抗原性生物标志物在结核分枝杆菌中的应用

Application of antigenic biomarkers for Mycobacterium tuberculosis 抗原性生物标志物在结核分枝杆菌中的应用 1 first-author †E-mail:rodriguez.elba@inifap.gob.mx †E-mail:rodriguez.elba@inifap.gob.mx rodriguez.elba@inifap.gob.mx 2 1 3 3 Instituto Nacional de Investigaciones Forestales, Agrícolas y Pecuarias (INIFAP), Centro Nacional de Investigación Disciplinaria en Fisiología y Mejoramiento Animal, Km. 1 Carretera a Colón, Ajuchitlán Colón, 76280, Colón, Querétaro, México Instituto Nacional de Investigaciones Forestales, Agrícolas y Pecuarias (INIFAP), Centro Nacional de Investigación Disciplinaria en Fisiología y Mejoramiento Animal, Km. 1 Carretera a Colón, Ajuchitlán Colón, 76280, Colón, Querétaro, México Universidad Mexiquense del Bicentenario, Unidad de Estudios Superiores de Tultitlán, Avenida Ex-Hacienda de Portales s/n, Villa Esmeralda, Tultitlán Estado de México, 54910, Tultitlán, México Universidad Mexiquense del Bicentenario, Unidad de Estudios Superiores de Tultitlán, Avenida Ex-Hacienda de Portales s/n, Villa Esmeralda, Tultitlán Estado de México, 54910, Tultitlán, México Universidad Autónoma de Querétaro, Facultad de Ciencias Naturales, Avenida de las Ciencias s/n, Juriquilla, Delegación Santa Rosa Jáuregui, 76230, Querétaro, México Universidad Autónoma de Querétaro, Facultad de Ciencias Naturales, Avenida de las Ciencias s/n, Juriquilla, Delegación Santa Rosa Jáuregui, 76230, Querétaro, México The study and characterization of biomolecules involved in the interaction between mycobacteria and their hosts are crucial to determine their roles in the invasion process and provide basic knowledge about the biology and pathogenesis of disease. Promising new biomarkers for diagnosis and immunotherapy have emerged recently. Mycobacterium is an ancient pathogen that has developed complex strategies for its persistence in the host and environment, likely based on the complexity of the network of interactions between the molecules involved in infection. Several biomarkers have received recent attention in the process of developing rapid and reliable detection techniques for tuberculosis. Among the most widely investigated antigens are CFP-10 (10-kDa culture filtrate protein), ESAT-6 (6-kDa early secretory antigenic target), Ag85A, Ag85B, CFP-7, and PPE18. Some of these antigens have been proposed as biomarkers to assess the key elements of the response to infection of both the pathogen and host. The design of novel and accurate diagnostic methods is essential for the control of tuberculosis worldwide. Presently, the diagnostic methods are based on the identification of molecules in the humoral response in infected individuals. Therefore, these tests depend on the capacity of the host to develop an immune response, which usually is heterogeneous. In the last 20 years, special attention has been given to the design of multiantigenic diagnostic methods to improve the levels of sensitivity and specificity. In this review, we summarize the state of the art in the study and use of mycobacterium biomolecules with the potential to support novel tuberculosis control strategies. 研究和鉴定分枝杆菌与其宿主间相互作用的生物分子,对于确定分枝杆菌在入侵过程中的作用至关重要,并为相关疾病生物学和发病机制提供基础知识.分枝杆菌是一种古老的病原体,它为自己在宿主和环境中生存发展了复杂的策略,这可能与其参与感染分子之间相互作用网络的复杂性有关.在结核病检测技术的发展过程中,一些生物标记物已经开始受到关注.研究最广泛的抗原有CFP-10、ESAT-6、Ag85A、Ag85B、CFP-7和PPE18.其中一些抗原已被作为生物标记来评估病原体和宿主感染反应.设计新颖而准确的诊断方法对控制全世界的结核病至关重要.目前,诊断方法是基于对感染个体体液反应分子的识别.因此,这些测试依赖于宿主产生免疫反应的能力,而免疫反应通常是异质的.在过去的20年里,人们特别重视多抗原诊断方法的设计,以提高敏感性和特异性水平.本文综述了结核分枝杆菌生物分子的研究和应用现状,以期为结核控制提供新的支持. Mycobacterium tuberculosis Recombinant antigen Diagnostics Biomarker 结核分枝杆菌 重组抗原 诊断 生物标志物 Introduction 1 Tuberculosis (TB) is an infectious disease caused by the bacillus Mycobacterium tuberculosis ( Mtb ), which can cause latent or active disease in humans. In 2018, about 10 million people had the disease, of whom 1.5 million died. Mtb is an obligate aerobic rod-shaped bacterium that contacts host cells and is subjected to the host immune response, resulting in latent TB infection (LTBI) or complete clearance of the bacteria. In LTBI, the Mtb reproduces inside the cellular host, developing a complex relationship that might progress to active TB infection (ATBI). Because TB is one of the top 10 causes of death from an infectious agent, prevention strategies and the development of better vaccines have been pursued (Kaufmann, 2020 2020 ). Also, about 2000 million people worldwide have LTBI that might progress to ATBI at any time (Weiner and Kaufmann, 2014 2014 ). Thus, the development of new treatments and vaccines is urgently needed (Kaufmann, 2020 2020 ). The most effective strategy, implemented by governments to control TB in humans, has been immunization with Bacille Calmette-Guérin (BCG) applied during childhood (Weiner and Kaufmann, 2014 2014 ). This vaccine induces a robust Th1 response and promotes the development of mycobacterial-specific polyfunctional and cytotoxic T cells. Although it confers a variable and incomplete protection against pulmonary TB in humans, it is highly effective in preventing the severe forms of TB in childhood (Fine, 2001 2001 ; Weiner and Kaufmann, 2014 2014 ). The variability in the vaccine’s efficacy is multifactorial, comprising genetic variation among vaccinated individuals, interference of the mycobacterial environment, and the use of different BCG strains (Weiner et al., 2013 2013 ; Weiner and Kaufmann, 2014 2014 ). Therefore, the goal is to develop a more efficient vaccine (Paylor, 2014 2014 ). The Mtb complex (MTC) includes M. tuberculosis , Mycobacterium bovis , Mycobacterium africanum , Mycobacterium pinnipedii , Mycobacterium avium , Mycobacterium canetti , Mycobacterium orygis , Mycobacterium caprae , Mycobacterium suricattlae , Mycobacterium mungi , and Mycobacterium microti , which are genetically related bacteria that cause similar clinical symptoms (Sinha et al., 2016 2016 ). The MTC species are genetically similar in nucleotide sequence, have identical sequences in their l6S ribosomal RNA (rRNA), and share > 95% homology in their genomes (Forrellad et al., 2013 2013 ; Banuls et al., 2015 2015 ). There are differences among these species in their host tropism, phenotype, and pathogenicity (Forrellad et al., 2013 2013 ; Banuls et al., 2015 2015 ). Among the MTC species, Mtb is the most important clinically, due to its relevance as the main causative agent of TB in human beings. Thus, in this review, we focus on Mtb . TB biomarkers are molecules or substances that can be found in the infected body when Mtb is present. They can be products of Mtb itself or of the body in response to infection caused by Mtb . Biomarkers play a crucial role in the detection, treatment, and control of TB. They are useful in diagnostic tests for the detection of ATBI, assisting in early diagnosis and facilitating surveillance during and after treatment. Therefore, the application of biomarkers provides an alternative to current methods that have low sensitivity and specificity. Antigen characterization leads to antibody-or antigen-directed assays that can improve the diagnosis or prognosis of TB. These approaches will improve detection strategies aimed at distinguishing between active and latent diseases and preventing dissemination of contagion, as well as informing decisions on appropriate treatments needed by patients. In this review, we discuss some of the most relevant Mtb antigenic biomarkers. TB diagnosis strategies 1 Diagnosis allows TB control in a simple, quick, and affordable manner (Amanfu, 2006 2006 ). However, about 36% of new TB cases are undiagnosed due to the limitations of current diagnostic methods (WHO, 2019 2019 ). As an initial test for TB diagnosis, acid-fast bacillus (AFB) smear microscopy and culture are used (Monkongdee et al., 2009 2009 ). Mtb isolation by culture is considered the “gold standard” for pulmonary TB and extrapulmonary TB (EPTB) (Bourassa, 2018 2018 ). However, the sensitivity and specificity of these methods are limited, and they are time-consuming (Swaminathan and Rekha, 2010 2010 ). Nonpulmonary TB is the most difficult to diagnose because of the lower mycobacteria concentration in samples (Delogu et al., 2013 2013 ). Culture is the most reliable test for TB diagnosis; however, the results are obtained between three and four weeks after sample collection due to the slow growth of Mtb . Moreover, the sensitivity and specificity of culture tests are limited (Swaminathan and Rekha, 2010 2010 ). Microbiological methods are the most conventional diagnostic tests, but depend on sputum samples, which are difficult to obtain (WHO, 2019 2019 ). The Ziehl-Neelsen stain detects the MTC, but is unreliable in detecting nontuberculous mycobacteria (Aitken et al., 2019 2019 ). Patients with a suspicious diagnosis or previous positive diagnosis with chronic fever and cough for more than two weeks and weight loss are evaluated by chest radiography. When the test results are inconclusive, patients must be monitored with additional tests (Ryu, 2015 2015 ). Because these tests are not quantitative, the monitoring of treatment efficiency is limited (Monkongdee et al., 2009 2009 ; Denkinger et al., 2013 2013 ). Molecular diagnosis tests based on polymerase chain reaction (PCR) using sputum samples have also been used for TB diagnosis. However, mycobacterial DNA is difficult to obtain in samples from asymptomatic patients and the tests have low sensitivity, leading to additional challenges in the diagnosis of EPTB. No “gold standard” test is available for LTBI diagnosis. Currently, LTBI is diagnosed indirectly by detecting specific antigens using the Mantoux tuberculin skin test or tuberculin skin test (TST) that uses a tuberculin-purified protein derivative, or by using the interferon-gamma release assay (IGRA) (Mack et al., 2009 2009 ; Dheda et al., 2016 2016 ). However, false-positive reactions with nontuberculous mycobacteria and BCG-vaccinated individuals are observed with TST (Huebner et al., 1993 1993 ; Seddon et al., 2016 2016 ). Some diagnostic tests do not distinguish between ATBI and LTBI. The TST, Ziehl-Neelsen stain assay, and IGRA are used for ATBI verification, while LTBI is confirmed by chest X-ray (Katial et al., 2001 2001 ). Furthermore, the detection of specific genes, such as guanylate-binding protein 5 ( GBP5 ), dual specificity phosphatase 3 ( DUSP3 ), and Kruppel-like factor 2 ( KLF2 ), can distinguish between ATBI and LTBI, but not EPTB (Sweeney et al., 2016 2016 ). Mycobacterial culture-filtering antigens have been proposed as excellent candidates to diagnose pulmonary TB (Manca et al., 1997 1997 ; Silva et al., 2003 