Rd7 genotyping of M. tuberculosis strains isolated from patients with lung tuberculosis in different areas of Kazakhstan

A three-primer PCR assay was designed for detection of possible deletions in the RD7 region of the Mycobacterium tuberculosis complex chromosome. The assay produced amplicons of different size depending on the presence or absence of the deletions. The PCR assay was applied to 176 isolates from patients with lung tuberculosis collected in different areas of Kazakhstan in summer 2004. The isolates were initially characterized by culture and biochemical tests. The RD7 genotyping results demonstrated no polymorphism and the absence of deletions in the RD7 genome region. Some strains were additionally characterized using PCR-RFLP analysis of gyrB and hsp64 genes. The RFLP-patterns obtained corresponded to the M. tuberculosis genotypes. The results of this work are consistent with certain previous studies, indicating population stability of the RD7 region in M. tuberculosis strains. Species characterization of the isolates shows that M. tuberculosis sensu stricto is the principal causative agent of human lung tuberculosis in Kazakhstan.     

Molecular analysis of genetic differences between Mycobacterium bovis BCG and virulent M. bovis.

The live attenuated bacillus Calmette-Guérin (BCG) vaccine for the prevention of disease associated with Mycobacterium tuberculosis was derived from the closely related virulent tubercle bacillus, Mycobacterium bovis. Although the BCG vaccine has been one of the most widely used vaccines in the world for over 40 years, the genetic basis of BCG's attenuation has never been elucidated. We employed subtractive genomic hybridization to identify genetic differences between virulent M. bovis and M. tuberculosis and avirulent BCG. Three distinct genomic regions of difference (designated RD1 to RD3) were found to be deleted from BCG, and the precise junctions and DNA sequence of each deletion were determined. RD3, a 9.3-kb genomic segment present in virulent laboratory strains of M. bovis and M. tuberculosis, was absent from BCG and 84% of virulent clinical isolates. RD2, a 10.7-kb DNA segment containing a novel repetitive element and the previously identified mpt-64 gene, was conserved in all virulent laboratory and clinical tubercle bacilli tested and was deleted only from substrains derived from the original BCG Pasteur strain after 1925. Thus, the RD2 deletion occurred after the original derivation of BCG. RD1, a 9.5-kb DNA segment found to be deleted from all BCG substrains, was conserved in all virulent laboratory and clinical isolates of M. bovis and M. tuberculosis tested. The reintroduction of RD1 into BCG repressed the expression of at least 10 proteins and resulted in a protein expression profile almost identical to that of virulent M. bovis and M. tuberculosis, as determined by two-dimensional gel electrophoresis. These data indicate a role for RD1 in the regulation of multiple genetic loci, suggesting that the loss of virulence by BCG is due to a regulatory mutation. These findings may be applicable to the rational design of a new attenuated tuberculosis vaccine and the development of new diagnostic tests to distinguish BCG vaccination from tuberculosis infection.     

Occurrence of RD9 region and 500 bp fragment among clinical isolates of Mycobacterium tuberculosis and Mycobacterium bovis

The present investigation dealt with the identification of Mycobacterium tuberculosis and M. bovis by RD9 region and 500 bp fragment PCR assays. Eight M. tuberculosis and 5 M. bovis characterized and identified from 40 human sputum and 41 bovine lung specimens and 20 M. tuberculosis and 9 M. bovis strains maintained at Mycobacteria Laboratory, Indian Veterinary Research Institute were included in this study. In this way, 28 M. tuberculosis and 14 M. bovis strains and, for comparison and control purpose, M. tuberculosis H37Rv, M. bovis BCG, M. canetti, M. smegmatis, M. phlei, M. chelonae, M. kansasii, M. xenopi and M. avium were subjected to RD9 and 500 bp amplification by PCR. All M. tuberculosis strains, M. tuberculosis H37 Rv and M. canetti yielded a product of 333 bp which showed presence of RD9 region in these strains, whereas all M. bovis yielded a product of 206 bp with RD9 PCR assay. There was no ampli-fication product found in M. bovis BCG, M. xenopi, M. smegmatis, M. phlei, M. chelonae, M. kansasii, and M. avium. PCR based on 500 bp fragment showed a product of 500 bp in all M. bovis strains and M. bovis BCG. There was no amplification product of 500 bp found in M. canetti, M. smegmatis, M. phlei, M. chelonae, M. avium, M. kansasii, M. xenopi and was absent in all M. tuberculosis strains. The PCR assay results correlated 100% with the culture and biochemical results of the isolates. Our study suggested that PCR based on RD9 and 500 bp may effectively identify two closely related species of M. tuberculosis and M. bovis.     

