Mycobacterium tuberculosis and the intimate discourse of a chronic infection

Mycobacterium tuberculosis is an extremely successful pathogen that demonstrates the capacity to modulate its host both at the cellular and tissue levels. At the cellular level, the bacterium enters its host macrophage and arrests phagosome maturation, thus avoiding many of the microbicidal responses associated with this phagocyte. Nonetheless, the intracellular environment places certain demands on the pathogen, which, in response, senses the environmental shifts and upregulates specific metabolic programs to allow access to nutrients, minimize the consequences of stress, and sustain infection. Despite its intracellular niche, Mycobacterium tuberculosis demonstrates a marked capacity to modulate the tissues surrounding infected cells through the release of potent, bioactive cell wall constituents. These cell wall lipids are released from the host cell by an exocytic process and induce physiological changes in neighboring phagocytes, which drives formation of a granuloma. This tissue response leads to the generation and accumulation of caseous debris and the progression of the human tuberculosis granuloma. Completion of the life cycle of tuberculosis requires damaging the host to release infectious bacteria into the airways to spread the infection. This damage reflects the pathogen's ability to subvert the host's innate and acquired immune responses to its own nefarious ends.     

Killing of Mycobacterium tuberculosis within human monocytes: activation by cytokines and calcitriol.

Human monocytes were isolated and their ability to harbour growth of virulent tubercle bacilli was assessed, in the presence or absence of various immunomodulators. Calcitriol (1,25(OH2), vitamin D3) alone, at doses of 10(-7)-10(-9) M endowed human monocytes with a significant ability to restrict intracellular growth of the tubercle bacilli. Crude immune lymphokines as well as recombinant interferon-gamma (IFN-gamma) endowed monocytes with no tuberculostatic activity. Similarly, other recombinant cytokines tested, notably colony-stimulating factor-1 (CSF-1), interleukin-1 (IL-1), interleukin-3 (IL-3) and interleukin-6 (IL-6) all failed to stimulate anti-tuberculous properties, and even increased growth of the tubercle bacilli in monocytes, in the case of CSF-1. Conversely, incubation of crude lymphokines in combination with calcitriol led to total stasis of the growth of M. tuberculosis. Experiments with recombinant cytokines and immunologically active vitamins showed that a combination of IFN-gamma tumour necrosis factor-alpha and calcitriol induced a significant amount of intramonocyte killing of M. tuberculosis. Addition of this cocktail of factors to already infected monocytes led to substantial killing of tubercle bacilli. These sets of experiments establish clearly that combinations of recombinant cytokines and vitamins may induce substantial intramonocyte killing of M. tuberculosis. The mechanism involved in this killing activity was not clarified.     

Toxicity effects of AgZnO nanoparticles and rifampicin on Mycobacterium tuberculosis into the macrophage

The World Health Organization acknowledges tuberculosis as a global threat. Tuberculosis infection is one of the top 10 causes of death worldwide. Nanotechnology and microbiology researchers are looking for new and safe nano drugs for eliminating Mycobacterium tuberculosis, the causative agent of tuberculosis. In this study, AgZnO nano‐crystals (AgZnONCs) is synthesized via the decomposition of the precursor of oxalate method. Characterization of AgZnONCs were evaluated. Next, various concentrations of AgZnONCs, as well AgZnONCs+Rifampicin, were prepared. The MTT assay was employed to study the viability of human macrophage cell lines (THP‐1) exposed to AgZnONCs. The bactericidal effects of AgZnONCs and AgZnONCs+Rifampicin were studied by Minimum Bactericidal Concentration (MBC) test. Subsequently, THP‐1 were infected by H₃₇Rv strain of M. tuberculosis (H₃₇RvMtb). Also, bactericidal effects of AgZnONCs and AgZnONCs+Rifampicin were compared with ex‐vivo conditions. The MBC of AgZnONCs and AgZnONCs+Rifampicin were ratios of 1:4 and 1:32 respectively (p‐value <0.05). Also, more than 50% and 80% of THP‐1 were alive in ratios of 1:4 and 1:32 in the presence of AgZnONCs, respectively. All phagocytic H₃₇RvMtb were killed in the presence of AgZnONCs+Rifampicin (p‐value <0.05), while AgZnONCs were not able to kill all the H₃₇RvMtb (p‐value >0.05). This study showed that, AgZnONCs+Rifampicin has the most anti‐tubercular behavior with respect to the macrophages.     

