Analysis of the role of Mycobacterium tuberculosis kasA gene mutations in isoniazid resistance

Previous studies have suggested that Mycobacterium tuberculosis kasA G312S and G269S gene mutations may represent sequence polymorphisms of the M. tuberculosis East-African-Indian (EAI) and T families, respectively, rather than relating to isoniazid resistance. The present study examined polymorphisms of these two codons in 98 drug-susceptible M. tuberculosis isolates (68 EAI and 30 T isolates). Twenty-eight isolates belonging to a sub-lineage of the EAI family had the kasA G312S mutation, but none of the 30 T isolates had the G269S mutation. The data suggest that the kasA G312S mutation is not related to isoniazid resistance, but represents a sequence polymorphism in a sub-lineage of the EAI family.     

Control of Mycobacterium tuberculosis growth by activated natural killer cells

We characterized the underlying mechanisms by which glutathione (GSH)‐enhanced natural killer (NK) cells inhibit the growth of Mycobacterium tuberculosis (M. tb) inside human monocytes. We observed that in healthy individuals, treatment of NK cells with N‐acetyl cysteine (NAC), a GSH prodrug in conjunction with cytokines such as interleukin (IL)‐2 + IL‐12, resulted in enhanced expression of NK cytotoxic ligands (FasL and CD40L) with concomitant stasis in the intracellular growth of M. tb. Neutralization of FasL and CD40L in IL‐2 + IL‐12 + NAC‐treated NK cells resulted in abrogation in the growth inhibition of M. tb inside monocytes. Importantly, we observed that the levels of GSH are decreased significantly in NK cells derived from individuals with HIV infection compared to healthy subjects, and this decrease correlated with a several‐fold increase in the growth of M. tb inside monocytes. This study describes a novel innate defence mechanism adopted by NK cells to control M. tb infection.     

Evaluation of the GenoType MTBDR assay for detection of rifampicin and isoniazid resistance in Mycobacterium tuberculosis complex isolates

Detection of drug resistance plays a critical role in tuberculosis treatment. The aim of this study was to evaluate the performance of GenoType Mycobacteria Drug Resistance (MTBDR) assay (Hain Lifescience, Germany) and to compare it with radiometric BACTEC 460 TB system (Becton Dickinson, USA) for the detection of rifampicin (RIF) and isoniazid (INH) resistance in 84 Mycobacterium tuberculosis complex (MTBC) isolates. RIF resistance was identified in 6 of 7 (85.7%) isolates and INH resistance was identified in 8 of 14 (57.1%) isolates by the GenoType MTBDR assay. Compared with BACTEC system, the sensitivity, specificity, positive predictive value and negative predictive values were 85.7%, 98.7%, 85.7% and 98.7% for RIF resistance; and 57.1%, 100%, 100% and 92.1% for INH resistance, respectively. GenoType MTBDR assay is reliable when tested specimen is resistant to the tested drugs. Although test was more successful in the detection of RIF resistance, it exhibited low sensitivity for the detection of INH resistance.     

Intestinal microbiota controls graft-versus-host disease independent of donor-host genetic disparity

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Mycobacterium tuberculosis,macrophages, and the innate immune response: does common variation matter?

Summary:  Despite the discovery of the tuberculosis (TB) bacillus over 100 years ago and the availability of effective drugs for over 50 years, there remain a number of formidable challenges for controlling Mycobacterium tuberculosis (MTb). Understanding the genetic and immunologic factors that influence human susceptibility could lead to novel insights for vaccine development as well as diagnostic advances to target treatment to those who are at risk for developing active disease. Although a series of studies over the past 50 years suggests that host genetics influences resistance to TB, a comprehensive understanding of which genes and variants are associated with susceptibility is only partially understood. In this article, we review recent advances in our understanding of human variation of the immune system and its effects on macrophage function and influence on MTb susceptibility. We emphasize recent discoveries in human genetic studies and correlate these findings with efforts to understand how these variants alter the molecular and cellular functions that regulate the macrophage response to MTb.     