Cytokines in response to proteins predicted in genomic regions of difference of Mycobacterium tuberculosis

Cellular immune responses are responsible for both protection and pathogenesis in tuberculosis, and are mediated/regulated by a complex network of pro-inflammatory, T helper (Th) type 1 and type 2 cytokines. In this study, the secretion of pro-inflammatory cytokines tumor necrosis factor-alpha (TNF-α), interleukin (IL)-6, IL-8 and IL-1β; Th1 cytokines interferon-gamma (IFN-γ), IL-2 and tumor necrosis factor-beta (TNF-β); and Th2 cytokines IL-4, IL-5 and IL-10 by the peripheral blood mononuclear cells (PBMCs) of pulmonary tuberculosis patients was studied. PBMCs were cultured in vitro in the absence and presence of complex mycobacterial antigens and peptides corresponding to 11 regions of difference (RD) of Mycobacterium tuberculosis that are deleted/absent in all vaccine strains of Mycobacterium bovis bacillus Calmette-Guérin (BCG). The culture supernatants were tested for secreted cytokines by FlowCytomix assay. PBMCs from the majority of patients (53-100%) spontaneously secreted detectable concentrations of all cytokines tested, except for IL2 (29%) and IL-10 (41%). The profiles of proinflammatory cytokines were largely similar for various complex antigens or RD peptides. However, with respect to Th1 and Th2 cytokines, the antigens could be divided into three groups; the first with Th1-bias (culture filtrate of M. tuberculosis, RD1, RD5, RD7, RD9 and RD10), the second with Th2-bias (whole cells and cell walls of M. tuberculosis, RD12, RD13 and RD15), and the third without Th1/Th2-bias (M. bovis BCG, RD4, RD6 and RD11). Complex mycobacterial antigens and RD proteins with Th1- and Th2-biases may have roles in protection and pathogenesis of tuberculosis, respectively.     

Modern and ancestral genotypes of Mycobacterium tuberculosis from Andhra Pradesh, India

Traditionally, the distribution of the Mycobacterium tuberculosis genotypes in India has been characterized by widespread prevalence of ancestral lineages (TbD1+ strains and variants) in the south and the modern forms (TbD1(-) CAS and variants) predominating in the north of India. The pattern was, however, not clearly known in the south-central region such as Hyderabad and the rest of the state of Andhra Pradesh where the prevalence of both tuberculosis (TB) and human immunodeficiency virus (HIV) infection is one of the highest in the country; this area has been the hotspot of TB vaccine trials. Spoligotyping of 101 clinical isolates obtained from Hyderabad and rural Andhra Pradesh confirmed the occurrence of major genogroups such as the ancestral (or the TbD1+ type or the East African Indian (EAI) type), the Central Asian (CAS) or Delhi type and the Beijing lineage in Andhra Pradesh. Sixty five different spoligotype patterns were observed for the isolates included in this study; these were further analyzed based on specific genetic signatures/mutations. It was found that the major genogroups, CAS and "ancestral," were almost equally prevalent in our collection but followed a north-south compartmentalization as was also reported previously. However, we observed a significant presence of MANU lineage in south Andhra Pradesh, which was earlier reported to be overwhelmingly present in Mumbai. This study portrays genotypic diversity of M. tuberculosis from the Indian state of Andhra Pradesh and provides a much needed snapshot of the strain diversity that will be helpful in devising effective TB control programs in this part of the world.     