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 infection within Warthin's tumor: report of two cases

We report two patients with Warthin's tumor who were also infected with Mycobacterium tuberculosis. Case 1 was a 75-year-old woman with Warthin's tumor and multiple small epithelioid granulomas with caseous necrosis involving the submandibular gland. This patient died of tuberculous meningitis 4 months after biopsy. Case 2 was a 78-year-old man with a 10-year history of a parotid mass which had enlarged rapidly over 2 months. Surgical excision revealed Warthin's tumor and epithelioid granulomas involving the left parotid gland. DNA extracted from paraffin sections was amplified by nested polymerase chain reaction (PCR) with primer sets for the mycobacterial 65-KDa antigen gene. Restriction enzyme digestion of the PCR products could differentiate Mycobacterium tuberculosis from other mycobacteria in both cases. Although the histogenesis of lymphoid components of Warthin's tumor is controversial, the frequent prevalence of inflammation or necrosis and our present findings suggest these components have a similar behavior to regional lymph nodes.     

结核分枝杆菌PhoPR双组分系统与不同毒力结核分枝杆菌致病性的相关性研究

目的：通过比较分析卡介苗株（BCG）、结核分枝杆菌国际标准无毒株（H37Ra）、结核分枝杆菌国际标准强毒株（ H37Rv）、新疆地区流行的优势强毒株结核分枝杆菌临床分离株（ XJ-MTB）的PhoP基因和PhoR基因的表达水平差异,探讨研究结核分枝杆菌PhoPR双组分系统与不同毒力结核分枝杆菌的致病性是否具有相关性。方法：首先提取上述四种不同毒力结核分枝杆菌菌株的总RNA,并进行完整性鉴定;再运用SYBR Green I实时荧光定量PCR技术检测各组菌株中PhoP基因和PhoR基因的表达水平;最后比较不同毒力结核分枝杆菌PhoP基因和PhoR基因的表达水平差异。结果：四种不同毒力菌株PhoP基因的相对表达量,由高到低依次为XJ-MTB（9.05）、H37Rv（1.00）、H37Ra（0.25）、BCG（0.08）,且四种菌株中PhoP基因的表达有显著的统计学差异（P＜0.05）;同时四种不同毒力菌株PhoR基因的相对表达量,由高到低依次为XJ-MTB（5.72）、H37Rv（1.00）、H37Ra（0.18）、BCG（0.07）,且四种菌株中PhoR基因的表达有显著的统计学差异（P＜0.05）。其中XJ-MTB菌株PhoP基因和PhoR基因的表达水平与BCG、H37Rv、H37Ra相比存在显著的统计学差异（P＜0.05）;H37Rv菌株PhoP基因和PhoR基因的表达与BCG、H37Ra相比存在显著的统计学差异（P＜0.05）;而BCG菌株PhoP基因和PhoR基因的表达与H37Ra相比统计学差异不显著（ P＞0.05）。结论：结核分枝杆菌PhoPR双组分系统中PhoP基因和PhoR基因在不同毒力结核分枝杆菌中的表达存在差异,并且该系统与不同毒力结核分枝杆菌的致病性存在相关性。     

结核杆菌H37Ra感染巨噬细胞的研究

目的:研究结核杆菌H37Ra感染巨噬细胞后氮氧化物的产生及细胞因子的分泌和表达情况.方法:结核杆菌H37Ra感染RAW264.7巨噬细胞株24h后收集上清液,用Griess法检测一氧化氮(NO),化学法检测过氧化氢(H2O2)的产生,逆转录-聚合酶链反应(RT-PCR)法检测感染后巨噬细胞IL-12和TNF-α mRNA的表达,ELISA法检测细胞因子IL-12和TNF-α的含量.结果:结核杆菌H37Ra感染巨噬细胞后产生的NO和H2 O2水平均显著升高,同时IL-12和TNF-α的分泌和表达也明显增加(P＜0.05).结论:结核杆菌H37Ra感染巨噬细胞后能产生大量的氮氧化物和抗结核细胞因子,从而产生有利于宿主的抗结核免疫应答.     

