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1.
In Mycobacterium tuberculosis (Mtb), surface-exposed Lipoarabinomannan (LAM) is a key determinant of immunogenicity, yet its intrinsic heterogeneity confounds typical structure–function analysis. Recently, LAM gained a strong foothold as a validated marker for active tuberculosis (TB) infection and has shown great potential in new diagnostic efforts. However, no efforts have yet been made to model or evaluate the impact of mixed polyclonal Mtb infections (infection with multiple strains) on TB diagnostic procedures other than antibiotic susceptibility testing. Here, we selected three TB clinical isolates (HN878, EAI, and IO) and purified LAM from these strains to present an integrated analytical approach of one-dimensional and two-dimensional Nuclear Magnetic Resonance (NMR) spectroscopy, as well as enzymatic digestion and site-specific mass spectrometry (MS) to probe LAM structure and behavior at multiple levels. Overall, we found that the glycan was similar in all LAM preparations, albeit with subtle variations. Succinates, lactates, hydroxybutyrate, acetate, and the hallmark of Mtb LAM-methylthioxylose (MTX), adorned the nonreducing terminal arabinan of these LAM species. Newly identified acetoxy/hydroxybutyrate was present only in LAM from EAI and IO Mtb strains. Notably, detailed LC/MS-MS unambiguously showed that all acyl modifications and the lactyl ether in LAM are at the 3-OH position of the 2-linked arabinofuranose adjacent to the terminal β-arabinofuranose. Finally, after sequential enzymatic deglycosylation of LAM, the residual glycan that has ∼50% of α−arabinofuranose -(1→5) linked did not bind to monoclonal antibody CS35. These data clearly indicate the importance of the arabinan termini arrangements for the antigenicity of LAM.  相似文献   

2.
Surfactant protein-D (SP-D) is a collectin produced in the distal lung airspaces that is believed to play an important role in innate pulmonary immunity. Naive immunologic responses to Mycobacterium tuberculosis (M.tb) are especially important in the lung, since entry of this inhaled pathogen into the alveolar macrophage is a pivotal event in disease pathogenesis. Here we investigated SP-D binding to M.tb and the effect of this binding on the adherence of M. tb to human macrophages. These studies demonstrate specific binding of SP-D to M.tb that is saturable, calcium dependent, and carbohydrate inhibitable. In addition to purified SP-D, SP-D in bronchoalveolar lavage fluids from healthy donors and patients with alveolar proteinosis also binds to M.tb. Incubation of M.tb with SP-D results in agglutination of the bacteria. In contrast to its binding to M.tb, SP-D binds minimally to the avirulent Mycobacterium smegmatis. SP-D binds predominantly to lipoarabinomannan from the virulent Erdman strain of M.tb, but not the lipoarabinomannan from M. smegmatis. The binding of SP-D to Erdman lipoarabinomannan is mediated by the terminal mannosyl oligosaccharides of this lipoglycan. Incubation of M.tb with subagglutinating concentrations of SP-D leads to reduced adherence of the bacteria to macrophages (62.7% of control adherence +/- 3.3% SEM, n = 8), whereas incubation of bacteria with surfactant protein A leads to significantly increased adherence to monocyte-derived macrophages. These data provide evidence for specific binding of SP-D to M. tuberculosis and indicate that SP-D and surfactant protein A serve different roles in the innate host response to this pathogen in the lung.  相似文献   

3.

Background  

The ability of Mycobacterium tuberculosis to survive and replicate in macrophages is crucial for the mycobacterium's ability to infect the host and cause tuberculosis. To identify Mycobacterium tuberculosis genes involved in survival in macrophages, a library of non-pathogenic Mycobacterium smegmatis bacteria, each carrying an individual integrated cosmid containing M. tuberculosis H37Rv genomic DNA, was passed through THP-1 human macrophages three times.  相似文献   