Comparison between agarose gel electrophoresis and capillary electrophoresis for variable numbers of tandem repeat typing of Mycobacterium tuberculosis

Variable numbers of tandem repeat (VNTR) typing of Mycobacterium tuberculosis was performed on 54 strains including 23 strains derived from 9 outbreaks. PCR amplicon sizes of 12 mycobacterial interspersed repetitive unit tandem repeat loci were measured using both agarose gel electrophoresis and capillary electrophoresis. Similarities using agarose gel electrophoresis of Euclidian distances among the 23 strains derived from the 9 outbreaks were significantly lower than that using capillary electrophoresis (Wilcoxon signed ranks test, P < 0.01). By clustering analysis using unweighted pair group method using arithmetic averages, all of the 23 strains derived from the 9 outbreaks were each clustered with more than 90% similarities based on the distance using capillary electrophoresis. In contrast, differential clusters with more than 90% similarity were observed with only 7 strains derived from 3 outbreaks when analyzed by agarose gel electrophoresis. These results indicated that measurement of PCR amplicon size of tandem repeat loci should be carried out using capillary electrophoresis and that agarose gel electrophoresis is not suitable for clustering analysis of M. tuberculosis VNTR typing.     

Identification of variable regions in the genomes of tubercle bacilli using bacterial artificial chromosome arrays

Whole-genome comparisons of the tubercle bacilli were undertaken using ordered bacterial artificial chromosome (BAC) libraries of Mycobacterium tuberculosis and the vaccine strain, Mycobacterium bovis BCG-Pasteur, together with the complete genome sequence of M. tuberculosis H37Rv. Restriction-digested BAC arrays of M. tuberculosis H37Rv were used in hybridization experiments with radiolabelled M. bovis BCG genomic DNA to reveal the presence of 10 deletions (RD1-RD10) relative to M. tuberculosis. Seven of these regions, RD4-RD10, were also found to be deleted from M. bovis, with the three M. bovis BCG-specific deletions being identical to the RD1-RD3 loci described previously. The distribution of RD4-RD10 in Mycobacterium africanum resembles that of M. tuberculosis more closely than that of M. bovis, whereas an intermediate arrangement was found in Mycobacterium microti, suggesting that the corresponding genes may affect host range and virulence of the various tubercle bacilli. Among the known products encoded by these loci are a copy of the proposed mycobacterial invasin Mce, three phospholipases, several PE, PPE and ESAT-6 proteins, epoxide hydrolase and an insertion sequence. In a complementary approach, direct comparison of BACs uncovered a third class of deletions consisting of two M. tuberculosis H37Rv loci, RvD1 and RvD2, deleted from the genome relative to M. bovis BCG and M. bovis. These deletions affect a further seven genes, including a fourth phospholipase, plcD. In summary, the insertions and deletions described here have important implications for our understanding of the evolution of the tubercle complex.     

Loss of RD1 contributed to the attenuation of the live tuberculosis vaccines Mycobacterium bovis BCG and Mycobacterium microti

Although large human populations have been safely immunized against tuberculosis with two live vaccines, Mycobacterium bovis BCG or Mycobacterium microti, the vole bacillus, the molecular basis for the avirulence of these vaccine strains remains unknown. Comparative genomics has identified a series of chromosomal deletions common to both virulent and avirulent species but only a single locus, RD1, that has been deleted from M. bovis BCG and M. microti. Restoration of RD1, by gene knock-in, resulted in a marked change in colonial morphology towards that of virulent tubercle bacilli. Three RD1-encoded proteins were localized in the cell wall, and two of them, the immunodominant T-cell antigens ESAT-6 and CFP-10, were also found in culture supernatants. The BCG::RD1 and M. microti::RD1 knock-ins grew more vigorously than controls in immunodeficient mice, inducing extensive splenomegaly and granuloma formation. Increased persistence and partial reversal of attenuation were observed when immunocompetent mice were infected with the BCG::RD1 knock-in, whereas BCG controls were cleared. Knocking-in five other RD loci did not affect the virulence of BCG. This study describes a genetic lesion that contributes to safety and opens new avenues for vaccine development.     