Mycobacterium tuberculosis Rv0652 stimulates production of tumour necrosis factor and monocytes chemoattractant protein-1 in macrophages through the Toll-like receptor 4 pathway

Mycobacterial proteins interact with host macrophages and modulate their functions and cytokine gene expression profile. The protein Rv0652 is abundant in culture filtrates of Mycobacterium tuberculosis K‐strain, which belongs to the Beijing family, compared with levels in the H37Rv and CDC1551 strains. Rv0652 induces strong antibody responses in patients with active tuberculosis. We investigated pro‐inflammatory cytokine production induced by Rv0652 in murine macrophages and the roles of signalling pathways. In RAW264.7 cells and bone marrow‐derived macrophages, recombinant Rv0652 induced predominantly tumour necrosis factor (TNF) and monocyte chemoattractant protein (MCP)‐1 production, which was dependent on mitogen‐activated protein kinases and nuclear factor‐κB. Specific signalling pathway inhibitors revealed that the extracellular signal‐regulated kinase 1/2 (ERK1/2), p38 and phosphatidylinositol 3‐kinase (PI3K) pathways were essential for Rv0652‐induced TNF production, whereas the ERK1/2 and PI3K pathways, but not the p38 pathway, were critical for MCP‐1 production in macrophages. Rv0652‐stimulated TNF and MCP‐1 secretion by macrophages occurred in a Toll‐like receptor 4‐dependent and MyD88‐dependent manner. In addition, Rv0652 significantly up‐regulated the expression of the mannose receptor, CD80, CD86 and MHC class II molecules. These results suggest that Rv0652 can induce a protective immunity against M. tuberculosis through the macrophage activation.     

Impacts of interleukin-12 on multiplication of Mycobacterium tuberculosis and Mycobacterium avium complex in mice

Objective: To evaluate the potential impacts of exogenous administration of murine recombinant interleukin-12 (IL-12) on multiplication of Mycobacterium tuberculosis and M. avium complex (MAC) in murine models.
Methods: Swiss or beige mice were infected intravenously with M. tuberculosis H37Rv or MAC respectively, and were treated by subcutaneous injection with various doses of IL-12, either alone or in combination with chemotherapy. Effectiveness of treatment was assessed by the enumeration of CFUs in the spleens and lungs, together with other indicators.
Results: Multiplication of M. tuberculosis was reduced by IL-12 in a dose-dependent manner if the treatment began at day 1, whereas no statistically significant suppression was observed if the treatment began at day 14. Combination with IL-12 did not enhance the bactericidal activity of antituberculosis chemotherapy. The growth curves of MAC in IL-12-treated mice were almost identical to those of untreated controls, indicating that IL-12 did not affect the multiplication of MAC in beige mice. In both experiments, the dosing of IL-12 approached levels of severe toxicity for the mouse strains used.
Conclusions: IL-12 had a positive affect on early multiplication of M. tuberculosis . It had no effect on early multiplication of M. avium complex.     

Immunophenotypic characterization of peripheral T lymphocytes in Mycobacterium tuberculosis infection and disease

The cellular immune response probably plays a pivotal role in determining the clinical outcome after exposure to Mycobacterium tuberculosis. We used multi‐parameter flow‐cytometry to evaluate the distribution of T‐lymphocyte subsets during infection and disease caused by M. tuberculosis. Samples were obtained from 71 volunteers to identify the T CD4⁺ and CD8⁺ lymphocyte numbers, and the activation plus memory/naïve phenotypes, as defined by CD38, HLA‐DR, CD45RA and CD27 markers. Subjects were divided into 18 healthy volunteers without detectable reaction to purified protein derivative (PPD^?), 18 health care workers with a recent conversion to PPD, 20 patients with active pulmonary tuberculosis (TBC) and 15 patients with treated TBC at 6 months of therapy. By multiple‐comparison analyses, the T CD4⁺ lymphocyte number of the TBC group was lower than the PPD^– group (P < 0·05). This difference was apparently lost after treatment. The higher and the lower number of naïve T CD4⁺ cells was observed in the PPD^– and TBC group, respectively. CD8⁺ T lymphocytes were also statistically different among the four groups (P = 0·0002), lower in the TBC group (P < 0·05). CD8⁺ T lymphocyte activation was evaluated by the CD38 and HLA‐DR surface expression. The percentage distribution of these markers was statistically different between the four groups (P = 0·0055). TBC patients had a higher percentage of CD38⁺ cells and mean fluorescence index, suggesting an overall increase of cell activation. These results suggest that peripheral T lymphocytes reflect cellular activation during TBC, along with possible redistribution of naïve, memory/effector and late differentiated memory/effector phenotypes in the peripheral blood after infection and disease caused by M. tuberculosis.     

