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1.
Nat Microbiol ; 4(8): 1378-1388, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31110366

RESUMO

Mycobacterium tuberculosis (Mtb)-derived components are usually recognized by pattern recognition receptors to initiate a cascade of innate immune responses. One striking characteristic of Mtb is their utilization of different type VII secretion systems to secrete numerous proteins across their hydrophobic and highly impermeable cell walls, but whether and how these Mtb-secreted proteins are sensed by host immune system remains largely unknown. Here, we report that MPT53 (Rv2878c), a secreted disulfide-bond-forming-like protein of Mtb, directly interacts with TGF-ß-activated kinase 1 (TAK1) and activates TAK1 in a TLR2- or MyD88-independent manner. MPT53 induces disulfide bond formation at C210 on TAK1 to facilitate its interaction with TRAFs and TAB1, thus activating TAK1 to induce the expression of pro-inflammatory cytokines. Furthermore, MPT53 and its disulfide oxidoreductase activity is required for Mtb to induce the host inflammatory responses via TAK1. Our findings provide an alternative pathway for host signalling proteins to sense Mtb infection and may favour the improvement of current vaccination strategies.

2.
BMC Biol ; 17(1): 7, 2019 01 25.
Artigo em Inglês | MEDLINE | ID: mdl-30683096

RESUMO

BACKGROUND: The emergence of drug-resistant strains of Mycobacterium tuberculosis (Mtb), especially those that are multidrug resistant poses a serious threat to global tuberculosis control. However, the mechanism underlying the occurrence of drug resistance against more than one drug is poorly understood. Given that the Beijing/W strains are associated with outbreaks and multidrug resistance, they may harbor a genetic advantage and provide useful insight into the disease. One marker found in all Beijing/W Mtb strains is a deletion of RD105 region that results in a gene fusion, Rv0071/74, with a variable number (3-9 m) of VDP (V: Val, D: Asp; P: Pro) repeats (coded by gtggacccg repeat sequences) at the N-terminal. Here, we report that this variable number of VDP repeats in Rv0071/74 regulates the development of multidrug resistance. RESULTS: We collected and analyzed 1255 Beijing/W clinical strains. The results showed that the number of VDP repeats in Rv0071/74 was related to the development of multidrug resistance, and the deletion of Rv0071/74-9 m from Beijing/W clinical strain restored drug susceptibility. Rv0071/74-9 m also increased resistance to multiple drugs when transferred to different mycobacterial strains. Cell-free assays indicate that the domain carrying 4-9 VDP repeats (4-9 m) showed a variable binding affinity with peptidoglycan and Rv0071/74 cleaves peptidoglycan. Furthermore, Rv0071/74-9 m increased cell wall thickness and reduced the intracellular concentration of antibiotics. CONCLUSIONS: These findings not only identify Rv0071/74 with VDP repeats as a newly identified multidrug resistance gene but also provide a new model for the development of multiple drug resistance.


Assuntos
Farmacorresistência Bacteriana Múltipla/genética , Mycobacterium tuberculosis/genética , Deleção de Sequência , Genótipo , Mycobacterium tuberculosis/efeitos dos fármacos
3.
Future Microbiol ; 14: 47-59, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30539658

RESUMO

AIM: To characterize a novel macrolide ATP binding cassette efflux pump encoding gene Rv1473 which might be involved in antibiotic resistance. METHODS: Mycobacterium smegmatis was used as a surrogate model for pathogenic mycobacteria, drug susceptibility assays and ethidium bromide accumulation assay were harnessed to verify drug resistance. The real-time quantitative PCR was used to evaluate the transcription levels of WhiB7 and Ms3140 upon exposure to macrolides. RESULTS: Rv1473 contributes to macrolides resistance via efflux mechanisms, and was positively regulated by the transcription factor WhiB7 upon macrolides exposure. CONCLUSION: Rv1473 is a novel ATP binding cassette efflux pump involved in mycobacterium intrinsic antibiotics resistance via efflux mechanism. This finding will facilitate novel antibiotic discovery and the treatment of pathogen, especially for nontuberculous mycobacteria.


