Mycobacterium tuberculosis CDC1551 induces a more vigorous host response in vivo and in vitro, but is not more virulent than other clinical isolates.

Mycobacterium tuberculosis CDC1551, a clinical isolate reported to be hypervirulent and to grow faster than other isolates, was compared with two other clinical isolates (HN60 and HN878) and two laboratory strains (H37Rv and Erdman). The initial (1-14 days) growth of CDC1551, HN60, HN878, and H37Rv was similar in the lungs of aerosol-infected mice, but growth of Erdman was slower. Thereafter, the growth rate of CDC1551 decreased relative to the other strains which continued to grow at comparable rates up to day 21. In the lungs of CDC1551-infected mice, small well-organized granulomas with high levels of TNF-alpha, IL-6, IL-10, IL-12, and IFN-gamma mRNA were apparent sooner than in lungs of mice infected with the other strains. CDC1551-infected mice survived significantly longer. These findings were confirmed in vitro. The growth rates of H37Rv and CDC1551 in human monocytes were the same, but higher levels of TNF-alpha, IL-10, IL-6, and IL-12 were induced in monocytes after infection with CDC1551 or by exposure of monocytes to lipid fractions from CDC1551. CD14 expression on the surface of the monocytes was up-regulated to a greater extent by exposure to the lipids of CDC1551. Thus, CDC1551 is not more virulent than other M. tuberculosis isolates in terms of growth in vivo and in vitro, but it induces a more rapid and robust host response.     

Unexpected Role for IL-17 in Protective Immunity against Hypervirulent Mycobacterium tuberculosis HN878 Infection

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), infects one third of the world''s population. Among these infections, clinical isolates belonging to the W-Beijing appear to be emerging, representing about 50% of Mtb isolates in East Asia, and about 13% of all Mtb isolates worldwide. In animal models, infection with W-Beijing strain, Mtb HN878, is considered “hypervirulent” as it results in increased mortality and causes exacerbated immunopathology in infected animals. We had previously shown the Interleukin (IL) -17 pathway is dispensable for primary immunity against infection with the lab adapted Mtb H37Rv strain. However, it is not known whether IL-17 has any role to play in protective immunity against infection with clinical Mtb isolates. We report here that lab adapted Mtb strains, such as H37Rv, or less virulent Mtb clinical isolates, such as Mtb CDC1551, do not require IL-17 for protective immunity against infection while infection with Mtb HN878 requires IL-17 for early protective immunity. Unexpectedly, Mtb HN878 induces robust production of IL-1β through a TLR-2-dependent mechanism, which supports potent IL-17 responses. We also show that the role for IL-17 in mediating protective immunity against Mtb HN878 is through IL-17 Receptor signaling in non-hematopoietic cells, mediating the induction of the chemokine, CXCL-13, which is required for localization of T cells within lung lymphoid follicles. Correct T cell localization within lymphoid follicles in the lung is required for maximal macrophage activation and Mtb control. Since IL-17 has a critical role in vaccine-induced immunity against TB, our results have far reaching implications for the design of vaccines and therapies to prevent and treat emerging Mtb strains. In addition, our data changes the existing paradigm that IL-17 is dispensable for primary immunity against Mtb infection, and instead suggests a differential role for IL-17 in early protective immunity against emerging Mtb strains.     

The Role of Lipid Raft Aggregation in the Infection of Type II Pneumocytes by Mycobacterium tuberculosis

