Final Report Summary - TB-VIR (Mycobacterium tuberculosis W-Beijing genetic diversity and differential virulence and host immune responses)
Despite tremendous efforts to fight tuberculosis (TB) this disease remains a major health problem, particularly in low-income developing countries. TB causes 1.7 million deaths annually worldwide. Up to one third of the global population is estimated to carry latent Mycobacterium tuberculosis infection. The notion that some M. tuberculosis lineages are more virulent than others is supported by results obtained with animal and cellular models. Such variations between strains probably result from genetic diversity, which seems to be greater in mycobacteria than previously thought. Recent reports have shown that different clinical isolates of M. tuberculosis produce different proteins and lipids, according to their genetic lineage, and this probably affects mycobacterial virulence and the host immune response to infection. W-Beijing-specific expression of a phenolic glycolipid, for instance, has been associated with inhibition of the host immune response and the 'hyperlethality' of this lineage.
The inhibition of the host immune response by mycobacterial strains of the W-Beijing family has recently been confirmed in vivo. The W-Beijing lineage is one of the most successful M. tuberculosis families worldwide in terms of morbidity and mortality. These strains are currently attracting considerable attention because they display important pathogenic features and are frequently associated with resistance to antimicrobial drugs. W-Beijing strains may be more able to acquire antimicrobial drug resistance than other strains due to mutations in putative mutator genes and a higher level of adaptability. Strains of the W-Beijing lineage are responsible for 80 to 90 % of TB cases in China, where TB incidence may exceed 50 per 100 000 in average. The remarkable ability of these strains to spread locally in large clonal lineages probably reflects their high level of fitness / transmissibility and their adaptation to the infection of humans. The predominance of the W-Beijing lineage probably results from genetic advantages, including unidentified virulence factors and the induction or modification of specific host cell responses not yet thoroughly investigated. The TB-VIR project aimed to unravel the links between differential host responses to M. tuberculosis infection and mycobacterial genetic diversity and virulence at the global genomic and post-genomic levels within the W-Beijing family, and between the W-Beijing family and other M. tuberculosis families, to improve our understanding of the epidemiological success of this lineage.
Important results have been obtained and published from the TB-VIR activity. This has helped increase understanding of M. tuberculosis virulence and anti-mycobacterial immune response, in particular regarding the W-Beijing family of strains. The genetic diversity within the family has been further elucidated using informative single nucleotide polymorphisms (SNP) and the role of transposable elements in the peculiar virulence of this family has been reported. Genotype-phenotype correlations were established. It was showed that host cell response to W-Beijing sublineages is rather homogeneous and clearly impaired as compared to the immune response to other mycobacterial lineages. This has been shown using a combination of innovative approaches such as global gene expression profiling and protein survey. Candidate vaccine strains have been isolated through TB-VIR activity and are now tested in animal models. In summary, the TB-VIR project has been successful not only to provide new knowledge on M. tuberculosis but also to generate new possible intervention tools and to promote Euro-Asian collaborations between laboratories in France, Germany and Spain, and laboratories in China and Korea.
Project context and objectives:
Despite tremendous efforts to fight tuberculosis (TB), this disease remains a major health problem, particularly in low-income developing countries. TB causes 1.7 million deaths annually worldwide, and up to one third of the global population is estimated to carry latent Mycobacterium tuberculosis infection (1). TB incidence varies from 7 per 100 000 in Northern Europe to up to 300 per 100 000 in parts of Africa. In eastern Europe, the incidence of TB now exceeds 100 per 100 000, and the proportion of multidrug-resistant (MDR) M. tuberculosis strains is high. Extensively drug-resistant (XDR) TB has emerged worldwide as a threat to public health and TB control, raising concerns about a possible future epidemic of virtually untreatable TB. The frequency and distribution of XDR TB cases recently described by the American Center for Disease Control (CDC) determined that during the 2000 to 2004 period, 20 % of the 17 690 TB isolates were MDR and 2 % were XDR (4). Population-based data on the drug susceptibility of M. tuberculosis isolates have been obtained from the United States (for 1993-2004), Latvia (for 2000-2002), and South Korea (for 2004), where 4 %, 19 %, and 15 % of MDR TB cases, respectively, were XDR.
Drug treatment has helped to decrease the incidence of TB in countries with effective public health systems, but vaccination is considered the most cost-effective intervention in most countries. The Mycobacterium bovis BCG vaccine currently in use, although effective against severe forms of TB in children, is of minimal and variable efficacy against the much more common and contagious pulmonary forms of TB in adults and requires improvement.
Following M. tuberculosis infection, only 5 to 10 % of immunocompetent individuals develop TB. Environmental factors probably play an important role, but it is increasingly thought that the virulence and load of the infecting strain, together with host genetic factors, such as polymorphisms in genes involved in anti-mycobacterial immunity, contribute to differences between infected individuals. On the host side, genetic variations in genes encoding key components of the immune system, such as the vitamin D receptor, monocyte chemoattractant protein (MCP)-1 and the C-type lectin DC-SIGN have been found to increase susceptibility to TB in certain parts of the world. On the pathogen side, several reports have strongly suggested that the severity and clinical manifestations of TB depend on the immunogenicity and pathogenicity of the infecting strain of M. tuberculosis, these properties varying considerably between strains. A recent study on almost 700 clinical isolates collected from 1996 to 2000 in Arkansas (US) concluded that patients infected with M. tuberculosis W-Beijing strains were three times more likely to develop extrathoracic TB than patients infected with strains of other lineages. However, some other studies reported no such association between M. tuberculosis genotype and clinical features of TB.
The notion that some M. tuberculosis lineages are more virulent than others is supported by results obtained with animal and cellular models. For instance, the W-Beijing isolates HN878 and W4 induce more IFN and less Th1 cytokines in vitro and in vivo in mice than another non-W-Beijing isolate, CDC1551 and are associated with lower survival rates in infected animals. Such variations between strains probably result from genetic diversity, which seems to be greater in mycobacteria than previously thought. Recent reports have shown that different clinical isolates of M. tuberculosis produce different proteins and lipids according to their genetic lineage, and this probably affects mycobacterial virulence and the host immune response to infection. The inhibition of the host immune response by mycobacterial strains of the W-Beijing family has recently been confirmed in vivo.
On the basis of IS6110 typing and other genotyping methods, including spoligotyping, SNPs, genotyping and large sequence polymorphism (LSP) analysis, M. tuberculosis strains of the W-Beijing family have been detected almost worldwide and new unexpected routes of transmission have been identified for these strains. This genotype was first identified in M. tuberculosis strains isolated in the Beijing area of China, hence its name. These strains are currently attracting considerable attention worldwide and are frequently associated with resistance to antimicrobial drugs. W-Beijing strains may be more able to acquire antimicrobial drug resistance than other strains, due to mutations in putative mutator genes and a higher level of adaptability. The W-Beijing genotype also contains a much larger number of IS6110 copies than other lineages. It has been demonstrated that IS6110 may increase the expression of neighbouring virulence genes by generating new promoter sequences capable of driving their expression.
MDR-TB hot spots worldwide include China, Russia and India. The high frequency of drug-resistant and MDR strains of M. tuberculosis remains a serious problem for TB control in China. Strains of the W-Beijing lineage are responsible for 80 to 90 % of TB cases in China. The remarkable ability of these strains to spread locally in large clonal lineages probably reflects their high level of fitness / transmissibility and their adaptation to the infection of human populations. The predominance of the W-Beijing lineage probably results from genetic advantages, including unidentified virulence factors and the induction or modification of specific host cell responses not yet thoroughly investigated.
The project aimed to unravel the links between differential host responses to M. tuberculosis infection and mycobacterial genetic diversity and virulence at the global genomic and post-genomic levels within the W-Beijing family, and between the W-Beijing family and other M. tuberculosis families, to improve understanding of the epidemiological success of this particular lineage.
Goals
1) Deciphering M. tuberculosis genetic diversity using highly discriminative technologies and extracting strains representative of the W-Beijing subgenotypes, and of other genotypes
Genetic diversity of M. tuberculosis clinical isolates collected in the Shanghai area, where nearly 90 % of the strains are expected to belong to the W-Beijing family, was investigated using spoligotyping, IS6110-RFLP profiling, MIRU-VNTR, RD-, and SNP-based genotyping. The Fudan University, in close collaboration with the CDC in Shanghai, has access to over 9000 clinical isolates collected since 1999. At least 1000 isolates were selected for highly discriminative genotyping. SNP-based genotyping, a new and very promising technique for the genotyping of mycobacteria and other pathogens, was carried out with the technical support of a small and medium-sized enterprise (SME), IntegraGen S.A. Diversity and evolution in the W-Beijing lineage have recently been correlated to SNPs in genes involved in DNA repair or other genes, and with LSPs or regions of difference. SNPs provide a unique tool for phylogenetic studies as they are less susceptible to the influence of selective pressure than other genetic markers and are unlikely to display the convergence sometimes observed with spoligotype or MIRU-VNTR markers. Selectively neutral SNPs should accumulate at a uniform rate and can thus be used to measure divergence. The project aimed to make full use of the power of SNP-based phylogenies as a 'gold standard' for assessing the accuracy of other DNA typing methods. This technique was compared with the most discriminative method currently in use, the MIRU-VNTR genotyping method, and with RD-based genotyping. The effect of IS6110 insertion on the expression of putative virulence genes was be investigated. Clinical isolates were clustered into lineages and sub-lineages and approximately 10 strains, representative of regional diversity, were selected for functional studies.
2) Understanding how host cells respond differentially to infection with M. tuberculosis W-Beijing subgenotypes and other genotypes
Comparative analysis of the changes in host cell gene expression following the infection of mouse and human macrophages with M. tuberculosis strains representative of the diversity of W-Beijing strains and with strains of other genotypes were realised. We carried out systematic analysis of changes in the host cell transcriptome in response to M. tuberculosis infection, using an approach combining the methods of experimental biology, bioinformatics and computational biology.
3) Identifying virulence genes in M. tuberculosis W-Beijing using functional genomics and investigating mycobacterial virulome diversity in W-Beijing subgenotypes and in other genotypes
A genome-covering M. tuberculosis W-Beijing mutant library, generated by Partner 12 (IPAS), was screened by partner 1 (CNRS) in host macrophages, using signature-tagged transposon mutagenesis (STM) and high-throughput confocal microscopy, a well-established technique in the laboratory of beneficiary 6 (IPK). partner 6 (IPK) does not belong to an ICPC, but the participation of this partner in the project is invaluable. The work allowed identifying virulence genes on a global, genome-wide level in a W-Beijing M. tuberculosis strain. Genetic diversity (SNPs, InDels, etc.) in the genes identified here were subsequently investigated in representative members of the W-Beijing subgenotypes and of other genotypes.
