Tuberculosis as a complex trait: impact of genetic epidemiological study design

Several studies have suggested a role for human genetic risk factors in the susceptibility to developing tuberculosis (TB). However, results of these studies have been inconsistent, and one potential reason for these inconsistencies is variation in aspects of study design. Specifically, phenotype definitions and population genetic factors have varied dramatically. Since TB is a complex trait, there are many challenges in designing studies to assess appropriately human genetic risk factors for the development of TB as opposed to the acquisition of latent M. tuberculosis infection. In this review we summarize these important study design differences, with illustrations from the TB genetics literature. We cite specific examples of studies of the NRAMP1 (SLC11A1) gene and present Fisher??s combined p values for different stratifications of these studies to further illustrate the impact of study design differences. Finally, we provide suggestions for the design of future genetic epidemiological studies of TB.     

Comparing epidemic tuberculosis in demographically distinct heterogeneous populations

There is wide variation in endemic tuberculosis (TB) levels between countries and we seek to identify possible causes of these differences. In this study we present an epidemiological model of Mycobacterium tuberculosis infection to investigate the effects of host genetics and demographic factors on epidemic TB. We discuss the general framework for this approach and present analytical results to identify important parameters affecting steady-state prevalence and incidence rates of TB disease. We then use numerical simulations of our model to observe the effects of a genetically susceptible subpopulation on TB disease dynamics at the population level. Finally, we simulate infection within a genetically heterogeneous population in two demographic settings: India (a typical population with high TB prevalence) and the USA (a typical population with low TB prevalence). Results show that changes in transmission parameters, the fraction of the population genetically susceptible to infection, and demographic factors strongly affect TB prevalence and incidence rates.     

Human genetics of tuberculosis: a long and winding road

Only a small fraction of individuals exposed to Mycobacterium tuberculosis develop clinical tuberculosis (TB). Over the past century, epidemiological studies have shown that human genetic factors contribute significantly to this interindividual variability, and molecular progress has been made over the past decade for at least two of the three key TB-related phenotypes: (i) a major locus controlling resistance to infection with M. tuberculosis has been identified, and (ii) proof of principle that severe TB of childhood can result from single-gene inborn errors of interferon-γ immunity has been provided; genetic association studies with pulmonary TB in adulthood have met with more limited success. Future genetic studies of these three phenotypes could consider subgroups of subjects defined on the basis of individual (e.g. age at TB onset) or environmental (e.g. pathogen strain) factors. Progress may also be facilitated by further methodological advances in human genetics. Identification of the human genetic variants controlling the various stages and forms of TB is critical for understanding TB pathogenesis. These findings should have major implications for TB control, in the definition of improved prevention strategies, the optimization of vaccines and clinical trials and the development of novel treatments aiming to restore deficient immune responses.     

Decreased plasma granulysin and increased interferon-gamma concentrations in patients with newly diagnosed and relapsed tuberculosis

Granulysin and interferon-gamma (IFN-γ) have broad antimicrobial activity which controls Mycobacterium tuberculosis (M. tuberculosis) infection. Circulating granulysin and IFN-γ concentrations were measured and correlated with clinical disease in Thai patients with newly diagnosed, relapsed and chronic tuberculosis (TB). Compared to controls, patients with newly diagnosed, relapsed and chronic TB had lower circulating granulysin concentrations, these differences being significant only in newly diagnosed and relapsed TB (P < 0.001 and 0.004, respectively). Granulysin concentrations in patients with newly diagnosed and relapsed TB were significantly lower than in those with chronic TB (P= 0.003 and P= 0.022, respectively). In contrast, significantly higher circulating IFN-γ concentrations were found in patients with newly diagnosed and relapsed TB compared to controls (P < 0.001). The IFN-γ concentrations in newly diagnosed and relapsed patients were not significantly different from those of patients with chronic TB. However, in vitro stimulation of peripheral blood mononuclear cells (PBMCs) from patients with newly diagnosed, relapsed and chronic TB with purified protein derivative (PPD) or heat killed M. tuberculosis (H37Ra) enhanced production of granulysin by PBMCs. In vitro, stimulation of PBMCs of newly diagnosed TB patients with PPD produced greater amounts of IFN-γ than did controls, while those stimulated with H37Ra did not. The results demonstrate that patients with active pulmonary TB have low circulating granulysin but high IFN-γ concentrations, suggesting possible roles in host defense against M. tuberculosis for these agents.     