Assuntos
Proteínas de Bactérias/metabolismo , Farmacorresistência Bacteriana Múltipla/genética , Macrolídeos/farmacologia , Proteínas de Membrana Transportadoras/metabolismo , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/genética , Fatores de Transcrição/metabolismo , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Eritromicina/farmacologia , Proteínas de Membrana Transportadoras/genética , Mycobacterium smegmatis/efeitos dos fármacos , Mycobacterium smegmatis/genética , Reação em Cadeia da Polimerase em Tempo Real , Roxitromicina/farmacologia , Deleção de Sequência/genética , Fatores de Transcrição/genética
4.
Infect Genet Evol ; 72: 86-92, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-30543940

RESUMO

Mycobacterium tuberculosis (MTB) infections rely on continued growth and division. Despite the substantial global burden of tuberculosis, the underlying mechanism governing growth is incompletely understood. Bifunctional penicillin-binding protein (PBP1), encoded by Rv0050 (ponA1) of MTB, is a key peptidoglycan synthase and plays a central role in mycobacterial growth and division by its interaction with Rpf-interacting protein A (RipA, peptidoglycan endopeptidase). Our previous work suggested that the hyper-variable proline repeats are located at the N end of PBP1. In this study, we prove that altered secondary structure resulting from polymorphic proline repeats modulates the interaction between PBP1 and RipA. Without proper coordination of peptidoglycan synthase and hydrolase, cell elongation and division is also altered resulting in phenotypic changes in the population as indicated by altered dispersion, slowed growth, or shortened cell length. Together, our data reveal that polymorphisms in Rv0050 induce mycobacterial growth and morphologic changes, and hence are responsible for giving bacteria their shape.

5.
Biol Sex Differ ; 9(1): 44, 2018 10 10.
Artigo em Inglês | MEDLINE | ID: mdl-30305157

RESUMO

BACKGROUND: Worldwide tuberculosis (TB) reports show a male bias in morbidity; however, the differences in pathogenesis between men and women with TB, as well as the mechanisms associated with such differences, are poorly investigated. We hypothesized that comparison of the degree of lung injury and clinical indices of well-matched men and women with newly diagnosed TB, and statistical analysis of the correlation between these indices and the extent of lung lesions, can provide insights into the mechanism of gender bias in TB. METHODS: We evaluated the acid-fast bacilli grading of sputum samples and compiled computed tomography (CT) data of the age-matched, newly diagnosed male and female TB patients without history of smoking or comorbidities. Inflammatory biomarker levels and routine haematological and coagulation-associated parameters were compared. Binary logistic regression analysis was used to define the association between the indices and lung lesions, and the influence of sex adjustment. RESULTS: Women with TB have a longer delay in seeking healthcare than men after onset of the TB-associated symptoms. Men with TB have significantly more severe lung lesions (cavities and healing-associated features) and higher bacterial counts compared to women with TB. Scoring of the CT images before and after anti-TB treatment showed a faster response to therapy in women than in men. Coagulation- and platelet-associated indices were in models from multivariate regression analysis with groups of males or females with TB or in combination. In univariate regression analysis, lower lymphocyte counts were associated with both cavity and more bacterial counts, independent of sex, age and BMI. The association of international normalized ratios (INR), prothrombin times (PTs), mean platelet volumes (MPVs) and fibrinogen (FIB) level with lung lesions was mostly influenced by sex adjustment. CONCLUSIONS: Sex influences the association between haemostasis and extent of TB lung lesions, which may be one mechanism involved in sex bias in TB pathogenesis.

6.
Nat Commun ; 9(1): 4072, 2018 10 04.
Artigo em Inglês | MEDLINE | ID: mdl-30287856

RESUMO

Tuberculosis (TB) is an infectious disease caused by Mycobacterium tuberculosis (Mtb), and remains a leading public health problem. Previous studies have identified host genetic factors that contribute to Mtb infection outcomes. However, much of the heritability in TB remains unaccounted for and additional susceptibility loci most likely exist. We perform a multistage genome-wide association study on 2949 pulmonary TB patients and 5090 healthy controls (833 cases and 1220 controls were genome-wide genotyped) from Han Chinese population. We discover two risk loci: 14q24.3 (rs12437118, Pcombined = 1.72 × 10-11, OR = 1.277, ESRRB) and 20p13 (rs6114027, Pcombined = 2.37 × 10-11, OR = 1.339, TGM6). Moreover, we determine that the rs6114027 risk allele is related to decreased TGM6 transcripts in PBMCs from pulmonary TB patients and severer pulmonary TB disease. Furthermore, we find that tgm6-deficient mice are more susceptible to Mtb infection. Our results provide new insights into the genetic etiology of TB.