4) Assessing differential host immune and pathological response to M. tuberculosis W-Beijing subgenotypes and other genotypes
Molecular and cell biology, biochemistry and immunology experiments were conducted in order to investigate the role of the host and mycobacterial genes identified by activities 1 to 3 in mycobacterial virulence and in the immune response to M. tuberculosis infection in vitro and in vivo. Mycobacterial virulence and the immune response to infection with various representative strains were assessed in vitro in cultured macrophages, and in vivo in the mouse model of M. tuberculosis infection. A meta-analysis of the data generated made it possible to describe fine genotype-phenotype associations between M. tuberculosis genotypes and subgenotypes, and mycobacterial virulence and immunopathogenicity.
5) Considering that a strong and integrated management of the above generated knowledge is an essential part of the success of this project, (WP1) was dedicated to scientific and knowledge management.
Project results
The following main scientific results were achieved:
- the phylogeny of M. tuberculosis (Mtb) Beijing strains has been constructed using polymorphisms in DNA replication, recombination and repair genes;
- the innate immune response to Mtb Beijing and other genotypes has been assessed;
- a high-density mutant library in Mtb Beijing has been screened visually at the phagosome level using high throughput confocal microscopy;
- the role of IS6110 in Mtb Beijing has been deciphered;
- novel techniques have been developed to study host cell promoter regions;
- non-Beijing strains of Mtb in China have been studied;
- mutations to study drug resistance in Mtb Beijing have been identified;
- fluoroquinolone resistance in Mtb Beijing has been studied;
- the complete genome of several Mtb Beijing and non-Beijing strains has been sequenced;
- the role of novel virulence genes involved in metal efflux has been studied in Mtb Beijing and non-Beijing.
The role of the NLRP3 inflammasome in immunity to Mtb Beijing and non-Beijing has been studied.
Main activities and achievements
WP 2 - Evaluation of M. tuberculosis genetic diversity in clinical isolates from the Shanghai area
The main objectives were to:
- identify strains representative of the genetic diversity observed in the Shanghai area, where over 90 % of the clinical isolates are expected to belong to the W-Beijing family;
- provide scientific community with know-how and proof of principle that SNP-genotyping can be successfully used to clusterise M. tuberculosis into genotypes and subgenotypes;
- sequence IS6110 insertion sites in various representative strains within the W-Beijing genotype and among mycobacterial genotypes;
- assess the influence of IS6110 insertion on driving the expression of neighbouring genes.
Study of Beijing strains of the Shanghai area together with Beijing strains from other countries all over the world (partners 5, 9 and 12) allowed to propose a phylogenetic tree that confirm the successive acquisition of point mutations that can be used as markers to differentiate Beijing strains (Mestre et al., PLoS One, 2011). The role of repetitive sequences in the evolution of Beijing strains was investigated by analysing IS6110 carrying regions (partner 4 and 12). They have been disseminated into the genomes either by transposition or homologous recombination (Alonso et al., Tuberculosis, 2011) Thanks to the complete sequence of several Beijing strains genomes and their comparison with genomes of other family strains (partners 10 and 12) new SNPs were identified which will be used for microevolution studies. The association of a limited number of SNPs and MIRU/VNTR loci was successfully tested in a collection of Beijing strains from the Shanghai area (partners 5, 9 and 12). The main goal was to study genetic diversity of W-Beijing strains by SNP genotyping. To reach this goal, genomes of three Beijing strains were sequined using the Illumina genome analyser GAII. Among these Beijing strains, a strain named GC1237 was included. In addition we sequenced two other Beijing strains: one ancient and one modern Beijing strain selected by taking into account a previously published work based on the sequential accumulation of SNPs in 3R genes. For each strain, all generated paired end (PE) reads have been analysed and aligned to the genome of the reference sequence H37Rv. This analysis led to the identification of 1211, 1287 and 1386 SNPs for the GC1237, R34-990172 and the W4 stain respectively.
After withdrawal of IntegraGen (partner 7) from the TB-VIR project the strategy was changed by retrieving all genome sequence data corresponding to five Beijing strains from different databases. In order to get more detailed insights in the genetic diversity of Beijing strains the genome sequence data of 15 non-Beijing strains were included in the analysis, including the complete genome sequence data of M. bovis and the vaccine strain, the BCG strain. The genomes of two M. africanum strains were analysed. M. africanum is situated in an intermediate position between M. tuberculosis and M. bovis. The analysis of all these genomes led to the identification of 35 073 SNPs whose comparison yielded a set of 12 475 polymorphic sites. These identified SNPs have been used to construct a phylogenetic tree using the maximum likelihood. For The Beijing strains, researchers were able to identify all specific SNP's for each strain and each Branch of the tree. These represent good candidates to perform genotyping tests for microevolution purposes. To identify the functions that have diversified to a larger extent in the MTBC complex, we analysed the distribution of SNPs in the genes using the clusters of orthologous groups (COG) gene classification and the Tuberculist nomenclature. Two thirds of the SNPs are non-synonymous (nsSNP) and one third is synonymous (sSNP). Researchers analysed all intragenic SNPs in the 16 COG categories. When accounting for the relative size of COGs it was found that genes playing a role in cell wall membrane and biogenesis are significantly enriched in SNPs (p = 0.0015). No other COG class significantly over-represents or under-represents SNP. The over-representation of SNP in the cell wall membrane is compatible with the previous observation that the most striking clusters of SNP occur in virulence-associated functions.
To determine the prevalence and transmission potential of non-Beijing family strains in mainland China, a population-based prospective study in three geographic areas was performed. 1004 M. tuberculosis clinical isolates from 988 culture positive pulmonary tuberculosis patients were collected between 2006 and 2008. Researchers selected the initial isolate from each patient for a total of 988 isolates that were analysed. Among the 988 clinical isolates, 304 (30.8 %) had non-Beijing strains of M. tuberculosis. The percentage of non-Beijing strains varied in different geographic areas; 45.9 % of the strains isolated from Sichuan were non-Beijing strains, while only 20.6 % of the strains in Shandong Province were non-Beijing strains (p < 0.0005). The proportion of non-Beijing family strains in Sichuan Province was significantly higher than that in Shandong Province (p < 0.0005) and Shanghai (p < 0.0005). The proportion of non-Beijing strains in Shanghai was also significantly higher than that in Shandong Province (p = 0.034). The prevalence of non-Beijing strains was different across the three study areas in mainland China. These differences may be due to factors such as human migration, transmission or diversification. Novel tools are urgently needed for the rapid, reliable detection of MDR and extensively drug-resistant (XDR) strains of Mycobacterium tuberculosis. To develop such tools information is needed about frequency and distribution of mycobacterial mutations and genotypes that are associated with phenotypic drug resistance. In a population-based study, researchers sequenced specific genes of M. tuberculosis that were associated with resistance to rifampin and isoniazid in 242 phenotypically MDR isolates and 50 phenotypically pan-susceptible isolates from tuberculosis (TB) cases in Shanghai.
They estimated the sensitivity and specificity of the mutations using the results of conventional, culture-based phenotypic drug susceptibility testing as the standard and detected mutations within the 81-bp core region of rpoB in 96.3 % of phenotypically MDR isolates. Mutations in two structural genes (katG, inhA) and two regulatory regions (the promoter of mabA-inhA and the intergenic region of oxyR-ahpC) were found in 89.3 % of the MDR isolates. In total, 88.0 % (213 / 242) of the phenotypic MDR strains were confirmed by mutations in the sequenced regions. Mutations in embB306 were considered a marker for MDR and significantly increased the sensitivity. Based on the findings, an approach that prospectively screens for mutations in 11 sites of the M. tuberculosis genome (rpoB531, 526, 516, 533, 513, katG315, inhA -15, ahpC -10, -6, -12, and embB306) could detect 86.8 % of MDR strains in Shanghai. This study lays the foundation for the development of a rapid, reliable molecular genetic test to detect MDR strains of M. tuberculosis in China. 15- and 24-locus VNTR genotype methods have been used to study the molecular epidemiology in settings dominated by Beijing strains but the reliability of 'clusters' defined by those methods has been questioned. SNP and VNTR were combined to genotype Beijing strains in Shanghai and found SNP genotyping can both increase the discriminatory power of VNTR and exclude VNTR homoplasy which caused by convergent evolution.
Other activities that were performed are as follows:
1. To determine the prevalence and transmission potential of non-Beijing family strains in mainland China, researchers performed a population-based prospective study in three geographic areas. They collected 1004 M. tuberculosis clinical isolates from 988 culture positive pulmonary tuberculosis patients. Among the 988 clinical isolates, 304 (30.8 %) had non-Beijing strains of M. tuberculosis. The percentage of non-Beijing strains varied in different geographic areas. These differences may be due to factors such as human migration, transmission, or diversification and adaptation of the mycobacteria to different hosts.
2. Novel tools are urgently needed for detection of multi-drug-resistant and extensively drug-resistant strains of Mycobacterium tuberculosis. To develop such tools, information about the frequency and distribution of the mycobacterial mutations and genotypes that are associated with phenotypic drug resistance is needed. The study done for this project lays the foundation for the development of a rapid, reliable molecular genetic test to detect MDR strains of M. tuberculosis in China. The 15- and 24-locus VNTR genotype methods have been used to study the molecular epidemiology in settings dominated by Beijing strains but the reliability of 'clusters' defined by those methods has been questioned, as lots of clustered strains were found no epidemiology links. We combined SNP and VNTR to genotype Beijing strains in Shanghai and found SNP genotyping can both increase the discriminatory power of VNTR and exclude VNTR homoplasy. Nineteen IS6110 copies were localised for this strain. Sixteen of the 19 copies present direct repeat sequences of 3 to 4 nucleotides in the flanking regions due to transposition events. The absence of DR in the other copies suggests rearrangments between IS6110 elements. One of these copies was localised in the hot spot between Rv0794 -Rv0797 in opposite orientation with respect to a copy in H37Rv. The other two locations correspond to the deleted regions of difference RD152 and RD207. Nine copies were in the same orientation as the downstream gene and four of them could act as promoter according to the distance to the next gene. Researchers investigated this location in other Beijing and non-Beijing strains and we observed that this insertion point was unique to GC 1237 strain. The expression of the genes preceded by IS6110 was studied by qRT-PCR and only a clear increase of about six fold in the case of Rv2179 gene was found. Researchers also studied the expression of this gene in macrophages and observed that the expression of this gene in GC1237 is higher than in H37Rv. The expression increases in intracellular conditions with respect to extracellular conditions. Researchers then studied the transcription of these genes in this region in GC1237 and H37Rv strains.
