genes encoding the phosphate binding proteins PstS1 and PstS2 is deficient in phosphate uptake and demonstrates reduced in vivo virulence,” Infection and Immunity, vol. 73, no. 3, pp. 1898–1902, 2005.
- K. Y. Dunphy, R. H. Senaratne, M. Masuzawa, L. V. Kendall, and L. W. Riley, “Attenuation of Mycobacterium tuberculosis functionally disrupted in a fatty acyl-coenzyme a synthetase gene fadD5,” Journal of Infectious Diseases, vol. 201, no. 8, pp. 1232–1239, 2010.
- E. L. Corbett, C. J. Watt, N. Walker et al., “The growing burden of tuberculosis: global trends and interactions with the HIV epidemic,” Archives of Internal Medicine, vol. 163, no. 9, pp. 1009–1021, 2003.
- K. E. Dooley and R. E. Chaisson, “Tuberculosis and diabetes mellitus: convergence of two epidemics,” The Lancet Infectious Diseases, vol. 9, no. 12, pp. 737–746, 2009.
- E. C. Hett, M. C. Chao, A. J. Steyn, S. M. Fortune, L. L. Deng, and E. J. Rubin, “A partner for the resuscitation-promoting factors of Mycobacterium tuberculosis,” Molecular Microbiology, vol. 66, no. 3, pp. 658–668, 2007.
- B. D. Kana, B. G. Gordhan, K. J. Downing et al., “The resuscitation-promoting factors of Mycobacterium tuberculosis are required for virulence and resuscitation from dormancy but are collectively dispensable for growth in vitro,” Molecular Microbiology, vol. 67, no. 3, pp. 672–684, 2008.
- E. Russell-Goldman, J. Xu, X. Wang, J. Chan, and J. M. Tufariello, “A Mycobacterium tuberculosis Rpf double-knockout strain exhibits profound defects in reactivation from chronic tuberculosis and innate immunity phenotypes,” Infection and Immunity, vol. 76, no. 9, pp. 4269–4281, 2008.
- P.-J. Cardona, “A dynamic reinfection hypothesis of latent tuberculosis infection,” Infection, vol. 37, no. 2, pp. 80–86, 2009.
- P.-J. Cardona, “New insights on the nature of latent tuberculosis infection and its treatment,” Inflammation and Allergy—Drug Targets, vol. 6, no. 1, pp. 27–39, 2007.
- D. Martínez, M. Vermeulen, E. Von Euw et al., “Extracellular acidosis triggers the maturation of human dendritic cells and the production of IL-12,” The Journal of Immunology, vol. 179, no. 3, pp. 1950–1959, 2007.
- P. Peyron, J. Vaubourgeix, Y. Poquet et al., “Foamy macrophages from tuberculous patients granulomas constitute a nutrient-rich reservoir for M. tuberculosis persistence,” PLoS Pathogens, vol. 4, no. 11, article e1000204, pp. 1–14, 2008.
- N. Cáceres, G. Tapia, I. Ojanguren et al., “Evolution of foamy macrophages in the pulmonary granulomas of experimental tuberculosis models,” Tuberculosis, vol. 89, no. 2, pp. 175–182, 2009.
- N. J. Garton, H. Christensen, D. E. Minnikin, R. A. Adegbola, and M. R. Barer, “Intracellular lipophilic inclusions of mycobacteria in vitro and in sputum,” Microbiology, vol. 148, no. 10, pp. 2951–2958, 2002.
- P. J. Cardona, R. Llatjos, S. Gordillo et al., “Evolution of granulomas in lungs of mice infected aerogenically with Mycobacterium tuberculosis,” Scandinavian Journal of Immunology, vol. 52, no. 2, pp. 156–163, 2000.
- M. Lipman and R. Breen, “Immune reconstitution inflammatory syndrome in HIV,” Current Opinion in Infectious Diseases, vol. 19, no. 1, pp. 20–25, 2006.
