Mycobacterium tuberculosis mammalian cell entry operon (mce) homologs in Mycobacterium other than tuberculosis (MOTT)

The cloned mammalian cell entry gene mce1a from Mycobacterium tuberculosis confers to non-pathogenic Escherichia coli the ability to invade and survive inside macrophages and HeLa cells. The aim of this work was to search for and characterize homologs of the four M. tuberculosis mammalian cell entry operons (mce1, mce2, mce3 and mce4) in mycobacteria other than tuberculosis (MOTT). The dot-blot and polymerase chain reaction (PCR) experiments performed on 24 clinical isolates representing 20 different mycobacterial species indicated that the mce operons were widely distributed throughout the genus Mycobacterium. BLAST search results showed the presence of mce1, mce2 and mce4 homologs in Mycobacterium bovis, Mycobacterium avium and Mycobacterium smegmatis. A homologous region for the mce3 operon was also found in M. avium and M. smegmatis. DNA and protein alignments were done to compare the M. tuberculosis mce operons and the deduced M. bovis, M. avium, and M. smegmatis homologs. The deduced proteins of M. bovis mce1, mce2 and mce4 operons had 99.6-100% homology with the respective M. tuberculosis mce proteins (MTmce). The similarity between M. avium mce proteins and the individual M. tuberculosis homologs ranged from 56.2 to 85.5%. The alignment results between M. smegmatis mce proteins and the respective MTmce proteins ranged from 58.5% to 68.5%. Primer sets were designed from the M. tuberculosis mce4a gene for amplification of 379-bp fragments. Amplification was successful in 14 strains representing 11 different mycobacterial species. The PCR fragments were sequenced from 10 strains representing eight species. Alignment of the sequenced PCR products showed that mce4a homologs are highly conserved in the genus Mycobacterium. In conclusions, the four mce operons in different mycobacterial species are generally organized in the same manner. The phylogenetic tree comparing the different mce operons showed that the mce1 operon was closely related to the mce2 operon and mce3 diverged from the other operons. The wide distribution of the mce operons in pathogenic and non-pathogenic mycobacteria implicates that the presence of these putative virulence genes is not an indicator for the pathogenicity of the bacilli. Instead, the pathogenicity of these factors might be determined by their expression.     

二代测序技术在结核分枝杆菌研究中的应用进展

结核病是由结核分枝杆菌引起的全球第二大传染病。二代测序技术为从基因组水平研究结核分枝杆菌提供了重要的研究方法。本文从结核病流行病学、结核分枝杆菌耐药和进化及相关生物信息学等方面,介绍二代测序技术在结核分枝杆菌研究中的应用进展。     

Tuberculosis patients co-infected with Mycobacterium bovis and Mycobacterium tuberculosis in an urban area of Brazil

In this cross-sectional study, mycobacteria specimens from 189 tuberculosis (TB) patients living in an urban area in Brazil were characterised from 2008-2010 using phenotypic and molecular speciation methods (pncA gene and oxyR pseudogene analysis). Of these samples, 174 isolates simultaneously grew on Löwenstein-Jensen (LJ) and Stonebrink (SB)-containing media and presented phenotypic and molecular profiles of Mycobacterium tuberculosis, whereas 12 had molecular profiles of M. tuberculosis based on the DNA analysis of formalin-fixed paraffin wax-embedded tissue samples (paraffin blocks). One patient produced two sputum isolates, the first of which simultaneously grew on LJ and SB media and presented phenotypic and molecular profiles of M. tuberculosis, and the second of which only grew on SB media and presented phenotypic profiles of Mycobacterium bovis. One patient provided a bronchial lavage isolate, which simultaneously grew on LJ and SB media and presented phenotypic and molecular profiles of M. tuberculosis, but had molecular profiles of M. bovis from paraffin block DNA analysis, and one sample had molecular profiles of M. tuberculosis and M. bovis identified from two distinct paraffin blocks. Moreover, we found a low prevalence (1.6%) of M. bovis among these isolates, which suggests that local health service procedures likely underestimate its real frequency and that it deserves more attention from public health officials.     

