Arabinogalactan enhances Mycobacterium marinum virulence by suppressing host innate immune responses.

Arabinogalactan (AG) participates in forming the cell wall core of mycobacteria, a structure known as the mAGP complex. Few studies have reported the virulence of inartificial AG or its interaction with the host immune system. Using clustered regularly interspaced short palindromic repeats interference gene editing technology, conditional Mycobacterium marinum mutants were constructed with a low expression of embA or glfT2 (EmbA_KD or GlfT2_KD), which are separately involved in the biosynthesis of AG arabinose and galactose domains. High-performance gel permeation chromatography and high-performance liquid chromatography assays confirmed that the EmbA_KD strain showed a remarkable decrease in AG content with fragmentary arabinose chains, and the GlfT2_KD strain displayed less reduction in content with cut-down galactose chains. Based on transmission and scanning electron microscopy observations, the cell walls of the two mutants were found to be dramatically thickened, and the boundaries of different layers were more distinct. Phenotypes including the over-secretion of extracellular substances and enhanced spreading motility with a concomitant decreased resistance to ethambutol appeared in the EmbA_KD strain. The EmbA_KD and GlfT2_KD strains displayed limited intracellular proliferation after infecting murine J774A.1 macrophages. The disease progression infected with the EmbA_KD or GlfT2_KD strain significantly slowed down in zebrafish/murine tail infection models as well. Through transcriptome profiling, macrophages infected by EmbA_KD/GlfT2_KD strains showed enhanced oxidative metabolism. The cell survival measured using the CCK8 assay of macrophages exposed to the EmbA_KD strain was upregulated and consistent with the pathway enrichment analysis of differentially expressed genes in terms of cell cycle/apoptosis. The overexpression of C/EBPß and the increasing secretion of proinflammatory cytokines were validated in the macrophages infected by the EmbA_KD mutant. In conclusion, the AG of Mycobacterium appears to restrain the host innate immune responses to enhance intracellular proliferation by interfering with oxidative metabolism and causing macrophage death. The arabinose chains of AG influence the Mycobacterium virulence and pathogenicity to a greater extent.

Transmission of extensively drug-resistant and multidrug resistant Mycobacterium tuberculosis in families identified by genotyping.

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.

[The study of inter-simple sequences repeat genotyping based on one pentanucleotide repeat sequences in mycobacteria].

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.

[Identification and evaluation of a new nucleic acid amplification test target for specific detection of Mycobacterium tuberculosis complex].

OBJECTIVE: To identify and evaluate a new nucleic acid amplification (NAA) test target for specific detection of Mycobacterium tuberculosis (MTB) complex (MTC). METHODS: MTC-specific fragment was obtained by ISSR genotyping technology. Primer pairs were designed based on the sequences of MTC-specific fragment and tested in 211 mycobacterial strains including 107 MTC strains and 104 nontuberculous mycobacteria (NTM) strains. IS6110 element (specific identification of MTC strains) and 16s rRNA gene (specific identification of Mycobacterium) amplification were used as a control to evaluate the efficacy of the NAA test target in the detection of MTC strains. RESULTS: One MTC-specific fragment with the length of 588 bp, located in 315947 - 316534 of the genome from MTB reference strain H(37) Rv, were obtained, cloned and sequenced. MTC-specific primer pairs MTCF/R were designed based on these sequences. All 211 mycobacterial strains accurately produced the genus-specific 16s rRNA amplicon. All MTC strains were positive in the MTCF/R PCR amplification while 99% MTC strains (106/107) were positive in the amplification of IS6110 sequences. All NTM strains were negative in both IS6110 and MTCF/R PCR amplification. CONCLUSIONS: The MTC-specific fragment developed in this study can be used as a new NAA test target to correctly distinguish MTC from NTM.

Selection and application of peptide mimotopes of MPT64 protein in Mycobacterium tuberculosis.