Interleukin-10 production by lung macrophages in CBA xid mutant mice infected with Mycobacterium tuberculosis

Mice on the CBA inbred strain background expressing the well characterized mutation designated xid in the cytoplasmic signalling enzyme Bruton's protein kinase have been previously noted to illustrate shifts in T helper type 1 (Th1)/Th2 immunity which is underlined by an apparent failure to produce the regulatory cytokine interleukin-10. In the current study we examined if this extended to infection with Mycobacterium tuberculosis, which also depends on Th1 immunity. Contrary to expectations, xid mice showed evidence of a transient early susceptibility to pulmonary infection, changes in macrophage morphology, and decreased activation of lung natural killer cells, while showing evidence of substantial IL-10 production and accumulation in lung lesions macrophages, but paradoxically this did not influence the course of the chronic disease. In addition, macrophages from the lungs of xid mice also expressed high levels of CD14. These observations suggest that the xid mutation in cellular signalling has much wider effects on the immune system than previously thought.     

Early clearance of Mycobacterium tuberculosis: a new frontier in prevention

Early clearance (EC) is the successful eradication of inhaled Mycobacterium tuberculosis before an adaptive immune response develops. Evidence for EC comes from case contact studies that consistently show that a proportion of heavily exposed individuals do not develop M. tuberculosis infection. Further support for the existence of this phenotype comes from genetic loci associated with tuberculin reactivity. In this review we discuss aspects of the innate response that may underpin EC and hypotheses that can be tested through field laboratory link studies in M. tuberculosis case contacts. Specifically, we consider mechanisms whereby alveolar macrophages recognize and kill intracellular M. tuberculosis, and how other cell types, such as neutrophils, natural killer T cells, mucosa‐associated invariant T cells and γδ T cells may assist. How EC may be impaired by HIV infection or vitamin D deficiency is also explored. As EC is a form of protective immunity, further study may advance the development of vaccines and immunotherapies to prevent M. tuberculosis infection.     

Characterization of the protective T-cell response generated in CD4-deficient mice by a live attenuated Mycobacterium tuberculosis vaccine

The global epidemic of tuberculosis, fuelled by acquired immune-deficiency syndrome, necessitates the development of a safe and effective vaccine. We have constructed a DeltaRD1DeltapanCD mutant of Mycobacterium tuberculosis (mc(2)6030) that undergoes limited replication and is severely attenuated in immunocompromised mice, yet induces significant protection against tuberculosis in wild-type mice and even in mice that completely lack CD4(+) T cells as a result of targeted disruption of their CD4 genes (CD4(-/-) mice). Ex vivo studies of T cells from mc(2)6030-immunized mice showed that these immune cells responded to protein antigens of M. tuberculosis in a major histocompatibility complex (MHC) class II-restricted manner. Antibody depletion experiments showed that antituberculosis protective responses in the lung were not diminished by removal of CD8(+), T-cell receptor gammadelta (TCR-gammadelta(+)) and NK1.1(+) T cells from vaccinated CD4(-/-) mice before challenge, implying that the observed recall and immune effector functions resulting from vaccination of CD4(-/-) mice with mc(2)6030 were attributable to a population of CD4(-) CD8(-) (double-negative) TCR-alphabeta(+), TCR-gammadelta(-), NK1.1(-) T cells. Transfer of highly enriched double-negative TCR-alphabeta(+) T cells from mc(2)6030-immunized CD4(-/-) mice into naive CD4(-/-) mice resulted in significant protection against an aerosol tuberculosis challenge. Enriched pulmonary double-negative T cells transcribed significantly more interferon-gamma and interleukin-2 mRNA than double-negative T cells from naive mice after a tuberculous challenge. These results confirmed previous findings on the potential for a subset of MHC class II-restricted T cells to develop and function without expression of CD4 and suggest novel vaccination strategies to assist in the control of tuberculosis in human immunodeficiency virus-infected humans who have chronic depletion of their CD4(+) T cells.     