7.
Emerg Microbes Infect ; 7(1): 34, 2018 Mar 21.
Artigo em Inglês | MEDLINE | ID: mdl-29559631

RESUMO

Tuberculosis caused by Mycobacterium tuberculosis (Mtb) infection remains a large global public health problem. One striking characteristic of Mtb is its ability to adapt to hypoxia and trigger the ensuing transition to a dormant state for persistent infection, but how the hypoxia response of Mtb is regulated remains largely unknown. Here we performed a quantitative acetylome analysis to compare the acetylation profile of Mtb under aeration and hypoxia, and showed that 377 acetylation sites in 269 Mtb proteins were significantly changed under hypoxia. In particular, deacetylation of dormancy survival regulator (DosR) at K182 promoted the hypoxia response in Mtb and enhanced the transcription of DosR-targeted genes. Mechanistically, recombinant DosRK182R protein demonstrated enhanced DNA-binding activity in comparison with DosRK182Q protein. Moreover, Rv0998 was identified as an acetyltransferase that mediates the acetylation of DosR at K182. Deletion of Rv0998 also promoted the adaptation of Mtb to hypoxia and the transcription of DosR-targeted genes. Mice infected with an Mtb strain containing acetylation-defective DosRK182R had much lower bacterial counts and less severe histopathological impairments compared with those infected with the wild-type strain. Our findings suggest that hypoxia induces the deacetylation of DosR, which in turn increases its DNA-binding ability to promote the transcription of target genes, allowing Mtb to shift to dormancy under hypoxia.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Lisina/metabolismo , Mycobacterium tuberculosis/metabolismo , Oxigênio/metabolismo , Proteínas Quinases/química , Proteínas Quinases/metabolismo , Tuberculose/microbiologia , Acetilação , Motivos de Aminoácidos , Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Humanos , Mycobacterium tuberculosis/química , Mycobacterium tuberculosis/genética , Proteínas Quinases/genética
8.
J Infect Dis ; 218(2): 312-323, 2018 Jun 20.
Artigo em Inglês | MEDLINE | ID: mdl-29228365

RESUMO

Tuberculosis, caused by Mycobacterium tuberculosis infection, remains a global threat to human health, but knowledge of the molecular mechanisms underlying the pathogenesis of tuberculosis is still limited. Although Notch4, a member of the Notch receptor family, is involved in the initiation of mammary tumors, its function in M. tuberculosis infection remains unclear. In this study, we found that Notch4-deficient mice were more resistant to M. tuberculosis infection, with a much lower bacterial burden and fewer pathological changes in the lungs. Notch4 inhibited M. tuberculosis-induced production of proinflammatory cytokines by interaction with TAK1 and inhibition of its activation. Furthermore, we found that Notch intracellular domain 4 prevented TRAF6 autoubiquitination and suppressed TRAF6-mediated TAK1 polyubiquitination. Finally, Notch inhibitors made mice more resistant to M. tuberculosis infection. These results suggest that Notch4 is a negative regulator of M. tuberculosis-induced inflammatory response, and treatment with a Notch inhibitor could serve as a new therapeutic strategy for tuberculosis.

9.
Chest ; 153(5): 1187-1200, 2018 May.
Artigo em Inglês | MEDLINE | ID: mdl-29224833

RESUMO

BACKGROUND: Exacerbated immunopathology is a frequent consequence of TB that is complicated by diabetes mellitus (DM); however, the underlying mechanisms are still poorly defined. METHODS: In the two groups of age- and sex-matched patients with TB and DM (DM-TB) and with TB and without DM, we microscopically evaluated the areas of caseous necrosis and graded the extent of perinecrotic fibrosis in lung biopsies from the sputum smear-negative (SN) patients. We scored acid-fast bacilli in sputum smear-positive (SP) patients and compiled CT scan data from both the SN and SP patients. We compared inflammatory biomarkers and routine hematologic and biochemical parameters. Binary logistic regression analyses were applied to define the indices associated with the extent of lung injury. RESULTS: Enlarged caseous necrotic areas with exacerbated fibrotic encapsulations were found in SN patients with DM-TB, consistent with the higher ratio of thick-walled cavities and more bacilli in the sputum from SP patients with DM-TB. Larger necrotic foci were detected in men compared with women within the SN TB groups. Significantly higher fibrinogen and lower high-density lipoprotein cholesterol (HDL-C) were observed in SN patients with DM-TB. Regression analyses revealed that diabetes, activation of the coagulation pathway (shown by increased platelet distribution width, decreased mean platelet volume, and shortened prothrombin time), and dyslipidemia (shown by decreased low-density lipoprotein cholesterol, HDL-C, and apolipoprotein A) are risk factors for severe lung lesions in both SN and SP patients with TB. CONCLUSIONS: Hemostasis and dyslipidemia are associated with granuloma necrosis and fibroplasia leading to exacerbated lung damage in TB, especially in patients with DM-TB.