Bovine tuberculosis: the genetic basis of host susceptibility

The prevalence of bovine tuberculosis (BTB) in the UK remains a significant economic burden and problem for the agri-food industry. Much effort has been directed towards improving diagnostics, finding vaccine candidates and assessing the usefulness of badger culling. The contribution that host genotype makes to disease outcome has, until recently, been overlooked; yet, it is biologically untenable that genetic variation does not play a role. In this review, we highlight the evidence, past and present, for a role of host genetics in determining susceptibility to BTB in livestock. We then address some of the major issues surrounding the design of future studies tasked with finding the exact causative genetic variation underpinning the TB susceptibility phenotype. Finally, we discuss some of the potential future benefits, and problems, that a knowledge of the genetic component to BTB resistance/susceptibility may bring to the agricultural industries and the wider scientific community.     

Mycobacterium marinum produces long-term chronic infections in medaka: a new animal model for studying human tuberculosis

Human infection by Mycobacterium tuberculosis is endemic, with approximately 2 billion infected and is the most common cause of adult death due to an infectious agent. Because of the slow growth rate of M. tuberculosis and risk to researchers, other species of Mycobacterium have been employed as alternative model systems to study human tuberculosis (TB). Mycobacterium marinum may be a good surrogate pathogen, conferring TB-like chronic infections in some fish. Medaka (Oryzias latipes) has been established for over five decades as a laboratory fish model for toxicology, genotoxicity, teratogenesis, carcinogenesis, classical genetics and embryology. We are investigating if medaka might also serve as a host for M. marinum in order to model human TB. We show that both acute and chronic infections are inducible in a dose dependent manner. Colonization of target organs and systemic granuloma formation has been demonstrated through the use of histology. M. marinum expressing green fluorescent protein (Gfp) was used to monitor bacterial colonization of these organs in fresh tissues as well as in intact animals. Moreover, we have employed the See-Through fish line, a variety of medaka devoid of major pigments, to monitor real-time disease progression, in living animals. We have also compared the susceptibility of another prominent fish model, zebrafish (Danio rerio), to our medaka-M. marinum model. We determined the course of infections in zebrafish is significantly more severe than in medaka. Together, these results indicate that the medaka-M. marinum model provides unique advantages for studying chronic mycobacteriosis.     

Spontaneous emergence of multiple drug resistance in tuberculosis before and during therapy

The emergence of drug resistance in M. tuberculosis undermines the efficacy of tuberculosis (TB) treatment in individuals and of TB control programs in populations. Multiple drug resistance is often attributed to sequential functional monotherapy, and standard initial treatment regimens have therefore been designed to include simultaneous use of four different antibiotics. Despite the widespread use of combination therapy, highly resistant M. tb strains have emerged in many settings. Here we use a stochastic birth-death model to estimate the probability of the emergence of multidrug resistance during the growth of a population of initially drug sensitive TB bacilli within an infected host. We find that the probability of the emergence of resistance to the two principal anti-TB drugs before initiation of therapy ranges from 10(-5) to 10(-4); while rare, this is several orders of magnitude higher than previous estimates. This finding suggests that multidrug resistant M. tb may not be an entirely "man-made" phenomenon and may help explain how highly drug resistant forms of TB have independently emerged in many settings.     

The polyketide Pks1 contributes to biofilm formation in Mycobacterium tuberculosis

Infections caused by biofilms are abundant and highly persistent, displaying phenotypic resistance to high concentrations of antimicrobials and modulating host immune systems. Tuberculosis (TB), caused by Mycobacterium tuberculosis, shares these qualities with biofilm infections. To identify genetic determinants of biofilm formation in M. tuberculosis, we performed a small-scale transposon screen using an in vitro pellicle biofilm assay. We identified five M. tuberculosis mutants that were reproducibly attenuated for biofilm production relative to that of the parent strain H37Rv. One of the most attenuated mutants is interrupted in pks1, a polyketide synthase gene. When fused with pks15, as in some M. tuberculosis isolates, pks1 contributes to synthesis of the immunomodulatory phenolic glycolipids (PGLs). However, in strains such as H37Rv with split pks15 and pks1 loci, PGL is not produced and pks1 has no previously defined role. We showed that pks1 complementation restores biofilm production independently of the known role of pks1 in PGL synthesis. We also assessed the relationship among biofilm formation, the pks15/1 genotype, and M. tuberculosis phylogeography. A global survey of M. tuberculosis clinical isolates revealed surprising sequence variability in the pks15/1 locus and substantial variation in biofilm phenotypes. Our studies identify novel M. tuberculosis genes that contribute to biofilm production, including pks1. In addition, we find that the ability to make pellicle biofilms is common among M. tuberculosis isolates from throughout the world, suggesting that this trait is relevant to TB propagation or persistence.     

