耐药结核分枝杆菌的适应性代价与补偿性进化

结核病耐药率的攀升是目前全球结核病防控面临的重大挑战。结核分枝杆菌主要通过其基因组中耐药相关基因发生点突变而获得耐药性。由于耐药相关基因通常具有重要的生理功能,其突变往往会导致结核分枝杆菌自身适应性下降,即产生“适应性代价”。然而,耐药结核分枝杆菌可通过进一步积累其他特定突变来回复其适应性,这种能使其适应性上升的突变称为“补偿性突变”。耐药结核分枝杆菌的补偿性进化被认为是耐药结核病广泛传播与流行的生物学基础。近年来,在结核病分子流行病学和基础研究领域,针对耐药结核分枝杆菌的补偿性进化开展了大量研究。本文从结核分枝杆菌的耐药分子机制、耐药突变的适应性代价与补偿性进化,以及补偿性进化如何影响耐药结核病传播等方面,综述耐药结核分枝杆菌补偿性进化的研究进展。     

耐药结核分枝杆菌基因组信息挖掘

<正>由于结核分枝杆菌的耐药性问题,使结核病这个古老的传染病死灰复燃,并成为全球性的严重公关问题。从1998年首个结核分枝杆菌(H37Rv)全基因组完成图的获得[1],到近年来采用新一代测序技术对多个菌株进行高通量的基因组测序与分析,对结核分枝杆菌进化及耐药机制的认识不断深入。关于结核分枝杆菌菌株的基因组比较分析有多篇报道,包括对中国12个省来源的161株结核分枝杆菌     

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。     

结核分枝杆菌毒力的研究进展

结核分枝杆菌可以在人体内缓慢增殖,释放代谢产物,造成细胞损伤,引起结核病。其致病机制可能与其复杂的细胞壁成分密切相关。结核分枝杆菌细胞壁中的脂质、糖类及蛋白类物质在构成屏障结构,保护并辅助结核分枝杆菌在细胞内的生长及迁移,调节宿主免疫应答,造成宿主组织细胞损伤等方面发挥重要生物学作用。其表达受众多基因的精细调控。本文将对结核分枝杆菌细胞壁中的脂质阿拉伯甘露聚糖和分枝菌酸在其毒力中的作用,以及sig基因对毒力的调节作用作一综述。     

耐药性结核分枝杆菌研究近况

结核分枝杆菌(简称结核杆菌,MTB)是结核痛的病原体.随着耐药性结核杆菌菌株的产生和播散,尤其是耐多药结核(MDR-TB)和广泛抗性结核菌株(XDR-TB)的出现,结核病的威胁在急速增长.现对结核杆菌的分子生物学、分型、耐药机制、致病机制、疫苗等方面作一概述.     

碱基切除修复与分枝杆菌基因组稳定性

维持基因组稳定是生物生存的基础。碱基切除修复(base excision repair,BER)是修复损伤DNA、维持基因组稳定的主要方式之一。碱基切除修复对结核分枝杆菌等胞内致病菌尤其重要。fpg编码碱基切除修复的关键酶。本文通过比较分枝杆菌的基因组,发现结核菌较其他非致病分枝杆菌具有更多的碱基切除修复基因。这提示碱基切除修复可能对结核菌在宿主体内存活和致病至关重要。这条途径也许是新结核病药物研发的重要靶标。     

DNA微阵列在结核分枝杆菌研究中的应用

高密度DNA微阵列技术可以通过一次杂交获得大量的基因组信息,广泛用于基因组结构和基因表达谱分析。本文简介DNA微阵列技术在结核分枝杆菌的功能基因组学,致病机理以及耐药机制,诊断等方面的应用。     

甘蔗宿根矮化病病原菌Leifsonia xyli subsp.xyli 基因组学研究进展

综述甘蔗宿根矮化病病原细菌Leifsonia xyli subsp.xyli(Lxx)基因组组成特征、基因组进化、基因组中致病相关基因与寄主适应性等方面的研究进展,并对该病原茵、引起番茄细菌性萎蔫和溃疡的病原细菌、马铃薯环腐病痛原细菌的基因组和致病相关基因进行比较,发现它们存在同源致病基因,如celA、pat1基因,其致病机理可能有相似性.     

