PhoPR双组分信号转导系统对结核分枝杆菌持留性的调控机制研究

目的 本研究通过诱导结核分枝杆菌在低氧环境中进入持留状态,来比较分析不同时间不同低氧环境下结核分枝杆菌中的PhoP基因和PhoR基因的表达水平差异,探讨研究PhoPR双组分信号转导系统对结核分枝杆菌持留性的调控机制。方法 首先对结核分枝杆菌国际标准无毒株(H37Ra)和结核分枝杆菌国际标准强毒株(H37Rv)在不同低氧环境下进行培养,提取每个样本菌株的总RNA,并进行完整性鉴定;运用SYBR Green I实时荧光定量PCR检测每个样本菌株中PhoP基因和PhoR基因的表达水平;比较分析不同时间不同低氧环境下的结核分枝杆菌菌株PhoP基因和PhoR基因的表达水平差异。结果 在不同时间点对低氧环境的结核分枝杆菌PhoP基因和PhoR基因的表达水平进行检测,结果显示:与第10 d相比,培养至第15 d时H37Rv菌株和H37Ra菌株中的PhoP基因和PhoR基因的表达水平均显著上调,差异有统计学意义(P＜0.05);并且培养至第25 d时,H37Rv菌株中的PhoP基因和PhoR基因的表达水平比H37Ra菌株表达均上调2.34倍,差异有统计学意义(P＜0.05)。结论 在不同时间点相同毒力的结核分枝杆菌的PhoPR双组分信号转导系统中PhoP基因和PhoR基因在不同低氧环境下的表达存在差异,而且在相同时间点不同毒力结核分枝杆菌的PhoPR双组分信号转导系统中PhoP基因和PhoR基因在不同低氧环境下的表达也存在差异,表明PhoPR双组分信号转导系统对结核分枝杆菌的持留性具有调控作用。     

In-vitro study of mouse peritoneal macrophage response to virulent and avirulent strains of mycobacteria

An immunological study of pathogenesis of tuberculosis was carried out in BALB-c mice in-vitro. Peritoneal macrophages obtained from BALB-c mice were challenged with virulent (H37Rv) and avirulent (H37Ra, BCG, M. phlei) strains of mycobacteria. Activated peritoneal macrophages showed enlargement, presence of intracellular bacteria and vacuolation. These significant changes in macrophage morphology were clearly evidenced in cells infected with virulent strains of Mycobacterium tuberculosis i.e. H37Rv while being absent in cells infected with avirulent H37Ra, BCG and M. phlei. Virulent mycobacteria (H37Rv) survive the phagocytic action of macrophages by residing inside the vacuoles. The capacity of virulent and avirulent strain to stimulate TNF-alpha production from peritoneal macrophage of BALB-c mice was also examined at different time interval i.e. 1,2,4,6 and 8th day by measuring cytolytic activity of culture supernatant against murine fibroblast cell line. The pattern of highest TNF release was in case of H37Rv and least with M. phlei as measured in culture supernatant after 1,2,4,6 and 8th day.     

Chemokine receptor 5 and its ligands in the immune response to murine tuberculosis