The Rv2180c, Rv2179c and aroG gene are cotranscribed in H37Rv. In the case of GC1237, amplification products were obtained from 3'end of IS6110 to aroG gene suggesting that the three genes from the insertion sequence are cotranscribed in an operon. researchers verified that IS6110 acts as a promoter by using the recombinant plasmids containing the promoterless gfp gene preceded by the region upstream Rv2179. Results showed that the fluorescence is about five fold higher when the IS is inserted indicating that the IS is acting as a promoter and the expression increased in intracellular conditions. The promotor activity of IS6110 was studied in different conditions and upregulation during stationary but not under hypoxia conditions was observed. In collaboration with Institut Pasteur (partner 6), the OPERA microscopy system was used to measure IS6110 promotor activity of different IS localisations. Using GFP as reporter gene, researchers have seen the strong promotor activity of the IS6110 located in the GC1237 strain upstream Rv2179c gene, confirming the data obtained by different techniques. The IS6110 upstream phoP gene was studied and a higher GFP intensity was measured in the cells infected with the construction including this IS6110. Researchers have studied the other 8 representative strains that present a variable pattern of MIRUS and RFLP. By IS6110 RFLP they observed that these strains contain 10 to 21 copies. They compared all locations from the nine strains and there are three common points including the characteristic in the DR region and between dnaA and dnaN region and two other insertions were observed in eight from the nine strains. In the common location between dnaA and dnaN genes, IS6110 doesn't increase the expression of the downstream dnaN gene.
WP3 - Differential host cell gene expression modifications upon infection
The main objectives were to:
- identify gene expression changes after infection with different mycobacterial strains using DNA chip technologies;
- investigate the host-microbe crosstalk on the single gene level as a function of microbial diversity;
- identify distinct functional roles for genes of each unit in host cell responses;
- identify transcription factors playing an important role in host responses and their potential target genes.
The transcription factor binding sites (TFBS) assays performed in period 1 pointed to the immunity- and development-related POU family TFs as putative regulators of genes differentially expressed in early phases of Mtb infection in THP-1 cells. It was shown in period 2 that THP-1 transcriptomic responses are time-specific, regardless of sub-lineages of Beijing family strains. These conserved host responses are dramatically modulated between 4 h and 18 h. Genes exhibiting consistent changes within the strains and differential expression between 4 h and 18 h were selected through LIMMA-based methodology. Five genes including CXCL10, CXCL11, TNFSF10, IL15 and CCL8 were validated for consistent expression by qRT-PCR approach. TFBS enrichment analysis indicates that STATs, IFR-1, IRF-7 and Oct-1 are putative regulators responsible for transcriptional immune responses of THP-1 cells to Mtb infection. The overall transcriptome profiles between BMDM cells infected by 10 different Mtb strains were similar at each time point, irrespective of the strain phylogeny. Due to the highly similar/conserved transcriptional responses induced by H37Rv and the investigated members of the W-Beijing family a selection of differentially regulated genes was not feasible.
Thus, validation by RT-PCR of potential genes of interest was not performed. During the second period of the project, WP3 aimed at deciphering the transcriptional signature of macrophages upon infection with representative W-Beijing Mtb strains. The goal was to perform clustering of genes and identify host cell gene expression modifications induced by Mtb genotypes. BMDMs were infected with M. tuberculosis (Mtb) H37Rv as laboratory control strain and nine different Mtb W-Beijing genotypes. Due to BSL3 capacity limitations, experiments were carried out in three consecutive series, each series comprising the H37Rv control strain and three different Beijing genotypes. Two technical replicates were generated by parallel sampling and performed two complete independent biological replicates at different days. Sample preparation for total RNA isolation took place at three different time points post infection (p.i.). In total 190 different samples were hybridised and analysed. For initial data analysis, all 41 174 microarray features of the whole genome microarray by divisive clustering were compared. The heat map of the differential expression shows that a similar pattern of expression was induced. In general it can be speculated that at the measured time points no significant and profound differences were identified between H37Rv and the investigated W-Beijing strains.
Due to the highly similar/conserved transcriptional responses induced by H37Rv and the investigated members of the W-Beijing family, a selection of differentially regulated genes was not feasible. Validation by RT-PCR of potential genes of interest was not performed. A follow-up investigation was initiated by partner 5 to explore differential transcriptional activity at early time-points between the lab strain in comparison with the Beijing isolates. Increasing sample size might provide significant power to the analysis. partner 5 performed the infection and provided the cell RNA to partner 8 for microarray assay. Differentiated THP-1 cells were infected with 12 representative M. tuberculosis clinical isolate from different sub-lineages of W-Beijing family Mtb using multiplicity of infection (MOI) 5 for 4 hours. The cell RNAs and supernatants were collected at 0 h, 4 h, 18 h and 48 h. RNAs were extracted and sent to partner 8 to perform global transcriptional analysis using microarray techniques. Host transcriptomic responses performed by partner 8 were shown to be time-specific, regardless of sub-lineages of the pathogen. These host-responsive genes are dramatically modulated between 4 h and 18 h. The robustness of host responses might be attributed to the involvement of response-relevant transcriptional factors and these regulators could be predicted from the observed transcriptomic changes. Genes differentially expressed between 4 h and 18 h may represent the common signatures of Mtb-infected THP-1 cells. A LIMMA-based methodology was applied to select genes exhibiting the consistent changes within the strains and differential expression between 4 h and 18 h. As validated in five randomly selected Mtb-infected samples by qRT-PCR approach, the CXCL10, CXCL11, TNFSF10, IL15 and CCL8 genes displayed consistent upregulation from 4 h to 18 h.
Differentially expressed genes identified can be categorised into two groups: Cluster 1 for induction at 18 h or 48 h compared with at 4 h, Cluster 2 for repression. Most of differentially expressed genes (92 %, 367 / 399) were induced. When enrichment analysis was applied to examine whether they shared functional or regulatory features, only biological features associated with genes in Cluster 1 were observed. No enrichment of biological relevance was observed in Cluster 2, which was thus excluded from further consideration in this study. The most significant gene ontology (GO) terms are immunity-related responses. Consistent with this, the most significant TFBS, represented in the form of position weight matrixes [PWMs]) are immunity-related transcription factors or putative immunity-related regulators, including IFN-stimulated response element (ISRE), IRF-1, IRF-7 and Oct-1. Through extensive literature mining it was found that each of the predicted regulators was biologically relevant. Transcription factors of STATs, IFR-1, IRF-7 and Oct-1 are collectively putative regulators responsible for transcriptional immune responses of THP-1 cells to Mtb infection.
WP4 - Screening of a genome-covering M. tuberculosis W-Beijing mutant library
The main objectives were to:
- infect murine macrophages with a 11 000-member genome-covering STM library generated by partner 12 (IPAS) in the W-Beijing strain GC1237;
- identify important mycobacterial virulence genes involved in host cell parasitism.
During the first 18 months of the project, researchers screened for the 10 mutants over a 11 000 Mycobacterium tuberculosis mutant library that traffic most frequently into acidified compartments early after phagocytosis, suggesting that they had lost their ability to arrest phagosomal maturation. Molecular analysis of these mutants revealed mainly disruptions in genes involved in cell envelope biogenesis (fadD28), the ESX-1 secretion system (espL/Rv3880), molybdopterin biosynthesis (moaC1 and moaD1), LppM and periplasmic phosphate-binding pstS3 lipoproteins, as well as in genes from novel locus, Rv1503c-Rv1506c and Rv2295c. All genes were present in the panel of M. tuberculosis Beijing strains sequenced in WP2. A number of non-synonymous (nsSNP) mutations were identified within the open reading frame of all genes. fadD28 has A to G nsSNP at position 1306 for four W/Beijing strains. A mutation in lppM-encoded protein was identified in M. tuberculosis Beijing GC1237. During the second period of the project, genotype / phenotype correlations were made for the Mtb virulence genes identified in WP4. The residual virulence of the mutants was assessed in vitro in cultured macrophages, and in vivo in the mouse model of Mtb infection. The ability of the different Beijing strains to induce cytokines responses in cultured human phagocytes was assessed, and researchers could identify specific signatures of the Beijing family in terms of cytokine and chemokine secretion and host cell response to infection. Novel TFs identified in WP03 are currently studied in future projects.
WP 5 - Understanding genotype / phenotype relationships between M. tuberculosis genetic diversity, differential virulence and immune responses in vitro and vivo
The main objectives were to identify:
- key genotype-phenotype relationships between M. tuberculosis genotypes and subgenotypes, in particular among the W-Beijing family;
- host cell responses at the transcriptome level;
- ability to overcome phagosome maturation and to parasitise host macrophages;
- host immune response to infection in vitro and vivo and mycobacterial immuno-pathogenicity.
Bone marrow derived macrophages (BMDM) were infected with M. tuberculosis (Mtb) H37Rv and nine different MtbW-Beijing genotypes. Due to BSL3 capacity limitations, experiments were carried out in three consecutive series, each series comprising the H37Rv control strain and three different Beijing genotypes. Researchers generated two technical replicates by parallel sampling and carried out two complete independent biological replicates. Cell-free supernatants from MTB-infected BMDM were collected at 4, 18 and 48 h p.i. In separate experiments BMDM were primed with IFN prior MTB infection. Triplicates were performed and collected cell-free supernatants at a single time-point, namely 24 h p.i. Up to 32 targets were measured using the bead-based assays and 21 cytokines / chemokines were detected. Researchers could not identify a significantly different secretion pattern for most cytokines. For several (but not all) W-Beijing members a trend to induce more abundant release of IL-6 (e.g. HM903, Cam22) was determined. The measurements at 24 h p.i. did not recapitulate the previously mentioned observations. Classically activated BMDM responded in a divergent way in terms of release of several cytokines and chemokines.