Antibody responses can be useful markers of tuberculosis (TB) infection, especially in the screening of extra-pulmonary TB. MPT64 is an important antigen in Mycobacterium tuberculosis (MTB) infection and is used in serological diagnosis. However, large variability in the diagnostic accuracy of MPT64 as a serological tool has limited its application. Phage-displayed random peptide libraries have emerged as a powerful technique to select peptides (epitopes) or mimotopes that may serve as surrogate diagnostic markers in serological tests. In the present study, this method was employed to identify mimotopes of the MPT64 protein of MTB by screening a linear heptapeptide library with rabbit antibodies raised against MPT64 protein. Two antigenic mimotopes (M2 and M6) resembling B-cell epitopes of MPT64 were identified that bound the affinity purified anti-MPT64 polyclonal antibodies and competed with MPT64 for antibody binding. From the results of sequence alignment and a structure modelling figure of MPT64, the sequence of the 2nd to 5th amino acids (DSML) of M2 was totally consistent with the sequence of the 224th to 227th amino acids of MPT64 and the peptide is located on the surface of the space structure of MPT64, suggesting that it might be a linear epitope of MPT64. The recognition of both phage-displayed and synthetic peptides of M2 by the anti-MPT64 polyclonal antibodies also supported this. Although no recurring sequence and no analogue to MPT64 of M6 were found for sequence alignment, the recognition of both phage-displayed and synthetic peptides of M6 by the anti-MPT64 polyclonal antibodies indicated that it might be a mimotope of a conformational epitope of MPT64. According to the results of the reactivity of human sera with synthetic M2 and M6 peptides and MPT64, M2 showed a significantly higher AUC and sensitivity than M6 and MPT64, especially for the sera from sputum-negative TB patients, suggesting that the M2 mimotope may be useful in serological diagnostic testing for TB.

[Associated factors for the infection of Beijing genotype Mycobacterium tuberculosis].

OBJECTIVE: to analyze the risk factors for the infection of Beijing genotype Mycobacterium tuberculosis (MTB) and the relationship to drug resistance and clinical symptoms. METHODS: sputum samples were collected from patients with pulmonary tuberculosis who were admitted to the hospital during July, 2007 to March, 2008. The sputum was cultured with L-J method, and then the bacterial DNA was isolated and genotyped with VNTR-7 (variable-number tandem repeats, VNTR) and RD105 deletion method respectively. The association between different genotypes and risk factors was analyzed. RESULTS: a hundred and sixteen clinical sputum isolates were obtained from 172 positive sputum cases. There were 112 isolates of MTB, and isolates of non-tuberculosis mycobacterium (NTM). Among the 97 isolates from Shanghai, Zhejiang and Jiangsu areas, Beijing genotype accounted for 86.6% (84/97), and non-Beijing genotype for 13.4% (13/97). The rates of MDR (multi-drug resistance), PDR (poly-drug resistance) and single drug resistance in Beijing genotype were not significantly higher than those in non-Beijing genotype. Among the risk factors, female gender, and CD(4)/CD(8)< 1 in patients with newly-treated tuberculosis, were associated with higher rate of Beijing genotype, chi(2) = 4.436, 4.494 and all P < 0.05, respectively. The Beijing genotype isolates were subdivided into 31 VNTR-7 types, and the distribution of quantity and resistance among different VNTR-7 genotypes was not even. A large number of MTB isolates (47.6%, 40/84) and drug resistant isolates (43.6%, 17/39) were among four VNTR-7 genotypes. CONCLUSION: Beijing genotype is the most prevalent MTB in Shanghai, Zhejian and Jiangsu areas. Female gender and low CD(4)/CD(8) ratio in patients with newly-treated TB are risk factors for infecting Beijing genotype MTB, which may have no relationship with drug resistance and clinical symptoms.

[Identification of the Mycobacterium tuberculosis complex by detecting recombinant secretory protein MPT64].

OBJECTIVE: To identify the Mycobacterium tuberculosis complex by detecting the secretory protein MPT64. METHODS: The gene mpt64 was amplified by polymerase chain reaction (PCR) from the genome of Mycobacterium tuberculosis H(37)Rv strain and cloned into expression vector. Immune sera from rabbits by recombinant proteins MPT64, were used to make enzyme-labeled antibodies and coated antibodies. A double antibody sandwich enzyme-linked immunosorbent assay (ELISA) was established to detect the secretory protein MPT64 of the culture supernatants in Mycobacterium strains. Results of ELISA were compared to those of the gene mpt64 amplified by PCR. RESULTS: A recombinant vector was constructed. The minimum detectable concentration of MPT64 was 0.01 mg/L. A total of 27 reference strains and 170 clinical isolate strains were evaluated. PCR for Mycobacterium tuberculosis reference strain, Mycobacterium bovis reference strain and Mycobacterium africanum reference strain was positive, but that for other reference stains was negative, consistent with the results of ELISA. In the 170 clinical isolate stains, the positive result of PCR and ELISA was 98.2% (111/113) and 97.3% (110/113) respectively, while the specificity of PCR and ELISA was both 100%, no positive result in non-tuberculosis mycobacterium strains. CONCLUSION: Identification of the Mycobacterium tuberculosis complex by detecting secretory protein MPT64 is rapid, sensitive, and specific, which can be used routinely in clinical laboratories.

[Construction of a human phage display ScFv library against Mycobacterium tuberculosis].