First Molecular Epidemiology Study of Mycobacterium tuberculosis in Kiribati

Tuberculosis incidence rates in Kiribati are among the highest in the Western Pacific Region, however the genetic diversity of circulating Mycobacterium tuberculosis complex strains (MTBC) and transmission dynamics are unknown. Here, we analysed MTBC strains isolated from culture positive pulmonary tuberculosis (TB) cases from the main TB referral centre between November 2007 and October 2009. Strain genotyping (IS6110 typing, spoligotyping, 24-loci MIRU-VNTR and SNP typing) was performed and demographic information collected. Among 73 MTBC strains analysed, we identified seven phylogenetic lineages, dominated by Beijing strains (49%). Beijing strains were further differentiated in two main branches, Beijing-A (n = 8) and -B (n = 28), that show distinct genotyping patterns and are characterized by specific deletion profiles (Beijing A: only RD105, RD207 deleted; Beijing B: RD150 and RD181 additionally deleted). Many Kiribati strains (59% based on IS6110 typing of all strains) occurred in clusters, suggesting ongoing local transmission. Beijing-B strains and over-crowded living conditions were associated with strain clustering (likely recent transmission), however little evidence of anti-tuberculous drug resistance was observed. We suggest enhanced case finding amongst close contacts and continued supervised treatment of all identified cases using standard first-line drugs to reduce TB burden in Kiribati. Beijing strains can be subdivided in different principle branches that might be associated with differential spreading patterns in the population.     

Ecotypes of the Mycobacterium tuberculosis complex

A phylogeny of the Mycobacterium tuberculosis complex has recently shown that the animal-adapted strains are found in a single lineage marked by the deletion of chromosomal region 9 (RD9) [Brosch et al., 2002. A new evolutionary scenario for the Mycobacterium tuberculosis complex. Proc. Natl Acad. Sci. USA 99 (6), 3684-3689]. We have obtained the spoligotype patterns of the RD9 deleted strains used to generate this new evolutionary scenario and we show that the presence of spoligotype spacers 3, 9, 16, 39, and 40-43 is phylogenetically informative in this lineage. We have used the phylogenetically informative spoligotype spacers to screen a database of spoligotype patterns and have identified further members of a group of strains apparently host-adapted to antelopes. The presence of the spoligotype spacers is congruent with the phylogeny generated by chromosomal deletions, suggesting that recombination is rare or absent between strains of this lineage. The phylogenetically informative spacers, in concert with the previously identified single nucleotide mutations and chromosomal deletions, can be used to identify a series of clades in the RD9 deleted lineage each with a separate host preference. Finally, we discuss the application of the ecotype concept to this series of clades and suggest that the M. tuberculosis complex may best be described as a series of host-adapted ecotypes.     

Presence of RD149 deletions in M. tuberculosis Central Asian Strain 1 isolates affect growth and TNFα induction in THP-1 monocytes

Central Asian Strain 1 (CAS1) is the prevalent Mycobacterium tuberculosis genogroup in South Asia. CAS1 strains carry deletions in RD149 and RD152 regions. Significance of these deletions is as yet unknown. We compared CAS1 strains with RD149 and concurrent RD149-RD152 deletions with CAS1 strains without deletions and with the laboratory reference strain, M. tuberculosis H37Rv for growth and for induction of TNFα, IL6, CCL2 and IL10 in THP-1 cells. Growth of CAS1 strains with deletions was slower in broth (RD149; p?=?0.024 and RD149-RD152; p?=?0.025) than that of strains without deletions. CAS1 strains with RD149 deletion strains further showed reduced intracellular growth (p?=?0.013) in THP-1 cells as compared with strains without deletions, and also as compared with H37Rv (p?=?0.007) and with CAS1 RD149-RD152 deletion strains (p?=?0.029). All CAS1 strains induced higher levels of TNFα and IL10 secretion in THP-1 cells than H37Rv. Additionally, CAS1 strains with RD149 deletions induced more TNFα secretion than those without deletions (p?=?0.013). CAS1 RD149 deletion strains from extrapulmonary sources showed more rapid growth and induced lower levels of TNFα and IL6 secretion in THP-1 cells than isolates from pulmonary sources. This data suggests that presence of RD149 reduces growth and increases the induction of TNFα in host cells by CAS1 strains. Differences observed for extrapulmonary strains may indicate an adaptation which increases potential for dissemination and tropism outside the lung. Overall, we hypothesise that RD149 deletions generate genetic diversity within strains and impact interactions of CAS1 strains with host cells with important clinical consequences.     