CXCL-10 levels were not significantly different between the tested bacterial strains. A similar tendency was reported for TNF, while IL-6 seemed to be less induced by several W-Beijing members at 24 h p.i. The discrepancies between kinetics measurements and the 24 h measurement might be explained by the fact that BMDM present receptors for several of the quantified cytokines / chemokines and target-receptor binding results in reduced concentration of the target soluble mediator in the cell culture supernatant. Infection is a dynamic process and autocrine sensing of secreted cytokine might result in changes in density of the cell surface receptors and synthesis of decoy receptors, which may bind the mediator as well. Activation of the BMDM with IFN resulted in increased levels of several cytokines compared with resting BMDM. IFN was able to restore the secretion of cytokine (TNF) for some Mtb strains while this propensity was not observed for IL-6.
Nitric oxide (NO) levels were quantified using the Griess reagent. As expected, IFN priming resulted in release of NO in culture supernatants. Abundance of the antimicrobial molecule was independent of the mycobacterial strain used to infect the BMDM. To investigate whether the W-Beijing strains have a growth advantage in macrophages we infected resting and IFN primed BMDM with H37Rv and the Beijing strains and followed bacterial replication using 3H-Uracil incorporation. The radioisotope labelled base is integrated in bacterial RNA and thus upon replication and metabolic activity the retained radioactive levels increased. Researchers incubated the lab strain and the W-Beijing strains in macrophage culture media and added the radionuclide labelled nucleic base. They observed that H37Rv and several of the tested Beijing strains grew well, showing a daily replication (GC1237, HNH5, N4, 990172), while other strains were defective in multiplication using macrophage media as substrate. Overall, results showed a conserved pattern of response of the lab Mtb strains and the W-Beijing members tested.
Behaviour recorded for isolated strains in a particular assay could not be extended to the whole subfamily or family. Based on the tools and results, a significant difference between H37Rv and the clinical W-Beijing strains was not observed and cannot be stated. Apoptosis is a physiological way of cell death by which multicellular organisms control homeostasis, cell transformation and intracellular infection with important implication in the host immune response. This process is triggered during the course of infections with intracellular facultative pathogens including M. tuberculosis. There have been proposed differences between Beijing and non-Beijing strains in terms of apoptosis induction on host cells. Beijing strains have been shown to induce more necrosis and less apoptosis than non-Beijing strains. However, these studies are incomplete. Thus, researchers used the nine Beijing reference strains to elucidate if this feature is intrinsic to Beijing family. Using different cell death markers to discern between apoptosis and necrosis they found that both Beijing and non-Beijing strains clearly induced apoptosis on macrophages. No global differences between both groups were measured.
Intracellular cytokine expression of proinflamatory IL-6 was studied in mouse MH-S macrophages infected with the panel of nine Beijing strains and with non-Beijing strains. It was found that none of the nine Beijing strains studied induced IL-6 expression, while all the non-Beijing strains did it. The 10 M. tuberculosis GC1237 mutants identified in WP4 were further studied. BMDM were inoculated with the various mutants and their growth was compared to that of wild type and of respective complemented strains. BMDM were infected at different MOI ranging from 1 to 5 and intracellular bacteria were counted using colony-forming unit enumeration method at day 0, 3 and 5 post-infection. pstS3, lppM, moaD1Tn mutants were degraded within the macrophages, Rv1503c, Rv1506c, moaC1, Rv3880c Tn mutants survived intracellularly, in contrast Rv2295c, fadD28 and ppe54 Tn mutants were able to actively multiply within the host cells similarly to the wild type strain. The cytokines release by BMDM that had been inoculated with the mutants was profiled at 24 hours using the Raybiotech Cytokine 32-Array and compared with that of their corresponding complemented strains and wild type. As expected M. tuberculosis GC1237 induced an increased secretion of IL-6 and GSCF compared to non-infected macrophages. The Rv1506c Tn mutant was able to further augment the secretion of IL-6. No specific pattern of cytokines expression using this technique was found among the different mutants.
The phenotype of the mutants in the mouse model in vivo was studies. All mutants but not fadD28 and Rv3880c were inoculated to Balb/C mice via the intranasal route and compared to the wild type GC1237 strain and complemented strain. Groups of five animals were sacrificed at three weeks and six weeks and CFU were enumerated. All eight mutants were attenuated by displaying at least one log less CFU in the lungs compared to wild type control. The wild type phenotype could be restored for Rv1503c, Rv1506c, ppe54 and Rv2295 Tn mutants. Concerning lppM Tn mutant, our hypothesis for explaining the lack of phenotype restoration is that complementation was performed with plasmid encoding M. tuberculosis H37Rv LppM which has a mutation compared to that of GC1237 protein. With regards to the others, moaC1, moaD1, pstS3 complemented Tn mutants, there is an issue of bacterial growth fitness in vitro on agar plates. The immune response generated by these mutants was investigated. Splenic T-cells from infected mice were analysed for CD3, CD4, CD8, CD44, CD45RB, CD62L, CD11b, CD11c, F4-80 and CD80 population. No significant variation had been found compared to wild type.
These data showed that mutants that are impaired in phagosome maturation arrest in vitro have all growth defects in vivo. Partners 6 and 5 have used OPERA microscopy system to perform a comparative study of the trafficking of the different mutants in H37Rv (H37Rv phoP, H37Rv phoP pSO5k and H37Rv phoP pSO7k) and GC1237 (GC1237 phoP, GC1237phoP pSO5k and GC1237 phoP pSO7k) inside macrophages and to analyse the influence of IS6110 as a promoter of phop gene (construction pSO7). DAPI was used, lysotraker-green and CypHer5 dyes. As a negative control non-infected cells were used, and as a positive heat-killed mutants. After 2 h of infection, nearly 100 % of the cells were found infected and preliminary results show that the fraction of bacteria co-localising with Lysotrake-positive staining are less than 10 % in all the mutants and more than 60 % of killed-bacteria were found to co-localise with acidified compartments. No significant difference between H37Rv and the GC1237 constructions were observed.
WP 1: Management and coordination of the consortium
The main objectives were to:
- set up an effective management framework for the TB-VIR consortium;
- ensure the usual and contractual administrative tasks;
- manage safety issues, legal, ethical and intellectual commercial / property-related issues, and gender equality promotion;
- coordinate the dissemination of knowledge inside and outside the network.
The TB-VIR coordination and management WP took care of the usual and contractual administrative tasks. The project management team focused on monitoring complex aspects related to the network's specific properties, the organisation of access to the key deliverables beyond the end of the European Commission (EC) contract, maintenance of the information hub, as well as identifying solutions for maintaining the momentum, added value of cooperation and collections obtained by the TB-VIR network thanks to the EC support. The management team acted as the interface between TB-VIR partners and EC, was in regular contact with EC Scientific Officer, and relayed problems and questions to the EC. The team was involved in the organisation of a variety of meetings, promotional materials and the maintenance of the project's website.
Potential impact
a. Potential impact and exploitation of results
Impact on the development of novel TB control strategies
TB remains a major threat to mankind and cannot be conquered without an effective vaccination strategy. No completely effective vaccination strategy has yet been developed. M. tuberculosis is one of the most effective human pathogens. The global incidence of TB increases by 2 % annually. The vast majority of cases occur in developing countries, especially in adults between 15 and 45 years of age. This situation necessitates the development of new types of intervention and improvement of the follow-up of TB patients in these countries. This project has contributed to the development of new interventions. A provisional patent application entitled 'Method for producing a vaccine for the treatment and/or prevention of tuberculosis' has been filed by partners 1 CNRS and 6 IPK. These vaccine candidates are currently under preclinical studies and will help provide novel intervention tools to combat TB.
Impact on pathogen research and pathogen-related diseases
The proposed work has identified the genes differentially expressed or regulated in response to various clinical isolates of M. tuberculosis with an emphasis on genes involved in the immune response. M. tuberculosis genes required for macrophage parasitism have been identified. Participation of beneficiary 6 has been invaluable to identify mycobacterial genes involved in intracellular trafficking. WP4 as a whole has been highly complementary with the activity of the EC-funded project TB-MACS. Findings of this study have increased understanding of M. tuberculosis / host interactions and have been an invaluable reference for TB research. Datasets produced in this study have been a valuable reference for TB research worldwide and have been made publicly available via publications.
Impact through innovation
The proposed project was highly innovative because it made use of new developments in functional genomics technology to investigate M. tuberculosis pathogenesis. This project brought together technologies from the fields of microarray analysis, functional genomics and transcriptomics, cell biology, confocal microscopy and bioinformatics. The technologies used and the know-how obtained in this project will be used by researchers working on other pathogens. The application of high-throughput screens as a means of connecting genotypes and complex phenotypes will be of considerable interest to researchers in functional genomics and studies of infection. The use of SNP genotyping to analyse M. tuberculosis W-Beijing population structure provides the international community working on TB and other infectious diseases invaluable information for further studies on pathogen population structure and genotype-phenotype relationships.
Impact through training and networking
Training the next generation of scientist has been an important achievement in TB-VIR (see http://www.moleculartb.org/index.html for details). The TB-VIR project also allowed for promoting and strengthening the links and scientific collaborations between European and Asian laboratories.
b. Dissemination activities
The project contributed significantly to the development of our knowledge base concerning M. tuberculosis virulence and anti-mycobacterial immunity. These achievements confirmed and extended the leading role of the European laboratories in mycobacterial research and the development of novel intervention tools. In addition to scientific publications, principal investigators from the TB-VIR consortium presented their work at a large number of national and international meetings. Fruitful collaborations with Asian research partners were established. Also many of the European partners are continuing the collaborations that were initiated under TB-VIR. The TB-VIR project resulted into 10 scientific publications in high-impact journals, 2 articles currently under submission, the participation in 13 conferences and symposiums and to 2 symposiums. A strong methodology has been set up to ensure the dissemination of the projects results. After verification that all possible steps have been taken for patent submission the data have been made available outside TB-VIR through meetings and various media supports.
Tools and ways of dissemination
- Adapted communication tools: the TB-VIR public website, presentation brochure and poster.
- Workshops and conference presentations: The participants presented the project and related results to scientific and clinical conferences and workshops in Europe and outside Europe.
- Publications in peer-reviewed and/or open access journals.
- Outreach the general public: In addition to our public website, a press release was written for the kick off meeting and disseminated at national level to newspapers and to public relations offices of the participant's institutions, but also at European level through the EC' communication services.