- G. Meintjes, S. D. Lawn, F. Scano et al., “Tuberculosis-associated immune reconstitution inflammatory syndrome: case definitions for use in resource-limited settings,” The Lancet Infectious Diseases, vol. 8, no. 8, pp. 516–523, 2008.
- H. G. Wiker, T. Mustafa, G. A. Bjune, and M. Harboe, “Evidence for waning of latency in a cohort study of tuberculosis,” BMC Infectious Diseases, vol. 10, article 37, pp. 1–10, 2010.
- D. A. Mitchison, “Basic mechanisms of chemotherapy,” Chest, vol. 76, no. 6, pp. 771–781, 1979.
- I. Brock, M. E. Munk, A. Kok-Jensen, and P. Andersen, “Performance of whole blood IFN-γ test for tuberculosis diagnosis based on PPD or the specific antigens ESAT-6 and CFP-10,” International Journal of Tuberculosis and Lung Disease, vol. 5, no. 5, pp. 462–467, 2001.
- A. Lalvani, A. A. Pathan, H. Durkan et al., “Enhanced contact tracing and spatial tracking of Mycobacterium tuberculosis infection by enumeration of antigen-specific T cells,” The Lancet, vol. 357, no. 9273, pp. 2017–2021, 2001.
- M. Pai, L. W. Riley, and J. M. Colford Jr., “Interferon-γ assays in the immunodiagnosis of tuberculosis: a systematic review,” The Lancet Infectious Diseases, vol. 4, no. 12, pp. 761–766, 2004.
- C. C. Whalen, “Diagnosis of latent tuberculosis infection measure for measure,” Journal of the American Medical Association, vol. 293, no. 22, pp. 2785–2787, 2005.
- P. Nahid, M. Pai, and P. C. Hopewell, “Advances in the diagnosis and treatment of tuberculosis,” Proceedings of the American Thoracic Society, vol. 3, no. 1, pp. 103–110, 2006.
- M. Pai, A. Zwerling, and D. Menzies, “Systematic review: T-cell-based assays for the diagnosis of latent tuberculosis infection: an update,” Annals of Internal Medicine, vol. 149, no. 3, pp. 177–184, 2008.
- S. Fuhrmann, M. Streitz, and F. Kern, “How flow cytometry is changing the study of TB immunology and clinical diagnosis,” Cytometry Part A, vol. 73, no. 11, pp. 1100–1106, 2008.
- S. Ahmad, H. A. Amoudy, J. E. R. Thole, D. B. Young, and A. S. Mustafa, “Identification of a novel protein antigen encoded by a Mycobacterium tuberculosis-specific RD1 region gene,” Scandinavian Journal of Immunology, vol. 49, no. 5, pp. 515–522, 1999.
- S. Ahmad, S. El-Shazly, A. S. Mustafa, and R. Al-Attiyah, “Mammalian cell-entry proteins encoded by the mce3 operon of Mycobacterium tuberculosis are expressed during natural infection in humans,” Scandinavian Journal of Immunology, vol. 60, no. 4, pp. 382–391, 2004.
- S. El-Shazly, A. S. Mustafa, S. Ahmad, and R. Al-Attiyah, “Utility of three mammalian cell entry proteins of Mycobacterium tuberculosis in the serodiagnosis of tuberculosis,” International Journal of Tuberculosis and Lung Disease, vol. 11, no. 6, pp. 676–682, 2007.
- K. K. Singh, Y. Dong, S. A. Patibandla, D. N. McMurray, V. K. Arora, and S. Laal, “Immunogenicity of the Mycobacterium tuberculosis PPE55 (Rv3347c) protein during incipient and clinical tuberculosis,” Infection and Immunity, vol. 73, no. 8, pp. 5004–5014, 2005.
- A. Lalvani, “Diagnosing tuberculosis infection in the 21st century: new tools to tackle an old enemy,” Chest, vol. 131, no. 6, pp. 1898–1906, 2007.