Dynamic, cholesterol-dense regions of the plasma membrane, known as lipid rafts (LR), have been observed to develop during and may be directly involved in infection of host cells by various pathogens. This study focuses on LR aggregation induced in alveolar epithelial cells during infection with Mycobacterium tuberculosis (Mtb) bacilli. We report dose- and time-dependent increases in LR aggregation after infection with three different strains at multiplicities of infection of 1, 10 and 100 from 2–24 hr post infection (hpi). Specific strain-dependent variations were noted among H37Rv, HN878 and CDC1551 with H37Rv producing the most significant increase from 15 aggregates per cell (APC) to 27 APC at MOI 100 during the 24 hour infection period. Treatment of epithelial cells with Culture Filtrate Protein, Total Lipids and gamma-irradiated whole cells from each strain failed to induce the level of LR aggregation observed during infection with any of the live strains. However, filtered supernatants from infected epithelial cells did produce comparable LR aggregation, suggesting a secreted mycobacterial product produced during infection of host cells is responsible for LR aggregation. Disruption of lipid raft formation prior to infection indicates that Mtb bacilli utilize LR aggregates for internalization and survival in epithelial cells. Treatment of host cells with the LR-disruption agent Filipin III produced a nearly 22% reduction in viable bacteria for strains H37Rv and HN878, and a 7% reduction for strain CDC1551 after 6 hpi. This study provides evidence for significant mycobacterial-induced changes in the plasma membrane of alveolar epithelial cells and that Mtb strains vary in their ability to facilitate aggregation and utilization of LR.     

The hypervirulent Mycobacterium tuberculosis strain HN878 induces a potent TH1 response followed by rapid down-regulation

The HN878 strain of Mycobacterium tuberculosis is regarded as "hypervirulent" due to its rapid growth and reduced survival of infected mice when compared with other clinical isolates. This property has been ascribed due to an early increase in type I IFNs and a failure to generate TH1-mediated immunity, induced by a response to an unusual cell wall phenolic glycolipid expressed by the HN878 isolate. We show, however, that although type I IFN does play an inhibitory role, this response was most apparent during the chronic disease stage and was common to all M. tuberculosis strains tested. In addition, we further demonstrate that the HN878 infection was associated with a potent TH1 response, characterized by the emergence of both CD4 and CD8 T cell subsets secreting IFN-gamma. However, where HN878 differed to the other strains tested was a subsequent reduction in TH1 immunity, which was temporally associated with the rapid emergence of a CD4+CD25+FoxP3+CD223+IL-10+ regulatory T cell population. This association may explain the paradoxical initial emergence of a TH1 response in these mice but their relatively short time of survival.     

Proteogenomic analysis of Mycobacterium tuberculosis by high resolution mass spectrometry

The genome sequencing of H37Rv strain of Mycobacterium tuberculosis was completed in 1998 followed by the whole genome sequencing of a clinical isolate, CDC1551 in 2002. Since then, the genomic sequences of a number of other strains have become available making it one of the better studied pathogenic bacterial species at the genomic level. However, annotation of its genome remains challenging because of high GC content and dissimilarity to other model prokaryotes. To this end, we carried out an in-depth proteogenomic analysis of the M. tuberculosis H37Rv strain using Fourier transform mass spectrometry with high resolution at both MS and tandem MS levels. In all, we identified 3176 proteins from Mycobacterium tuberculosis representing ~80% of its total predicted gene count. In addition to protein database search, we carried out a genome database search, which led to identification of ~250 novel peptides. Based on these novel genome search-specific peptides, we discovered 41 novel protein coding genes in the H37Rv genome. Using peptide evidence and alternative gene prediction tools, we also corrected 79 gene models. Finally, mass spectrometric data from N terminus-derived peptides confirmed 727 existing annotations for translational start sites while correcting those for 33 proteins. We report creation of a high confidence set of protein coding regions in Mycobacterium tuberculosis genome obtained by high resolution tandem mass-spectrometry at both precursor and fragment detection steps for the first time. This proteogenomic approach should be generally applicable to other organisms whose genomes have already been sequenced for obtaining a more accurate catalogue of protein-coding genes.     

Whole-genome comparison of Mycobacterium tuberculosis clinical and laboratory strains