SETTING: The ability of chemokines such as macrophage inflammatory protein (MIP)-1alpha, MIP-1beta, and regulated-upon-activation, normal T cell expressed and secreted (RANTES), to attract and activate T cells and monocytes, the building blocks of the granuloma, suggests that these chemokines may have a role in modulating immune responses to Mycobacterium tuberculosis infection. OBJECTIVE: We hypothesized that the chemokine receptor 5 (CCR5) ligands, MIP-1alpha, MIP-1beta and RANTES, are virulence correlates in M. tuberculosis infection and are indispensable to granuloma formation. DESIGN: The ability of virulent (H37Rv) and avirulent (H37Ra) strains of M. tuberculosis to induce chemokine production in vivo and in vitro was determined at protein and mRNA levels. We also compared bacterial burden, and granuloma numbers and size in H37Rv-infected CCR5-/- or wild-type C57BL/6 mice. RESULTS: In vivo, lung mRNA and protein measurements of MIP-1alpha, MIP-1beta and RANTES indicate significantly higher (p<0.05) values (days 14-28) in the H37Rv-infected than the H37Ra-infected mice. This is consistent with a higher infection burden of the virulent strain. However, in vitro alveolar macrophage stimulation by H37Rv or H37Ra yielded no significant differences in production of the three chemokines at all time points. Histological analysis of granulomas did not show any significant differences in granuloma numbers, size and M. tuberculosis growth in CCR5-/- compared to wild-type mice. CONCLUSIONS: The production of the CCR5 ligands, MIP-1alpha, MIP-1beta, and RANTES, does not clearly correlate with virulence of M. tuberculosis. These ligands and their receptors may not be indispensable to the development of granulomas in murine tuberculosis.     

Comparative expression studies of a complex phenotype: cord formation in Mycobacterium tuberculosis

The aggregation of mycobacteria into structures known as cords is an intrinsic property of the human tubercle bacillus. This property is thought to be determined by the lipid composition of the bacterial cell surface and may contribute to the virulence of the organism. Using microarray technology, we compared the pattern of gene expression of H37Rv, a virulent, cording strain of Mycobacterium tuberculosis, with H37Ra, an avirulent, non-cording strain derived from the same original patient isolate, under five different nutrient combinations and growth conditions. Under all of these conditions, H37Rv formed cords and H37Ra did not. By focusing our analysis only on genes that were differentially expressed under all conditions, we hoped to enrich the resulting gene list for genes associated with cording. We identified 22 genes that were consistently expressed at higher levels in H37Rv than in H37Ra under all conditions tested. Genes involved in lipid metabolism and the cell membrane were significantly enriched in our gene list, indicating that the cell wall and the cell membrane may be the major sites of difference between these two strains. This work represents a new strategy for enriching gene lists for relevant genes, which may also be applicable for other types of problems.     

The invasion of Mycobacterium tuberculosis into non-phagocytic cells

To explore the ability of tubercle bacilli to invade and survive within non-phagocytic cells, we used in this study a human fibroblast cell line, WI-38, derived from normal embryonic lung and a human epithelial cell line, SQ-5, derived from lung squamous cell carcinoma. Live M. tuberculosis Erdman and M. tuberculosis H37Rv invaded WI-38 cells more efficiently than live M. tuberculosis H37Ra, M. bovis Ravenel, M. bovis BCG Tokyo and M. bovis BCG Pasteur. The capability of tubercle bacilli to invade WI-38 cells was Erdman > or = H37Rv > BCG Pasteur [symbol: see text] M. bovis Ravenel [symbol: see text] BCG Tokyo > H37Ra. A similar invasive ability was observed using SQ-5 cells. In contrast with live bacilli, heat-killed bacilli failed to invade WI-38 cells, whereas they were detected within SQ-5 cells. These results and incorporation of latex beads suggest that SQ-5 cells, but not WI-38 cells, possess phagocytic activity. H37Rv multiplied most actively within WI-38 cells when compared to H37Ra and BCG Tokyo, suggesting that the ability to invade and survive within non-phagocytic cells reflects the more active invasion of virulent M. tuberculosis than avirulent M. tuberculosis. The assay system used in this study may help us to clarify the virulence of tubercle bacilli in vitro.     