Analysis of chromosome deletions of TbDl, RD6 and pks15/1 in clinical strains of Mycobacterium tuberculosis

Deletions are very important sources of the variability among members of the mycobacterial tuberculosis complex (MTC). Deletion analysis of MTC clinical isolates was performed to clarify phylogenetic relationships and help to identify epidemiologically significant groups of the MTC. In this study, the variability of the TbDl, RD6 and pks15/1 chromosome loci in clinical MTC strains and comparison of those results with IS6110-RFLP (restriction fragment length polymorphism), sSNP (synonymous single nucleotide polymorphism), PGG (Principal Genetic Group) typing data were used to determine if these chromosome regions constitute good molecular markers for some of the epidemiologically important groups of the MTC. In the present study, 122, 61 and 294 clinical isolates were tested for the TbDl, RD6 and pks15/1 deletions, respectively. Specific probes were designed and used in RFLP analysis as well as sequencing techniques were applied. We found that all strains with intact TbDl region belonged to the sSNP cluster I, PGG 1 (katG463Leu and gyrA95Thr). The RD6 deletion was not determined to be a strict characteristic feature of any specific genetic group of the tested M.tb strains, but presence of this deletion is presumed for strains of high virulence, and associated with principal genetic groups 2 or 3. The genetic event that led to this deletion likely occurred in the strain that belongs to PGG 1. Identification of strains with an intact pksl5/1 gene cluster provided a potential marker for virulence. An intact pks15/1 gene cluster is required for the biosynthesis of the phenolic glycolipids (PGL-tb), production of which by clinical isolates was correlated with virulence.     

The Genotypic Population Structure of Mycobacterium tuberculosis Complex from Moroccan Patients Reveals a Predominance of Euro-American Lineages

Background

Tuberculosis (TB) remains a major health problem in Morocco. Characterization of circulating Mycobacterium tuberculosis genotypic lineages, important to understand the dynamic of the disease, was hereby addressed for the first time at a national level.

Methodology/Principal Findings

Spoligotyping was performed on a panel of 592 M. tuberculosis complex strains covering a 2-year period (2004–2006). It identified 129 patterns: 105 (n = 568 strains) corresponded to a SIT number in the SITVIT2 database, while 24 patterns were labeled as orphan. A total of 523 (88.3%) strains were clustered vs. 69 or 11.7% unclustered. Classification of strains within 3 large phylogenetical groups was as follows: group 1– ancestral/TbD1+/PGG1 (EAI, Bovis, Africanum), group 2– modern/TbD1−/PGG1 group (Beijing, CAS), group 3– evolutionary recent/TbD1−/PGG2/3 (Haarlem, X, S, T, LAM; alternatively designated as the Euro-American lineage). As opposed to group 3 strains (namely LAM, Haarlem, and T) that predominated (86.5% of all isolates), 6 strains belonged to group 2 (Beijing n = 5, CAS n = 1), and 3 strains (BOV_1 n = 2, BOV_4-CAPRAE) belonged to ancestral group 1 (EAI and AFRI lineage strains were absent). 12-loci MIRU-VNTR typing of the Casablanca subgroup (n = 114 strains) identified 71 patterns: 48 MITs and 23 orphan patterns; it allowed to reduce the clustering rate from 72.8% to 29.8% and the recent transmission rate from 64% to 20.2%.

Conclusion

The M. tuberculosis population structure in Morocco is highly homogeneous, and is characterized by the predominance of the Euro-American lineages, namely LAM, Haarlem, and T, which belong to the “evolutionary recent” TbD1−/PGG2/3 phylogenetic group. The combination of spoligotyping and MIRUs decreased the clustering rate significantly, and should now be systematically applied in larger studies. The methods used in this study appear well suited to monitor the M. tuberculosis population structure for an enhanced TB management program in Morocco.     

Highly polymorphic variable-number tandem repeats loci for differentiating Beijing genotype strains of Mycobacterium tuberculosis in Shanghai, China

The PCR-based variable-number tandem repeats (VNTR) typing method is a very promising tool for the molecular epidemiological study of Mycobacterium tuberculosis. The discriminatory power of the VNTR loci that were optimized in many previous studies has not been evaluated in Shanghai, an area where Beijing genotype strains dominate. In the present study, we first performed a literature search to identify VNTR loci that were at least 45 bp in length. Second, we determined the Hunter-Gaston discriminatory index (HGI) values of each of the 45 VNTR loci that we identified, using Beijing genotype strains from a 'test set' of isolates from a population with low migration in Chongming Island, Shanghai, China. Third, we optimized two sets of VNTR loci, which we named VNTR-7 and VNTR-16. The HGI value of VNTR-7 was slightly lower than that of IS6110 restriction fragment length polymorphisms (RFLP), and the HGI values of VNTR-16 and IS6110 RFLP were comparable. Our results suggest that VNTR-7, followed by VNTR-16 and IS6110 RFLP, can be used routinely as a tool to discriminate between M. tuberculosis isolates in population-based epidemiologic studies of M. tuberculosis transmission in Shanghai, China.     