10.
Acta Derm Venereol ; 97(4): 472-477, 2017 Apr 06.
Artigo em Inglês | MEDLINE | ID: mdl-27840887

RESUMO

Cutaneous tuberculosis (CTB) is probably underreported due to difficulties in detection and diagnosis. To address this issue, genotypes of Mycobacterium tuberculosis strains isolated from 30 patients with CTB were mapped at multiple loci, namely, RD105 deletions, spacer oligonucleotides, and Mycobacterial Interspersed Repetitive Unit-Variable Number Tandem Repeats (MIRU-VNTRs). Fifty-eight strains of pulmonary tuberculosis (PTB) were mapped as experimental controls. Drug resistance-associated gene mutations were determined by amplicon sequencing of target regions within 7 genes. Beijing family isolates were the most prevalent strains in CTB and PTB. MIRU-VNTR typing separated the Beijing strains from the non-Beijing strains, and the majority of CTB could be separated from PTB counterparts. Drug resistance determining regions showed only one CTB strain expressing isomazid resistance. Thus, while the CTB strains belonged to the same phylogenetic lineages and sub-lineages as the PTB strains, they differed at the level of several MIRU-VNTRs and in the proportion of drug resistance.


Assuntos
DNA Bacteriano/genética , Mycobacterium/genética , Pele/microbiologia , Tuberculose Cutânea/microbiologia , Adulto , Antituberculosos/uso terapêutico , Estudos de Casos e Controles , China/epidemiologia , DNA Bacteriano/isolamento & purificação , Farmacorresistência Bacteriana/genética , Feminino , Genótipo , Humanos , Sequências Repetitivas Dispersas , Masculino , Testes de Sensibilidade Microbiana , Pessoa de Meia-Idade , Repetições Minissatélites , Técnicas de Diagnóstico Molecular , Mycobacterium/efeitos dos fármacos , Mycobacterium/isolamento & purificação , Fenótipo , Filogenia , Tuberculose Cutânea/diagnóstico , Tuberculose Cutânea/tratamento farmacológico , Tuberculose Cutânea/epidemiologia
11.
PLoS One ; 11(11): e0166052, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27835653

RESUMO

Mycobacterium tuberculosis (MTB) is a specific aerobic bacterium, but can survive under hypoxic conditions, such as those in lung cheese necrosis, granulomas, or macrophages. It is not clear whether the drug sensitivity and growth characteristics of MTB under hypoxic conditions are different from those under aerobic conditions. In this study, we examined the drug resistance and growth characteristics of MTB clinical isolates by a large sample of in vitro drug susceptibility tests, using an automatic growth instrument. Under hypoxic conditions, variance in drug resistance was observed in nearly one-third of the MTB strains and was defined as MTB strains with changed drug sensitivity (MTB-CDS). Among these strains, resistance in a considerable proportion of clinical strains was significantly increased, and some strains emerged as multi-drug resistant. Growth test results revealed a high growth rate and large survival number in macrophages under hypoxia in MTB-CDS. According to the results of fluorescence quantitative PCR, the expression of some genes, including RegX3 (involving RIF resistance), Rv0194 (efflux pump gene), four genes related to transcription regulation (KstR, DosR, Rv0081 and WhiB3) and gene related to translation regulation (DATIN), were upregulated significantly under hypoxic conditions compared to that under aerobic conditions (p < 0.05). Thus, we concluded that some MTB clinical isolates can survive under hypoxic conditions and their resistance could change. As for poor clinical outcomes in patients, based on routine drug susceptibility testing, drug susceptibility tests for tuberculosis under hypoxic conditions should also be recommended. However, the detailed mechanisms of the effect of hypoxia on drug sensitivity and growth characteristics of MTB clinical isolates still requires further study.


Assuntos
Proteínas de Bactérias/genética , Farmacorresistência Bacteriana/genética , Regulação Bacteriana da Expressão Gênica , Mycobacterium tuberculosis/genética , Aerobiose , Anaerobiose , Farmacorresistência Bacteriana Múltipla/genética , Humanos , Testes de Sensibilidade Microbiana , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/fisiologia , Proteínas Quinases/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição/genética , Tuberculose/microbiologia
12.
Appl Microbiol Biotechnol ; 100(5): 2279-87, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26577672

RESUMO

Although serological detection is a practical strategy for early detection and diagnosis of tuberculosis (TB), inconsistent and imprecise estimates of sensitivity and specificity block its development and application for clinic. New or alternative serological antigens with improved accuracy are urgently needed. A phage-displayed random peptide library was employed to screen for immunoactive peptides using specific immunoglobulin G (IgG) of TB patients as target molecules. With two screening strategies, 20 single phages displaying different sequences were obtained and no sequence homology was found among these phages. From the results of phage-ELISA, H12, TB6, TB15, and TB18 phages showed higher affinity to IgGs from TB patients(S/N ≥2.1) and were identified as the positive clones. Significant differences in the detection values of sera from 47 TB patients and 37 healthy individuals were found for these four phage clones. According to the reactivity of 284 human sera to synthetic H12, TB6, TB15, and TB18 peptides as determined by ELISA, TB15 showed significantly higher areas under the curve (AUC) and sensitivity than other peptides, providing a lead molecule for the development of new serology diagnostic strategies for TB.