Disruption of phagosomal membranes of normal alveolar macrophages by the H37Rv strain of Mycobacterium tuberculosis. A correlate of virulence

The H37Rv and H37Ra strains of Mycobacterium tuberculosis were incubated with normal rabbit alveolar macrophages and examined by electron microscopy at 5 to 6 and 18 to 24 h of incubation. At the 18- to 24-h incubation interval, 60 to 100% of the endocytosed organisms of the H37Rv strain disrupted the phagosomal membranes and appeared free in the cytoplasm of the macrophages. In contrast, the H37Ra strain lacked this putative virulence characteristic, and greater than 99% of the organisms appeared within intact phagosomes. Heating the organisms of the H37Rv strain abrogated to a large extent, but not completely, their capacity to disrupt phagosomal membranes. In the course of the interaction of organisms of the virulent H37Rv strain with phagosomal membranes of normal rabbit alveolar macrophages, adherence of the phagosomal membrane to the surface of the organisms was a prominent feature that was followed by fragmentation and apparent disintegration of the membrane. This potential virulence characteristic could explain why there is essentially no resistance expressed in the lung during the early postinfectious period of primary infection to M. tuberculosis.     

Changes in antibacterial activity of murine peritoneal macrophages against Mycobacterium tuberculosis after prolonged in vitro precultivation

We examined profiles of intramacrophagial growth of M. tuberculosis (MTB) when mouse peritoneal macrophages (M phi s) were infected with the organisms at day 0 or day 7 after in vitro precultivation, and obtained the following results. First, the growth rate of the virulent MTB H37Rv strain as well as attenuated H37Ra strain was slower in M phi s which had been precultured for 7 days (M phi s [day 7]) than in freshly prepared M phi s without precultivation (M phi s [day 0]). The doubling time of MTB H37Rv was 2.2 and 2.9 days in M phi s [day 0] and M phi s [day 7], respectively, and that of MTB H37Ra was 2.9 and 3.6 days in M phi s [day 0] and M phi s [day 7], respectively. Second, MTB-mediated cytotoxicity in terms of the LDH release from infected M phi s was less marked in M phi s [day 7] than in M phi s [day 0], when they were infected with MTB of either the H37Rv or H37Ra strain. MTB H37Ra strain exhibited much weaker cytotoxic effects on host M phi s than did H37Rv strain. Third, when M phi s [day 7] were infected with MTB of either the H37Rv or H37Ra strain, they showed markedly lowered levels of reactive oxygen intermediate (ROI) production than did M phi s [day 0]. In contrast, the reactive nitrogen intermediate (RNI) producing ability of M phi s in response to MTB infection was not so markedly reduced in M phi s [day 7] from that of M phi s [day 0]. As mentioned above, the M phi s [day 7] did not permit accelerated growth of infected MTB, compared to the MTB growth in the M phi s [day 0]. It thus appears that ROI played a trivial role in the antimicrobial activity against MTB of murine peritoneal M phi s which had been precultured for long periods. Although it is regarded that RNI played more critical roles in M phi anti-MTB activity than did ROI, the present results also suggest that other kinds of antimicrobial effectors are required in M phi antimicrobial activity against MTB organisms, particularly in the case of M phi s after prolonged in vitro cultivation.     

结核分枝杆菌耐利福平药物机理的初步研究

目的通过诱导试验观察结核菌产生耐利福平药物的全过程,初步分析结核菌耐该药的机理。方法采用诱导试验、聚合酶链反应—单链构象多态性(PCR-SSCP)和序列分析,对经不同药物浓度传代培养后的结核菌强毒株（H₃₇Rv）和36株临床耐药分离株进行分析。结果在诱导到第7代时,H₃₇Rv的PCR-SSCP电泳出现异常,经测序证实在513位点发生基因突变。36株临床耐药分离株中有23株发生突变,为63.9% (23/36);其中有5株与诱导株基因突变位点相同。结论用药物不间断的刺激结核菌,是产生结核菌耐利福平药物的重要原因。     