结核分枝杆菌Rv0901基因重组耻垢分枝杆菌的构建及其细胞作用

目的对结核分枝杆菌Rv0901基因的功能进行研究。方法以PCR扩增Rv0901基因编码序列,定向克隆人穿梭表达质粒pMV261获得重组穿梭表达质粒,将重组质粒电穿孔进入耻垢分枝杆菌,构建重组Rv0901基因的耻垢分枝杆菌,对重组耻垢分枝杆菌进行诱导表达,用SDS-PAGE检测表达结果。比较耻垢分枝杆菌和重组耻垢分枝杆菌对THP-1细胞的不同作用。结果成功构建重组穿梭表达质粒及重组Rv0901基因的耻垢分枝杆菌,重组菌诱导THP-1细胞的凋亡率高于耻垢分枝杆菌,其感染THP-1细胞后细胞的存活率低于耻垢分枝杆菌的感染,重组菌感染THP-1细胞后细胞培养液中NO(一氧化氮)的产生高于耻垢分枝杆菌的感染。结论Rv0901基因可能与结核毒力存在一定关系。     

不同毒力结核分枝杆菌感染小鼠对肺泡巨噬细胞的凋亡率及其时相性变化的影响

目的:探讨不同毒力结核分枝杆菌感染小鼠肺泡巨噬细胞的凋亡率及其时相性变化。方法:不同毒力结核分枝杆菌悬液分别经小鼠尾静脉注射、复制及鉴定各组小鼠感染模型,各组小鼠感染模型复制成功后第1、3、5、7、9、11、13、15天,进行肺泡灌洗,收集小鼠肺泡灌洗液,获取感染小鼠肺泡巨噬细胞,以激光共聚焦显微镜技术检测及鉴定结核分枝杆菌感染小鼠肺泡巨噬细胞,流式细胞术检测上述各时间点、各组感染小鼠肺泡巨噬细胞的凋亡率,比较各组感染小鼠肺泡巨噬细胞凋亡率的时相性变化。结果:激光共聚焦显微镜检测结果显示,结核分枝杆菌国际标准强毒株H37Rv株和卡介苗菌株(BCG)均被小鼠肺泡巨噬细胞大量吞噬。流式细胞技术检测结果显示:结核分枝杆菌国际标准强毒株H37Rv株感染小鼠模型组和卡介苗菌株(BCG)感染小鼠模型组,在小鼠感染模型复制成功后1～9天,小鼠肺泡巨噬细胞的凋亡率逐渐升高,9天时两组小鼠肺泡巨噬细胞的凋亡率均达最高,随着时间的延长,两组小鼠肺泡巨噬细胞的凋亡率呈现逐渐降低趋势。结核分枝杆菌国际标准强毒株H37Rv株感染小鼠组,感染小鼠的肺泡巨噬细胞的凋亡率明显高于卡介苗菌株(BCG)感染小鼠组,差异有统计学意义(P<0.05)。结论:感染小鼠的肺泡巨噬细胞的凋亡率与结核分枝杆菌的毒力强弱呈正相关。     

Expression,purification and characterization of Mycobacterium tuberculosis RpfE protein

Resuscitation promoting factor E(RpfE)is one of the five Rpf-like proteins in Mycobacterium tuberculosis(M.tuberculosis).These Rpf-like proteins are secretory,which make them candidates for recognition by the host immune system.In this study,the RpfE gene was amplified from M.tuberculosis,cloned into the expression vectors pDE22 and pPRO EXHT,and were expressed in Mycobacterium vaccae(M.vaccae)and Escherichia coli DH5α,respectively.Both recombinant RpfE proteins were purified by Ni-Sepharose affinity chromatography,and were given to C57BL/6 mice.The RpfE proteins elicited T cell proliferation,and stimulated the production of gamma interferon(IFN-γ),interleukin-10(IL-10)and IL-12.Our results indicated that the RpfE protein expressed in M.vaccae could more efficiently stimulate cellular immune response,making it a promising candidate as a subunit vaccine.     