4.
目的利用流式细胞技术检测耐多药结核分枝杆菌(Multidrug resistant mycobacterium tuberculosis,MDRTB)临床分离株和结核分枝杆菌一、二线药物敏感株分别感染U937细胞后的凋亡率及其时相性变化。方法用两组细菌分别感染U937细胞,在感染后2、4、8及12 h用流式细胞技术检测各组感染U937细胞的凋亡率,并分析其时相性变化。结果结核分枝杆菌MDR株和敏感株均能诱导U937细胞凋亡,两组细胞凋亡率比较差异有统计学意义(P0.01),MDR株诱导U937细胞凋亡率显著低于敏感株诱导的凋亡率,且在诱导后2、4、8及12 h各时间点的凋亡率都显著低于敏感株组,诱导早期凋亡率升高显著,特别是诱导后2 h凋亡率升高最显著,4 h以后凋亡率升高逐渐变慢。结论结核分枝杆菌临床分离株感染U937细胞后能快速诱导其凋亡,凋亡率与诱导菌株的耐药表型相关,敏感株诱导的凋亡率显著高于MDR株,诱导早期凋亡率升高最显著,4 h以后凋亡率升高减慢。  相似文献   

5.
Antimicrobial resistance was studied in 100 Mycobacterium tuberculosis strains selected randomly from sputum cultures of newly diagnosed tuberculosis patients. Resistance of the isolates to rifampicin, isoniazid, and ethambutol was tested by both drug susceptibility testing (DST) and allele-specific PCR (AS-PCR). A total of 19 (19%) isolates were found resistant to at least one of the antituberculosis drugs investigated by PCR compared with 14 (14%) resistant isolates detected by DST. Eleven mutations were detected by AS-PCR in the rpoB gene (codons 516, 526, and 531), associated with rifampicin resistance, a marker of multidrug-resistant tuberculosis (MDR-TB), 14 mutations in the katG gene codon 315 that confers resistance to isoniazid, and nine mutations in the embB gene codon 306 that confers resistance to ethambutol. Mutations in the six multidrug-resistant isolates were confirmed by DNA sequencing. Results were compared with phenotypic DST data. Nineteen different mutation types to at least one of the drugs were found; six isolates (6%) were classified as MDR-TB, defined as resistance to at least rifampicin and isoniazid. The rates of concordance of the PCR with the phenotypic susceptibility test were 71.4, 54.5, and 44.4 for isoniazid, rifampicin, and ethambutol, respectively. These results highlight the importance of molecular epidemiology studies of tuberculosis in understudied regions with a tuberculosis burden to uncover the true prevalence of the MDR-TB.  相似文献   

6.
The invasiveness of 2 grud sensitive and 8 holy-resistant M. tuberculosis clinical isolated was evaluated in experiments on BALB/c mice. Mycobacterial suspension was injected into the caudal vein of experimental animals. The results were evaluated by the degree of contamination of lungs and spleen of infected animals euthanized at different periods on time. The study revealed high variability in the degree of contamination of the organs of the animals infected with M. tuberculosis drug-resistant clinical isolates.  相似文献   

7.

Background

Due to excessive antibiotic use, drug-resistant Mycobacterium tuberculosis has become a serious public health threat and a major obstacle to disease control in many countries. To better understand the evolution of drug-resistant M. tuberculosis strains, we performed whole genome sequencing for 7 M. tuberculosis clinical isolates with different antibiotic resistance profiles and conducted comparative genomic analysis of gene variations among them.

Results

We observed that all 7 M. tuberculosis clinical isolates with different levels of drug resistance harbored similar numbers of SNPs, ranging from 1409–1464. The numbers of insertion/deletions (Indels) identified in the 7 isolates were also similar, ranging from 56 to 101. A total of 39 types of mutations were identified in drug resistance-associated loci, including 14 previously reported ones and 25 newly identified ones. Sixteen of the identified large Indels spanned PE-PPE-PGRS genes, which represents a major source of antigenic variability. Aside from SNPs and Indels, a CRISPR locus with varied spacers was observed in all 7 clinical isolates, suggesting that they might play an important role in plasticity of the M. tuberculosis genome. The nucleotide diversity (Л value) and selection intensity (dN/dS value) of the whole genome sequences of the 7 isolates were similar. The dN/dS values were less than 1 for all 7 isolates (range from 0.608885 to 0.637365), supporting the notion that M. tuberculosis genomes undergo purifying selection. The Л values and dN/dS values were comparable between drug-susceptible and drug-resistant strains.