Glycolipid-patterns of Mycobacterium tuberculosis as an aid for sub-typing

Total lipids were extracted from Mycobacterium tuberculosis with chloroform-methanol (2:1), applied on a silica-gel thin-layer plate, and developed with chloroform-methanol-acetone (90:10:5). Glycolipids were detected by spraying Anthrone-reagent and heating. Strain H37Rv of M. tuberculosis showed four Anthrone-positive spots, namely trehalose-monomycolate, unidentified glycolipid, trehalose-dimycolate and GL-Rv, and strain H37Ra showed only two spots corresponding to trehalose-monomycolate and trehalose-dimycolate. Other 4 laboratory-stock strains of M. tuberculosis showed glycolipid-pattern identical with either of these two patterns. One hundred and fifty-eight strains of M. tuberculosis, isolated clinically from tuberculosis patients, were classified into 7 types according to their glycolipid-pattern. Twenty-seven strains contained one more Anthrone-positive spot other than those of strain H37Rv. Pattern II was most frequently observed (60 strains), and then pattern I (33 strains), VI (29 strains), IV (13 strains), V (9 strains), VII (8 strains), and III (6 strains). Pattern I corresponded to that of strain H37Ra and pattern VI corresponded to that of strain H37Rv. Glycolipid-pattern did not correlate to clinical features of patients from whom the bacilli had been isolated. A glycolipid, which moved to just under the solvent front, was a new glycolipid which has been found by us and designated as GL-Rv. Chemical structure of GL-Rv was clarified by us as trehalose-polyacyl derivatives (no mycolic acid as the acyl residue). Glycolipid-pattern was very stable and reproducible for each strain of M. tuberculosis.(ABSTRACT TRUNCATED AT 250 WORDS)     

流式细胞术检测不同毒力结核分枝杆菌感染巨噬细胞的凋亡率及其时相性变化

目的利用流式细胞技术检测结核分枝杆菌国际标准强毒株H37Rv株和卡介苗菌株(BCG)分别感染巨噬细胞的凋亡率及其时相性变化。方法分别用结核分枝杆菌H37Rv株和BCG菌株感染巨噬细胞RAW264.7细胞株,于感染后1、3、6及12h,用流式细胞技术检测各组感染巨噬细胞的凋亡率,并分析其时相性变化。结果结核分枝杆菌H37Rv株和BCG菌株感染均使巨噬细胞凋亡率升高,其中在感染后1h和12h凋亡率升高更显著,两组比较凋亡率差异有统计学意义(P<0.05)。结论结核分枝杆菌感染导致巨噬细胞凋亡,凋亡率与菌株毒力强弱相关,毒力弱的结核分枝杆菌感染早期巨噬细胞升高更显著。     

结核分支杆菌H37Rv与H36Ra株mRNA基因差异显示研究

目的 采用ｍＲＮＡ差异显示技术研究Ｈ３７Ｒｖ珠与Ｈ３７Ｒａ株的基因差异，为获取结核分支杆菌毒力相关基因奠定基础。方法 提取结核分支杆菌Ｈ３７Ｒｖ株和Ｈ３７Ｒａ株ＲＮＡ，经逆转录、ＰＣＲ扩增、聚丙烯酰胺凝胶电泳和放射自显影进行ｍＲＮＡ的差异显示。回收差异条带、再扩增和序列分析，进行同源性查询。结果 经差异显示后，共回收与Ｈ３７Ｒｖ株ｍＲＮＡ差异条带１０条。经结果查询，发现其中２条差异条带与结核分支杆     

结核分支杆菌H37Rv与H37Ra株mRNA基因差异显示研究

目的　采用mRNA差异显示技术研究H3 7Rv株与H3 7Ra株的基因差异 ,为获取结核分支杆菌毒力相关基因奠定基础。方法　提取结核分支杆菌H3 7Rv株和H3 7Ra株RNA ,经逆转录、PCR扩增、聚丙烯酰胺凝胶电泳和放射自显影进行mRNA的差异显示。回收差异条带、再扩增和序列分析 ,进行同源性查询。结果　经差异显示后 ,共回收与H3 7Ra株不同的H3 7Rv株mRNA差异条带 10条。经结果查询 ,发现其中 2条差异条带与结核分支杆菌H3 7Rv株DNA序列具有同源性 ,而与H3 7Ra无同源性。结论　mRNA差异显示方法具有高效、灵敏等优点 ,可在基因水平掌握同一种属或不同种属不同株间的差异 ,用于结核分支杆菌相关毒力基因的研究。研究获得的H3 7Rv株与H3 7Ra株的差异片段可能含有与结核分支杆菌毒力相关的基因     

Expression, characterization and subcellular localization of the Mycobacterium tuberculosis PPE gene Rv1917c.