结核分枝杆菌ABC转运蛋白与物质的跨膜转运

结核分枝杆菌作为一种胞内寄生菌,主要存在于巨噬细胞吞噬体内,并且通过与宿主细胞竞争摄取营养物质、主动排出有毒物质来维持生存。因此,参与上述过程的ABC转运蛋白在结核分枝杆菌的致病中发挥着举足轻重的作用。已有报道结核分枝杆菌基因组编码了38个ABC转运蛋白。这类蛋白质有着广泛的底物结合谱,参与了无机离子、糖类、氨基酸、寡肽、药物等多种物质的跨膜转运。本文将对结核分枝杆菌编码的ABC转运蛋白超家族中的不同成员及其底物特异性、转运机制以及与毒力的关系的研究进展进行综述。     

结核分枝杆菌Rv1057基因对巨噬细胞感染早期细胞因子表达的影响分析

Rv1057是结核分枝杆菌中唯一的7-折叠片β-螺旋蛋白,其生物学功能尚不清楚。为探讨Rv1057基因在结核分枝杆菌感染初期对巨噬细胞免疫应答的影响,利用Rv1057基因缺失菌株D1057和野生型H37Rv菌株感染巨噬细胞,检测结核分枝杆菌在巨噬细胞内的增殖速度,分析巨噬细胞在感染初期的细胞因子表达量变化。研究结果表明:D1057菌株在巨噬细胞内的活菌数量和增殖速度都显著低于H37Rv,被D1057感染的巨噬细胞中IL-1β、IL-10、TNF-α和IFN-γ表达量显著降低,但IL-8大量表达且无显著差异。上述研究结果证实,缺失Rv1057会降低结核分枝杆菌刺激巨噬细胞产生某些细胞因子的能力,明确了Rv1057基因参与结核分枝杆菌与巨噬细胞之间的免疫应答,为进一步解析Rv1057基因的生物学功能、结核分枝杆菌的致病机制奠定了基础。     

Differentiation of the Mycobacterium tuberculosis W-Beijing family strains from the Russian Federation by the VNTR-typing

Recent phylogenetic studies allowed the Mycobacterium tuberculosis complex to be divided into a number of the strain families. The W-Beijing family is one of most widespread M. tuberculosis variants frequently causing epidemic outbreaks. This family is genetically homogenous and conserved so that ETR A, B, C, D, E - typing is insufficient for the W-Beijing differentiation. All W-Beijing isolates have common profile (42435). This led to the false clustering in the molecular epidemiology study, especially in the region of predominance of the W-Beijing family. In this investigation we searched for the VNTR loci with high evolution rate, which were polymorphic in the W-Beijing genome. Eleven VNTR-loci were assayed in the DNA panel of 99 M. tuberculosis isolates from the tuberculosis patients in North-West and West-Siberian regions of Russia during the period from 2000 to 2001. Ninety nine strains of M. tuberculosis were divided into 74 VNTR-types, 51 isolates of the W-Beijing family were subdivided into 30 VNTR-types. The Hunter-Gudson index (HGDI) for all studied loci (ETR-A, ETR-C, ETR-E, V, V2, V3, V4, V5, V6, V10, V11) was close to one of the IS6110 RFLP indices being "the gold standard" of the M. tuberculosis complex genotyping. The V2, V3 loci located in the sequences of the PPE gene family, were highly polymorphic and more discriminative then others (HGDI is about 0.8). The congruence between the IS6110 RFLP-typing and 11 loci VNTR-typing was measured during genotyping for 23 isolates of the W-Beijing family. The isolates were divided into 9 genotypes by the IS6110 RFLP and into 13 variants by the VNTR-typing. The profiles correlation coefficient was 0.767689 that reflected the differences in the rate and type of the given genome target evolution.     

Human diseases as representation of interspecies competition

Different aspects of competition between human and pathogenic microorganisms as well as their role in evolution of the human as biological species and development of its polymorphism were reviewed. Biological consequences of interaction between human and pathogenic microorganisms and also main factors of genetic selection such as tuberculosis, malaria, etc. were characterized. Numerous forms of polymorphism, which determine level of susceptibility and features of clinical course of many infections in humans, were described. It has been concluded that selective pressure of microorganisms results from direct selection due to excessive mortality of infected persons before integration in human genome as well as that immunologic pressure of mass immunization results in changes of pathogenic microorganisms populations leading to emergence of their antigenically distinct variants. New knowledge about competition between human and pathogenic microorganisms requires development of fundamentally new approaches in fight with infectious diseases.     