Conclusions

In this study, we show that clinical M. tuberculosis isolates exhibit distinct variations in terms of the distribution of SNP, Indels, CRISPR-cas locus, as well as the nucleotide diversity and selection intensity, but there are no generalizable differences between drug-susceptible and drug-resistant isolates on the genomic scale. Our study provides evidence strengthening the notion that the evolution of drug resistance among clinical M. tuberculosis isolates is clearly a complex and diversified process.

Electronic supplementary material

The online version of this article (doi: 10.1186/1471-2164-15-469) contains supplementary material, which is available to authorized users.  相似文献   

8.

Background

Mycobacterium tuberculosis phoP mutant SO2 derived from a clinical isolate was shown to be attenuated in mouse bone marrow-derived macrophages and in vivo mouse infection model and has demonstrated a high potential as attenuated vaccine candidate against tuberculosis.

Methodology/Principal Findings

In this study, we analyze the adhesion and the intracellular growth and trafficking of SO2 in human macrophages. Our results indicate an enhanced adhesion to phagocitic cells and impaired intracellular replication of SO2 in both monocyte-derived macrophages and human cell line THP-1 in comparison with the wild type strain, consistent with murine model. Intracellular trafficking analysis in human THP-1 cells suggest that attenuation of SO2 within macrophages could be due to an impaired ability to block phagosome-lysosome fusion compared with the parental M. tuberculosis strain. No differences were found between SO2 and the wild-type strains in the release and mycobacterial susceptibility to nitric oxide (NO) produced by infected macrophages.

Conclusions/Significance

SO2 has enhanced ability to bind human macrophages and differs in intracellular trafficking as to wild-type M. tuberculosis. The altered lipid profile expression of the phoP mutant SO2 and its inability to secrete ESAT-6 is discussed.  相似文献   

9.
Phagocytosis by macrophages represents the early step of the mycobacterial infection. It is governed both by the nature of the host receptors used and the ligands exposed on the bacteria. The outermost molecules of the nonpathogenic Mycobacterium smegmatis were extracted by a mechanical treatment and found to specifically and dose dependently inhibit the phagocytosis of both M. smegmatis and the opportunistic pathogen M. kansasii by human macrophages derived from monocytes. The inhibitory activity was attributed to surface lipids because it is extracted by chloroform and reduced by alkaline hydrolysis but not by protease treatment. Fractionation of surface lipids by adsorption chromatography indicated that the major inhibitory compounds consisted of phospholipids and glycopeptidolipids (GPLs). Mass spectrometry and nuclear magnetic resonance spectroscopy analyses, combined with chemical degradation methods, demonstrated the existence of a novel family of GPLs that consists of a core composed of the long-chain tripeptidyl amino-alcohol with a di-O-acetyl-6-deoxytalosyl unit substituting the allo-threoninyl residue and a 2-succinyl-3,4-di-O-CH3-rhamnosyl unit linked to the alaninol end of the molecules. These compounds, as well as diglycosylated GPLs at the alaninol end and de-O-acylated GPLs, but not the non-serovar-specific di-O-acetylated GPLs, inhibited the phagocytosis of M. smegmatis and M. avium by human macrophages at a few nanomolar concentration without affecting the rate of zymosan internalization. At micromolar concentrations, the native GPLs also inhibit the uptake of both M. tuberculosis and M. kansasii. De-O-acylation experiments established the critical roles of both the succinyl and acetyl substituents. Collectively, these data provide evidence that surface-exposed mycobacterial glycoconjugates are efficient competitors of the interaction between macrophages and mycobacteria and, as such, could represent pharmacological tools for the control of mycobacterial infections.  相似文献   

10.
In the current study we aimed to execute a rather less complicated molecular tying method, i.e. the random amplification of polymorphic DNA (RAPD) analysis to find the heterogeneity of Iranian strains of Mycobacterium tuberculosis. The isolates comprised a total of 96 strains of M. tuberculosis collected from clinical specimens of patients in Isfahan and Tehran. The isolates were assigned to the species M. tuberculosis by the key conventional and molecular methods. They were then subjected to RAPD analysis by four arbitrary primers, namely, the primers 27F, 1525R, MS- GF and INS-2. They were then evaluated for the number and intensity of the band patterns. The RAPD profiles of the Iranian isolates showed a degree of heterogeneity which varied based on the primer used. However, analysis of the isolates by primer INS-2 revealed the highest degree of diversity yielding 31 distinguishable RAPD types. RAPD analysis provides a rapid and easy means of identifying heterogeneity among the M. tuberculosis isolates. This typing system might be considered a valuable alternative molecular typing for countries with limited resources provided that the reproducibility and reliability of the method is carefully assured.  相似文献   