Assuntos
Anticorpos Antibacterianos/sangue , Imunoglobulina G/sangue , Mycobacterium tuberculosis/imunologia , Peptídeos/imunologia , Peptídeos/isolamento & purificação , Testes Sorológicos/métodos , Tuberculose/diagnóstico , Ensaio de Imunoadsorção Enzimática/métodos , Humanos , Programas de Rastreamento , Biblioteca de Peptídeos
13.
Appl Microbiol Biotechnol ; 99(21): 9073-83, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26194558

RESUMO

Microscopy of direct smear with the Ziehl-Neelsen stain is still broadly used in tuberculosis diagnosis. However, this method suffers from low specificity and is difficult to distinguish Mycobacterium tuberculosis (MTB) from nontuberculosis mycobacterial (NTM), since all mycobacterial species are positive in Ziehl-Neelsen stain. In this study, we utilized whole cell SELEX to obtain species-specific aptamers for increasing the specificity of MTB detection. Whole cell SELEX was performed in MTB reference strain H37Rv by two selection processes based on enzyme-linked plate or Eppendorf tube, respectively. To increase success rate of generating aptamers, the selection processes were systematically monitored to understand the dynamic evolution of aptamers against complex structure of target bacteria. Two preponderant groups and ten high-affinity aptamers were obtained by analyzing the dynamic evolution. Preponderant aptamer MA1 from group I showed relatively high binding affinity with apparent dissociation constant (KD value) of 12.02 nM. Sandwich ELISA assay revealed five aptamer combinations effectively bound MTB strains in preliminary evaluation, especially the combination based on aptamer MA2 (another preponderant aptamer from group II) and MA1. Further evaluated in many other strains, MA2/MA1 combination effectively identified MTB from NTM or other pathogenic bacteria, and displayed the high specificity and sensitivity. Binding analysis of aptamer MA1 or MA2 by fluorescence microscopy observation showed high binding reactivity with H37Rv, low apparent cross-reactivity with M. marinum, and no apparent cross-reactivity with Enterobacter cloacae. Taken together, this study provides attractive candidate species-specific aptamers to effectively capture or discriminate MTB strains.


Assuntos
Aptâmeros de Nucleotídeos/metabolismo , Técnicas Bacteriológicas/métodos , Técnicas de Diagnóstico Molecular/métodos , Mycobacterium tuberculosis/isolamento & purificação , Tuberculose/diagnóstico , Aptâmeros de Nucleotídeos/genética , Reações Cruzadas , Mycobacterium tuberculosis/genética , Técnica de Seleção de Aptâmeros , Sensibilidade e Especificidade
14.
Tuberculosis (Edinb) ; 95(4): 497-504, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-25937126

RESUMO

Tuberculosis (TB) remains a major global health problem and host genetic factors play a critical role in susceptibility and resistance to TB. The aim of this study was to identify novel candidate genes associated with TB susceptibility. We performed a population-based case-control study to genotype 13 tag SNPs spanning Epstein-Barr virus-induced gene 3 (EBI3), colony stimulating factor 2 (CSF2), IL-4, interferon beta 1 (IFNB1), chemokine (C-X-C motif) ligand 14 (CXCL14) and myeloid differentiation primary response gene 88 (Myd88) genes in 435 pulmonary TB patients and 375 health donors from China. We observed that EBI3 gene rs4740 polymorphism was associated with susceptibility to pulmonary tuberculosis (PTB) and the allele G was associated with a protective effect against PTB. Furthermore, EBI3 deficiency led to reduced bacterial burden and histopathological impairment in the lung of mice infected with Mycobacterium bovis BCG. Meanwhile, higher abundance of EBI3 was observed in the granuloma of PTB patients and in the lung tissue of BCG-infected mice. Of note, the expression of EBI3 in macrophages was remarkably induced by mycobacteria infection at both mRNA and protein level. In conclusion, EBI3 gene rs4740 polymorphism is closely associated with susceptibility to PTB and the elevation and enrichment of EBI3 in the lung which at least partially derived from macrophages may contribute to the exacerbation of mycobacterial infection.