结核分枝杆菌脂蛋白Rv1016c增强细菌成膜能力和抑制自噬

目的研究结核分枝杆菌(Mycobacterium tuberculosis, Mtb)脂蛋白Rv1016c在Mtb感染和结核病发病中的作用和机制。方法将Mtb脂蛋白Rv1016c基因导入野生耻垢分枝杆菌(Mycobacterium smegmatis, MS)构建重组菌株MS-Rv1016c,比较脂蛋白Rv1016c对菌体生长、成膜能力、细菌聚集、毒力等方面的影响,评估重组菌株MS-Rv1016c对自噬的影响。结果 Rv1016c基因的导入,因过表达脂蛋白使得MS的菌落变大、褶皱增加,使菌体聚集度降低,使细菌成膜速度加快、生物被膜产量增加;Rv1016c显著抑制巨噬细胞自噬,促进细菌在细胞内持留。结论 Rv1016c能够促进MS生物被膜形成,抑制细胞自噬,增强细菌毒力。为研究脂蛋白在Mtb致病机理中的作用提供理论依据。     

Differences between tuberculosis cases infected with Mycobacterium africanum, West African type 2, relative to Euro-American Mycobacterium tuberculosis: an update

Mycobacterium africanum (MAF) is a common cause of human pulmonary tuberculosis in West Africa. We previously described phenotypic differences between MAF and Mycobacterium tuberculosis (MTB) among 290 patients. In the present analysis, we compared 692 tuberculosis patients infected with the two most common lineages within the (MTB) complex found in the Gambia, namely MAF West African type 2 (39% prevalence) and Euro-American MTB (55% prevalence). We identified additional phenotypic differences between infections with these two organisms. MAF patients were more likely to be older and HIV infected. In addition, they had worse disease on chest X-ray, despite complaining of cough for an equal duration, and were more likely severely malnourished. In this cohort, the prevalence of MAF did not change significantly over a 7-year period.     

结核分枝杆菌Rv1009的生物信息学分析及克隆、表达

以往在对藤黄微球菌的研究中发现了促进复活因子(RPF),该因子可以有效促进休眠期的多种革兰氏阳性细菌复活和生长。生物信息学分析表明在多种革兰氏阳性细菌的基因组中均存在编码RPF样蛋白的基因。从GeneBank中获得了结核分枝杆菌、麻风分枝杆菌、谷氨酸棒杆菌和微链霉菌等革兰氏阳性细菌的多种RPF样蛋白相关信息,通过同源性分析发现这些蛋白均含有一个由75个氨基酸残基构成的高度保守序列,即RPF作用域。进一步对结核分枝杆菌H37Rv株的Rv1009进行了分析,并用PCR法克隆了其编码基因全长,定向克隆至pGEX 4T-2,在大肠杆菌BL21 (DE3)中获得了Rv1009-GST融合蛋白的高效表达,为深入探讨其生物学功能奠定了基础。     

结核分枝杆菌的致病机理

结核病仍是全球健康的主要威胁,其致病菌结核分枝杆菌的致闰机理与众不同。脂类代谢在致病中具有重要的作用。巨噬细胞被入侵的细菌修的机理也是研究细菌持续感染致病的重要突破点,研究细菌的致病机理可以为开发新的疫苗和治疗药物奠定坚实的基础。     

Electron microscopy analysis of Mycobacterium tuberculosis cell division

The ultrastructure of Mycobacterium tuberculosis cells undergoing division was examined by electron microscopy. Two features of cell division were observed and are described here. First, cells are capable of undergoing a type of "snapping" postfission movement. This movement is likely due to a multi-layered cell wall in which the inner layer participates in septum formation while the outer layer ruptures first on one side. A second feature related to cell division is the ability of dividing cells to form transient branching structures.     