Identification of Tuberculosis Susceptibility Genes with Human Macrophage Gene Expression Profiles

Although host genetics influences susceptibility to tuberculosis (TB), few genes determining disease outcome have been identified. We hypothesized that macrophages from individuals with different clinical manifestations of Mycobacterium tuberculosis (Mtb) infection would have distinct gene expression profiles and that polymorphisms in these genes may also be associated with susceptibility to TB. We measured gene expression levels of >38,500 genes from ex vivo Mtb-stimulated macrophages in 12 subjects with 3 clinical phenotypes: latent, pulmonary, and meningeal TB (n = 4 per group). After identifying differentially expressed genes, we confirmed these results in 34 additional subjects by real-time PCR. We also used a case-control study design to examine whether polymorphisms in differentially regulated genes were associated with susceptibility to these different clinical forms of TB. We compared gene expression profiles in Mtb-stimulated and unstimulated macrophages and identified 1,608 and 199 genes that were differentially expressed by >2- and >5-fold, respectively. In an independent sample set of 34 individuals and a subset of highly regulated genes, 90% of the microarray results were confirmed by RT-PCR, including expression levels of CCL1, which distinguished the 3 clinical groups. Furthermore, 6 single nucleotide polymorphisms (SNPs) in CCL1 were found to be associated with TB in a case-control genetic association study with 273 TB cases and 188 controls. To our knowledge, this is the first identification of CCL1 as a gene involved in host susceptibility to TB and the first study to combine microarray and DNA polymorphism studies to identify genes associated with TB susceptibility. These results suggest that genome-wide studies can provide an unbiased method to identify critical macrophage response genes that are associated with different clinical outcomes and that variation in innate immune response genes regulate susceptibility to TB.     

Evidence for a major gene influence on tumor necrosis factor-alpha expression in tuberculosis: path and segregation analysis

OBJECTIVE: Tuberculosis (TB) is a growing global public health problem. Several studies suggest a role for host genetics in disease susceptibility, but studies to date have been inconsistent and a comprehensive genetic model has not emerged. A limitation of previous genetic studies is that they only analyzed the binary trait TB, which does not reflect disease heterogeneity. Furthermore, these studies have not accounted for the influence of shared environment within households on TB risk, which may spuriously inflate estimates of heritability. METHODS: We conducted a household contact study in a TB-endemic community in Uganda. Antigen-induced tumor necrosis factor-alpha (TNFalpha) expression, a key component of the underlying immune response to TB, was used as an endophenotype for TB. RESULTS: Path analysis, conducted to assess the effect of shared environment, suggested that TNFalpha is heritable (narrow sense heritability = 34-66%); the effect of shared environment is minimal (1-14%), but gene-environment interaction may be involved. Segregation analysis of TNFalpha suggested a major gene model that explained one-third of the phenotypic variance, and provided putative evidence of natural selection acting on this phenotype. CONCLUSION: Our data further support TNFalpha as an endophenotype for TB, as it may increase power to detect disease-predisposing loci.     

Determinants of the Sympatric Host-Pathogen Relationship in Tuberculosis

Major contributions from pathogen genome analysis and host genetics have equated the possibility of Mycobacterium tuberculosis co-evolution with its human host leading to more stable sympatric host–pathogen relationships. However, the attribution to either sympatric or allopatric categories depends on the resolution or grain of genotypic characterization. We explored the influence on the sympatric host-pathogen relationship of clinical (HIV infection and multidrug-resistant tuberculosis [MDRTB]) and demographic (gender and age) factors in regards to the genotypic grain by using spacer oligonucleotide typing (spoligotyping) for classification of M. tuberculosis strains within the Euro-American lineage. We analyzed a total of 547 tuberculosis (TB) cases, from six year consecutive sampling in a setting with high TB-HIV coinfection (32.0%). Of these, 62.0% were caused by major circulating pathogen genotypes. The sympatric relationship was defined according to spoligotype in comparison to the international spoligotype database SpolDB4. While no significant association with Euro-American lineage was observed with any of the factors analyzed, increasing the resolution with spoligotyping evidenced a significant association of MDRTB with sympatric strains, regardless of the HIV status. Furthermore, distribution curves of the prevalence of sympatric and allopatric TB in relation to patients’ age showed an accentuation of the relevance of the age of onset in the allopatric relationship, as reflected in the trimodal distribution. On the contrary, sympatric TB was characterized by the tendency towards a typical (standard) distribution curve. Our results suggest that within the Euro-American lineage a greater degree of genotyping fine-tuning is necessary in modeling the biological processes behind the host-pathogen interplay. Furthermore, prevalence distribution of sympatric TB to age was suggestive of host genetic determinisms driven by more common variants.     