Virulence and immunity are poorly understood in Mycobacterium tuberculosis. We sequenced the complete genome of the M. tuberculosis clinical strain CDC1551 and performed a whole-genome comparison with the laboratory strain H37Rv in order to identify polymorphic sequences with potential relevance to disease pathogenesis, immunity, and evolution. We found large-sequence and single-nucleotide polymorphisms in numerous genes. Polymorphic loci included a phospholipase C, a membrane lipoprotein, members of an adenylate cyclase gene family, and members of the PE/PPE gene family, some of which have been implicated in virulence or the host immune response. Several gene families, including the PE/PPE gene family, also had significantly higher synonymous and nonsynonymous substitution frequencies compared to the genome as a whole. We tested a large sample of M. tuberculosis clinical isolates for a subset of the large-sequence and single-nucleotide polymorphisms and found widespread genetic variability at many of these loci. We performed phylogenetic and epidemiological analysis to investigate the evolutionary relationships among isolates and the origins of specific polymorphic loci. A number of these polymorphisms appear to have occurred multiple times as independent events, suggesting that these changes may be under selective pressure. Together, these results demonstrate that polymorphisms among M. tuberculosis strains are more extensive than initially anticipated, and genetic variation may have an important role in disease pathogenesis and immunity.     

A new high-throughput AFLP approach for identification of new genetic polymorphism in the genome of the clonal microorganism Mycobacterium tuberculosis

We have here applied high-throughput amplified fragment length polymorphism (htAFLP) analysis to strains belonging to the five classical species of the Mycobacterium tuberculosis complex. Using 20 strains, three enzyme combinations and eight selective amplification primer pairs, 24 AFLP reactions were performed per strain. Overall, this resulted in 480 DNA fingerprints and more than 1200 htAFLP-amplified PCR fragments were visualised per strain. The cumulative dendrogram correctly clustered strains from the various species, albeit within a distance of 6.5% for most of them. The single isolate of Mycobacterium canettii presented separately at 19% distance. All over, 169 fragments (14%) appeared to be polymorphic. Sixty-eight were specific for M. canetti and forty-five for Mycobacterium bovis. For the 10 different M. tuberculosis strains included in the present analysis, 56 polymorphic markers were identified. Upon sequencing 20 of these marker regions and comparisons with the H37Rv genome sequence, 25% appeared to share homology to members of the antigenically variable PE/PPE surface protein encoding gene family confirming previous findings on the genetic heterogeneity within these genes. In addition, homologues for phage genes and insertion element-encoded genes were detected. Forty-five percent of the sequences derived from ORFs with a currently unknown function, which was corroborated by genome sequence comparison for the clinical M. tuberculosis CD 1551 isolate. Sequence variation in M. tuberculosis was assessed in more detail for a subset of these loci by newly designed PCR restriction fragment length polymorphism (RFLP) tests and direct sequencing. Fourteen novel PCR RFLP tests were developed and twelve novel single nucleotide polymorphisms (SNPs) were identified, all suited for epidemiological analysis of M. tuberculosis. The tests allowed for identification of the major Mycobacterium species and M. tuberculosis variants and clones.     

Genetic basis for the synthesis of the immunomodulatory mannose caps of lipoarabinomannan in Mycobacterium tuberculosis

Lipoarabinomannan (LAM) is a high molecular weight, heterogenous lipoglycan present in abundant quantities in Mycobacterium tuberculosis and many other actinomycetes. In M. tuberculosis, the non-reducing arabinan termini of the LAM are capped with alpha1-->2 mannose residues; in some other species, the arabinan of LAM is not capped or is capped with inositol phosphate. The nature and extent of this capping plays an important role in disease pathogenesis. MT1671 in M. tuberculosis CDC1551 was identified as a glycosyltransferase that could be involved in LAM capping. To determine the function of this protein a mutant strain of M. tuberculosis CDC1551 was studied, in which MT1671 was disrupted by transposition. SDS-PAGE analysis showed that the LAM of the mutant strain migrated more rapidly than that of the wild type and did not react with concanavalin A as did wild-type LAM. Structural analysis using NMR, gas chromatography/mass spectrometry, endoarabinanase digestion, Dionex high pH anion exchange chromatography, and matrix-assisted laser desorption ionization-time-of-flight mass spectrometry demonstrated that the LAM of the mutant strain was devoid of mannose capping. Since an ortholog of MT1671 is not present in Mycobacterium smegmatis mc(2)155, a recombinant strain was constructed that expressed this protein. Analysis revealed that the LAM of the recombinant strain was larger than that of the wild type, had gained concanavalin A reactivity, and that the arabinan termini were capped with a single mannose residue. Thus, MT1671 is the mannosyltransferase involved in deposition of the first of the mannose residues on the non-reducing arabinan termini and the basis of much of the interaction between the tubercle bacillus and the host cell.     

