- [Show abstract] [Hide abstract] ABSTRACT: Three hypervirulent strains of Mycobacterium tuberculosis isolated from patients suffering from tuberculous meningitis were shown to grow more rapidly inside human macrophages in our previous study. In the current investigation, genomic polymorphisms in these hypervirulent strains were examined using microarray-based comparative genomic hybridization. Among the five genomic polymorphisms identified, two are in-frame deletion (Rv0071/4 and Rv0613c/6c), two are frameshift deletion (Rv1758' and Rv2820c'), and one is gene replacement (Mb3159). The five genomic polymorphisms were transformed into Mycobacterium smegmatis strain mc(2)155 and the survivability of recombinants inside the human monocytic cell line THP-1 was measured. Interestingly, only the recombinant possessing the Rv2820c' survived significantly better than the vector control after 6 h of ex vivo infection (P < 0.001, one-way ANOVA). The Rv2820c' was later transformed into Mycobacterium marinum strain M and the recombinant was used to infect zebrafish. The in vivo infection also showed that the zebrafish infected with the recombinant possessing the Rv2820c' died significantly faster than the vector control (P = 0.006, log-rank test). The 3' truncation in the Rv2820c' was caused by the Beijing/W-defining deletion RD207 and is commonly found in the Beijing/W strains. The current study demonstrated that the truncated Rv2820c of Beijing/W strains could enhance mycobacterial virulence ex vivo and in vivo. This enhancement, however, was not observed for the intact Rv2820c of the non-Beijing/W strains. The presence of the 3' truncated portion of Rv2820c may interfere with overall protein folding and render the Rv2820c of the non-Beijing/W strains non-functional.
- [Show abstract] [Hide abstract] ABSTRACT: Objective: To study the different response in macrophages treated with different agonists (H37Rv-PPD and BCG-PPD) related with Mycobacterium tuberculosis and the relationship with TNF-α, IL-1β and IL-10. Methods: Using H37Rv-PPD and BCG-PPD to stimulate THP-1 cell for 3 h, 8 h, 15 h, 24 h respectively. Cells were ananlyzed by Hochest staining under fluorescence microscopy to assay cell death (apoptosis and necrosis). At each stimulating time, TNF-α, IL-1β (3 and IL-10 were examined by ELISA. Results: Under fluorescence microscopy, it could easily see oval apoptotic bodies of THP-1 stimulated by BCG-PPD. However, the nucleus were often isolated and necrosis-like when cells were stimulated by H37Rv-PPD. In a word , BCG-PPD tend to induce THP-1 cells to apoptosis, but H37Rv-PPD inclined to induce cells to undergo necrosis. In supernatant of cells stimulated by BCG-PPD, the expression of TNF-α and IL-10 were lower than the cells stimulated by H37Rv-PPD, but the expression of IL-1β was higher than the latter. Conclusion: It indicated that the necrosis of cells stimulated by H37Rv-PPD was asossiated with the excessive expression of TNF-α and IL-10, and the apoptosis of cells induced by BCG-PPD was IL-1β related. Perhaps the mechanism of differences in virulence exist in protein of strain, and associated with cytokines IL-1β, TNF-α and IL-10.
- [Show abstract] [Hide abstract] ABSTRACT: Ability to persist in human macrophages is central to the virulence of Mycobacterium tuberculosis and is not invariable among various strains. Differential gene expression that is associated with phenotypic virulence may provide additional information of virulent genes involved in the pathogenesis of M. tuberculosis, which is not fully elucidated. Three hypervirulent strains of M. tuberculosis isolated from patients suffering with tuberculous meningitis were shown to grow more rapidly inside human macrophages in a previous study. In the current investigation, expression of 7 mycobacterial genes (fadE28, mce1A, mymA, acr, sigA, sugC, and Rv3723) of these strains during ex vivo macrophage challenge and in vitro acid shock was quantified by real-time PCR. Using rrs gene as a normalisation gene, fadE28 gene exhibited differential gene expression that is associated with phenotypic virulence, whereas the other 6 genes showed indistinguishable expression patterns. Up-regulation of fadE28 gene in the hypervirulent strains may account for virulence by increasing the efficiency of beta-oxidation, which is important for the persistence in macrophages as M. tuberculosis uses fatty acids preferably inside phagosome of macrophages. The fadE28 gene, together with its adjacent genes may also be critical in the process of lipid modification that could facilitate parasitism in human macrophages.
- [Show abstract] [Hide abstract] ABSTRACT: Among 125 clinical isolates of Mycobacterium tuberculosis collected in Hong Kong and Shanghai, China, between 2002 and 2004, IS6110 typing revealed that 71 strains (57%) belonged to the Beijing family. The intracellular growth of the strains in human peripheral blood monocyte-derived macrophages was measured ex vivo on days 0, 3, 6, and 10. Among all tested strains, three hypervirulent strains showed significant increases in intracellular growth after 10 days of incubation. With an initial bacterial load of 10(4) CFU, most of the clinical isolates and H37Ra (an avirulent strain) exhibited no intracellular survival on day 10, while the three hypervirulent strains together with H37Rv (a virulent strain) showed on average a two- to fourfold rise in CFU count. These three hypervirulent strains belonging to a non-Beijing family were isolated from patients suffering from tuberculosis meningitis. Cytokines secreted by gamma interferon-activated macrophages were measured daily after challenge with selected strains of M. tuberculosis. The levels of tumor necrosis factor alpha were elevated after 24 h of infection among all strains, but the levels were significantly lower among the three hypervirulent strains, whereas interleukin 10 (IL-10) and IL-12 were not detected. Results were concordant with the differential expression of the corresponding cytokine genes in activated macrophages, as monitored by real-time PCR. Our findings highlighted that these three hypervirulent strains may possess an innate mechanism for escaping host immunity, which accounts for their characteristic virulence in patients presenting with a more severe form of disease.