Assuntos
Interleucinas/genética , Pulmão/metabolismo , Mycobacterium tuberculosis/patogenicidade , Polimorfismo de Nucleotídeo Único , Receptores de Citocinas/genética , Tuberculose Pulmonar/genética , Animais , Carga Bacteriana , Estudos de Casos e Controles , Linhagem Celular , China , Modelos Animais de Doenças , Predisposição Genética para Doença , Interações Hospedeiro-Patógeno , Humanos , Interleucinas/metabolismo , Pulmão/microbiologia , Macrófagos/microbiologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Antígenos de Histocompatibilidade Menor , Mycobacterium bovis/patogenicidade , Fenótipo , Fatores de Proteção , Receptores de Citocinas/deficiência , Fatores de Risco , Tuberculose Pulmonar/diagnóstico , Tuberculose Pulmonar/metabolismo , Tuberculose Pulmonar/microbiologia , Tuberculose Pulmonar/prevenção & controle
15.
Int J Nanomedicine ; 10: 77-88, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25565805

RESUMO

Despite suffering from the major disadvantage of low sensitivity, microscopy of direct smear with the Ziehl-Neelsen stain is still broadly used for detection of acid-fast bacilli and diagnosis of tuberculosis. Here, we present a unique detection method of Mycobacterium tuberculosis (MTB) using surface functionalized magnetic microspheres (MMSs) coupled with quantum dots (QDs), conjugated with various antibodies and phage display-derived peptides. The principle is based upon the conformation of the sandwich complex composed of bacterial cells, MMSs, and QDs. The complex system is tagged with QDs for providing the fluorescent signal as part of the detection while magnetic separation is achieved by MMSs. The peptide ligand H8 derived from the phage display library Ph.D.-7 is developed for MTB cells. Using the combinations of MMS-polyclonal antibody+QD-H8 and MMS-H8+QD-H8, a strong signal of 10(3) colony forming units (CFU)/mL H37R(v) was obtained with improved specificity. MS-H8+QD-H8 combination was further optimized by adjusting the concentrations of MMSs, QDs, and incubation time for the maximum detection signal. The limit of detection for MTB was found to reach 10(3) CFU/mL even for the sputum matrices. Positive sputum samples could be distinguished from control. Thus, this novel method is shown to improve the detection limit and specificity of MTB from the sputum samples, and to reduce the testing time for accurate diagnosis of tuberculosis, which needs further confirmation of more clinical samples.


Assuntos
Hidrolases/isolamento & purificação , Nanopartículas de Magnetita/química , Microesferas , Mycobacterium tuberculosis/isolamento & purificação , Pontos Quânticos , Tuberculose/diagnóstico , Proteínas de Transporte , Contagem de Colônia Microbiana , Humanos , Limite de Detecção , Sensibilidade e Especificidade , Escarro/microbiologia
16.
BMC Infect Dis ; 14: 200, 2014 Apr 13.
Artigo em Inglês | MEDLINE | ID: mdl-24725975

RESUMO

BACKGROUND: Multidrug-resistant tuberculosis (MDR-TB) is a major public health problem. Early diagnosis of MDR-TB patients is essential for minimizing the risk of Mycobacterium tuberculosis (MTB) transmission. The conventional drug susceptibility testing (DST) methods for detection of drug-resistant M. tuberculosis are laborious and cannot provide the rapid detection for clinical practice. METHODS: The aim of this study was to develop a pyrosequencing approach for the simultaneous detection of resistance to rifampin (RIF), isoniazid (INH), ethambutol (EMB), streptomycin (SM), ofloxacin (OFL) and amikacin (AMK) in M. tuberculosis clinical isolates and sputum samples from re-treatment pulmonary tuberculosis (PTB) patients. We identified the optimum conditions for detection mutation of rpoB, katG, rpsl, embB, gyrA and rrs gene by pyrosequencing. Then this approach was applied to detect 205 clinical isolates and 24 sputum samples of M. tuberculosis from re-treatment PTB patients. RESULTS: The mutations of rpoB and gyrA gene were detected by pyrosequencig with the SQA mode, and the mutations of katG, rpsl, embB, gyrA and rrs gene were detected by pyrosequencing with SNP mode. Compared with the Bactec MGIT 960 mycobacterial detection system, the accuracy of pyrosequencing for the detection of RIF, INH, EMB, SM, AMK and OFL resistance in clinical isolates was 95.0%, 79.2%, 70.3%, 84.5%, 96.5% and 91.1%, respectively. In sputum samples the accuracy was 83.3%, 83.3%, 60.9%, 83.3%, 87.5% and 91.7%, respectively. CONCLUSIONS: The newly established pyrosequencing assay is a rapid and high-throughput method for the detection of resistance to RIF, INH, SM, EMB, OFL and AMK in M. tuberculosis. Pyrosequencing can be used as a practical molecular diagnostic tool for screening and predicting the resistance of re-treatment pulmonary tuberculosis patients.