Genome comparison of Mycobacterium tuberculosis and other bacteria

The availability of the complete genome sequence of Mycobacterium tuberculosis allows its phylogenetic analysis based on the whole genome rather than single genes. As a genome-based tree is more representative of whole organisms and less inconsistent than single-gene trees, it could provide a better index for interpretation and inference about the origin and nature of species. The standard bacterial phylogeny based on 16S ribosomal RNA sequence comparison shows that M. tuberculosis is more related to Gram-positive than to Gram-negative bacteria. Our results based on genome comparison in terms of shared orthologous genes challenge this implication. We demonstrate that M. tuberculosis is more related to Gram-negative than to Gram-positive bacteria by a quantitative analysis on the genome tree. The numerical distance data derived from genome comparison and those from 16S rRNA comparison show high significant correlation, implying that conserved gene content carries a strong phylogenetic signature in evolution.     

Biosynthesis of the arabinogalactan-peptidoglycan complex of Mycobacterium tuberculosis

The compositional complexity of the mycobacterial cell envelope differentiates Mycobacterium species from most other prokaryotes. Historically, research in this area has focused on the elucidation of the structure of the mycobacterial cell envelope with the result that the structures of the mycolic acid-arabinogalactan-peptidoglycan complex from M. tuberculosis are fairly well understood. However, the current impetus for studying M. tuberculosis and other pathogenic mycobacteria is the need to identify targets for the development of new drugs. Therefore, emphasis has been shifting to the study of cell envelope biosynthesis and the identification of enzymes that are essential to the viability of M. tuberculosis. The publication of the complete M. tuberculosis genome in 1998 has greatly aided these studies. To date, thirteen enzymes involved in the synthesis of the arabinogalactan-peptidoglycan complex of M. tuberculosis have been identified and at least partially characterized. Eleven of these enzymes were reported subsequent to the publication of the M. tuberculosis genome, a clear indication of the rapid evolution of knowledge stimulated by the sequencing of the genome. In this article we review the current understanding of M. tuberculosis arabinogalactan-peptidoglycan structure and biosynthesis.     

Identification of variable regions in the genomes of tubercle bacilli using bacterial artificial chromosome arrays

Whole-genome comparisons of the tubercle bacilli were undertaken using ordered bacterial artificial chromosome (BAC) libraries of Mycobacterium tuberculosis and the vaccine strain, Mycobacterium bovis BCG-Pasteur, together with the complete genome sequence of M. tuberculosis H37Rv. Restriction-digested BAC arrays of M. tuberculosis H37Rv were used in hybridization experiments with radiolabelled M. bovis BCG genomic DNA to reveal the presence of 10 deletions (RD1-RD10) relative to M. tuberculosis. Seven of these regions, RD4-RD10, were also found to be deleted from M. bovis, with the three M. bovis BCG-specific deletions being identical to the RD1-RD3 loci described previously. The distribution of RD4-RD10 in Mycobacterium africanum resembles that of M. tuberculosis more closely than that of M. bovis, whereas an intermediate arrangement was found in Mycobacterium microti, suggesting that the corresponding genes may affect host range and virulence of the various tubercle bacilli. Among the known products encoded by these loci are a copy of the proposed mycobacterial invasin Mce, three phospholipases, several PE, PPE and ESAT-6 proteins, epoxide hydrolase and an insertion sequence. In a complementary approach, direct comparison of BACs uncovered a third class of deletions consisting of two M. tuberculosis H37Rv loci, RvD1 and RvD2, deleted from the genome relative to M. bovis BCG and M. bovis. These deletions affect a further seven genes, including a fourth phospholipase, plcD. In summary, the insertions and deletions described here have important implications for our understanding of the evolution of the tubercle complex.     