Host-pathogen coevolution in human tuberculosis

Tuberculosis (TB) is a disease of antiquity. Yet TB today still causes more adult deaths than any other single infectious disease. Recent studies show that contrary to the common view postulating an animal origin for TB, Mycobacterium tuberculosis complex (MTBC), the causative agent of TB, emerged as a human pathogen in Africa and colonized the world accompanying the Out-of-Africa migrations of modern humans. More recently, evolutionarily 'modern' lineages of MTBC expanded as a consequence of the global human population increase, and spread throughout the world following waves of exploration, trade and conquest. While epidemiological data suggest that the different phylogenetic lineages of MTBC might have adapted to different human populations, overall, the phylogenetically 'modern' MTBC lineages are more successful in terms of their geographical spread compared with the 'ancient' lineages. Interestingly, the global success of 'modern' MTBC correlates with a hypo-inflammatory phenotype in macrophages, possibly reflecting higher virulence, and a shorter latency in humans. Finally, various human genetic variants have been associated with different MTBC lineages, suggesting an interaction between human genetic diversity and MTBC variation. In summary, the biology and the epidemiology of human TB have been shaped by the long-standing association between MTBC and its human host.     

Comparison of the predicted population coverage of tuberculosis vaccine candidates Ag85B-ESAT-6, Ag85B-TB10.4, and Mtb72f via a bioinformatics approach

The Bacille-Calmette Guérin (BCG) vaccine does not provide consistent protection against adult pulmonary tuberculosis (TB) worldwide. As novel TB vaccine candidates advance in studies and clinical trials, it will be critically important to evaluate their global coverage by assessing the impact of host and pathogen variability on vaccine efficacy. In this study, we focus on the impact that host genetic variability may have on the protective effect of TB vaccine candidates Ag85B-ESAT-6, Ag85B-TB10.4, and Mtb72f. We use open-source epitope binding prediction programs to evaluate the binding of vaccine epitopes to Class I HLA (A, B, and C) and Class II HLA (DRB1) alleles. Our findings suggest that Mtb72f may be less consistently protective than either Ag85B-ESAT-6 or Ag85B-TB10.4 in populations with a high TB burden, while Ag85B-TB10.4 may provide the most consistent protection. The findings of this study highlight the utility of bioinformatics as a tool for evaluating vaccine candidates before the costly stages of clinical trials and informing the development of new vaccines with the broadest possible population coverage.     

Not to wake a sleeping giant: new insights into host-pathogen interactions identify new targets for vaccination against latent Mycobacterium tuberculosis infection

Mycobacterium tuberculosis is one of the worlds' most successful and sophisticated pathogens. It is estimated that over 2 billion people today harbour latent M. tuberculosis infection without any clinical symptoms. As most new cases of active tuberculosis (TB) arise from this (growing) number of latently infected individuals, urgent measures to control TB reactivation are required, including post-exposure/therapeutic vaccines. The current bacille Calmette-Guérin (BCG) vaccine and all new generation TB vaccines being developed and tested are essentially designed as prophylactic vaccines. Unfortunately, these vaccines are unlikely to be effective in individuals already latently infected with M. tuberculosis. Here, we argue that detailed analysis of M. tuberculosis genes that are switched on predominantly during latent stage infection may lead to the identification of new antigenic targets for anti-TB strategies. We will describe essential host-pathogen interactions in TB with particular emphasis on TB latency and persistent infection. Subsequently, we will focus on novel groups of late-stage specific genes, encoded amongst others by the M. tuberculosis dormancy (dosR) regulon, and summarise recent studies describing human T-cell recognition of these dormancy antigens in relation to (latent) M. tuberculosis infection. We will discuss the possible relevance of these new classes of antigens for vaccine development against TB.     