Assuntos
Antituberculosos/farmacologia , Mycobacterium tuberculosis/genética , Escarro/microbiologia , Tuberculose Resistente a Múltiplos Medicamentos/microbiologia , Tuberculose Pulmonar/microbiologia , DNA Bacteriano/genética , Genes Bacterianos/genética , Humanos , Testes de Sensibilidade Microbiana/métodos , Tipagem Molecular/métodos , Mutação , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/isolamento & purificação , Análise de Sequência de DNA/métodos , Tuberculose Resistente a Múltiplos Medicamentos/diagnóstico , Tuberculose Resistente a Múltiplos Medicamentos/tratamento farmacológico , Tuberculose Pulmonar/diagnóstico , Tuberculose Pulmonar/tratamento farmacológico
17.
PLoS One ; 8(9): e73955, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24058507

RESUMO

BACKGROUND: Accurate and early diagnosis of tuberculosis (TB) is of major importance in the control of TB. One of the most important technical advances in diagnosis of tuberculosis is the development of nucleic acid amplification (NAA) tests. However, the choice of the target sequence remains controversial in NAA tests. Recently, interesting alternatives have been found in hypothetical protein coding sequences from mycobacterial genome. METHODOLOGY/PRINCIPAL FINDINGS: To obtain rational biomarker for TB diagnosis, the conservation of three hypothetical genes was firstly evaluated in 714 mycobacterial strains. The results showed that SCAR1 (Sequenced Characterized Amplified Region) based on Rv0264c coding gene showed the highest conservation (99.8%) and SCAR2 based on Rv1508c gene showed the secondary high conservation (99.7%) in M. tuberculosis (MTB) strains. SCAR3 based on Rv2135c gene (3.2%) and IS6110 (8%) showed relatively high deletion rate in MTB strains. Secondly, three SCAR markers were evaluated in 307 clinical sputum from patients in whom TB was suspected or patients with diseases other than TB. The amplification of IS6110 and 16SrRNA sequences together with both clinical and bacteriological identification was as a protocol to evaluate the efficacy of SCAR markers. The sensitivities and specificities, positive predictive value (PPV) and negative predictive value (NPV) of all NAA tests were higher than those of bacteriological detection. In four NAA tests, IS6110 and SCAR3 showed the highest PPV (100%) and low NPV (70% and 68.8%, respectively), and SCAR1 and SCAR2 showed the relatively high PPV and NPV (97% and 82.6%, 95.6% and 88.8%, respectively). CONCLUSIONS/SIGNIFICANCE: Our result indicated that SCAR1 and SCAR2 with a high degree of sequence conservation represent efficient and promising alternatives as NAA test targets in identification of MTB. Moreover, the targets developed from this study may provide more alternative targets for the development of a multisite system to effectively detect MTB in samples.


Assuntos
DNA Bacteriano/genética , Tipagem de Sequências Multilocus/métodos , Mycobacterium tuberculosis/genética , Fases de Leitura Aberta , Tuberculose/diagnóstico , Técnicas de Tipagem Bacteriana , Sequência de Bases , Sequência Conservada , Primers do DNA , DNA Bacteriano/química , Humanos , Dados de Sequência Molecular , Mycobacterium tuberculosis/isolamento & purificação , Reação em Cadeia da Polimerase em Tempo Real , Sensibilidade e Especificidade , Escarro/microbiologia , Tuberculose/microbiologia
18.
Chin Med J (Engl) ; 126(3): 521-5, 2013 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-23422118

RESUMO

BACKGROUND: Diagnosis and appropriate treatment of multidrug-resistant tuberculosis (MDR-TB) remain major challenges. We sought to elucidate that persons who share a household with drug resistance tuberculosis patients are at high risk for primary drug resistance tuberculosis and how to prevent these outbreaks. METHODS: We used 12-locus mycobacterial interspersed repetitive unit and 7-locus variable-number tandem repeat to identify household transmission of extensively drug resistant and multiple drug resistant Mycobacterium tuberculosis in three families admitted in Shanghai Pulmonary Hospital affiliated with Tongji University. Drug susceptibility tests were done by the modified proportion method in the MGIT 960 system in the same time. Clinical data were also obtained from the subjects' medical records. RESULTS: All of the six strains were defined as Beijing genotype by the deletion-targeted multiplex PCR (DTM-PCR) identification on the genomic deletion RD105. Strains from family-1 had the same minisatellite interspersed repetitive unit (MIRU) pattern (232225172531) and the same MIRU pattern (3677235). Strains from family-2 had the same MIRU pattern (2212261553323) and the same MIRU pattern (3685134). Strains from family-3 did not have the same MIRU pattern and they differed at only one locus (223326173533, 223325173533), and did not have the same VNTR pattern with two locus differed (3667233, 3677234). CONCLUSIONS: Household transmission exists in the three families. A clear chain of tuberculosis transmission within family exists. Tuberculosis susceptibility should be considered when there is more than one tuberculosis patients in a family. Household tuberculosis transmission could be prevented with adequate treatment of source patients.