11.
Intracellular survival plays a central role in the pathogenesis of Mycobacterium tuberculosis. To identify M. tuberculosis genes required for intracellular survival within macrophages, an M. tuberculosis H37Rv plasmid library was constructed by using the shuttle vector pOLYG. This plasmid library was electroporated into Mycobacterium smegmatis 1-2c, and the transformants were used to infect the human macrophage-like cell line U-937. Because M. smegmatis does not readily survive within macrophages, any increased intracellular survival is likely due to cloned M. tuberculosis H37Rv DNA. After six sequential passages of M. smegmatis transformants through U-937 cells, one clone (p69) was enriched more than 70% as determined by both restriction enzyme and PCR analyses. p69 demonstrated significantly enhanced survival compared to that of the vector control, ranging from 2.4- to 5.3-fold at both 24 and 48 h after infection. DNA sequence analysis revealed three open reading frames (ORFs) in the insert of p69. ORF2 (1.2 kb) was the only one which contained a putative promoter region and a ribosome-binding site. Deletion analysis of the p69 insert DNA showed that disruption of ORF2 resulted in complete loss of the enhanced intracellular survival phenotype. This gene was named the enhanced intracellular survival (eis) gene. By using an internal region of eis as a probe for Southern analysis, eis was found in the genomic DNA of various M. tuberculosis strains and of Mycobacterium bovis BCG but not in that of M. smegmatis or 10 other nonpathogenic mycobacterial species. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis showed that all M. smegmatis eis-containing constructs expressed a unique protein of 42 kDa, the predicted size of Eis. The expression of this 42-kDa protein directly correlated to the enhanced survival of M. smegmatis p69 in U-937 cells. These results suggest a possible role for eis and its protein product in the intracellular survival of M. tuberculosis.  相似文献   

12.
13.

Background

Tuberculosis (TB) is the most threatening infectious disease globally. Although progress has been made to reduce global incidence of TB, emergence of multidrug resistant (MDR) TB threatens to undermine these advances. To combat the disease, novel intervention strategies effective against drug resistant and sensitive subpopulations of M. tuberculosis are urgently required as adducts in the present treatment regimen. Using THP-1 cells we have analyzed and compared the global protein expression profile of broth-cultured and intraphagosomally grown drug resistant and sensitive M.tuberculosis clinical isolates.

Results

On comparing the two dimensional (2-DE) gels, many proteins were found to be upregulated/expressed during intracellular state which were identified by matrix assisted laser desorption/ionization mass spectrometry (MALDI-MS). Four proteins (adenosylhomocysteinase, aspartate carbomyltransferase, putatitive thiosulfate sulfurtransferase and universal stress protein) were present in both intracellular MDR and sensitive isolates and three of these belonged to intermediary metabolism and respiration category. Two proteins (alanine dehydrogenase and adenosine kinase) of intracellular MDR isolate and two (glucose-6-phosphate isomerase and ATP synthase epsilon chain) of intracellular sensitive isolate belonged to intermediary metabolism and respiration category. One protein (Peroxidase/Catalase) of intracellular MDR and three (HSPX, 14 kDa antigen and 10 kDa chaperonin) of sensitive isolate belonged to virulence, detoxification and adaptation category. ESAT-6 of intracellular MDR belonged to cell wall and cell processes category. Two proteins (Antigen 85-C and Antigen 85-A) of intracellular sensitive isolate were involved in lipid metabolism while probable peptidyl-prolyl cis-trans isomerase A was involved in information pathways. Four (Rv0635, Rv1827, Rv0036c and Rv2032) of intracellular MDR and two proteins (Rv2896c and Rv2558c) of sensitive isolate were hypothetical proteins which were functionally characterized using bioinformatic tools. Bioinformatic findings revealed that the proteins encoded by Rv0036, Rv2032c, Rv0635, Rv1827 and Rv2896c genes are involved in cellular metabolism and help in intracellular survival.