结核分枝杆菌脂阿拉伯甘露聚糖的提纯及活性初步鉴定

目的:提取纯化结核分枝杆菌(MTB)脂阿拉伯甘露聚糖(LAM)。方法:MTB菌体彻底破碎后,去脂,去蛋白,上清液经苯酚萃取,酒精沉淀,得到LAM;以提取的LAM作为包被抗原检测血清中的LAM抗体。结果和结论:提取到LAM抗原,免疫印迹表明,LAM迁移范围相对分子质量为25×103~40×103,主要集中在35×103处。在64例肺结核患者中,有43例LAM-ELISA检测阳性(敏感性为67.19%);在67例健康志愿者中,有64例LAM-ELISA检测阴性(特异性为95.52%)。     

Revised structure of a trehalose-containing immunoreactive glycolipid of Mycobacterium tuberculosis

Nuclear magnetic resonance spectroscopy, fast-atom bombardment mass spectrometry as well as various chemical degradations and chromatographic techniques were used to re-examine the structure of a highly immunoreactive glycolipid previously described in Mycobacterium tuberculosis (strain Canetti) as a 2,3-diacyl trehalose 2'-sulfate (labelled SL-IV). Ion exchange chromatography allowed the recognition of a neutral and an acidic glycolipid, indistinguishable on conventional silica gel. The neutral glycolipid was shown to be serologically identical to SL-IV and its structure was established as 2,3-diacyl trehalose. It corresponded to the non-chemically defined highly observed immunoreactive lipid previously recognized by others in M. tuberculosis (H37Rv).     

Advances in antibody-mediated immunity against Mycobacterium tuberculosis: implications for a novel vaccine strategy

Cell-mediated immunity is considered to be the major component of the host response against Mycobacterium tuberculosis, whereas antibody-mediated immunity historically has been considered inconsequential. In recent years, studies from several groups have challenged the traditional dogma and demonstrated that monoclonal antibodies can modify various aspects of mycobacterial infections. This review describes the experimental evidence supporting a role for antibodies in defense against mycobacterial infections and outlines future challenges to the field of antibody-mediated immunity against M. tuberculosis, with particular emphasis on the implications of these findings for a novel vaccine strategy.     

Electrochemical-based biosensors for detection of Mycobacterium tuberculosis and tuberculosis biomarkers

Abstract

Early detection of tuberculosis (TB) reduces the interval between infection and the beginning of treatment. However, commercially available tests cannot discriminate between BCG-vaccinated healthy persons and patients. Also, they are not suitable to be used for immunocompromised persons. In recent years, biosensors have attracted great attention due to their simple utility, accessibility, and real-time outputs. These sensors are increasingly being considered as pioneering tools for point-of-care diagnostics in communities with a high burden of TB and limited accessibility to reference laboratories. Among other types of biosensors, the electrochemical sensors have the advantages of low-cost operation, fast processing, simultaneous multi-analyte analyzing, operating with turbid samples, comparable sensitivity and readily available miniaturization. Electrochemical biosensors are sub-divided into several categories including: amperometric, impedimetric, potentiometric, and conductometric biosensors. The biorecognition element in electrochemical biosensors is usually based on antibodies (immunosensors), DNAs or PNAs (genosensors), and aptamers (aptasensors). In either case, whether an interaction of the antigen–antibody/aptamer or the hybridization of probe with target mycobacterial DNA is detected, a change in the electrical current occurs that is recorded and displayed as a plot. Therefore, impedimetric-based methods evaluate resistance to electron transfer toward an electrode by a Nyquist plot and amperometric/voltammetric-based methods weigh the electrical current by means of cyclic voltammetry, square wave voltammetry, and differential pulse voltammetry. Electrochemical biosensors provide a promising scope for the new era of diagnostics. As a consequence, they can improve detection of Mycobacterium tuberculosis traces even in attomolar scales.     

cAMP levels within Mycobacterium tuberculosis and Mycobacterium bovis BCG increase upon infection of macrophages