Interplay of innate lymphoid cells and the microbiota

Innate lymphoid cells (ILC) are a recently identified group of innate lymphocytes that are preferentially located at barrier surfaces. Barrier surfaces are in direct contact with complex microbial ecosystems, collectively referred to as the microbiota. It is now believed that the interplay of the microbiota with host components (i.e. epithelial cells and immune cells) promotes host fitness by regulating organ homeostasis, metabolism, and host defense against pathogens. In this review, we will give an overview of this multifaceted interplay between ILC and components of the microbiota.     

Sensing of Mycobacterium tuberculosis and consequences to both host and bacillus

Mycobacterium tuberculosis (Mtb), the primary causative agent of human tuberculosis, has killed more people than any other bacterial pathogen in human history and remains one of the most important transmissible diseases worldwide. Because of the long-standing interaction of Mtb with humans, it is no surprise that human mucosal and innate immune cells have evolved multiple mechanisms to detect Mtb during initial contact. To that end, the cell surface of human cells is decorated with numerous pattern recognition receptors for a variety of mycobacterial ligands. Furthermore, once Mtb is ingested into professional phagocytes, other host molecules are engaged to report on the presence of an intracellular pathogen. In this review, we discuss the role of specific mycobacterial products in modulating the host's ability to detect Mtb. In addition, we describe the specific host receptors that mediate the detection of mycobacterial infection and the role of individual receptors in mycobacterial pathogenesis in humans and model organisms.     

Immunological consequences of strain variation within the Mycobacterium tuberculosis complex

In 2015, there were an estimated 10.4 million new cases of tuberculosis (TB) globally, making it one of the leading causes of death due to an infectious disease. TB is caused by members of the Mycobacterium tuberculosis complex (MTBC), with human disease resulting from infection by M. tuberculosis sensu stricto and M. africanum. Recent progress in genotyping techniques, in particular the increasing availability of whole genome sequence data, has revealed previously under appreciated levels of genetic diversity within the MTBC. Several studies have shown that this genetic diversity may translate into differences in TB transmission, clinical manifestations of disease, and host immune responses. This suggests the existence of MTBC genotype‐dependent host–pathogen interactions which may influence the outcome of infection and progression of disease. In this review, we highlight the studies demonstrating differences in innate and adaptive immunological outcomes consequent on MTBC genetic diversity, and discuss how these differences in immune response might influence the development of TB vaccines, diagnostics and new therapies.     

Mycobacterium tuberculosis PPE18 protein inhibits MHC class II antigen presentation and B cell response in mice

PPE18 protein belongs to PE/PPE family of Mycobacterium tuberculosis. We reported earlier that PPE18 protein provides survival advantage to M. tuberculosis during infection. In the current study, we found that PPE18 inhibits MHC class II-mediated antigen presentation by macrophages in a dose-dependent manner without affecting the surface level of MHC class II or co-stimulatory molecules. PPE18 does not affect antigen uptake or presentation of preprocessed peptide by macrophages. Antigen degradation was found to be inhibited by PPE18 protein due to perturbation in phagolysosomal acidification. PPE18-mediated inhibition of MHC class II antigen presentation caused poorer activation of CD4 T cells. Mice infected with M. smegmatis expressing PPE18 exhibited reduced maturation and activation of B cells and had decreased Mycobacteria-specific antibody titers. Thus M. tuberculosis probably utilizes PPE18 to inhibit MHC class II antigen presentation causing poorer activation of adaptive immune responses. This study may be useful in understanding host–pathogen interaction and open up directions of designing novel therapeutics targeting PPE18 to tackle this nefarious pathogen.     