Conclusions

Mass spectrometry and bioinformatic analysis of both MDR and sensitive isolates of M. tuberculosis during intraphagosomal growth showed that majority of commonly upregulated/expressed proteins belonged to the cellular metabolism and respiration category. Inhibitors of the metabolic enzymes/intermediate can therefore serve as suitable drug targets against drug-resistant and sensitive subpopulations of M. tuberculosis.  相似文献   

14.
Isoniazid (INH) resistance was genotypically assessed in 104 (37 INH-susceptible, 67 INH-resistant) genetically unrelated Mycobacterium tuberculosis strains cultured in North Italy. The PCR products of selected regions of the katG gene, the oxyR-ahpC intergenic region, and the inhA regulatory region were analyzed utilizing the double gradient-denaturing gradient gel electrophoresis (DG-DGGE) technique and confirmed by DNA sequencing. Mutations were detected in 61 (91%) of the INH-resistant strains, the relative frequency of the mutations being 65.7% in katG, 23.9% in oxyR-ahpC, and 13.4% in inhA. Previously described alterations, invariably associated with drug resistance, accounted for 95.1% of the mutations. No alterations were found in the INH-susceptible strains. DG-DGGE analysis and DNA sequencing were equally sensitive, but the former is cheaper, easier and more robust. Rapid genotypic assessment of INH resistance by means of the methodology described here could reasonably be used in clinical mycobacteriology laboratories.  相似文献   

15.
Mycobacterium tuberculosis successfully parasitizes macrophages by disrupting the maturation of its phagosome, creating an intracellular compartment with endosomal rather than lysosomal characteristics. We have recently demonstrated that live M. tuberculosis infect human macrophages in the absence of an increase in cytosolic Ca(2+) ([Ca(2+)](c)), which correlates with inhibition of phagosome-lysosome fusion and intracellular viability. In contrast, killed M. tuberculosis induces an elevation in [Ca(2+)](c) that is coupled to phagosome-lysosome fusion. We tested the hypothesis that defective activation of the Ca(2+)-dependent effector proteins calmodulin (CaM) and CaM-dependent protein kinase II (CaMKII) contributes to the intracellular pathogenesis of tuberculosis. Phagosomes containing live M. tuberculosis exhibited decreased levels of CaM and the activated form of CaMKII compared with phagosomes encompassing killed tubercle bacilli. Furthermore, ionophore-induced elevations in [Ca(2+)](c) resulted in recruitment of CaM and activation of CaMKII on phagosomes containing live M. tuberculosis. Specific inhibitors of CaM or CaMKII blocked Ca(2+) ionophore-induced phagosomal maturation and enhanced the bacilli's intracellular viability. These results demonstrate a novel role for CaM and CaMKII in the regulation of phagosome-lysosome fusion and suggest that defective activation of these Ca(2+)-activated signaling components contributes to the successful parasitism of human macrophages by M. tuberculosis.  相似文献   

16.
A total of 230 clinically drug-resistant and 3 drug-sensitive isolates of M. tuberculosis obtained from patients in Tula and Tula region in 1998-2001 were studied. The RFLP-IS6110 genotyping showed that 52 (30.2%) of isolates had unique patterns, and 120 (69.8%) of them were grouped to form 16 clusters. 95 (55.2%) and 53 (30.8%) of isolates were attributed to groups A1 and W (Beijing), respectively. Double mycobacterium cultures were detected in 4.1% of isolates. 2 to 4 clinical isolates were obtained from each of 55 patients during 1.5 years. A replacement of mycobacterium isolates was registered in the course of treatment in 12 (21.8%) patients. No replacement of clinical isolates occurred in 43 (78.2%) patients during the whole follow-up period. Repeatedly obtained isolates acquired the determinants of drug-resistance in 5 (11.6%) patients. Changes in the quantity of IS6110 elements were registered only in 1.05% of isolates during a 3-year follow-up.  相似文献   