OBJECTIVE: To construct a human phage display single-chain Fv (ScFv) antibody library against Mycobacterium tuberculosis (MTB), for specific ScFv antibody cloning. METHODS: Total RNA was isolated from the lymphocytes of patients with positive serum antibody against MTB and reverse transcribed into cDNA. The heavy chain and light chain variable region gene of human immunoglobulin were amplified individually by PCR and then assembled into ScFv genes. The ScFv genes were ligated into phagemid pCANTAB5S. The human phage display ScFv library against MTB was constructed by transforming the recombinant phagemid into E. coli TG1 with the presence of helper phage M13K07. RESULTS: The human phage display ScFv library containing 10(7) different clones was constructed successfully. CONCLUSIONS: A phage display ScFv library against MTB has been constructed based on the variable region gene of immunoglobulin of the lymphocytes of TB patients and phagemid pCANTAB5S. The specific ScFv antibodies can be screened from this library.

[Selecting and affinity analysis of DNA aptamers to MPT64 protein from Mycobacterium tuberculosis].

OBJECTIVE: To obtain DNA oligonucleotide aptamers which can specifically bind to MPT64 protein from Mycobacterium tuberculosis by SELEX technology. METHODS: An in vitro synthesized 78 per random DNA library was subjected to 12 rounds of selection by SELEX (Systematic evolution of ligands by exponential enrichment) method against MPT64 protein. Binding of the aptamers to the protein was examined by biotin-streptavidin-horseradish peroxidase system. DNAMAN package was employed to analyze the sequences and the second structures of the aptamers. Moreover, target protein was bound to one aptamer and another aptamer modified with biotin together forming a sandwich-like complex, which was captured in microwell, to be tested in negative group including BCG and reference strains from nontuberculous mycobacteria, and positive group including H37Rv, Mycobacterium bovis reference strain, and clinical strains from Mycobacterium tuberculosis. RESULTS: After 12 rounds of selection, high-affinity aptamers to MPT64 was obtained. The OD value at 450 nm of affinity of aptamers to MPT64 protein was from 0. 492 to 1.243, in which 73.3% was over 1.0. Pocket and stem-loops was the basis of aptamers binding to MPT64 protein by the analysis of structures,with several GC pairs among bridges between pocket and stem-loops. The analysis of the sandwich-like complex system based on two aptamers and protein showed that the positive percentage was 87. 9% in the positive group while the negative percentage was 85.7% in the negative group, with positive H37Rv and Mycobacterium bovis, and negative BCG, when the cut-off value for a positive response was 0.61 OD. CONCLUSION: A set of aptamers with considerable binding affinity to MPT64 protein were successfully selected from the initial random DNA library.

[Evaluation of pyrosequencing for the detection of rpoB gene mutation in Mycobacterium tuberculosis].

OBJECTIVE: To detect the mutations of rpoB gene in Mycobacterium tuberculosis by pyrosequencing and to evaluate the values on detection of rifampin resistance in clinical isolates. METHODS: Using the new technology of pyrosequencing, the mutations in the rifampin resistance determining region (RRDR) of rpoB gene were analyzed. The results were compared with those obtained from methods of the absolute concentration and the minimum inhibitory concentration (MIC). RESULTS: Among the 150 Mycobacterium tuberculosis clinical isolates, 84 were susceptible and 66 resistant to RIF. 54 of the 66 resistant isolates were multidrug-resistant (MDR) strains. Ser531Leu and His526Asp or Tyr, including twelve different genotypes and six codons, were the most common mutations. In the drug susceptibility testing, the accordance rates of the pyrosequencing and the absolute concentration method as well as MIC were 92.7% and 97.8% respectively. CONCLUSION: Not only is the pyrosequencing technology a fast, sensitive and high throughput method in detecting rifampin resistance in Mycobacterium tuberculosis, but also a useful tool in the research of rifampin resistance mechanism.

Use of intersimple sequence repeats markers to develop strain-specific SCAR markers for Lentinula edodes.

Although Lentinula edodes is the second most important cultivated mushroom worldwide, most industrially cultivated strains have been identified only through traditional phenotypic analysis. Here, we report for the first time the use of sequence characterized amplified region (SCAR) markers for strain differentiation. SCAR markers were created by first generating and sequencing single intersimple sequence repeats fragments, and then designing primers based on these sequences to amplify strain-specific fragments of a certain size. One SCAR primer pair, ISL450F/R7 (amplifying a band of c. 450 bp), was designed to identify one strain of L. edodes (strain No. 7). The SCAR primer pair was then used to correctly amplify the single unique fragment from DNA samples taken from a total of 85 strains representing three separate species. Our data provide the foundation for a precise and rapid PCR-based strain-diagnostic system for L. edodes.