结核分枝杆菌感染实验模型

结核分枝杆菌是引起人结核病的主要病原,全世界约有1/3人口感染结核分枝杆菌。尽管该病原可感染并引起许多动物疾病,但人类是其中心宿主。为研究结核分枝杆菌的致病机理及宿主对本病原的保护性和免疫病理学反应,选择合适的动物模型非常必要。本文阐述了结核病研究中常用的实验模型及各种模型的优缺点。实验模型的合理应用将促进我们对结核病的认识,从中获取的资料将有助于我们发现更好的预防和治疗方案。     

结核分枝杆菌在动物中的流行与传播

结核分枝杆菌(Mycobacterium tuberculosis)是一种重要的人兽共患病病原菌,人类是其主要宿主。千百年来,由于动物与人类关系密切,动物也成为结核分枝杆菌的重要宿主,且感染结核分枝杆菌的动物还能成为传染源,将其传播至人类和其他动物。关于动物感染结核分枝杆菌已有大量报道,包括大象、非人灵长类动物、貘、海狮、犬、猫、牛、鸟等。本文就结核分枝杆菌在野生动物、家畜中的流行与传播进行介绍,总结其在动物间传播的主要途径。     

Variant G57E of mannose binding lectin associated with protection against tuberculosis caused by Mycobacterium africanum but not by M. tuberculosis

Structural variants of the Mannose Binding Lectin (MBL) cause quantitative and qualitative functional deficiencies, which are associated with various patterns of susceptibility to infectious diseases and other disorders. We determined genetic MBL variants in 2010 Ghanaian patients with pulmonary tuberculosis (TB) and 2346 controls and characterized the mycobacterial isolates of the patients. Assuming a recessive mode of inheritance, we found a protective association between TB and the MBL2 G57E variant (odds ratio 0.60, confidence interval 0.4-0.9, P 0.008) and the corresponding LYQC haplotype (P(corrected) 0.007) which applied, however, only to TB caused by M. africanum but not to TB caused by M. tuberculosis. In vitro, M. africanum isolates bound recombinant human MBL more efficiently than did isolates of M. tuberculosis. We conclude that MBL binding may facilitate the uptake of M. africanum by macrophages, thereby promoting infection and that selection by TB may have favoured the spread of functional MBL deficiencies in regions endemic for M. africanum.     

Crystallographic and pre-steady-state kinetics studies on binding of NADH to wild-type and isoniazid-resistant enoyl-ACP(CoA) reductase enzymes from Mycobacterium tuberculosis

An understanding of isoniazid (INH) drug resistance mechanism in Mycobacterium tuberculosis should provide significant insight for the development of newer anti-tubercular agents able to control INH-resistant tuberculosis (TB). The inhA-encoded 2-trans enoyl-acyl carrier protein reductase enzyme (InhA) has been shown through biochemical and genetic studies to be the primary target for INH. In agreement with these results, mutations in the inhA structural gene have been found in INH-resistant clinical isolates of M.tuberculosis, the causative agent of TB. In addition, the InhA mutants were shown to have higher dissociation constant values for NADH and lower values for the apparent first-order rate constant for INH inactivation as compared to wild-type InhA. Here, in trying to identify structural changes between wild-type and INH-resistant InhA enzymes, we have solved the crystal structures of wild-type and of S94A, I47T and I21V InhA proteins in complex with NADH to resolutions of, respectively, 2.3A, 2.2A, 2.0 A, and 1.9A. The more prominent structural differences are located in, and appear to indirectly affect, the dinucleotide binding loop structure. Moreover, studies on pre-steady-state kinetics of NADH binding have been carried out. The results showed that the limiting rate constant values for NADH dissociation from the InhA-NADH binary complexes (k(off)) were eleven, five, and tenfold higher for, respectively, I21V, I47T, and S94A INH-resistant mutants of InhA as compared to INH-sensitive wild-type InhA. Accordingly, these results are proposed to be able to account for the reduction in affinity for NADH for the INH-resistant InhA enzymes.     

Ancient origin and gene mosaicism of the progenitor of Mycobacterium tuberculosis

The highly successful human pathogen Mycobacterium tuberculosis has an extremely low level of genetic variation, which suggests that the entire population resulted from clonal expansion following an evolutionary bottleneck around 35,000 y ago. Here, we show that this population constitutes just the visible tip of a much broader progenitor species, whose extant representatives are human isolates of tubercle bacilli from East Africa. In these isolates, we detected incongruence among gene phylogenies as well as mosaic gene sequences, whose individual elements are retrieved in classical M. tuberculosis. Therefore, despite its apparent homogeneity, the M. tuberculosis genome appears to be a composite assembly resulting from horizontal gene transfer events predating clonal expansion. The amount of synonymous nucleotide variation in housekeeping genes suggests that tubercle bacilli were contemporaneous with early hominids in East Africa, and have thus been coevolving with their human host much longer than previously thought. These results open novel perspectives for unraveling the molecular bases of M. tuberculosis evolutionary success.