Dual mechanism for Mycobacterium tuberculosis cytotoxicity on lung epithelial cells

Mycobacterium tuberculosis strains CDC1551 and Erdman were used to assess cytotoxicity in infected A549 human alveolar epithelial cell monolayers. Strain CDC1551 was found to induce qualitatively greater disruption of A549 monolayers than was strain Erdman, although total intracellular and cell-associated bacterial growth rates over the course of the infections were not significantly different. Cell-free culture supernatants from human monocytic cells infected with either of the 2 M. tuberculosis strains produced a cytotoxic effect on A549 cells, correlating with the amount of tumor necrosis factor alpha (TNF-α) released by the infected monocytes. The addition of TNF-α-neutralizing antibodies to the supernatants from infected monocyte cultures did prevent the induction of a cytotoxic effect on A549 cells overlaid with this mixture but did not prevent the death of epithelial cells when added prior to infection with M. tuberculosis bacilli. Thus, these data agree with previous observations that lung epithelial cells infected with M. tuberculosis bacilli are rapidly killed in vitro. In addition, the data indicate that some of the observed epithelial cell killing may be collateral damage; the result of TNF-α released from M. tuberculosis-infected monocytes.     

Screening and comparison of differentially expressed genes between one MDR-TB strain and the virulent M. tuberculosis H37Rv

To screen and compare the differentially expressed genes between one MDR-TB strain separated from one child patient and the virulent Mycobacterium tuberculosis H37Rv, suppression subtractive hybridization (SSH) technology was used to build a library of cDNAs that were differentially expressed in the MDR and H37Rv. From this cDNA library, genes that were expressed in the MDR-TB but not in the H37Rv were selected for gene sequencing and homology analysis; 113 positive clones were obtained, their cDNA fragments were sequenced, and homology analysis was performed. Four novel sequences were identified. The results provide a partial list of genes differentially expressed in MDR-TB and four novel genes were found. Identification of these genes may contribute to our understanding of MDR-TB development.     

Mycobacterium tuberculosis: a model system for structural genomics

Over the past five years, genomics has had a major impact on Mycobacterium tuberculosis research. With the publication of the sequences of two virulent strains (H37Rv and CDC1551) and three closely related sequences, M. tuberculosis is becoming a model system for proteomics and structural genomics initiatives. Together with the promise of structures of proteins with novel folds, high-resolution structures of drug targets are providing the basis for rational inhibitor design, with the goal of the development of novel anti-tuberculars. In addition, this work is aiding scientists in the quest for an effective vaccine against this persistent pathogen.     

猪繁殖与呼吸综合征病毒cDNA文库的构建及基因组序列的测定

猪繁殖与呼吸综合征自1987年首次在美国发现以来,几年之内便席卷了北美洲和欧洲大陆,后蔓延至许多亚太国家和地区 .我国1995年首次暴发此病,该病在我国普遍存在,给我国养猪业的健康发展造成巨大障碍.目前国内外尚无理想防疫疫苗.当前用于预防的猪繁殖与呼吸综合征的主要疫苗是弱毒苗和灭活疫苗.灭活疫苗免疫效果差,弱毒苗能提供较好的免疫保护,但毒力返强的几率相当高,这一点已在几年前丹麦等国因广泛使用弱毒苗而导致该病大暴发中得以证实.     

Development of a physical and genetic map of the virulent Wolbachia strain wMelPop

We report here the construction of a physical and genetic map of the virulent Wolbachia strain, wMelPop. This map was determined by ordering 28 chromosome fragments that resulted from digestion with the restriction endonucleases FseI, ApaI, SmaI, and AscI and were resolved by pulsed-field gel electrophoresis. Southern hybridization was done with 53 Wolbachia-specific genes as probes in order to determine the relative positions of these restriction fragments and use them to serve as markers. Comparison of the resulting map with the whole genome sequence of the closely related benign Wolbachia strain, wMel, shows that the two genomes are largely conserved in gene organization with the exception of a single inversion in the chromosome.