Website of the project: http://www.tb-vir.org
Contacts:
Project coordinator: Dr Olivier Neyrolles
IPBS - CNRS UMR 5089
205 Route de Narbonne, F-31077 Toulouse, FRANCE
Phone: +33-561-175475
Fax: +33-561-175994
E-mail: olivier.neyrolles@ipbs.fr
- General manager: Dr Jerome Weinbach
Inserm-Transfert
Paris BioPark
7 Rue Watt, F-75013 Paris, FRANCE
Phone: +33-155-030139
Mobile: +33-676-630550
Fax: + 33-155-030160
E-mail: jerome.weinbach@inserm-transfert.fr
The inhibition of the host immune response by mycobacterial strains of the W-Beijing family has recently been confirmed in vivo. The W-Beijing lineage is one of the most successful M. tuberculosis families worldwide in terms of morbidity and mortality. These strains are currently attracting considerable attention because they display important pathogenic features and are frequently associated with resistance to antimicrobial drugs. W-Beijing strains may be more able to acquire antimicrobial drug resistance than other strains due to mutations in putative mutator genes and a higher level of adaptability. Strains of the W-Beijing lineage are responsible for 80 to 90 % of TB cases in China, where TB incidence may exceed 50 per 100 000 in average. The remarkable ability of these strains to spread locally in large clonal lineages probably reflects their high level of fitness / transmissibility and their adaptation to the infection of humans. The predominance of the W-Beijing lineage probably results from genetic advantages, including unidentified virulence factors and the induction or modification of specific host cell responses not yet thoroughly investigated. The TB-VIR project aimed to unravel the links between differential host responses to M. tuberculosis infection and mycobacterial genetic diversity and virulence at the global genomic and post-genomic levels within the W-Beijing family, and between the W-Beijing family and other M. tuberculosis families, to improve our understanding of the epidemiological success of this lineage.
Important results have been obtained and published from the TB-VIR activity. This has helped increase understanding of M. tuberculosis virulence and anti-mycobacterial immune response, in particular regarding the W-Beijing family of strains. The genetic diversity within the family has been further elucidated using informative single nucleotide polymorphisms (SNP) and the role of transposable elements in the peculiar virulence of this family has been reported. Genotype-phenotype correlations were established. It was showed that host cell response to W-Beijing sublineages is rather homogeneous and clearly impaired as compared to the immune response to other mycobacterial lineages. This has been shown using a combination of innovative approaches such as global gene expression profiling and protein survey. Candidate vaccine strains have been isolated through TB-VIR activity and are now tested in animal models. In summary, the TB-VIR project has been successful not only to provide new knowledge on M. tuberculosis but also to generate new possible intervention tools and to promote Euro-Asian collaborations between laboratories in France, Germany and Spain, and laboratories in China and Korea.
Project context and objectives:
Despite tremendous efforts to fight tuberculosis (TB), this disease remains a major health problem, particularly in low-income developing countries. TB causes 1.7 million deaths annually worldwide, and up to one third of the global population is estimated to carry latent Mycobacterium tuberculosis infection (1). TB incidence varies from 7 per 100 000 in Northern Europe to up to 300 per 100 000 in parts of Africa. In eastern Europe, the incidence of TB now exceeds 100 per 100 000, and the proportion of multidrug-resistant (MDR) M. tuberculosis strains is high. Extensively drug-resistant (XDR) TB has emerged worldwide as a threat to public health and TB control, raising concerns about a possible future epidemic of virtually untreatable TB. The frequency and distribution of XDR TB cases recently described by the American Center for Disease Control (CDC) determined that during the 2000 to 2004 period, 20 % of the 17 690 TB isolates were MDR and 2 % were XDR (4). Population-based data on the drug susceptibility of M. tuberculosis isolates have been obtained from the United States (for 1993-2004), Latvia (for 2000-2002), and South Korea (for 2004), where 4 %, 19 %, and 15 % of MDR TB cases, respectively, were XDR.
Drug treatment has helped to decrease the incidence of TB in countries with effective public health systems, but vaccination is considered the most cost-effective intervention in most countries. The Mycobacterium bovis BCG vaccine currently in use, although effective against severe forms of TB in children, is of minimal and variable efficacy against the much more common and contagious pulmonary forms of TB in adults and requires improvement.
Following M. tuberculosis infection, only 5 to 10 % of immunocompetent individuals develop TB. Environmental factors probably play an important role, but it is increasingly thought that the virulence and load of the infecting strain, together with host genetic factors, such as polymorphisms in genes involved in anti-mycobacterial immunity, contribute to differences between infected individuals. On the host side, genetic variations in genes encoding key components of the immune system, such as the vitamin D receptor, monocyte chemoattractant protein (MCP)-1 and the C-type lectin DC-SIGN have been found to increase susceptibility to TB in certain parts of the world. On the pathogen side, several reports have strongly suggested that the severity and clinical manifestations of TB depend on the immunogenicity and pathogenicity of the infecting strain of M. tuberculosis, these properties varying considerably between strains. A recent study on almost 700 clinical isolates collected from 1996 to 2000 in Arkansas (US) concluded that patients infected with M. tuberculosis W-Beijing strains were three times more likely to develop extrathoracic TB than patients infected with strains of other lineages. However, some other studies reported no such association between M. tuberculosis genotype and clinical features of TB.
The notion that some M. tuberculosis lineages are more virulent than others is supported by results obtained with animal and cellular models. For instance, the W-Beijing isolates HN878 and W4 induce more IFN and less Th1 cytokines in vitro and in vivo in mice than another non-W-Beijing isolate, CDC1551 and are associated with lower survival rates in infected animals. Such variations between strains probably result from genetic diversity, which seems to be greater in mycobacteria than previously thought. Recent reports have shown that different clinical isolates of M. tuberculosis produce different proteins and lipids according to their genetic lineage, and this probably affects mycobacterial virulence and the host immune response to infection. The inhibition of the host immune response by mycobacterial strains of the W-Beijing family has recently been confirmed in vivo.
On the basis of IS6110 typing and other genotyping methods, including spoligotyping, SNPs, genotyping and large sequence polymorphism (LSP) analysis, M. tuberculosis strains of the W-Beijing family have been detected almost worldwide and new unexpected routes of transmission have been identified for these strains. This genotype was first identified in M. tuberculosis strains isolated in the Beijing area of China, hence its name. These strains are currently attracting considerable attention worldwide and are frequently associated with resistance to antimicrobial drugs. W-Beijing strains may be more able to acquire antimicrobial drug resistance than other strains, due to mutations in putative mutator genes and a higher level of adaptability. The W-Beijing genotype also contains a much larger number of IS6110 copies than other lineages. It has been demonstrated that IS6110 may increase the expression of neighbouring virulence genes by generating new promoter sequences capable of driving their expression.
MDR-TB hot spots worldwide include China, Russia and India. The high frequency of drug-resistant and MDR strains of M. tuberculosis remains a serious problem for TB control in China. Strains of the W-Beijing lineage are responsible for 80 to 90 % of TB cases in China. The remarkable ability of these strains to spread locally in large clonal lineages probably reflects their high level of fitness / transmissibility and their adaptation to the infection of human populations. The predominance of the W-Beijing lineage probably results from genetic advantages, including unidentified virulence factors and the induction or modification of specific host cell responses not yet thoroughly investigated.
The project aimed to unravel the links between differential host responses to M. tuberculosis infection and mycobacterial genetic diversity and virulence at the global genomic and post-genomic levels within the W-Beijing family, and between the W-Beijing family and other M. tuberculosis families, to improve understanding of the epidemiological success of this particular lineage.
Goals
1) Deciphering M. tuberculosis genetic diversity using highly discriminative technologies and extracting strains representative of the W-Beijing subgenotypes, and of other genotypes
Genetic diversity of M. tuberculosis clinical isolates collected in the Shanghai area, where nearly 90 % of the strains are expected to belong to the W-Beijing family, was investigated using spoligotyping, IS6110-RFLP profiling, MIRU-VNTR, RD-, and SNP-based genotyping. The Fudan University, in close collaboration with the CDC in Shanghai, has access to over 9000 clinical isolates collected since 1999. At least 1000 isolates were selected for highly discriminative genotyping. SNP-based genotyping, a new and very promising technique for the genotyping of mycobacteria and other pathogens, was carried out with the technical support of a small and medium-sized enterprise (SME), IntegraGen S.A. Diversity and evolution in the W-Beijing lineage have recently been correlated to SNPs in genes involved in DNA repair or other genes, and with LSPs or regions of difference. SNPs provide a unique tool for phylogenetic studies as they are less susceptible to the influence of selective pressure than other genetic markers and are unlikely to display the convergence sometimes observed with spoligotype or MIRU-VNTR markers. Selectively neutral SNPs should accumulate at a uniform rate and can thus be used to measure divergence. The project aimed to make full use of the power of SNP-based phylogenies as a 'gold standard' for assessing the accuracy of other DNA typing methods. This technique was compared with the most discriminative method currently in use, the MIRU-VNTR genotyping method, and with RD-based genotyping. The effect of IS6110 insertion on the expression of putative virulence genes was be investigated. Clinical isolates were clustered into lineages and sub-lineages and approximately 10 strains, representative of regional diversity, were selected for functional studies.
2) Understanding how host cells respond differentially to infection with M. tuberculosis W-Beijing subgenotypes and other genotypes
Comparative analysis of the changes in host cell gene expression following the infection of mouse and human macrophages with M. tuberculosis strains representative of the diversity of W-Beijing strains and with strains of other genotypes were realised. We carried out systematic analysis of changes in the host cell transcriptome in response to M. tuberculosis infection, using an approach combining the methods of experimental biology, bioinformatics and computational biology.
3) Identifying virulence genes in M. tuberculosis W-Beijing using functional genomics and investigating mycobacterial virulome diversity in W-Beijing subgenotypes and in other genotypes
A genome-covering M. tuberculosis W-Beijing mutant library, generated by Partner 12 (IPAS), was screened by partner 1 (CNRS) in host macrophages, using signature-tagged transposon mutagenesis (STM) and high-throughput confocal microscopy, a well-established technique in the laboratory of beneficiary 6 (IPK). partner 6 (IPK) does not belong to an ICPC, but the participation of this partner in the project is invaluable. The work allowed identifying virulence genes on a global, genome-wide level in a W-Beijing M. tuberculosis strain. Genetic diversity (SNPs, InDels, etc.) in the genes identified here were subsequently investigated in representative members of the W-Beijing subgenotypes and of other genotypes.