- D. Menzies, M. Pai, and G. Comstock, “Meta-analysis: new tests for the diagnosis of latent tuberculosis infection: areas of uncertainty and recommendations for research,” Annals of Internal Medicine, vol. 146, no. 5, pp. 340–354, 2007.
- M. Pai and R. O'Brien, “New diagnostics for latent and active Tuberculosis: state of the art and future prospects,” Seminars in Respiratory and Critical Care Medicine, vol. 29, no. 5, pp. 560–568, 2008.
- M. Pai, J. Minion, K. Steingart, and A. Ramsay, “New and improved tuberculosis diagnostics: evidence, policy, practice, and impact,” Current Opinion in Pulmonary Medicine, vol. 16, no. 3, pp. 271–284, 2010.
- A. Strassburg, C. Jafari, M. Ernst, W. Lotz, and C. Lange, “Rapid diagnosis of pulmonary TB by BAL enzyme-linked immunospot assay in an immunocompromised host,” European Respiratory Journal, vol. 31, no. 5, pp. 1132–1135, 2008.
- S.-H. Kim, K. Chu, S.-J. Choi et al., “Diagnosis of central nervous system tuberculosis by T-cell-based assays on peripheral blood and cerebrospinal fluid mononuclear cells,” Clinical and Vaccine Immunology, vol. 15, no. 9, pp. 1356–1362, 2008.
- R. N. van Zyl-Smit, A. Zwerling, K. Dheda, and M. Pai, “Within-subject variability of interferon-g assay results for tuberculosis and boosting effect of tuberculin skin testing: a systematic review,” PLoS ONE, vol. 4, no. 12, article e8517, pp. 1–8, 2009.
- R. M. Jasmer, P. Nahid, and P. C. Hopewell, “Latent tuberculosis infection,” The New England Journal of Medicine, vol. 347, no. 23, pp. 1860–1866, 2002.
- Centers for Disease Control and Prevention, “Guidelines for the investigation of contacts with infectious tuberculosis: recommendations from the National Tuberculosis Controllers Association and CDC,” Morbidity and Mortality Weekly Report Recommendation Report, vol. 54, no. RR-15, pp. 1–47, 2005.
- A. T. Abal, S. Ahmad, and E. Mokaddas, “Variations in the occurrence of the S315T mutation within the katG gene in isoniazid-resistant clinical Mycobacterium tuberculosis isolates from Kuwait,” Microbial Drug Resistance, vol. 8, no. 2, pp. 99–105, 2002.
- S. Ahmad, E. Mokaddas, and E. Fares, “Characterization of rpoB mutations in rifampin-resistant clinical Mycobacterium tuberculosis isolates from Kuwait and Dubai,” Diagnostic Microbiology and Infectious Disease, vol. 44, no. 3, pp. 245–252, 2002.
- S. Ahmad, E. Fares, G. F. Araj, T. D. Chugh, and A. S. Mustafa, “Prevalence of S315T mutation within the katG gene in isoniazid-resistant clinical Mycobacterium tuberculosis isolates from Dubai and Beirut,” International Journal of Tuberculosis and Lung Disease, vol. 6, no. 10, pp. 920–926, 2002.
- S. Ahmad and E. Mokaddas, “Contribution of AGC to ACC and other mutations at codon 315 of the katG gene in isoniazid-resistant Mycobacterium tuberculosis isolates from the Middle East,” International Journal of Antimicrobial Agents, vol. 23, no. 5, pp. 473–479, 2004.
- S. Ahmad, A.-A. Jaber, and E. Mokaddas, “Frequency of embB codon 306 mutations in ethambutol-susceptible and—resistant clinical Mycobacterium tuberculosis isolates in Kuwait,” Tuberculosis, vol. 87, no. 2, pp. 123–129, 2007.
- M. Al-Zarouni, N. Dash, M. Al Ali, F. Al-Shehhi, and D. Panigrahi, “Tuberculosis and MDR-TB in the northern Emirates of United Arab emirates: a 5-year study,” Southeast Asian Journal of Tropical Medicine and Public Health, vol. 41, no. 1, pp. 163–168, 2010.