17.
The biosynthesis of lipoarabinomannan (LAM), a key mycobacterial lipoglycan that has been implicated in numerous immunoregulatory functions, was examined utilizing D-mannosamine (ManN) as a tool to identify mannosyltransferase genes involved in LAM synthesis. Cell-free reactions utilizing cellular membranes of mycobacteria as the enzyme source indicated that ManN inhibited the synthesis of phosphatidylinositol mannosides, early precursors to LAM. A selection strategy was devised to screen a Mycobacterium tuberculosis genomic library in Mycobacterium smegmatis for clones conferring conditional resistance to ManN, with the rationale that overexpression of the gene(s) encoding a target of ManN would impart a ManN-resistant phenotype under these conditions. This strategy led to the identification of pimB, whose deduced amino acid sequence shows similarity to mannosyltransferases and other glycosyltransferases. Partially purified recombinant PimB protein from Escherichia coli or membranes from M. smegmatis overexpressing the pimB gene were used in cell-free assays to show that PimB catalyzes the formation of triacylphosphatidylinositol dimannoside from GDP-mannose and triacylphosphatidylinositol monomannoside.  相似文献   

18.
结核分枝杆菌ESX-1分泌蛋白ESAT-6增强巨噬细胞吞噬功能   总被引:1,自引:0,他引:1  
【目的】研究结核分枝杆菌(Mycobacterium tuberculosis)分泌蛋白ESAT-6(early secreted antigenictarget of 6 kDa)对巨噬细胞吞噬功能的影响。【方法】用重组质粒pFLAG-ESAT-6和pFLAG-EGFP转染RAW264.7细胞,经G418筛选,PCR、RT-PCR和Western blot鉴定,获得稳定表达flag-ESAT-6和flag-EGFP的RAW细胞系,然后用流式细胞术观察各稳转细胞系吞噬荧光微球的能力,并用共聚焦显微镜和菌落计数法检测稳转细胞系吞噬大肠杆菌(Escherichia coli)的能力。【结果】获得了稳定表达flag-ESAT-6的RAW-E6细胞系和表达flag-EGFP的RAW-EGFP细胞系;流式细胞术检测结果表明RAW-E6吞噬荧光微球的能力显著强于野生型细胞系RAW264.7和对照细胞系RAW-EGFP;菌落计数和激光共聚焦分析表明RAW-E6细胞系吞噬E.coli的能力也显著强于RAW264.7和RAW-EGFP。【结论】通过胞内表达发现结核分枝杆菌分泌蛋白ESAT-6能够增强巨噬细胞的吞噬功能,这将为深入理解结核分枝杆菌的致病机制提供新的思路。  相似文献   

19.
杨瑞丽  孙佳楠  陆伟 《生命科学》2013,(11):1084-1088
结核分枝杆菌(Mycobacterium tuberculosis,Mtb)感染后能抑制宿主巨噬细胞(M西)的免疫反应,并在其中生存、复制。研究表明Mtb减毒株感染主要诱导宿主Mφ凋亡,凋亡能抑制胞内Mtb的活力;而Mtb毒力株感染能抑制凋亡的完成,诱导Mφ坏死,最终导致Mtb扩散、感染临近细胞。通过对Mtb感染诱导宿主Mφ不同死亡方式的讨论,进一步认识Mtb的致病机制。  相似文献   

20.
The phagosome is a central mediator of both the homeostatic and microbicidal functions of a macrophage. Following phagocytosis, Mycobacterium tuberculosis (Mtb) is able to establish infection through arresting phagosome maturation and avoiding the consequences of delivery to the lysosome. The infection of a macrophage by Mtb leads to marked changes in the behaviour of both the macrophage and the surrounding tissue as the bacterium modulates its environment to promote its survival. In this study, we use functional physiological assays to probe the biology of the phagosomal network in Mtb‐infected macrophages. The resulting data demonstrate that Mtb modifies phagosomal function in a TLR2/TLR4‐dependent manner, and that most of these modifications are consistent with an increase in the activation status of the cell. Specifically, superoxide burst is enhanced and lipolytic activity is decreased upon infection. There are some species‐ or cell type‐specific differences between human and murine macrophages in the rates of acidification and the degree of proteolysis. However, the most significant modification is the marked reduction in intra‐phagosomal lipolysis because this correlates with the marked increase in the retention of host lipids in the infected macrophage, which provides a potential source of nutrients that can be accessed by Mtb.  相似文献   

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