4) Assessing differential host immune and pathological response to M. tuberculosis W-Beijing subgenotypes and other genotypes
Molecular and cell biology, biochemistry and immunology experiments were conducted in order to investigate the role of the host and mycobacterial genes identified by activities 1 to 3 in mycobacterial virulence and in the immune response to M. tuberculosis infection in vitro and in vivo. Mycobacterial virulence and the immune response to infection with various representative strains were assessed in vitro in cultured macrophages, and in vivo in the mouse model of M. tuberculosis infection. A meta-analysis of the data generated made it possible to describe fine genotype-phenotype associations between M. tuberculosis genotypes and subgenotypes, and mycobacterial virulence and immunopathogenicity.
5) Considering that a strong and integrated management of the above generated knowledge is an essential part of the success of this project, (WP1) was dedicated to scientific and knowledge management.
Project results
The following main scientific results were achieved:
- the phylogeny of M. tuberculosis (Mtb) Beijing strains has been constructed using polymorphisms in DNA replication, recombination and repair genes;
- the innate immune response to Mtb Beijing and other genotypes has been assessed;
- a high-density mutant library in Mtb Beijing has been screened visually at the phagosome level using high throughput confocal microscopy;
- the role of IS6110 in Mtb Beijing has been deciphered;
- novel techniques have been developed to study host cell promoter regions;
- non-Beijing strains of Mtb in China have been studied;
- mutations to study drug resistance in Mtb Beijing have been identified;
- fluoroquinolone resistance in Mtb Beijing has been studied;
- the complete genome of several Mtb Beijing and non-Beijing strains has been sequenced;
- the role of novel virulence genes involved in metal efflux has been studied in Mtb Beijing and non-Beijing.
The role of the NLRP3 inflammasome in immunity to Mtb Beijing and non-Beijing has been studied.
Main activities and achievements
WP 2 - Evaluation of M. tuberculosis genetic diversity in clinical isolates from the Shanghai area
The main objectives were to:
- identify strains representative of the genetic diversity observed in the Shanghai area, where over 90 % of the clinical isolates are expected to belong to the W-Beijing family;
- provide scientific community with know-how and proof of principle that SNP-genotyping can be successfully used to clusterise M. tuberculosis into genotypes and subgenotypes;
- sequence IS6110 insertion sites in various representative strains within the W-Beijing genotype and among mycobacterial genotypes;
- assess the influence of IS6110 insertion on driving the expression of neighbouring genes.
Study of Beijing strains of the Shanghai area together with Beijing strains from other countries all over the world (partners 5, 9 and 12) allowed to propose a phylogenetic tree that confirm the successive acquisition of point mutations that can be used as markers to differentiate Beijing strains (Mestre et al., PLoS One, 2011). The role of repetitive sequences in the evolution of Beijing strains was investigated by analysing IS6110 carrying regions (partner 4 and 12). They have been disseminated into the genomes either by transposition or homologous recombination (Alonso et al., Tuberculosis, 2011) Thanks to the complete sequence of several Beijing strains genomes and their comparison with genomes of other family strains (partners 10 and 12) new SNPs were identified which will be used for microevolution studies. The association of a limited number of SNPs and MIRU/VNTR loci was successfully tested in a collection of Beijing strains from the Shanghai area (partners 5, 9 and 12). The main goal was to study genetic diversity of W-Beijing strains by SNP genotyping. To reach this goal, genomes of three Beijing strains were sequined using the Illumina genome analyser GAII. Among these Beijing strains, a strain named GC1237 was included. In addition we sequenced two other Beijing strains: one ancient and one modern Beijing strain selected by taking into account a previously published work based on the sequential accumulation of SNPs in 3R genes. For each strain, all generated paired end (PE) reads have been analysed and aligned to the genome of the reference sequence H37Rv. This analysis led to the identification of 1211, 1287 and 1386 SNPs for the GC1237, R34-990172 and the W4 stain respectively.
After withdrawal of IntegraGen (partner 7) from the TB-VIR project the strategy was changed by retrieving all genome sequence data corresponding to five Beijing strains from different databases. In order to get more detailed insights in the genetic diversity of Beijing strains the genome sequence data of 15 non-Beijing strains were included in the analysis, including the complete genome sequence data of M. bovis and the vaccine strain, the BCG strain. The genomes of two M. africanum strains were analysed. M. africanum is situated in an intermediate position between M. tuberculosis and M. bovis. The analysis of all these genomes led to the identification of 35 073 SNPs whose comparison yielded a set of 12 475 polymorphic sites. These identified SNPs have been used to construct a phylogenetic tree using the maximum likelihood. For The Beijing strains, researchers were able to identify all specific SNP's for each strain and each Branch of the tree. These represent good candidates to perform genotyping tests for microevolution purposes. To identify the functions that have diversified to a larger extent in the MTBC complex, we analysed the distribution of SNPs in the genes using the clusters of orthologous groups (COG) gene classification and the Tuberculist nomenclature. Two thirds of the SNPs are non-synonymous (nsSNP) and one third is synonymous (sSNP). Researchers analysed all intragenic SNPs in the 16 COG categories. When accounting for the relative size of COGs it was found that genes playing a role in cell wall membrane and biogenesis are significantly enriched in SNPs (p = 0.0015). No other COG class significantly over-represents or under-represents SNP. The over-representation of SNP in the cell wall membrane is compatible with the previous observation that the most striking clusters of SNP occur in virulence-associated functions.
To determine the prevalence and transmission potential of non-Beijing family strains in mainland China, a population-based prospective study in three geographic areas was performed. 1004 M. tuberculosis clinical isolates from 988 culture positive pulmonary tuberculosis patients were collected between 2006 and 2008. Researchers selected the initial isolate from each patient for a total of 988 isolates that were analysed. Among the 988 clinical isolates, 304 (30.8 %) had non-Beijing strains of M. tuberculosis. The percentage of non-Beijing strains varied in different geographic areas; 45.9 % of the strains isolated from Sichuan were non-Beijing strains, while only 20.6 % of the strains in Shandong Province were non-Beijing strains (p < 0.0005). The proportion of non-Beijing family strains in Sichuan Province was significantly higher than that in Shandong Province (p < 0.0005) and Shanghai (p < 0.0005). The proportion of non-Beijing strains in Shanghai was also significantly higher than that in Shandong Province (p = 0.034). The prevalence of non-Beijing strains was different across the three study areas in mainland China. These differences may be due to factors such as human migration, transmission or diversification. Novel tools are urgently needed for the rapid, reliable detection of MDR and extensively drug-resistant (XDR) strains of Mycobacterium tuberculosis. To develop such tools information is needed about frequency and distribution of mycobacterial mutations and genotypes that are associated with phenotypic drug resistance. In a population-based study, researchers sequenced specific genes of M. tuberculosis that were associated with resistance to rifampin and isoniazid in 242 phenotypically MDR isolates and 50 phenotypically pan-susceptible isolates from tuberculosis (TB) cases in Shanghai.
They estimated the sensitivity and specificity of the mutations using the results of conventional, culture-based phenotypic drug susceptibility testing as the standard and detected mutations within the 81-bp core region of rpoB in 96.3 % of phenotypically MDR isolates. Mutations in two structural genes (katG, inhA) and two regulatory regions (the promoter of mabA-inhA and the intergenic region of oxyR-ahpC) were found in 89.3 % of the MDR isolates. In total, 88.0 % (213 / 242) of the phenotypic MDR strains were confirmed by mutations in the sequenced regions. Mutations in embB306 were considered a marker for MDR and significantly increased the sensitivity. Based on the findings, an approach that prospectively screens for mutations in 11 sites of the M. tuberculosis genome (rpoB531, 526, 516, 533, 513, katG315, inhA -15, ahpC -10, -6, -12, and embB306) could detect 86.8 % of MDR strains in Shanghai. This study lays the foundation for the development of a rapid, reliable molecular genetic test to detect MDR strains of M. tuberculosis in China. 15- and 24-locus VNTR genotype methods have been used to study the molecular epidemiology in settings dominated by Beijing strains but the reliability of 'clusters' defined by those methods has been questioned. SNP and VNTR were combined to genotype Beijing strains in Shanghai and found SNP genotyping can both increase the discriminatory power of VNTR and exclude VNTR homoplasy which caused by convergent evolution.
Other activities that were performed are as follows:
1. To determine the prevalence and transmission potential of non-Beijing family strains in mainland China, researchers performed a population-based prospective study in three geographic areas. They collected 1004 M. tuberculosis clinical isolates from 988 culture positive pulmonary tuberculosis patients. Among the 988 clinical isolates, 304 (30.8 %) had non-Beijing strains of M. tuberculosis. The percentage of non-Beijing strains varied in different geographic areas. These differences may be due to factors such as human migration, transmission, or diversification and adaptation of the mycobacteria to different hosts.
2. Novel tools are urgently needed for detection of multi-drug-resistant and extensively drug-resistant strains of Mycobacterium tuberculosis. To develop such tools, information about the frequency and distribution of the mycobacterial mutations and genotypes that are associated with phenotypic drug resistance is needed. The study done for this project lays the foundation for the development of a rapid, reliable molecular genetic test to detect MDR strains of M. tuberculosis in China. The 15- and 24-locus VNTR genotype methods have been used to study the molecular epidemiology in settings dominated by Beijing strains but the reliability of 'clusters' defined by those methods has been questioned, as lots of clustered strains were found no epidemiology links. We combined SNP and VNTR to genotype Beijing strains in Shanghai and found SNP genotyping can both increase the discriminatory power of VNTR and exclude VNTR homoplasy. Nineteen IS6110 copies were localised for this strain. Sixteen of the 19 copies present direct repeat sequences of 3 to 4 nucleotides in the flanking regions due to transposition events. The absence of DR in the other copies suggests rearrangments between IS6110 elements. One of these copies was localised in the hot spot between Rv0794 -Rv0797 in opposite orientation with respect to a copy in H37Rv. The other two locations correspond to the deleted regions of difference RD152 and RD207. Nine copies were in the same orientation as the downstream gene and four of them could act as promoter according to the distance to the next gene. Researchers investigated this location in other Beijing and non-Beijing strains and we observed that this insertion point was unique to GC 1237 strain. The expression of the genes preceded by IS6110 was studied by qRT-PCR and only a clear increase of about six fold in the case of Rv2179 gene was found. Researchers also studied the expression of this gene in macrophages and observed that the expression of this gene in GC1237 is higher than in H37Rv. The expression increases in intracellular conditions with respect to extracellular conditions. Researchers then studied the transcription of these genes in this region in GC1237 and H37Rv strains.
