Characterization of the highly abundant polymorphic GC-rich-repetitive sequence (PGRS) present in Mycobacterium tuberculosis

The polymorphic GC-rich repetitive sequence (PGRS) found on the chromosome of Mycobacterium tuberculosis was characterized by means of mapping, cloning and sequencing. PGRS was present in at least 26 loci and consisted of many tandem repeats of the consensus sequence CGGCGGCAA. As the core of the consensus motif was the triplet CGG, or CRR (where R is a purine), it seems likely that PGRS arose by means of triplet expansion, accounting for its polymorphism. Several copies of PGRS were linked to a conserved open reading frame. PGRS was used as the target sequence for the polymerase chain reaction in an attempt to develop a new typing technique.     

Impact of Protein Domains on PE_PGRS30 Polar Localization in Mycobacteria

PE_PGRS proteins are unique to the Mycobacterium tuberculosis complex and a number of other pathogenic mycobacteria. PE_PGRS30, which is required for the full virulence of M. tuberculosis (Mtb), has three main domains, i.e. an N-terminal PE domain, repetitive PGRS domain and the unique C-terminal domain. To investigate the role of these domains, we expressed a GFP-tagged PE_PGRS30 protein and a series of its functional deletion mutants in different mycobacterial species (Mtb, Mycobacterium bovis BCG and Mycobacterium smegmatis) and analysed protein localization by confocal microscopy. We show that PE_PGRS30 localizes at the mycobacterial cell poles in Mtb and M. bovis BCG but not in M. smegmatis and that the PGRS domain of the protein strongly contributes to protein cellular localization in Mtb. Immunofluorescence studies further showed that the unique C-terminal domain of PE_PGRS30 is not available on the surface, except when the PGRS domain is missing. Immunoblot demonstrated that the PGRS domain is required to maintain the protein strongly associated with the non-soluble cellular fraction. These results suggest that the repetitive GGA-GGN repeats of the PGRS domain contain specific sequences that contribute to protein cellular localization and that polar localization might be a key step in the PE_PGRS30-dependent virulence mechanism.     

The PGRS domain is responsible for translocation of PE_PGRS30 to cell poles while the PE and the C-terminal domains localize it to the cell wall

PE_PGRS proteins localize in the mycobacterial cell wall and the cell wall localization of PE_PGRS33 has been shown to be attributed to its PE domain. In this study, we expressed deletion mutants of PE_PGRS30 in Mycobacterium smegmatis to characterize the role of its domains in protein localization. It was revealed that, apart from the PE domain, the C-terminal domain present in few PE_PGRS proteins carries individual cell wall localization signals. Proteinase K sensitivity assay showed that PE_PGRS30 is exposed on the mycobacterial surface through its PGRS domain. PGRS domain was also shown to be responsible for polar localization of PE_PGRS30.     

Mycobacterial PE, PPE and ESX clusters: novel insights into the secretion of these most unusual protein families

The human pathogen Mycobacterium tuberculosis harbours a large number of genes that encode proteins whose N-termini contain the characteristic motifs Pro–Glu (PE) or Pro–Pro–Glu (PPE). A subgroup of the PE proteins contains polymorphic GC-rich sequences (PGRS), while a subgroup of the PPE proteins contains major polymorphic tandem repeats (MPTR). The function of most of these proteins remains unknown. However, in this issue of Molecular Microbiology , Abdallah and colleagues show that PE_PGRS proteins from the model organism Mycobacterium marinum are secreted by components of the ESX-5 system that belongs to the recently defined type VII secretion systems. These observations, which now need to be addressed and confirmed in M. tuberculosis , open new perspectives on the function of these highly abundant proteins.     

Execution of macrophage apoptosis by PE_PGRS33 of Mycobacterium tuberculosis is mediated by Toll-like receptor 2-dependent release of tumor necrosis factor-alpha

Combating tuberculosis requires a detailed understanding of how mycobacterial effectors modulate the host immune response. The role of the multigene PE family of proteins unique to mycobacteria in the pathogenesis of tuberculosis is still poorly understood, although certain PE_PGRS genes have been linked to virulence. Tumor necrosis factor-alpha (TNF-alpha) is essential for successfully combating tuberculosis. In this study we provide evidence that PE_PGRS33, a surface exposed protein, elicits TNF-alpha release from macrophages in a TLR2 (Toll-like receptor 2)-dependent manner. ASK1 (apoptosis signal-regulating kinase 1) is activated downstream of TLR2. ASK1 activates the MAPKs p38 and JNK. PE_PGRS33-induced signaling leads to enhanced expression of TNF-alpha and TNF receptor I (TNFRI) genes. Mycobacterium smegmatis expressing PE_ PGRS33 elicits the same effects as purified PE_PGRS33. TNF-alpha release occurs even when internalization of the bacteria is blocked by cytochalasin D, suggesting that interaction of PE_ PGRS33 with TLR2 is sufficient to trigger the effects described. Release of TNF-alpha plays the determining role in triggering apoptosis in macrophages challenged with PE_PGRS33. The death receptor-dependent signals are amplified through classical caspase 8-dependent mitochondrial release of cytochrome c, leading to the activation of caspases 9 and 3. An important aspect of our findings is that deletions within the PGRS domain (simulating those occurring in clinical strains) attenuate the TNF-alpha-inducing ability of PE_PGRS33. These results provide the first evidence that variations in the polymorphic repeats of the PGRS domain modulate the innate immune response.     