巨噬细胞的自噬及其在抗结核分枝杆菌感染中的作用

白噬是广泛存在于真核细胞内的一种溶酶体依赖性的自降解途径,可通过降解长寿蛋白和受损细胞器维持细胞内的平衡。近年来的研究发现,巨噬细胞的自噬还是固有免疫和适应性免疫的重要组成部分,可参与胞内感染病原体的清除。目前已发现有多种途径参与自噬的诱导和调节。在感染的巨噬细胞内,诱导自噬的发生能促进吞噬体和溶酶体的融合,抑制胞内结核分枝杆菌（Mtb）的存活。但同时Mtb也可通过某些机制抑制巨噬细胞自噬的发生以逃避巨噬细胞的杀伤,进而长期持留于巨噬细胞内。深入了解巨噬细胞自噬与胞内Mtb相互作用的机制,有助于人类更好的预防和控制结核病。     

Spoligotype patterns of Mycobacterium tuberculosis isolated from extra pulmonary tuberculosis patients in Puducherry,India

Purpose: Genotyping studies like spoligotyping are valuable tools in understanding the genetic diversity and epidemiology of Mycobacterium tuberculosis. Though there are reports of spoligotyping of M. tuberculosis isolates from pulmonary specimens from different parts of India, spoligotyping of extra pulmonary tuberculosis isolates are very few. Puducherry has not yet recorded spoligopatterns of M. tuberculosis from either pulmonary or extra pulmonary (EPTB) specimens. The aim of this study is to analyze the spoligotype patterns of EPTB strains circulating in Puducherry and neighboring districts of Tamil Nadu. Materials and Methods: During June 2011 to December 2013, 570 EPTB specimens were processed by culturing on to Lowenstein Jensen (LJ) medium and automated Mycobacterium Growth Indicator Tube system (MGIT960). Identification of M. tuberculosis was carried out as per standard procedures, and MPT 64 antigen positivity in a commercial immunochromatography kit. Spoligotyping was carried out at National Institute of Research in Tuberculosis (ICMR), Chennai. Results: M. tuberculosis was isolated from 67 single EPTB specimens (11.8%) like pus/cold abscess (34), TB spine (10), pleural fluid (10), urine (5), tissue bit (2), lymph nodes (2), ascitic fluid (2), synovial fluid (1) and endometrial curetting (1). Among 67 isolates with 41 spoligopatterns, EAI lineage with 28 isolates (41.8%) predominated followed by 18 orphans (26.9%), 10 Beijing (14.9%) and 8 U (11.9%). BOVIS1_BCG (ST482), T1-T2 (ST78) and H3 (ST50) were represented by one strain each (1.5%). Conclusions: Spoligotyping plays a significant role in the epidemiology of tuberculosis. Three spoligotypes, T1-T2 (ST78), EAI6 (ST292) and U (ST1429) are reported for the first time in India.     

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.     

Exosomes carrying mycobacterial antigens can protect mice against Mycobacterium tuberculosis infection

Approximately 2 billion people are infected with Mycobacterium tuberculosis, the etiological agent of tuberculosis (TB), and an estimated 1.5 million individuals die annually from TB. Presently, Mycobacterium bovis BCG remains the only licensed TB vaccine; however, previous studies suggest its protective efficacy wanes over time and fails in preventing pulmonary TB. Therefore, a safe and effective vaccine is urgently required to replace BCG or boost BCG immunizations. Our previous studies revealed that mycobacterial proteins are released via exosomes from macrophages infected with M. tuberculosis or pulsed with M. tuberculosis culture filtrate proteins (CFP). In the present study, exosomes purified from macrophages treated with M. tuberculosis CFP were found to induce antigen‐specific IFN‐γ and IL‐2‐expressing CD4⁺ and CD8⁺ T cells. In exosome‐vaccinated mice, there was a similar T_H1 immune response but a more limited T_H2 response compared to BCG‐vaccinated mice. Using a low‐dose M. tuberculosis mouse aerosol infection model, exosomes from CFP‐treated macrophages were found to both prime a protective immune response as well as boost prior BCG immunization. The protection was equal to or superior to BCG. In conclusion, our findings suggest that exosomes might serve as a novel cell‐free vaccine against an M. tuberculosis infection.