2003 ). Mtb -specific peptide fragments, such as ESAT-6 (6-kDa early secretory antigenic target) and CFP-10 (10-kDa culture filtrate protein), are involved in mycobacterial virulence and are recognized by T cells during human infection (Forrellad et al., 2013 2013 ; Liu et al., 2017 2017 ). For TB diagnosis, specific peptide fragments are quantified from serum samples using antibody-labeled silicon nanoparticles and high-throughput mass spectrometry (NanoDisk-MS) with high specificity (87%–100%) (Liu et al., 2017 2017 ). The Mtb cell wall has a thick envelope comprising lipoarabinomannan (LAM) that modulates the host immune response (Briken et al., 2004 2004 ). Purified LAM material has been used for immunoassays using sputum, blood, and urine samples from ATBI with promising results (Sigal et al., 2018 2018 ; Bulterys et al., 2019 2019 ; Broger et al., 2019 2019 ; Kawasaki et al., 2019 2019 ; Brock et al., 2020 2020 ). Despite the significant advances in TB diagnosis, a better diagnostic method that allows accurate identification among infected individuals, infected vaccinated patients, and healthy individuals is needed. Following the World Health Organization (WHO) call, scientific efforts are being directed to develop a rapid test based on biomarkers to detect ATBI (WHO, 2014 2014 ) in a point-of-care (POC) format such as that based on the lateral flow urine LAM assay (LF-LAM) with high specificity and sensitivity, allowing immediate administration of treatment if required (Lawn et al., 2012 2012 , 2015 2015 ; WHO, 2015 2015 ; Shah et al., 2016 2016 ). The production of soluble recombinant molecules is necessary to validate their use in the different types of diagnostic tests to ensure high yields of mycobacterial antigens. Therefore, the cloning, expression, and purification of these antigens should be generally and ideally optimized in Escherichia coli as a host that will allow easy and low-cost handling. Mtb -secreted antigens mediate important biological functions through interaction with the cells they infect. An important aspect of their function is that these antigens contribute to the virulence and pathogenesis of the disease (Beatty and Russell, 2000 2000 ; Pieters and Gatfield, 2002 2002 ; Adlakha et al., 2012 2012 ). Little is known about the actions or exact destination of mycobacterial antigens during infection. However, several extracellular proteins are known to be secreted in a growing culture and are vital molecules to contact host cells in the initial phase of infection in humans and animals (Pollock and Andersen, 1997 1997 ). Serological tests are an important resource for diagnosing most infectious diseases, as they are simple, fast, and often inexpensive. These tests are useful for detecting EPTB, which is difficult to detect by conventional methods. Antigens are used to detect circulating antibodies in these assays. Among the tests used are complement fixation, hemagglutination, radioimmunoassay, and enzyme immunoadsorption tests (Nurwidya et al., 2018 2018 ). Some of these assays are commercially available, but their specificity and sensitivity are low. Antigenic components involved in Mtb pathogenesis 1 Mtb H37Ra is an attenuated strain that is used as a model to elucidate virulence mechanisms. When tubercle bacilli from the air reach the alveoli of an uninfected host, TB infection is initiated. The recognition between macrophages and the mycobacterium is carried out through direct interaction of surface components, such as the cholesterol-rich domains of the host cell plasma membrane (Gatfield and Pieters, 2000 2000 ). A macrophage-inducible C-type lectin (CLEC4E), called mincle, is an important factor in the interaction between mycobacteria and macrophages, leading to granuloma formation (Feinberg et al., 2013 2013 ). In the initial infection site, phagocytosis through the direct interaction of the macrophage with the mycobacterium is crucial for the lung host defense, because opsonization does not occur efficiently in noninflammatory bronchoalveolar fluid (Fenton and Vermeulen, 1996 1996 ). At this point, alveolar macrophages phagocytize mycobacteria; however, if bacilli survive, they replicate and diffuse into nearby cells (Wolf et al., 2008 2008 ). Macrophages have specific receptors for various opsonins that coat the mycobacteria. Through the opsonization of the mycobacterium with complement molecules, immunoglobulins, mannose-binding proteins, and surfactant factor A, recognition is made, and the mycobacterium enters the macrophage efficiently (Schlesinger et al., 1990 1990 ; Fenton and Vermeulen, 1996 1996 ). These receptors bind to nonopsonized Mtb or recognize opsonins on the bacilli surface. Among the receptors are C-type lectins such as the mannose receptor (MR), dendritic cell-specific intercellular adhesion molecule-3, grabbing nonintegrin (DC-SIGN) and dectin-1, complement receptors, surfactant protein receptors, scavenger receptors, glycosylphosphatidylinositol (GPI)-anchored receptors such as CD14, and Toll-like receptors (TLRs) (Ehlers and Daffé, 1998 1998 ; Ernst, 1998 1998 ; Means et al., 1999 1999 ). Thereafter, at the primary infection site, neutrophils and lymphocytes form a cellular infiltrate encapsulating the bacilli (Ghon complex) (Ghon, 1923 1923 ). Mtb can prevent phagosome maturation due to its interference with Rab-controlled membrane trafficking (Vergne et al., 2004 2004 ). The Ghon complex maintains bacilli in a dormant, nonmetabolic active state for many years or a lifetime. Occasionally, for unknown reasons, mycobacteria start replicating inside this primary lesion, and this might progress to active infection (Bishai, 2000 2000 ). However, in LTBI patients, bacilli persist in tissues and cells not associated with the Ghon complex (Hernández-Pando et al., 2000 2000 ), suggesting that the site of primary infection is not always associated with ATBI. Many molecular factors, which might trigger an immune response, are involved in the bacterial pathogenesis and correspond to major cellular components. In the mycobacteria–host interaction, the components of the capsule and those that are secreted by the mycobacterium are decisive for the invasion, replication, and survival of the bacillus in the new environment. The functional and structural components of the mycobacterial plasma membrane, as well as the cell envelope, are the main strategic targets for developing effective drugs and synthesizing candidate molecules for diagnosis and vaccines (Santos et al., 2017 2017 ). Moreover, carbohydrates are the most abundant structural molecules in mycobacteria, and they are an important factor for immune evasion. However, carbohydrates are recognized by macrophages, T lymphocytes, and other host immune response cells (Schlesinger, 1993 1993 ). Mycobacteria comprise a plasma membrane, the peptidoglycan-arabinogalactan complex (AGP) asymmetric outer membrane or “mycomembrane,” covalently bound to AGP through mycolic acids, and an outer capsule (Kalscheuer et al., 2019 2019 ). The cell envelope is the key factor for the survival of the mycobacterium, and the lipids and polysaccharides comprising it have unique chemical structures. Bacilli have a thick wall and a cell membrane separated by a periplasmic space (Kalscheuer et al., 2019 2019 ). When discussing mycobacterial composition, the molecules that activate the host’s immune system are important; wall biomolecules play a major role in infection pathogenesis. Because effective drugs against TB target the synthesis of cell wall components (Delogu et al., 2013 2013 ), the cellular composition of Mycobacteria helps to identify novel biomarkers for diagnosis or treatment. The cell envelope of Mtb comprises the plasma membrane, cell wall core (a covalently linked mycolic acid, arabinogalactan, and peptidoglycan complex (MAPc)), and the outermost layer (polysaccharide-rich, capsule-like material) (Brennan and Nikaido, 1995 1995 ). Structural and functional molecules from the plasma membrane and cellular envelope are targets for new anti-TB drugs (Santos et al., 2017 2017 ). The capsule is formed by polysaccharides and proteins, but also contains specific lipids that form the outer leaflet and outer membrane whose inner leaflet has mycolic acid residues (Brennan, 2003 2003 ). The resistance of Mtb to antibiotics and other therapeutic agents is due mainly to this asymmetric permeability barrier, which allows molecular traffic in both directions. Capsule proteins such as LpqH (lipoprotein) and PstS1 (phosphate-binding protein), Ag85B (secreted antigen) and capsule polysaccharides have been proposed as target molecules for vaccine development (Kalscheuer et al., 2019 2019 ). Mtb has a peculiar cell wall core consisting of N -glycosylated peptidoglycan linked to D -arabino- D - galactan (AG) via phosphoryl- N -acetylglucosaminosyl-rhamnosyl linkage units. AG is esterified by α-alkyl-β-hydroxy mycolic acids (Delogu et al., 2013 2013 ). A thin layer of peptidoglycan is covalently linked to arabinogalactan and LAM, which are bound to mycolic acid, forming the AGP (Kalscheuer et al., 2019 2019 ). AG strands and peptidoglycan are coiled and perpendicular to the plane of the plasma membrane (Dmitriev et al., 2000 2000 ). The mycobacteria phosphatidylinositol phosphate synthase (PgsA1), which is involved in the synthesis of inositol-derived phospholipids that form the bacilli cell wall (Guerin et al., 2010 2010 ), has been used to develop novel antibacterial drugs with promising results. The major components of the wall are LAM, arabinogalactan, and other carbohydrates: mycolic acids, glycolipids, phenolic lipids, and peptidoglycan. Peptidoglycans in the first layer of the cell envelope allow rigidity and osmotic stability (Jankute et al., 2015 2015 ). The cell wall is a porous membrane that surrounds the surface of the bacterium, allowing the transport of molecules into and out of the cell. It is a heteropolymer formed by short peptide chains and glycans that crosslink to produce a macromolecule with great elastic capacity. Also, the cell wall comprises N -acetylglucosamine (GlcNAc) and N- acetylmuramic acid (MurNAc) residues linked by β-1 > 4 bonds (Egan et al., 2018 2018 ). Catalytic enzymes and secretion products are released from the Mtb cell wall, and many of these are virulence factors. Among the secretion products are CFPs, such as KatG (catalase-peroxidase), SodA (superoxide dismutase), HspX (alpha-crystallin), ESAT-6/CFP-10, and glutamine synthase. ESAT secretion systems (ESXs) are also important mycobacterial virulence factors (Abdallah et al., 2007 2007 ). In Mtb , ESX1 secretes many antigens, such as ESAT-6 and CFP-10, which are highly immunogenic secreted proteins used in TB diagnosis. In culture, ESAT-6 and its chaperone CFP-10 are abundantly secreted highly immunogenic proteins that are involved in different mycobacterial virulence mechanisms (Dillon et al., 2000 2000 ). CFP-10 and ESAT-6 form a 1:1 complex that specifically binds to the surface of macrophages and monocytes, suggesting a role in the host signaling mechanisms for the advantage of the mycobacteria that might lead to the modulation of host behavior (Renshaw et al., 2005 2005 ). CFP-10 and ESAT-6 are encoded by pair-arranged genes in a single locus (RD1), which includes a large family of mycobacterial proteins (Renshaw et al., 2002 2002 ). These genes are coordinately regulated, and despite lacking a signal sequence, both proteins are secreted (Pym et al., 2003 2003 ). Together, these proteins modulate innate and adaptive immune responses. ESAT-6 inactivation results in an important reduction of virulence (Pym et al., 2002 2002 ). Furthermore, ESAT-6 interacts with other proteins. When ESAT-6 interacts with TLR2, a decrease in interleukin-12 (IL-12) is observed, leading to the production of Th2 lymphocytes, which help Mtb survival in the host cells. ESAT-6 also interacts with host protein β-2-microglobulin (β2M), resulting in the downregulation of class 1-mediated antigen presentation (Sreejit et al., 2014 2014 ). Also, several proteins of the mycobacterial secretory proteome include those between 35 and 45 kDa bound to host cells (Adlakha et al., 2012 2012 ). However, some of the virulence factors are not secretion products, but are constituents of the cell wall, such as ERP (exported repetitive protein), FadD26 (long-chain-fatty-acid–AMP ligase), FadD28, MmpL7 (phthiocerol dimycocerosate exporter), FbpA (diacylglycerol acyltransferase/mycolyltransferase Ag85A), MmaA4 (hydroxymycolate synthase), PcaA (cyclopropane mycolic acid synthase 3), OmpA (outer membrane protein A), HBHA (heparin-binding hemagglutinin), and LAM. HBHA is a mycobacterial adhesin (Delogu and Brennan, 1999 1999 ) that, together with PE_PGRS (highly homologous gene families encoding complex proteins) (Iantomasi et al., 2012 2012 ), is a surface Mtb constituent involved in the interaction of mycobacteria with the host cell and might be a possible target for diagnosis. Moreover, PhoP (possible two-component system response positive transcriptional regulator) involved in bacilli pathogenesis (Ryndak et al., 2008 2008 ) and enzymes that combat the reactive oxygen intermediates, such as SodA, SocC (superoxide dismutase [Cu–Zn]), KatG (catalase-peroxidase) and AhpC (alkyl hydroperoxide reductase C) (Smith, 2003 2003 ), might be promising targets for vaccine design. Omics approaches for identifying TB biomarkers 1 Genome sequencing of mycobacteria such as Mtb has made it possible to characterize and study the genes important in TB’s pathogenesis, such as immunogenic antigens, which have been analyzed using various approaches (Cole et al., 1998 1998 ; Garnier et al., 2003 2003 ). The Mtb genome showed that almost 100 genes encode large proteins with a sequence highly homologous with Pro-Glu (PE), and many also contain a repetitive domain rich in Gly and Ala, referred to as the polymorphic GC-rich repetitive sequence (PGRS) region. These PE and PE_PGRS protein families are present in the cell walls of mycobacteria and might be important in controlling TB infection (Brennan and Delogu, 2002 2002 ). Studies have reported several mycobacterial secretory proteins of the proteome that are candidate molecules for studying interaction with the host cell. Adlakha et al. ( 2012 2012 ) investigated the interactions between mycobacterial secretion proteins in a human alveolar cell line and demonstrated that various mycobacterial proteins between 35 and 45 kDa bind to the cells with high affinity, resulting in lysis, guiding the cells to release important cytokines of anti-mycobacterial immunity. The expression of virulence molecules in bacteria is regulated by various environmental and host factors. In the host–pathogen interaction, some genes are stimulated and expressed by the host during infection (Mekalanos, 1992 1992 ; Heithoff et al., 1997 1997 ). Therefore, the identification of antigens in infectious microorganisms is crucial because the interaction of antigens with molecules involved in the immune response, and their potential value can be studied to aid the design of recombinant vaccines and diagnostic methods (Vir et al., 2014 2014 ). The description of cell molecular mechanisms has allowed the development of techniques to manipulate macromolecules. It is now quite easy to synthesize heterologous proteins. Protein synthesis through genetic engineering allows a detailed study of the binding sites of many protein structures through molecular dynamics and modeling. To understand the dynamics of the host–mycobacteria interaction, “omics” was used to integrate large-scale experimental data. Transcriptomic studies of Mycobacterium allowed the construction of metabolic pathways based on genomic information (Boshoff and Lun, 2010 2010 ). The transcriptional profile of Mtb in the macrophage infection phase provides information about regulatory and catalytic routes, helping to identify the roles of key components, such as WhiB3, which maintains redox homeostasis, the transcriptional regulator Rv3574, RshA-PknB-SigH involved in the regulatory pathway for stress adaptation, IdeR related to iron regulation, RelA, and PhoP (Rodriguez, 2006 2006 ; Kendall et al., 2007 2007 ; Park et al., 2008 2008 ; Singh et al., 2009 2009 ). Transcriptional analyses performed on different infected tissues demonstrated that Mycobacteria responded differently to external signals, making it difficult to understand the infection dynamics. Analysis of the Mtb H37Rv transcriptome in a lipid-rich environment using RNAseq revealed the expression of 368 genes involved in the response of the efflux systems, iron capture, and sulfur reduction (Aguilar-Ayala et al., 2017 2017 ). Comparative genomic studies have revealed genomic differences in Mtb lineages, particularly in virulence and drug resistance. For example, methylation in RNA expression is associated with the pathogenesis of Mtb . Sequence-based genomic sequence analysis of 22 isolates of Mtb demonstrated lineage-specific differential gene expression linked to single-nucleotide polymorphism (SNP)-based quantitative expression trait loci, including SNP promoter regions and transcription initiation sites (Gomez-Gonzalez et al., 2019 2019 ). Proteomic studies have increased our knowledge about possible proteins for diagnosis. Thus far, 418 proteins have been identified as serum protein biomarkers from patients with ATBI and LTBI using the LC-Orbitrap Elite platform and enzyme-linked immunosorbent assay (ELISA). Patients with ATBI display serum markers such as the complement system and those related to inflammation and modulation of the immune response, such as C-reactive protein, haptoglobin, alpha-1 acid glycoprotein, complement component C9, neutrophilic defensin 1, and amyloid P component. Furthermore, in those patients, a decrease in proteins, such as apolipoprotein A, plasma kallikrein, and serotransferrin, was observed, suggesting the importance of lipid transport and iron assimilation in TB infection (Mateos et al., 2020 2020 ). Proteomic and lipidomic studies have helped elucidate the molecular composition of the mycobacterial capsule. Among the lipoproteins found are LpqH and PstS1, as well as proteins of the Ag85 complex. The enzymatic activity of BlaC, Ald, Adh, KatG, GlnA1, PncA, and SodA has also been measured (Kalscheuer et al., 2019 2019 ). Capsule polysaccharides have been proposed as target molecules for vaccine development, because they are located on the surface of the mycobacterium and differ from human glycans. The mycobacterial capsule prevents dehydration, helps adherence to the host cell, and provides resistance to the host’s immunity. Several different transcriptomic and proteomic studies have analyzed the differences between virulent strains of Mtb (Jena et al., 2013 2013 ; Jhingan et al., 2016 2016 ; Verma et al., 2017 2017 ). These studies identified proteins that were differentially expressed among strains, and mutations that could be associated with the attenuation of virulence of the H37Ra strain. Differences are also mediated by phosphorylation in the bacterial secretion system VII, regulatory system, and fatty acid biosynthesis in the same strain compared with H37Rv. Through multi-omics approaches, nine genes present in the H37Rv strain were shown to be absent in the H37Ra strain. Two putative virulence factors belonging to the ESAT-6 type protein family stood out and 408 new proteins have been identified (Pinto et al., 2018 2018 ). M. tuberculosis antigenic biomarkers and their application to prevent TB 1 The accurate identification of mycobacterial infection is the first step in determining the appropriate therapeutic regimen for a patient. The diagnosis of pulmonary TB or EPTB is challenging. Therefore, innovative strategies are currently being developed using in vitro, in vivo, and in silico approaches to identify new immunogenic proteins of the mycobacterium. One of these strategies is the synthetic production and validation of highly immunogenic mycobacterial antigens which have different uses, such as in diagnosis or therapy, and to design recombinant vaccines (Table 1 1 ) (Meier et al., 2018 2018 ; Zhang et al., 2018 2018 ). The antigens available for experimental research are varied. However, none of these synthetic antigens are approved for mass use in the diagnosis of TB. Currently, TB is prevented using vaccines which could be enriched with recombinant immunogenic antigens. Some vaccines designed and tested in infants have revealed an excellent safety profile, but failed to promote protective efficacy in that population (Weiner and Kaufmann, 2014 2014 ). The antigenic components of Mtb are complex and highly variable, and many are not well defined. ESAT-6, 6-kDa early secretory antigenic target; CFP, culture filtrate protein; rBCG, recombinant bacille Calmette-Guérin; ELISPOT, enzyme-linked immunospot assay; CMX, Ag85C-MPT51-HspX fusion protein; SPCE, screen-printed carbon electrode. For further details on Mycobacterium tuberculosis ( Mtb ) proteins, see the database: http://www.tbdb.org http://www.tbdb.org and http://tuberculist.epfl.ch http://tuberculist.epfl.ch  Protein Vaccine/assay candidate Reference  