The Rv2180c, Rv2179c and aroG gene are cotranscribed in H37Rv. In the case of GC1237, amplification products were obtained from 3'end of IS6110 to aroG gene suggesting that the three genes from the insertion sequence are cotranscribed in an operon. researchers verified that IS6110 acts as a promoter by using the recombinant plasmids containing the promoterless gfp gene preceded by the region upstream Rv2179. Results showed that the fluorescence is about five fold higher when the IS is inserted indicating that the IS is acting as a promoter and the expression increased in intracellular conditions. The promotor activity of IS6110 was studied in different conditions and upregulation during stationary but not under hypoxia conditions was observed. In collaboration with Institut Pasteur (partner 6), the OPERA microscopy system was used to measure IS6110 promotor activity of different IS localisations. Using GFP as reporter gene, researchers have seen the strong promotor activity of the IS6110 located in the GC1237 strain upstream Rv2179c gene, confirming the data obtained by different techniques. The IS6110 upstream phoP gene was studied and a higher GFP intensity was measured in the cells infected with the construction including this IS6110. Researchers have studied the other 8 representative strains that present a variable pattern of MIRUS and RFLP. By IS6110 RFLP they observed that these strains contain 10 to 21 copies. They compared all locations from the nine strains and there are three common points including the characteristic in the DR region and between dnaA and dnaN region and two other insertions were observed in eight from the nine strains. In the common location between dnaA and dnaN genes, IS6110 doesn't increase the expression of the downstream dnaN gene.
WP3 - Differential host cell gene expression modifications upon infection
The main objectives were to:
- identify gene expression changes after infection with different mycobacterial strains using DNA chip technologies;
- investigate the host-microbe crosstalk on the single gene level as a function of microbial diversity;
- identify distinct functional roles for genes of each unit in host cell responses;
- identify transcription factors playing an important role in host responses and their potential target genes.
The transcription factor binding sites (TFBS) assays performed in period 1 pointed to the immunity- and development-related POU family TFs as putative regulators of genes differentially expressed in early phases of Mtb infection in THP-1 cells. It was shown in period 2 that THP-1 transcriptomic responses are time-specific, regardless of sub-lineages of Beijing family strains. These conserved host responses are dramatically modulated between 4 h and 18 h. Genes exhibiting consistent changes within the strains and differential expression between 4 h and 18 h were selected through LIMMA-based methodology. Five genes including CXCL10, CXCL11, TNFSF10, IL15 and CCL8 were validated for consistent expression by qRT-PCR approach. TFBS enrichment analysis indicates that STATs, IFR-1, IRF-7 and Oct-1 are putative regulators responsible for transcriptional immune responses of THP-1 cells to Mtb infection. The overall transcriptome profiles between BMDM cells infected by 10 different Mtb strains were similar at each time point, irrespective of the strain phylogeny. Due to the highly similar/conserved transcriptional responses induced by H37Rv and the investigated members of the W-Beijing family a selection of differentially regulated genes was not feasible.
Thus, validation by RT-PCR of potential genes of interest was not performed. During the second period of the project, WP3 aimed at deciphering the transcriptional signature of macrophages upon infection with representative W-Beijing Mtb strains. The goal was to perform clustering of genes and identify host cell gene expression modifications induced by Mtb genotypes. BMDMs were infected with M. tuberculosis (Mtb) H37Rv as laboratory control strain and nine different Mtb W-Beijing genotypes. Due to BSL3 capacity limitations, experiments were carried out in three consecutive series, each series comprising the H37Rv control strain and three different Beijing genotypes. Two technical replicates were generated by parallel sampling and performed two complete independent biological replicates at different days. Sample preparation for total RNA isolation took place at three different time points post infection (p.i.). In total 190 different samples were hybridised and analysed. For initial data analysis, all 41 174 microarray features of the whole genome microarray by divisive clustering were compared. The heat map of the differential expression shows that a similar pattern of expression was induced. In general it can be speculated that at the measured time points no significant and profound differences were identified between H37Rv and the investigated W-Beijing strains.
Due to the highly similar/conserved transcriptional responses induced by H37Rv and the investigated members of the W-Beijing family, a selection of differentially regulated genes was not feasible. Validation by RT-PCR of potential genes of interest was not performed. A follow-up investigation was initiated by partner 5 to explore differential transcriptional activity at early time-points between the lab strain in comparison with the Beijing isolates. Increasing sample size might provide significant power to the analysis. partner 5 performed the infection and provided the cell RNA to partner 8 for microarray assay. Differentiated THP-1 cells were infected with 12 representative M. tuberculosis clinical isolate from different sub-lineages of W-Beijing family Mtb using multiplicity of infection (MOI) 5 for 4 hours. The cell RNAs and supernatants were collected at 0 h, 4 h, 18 h and 48 h. RNAs were extracted and sent to partner 8 to perform global transcriptional analysis using microarray techniques. Host transcriptomic responses performed by partner 8 were shown to be time-specific, regardless of sub-lineages of the pathogen. These host-responsive genes are dramatically modulated between 4 h and 18 h. The robustness of host responses might be attributed to the involvement of response-relevant transcriptional factors and these regulators could be predicted from the observed transcriptomic changes. Genes differentially expressed between 4 h and 18 h may represent the common signatures of Mtb-infected THP-1 cells. A LIMMA-based methodology was applied to select genes exhibiting the consistent changes within the strains and differential expression between 4 h and 18 h. As validated in five randomly selected Mtb-infected samples by qRT-PCR approach, the CXCL10, CXCL11, TNFSF10, IL15 and CCL8 genes displayed consistent upregulation from 4 h to 18 h.
Differentially expressed genes identified can be categorised into two groups: Cluster 1 for induction at 18 h or 48 h compared with at 4 h, Cluster 2 for repression. Most of differentially expressed genes (92 %, 367 / 399) were induced. When enrichment analysis was applied to examine whether they shared functional or regulatory features, only biological features associated with genes in Cluster 1 were observed. No enrichment of biological relevance was observed in Cluster 2, which was thus excluded from further consideration in this study. The most significant gene ontology (GO) terms are immunity-related responses. Consistent with this, the most significant TFBS, represented in the form of position weight matrixes [PWMs]) are immunity-related transcription factors or putative immunity-related regulators, including IFN-stimulated response element (ISRE), IRF-1, IRF-7 and Oct-1. Through extensive literature mining it was found that each of the predicted regulators was biologically relevant. Transcription factors of STATs, IFR-1, IRF-7 and Oct-1 are collectively putative regulators responsible for transcriptional immune responses of THP-1 cells to Mtb infection.
WP4 - Screening of a genome-covering M. tuberculosis W-Beijing mutant library
The main objectives were to:
- infect murine macrophages with a 11 000-member genome-covering STM library generated by partner 12 (IPAS) in the W-Beijing strain GC1237;
- identify important mycobacterial virulence genes involved in host cell parasitism.
During the first 18 months of the project, researchers screened for the 10 mutants over a 11 000 Mycobacterium tuberculosis mutant library that traffic most frequently into acidified compartments early after phagocytosis, suggesting that they had lost their ability to arrest phagosomal maturation. Molecular analysis of these mutants revealed mainly disruptions in genes involved in cell envelope biogenesis (fadD28), the ESX-1 secretion system (espL/Rv3880), molybdopterin biosynthesis (moaC1 and moaD1), LppM and periplasmic phosphate-binding pstS3 lipoproteins, as well as in genes from novel locus, Rv1503c-Rv1506c and Rv2295c. All genes were present in the panel of M. tuberculosis Beijing strains sequenced in WP2. A number of non-synonymous (nsSNP) mutations were identified within the open reading frame of all genes. fadD28 has A to G nsSNP at position 1306 for four W/Beijing strains. A mutation in lppM-encoded protein was identified in M. tuberculosis Beijing GC1237. During the second period of the project, genotype / phenotype correlations were made for the Mtb virulence genes identified in WP4. The residual virulence of the mutants was assessed in vitro in cultured macrophages, and in vivo in the mouse model of Mtb infection. The ability of the different Beijing strains to induce cytokines responses in cultured human phagocytes was assessed, and researchers could identify specific signatures of the Beijing family in terms of cytokine and chemokine secretion and host cell response to infection. Novel TFs identified in WP03 are currently studied in future projects.
WP 5 - Understanding genotype / phenotype relationships between M. tuberculosis genetic diversity, differential virulence and immune responses in vitro and vivo
The main objectives were to identify:
- key genotype-phenotype relationships between M. tuberculosis genotypes and subgenotypes, in particular among the W-Beijing family;
- host cell responses at the transcriptome level;
- ability to overcome phagosome maturation and to parasitise host macrophages;
- host immune response to infection in vitro and vivo and mycobacterial immuno-pathogenicity.
Bone marrow derived macrophages (BMDM) were infected with M. tuberculosis (Mtb) H37Rv and nine different MtbW-Beijing genotypes. Due to BSL3 capacity limitations, experiments were carried out in three consecutive series, each series comprising the H37Rv control strain and three different Beijing genotypes. Researchers generated two technical replicates by parallel sampling and carried out two complete independent biological replicates. Cell-free supernatants from MTB-infected BMDM were collected at 4, 18 and 48 h p.i. In separate experiments BMDM were primed with IFN prior MTB infection. Triplicates were performed and collected cell-free supernatants at a single time-point, namely 24 h p.i. Up to 32 targets were measured using the bead-based assays and 21 cytokines / chemokines were detected. Researchers could not identify a significantly different secretion pattern for most cytokines. For several (but not all) W-Beijing members a trend to induce more abundant release of IL-6 (e.g. HM903, Cam22) was determined. The measurements at 24 h p.i. did not recapitulate the previously mentioned observations. Classically activated BMDM responded in a divergent way in terms of release of several cytokines and chemokines.
CXCL-10 levels were not significantly different between the tested bacterial strains. A similar tendency was reported for TNF, while IL-6 seemed to be less induced by several W-Beijing members at 24 h p.i. The discrepancies between kinetics measurements and the 24 h measurement might be explained by the fact that BMDM present receptors for several of the quantified cytokines / chemokines and target-receptor binding results in reduced concentration of the target soluble mediator in the cell culture supernatant. Infection is a dynamic process and autocrine sensing of secreted cytokine might result in changes in density of the cell surface receptors and synthesis of decoy receptors, which may bind the mediator as well. Activation of the BMDM with IFN resulted in increased levels of several cytokines compared with resting BMDM. IFN was able to restore the secretion of cytokine (TNF) for some Mtb strains while this propensity was not observed for IL-6.