Assuntos
Mycobacterium tuberculosis/genética , Tuberculose Resistente a Múltiplos Medicamentos/transmissão , Adulto , Feminino , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase Multiplex , Mycobacterium tuberculosis/classificação , Mycobacterium tuberculosis/patogenicidade , Radiografia , Tuberculose Resistente a Múltiplos Medicamentos/diagnóstico por imagem , Adulto Jovem
19.
PLoS One ; 8(1): e52848, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23308124

RESUMO

BACKGROUND: The 10-kDa culture filtrate protein (CFP10) and 6-kDa early-secreted target antigen (ESAT-6) play important roles in mycobacterial virulence and pathogenesis through a 1:1 complex formation (CFP10/ESAT-6 protein, CE protein), which have been used in discriminating TB patients from BCG-vaccinated individuals. The B-cell epitopes of CFP10 and ESAT-6 separately have been analyzed before, however, the epitopes of the CE protein are unclear and the precise epitope in the positions 40 to 62 of ESAT-6 is still unknown. METHODS: In the present study, we searched for the B-cell epitopes of CE protein by using phage-display library biopanning with the anti-CE polyclonal antibodies. The epitopes were identified by sequence alignment, binding affinity and specificity detection, generation of polyclonal mouse sera and detection of TB patient sera. RESULTS: One linear B-cell epitope (KWDAT) consistent with the 162(nd)-166(th) sequence of CE and the 57(th)-61(st) sequence of ESAT-6 protein was selected and identified. Significantly higher titers of E5 peptide-binding antibodies were found in the sera of TB patients compared with those of healthy individuals. CONCLUSION: There was a B-cell epitope for CE and ESAT-6 protein in the position 40 to 62 of ESAT-6. E5 peptide may be useful in the serodiagnosis of tuberculosis, which need to be further confirmed by more sera samples.


Assuntos
Antígenos de Bactérias/imunologia , Epitopos de Linfócito B/análise , Epitopos de Linfócito B/imunologia , Mycobacterium tuberculosis/imunologia , Mycobacterium tuberculosis/patogenicidade , Tuberculose/microbiologia , Sequência de Aminoácidos , Animais , Anticorpos Antibacterianos/sangue , Anticorpos Antibacterianos/imunologia , Antígenos de Bactérias/análise , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Modelos Moleculares , Dados de Sequência Molecular , Mycobacterium tuberculosis/química , Biblioteca de Peptídeos , Peptídeos/química , Peptídeos/imunologia , Alinhamento de Sequência , Tuberculose/sangue , Tuberculose/imunologia
20.
Zhonghua Jie He He Hu Xi Za Zhi ; 35(8): 592-5, 2012 Aug.
Artigo em Chinês | MEDLINE | ID: mdl-23158007

RESUMO

OBJECTIVE: To establish inter-simple sequences repeat (ISSR) molecular makers based on (CAGCG)n repeat sequence in mycobacteria. METHODS: The distribution of pentanucleotide repeat sequence (CAGCG)n in mycobacterial genomes was analyzed by MICdb 2.0 software in the microsatellite database. ISSR primer MISP6 based on (CAGCG)n sequences was designed and tested in mycobacterial strains, which included 17 mycobacterial strains and 41 Mycobacterium tuberculosis clinical strains. RESULTS: The abundances of pentanucleotide repeat sequences (CAGCG)n were high in most of the mycobacterial genomes and they were mainly located in the coding regions. The results of ISSR analysis in mycobacteria showed that 15 reference strains from mycobacteria were clustered into 2 major clusters. The first cluster contained 2 subtypes and the second cluster contained 4 subtypes. Forty-one clinical strains from Mycobacterium tuberculosis were divided into 2 major clusters by the analysis of MISP6 primer, and each cluster had 2 subtypes. CONCLUSION: ISSR primer MISP6 based on (CAGCG)n sequences can be used as a genetic marker to genotype mycobacterial strains.


Assuntos
Genoma Bacteriano , Mycobacterium/genética , Sequências Repetitivas de Ácido Nucleico , Técnicas de Tipagem Bacteriana , Primers do DNA/genética , DNA Bacteriano/genética , Marcadores Genéticos , Genótipo , Mycobacterium/classificação
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