Ag85A MVA85A, Crucell Ad35, Ad5Ag85A, ChAdOx1.85A+MVA85A Weiner and Kaufmann, 2014; Kaufmann, 2020 2020  Ag85B H1, H4, H56, Crucell Ad35 Weiner and Kaufmann, 2014; Kaufmann, 2020 2020  Ag85B and ESAT-6 Hybrid 1 (H1), TB-FLU-04L Sable et al., 2019 2019 ; Kaufmann, 2020 2020  ESAT-6 H1, H56 Weiner and Kaufmann, 2014; Kaufmann, 2020 2020  CFP-7 H4, Crucell Ad35 Weiner and Kaufmann, 2014; Kaufmann, 2020 2020   Mtb 39a (PPE18) and Mtb 32a M72 Weiner and Kaufmann, 2014; Kaufmann, 2020 2020  Rv2660 H56 Weiner and Kaufmann, 2014; Kaufmann, 2020 2020  Rv2608, Rv3619, Rv3620, and Rv1813 ID93 Weiner and Kaufmann, 2014; Kaufmann, 2020 2020  Ag85A, PPE, and pfkB Multistage DNA vaccine, A39 Su et al., 2017 2017  Ag85C, MPT5, and HspX rBCG-CMX da Costa et al., 2017 2017  Ag85B and ESAT-6 Lipo-AE vaccine formulation with the PolyIC adjuvant Diogo et al., 2019 2019  ESAT-6, CFP-10, TB10.3, AlaDH, Ag85B, PstS1, and HspX ELISPOT assay Chiappini et al., 2012 2012  38kD, ESAT-6, and CFP-10 Multiple-antigen detection assay Dai et al., 2017 2017  CFP-10 and ESAT-6 Electrochemical-based SPCE Bakhori et al., 2020 2020 The use of protective antigens in TB diagnosis or treatment is a topic that is widely addressed. TB bacilli at the time of infection in the host synthesize proteins that can be ignored by the immune system. However, some others are dominant antigens that activate a response through antigen-specific T cells (Aagaard et al., 2009 2009 ). There is an arsenal of antigens that stimulate the T-cell-mediated immune response; however, these antigens may have subdominant or masked epitopes, inactivating the function of the antigen (Comas et al., 2010 2010 ). Currently, numerous biomarkers have been identified and proposed to diagnose or treat TB. In the following paragraphs, we review some of the most relevant biomarkers. The effectiveness of recombinant TB antigens in the diagnosis of TB has been evaluated on a large scale using microarrays and ELISA. A study showed that 42 TB antigens of 103 analyzed induce a specific immune response in infected patients. Three new polyepitope fusion proteins were designed with polyepitopes, and the characterization of these antigens was carried out by multiantigen print immunoassay (MAPIA); 90% of the patients infected with TB had specific antibodies. These antigens could be used to detect infection (Ireton et al., 2010 2010 ). Most of the identified biomarkers are used to determine ATBIs. These biomarkers are Mtb products that are detected in blood, saliva, or urine samples from an infected individual. For example, Ag85 complex antigens have been identified in blood. Thus, proposed diagnostic tests based on a biomarker combination to detect TB could provide better surveillance and control of the disease. Different types of TB biomarkers have been identified, such as antibodies, cytokines, metabolic activity markers, volatile organic compounds, and mycobacterial antigens. Antigenic biomarkers are of special importance in the design of novel methods to diagnose TB. New vaccination strategies are needed to control TB. Several studies have identified candidate molecules for recombinant vaccines, including Rv0287, Rv1174, Rv1196, and Rv3616c, which have been tested in vaccines (Mustafa et al., 2006 2006 ). Genetically recombined modified vaccines have also been designed, such as Mtb 72f from Glaxo-Smith-Kline, that combines two Mtb immunogenic antigens— Mtb 39a (Rv1196) and Mtb 32a (Rv0125)—with the adjuvant ASO2a or ASOIb. This combination was immunogenic with no side effects (Morandi et al., 2013 2013 ). The CFP-21 antigen (Rv1984c) induces markedly high interferon-γ (IFN-γ) responses in patients with TB that can be expressed by recombinant techniques to test its usefulness in future vaccines. A recent study showed that CFP-10 and CFP-21 antigenic proteins encapsulated in chitosan nanoparticles (ChNs) have protective and therapeutic potential against Mtb in immunized mice (Pandey et al., 2020 2020 ). A computational approach was used recently to predict the epitope affinity of HLA molecules for ten Mtb antigens ( Mtb 39a, Mtb 32a, Ag85B, ESAT-6, TB10.4, Rv2660, Rv2608, Rv3619, Rv3620, and Rv1813) representing the M72, H1, H4, H56, and ID93 vaccines found in clinical trials. The results showed that the ID93 vaccine, comprising the Rv2608, Rv1813, Rv3619, and Rv3620 antigens, had the best potential to prevent ATBI and LTBI (Ong et al., 2020 2020 ). A current challenge in medicine is the validation and use of recombinant vaccines. Various recombinant vaccines have been investigated. da Costa et al. ( 2014 2014 ) designed a recombinant BCG vaccine (rBCG) with the capacity to express the immune-dominant epitopes of Ag85C, MPT51, and HspX. The vaccine was stable for more than 20 d after vaccination. In mice, this vaccine produced an immune response specific to each of the recombinant proteins used. A vaccine was recently developed with Ag85B and ESAT-6 antigens encapsulated in liposomes. The lipo-AE vaccine formulation in combination with the PolyIC adjuvant induced a combined Th1/Th17-Th2 immune response to the Ag85B antigen, but a weak immune response to ESAT-6 (Diogo et al., 2019 2019 ). Using recombinant technology represents a new platform for the validation of new vaccines against TB. Cytomegalovirus (CMV)-based vectors capable of expressing multiple Mtb antigens are a good strategy for vaccine development. CMV vectors can be genetically altered to stimulate highly diverse CD8 + T-cell responses that differ in their intended epitope, making it possible to uniquely tailor the CD8 + T-cell response for each disease to maximize therapeutic or prophylactic protection. This vaccine has shown protection in preclinical studies, with sustained antigenic expression representing long-term immunity, an attractive strategy for a new TB vaccine (Früh and Picker, 2017 2017 ; Liu et al., 2019 2019 ). Another strategy is the use of messenger RNA (mRNA) as a vaccine vector. This is a simple technology that could express antigens with good performance and could be used against different pathogens, including Mtb (Lorenzi et al., 2010 2010 ). Promising vaccines exist in preclinical studies, but better models are needed to standardize and predict vaccine efficacy in humans. Among the most studied antigens of Mycobacterium are ESAT-6, CFP-10, TB10.3, TB10.4, MTSP11, MPT70, Ag85, and MPT83. Regarding the inactivity of Mtb bacilli, transcriptomic analyses have shown that exposure to nitric oxide induces their inactivity, and differential expression of a set of DosR/S regulon genes of the dormancy regulon has been demonstrated (Voskuil et al., 2003 2003 ; Chiappini et al., 2012 2012 ). These genes induce bacillus inactivation, a state in which there is no replication of the mycobacterium (Murphy and Brown, 2008 2008 ; Rustad et al., 2008 2008 ). Inactivation or dormancy of Mtb bacilli within the host is important for mycobacterial pathogenesis because it allows the bacillus to remain in the body without being actively attacked by the host’s immune system. The inactive bacillus resides in a granuloma under hypoxic conditions and with limited access to nutrients. The molecular mechanism through which the bacilli reactivate is unknown. Mycobacterial antigens, such as Ag85A, ESAT-6, and CFP-10, are expressed in different growth phases of the bacillus. ESAT-6 and CFP-10 are also expressed in the stationary phase. MDP1, Acr, and DosS antigens are upregulated from the stationary phase to the dormancy phase, suggesting that they may be present in the growth phase and in dormancy in individuals with previous TB. This knowledge could be used to identify antibodies against these antigens and determine whether a patient has an LTBI or ATBI (Belay et al., 2015 2015 ). Conclusions 1 We have reviewed progress in the study of Mtb immunogenic antigens and the potential use of recombinant antigens in TB control. Various antigens have been identified and characterized, and more are yet to be uncovered. These study targets have been evaluated using different platforms, and the rapidly accumulating data are being used to understand and integrate the information and apply it effectively in the control of human and animal TB. New live vaccines are currently being investigated and developed, as well as recombinant antigens and vector-based vaccines that express immunogenic Mtb antigens. The massive synthesis of antigens or molecules involved in Mtb infection allows the study and validation of new biomarkers to diagnose ATBI or LTBI. New vaccine candidates have already emerged that target patients diagnosed with LTBI. Despite the advances, more experimental data, clinical studies, and system biology are required to integrate the information and decipher the network of interactions between the components of the host immune mechanism and mycobacterial antigens. In this challenge, multidisciplinary research is expected to provide the tools to develop new diagnostic methods, vaccines, and therapies against TB. Biomarker technologies, such as recombinant diagnostics or vaccine antigens, have proven to be a valuable platform for producing large-scale tests, simplifying test validation, and accelerating entry into the market. In theory, the current methods of TB control reduce the number of infections in the population. In highly developed countries, the TB incidence is low and the disease is almost eradicated (Gordejo and Vermeersch, 2006 2006 ). However, in developing countries, TB is still considered a serious problem and a potential global risk. Therefore, it is crucial to invest in the development of biomarker technology and the study of TB. The Mtb genome was sequenced over 20 years ago, and most of the proteins encoded within its genome have been explored in detail. Precise functions have been attributed to more than 40% of the proteins. Immunogenic antigens have great relevance. Through years of research and observation, it was observed that these antigens cause variations in the patterns of the host immune response. Due to their potential use as biomarkers, designing a multigene cocktail to increase test sensitivity and improve diagnosis is feasible. Advances in the characterization of the molecules involved in the pathogenesis of TB and understanding the complex network of interactions between mycobacterial antigens and the host immune system will allow the future improvement of disease prevention and control strategies. Many points still need to be addressed concerning this disease. We need to validate better models to predict the efficacy of TB testing in humans without violating established ethical and regulatory issues. TB will be eradicated only if optimal sanitary measures are strictly implemented, eliminating the precarious living conditions of populations worldwide. The detection methods for Mtb do not always guarantee a reliable diagnosis. However, many studies have demonstrated the usefulness of mycobacterial biomarkers, with important implications for TB diagnosis and treatment. More rigorous validation studies of these biomarkers are required for them to become successful diagnostic tools or vaccines against the disease. References CS Aagaard , TTKT Hoang , C Vingsbo-Lundberg , 等 . Quality and vaccine efficacy of CD4 + T cell responses directed to dominant and subdominant epitopes in ESAT-6 from Mycobacterium tuberculosis . J Immunol , 2009 . 