SETTING: The PPE gene family of Mycobacterium tuberculosis is thought to be of immunological significance. One member, Rv1917c, is highly polymorphic in clinical isolates. OBJECTIVE: To characterize Rv1917c gene polymorphism and expression, and to determine the cellular location and glycosylation status of the encoded protein. DESIGN: Tandem repeat regions of Rv1917c were amplified and sequenced to determine the molecular basis for the gene polymorphism. RT-PCR analysis was utilized to detect expression of Rv1917c mRNA in liquid cultures of M. tuberculosis H37Rv. The gene was cloned as a 3'-terminal green fluorescent protein (GFP) fusion, downstream of an acetamide-inducible promoter, and expressed in Mycobacterium smegmatis and Mycobacterium bovis BCG. The expression product was characterized in terms of cellular location and glycosylation status. RESULTS: PCR and sequence data demonstrated that variable numbers of tandem repeats within Rv1917c contribute to gene polymorphism. RT-PCR analysis demonstrated that Rv1917c mRNA is expressed in liquid cultures of M. tuberculosis H37Rv. Expression of the recombinant protein in M. smegmatis and M. bovis BCG was visualized by fluorescence microscopy and flow cytometry. A protein of the predicted size (166 kDa) was confirmed by Western blotting. Cell fractionation studies demonstrated that the recombinant protein is hydrophobic, suggestive of cell wall-association, while flow cytometric data derived from antibody binding experiments suggested that it is surface exposed. Analysis of the glycosylation status of the expressed protein failed to demonstrate glycosylation. CONCLUSION: Rv1917c mRNA is expressed in M. tuberculosis H37Rv, and Rv1917c gene polymorphism is associated with variable numbers of tandem repeats. The recombinant Rv1917c protein is surface exposed.     

Comparative analysis of mycobacterial infections in susceptible I/St and resistant A/Sn inbred mice.

SETTING: The availability and appropriate use of animal models is of significant importance for a better and more detailed understanding of the genetic, immunological and pathological mechanisms underlying the development of mycobacterial disease in humans. OBJECTIVE: To define a mouse model for tuberculosis severity that can be easily adapted to genetic and immunological analysis of host response to Mycobacterium tuberculosis infection. DESIGN: We describe here two inbred strains of mice, I/St and A/Sn (both Nramp1'), that differ vastly in commonly used parameters of susceptibility to infection with virulent and attenuated strains of M. tuberculosis. RESULTS: Following infection with a high dose of virulent H37Rv. M. tuberculosis and compared to their resistant A/Sn counterparts, I/St mice displayed more than a 2-fold shorter mean survival time and a more rapid onset and progression of severe body weight loss (cachexia). Moreover, I/St mice supported 20-100-fold higher multiplication of M. tuberculosis following challenge with H37Rv over a large range of infectious inocula. The high susceptibility of I/St mice was also reflected by more severe lung histopathology as evidenced by larger and more numerous lung granuloma and macrophage dominated cellular infiltrates. Finally, we determined that I/St are also unable to control infection with attenuated H37Ra M. tuberculosis and two strains of M. bovis (BCG and Ravenel) indicating hyper-susceptibility of the I/St mouse strain to mycobacterial infections. CONCLUSIONS: The results of our experiments suggest that comparative analysis of resistant A/Sn and susceptible I/St mice provides an ideal way to study host dependent aspects of tuberculosis susceptibility under the controlled conditions provided by an animal model.