Nitric oxide (NO) levels were quantified using the Griess reagent. As expected, IFN priming resulted in release of NO in culture supernatants. Abundance of the antimicrobial molecule was independent of the mycobacterial strain used to infect the BMDM. To investigate whether the W-Beijing strains have a growth advantage in macrophages we infected resting and IFN primed BMDM with H37Rv and the Beijing strains and followed bacterial replication using 3H-Uracil incorporation. The radioisotope labelled base is integrated in bacterial RNA and thus upon replication and metabolic activity the retained radioactive levels increased. Researchers incubated the lab strain and the W-Beijing strains in macrophage culture media and added the radionuclide labelled nucleic base. They observed that H37Rv and several of the tested Beijing strains grew well, showing a daily replication (GC1237, HNH5, N4, 990172), while other strains were defective in multiplication using macrophage media as substrate. Overall, results showed a conserved pattern of response of the lab Mtb strains and the W-Beijing members tested.
Behaviour recorded for isolated strains in a particular assay could not be extended to the whole subfamily or family. Based on the tools and results, a significant difference between H37Rv and the clinical W-Beijing strains was not observed and cannot be stated. Apoptosis is a physiological way of cell death by which multicellular organisms control homeostasis, cell transformation and intracellular infection with important implication in the host immune response. This process is triggered during the course of infections with intracellular facultative pathogens including M. tuberculosis. There have been proposed differences between Beijing and non-Beijing strains in terms of apoptosis induction on host cells. Beijing strains have been shown to induce more necrosis and less apoptosis than non-Beijing strains. However, these studies are incomplete. Thus, researchers used the nine Beijing reference strains to elucidate if this feature is intrinsic to Beijing family. Using different cell death markers to discern between apoptosis and necrosis they found that both Beijing and non-Beijing strains clearly induced apoptosis on macrophages. No global differences between both groups were measured.
Intracellular cytokine expression of proinflamatory IL-6 was studied in mouse MH-S macrophages infected with the panel of nine Beijing strains and with non-Beijing strains. It was found that none of the nine Beijing strains studied induced IL-6 expression, while all the non-Beijing strains did it. The 10 M. tuberculosis GC1237 mutants identified in WP4 were further studied. BMDM were inoculated with the various mutants and their growth was compared to that of wild type and of respective complemented strains. BMDM were infected at different MOI ranging from 1 to 5 and intracellular bacteria were counted using colony-forming unit enumeration method at day 0, 3 and 5 post-infection. pstS3, lppM, moaD1Tn mutants were degraded within the macrophages, Rv1503c, Rv1506c, moaC1, Rv3880c Tn mutants survived intracellularly, in contrast Rv2295c, fadD28 and ppe54 Tn mutants were able to actively multiply within the host cells similarly to the wild type strain. The cytokines release by BMDM that had been inoculated with the mutants was profiled at 24 hours using the Raybiotech Cytokine 32-Array and compared with that of their corresponding complemented strains and wild type. As expected M. tuberculosis GC1237 induced an increased secretion of IL-6 and GSCF compared to non-infected macrophages. The Rv1506c Tn mutant was able to further augment the secretion of IL-6. No specific pattern of cytokines expression using this technique was found among the different mutants.
The phenotype of the mutants in the mouse model in vivo was studies. All mutants but not fadD28 and Rv3880c were inoculated to Balb/C mice via the intranasal route and compared to the wild type GC1237 strain and complemented strain. Groups of five animals were sacrificed at three weeks and six weeks and CFU were enumerated. All eight mutants were attenuated by displaying at least one log less CFU in the lungs compared to wild type control. The wild type phenotype could be restored for Rv1503c, Rv1506c, ppe54 and Rv2295 Tn mutants. Concerning lppM Tn mutant, our hypothesis for explaining the lack of phenotype restoration is that complementation was performed with plasmid encoding M. tuberculosis H37Rv LppM which has a mutation compared to that of GC1237 protein. With regards to the others, moaC1, moaD1, pstS3 complemented Tn mutants, there is an issue of bacterial growth fitness in vitro on agar plates. The immune response generated by these mutants was investigated. Splenic T-cells from infected mice were analysed for CD3, CD4, CD8, CD44, CD45RB, CD62L, CD11b, CD11c, F4-80 and CD80 population. No significant variation had been found compared to wild type.
These data showed that mutants that are impaired in phagosome maturation arrest in vitro have all growth defects in vivo. Partners 6 and 5 have used OPERA microscopy system to perform a comparative study of the trafficking of the different mutants in H37Rv (H37Rv phoP, H37Rv phoP pSO5k and H37Rv phoP pSO7k) and GC1237 (GC1237 phoP, GC1237phoP pSO5k and GC1237 phoP pSO7k) inside macrophages and to analyse the influence of IS6110 as a promoter of phop gene (construction pSO7). DAPI was used, lysotraker-green and CypHer5 dyes. As a negative control non-infected cells were used, and as a positive heat-killed mutants. After 2 h of infection, nearly 100 % of the cells were found infected and preliminary results show that the fraction of bacteria co-localising with Lysotrake-positive staining are less than 10 % in all the mutants and more than 60 % of killed-bacteria were found to co-localise with acidified compartments. No significant difference between H37Rv and the GC1237 constructions were observed.
WP 1: Management and coordination of the consortium
The main objectives were to:
- set up an effective management framework for the TB-VIR consortium;
- ensure the usual and contractual administrative tasks;
- manage safety issues, legal, ethical and intellectual commercial / property-related issues, and gender equality promotion;
- coordinate the dissemination of knowledge inside and outside the network.
The TB-VIR coordination and management WP took care of the usual and contractual administrative tasks. The project management team focused on monitoring complex aspects related to the network's specific properties, the organisation of access to the key deliverables beyond the end of the European Commission (EC) contract, maintenance of the information hub, as well as identifying solutions for maintaining the momentum, added value of cooperation and collections obtained by the TB-VIR network thanks to the EC support. The management team acted as the interface between TB-VIR partners and EC, was in regular contact with EC Scientific Officer, and relayed problems and questions to the EC. The team was involved in the organisation of a variety of meetings, promotional materials and the maintenance of the project's website.
Potential impact
a. Potential impact and exploitation of results
Impact on the development of novel TB control strategies
TB remains a major threat to mankind and cannot be conquered without an effective vaccination strategy. No completely effective vaccination strategy has yet been developed. M. tuberculosis is one of the most effective human pathogens. The global incidence of TB increases by 2 % annually. The vast majority of cases occur in developing countries, especially in adults between 15 and 45 years of age. This situation necessitates the development of new types of intervention and improvement of the follow-up of TB patients in these countries. This project has contributed to the development of new interventions. A provisional patent application entitled 'Method for producing a vaccine for the treatment and/or prevention of tuberculosis' has been filed by partners 1 CNRS and 6 IPK. These vaccine candidates are currently under preclinical studies and will help provide novel intervention tools to combat TB.
Impact on pathogen research and pathogen-related diseases
The proposed work has identified the genes differentially expressed or regulated in response to various clinical isolates of M. tuberculosis with an emphasis on genes involved in the immune response. M. tuberculosis genes required for macrophage parasitism have been identified. Participation of beneficiary 6 has been invaluable to identify mycobacterial genes involved in intracellular trafficking. WP4 as a whole has been highly complementary with the activity of the EC-funded project TB-MACS. Findings of this study have increased understanding of M. tuberculosis / host interactions and have been an invaluable reference for TB research. Datasets produced in this study have been a valuable reference for TB research worldwide and have been made publicly available via publications.
Impact through innovation
The proposed project was highly innovative because it made use of new developments in functional genomics technology to investigate M. tuberculosis pathogenesis. This project brought together technologies from the fields of microarray analysis, functional genomics and transcriptomics, cell biology, confocal microscopy and bioinformatics. The technologies used and the know-how obtained in this project will be used by researchers working on other pathogens. The application of high-throughput screens as a means of connecting genotypes and complex phenotypes will be of considerable interest to researchers in functional genomics and studies of infection. The use of SNP genotyping to analyse M. tuberculosis W-Beijing population structure provides the international community working on TB and other infectious diseases invaluable information for further studies on pathogen population structure and genotype-phenotype relationships.
Impact through training and networking
Training the next generation of scientist has been an important achievement in TB-VIR (see http://www.moleculartb.org/index.html for details). The TB-VIR project also allowed for promoting and strengthening the links and scientific collaborations between European and Asian laboratories.
b. Dissemination activities
The project contributed significantly to the development of our knowledge base concerning M. tuberculosis virulence and anti-mycobacterial immunity. These achievements confirmed and extended the leading role of the European laboratories in mycobacterial research and the development of novel intervention tools. In addition to scientific publications, principal investigators from the TB-VIR consortium presented their work at a large number of national and international meetings. Fruitful collaborations with Asian research partners were established. Also many of the European partners are continuing the collaborations that were initiated under TB-VIR. The TB-VIR project resulted into 10 scientific publications in high-impact journals, 2 articles currently under submission, the participation in 13 conferences and symposiums and to 2 symposiums. A strong methodology has been set up to ensure the dissemination of the projects results. After verification that all possible steps have been taken for patent submission the data have been made available outside TB-VIR through meetings and various media supports.
Tools and ways of dissemination
- Adapted communication tools: the TB-VIR public website, presentation brochure and poster.
- Workshops and conference presentations: The participants presented the project and related results to scientific and clinical conferences and workshops in Europe and outside Europe.
- Publications in peer-reviewed and/or open access journals.
- Outreach the general public: In addition to our public website, a press release was written for the kick off meeting and disseminated at national level to newspapers and to public relations offices of the participant's institutions, but also at European level through the EC' communication services.
Website of the project: http://www.tb-vir.org
Contacts:
Project coordinator: Dr Olivier Neyrolles
IPBS - CNRS UMR 5089
205 Route de Narbonne, F-31077 Toulouse, FRANCE
Phone: +33-561-175475
Fax: +33-561-175994
E-mail: olivier.neyrolles@ipbs.fr
- General manager: Dr Jerome Weinbach
Inserm-Transfert
Paris BioPark
7 Rue Watt, F-75013 Paris, FRANCE
Phone: +33-155-030139
Mobile: +33-676-630550
Fax: + 33-155-030160
E-mail: jerome.weinbach@inserm-transfert.fr