183 ( 4 ): 2659 - 2668 . 19620314 DOI: 10.4049/jimmunol.0900947 http://doi.org/10.4049/jimmunol.0900947 . Quality and vaccine efficacy of CD4+ T cell responses directed to dominant and subdominant epitopes in ESAT-6 from Mycobacterium tuberculosis AM Abdallah , NCG van Pittius , PAD Champion , 等 . Type VII secretion–mycobacteria show the way . Nat Rev Microbiol , 2007 . 5 ( 11 ): 883 - 891 . 17922044 DOI: 10.1038/nrmicro1773 http://doi.org/10.1038/nrmicro1773 . Type VII secretion–mycobacteria show the way N Adlakha , P Vir , I Verma . Effect of mycobacterial secretory proteins on the cellular integrity and cytokine profile of type II alveolar epithelial cells . Lung India , 2012 . 29 ( 4 ): 313 - 318 . 23243342 DOI: 10.4103/0970-2113.102796 http://doi.org/10.4103/0970-2113.102796 . Effect of mycobacterial secretory proteins on the cellular integrity and cytokine profile of type II alveolar epithelial cells DA Aguilar-Ayala , L Tilleman , F van Nieuwerburgh , 等 . The transcriptome of Mycobacterium tuberculosis in a lipid-rich dormancy model through RNAseq analysis . Sci Rep, 7:17665 , 2017 . DOI: 10.1038/s41598-017-17751-x http://doi.org/10.1038/s41598-017-17751-x . The transcriptome of Mycobacterium tuberculosis in a lipid-rich dormancy model through RNAseq analysis JM Aitken , TJ Borody , G Agrawal . A revaluation of the use of conventional Ziehl-Neelsen stain for detection of non-tuberculous mycobacteria . N Z J Med Lab Sci , 2019 . 73 ( 2 ): 85 . A revaluation of the use of conventional Ziehl-Neelsen stain for detection of non-tuberculous mycobacteria W Amanfu . The situation of tuberculosis and tuberculosis control in animals of economic interest . Tuberculosis , 2006 . 86 ( 3-4 ): 330 - 335 . 16644282 DOI: 10.1016/j.tube.2006.01.007 http://doi.org/10.1016/j.tube.2006.01.007 . The situation of tuberculosis and tuberculosis control in animals of economic interest NM Bakhori , NA Yusof , J Abdullah , 等 . Surface enhanced CdSe/ZnS QD/SiNP electrochemical immunosensor for the detection of Mycobacterium tuberculosis by combination of CFP10-ESAT6 for better diagnostic specificity . Materials (Basel) , 2020 . 13 ( 1 ): 149 DOI: 10.3390/ma13010149 http://doi.org/10.3390/ma13010149 . Surface enhanced CdSe/ZnS QD/SiNP electrochemical immunosensor for the detection of Mycobacterium tuberculosis by combination of CFP10-ESAT6 for better diagnostic specificity AL Banuls , A Sanou , NT van Anh , 等 . Mycobacterium tuberculosis : ecology and evolution of a human bacterium . J Med Microbiol , 2015 . 64 ( 11 ): 1261 - 1269 . 26385049 DOI: 10.1099/jmm.0.000171 http://doi.org/10.1099/jmm.0.000171 . Mycobacterium tuberculosis: ecology and evolution of a human bacterium WL Beatty , DG Russell . Identification of mycobacterial surface proteins released into subcellular compartments of infected macrophages . Infect Immun , 2000 . 68 ( 12 ): 6997 - 7002 . 11083824 DOI: 10.1128/IAI.68.12.6997-7002.2000 http://doi.org/10.1128/IAI.68.12.6997-7002.2000 . Identification of mycobacterial surface proteins released into subcellular compartments of infected macrophages M Belay , M Legesse , A Mihret , 等 . Pro- and anti-inflammatory cytokines against Rv2031 are elevated during latent tuberculosis: a study in cohorts of tuberculosis patients, household contacts and community controls in an endemic setting . PLoS ONE , 2015 . 10 ( 4 ): e0124134 25897840 DOI: 10.1371/journal.pone.0124134 http://doi.org/10.1371/journal.pone.0124134 . Pro- and anti-inflammatory cytokines against Rv2031 are elevated during latent tuberculosis: a study in cohorts of tuberculosis patients, household contacts and community controls in an endemic setting WR Bishai . Rekindling old controversy on elusive lair of latent tuberculosis . Lancet , 2000 . 356 ( 9248 ): 2113 - 2114 . 11191532 DOI: 10.1016/S0140-6736(00)03485-1 http://doi.org/10.1016/S0140-6736(00)03485-1 . Rekindling old controversy on elusive lair of latent tuberculosis HIM Boshoff , DS Lun . Systems biology approaches to understanding mycobacterial survival mechanisms . Drug Discov Today Dis Mech , 2010 . 7 ( 1 ): e75 - e82 . 21072257 DOI: 10.1016/j.ddmec.2010.09.008 http://doi.org/10.1016/j.ddmec.2010.09.008 . Systems biology approaches to understanding mycobacterial survival mechanisms L Bourassa . The NanoDisk-MS assay: a new frontier in biomarker-based tuberculosis diagnostics? . Clin Chem , 2018 . 64 ( 5 ): 763 - 765 . 29483104 DOI: 10.1373/clinchem.2017.285460 http://doi.org/10.1373/clinchem.2017.285460 . The NanoDisk-MS assay: a new frontier in biomarker-based tuberculosis diagnostics? MJ Brennan , G Delogu . The PE multigene family: a ‘molecular mantra’ for mycobacteria . Trends Microbiol , 2002 . 10 ( 5 ): 246 - 249 . 11973159 DOI: 10.1016/S0966-842X(02)02335-1 http://doi.org/10.1016/S0966-842X(02)02335-1 . The PE multigene family: a ‘molecular mantra’ for mycobacteria PJ Brennan . Structure, function, and biogenesis of the cell wall of Mycobacterium tuberculosis . Tuberculosis , 2003 . 83 ( 1-3 ): 91 - 97 . 12758196 DOI: 10.1016/S1472-9792(02)00089-6 http://doi.org/10.1016/S1472-9792(02)00089-6 . Structure, function, and biogenesis of the cell wall of Mycobacterium tuberculosis PJ Brennan , H Nikaido . The envelope of mycobacteria . Ann Rev Biochem , 1995 . 64 ( 1 ): 29 - 63 . 7574484 DOI: 10.1146/annurev.bi.64.070195.000333 http://doi.org/10.1146/annurev.bi.64.070195.000333 . The envelope of mycobacteria V Briken , SA Porcelli , GS Besra , 等 . Mycobacterial lipoarabinomannan and related lipoglycans: from biogenesis to modulation of the immune response . Mol Microbiol , 2004 . 53 ( 2 ): 391 - 403 . 15228522 DOI: 10.1111/j.1365-2958.2004.04183.x http://doi.org/10.1111/j.1365-2958.2004.04183.x . Mycobacterial lipoarabinomannan and related lipoglycans: from biogenesis to modulation of the immune response M Brock , D Hanlon , MW Zhao , 等 . Detection of mycobacterial lipoarabinomannan in serum for diagnosis of active tuberculosis . Diagn Microbiol Infect Dis , 2020 . 96 ( 2 ): 114937 31785971 DOI: 10.1016/j.diagmicrobio.2019.114937 http://doi.org/10.1016/j.diagmicrobio.2019.114937 . Detection of mycobacterial lipoarabinomannan in serum for diagnosis of active tuberculosis T Broger , M Tsionksy , A Mathew , 等 . Sensitive electrochemiluminescence (ECL) immunoassays for detecting lipoarabinomannan (LAM) and ESAT-6 in urine and serum from tuberculosis patients . PLoS ONE , 2019 . 14 ( 4 ): e0215443 30998715 DOI: 10.1371/journal.pone.0215443 http://doi.org/10.1371/journal.pone.0215443 . Sensitive electrochemiluminescence (ECL) immunoassays for detecting lipoarabinomannan (LAM) and ESAT-6 in urine and serum from tuberculosis patients MA Bulterys , B Wagner , M Redard-Jacot , 等 . Point-of-Care Urine LAM tests for tuberculosis diagnosis: a mini-review . J Clin Med, 9:111 , 2019 . Point-of-Care Urine LAM tests for tuberculosis diagnosis: a mini-review E Chiappini , CD Bella , F Bonsignori , 等 . Potential role of M. tuberculosis specific IFN-γ and IL-2 ELISPOT assays in discriminating children with active or latent tuberculosis . PLoS ONE , 2012 . 7 ( 9 ): e46041 23029377 DOI: 10.1371/journal.pone.0046041 http://doi.org/10.1371/journal.pone.0046041 . Potential role of M. tuberculosis specific IFN-γ and IL-2 ELISPOT assays in discriminating children with active or latent tuberculosis ST Cole , R Brosch , J Parkhill , 等 . Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence . Nature , 1998 . 393 ( 6685 ): 537 - 544 . 9634230 DOI: 10.1038/31159 http://doi.org/10.1038/31159 . Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence I Comas , J Chakravartti , PM Small , 等 . Human T cell epitopes of Mycobacterium tuberculosis are evolutionarily hyperconserved . Nat Genet , 2010 . 42 ( 6 ): 498 - 503 . 20495566 DOI: 10.1038/ng.590 http://doi.org/10.1038/ng.590 . Human T cell epitopes of Mycobacterium tuberculosis are evolutionarily hyperconserved AC da Costa , A de Oliveira Costa-Júnior , FM de Oliveira , 等 . A new recombinant BCG vaccine induces specific Th17 and Th1 effector cells with higher protective efficacy against tuberculosis . PLoS ONE , 2014 . 9 ( 11 ): e112848 25398087 DOI: 10.1371/journal.pone.0112848 http://doi.org/10.1371/journal.pone.0112848 . A new recombinant BCG vaccine induces specific Th17 and Th1 effector cells with higher protective efficacy against tuberculosis AC da Costa , DP de Resende , BDP de Santos , 等 . Modulation of macrophage responses by CMX, a fusion protein composed of Ag85C, MPT51, and HspX from Mycobacterium tuberculosis . Front Microbiol, 8:623 , 2017 . DOI: 10.3389/fmicb.2017.00623 http://doi.org/10.3389/fmicb.2017.00623 . Modulation of macrophage responses by CMX, a fusion protein composed of Ag85C, MPT51, and HspX from Mycobacterium tuberculosis ZH Dai , ZQ Liu , BS Xiu , 等 . A multiple-antigen detection assay for tuberculosis diagnosis based on broadly reactive polyclonal antibodies . Iran J Basic Med Sci , 2017 . 20 ( 4 ): 360 - 367 . 28804606 . A multiple-antigen detection assay for tuberculosis diagnosis based on broadly reactive polyclonal antibodies G Delogu , MJ Brennan . Functional domains present in the mycobacterial hemagglutinin, HBHA . J Bacteriol , 1999 . 181 ( 24 ): 7464 - 7469 . 10601202 DOI: 10.1128/JB.181.24.7464-7469.1999 http://doi.org/10.1128/JB.181.24.7464-7469.1999 . Functional domains present in the mycobacterial hemagglutinin, HBHA G Delogu , M Sali , G Fadda . The biology of Mycobacterium tuberculosis infection . Mediterr J Hematol Infect Dis , 2013 . 5 ( 1 ): e2013070 24363885 DOI: 10.4084/mjhid.2013.070 http://doi.org/10.4084/mjhid.2013.070 . The biology of Mycobacterium tuberculosis infection CM Denkinger , M Pai , M Patel , 等 . Gamma interferon release assay for monitoring of treatment response for active tuberculosis: an explosion in the spaghetti factory . J Clin Microbiol , 2013 . 51 ( 2 ): 607 - 610 . 