Adenosine 3',5'-cyclic monophosphate (cAMP)-mediated signal transduction is common in both prokaryotes and eukaryotes, and several bacterial pathogens modulate cAMP signaling pathways of their mammalian hosts during infection. In this study, cAMP levels associated with Mycobacterium tuberculosis and Mycobacterium bovis BCG were measured during macrophage infection. cAMP levels within both bacteria increased c . 50-fold during infection of J774.16 macrophages, relative to the cAMP levels within bacteria incubated in tissue culture media alone. cAMP levels also increased within the macrophage cytoplasm upon uptake of live, but not dead, mycobacteria. The presence of albumin in the absence of oleic acid significantly decreased cAMP secretion and production by both M. tuberculosis and M. bovis BCG. These results suggest that cAMP signaling plays a role in the interaction of tuberculosis-complex mycobacteria with macrophages during infection, and that albumin may be a physiological indicator differentiating host environments during infection.     

结核分枝杆菌RD1区研究进展

RD1区是结核分枝杆菌(MTB)在长期传代过程中丢失的重要保护性抗原,RD1区仅存在于致病性分枝杆菌中,而在卡介苗(BCG)及环境分枝杆菌中缺失。RD1区基因全长9.5kb,共有9个开放读码框,分别编码9个蛋白。RD1区是MTB毒力的关键因素之一,同时RD1区存在一种新分泌表达体系,能保证ESAT6和CFP10蛋白分泌表达。RD1区蛋白有较强的免疫原性,在MTB的预防和诊断中将可能发挥巨大作用,并有可能成为筛选抗MTB药物的理想靶抗原。     

结核分枝杆菌免疫优势抗原研究进展

结核病是当今影响人类健康、流行性最广、病死率最高的感染性疾病之一。结核病的诊断和疫苗的构建成为当前的研究热点,筛选出结核分枝杆菌免疫优势抗原是快速准确的诊断结核病及研制安全有效的疫苗的关键。拟对近年来国内外学者发现的结核分枝杆菌免疫优势抗原的分子生物学特性研究进展进行综述。     

结核分枝杆菌滤过型的初步研究

目的研究结核分枝杆菌滤过型的生物学特性与检测方法。方法菌阴肺结核患者的结核分枝杆菌及其L型的液体培养物经0.45μm滤膜过滤后,涂片观察细菌形态,滤液分别进行分枝杆菌及其L型培养,并采用荧光基因定量法进行结核菌DNA检测。培养物用免疫组化染色鉴定,并采用透射电镜观察。结果30例痰、血滤前、滤后FQ-PCR检测同时阳性为53%,同时阴性为13%,滤前、滤后FQ-PCR结果符合率为67%。滤过后液体培养9例涂片见少许抗酸颗粒或椭圆性球菌。透射电镜见细胞壁缺失的"致密体"样细菌和细胞壁缺如菌。结论菌阴肺结核痰及血培养物中存在结核菌滤过型,滤过型携带遗传信息并可自我复制,采用生物学方法可以对其进行检测。     

结核分枝杆菌eis基因对鼠巨噬细胞Raw264．7白噬影响的研究

目的探讨结核分枝杆菌eis基因对巨噬细胞自噬的影响。方法将鼠巨噬细胞Raw264．7以自噬体荧光表达质粒GFP-LC3转染,将含eis基因的重组耻垢分枝杆菌MS—pmv261-eis与不含eis基因的耻垢分枝杆菌MS—pmv261分别感染宿主巨噬细胞,透射电镜下观察自噬小体形成情况,荧光显微镜下观察自噬荧光并计数,Westernblot检测如基因表达的蛋白及自噬蛋白LC3-Ⅱ的表达水平。结果结核分枝杆菌eis基因可抑制感染宿主细胞自噬小体的形成,并显著抑制自噬荧光小点形成（P〈0．05）,显著降低了自噬蛋白LC3-Ⅱ表达水平。结论结核分枝杆菌e曲基因对Raw264．7细胞自噬有抑制作用。