The RD1 proteins of Mycobacterium tuberculosis: expression in Mycobacterium smegmatis and biochemical characterization

A 9.5-kb section of DNA called region of deletion 1 (RD1) is present in virulent Mycobacterium tuberculosis strains but is deleted in all attenuated Mycobacterium bovis BCG vaccine strains. This region codes for at least nine genes. Some or all RD1 gene products may be involved in virulence and pathogenesis, and at least two, ESAT-6 and CFP-10, represent potent T- and B-cell antigens. In order to produce the entire set of RD1 proteins with their natural posttranslational modifications, a robust expression system for M. tuberculosis proteins in the fast-growing saprophytic strain Mycobacterium smegmatis was developed. Our system employs the inducible acetamidase promoter and allows translational fusion of recombinant M. tuberculosis proteins with polyhistidine or influenza hemagglutinin epitope tags for affinity purification. Using eGFP as reporter gene, we showed that the acetamidase promoter is tightly regulated in M. smegmatis and that this promoter is much stronger than the widely used constitutive groEL2 promoter. We then cloned 11 open reading frames (ORFs) found within RD1 and successfully expressed and purified the respective proteins. Sera from tuberculosis patients and M. tuberculosis-infected mice reacted with 10 purified RD1 proteins, thus demonstrating that Rv3871, Rv3872, Rv3873, CFP-10, ESAT-6, Rv3876, Rv3878, Rv3879c and ORF-14 are expressed in vivo. Finally, glycosylation of the RD1 proteins was analyzed. We present preliminary evidence that the PPE protein Rv3873 is glycosylated at its C terminus, thus highlighting the ability of M. smegmatis to produce M. tuberculosis proteins bearing posttranslational modifications.     

Genomic interrogation of the dassie bacillus reveals it as a unique RD1 mutant within the Mycobacterium tuberculosis complex

Despite their remarkable genetic homology, members of the Mycobacterium tuberculosis complex express very different phenotypes, most notably in their spectra of clinical presentation. For example, M. tuberculosis is regarded as pathogenic to humans, whereas members having deleted RD1, such as Mycobacterium microti and Mycobacterium bovis BCG, are not. The dassie bacillus, an infrequent variant of the M. tuberculosis complex characterized as being most similar to M. microti, is the causative agent of tuberculosis (TB) in the dassie (Procavia capensis). Intriguingly, the dassie bacillus is not pathogenic to rabbits or guinea pigs and has never been documented to infect humans. Although it was identified more than a half-century ago, the reasons behind its attenuation are unknown. Because large sequence polymorphisms have presented themselves as the most obvious genomic distinction among members of the M. tuberculosis complex, the DNA content of the dassie bacillus was interrogated by Affymetrix GeneChip to identify regions that are absent from it but present in M. tuberculosis H37Rv. Comparison has led to the identification of nine regions of difference (RD), five of which are shared with M. microti (RDs 3, 7, 8, 9, and 10). Although the dassie bacillus does not share the other documented deletions in M. microti (RD1(mic), RD5(mic), MID1, MID2, and MID3), it has endured unique deletions in the regions of RD1, RD5, N-RD25, and Rv3081-Rv3082c (virS). RD1(das), affecting only Rv3874-Rv3877, is the smallest natural deletion of the RD1 region uncovered and points to genes within this region that are likely implicated in virulence. Newfound deletions from the dassie bacillus are discussed in relation to their evolutionary and biological significance.     