23175268 DOI: 10.1128/JCM.02278-12 http://doi.org/10.1128/JCM.02278-12 . Gamma interferon release assay for monitoring of treatment response for active tuberculosis: an explosion in the spaghetti factory K Dheda , CE Barry , G Maartens . Tuberculosis . Lancet , 2016 . 387 ( 10024 ): 1211 - 1226 . 26377143 DOI: 10.1016/S0140-6736(15)00151-8 http://doi.org/10.1016/S0140-6736(15)00151-8 . Tuberculosis DC Dillon , MR Alderson , CH Day , 等 . Molecular and immunological characterization of Mycobacterium tuberculosis CFP-10, an immunodiagnostic antigen missing in Mycobacterium bovis BCG . J Clin Microbiol , 2000 . 38 ( 9 ): 3285 - 3290 . 10970372 DOI: 10.1128/JCM.38.9.3285-3290.2000 http://doi.org/10.1128/JCM.38.9.3285-3290.2000 . Molecular and immunological characterization of Mycobacterium tuberculosis CFP-10, an immunodiagnostic antigen missing in Mycobacterium bovis BCG GR Diogo , P Hart , A Copland , 等 . Immunization with Mycobacterium tuberculosis antigens encapsulated in phosphatidylserine liposomes improves protection afforded by BCG . Front Immunol, 10:1349 , 2019 . DOI: 10.3389/fimmu.2019.01349 http://doi.org/10.3389/fimmu.2019.01349 . Immunization with Mycobacterium tuberculosis antigens encapsulated in phosphatidylserine liposomes improves protection afforded by BCG BA Dmitriev , S Ehlers , ET Rietschel , 等 . Molecular mechanics of the mycobacterial cell wall: from horizontal layers to vertical scaffolds . Int J Med Microbiol , 2000 . 290 ( 3 ): 251 - 258 . 10959727 DOI: 10.1016/S1438-4221(00)80122-8 http://doi.org/10.1016/S1438-4221(00)80122-8 . Molecular mechanics of the mycobacterial cell wall: from horizontal layers to vertical scaffolds AJF Egan , R Maya–Martinez , I Ayala , 等 . Induced conformational changes activate the peptidoglycan synthase PBP1B . Mol Microbiol , 2018 . 110 ( 3 ): 335 - 356 . 30044025 DOI: 10.1111/mmi.14082 http://doi.org/10.1111/mmi.14082 . Induced conformational changes activate the peptidoglycan synthase PBP1B MRW Ehlers , M Daffé . Interactions between Mycobacterium tuberculosis and host cells: are mycobacterial sugars the key? . Trends Microbiol , 1998 . 6 ( 8 ): 328 - 335 . 9746944 DOI: 10.1016/S0966-842X(98)01301-8 http://doi.org/10.1016/S0966-842X(98)01301-8 . Interactions between Mycobacterium tuberculosis and host cells: are mycobacterial sugars the key? JD Ernst . Macrophage receptors for Mycobacterium tuberculosis . Infect Immun , 1998 . 66 ( 4 ): 1277 - 1281 . 9529042 DOI: 10.1128/IAI.66.4.1277-1281.1998 http://doi.org/10.1128/IAI.66.4.1277-1281.1998 . Macrophage receptors for Mycobacterium tuberculosis H Feinberg , SAF Jégouzo , TJW Rowntree , 等 . Mechanism for recognition of an unusual mycobacterial glycolipid by the macrophage receptor mincle . J Biol Chem , 2013 . 288 ( 40 ): 28457 - 28465 . 23960080 DOI: 10.1074/jbc.M113.497149 http://doi.org/10.1074/jbc.M113.497149 . Mechanism for recognition of an unusual mycobacterial glycolipid by the macrophage receptor mincle MJ Fenton , MW Vermeulen . Immunopathology of tuberculosis: roles of macrophages and monocytes . Infect Immun , 1996 . 64 ( 3 ): 683 - 690 . 8641767 DOI: 10.1128/IAI.64.3.683-690.1996 http://doi.org/10.1128/IAI.64.3.683-690.1996 . Immunopathology of tuberculosis: roles of macrophages and monocytes PEM Fine . BCG: the challenge continues . Scand J Infect Dis , 2001 . 33 ( 1 ): 58 - 60 . DOI: 10.1080/003655401753382576 http://doi.org/10.1080/003655401753382576 . BCG: the challenge continues MA Forrellad , LI Klepp , A Gioffré , 等 . Virulence factors of the Mycobacterium tuberculosis complex . Virulence , 2013 . 4 ( 1 ): 3 - 66 . 23076359 DOI: 10.4161/viru.22329 http://doi.org/10.4161/viru.22329 . Virulence factors of the Mycobacterium tuberculosis complex K Früh , L Picker . CD8+ T cell programming by cytomegalovirus vectors: applications in prophylactic and therapeutic vaccination . Curr Opin Immunol , 2017 . 47 52 - 56 . 28734175 DOI: 10.1016/j.coi.2017.06.010 http://doi.org/10.1016/j.coi.2017.06.010 . CD8+ T cell programming by cytomegalovirus vectors: applications in prophylactic and therapeutic vaccination T Garnier , K Eiglmeier , JC Camus , 等 . The complete genome sequence of Mycobacterium bovis . Proc Natl Acad Sci USA , 2003 . 100 ( 13 ): 7877 - 7882 . DOI: 10.1073/pnas.1130426100 http://doi.org/10.1073/pnas.1130426100 . The complete genome sequence of Mycobacterium bovis J Gatfield , J Pieters . Essential role for cholesterol in entry of mycobacteria into macrophages . Science , 2000 . 288 ( 5471 ): 1647 - 1651 . 10834844 DOI: 10.1126/science.288.5471.1647 http://doi.org/10.1126/science.288.5471.1647 . Essential role for cholesterol in entry of mycobacteria into macrophages A Ghon . The primary complex in human tuberculosis and its significance . Am Rev Tuberc , 1923 . 7 ( 5 ): 314 - 317 . . The primary complex in human tuberculosis and its significance PJ Gomez-Gonzalez , N Andreu , JE Phelan , 等 . An integrated whole genome analysis of Mycobacterium tuberculosis reveals insights into relationship between its genome, transcriptome and methylome . Sci Rep, 9:5204 , 2019 . DOI: 10.1038/s41598-019-41692-2 http://doi.org/10.1038/s41598-019-41692-2 . An integrated whole genome analysis of Mycobacterium tuberculosis reveals insights into relationship between its genome, transcriptome and methylome FJR Gordejo , JP Vermeersch . Towards eradication of bovine tuberculosis in the European Union . Vet Microbiol , 2006 . 112 ( 2-4 ): 101 - 109 . 16388921 DOI: 10.1016/j.vetmic.2005.11.034 http://doi.org/10.1016/j.vetmic.2005.11.034 . Towards eradication of bovine tuberculosis in the European Union ME Guerin , J Korduláková , PM Alzari , 等 . Molecular basis of phosphatidyl- myo -inositol mannoside biosynthesis and regulation in mycobacteria . J Biol Chem , 2010 . 285 ( 44 ): 33577 - 33583 . 20801880 DOI: 10.1074/jbc.R110.168328 http://doi.org/10.1074/jbc.R110.168328 . Molecular basis of phosphatidyl-myo-inositol mannoside biosynthesis and regulation in mycobacteria DM Heithoff , CP Conner , PC Hanna , 等 . Bacterial infection as assessed by in vivo gene expression . Proc Natl Acad Sci , 1997 . 94 ( 3 ): 934 - 939 . DOI: 10.1073/pnas.94.3.934 http://doi.org/10.1073/pnas.94.3.934 . Bacterial infection as assessed by in vivo gene expression R Hernández-Pando , M Jeyanathan , G Mengistu , 等 . Persistence of DNA from Mycobacterium tuberculosis in superficially normal lung tissue during latent infection . Lancet , 2000 . 356 ( 9248 ): 2133 - 2138 . 11191539 DOI: 10.1016/S0140-6736(00)03493-0 http://doi.org/10.1016/S0140-6736(00)03493-0 . Persistence of DNA from Mycobacterium tuberculosis in superficially normal lung tissue during latent infection RE Huebner , MF Schein , Jr JB Bass . The tuberculin skin test . Clin Infect Dis , 1993 . 17 ( 6 ): 968 - 975 . 8110954 DOI: 10.1093/clinids/17.6.968 http://doi.org/10.1093/clinids/17.6.968 . The tuberculin skin test R Iantomasi , M Sali , A Cascioferro , 等 . PE_PGRS30 is required for the full virulence of Mycobacterium tuberculosis . Cell Microbiol , 2012 . 14 ( 3 ): 356 - 367 . 22050772 DOI: 10.1111/j.1462-5822.2011.01721.x http://doi.org/10.1111/j.1462-5822.2011.01721.x . PE_PGRS30 is required for the full virulence of Mycobacterium tuberculosis GC Ireton , R Greenwald , H Liang , 等 . Identification of Mycobacterium tuberculosis antigens of high serodiagnostic value . Clin Vaccine Immunol , 2010 . 17 ( 10 ): 1539 - 1547 . 20719988 DOI: 10.1128/CVI.00198-10 http://doi.org/10.1128/CVI.00198-10 . Identification of Mycobacterium tuberculosis antigens of high serodiagnostic value M Jankute , JAG Cox , J Harrison , 等 . Assembly of the mycobacterial cell wall . Ann Rev Microbiol , 2015 . 69 405 - 423 . DOI: 10.1146/annurev-micro-091014-104121 http://doi.org/10.1146/annurev-micro-091014-104121 . Assembly of the mycobacterial cell wall L Jena , S Kashikar , S Kumar , 等 . Comparative proteomic analysis of Mycobacterium tuberculosis strain H37Rv versus H37Ra . Int J Mycobacteriol , 2013 . 2 ( 4 ): 220 - 226 . 26786126 DOI: 10.1016/j.ijmyco.2013.10.004 http://doi.org/10.1016/j.ijmyco.2013.10.004 . Comparative proteomic analysis of Mycobacterium tuberculosis strain H37Rv versus H37Ra GD Jhingan , S Kumari , SV Jamwal , 等 . Comparative proteomic analyses of avirulent, virulent, and clinical strains of Mycobacterium tuberculosis identify strain-specific patterns . J Biol Chem , 2016 . 291 ( 27 ): 14257 - 14273 . 27151218 DOI: 10.1074/jbc.M115.666123 http://doi.org/10.1074/jbc.M115.666123 . Comparative proteomic analyses of avirulent, virulent, and clinical strains of Mycobacterium tuberculosis identify strain-specific patterns R Kalscheuer , A Palacios , I Anso , 等 . The Mycobacterium tuberculosis capsule: a cell structure with key implications in pathogenesis . Biochem J , 2019 . 476 ( 14 ): 1995 - 2016 . 31320388 DOI: 10.1042/BCJ20190324 http://doi.org/10.1042/BCJ20190324 . The Mycobacterium tuberculosis capsule: a cell structure with key implications in pathogenesis RK Katial , J Hershey , T Purohit-Seth , 等 . Cell-mediated immune response to tuberculosis antigens: comparison of skin testing and measurement of in vitro gamma interferon production in whole-blood culture . Clin Diagn Lab Immunol , 2001 . 8 ( 2 ): 339 - 345 . 11238218 DOI: 10.1128/CDLI.8.2.339-345.2001 http://doi.org/10.1128/CDLI.8.2.339-345.2001 . Cell-mediated immune response to tuberculosis antigens: comparison of skin testing and measurement of in vitro gamma interferon production in whole-blood culture SHE Kaufmann . Vaccination against tuberculosis: revamping BCG by molecular genetics guided by immunology . Front Immunol, 11:316 , 2020 . DOI: 10.3389/fimmu.2020.00316 http://doi.org/10.3389/fimmu.2020.00316 . Vaccination against tuberculosis: revamping BCG by molecular genetics guided by immunology M Kawasaki , C Echiverri , L Raymond , 等 . Lipoarabinomannan in sputum to detect bacterial load and treatment response in patients with pulmonary tuberculosis: analytic validation and evaluation in two cohorts . PLoS Med , 2019 . 16 ( 4 ): e1002780 30978194 DOI: 10.1371/journal.pmed.1002780 http://doi.org/10.1371/journal.pmed.1002780 . Lipoarabinomannan in sputum to detect bacterial load and treatment response in patients with pulmonary tuberculosis: analytic validation and evaluation in two cohorts SL Kendall , M Withers , CN Soffair , 等 . A highly conserved transcriptional repressor controls a large regulon involved in lipid degradation in Mycobacterium smegmatis and Mycobacterium tuberculosis . Mol Microbiol , 2007 . 65 ( 3 ): 684 - 699 . 