异烟肼耐药和敏感结核分枝杆菌的比较蛋白质组学研究

目的　研究结核分枝杆菌异烟肼耐药菌株与敏感菌株的蛋白质表达差异 ,鉴定结核分枝杆菌中与异烟肼耐药相关的菌体蛋白。方法　应用双向电泳技术比较 2株异烟肼耐药菌株与 2株敏感菌株的全菌蛋白表达图谱差异 ,发现异烟肼耐药结核分枝杆菌表达量显著增加 2 6个蛋白质斑点 ,通过基质辅助激光解吸 /电离飞行时间质谱仪进行质谱分析 ,获得 6个明确的肽质量指纹谱 ,通过蛋白质数据库进行检索分析。结果　 6个蛋白分别为海藻糖磷酸磷酸酶、铁钼蛋白A、莽草酸 5脱氢酶、葡萄糖胺果糖 6磷酸氨基转移酶、吲哚 3甘油磷酸合成酶、TetR家族转录调控因子。结论　上述蛋白的鉴定将对发现新型抗结核药物靶位和异烟肼耐药结核病诊断的分子标志物可能有所裨益     

Acquired drug resistance in Mycobacterium tuberculosis isolates from treated patients in the province of Bursa, Turkey

Objective: To evaluate the distribution of acquired resistance in isolates of Mycobacterium tuberculosis from treated patients in two periods, 1984–89 and 1990–95, in the Bursa (Southern Marmara) region.
Method: Susceptibility of 531 M. tuberculosis isolates to four commonly used drugs (isoniazid (INH), streptomycin (SM), ethambutol (EMB) and rifampin (RMP)) was determined by the absolute concentration method of Canetti et al.
Results: In 203 strains isolated in the years 1984–89, the total acquired resistance was 32.5%, and it was 37.5% in 328 strains isolated in 1990–95 ( p >0.05). Resistance to INH, SM, RMP and EMB was found in 23.6%, 16.7%, 6.4% and 3.9%, respectively, in the first period (1984–89), and in 26.2%, 20.4%, 25.3% and 8.2%, respectively, in the second period (1990–95). The increase in RMP resistance was statistically significant ( p <0.001). The incidence of multidrug-resistant strains was 12.3% in the first period, and 24.4% in the second period, a significant increase ( p <0.001).
Conclusions: We believe that progressive emergence of phenotypes resistant to INH+RMP in our region is caused by inadequate treatment for various reasons. In the present study, the fact that multidrug resistance occurred in nearly 25% of patients treated previously but still infective suggests that the approach to surveillance, patient therapy and follow-up programs should be fundamentally reconsidered in our region.     

Gut microbiota alterations affect glioma growth and innate immune cells involved in tumor immunosurveillance in mice

Glioma is a CNS tumor with few therapeutic options. Recently, host microbiota has been involved in the immune modulation of different tumors, but no data are available on the possible effects of the gut–immune axis on brain tumors. Here, we investigated the effect of gut microbiota alteration in a syngeneic (GL261) mouse model of glioma, treating mice with two antibiotics (ABX) and evaluating the effects on tumor growth, microbe composition, natural killer (NK) cells and microglia phenotype. We report that ABX treatment (i) altered the intestinal microbiota at family level, (ii) reduced cytotoxic NK cell subsets, and (iii) altered the expression of inflammatory and homeostatic proteins in microglia. All these findings could contribute to the increased growth of intracranial glioma that was observed after ABX treatment. These results demonstrate that chronic ABX administration alters microbiota composition and contributes to modulate brain immune state paving the way to glioma growth.     

Differences in T‐cell responses between Mycobacterium tuberculosis and Mycobacterium africanum‐infected patients