A non-RD1 gene cluster is required for Snm secretion in Mycobacterium tuberculosis

The Snm secretion system is a crucial virulence determinant of Mycobacterium tuberculosis. Genes encoding all known components of this alternative secretion pathway are clustered at the same genetic locus, known as RD1. Here, we show that a mutant M. tuberculosis strain containing a transposon insertion in the Rv3615c gene, which is situated outside the RD1 locus, results in loss of Snm secretion. Complementation analysis revealed that both Rv3615c and the downstream gene Rv3614c are required for Snm secretion. Thus, we have renamed the two genes snm9 and snm10 respectively. The snm9::Tn mutant phenocopies bona fide snm mutants, exhibiting attenuation in mice, macrophage growth defects and failure to suppress cytokine induction. Furthermore, yeast two-hybrid analysis revealed a physical interaction between Snm10 and Snm7 (Rv3882c), suggesting that Snm10 may function in complex with other Snm proteins during secretion. Thus, snm9 and snm10 are the first genes located outside the RD1 locus identified as critical components of Snm secretion. These data indicate that Snm secretion consists of an elaborate network of interactions that likely arose from multiple duplication events during the evolution of M. tuberculosis.     

结核分枝杆菌RD1区研究进展

RD1区是结核分枝杆菌(MTB)在长期传代过程中丢失的重要保护性抗原,RD1区仅存在于致病性分枝杆菌中,而在卡介苗(BCG)及环境分枝杆菌中缺失。RD1区基因全长9.5kb,共有9个开放读码框,分别编码9个蛋白。RD1区是MTB毒力的关键因素之一,同时RD1区存在一种新分泌表达体系,能保证ESAT6和CFP10蛋白分泌表达。RD1区蛋白有较强的免疫原性,在MTB的预防和诊断中将可能发挥巨大作用,并有可能成为筛选抗MTB药物的理想靶抗原。     

多重PCR方法特异性鉴定卡介苗菌株多糖核酸的初探

与结核分枝杆菌H37Rv菌株进行比较,BCG菌株可找到一个特殊的缺失片段RD1,它存在于所有有毒分枝杆菌中,而在所有的卡介苗菌株中均缺失。应用多重PCR方法检测RD1区的存在与否,可以区别BCG和其它有毒的分枝杆菌。卡介菌多糖核酸来源于卡介菌,检测成品中DNA是否含有RD1区,能特异性地鉴别该制品。结果显示牛分枝杆菌标准株和结核分枝杆菌H37Rv存在RD1区;而卡介菌多糖核酸注射液和国内皮内注射用BCG疫苗生产用菌株扩增产物一致,提示均缺失RD1区。因此,这种多重PCR方法适用于卡介菌多糖核酸注射液的特异性鉴别试验。     

The genetics of reading disability in an often excluded sample: novel loci suggested for reading disability in rolandic epilepsy

Background

Reading disability (RD) is a common neurodevelopmental disorder with genetic basis established in families segregating “pure” dyslexia. RD commonly occurs in neurodevelopmental disorders including Rolandic Epilepsy (RE), a complex genetic disorder. We performed genomewide linkage analysis of RD in RE families, testing the hypotheses that RD in RE families is genetically heterogenenous to pure dyslexia, and shares genetic influences with other sub-phenotypes of RE.

Methods

We initially performed genome-wide linkage analysis using 1000 STR markers in 38 US families ascertained through a RE proband; most of these families were multiplex for RD. We analyzed the data by two-point and multipoint parametric LOD score methods. We then confirmed the linkage evidence in a second US dataset of 20 RE families. We also resequenced the SEMA3C gene at the 7q21 linkage locus in members of one multiplex RE/RD pedigree and the DISC1 gene in affected pedigrees at the 1q42 locus.

Results

In the discovery dataset there was suggestive evidence of linkage for RD to chromosome 7q21 (two-point LOD score 3.05, multipoint LOD 3.08) and at 1q42 (two-point LOD 2.87, multipoint LOD 3.03). Much of the linkage evidence at 7q21 derived from families of French-Canadian origin, whereas the linkage evidence at 1q42 was well distributed across all the families. There was little evidence for linkage at known dyslexia loci. Combining the discovery and confirmation datasets increased the evidence at 1q42 (two-point LOD = 3.49, multipoint HLOD = 4.70), but decreased evidence at 7q21 (two-point LOD = 2.28, multipoint HLOD  = 1.81), possibly because the replication sample did not have French Canadian representation.

Discussion

Reading disability in rolandic epilepsy has a genetic basis and may be influenced by loci at 1q42 and, in some populations, at 7q21; there is little evidence of a role for known DYX loci discovered in “pure” dyslexia pedigrees. 1q42 and 7q21 are candidate novel dyslexia loci.