17635188 DOI: 10.1111/j.1365-2958.2007.05827.x http://doi.org/10.1111/j.1365-2958.2007.05827.x . A highly conserved transcriptional repressor controls a large regulon involved in lipid degradation in Mycobacterium smegmatis and Mycobacterium tuberculosis SD Lawn , AD Kerkhoff , M Vogt , 等 . Diagnostic accuracy of a low-cost, urine antigen, point-of-care screening assay for HIV-associated pulmonary tuberculosis before antiretroviral therapy: a descriptive study . Lancet Infect Dis , 2012 . 12 ( 3 ): 201 - 209 . 22015305 DOI: 10.1016/S1473-3099(11)70251-1 http://doi.org/10.1016/S1473-3099(11)70251-1 . Diagnostic accuracy of a low-cost, urine antigen, point-of-care screening assay for HIV-associated pulmonary tuberculosis before antiretroviral therapy: a descriptive study SD Lawn , AD Kerkhoff , MP Nicol , 等 . Underestimation of the true specificity of the urine lipoarabinomannan point-of-care diagnostic assay for HIV-associated tuberculosis . J Acquir Immune Defic Syndr , 2015 . 69 ( 4 ): e144 - e146 . 25961395 DOI: 10.1097/QAI.0000000000000672 http://doi.org/10.1097/QAI.0000000000000672 . Underestimation of the true specificity of the urine lipoarabinomannan point-of-care diagnostic assay for HIV-associated tuberculosis C Liu , Z Zhao , J Fan , 等 . Quantification of circulating Mycobacterium tuberculosis antigen peptides allows rapid diagnosis of active disease and treatment monitoring . Proc Natl Acad Sci USA , 2017 . 114 ( 15 ): 3969 - 3974 . DOI: 10.1073/pnas.1621360114 http://doi.org/10.1073/pnas.1621360114 . Quantification of circulating Mycobacterium tuberculosis antigen peptides allows rapid diagnosis of active disease and treatment monitoring J Liu , DK Jaijyan , QY Tang , 等 . Promising cytomegalovirus-based vaccine vector induces robust CD8 + T-cell response . Int J Mol Sci , 2019 . 20 ( 18 ): 4457 DOI: 10.3390/ijms20184457 http://doi.org/10.3390/ijms20184457 . Promising cytomegalovirus-based vaccine vector induces robust CD8+ T-cell response JC Lorenzi , AP Trombone , CD Rocha , 等 . Intranasal vaccination with messenger RNA as a new approach in gene therapy: use against tuberculosis . BMC Biotechnol, 10:77 , 2010 . DOI: 10.1186/1472-6750-10-77 http://doi.org/10.1186/1472-6750-10-77 . Intranasal vaccination with messenger RNA as a new approach in gene therapy: use against tuberculosis U Mack , GB Migliori , M Sester , 等 . LTBI: latent tuberculosis infection or lasting immune responses to M. tuberculosis ? A TBNET consensus statement . Eur Respir J , 2009 . 33 ( 5 ): 956 - 973 . 19407047 DOI: 10.1183/09031936.00120908 http://doi.org/10.1183/09031936.00120908 . LTBI: latent tuberculosis infection or lasting immune responses to M. tuberculosis? A TBNET consensus statement C Manca , K Lyashchenko , HG Wiker , 等 . Molecular cloning, purification, and serological characterization of MPT63, a novel antigen secreted by Mycobacterium tuberculosis . Infect Immun , 1997 . 65 ( 1 ): 16 - 23 . 8975887 DOI: 10.1128/IAI.65.1.16-23.1997 http://doi.org/10.1128/IAI.65.1.16-23.1997 . Molecular cloning, purification, and serological characterization of MPT63, a novel antigen secreted by Mycobacterium tuberculosis J Mateos , O Estévez , Á González-Fernández , 等 . Serum proteomics of active tuberculosis patients and contacts reveals unique processes activated during Mycobacterium tuberculosis infection . Sci Rep, 10:3844 , 2020 . DOI: 10.1038/s41598-020-60753-5 http://doi.org/10.1038/s41598-020-60753-5 . Serum proteomics of active tuberculosis patients and contacts reveals unique processes activated during Mycobacterium tuberculosis infection TK Means , SY Wang , E Lien , 等 . Human Toll-like receptors mediate cellular activation by Mycobacterium tuberculosis . J Immunol , 1999 . 163 ( 7 ): 3920 - 3927 . 10490993 . Human Toll-like receptors mediate cellular activation by Mycobacterium tuberculosis NR Meier , M Jacobsen , THM Ottenhoff , 等 . A systematic review on novel Mycobacterium tuberculosis antigens and their discriminatory potential for the diagnosis of latent and active tuberculosis . Front Immunol, 9:2476 , 2018 . DOI: 10.3389/fimmu.2018.02476 http://doi.org/10.3389/fimmu.2018.02476 . A systematic review on novel Mycobacterium tuberculosis antigens and their discriminatory potential for the diagnosis of latent and active tuberculosis JJ Mekalanos . Environmental signals controlling expression of virulence determinants in bacteria . J Bacteriol , 1992 . 174 ( 1 ): 1 - 7 . 1729202 DOI: 10.1128/jb.174.1.1-7.1992 http://doi.org/10.1128/jb.174.1.1-7.1992 . Environmental signals controlling expression of virulence determinants in bacteria P Monkongdee , KD McCarthy , KP Cain , 等 . Yield of acid-fast smear and mycobacterial culture for tuberculosis diagnosis in people with human immunodeficiency virus . Am J Respir Crit Care Med , 2009 . 180 ( 9 ): 903 - 908 . 19628775 DOI: 10.1164/rccm.200905-0692OC http://doi.org/10.1164/rccm.200905-0692OC . Yield of acid-fast smear and mycobacterial culture for tuberculosis diagnosis in people with human immunodeficiency virus M Morandi , M Sali , R Manganelli , 等 . Exploiting the mycobacterial cell wall to design improved vaccines against tuberculosis . J Infect Dev Ctries , 2013 . 7 ( 3 ): 169 - 181 . 23492994 DOI: 10.3855/jidc.3114 http://doi.org/10.3855/jidc.3114 . Exploiting the mycobacterial cell wall to design improved vaccines against tuberculosis DJ Murphy , JR Brown . Novel drug target strategies against Mycobacterium tuberculosis . Curr Opin Microbiol , 2008 . 11 ( 5 ): 422 - 427 . 18801459 DOI: 10.1016/j.mib.2008.08.001 http://doi.org/10.1016/j.mib.2008.08.001 . Novel drug target strategies against Mycobacterium tuberculosis AS Mustafa , YA Skeiky , R Al-Attiyah , 等 . Immunogenicity of Mycobacterium tuberculosis antigens in Mycobacterium bovis BCG-vaccinated and M. bovis- infected cattle . Infect Immun , 2006 . 74 ( 8 ): 4566 - 4572 . 16861643 DOI: 10.1128/IAI.01660-05 http://doi.org/10.1128/IAI.01660-05 . Immunogenicity of Mycobacterium tuberculosis antigens in Mycobacterium bovis BCG-vaccinated and M. bovis-infected cattle F Nurwidya , D Handayani , E Burhan , 等 . Molecular diagnosis of tuberculosis . Chonnam Med J , 2018 . 54 ( 1 ): 1 - 9 . 29399559 DOI: 10.4068/cmj.2018.54.1.1 http://doi.org/10.4068/cmj.2018.54.1.1 . Molecular diagnosis of tuberculosis E Ong , YQ He , ZH Yang . Epitope promiscuity and population coverage of Mycobacterium tuberculosis protein antigens in current subunit vaccines under development . Infect Genet Evol, 80:104186 , 2020 . DOI: 10.1016/j.meegid.2020.104186 http://doi.org/10.1016/j.meegid.2020.104186 . Epitope promiscuity and population coverage of Mycobacterium tuberculosis protein antigens in current subunit vaccines under development RP Pandey , S Kumar , S Ahmad , 等 . Use Chou’s 5-steps rule to evaluate protective efficacy induced by antigenic proteins of Mycobacterium tuberculosis encapsulated in chitosan nanoparticles . Life Sci, 256:117961 , 2020 . DOI: 10.1016/j.lfs.2020.117961 http://doi.org/10.1016/j.lfs.2020.117961 . Use Chou’s 5-steps rule to evaluate protective efficacy induced by antigenic proteins of Mycobacterium tuberculosis encapsulated in chitosan nanoparticles ST Park , CM Kang , RN Husson . Regulation of the SigH stress response regulon by an essential protein kinase in Mycobacterium tuberculosis . Proc Natl Acad Sci USA , 2008 . 105 ( 35 ): 13105 - 13110 . DOI: 10.1073/pnas.0801143105 http://doi.org/10.1073/pnas.0801143105 . Regulation of the SigH stress response regulon by an essential protein kinase in Mycobacterium tuberculosis R Paylor . Mouser P . Bovine tuberculosis . Michigan State University Veterinary Student, Graduate Student , 2014 . Michigan State University , 1 - 3 . . Bovine tuberculosis J Pieters , J Gatfield . Hijacking the host: survival of pathogenic mycobacteria inside macrophages . Trends Microbiol , 2002 . 10 ( 3 ): 142 - 146 . 11864824 DOI: 10.1016/S0966-842X(02)02305-3 http://doi.org/10.1016/S0966-842X(02)02305-3 . Hijacking the host: survival of pathogenic mycobacteria inside macrophages SM Pinto , R Verma , J Advani , 等 . Integrated multi-omic analysis of Mycobacterium tuberculosis H37Ra redefines virulence attributes . Front Microbiol, 9:1314 , 2018 . DOI: 10.3389/fmicb.2018.01314 http://doi.org/10.3389/fmicb.2018.01314 . Integrated multi-omic analysis of Mycobacterium tuberculosis H37Ra redefines virulence attributes JM Pollock , P Andersen . Predominant recognition of the ESAT-6 protein in the first phase of interferon with Mycobacterium bovis in cattle . Infect Immun , 1997 . 65 ( 7 ): 2587 - 2592 . 9199424 DOI: 10.1128/IAI.65.7.2587-2592.1997 http://doi.org/10.1128/IAI.65.7.2587-2592.1997 . Predominant recognition of the ESAT-6 protein in the first phase of interferon with Mycobacterium bovis in cattle AS Pym , P Brodin , R Brosch , 等 . Loss of RD1 contributed to the attenuation of the live tuberculosis vaccines Mycobacterium bovis BCG and Mycobacterium microti . Mol Microbiol , 2002 . 46 ( 3 ): 709 - 717 . 12410828 DOI: 10.1046/j.1365-2958.2002.03237.x http://doi.org/10.1046/j.1365-2958.2002.03237.x . Loss of RD1 contributed to the attenuation of the live tuberculosis vaccines Mycobacterium bovis BCG and Mycobacterium microti AS Pym , P Brodin , L Majlessi , 等 . Recombinant BCG exporting ESAT-6 confers enhanced protection against tuberculosis . Nat Med , 2003 . 9 ( 5 ): 533 - 539 . 12692540 DOI: 10.1038/nm859 http://doi.org/10.1038/nm859 . Recombinant BCG exporting ESAT-6 confers enhanced protection against tuberculosis PS Renshaw , P Panagiotidou , A Whelan , 等 . 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 . J Biol Chem , 2002 . 277 ( 24 ): 21598 - 21603 . 11940590 DOI: 10.1074/jbc.M201625200 http://doi.org/10.1074/jbc.M201625200 . 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 PS Renshaw , KL Lightbody , V Veverka , 等 . Structure and function of the complex formed by the tuberculosis virulence factors CFP–10 and ESAT–6 . EMBO J , 2005 . 24 ( 14 ): 2491 - 2498 . 15973432 DOI: 10.1038/sj.emboj.7600732 http://doi.org/10.1038/sj.emboj.7600732 . Structure and function of the complex formed by the tuberculosis virulence factors CFP–10 and ESAT–6 GM Rodriguez . Control of iron metabolism in Mycobacterium tuberculosis . Trends Microbiol , 2006 . 14 ( 7 ): 320 - 327 . 16759864 DOI: 10.1016/j.tim.2006.05.006 http://doi.org/10.1016/j.tim.2006.05.006 . Control of iron metabolism in Mycobacterium tuberculosis TR Rustad , MI Harrell , RL Liao , 等 . The enduring hypoxic response of Mycobacterium tuberculosis . PLoS ONE , 2008 . 3 ( 1 ): e1502 18231589 DOI: 10.1371/journal.pone.0001502 http://doi.org/10.1371/journal.pone.0001502 . The enduring hypoxic response of Mycobacterium tuberculosis M Ryndak , SS Wang , I Smith . PhoP, a key player in Mycobacterium tuberculosis virulence . Trends Microbiol , 2008 . 