In The Gambia, Mycobacterium tuberculosis (Mtb) and Mycobacterium africanum (Maf) are major causes of tuberculosis (TB). Maf is more likely to cause TB in immune suppressed individuals, implying differences in virulence. Despite this, few studies have assessed the underlying immunity to the two pathogens in human. In this study, we analyzed T‐cell responses from 19 Maf‐ and 29 Mtb‐infected HIV‐negative patients before and after TB chemotherapy following overnight stimulation of whole blood with TB‐specific antigens. Before treatment, percentages of early secreted antigenic target‐6(ESAT‐6)/culture filtrate protein‐10(CFP‐10) and purified protein derivative‐specific single‐TNF‐α‐producing CD4⁺ and CD8⁺ T cells were significantly higher while single‐IL‐2‐producing T cells were significantly lower in Maf‐ compared with Mtb‐infected patients. Purified protein derivative‐specific polyfunctional CD4⁺ T cells frequencies were significantly higher before than after treatment, but there was no difference between the groups at both time points. Furthermore, the proportion of CD3⁺CD11b⁺ T cells was similar in both groups pretreatment, but was significantly lower with higher TNF‐α, IL‐2, and IFN‐γ production in Mtb‐ compared with that of Maf‐infected patients posttreatment. Our data provide evidence of differences in T‐cell responses to two mycobacterial strains with differing virulence, providing some insight into TB pathogenesis with different Mtb strains that could be prospectively explored as biomarkers for TB protection or susceptibility.     

A role for interleukin‐17A in modulating intracellular survival of Mycobacterium bovis bacillus Calmette–Guérin in murine macrophages

Interleukin 17A IL‐17A is a crucial immunomodulator in various chronic immunological diseases including rheumatoid arthritis and inflammatory bowel disease. The cytokine has also been demonstrated to control the pathogenesis of the Mycobacterium tuberculosis by dysregulating production of cytokines and chemokines and promoting granuloma formation. Whether IL‐17A regulates innate defence mechanisms of macrophages in response to mycobacterial infection remains to be elucidated. In the current report, we investigated the effects of IL‐17A on modulating the intracellular survival of Mycobacterium bovis bacillus Calmette–Guérin (BCG) in RAW264.7 murine macrophages. We observed that IL‐17A pre‐treatment for 24 hr was able to synergistically enhance BCG‐induced nitric oxide (NO) production and inducible nitric oxide synthase expression in dose‐ and time‐dependent manners. We further delineated the mechanisms involved in this synergistic reaction. IL‐17A was found to specifically enhanced BCG‐induced phosphorylation of Jun N‐terminal kinase (JNK), but not of extracellular signal‐regulated kinase 1/2 and p38 mitogen‐activated protein kinase. By using a specific JNK inhibitor (SP600125), we found that the production of NO in BCG‐infected macrophages was significantly suppressed. Taken together, we confirmed the involvement of the JNK pathway in IL‐17A‐enhanced NO production in BCG‐infected macrophages. We further demonstrated that IL‐17A significantly enhanced the clearance of intracellular BCG by macrophages through an NO‐dependent killing mechanism. In conclusion, our study revealed an anti‐mycobacterial role of IL‐17A through priming the macrophages to produce NO in response to mycobacterial infection.     

New tricks for old dogs: countering antibiotic resistance in tuberculosis with host-directed therapeutics

Despite the availability of Mycobacterium tuberculosis (Mtb) drugs for over 50 years, tuberculosis (TB) remains at pandemic levels. New drugs are urgently needed for resistant strains, shortening duration of treatment, and targeting different stages of the disease, especially for treatment during human immunodeficiency virus co-infection. One solution to the conundrum that antibiotics kill the bacillus yet select for resistance is to target the host rather than the pathogen. Here, we discuss recent progress in so-called ‘host-directed therapeutics’ (HDTs), focusing on two general mechanistic strategies: (i) HDTs that disrupt Mtb pathogenesis in macrophages and (ii) immunomodulatory HDTs that facilitate protective immune responses that kill Mtb or reduce deleterious responses that exacerbate disease. HDTs hold significant promise as adjunctive therapies in that they are less likely to engender resistance, will likely have efficacy against antibiotic-resistant strains, and may have activity against non-replicating Mtb. However, TB is a complex and variegated disease, and human populations exhibit significant diversity in their immune responses to it, which presents a complicated landscape for HDTs to navigate. Nevertheless, we suggest that a detailed mechanistic understanding of drug action, together with careful selection of disease stage targets and dosing strategies may overcome such limitations and allow the development of HDTs as effective adjunctive treatment options for TB.