Independent large scale duplications in multiple M. tuberculosis lineages overlapping the same genomic region

Mycobacterium tuberculosis, the causative agent of most human tuberculosis, infects one third of the world's population and kills an estimated 1.7 million people a year. With the world-wide emergence of drug resistance, and the finding of more functional genetic diversity than previously expected, there is a renewed interest in understanding the forces driving genome evolution of this important pathogen. Genetic diversity in M. tuberculosis is dominated by single nucleotide polymorphisms and small scale gene deletion, with little or no evidence for large scale genome rearrangements seen in other bacteria. Recently, a single report described a large scale genome duplication that was suggested to be specific to the Beijing lineage. We report here multiple independent large-scale duplications of the same genomic region of M. tuberculosis detected through whole-genome sequencing. The duplications occur in strains belonging to both M. tuberculosis lineage 2 and 4, and are thus not limited to Beijing strains. The duplications occur in both drug-resistant and drug susceptible strains. The duplicated regions also have substantially different boundaries in different strains, indicating different originating duplication events. We further identify a smaller segmental duplication of a different genomic region of a lab strain of H37Rv. The presence of multiple independent duplications of the same genomic region suggests either instability in this region, a selective advantage conferred by the duplication, or both. The identified duplications suggest that large-scale gene duplication may be more common in M. tuberculosis than previously considered.     

Mapping of IS6110 flanking regions in clinical isolates of Mycobacterium tuberculosis demonstrates genome plasticity

Southern hybridization was used in combination with IS6110 insertion-locus-specific probes in a comparative study to determine the structure of chromosomal domains flanking IS6110 elements in clinical isolates of Mycobacterium tuberculosis. The resulting restriction fragment length polymorphism (RFLP) data demonstrated three mutational mechanisms responsible for the polymorphisms observed: IS6110 insertion, chromosomal mutation and deletion. The frequency of IS6110 insertion within many of the chromosomal regions demonstrates that preferential integration regions are common in M. tuberculosis. Mapping the IS6110 insertion positions and chromosomal deletions in relation to the M. tuberculosis H37Rv and M. bovis BCG genome sequences reveals numerous disruptions of predicted open reading frames (ORFs). A phylogenetic tree, based on the mutational data, showed a number of independently evolving lineages of M. tuberculosis, while analysis of the mutational events occurring at each branch point suggests both divergent and convergent evolution. A significant positive correlation was demonstrated between the mutation rate and the frequency of occurrence of different isolates in families of strains, suggesting that evolution may impact on strain 'fitness' or that strain proliferation may increase the chance of mutation. We conclude that the genome of clinical isolates of M. tuberculosis continues to evolve.     

Development of vaccines to control bovine tuberculosis in cattle and relationship to vaccine development for other intracellular pathogens

Vaccination of cattle against bovine tuberculosis could be an important strategy for the control of disease either where there is a wildlife reservoir of Mycobacterium bovis infection or in developing countries where it is not economically feasible to implement a 'test and slaughter' control program. Advances in the understanding of protective immune responses to M. bovis infection in cattle and the advent of new molecular biological techniques, coupled with the sequencing of the M. bovis genome have provided opportunities for the rational development of improved tuberculosis vaccines. A number of new tuberculosis vaccines including attenuated M. bovis strains, killed mycobacteria, protein and DNA vaccines are under development and many are being assessed in cattle. Recent results have revealed several promising vaccine candidates and vaccination strategies. Ways of distinguishing between vaccinated and infected cattle are becoming available and the possibility of new approaches to the eradication of tuberculosis from domestic livestock is discussed. Similarities between the mechanisms of protective immunity against M. bovis and against other intracellular parasites continue to be found and discoveries from vaccine studies on bovine tuberculosis may provide helpful insights into requirements for vaccines against other intracellular pathogens.     

Eukaryotic genes in Mycobacterium tuberculosis could have a role in pathogenesis and immunomodulation.

Acquisition of new genetic material through horizontal gene transfer has been an important feature in the evolution of many pathogenic bacteria. Here, we report the presence of 19 genes of eukaryotic origin in the genome of Mycobacterium tuberculosis, some of which are unique to the M. tuberculosis complex. These genes, having been retained in the genome through selective advantage, most probably have key functions in the organism and in mammalian tuberculosis. We explore the role these genes might have in manipulation of the host immune system by altering the balance of steroid hormones.