16 ( 11 ): 528 - 534 . 18835713 DOI: 10.1016/j.tim.2008.08.006 http://doi.org/10.1016/j.tim.2008.08.006 . PhoP, a key player in Mycobacterium tuberculosis virulence YJ Ryu . Diagnosis of pulmonary tuberculosis: recent advances and diagnostic algorithms . Tuber Respir Dis , 2015 . 78 ( 2 ): 64 - 71 . DOI: 10.4046/trd.2015.78.2.64 http://doi.org/10.4046/trd.2015.78.2.64 . Diagnosis of pulmonary tuberculosis: recent advances and diagnostic algorithms SB Sable , JE Posey , TJ Scriba . Tuberculosis vaccine development: progress in clinical evaluation . Clin Microbiol Rev , 2019 . 33 ( 1 ): e00100 - 19 . 31666281 DOI: 10.1128/CMR.00100-19 http://doi.org/10.1128/CMR.00100-19 . Tuberculosis vaccine development: progress in clinical evaluation R Santos , O Ursu , A Gaulton , 等 . A comprehensive map of molecular drug targets . Nat Rev Drug Dis , 2017 . 16 ( 1 ): 19 - 34 . DOI: 10.1038/nrd.2016.230 http://doi.org/10.1038/nrd.2016.230 . A comprehensive map of molecular drug targets LS Schlesinger . Macrophage phagocytosis of virulent but not attenuated strains of Mycobacterium tuberculosis is mediated by mannose receptors in addition to complement receptors . J Immunol , 1993 . 150 ( 7 ): 2920 - 2930 . 8454864 . Macrophage phagocytosis of virulent but not attenuated strains of Mycobacterium tuberculosis is mediated by mannose receptors in addition to complement receptors LS Schlesinger , CG Bellinger-Kawahara , NR Payne , 等 . Phagocytosis of Mycobacterium tuberculosis is mediated by human monocyte complement receptors and complement component C3 . J Immunol , 1990 . 144 ( 7 ): 2771 - 2780 . 2108212 . Phagocytosis of Mycobacterium tuberculosis is mediated by human monocyte complement receptors and complement component C3 JA Seddon , J Paton , Z Nademi , 等 . The impact of BCG vaccination on tuberculin skin test responses in children is age dependent: evidence to be considered when screening children for tuberculosis infection . Thorax , 2016 . 71 ( 10 ): 932 - 939 . 27335104 DOI: 10.1136/thoraxjnl-2015-207687 http://doi.org/10.1136/thoraxjnl-2015-207687 . The impact of BCG vaccination on tuberculin skin test responses in children is age dependent: evidence to be considered when screening children for tuberculosis infection M Shah , C Hanrahan , ZY Wang , 等 . Lateral flow urine lipoarabinomannan assay for detecting active tuberculosis in HIV–positive adults . Cochrane Database Syst Rev , 2016 . 2016 ( 5 ): CD011420 DOI: 10.1002/14651858.CD011420.pub2 http://doi.org/10.1002/14651858.CD011420.pub2 . Lateral flow urine lipoarabinomannan assay for detecting active tuberculosis in HIV–positive adults GB Sigal , A Pinter , TL Lowary , 等 . A novel sensitive immunoassay targeting the 5-methylthio- D -xylofuranose–lipoarabinomannan epitope meets the WHO’s performance target for tuberculosis diagnosis . J Clin Microbiol , 2018 . 56 ( 12 ): e01338 - 18 . 30257899 DOI: 10.1128/JCM.01338-18 http://doi.org/10.1128/JCM.01338-18 . A novel sensitive immunoassay targeting the 5-methylthio-D-xylofuranose–lipoarabinomannan epitope meets the WHO’s performance target for tuberculosis diagnosis VMC Silva , G Kanaujia , ML Gennaro , 等 . Factors associated with humoral response to ESAT-6, 38 kDa and 14 kDa in patients with a spectrum of tuberculosis . Int J Tuber Lung Dis , 2003 . 7 ( 5 ): 478 - 484 . . Factors associated with humoral response to ESAT-6, 38 kDa and 14 kDa in patients with a spectrum of tuberculosis A Singh , DK Crossman , D Mai , 等 . Mycobacterium tuberculosis WhiB3 maintains redox homeostasis by regulating virulence lipid anabolism to modulate macrophage response . PLoS Pathog , 2009 . 5 ( 8 ): e1000545 19680450 DOI: 10.1371/journal.ppat.1000545 http://doi.org/10.1371/journal.ppat.1000545 . Mycobacterium tuberculosis WhiB3 maintains redox homeostasis by regulating virulence lipid anabolism to modulate macrophage response P Sinha , A Gupta , P Prakash , 等 . Differentiation of Mycobacterium tuberculosis complex from non-tubercular mycobacteria by nested multiplex PCR targeting IS6110 , MTP40 and 32kD alpha antigen encoding gene fragments . BMC Infect Dis, 16:123 , 2016 . DOI: 10.1186/s12879-016-1450-1 http://doi.org/10.1186/s12879-016-1450-1 . Differentiation of Mycobacterium tuberculosis complex from non-tubercular mycobacteria by nested multiplex PCR targeting IS6110, MTP40 and 32kD alpha antigen encoding gene fragments I Smith . Mycobacterium tuberculosis pathogenesis and molecular determinants of virulence . Clin Microbiol Rev , 2003 . 16 ( 3 ): 463 - 496 . 12857778 DOI: 10.1128/CMR.16.3.463-496.2003 http://doi.org/10.1128/CMR.16.3.463-496.2003 . Mycobacterium tuberculosis pathogenesis and molecular determinants of virulence G Sreejit , A Ahmed , N Parveen , 等 . The ESAT-6 protein of Mycobacterium tuberculosis interacts with beta-2-microglobulin (β2M) affecting antigen presentation function of macrophage . PLoS Pathog , 2014 . 10 ( 10 ): e1004446 25356553 DOI: 10.1371/journal.ppat.1004446 http://doi.org/10.1371/journal.ppat.1004446 . The ESAT-6 protein of Mycobacterium tuberculosis interacts with beta-2-microglobulin (β2M) affecting antigen presentation function of macrophage HB Su , SL Zhu , L Zhu , 等 . Mycobacterium tuberculosis latent antigen Rv2029c from the multistage DNA vaccine A39 drives TH1 responses via TLR-mediated macrophage activation . Front Microbiol, 8:2266 , 2017 . DOI: 10.3389/fmicb.2017.02266 http://doi.org/10.3389/fmicb.2017.02266 . Mycobacterium tuberculosis latent antigen Rv2029c from the multistage DNA vaccine A39 drives TH1 responses via TLR-mediated macrophage activation S Swaminathan , B Rekha . Pediatric tuberculosis: global overview and challenges . Clin Infect Dis , 2010 . 50 ( S3 ): S184 - S194 . 20397947 DOI: 10.1086/651490 http://doi.org/10.1086/651490 . Pediatric tuberculosis: global overview and challenges TE Sweeney , L Braviak , CM Tato , 等 . Genome-wide expression for diagnosis of pulmonary tuberculosis: a multicohort analysis . Lancet Respir Med , 2016 . 4 ( 3 ): 213 - 224 . 26907218 DOI: 10.1016/S2213-2600(16)00048-5 http://doi.org/10.1016/S2213-2600(16)00048-5 . Genome-wide expression for diagnosis of pulmonary tuberculosis: a multicohort analysis I Vergne , J Chua , SB Singh , 等 . Cell biology of Mycobacterium tuberculosis phagosome . Annu Rev Cell Dev Biol , 2004 . 20 367 - 394 . 15473845 DOI: 10.1146/annurev.cellbio.20.010403.114015 http://doi.org/10.1146/annurev.cellbio.20.010403.114015 . Cell biology of Mycobacterium tuberculosis phagosome R Verma , SM Pinto , AH Patil , 等 . Quantitative proteomic and phosphoproteomic analysis of H37Ra and H37Rv strains of Mycobacterium tuberculosis . J Proteome Res , 2017 . 16 ( 4 ): 1632 - 1645 . 28241730 DOI: 10.1021/acs.jproteome.6b00983 http://doi.org/10.1021/acs.jproteome.6b00983 . Quantitative proteomic and phosphoproteomic analysis of H37Ra and H37Rv strains of Mycobacterium tuberculosis P Vir , D Gupta , R Agarwal , 等 . Interaction of alveolar epithelial cells with CFP21, a mycobacterial cutinase-like enzyme . Mol Cell Biochem , 2014 . 396 ( 1-2 ): 187 - 199 . 25091806 DOI: 10.1007/s11010-014-2154-8 http://doi.org/10.1007/s11010-014-2154-8 . Interaction of alveolar epithelial cells with CFP21, a mycobacterial cutinase-like enzyme MI Voskuil , D Schnappinger , KC Visconti , 等 . Inhibition of respiration by nitric oxide induces a Mycobacterium tuberculosis dormancy program . J Exp Med , 2003 . 198 ( 5 ): 705 - 713 . 12953092 DOI: 10.1084/jem.20030205 http://doi.org/10.1084/jem.20030205 . Inhibition of respiration by nitric oxide induces a Mycobacterium tuberculosis dormancy program III Weiner J , SHE Kaufmann . Recent advances towards tuberculosis control: vaccines and biomarkers . J Int Med , 2014 . 275 ( 5 ): 467 - 480 . DOI: 10.1111/joim.12212 http://doi.org/10.1111/joim.12212 . Recent advances towards tuberculosis control: vaccines and biomarkers J Weiner , J Maertzdorf , SHE Kaufmann . The dual role of biomarkers for understanding basic principles and devising novel intervention strategies in tuberculosis . Ann New York Acad Sci , 2013 . 1283 ( 1 ): 22 - 29 . DOI: 10.1111/j.1749-6632.2012.06802.x http://doi.org/10.1111/j.1749-6632.2012.06802.x . The dual role of biomarkers for understanding basic principles and devising novel intervention strategies in tuberculosis (World Health Organization) WHO . High-priority target product profiles for new tuberculosis diagnostics: report of a consensus meeting , 2014 . Geneva, Switzerland, WHO. https://apps.who.int/iris/handle/10665/135617 , . High-priority target product profiles for new tuberculosis diagnostics: report of a consensus meeting HO W . The use of lateral flow urine lipoarabinomannan assay (LF-LAM) for the diagnosis and screening of active tuberculosis in people living with HIV: policy guidance . 2015 . WHO. https://apps.who.int/iris/handle/10665/193633 . . The use of lateral flow urine lipoarabinomannan assay (LF-LAM) for the diagnosis and screening of active tuberculosis in people living with HIV: policy guidance HO W . Global tuberculosis report 2019 . 2019 . WHO. https:// apps.who.int/iris/handle/10665/329368 . . Global tuberculosis report 2019 AJ Wolf , L Desvignes , B Linas , 等 . Initiation of the adaptive immune response to Mycobacterium tuberculosis depends on antigen production in the local lymph node, not the lungs . J Exp Med , 2008 . 205 ( 1 ): 105 - 115 . 18158321 DOI: 10.1084/jem.20071367 http://doi.org/10.1084/jem.20071367 . Initiation of the adaptive immune response to Mycobacterium tuberculosis depends on antigen production in the local lymph node, not the lungs W Zhang , QB Shu , Z Zhao , 等 . Antigen 85B peptidomic analysis allows species-specific mycobacterial identification . Clin Proteomics , 2018 . 15 ( 1 ): 1 29321721 DOI: 10.1186/s12014-017-9177-6 http://doi.org/10.1186/s12014-017-9177-6 . Antigen 85B peptidomic analysis allows species-specific mycobacterial identification Compliance with ethics guidelines Elba RODRÍGUEZ-HERNÁNDEZ, Laura Itzel QUINTAS-GRANADOS, Susana FLORES-VILLALVA, Jorge Germinal CANTÓ-ALARCÓN, and Feliciano MILIÁN-SUAZO declare that they have no conflict of interest. This article does not contain any studies with human or animal subjects performed by any of the authors. 10.1631/jzus.B2000325 Project supported by the Consejo Nacional de Ciencia y Tecnología (CONACYT; No. 284118), México 2020-06-16 2020-08-19 2022-01-05 Copyright © Zhejiang University and Springer-Verlag GmbH Germany, part of Springer Nature 2020 2020 Journal of Zhejiang University-SCIENCE B (Biomedicine and Biotechnology) 11 21