Molecular methods for Mycobacterium tuberculosis strain typing: a users guide

There are now a wide range of techniques available to type Mycobacterium tuberculosis, the problem is to choose the correct technique. For large scale epidemiological studies the portability and standardization of IS6110 restriction fragment length polymorphism (RFLP) means that this remains the gold standard technique. In the next few years the internationally standard mycobacterial interspersed repetitive unit (MIRU) may come to challenge this primacy. Low copy number stains remain a problem and these can be typed by either polymorphic Guanine cytosine-rich repetitive sequence (PGRS) or MIRU-variable numbers of tandem repeat (VNTR). To confirm whether strains are part of a true cluster PGRS remains the method of choice. For local outbreaks and investigations of laboratory cross contamination where speed is of greatest importance suspect strains should be initially investigated using a PCR-based method. The superior reproducibility and discrimination of MIRU-VNTR means that these methods should be favoured. If matches are found, then further confirmation of identity can be achieved using IS6110 RFLP or PGRS if the strains prove to have a low IS6110 copy number.     

IS902, an insertion element of the chronic-enteritis-causing Mycobacterium avium subsp. silvaticum.

An insertion sequence element of Mycobacterium avium subsp. silvaticum was isolated and its complete nucleotide sequence determined. IS902 is 1470 bp in size and is repeated 10-12 times per genome. An open reading frame of 1200 bp was identified, encoding a protein product of Mr 43932. This protein is highly similar to the predicted proteins of IS900 of Mycobacterium paratuberculosis, IS116 of Streptomyces clavuligerus and IS110 of Streptomyces coelicolor. IS902 lacks terminal inverted repeats and flanking direct repeats but displays insertion site specificity.     

Simple sequence repeats (microsatellites): mutational mechanisms and contributions to bacterial pathogenesis. A meeting review

This review summarises the presentations and discussions that took place during a European Science Foundation-funded workshop whose purpose was to gain current perspectives on the mutational mechanisms of simple sequence repeats and the contribution of localised hypermutation in such repeats to bacterial pathogenesis. In vitro biophysical and biochemical assays of mutational mechanisms were covered as well as genetic studies in various eukaryotic and prokaryotic organisms. Presentations on bacterial pathogenesis elaborated investigations of the use of repeats for typing of strains, epidemiological investigations of mutation rates and functions of loci whose expression is controlled by simple sequence repeats. This review tabulates current perspectives on the cis- and trans-acting factors for mutation of simple sequence repeats and the orientations of mononucleotide repeats in some bacterial species that utilise repeats for adaptation.     

Replication from one of the three origins of the plasmid R6K requires coupled expression of two plasmid-encoded proteins

The minimal beta-replicon of plasmid R6K contains an open reading frame for a 151-amino acid protein in addition to the seven 22-base pair direct nucleotide sequence repeats, the structural gene (pir) for the pi initiation protein, and the beta-origin sequence that have been shown to be required for replication activity. In this work, a site-specific mutation by linker insertion in this putative coding sequence, designated bis, resulted in a nonfunctional beta-replicon. The nonfunctional beta-replicon was complementable in trans and the protein coded by the bis sequence was detected in an immunoblot assay as a hybrid product from a bis-lac z fused gene. The bis gene is not required for a functional alpha or gamma origin replication origin of R6K. A site-specific mutation in the upstream pir gene was shown to lead to a loss of synthesis of the bis product and inactivation of the beta-replicon. Trans-complementation of this mutation for beta-replicon activity required the wild-type sequence of the pir gene joined to the intact bis sequence. These results indicate that the bis product is required for activity specifically of the beta-origin, and its synthesis is coupled in cis to the expression of pi protein from an unaltered pir gene.     

Mycobacterial PE_PGRS proteins contain calcium-binding motifs with parallel beta-roll folds

The PE_PGRS family of proteins unique to mycobacteria is demonstrated to contain multiple calcium-binding and glycine-rich sequence motifs GGXGXD/NXUX. This sequence repeat constitutes a calcium-binding parallel beta-roll or parallel beta-helix structure and is found in RTX toxins secreted by many Gram-negative bacteria. It is predicted that the highly homologous PE PGRS proteins containing multiple copies of the nona-peptide motif could fold into similar calcium-binding structures. The implication of the predicted calcium-binding property of PE PGRS proteins in the light of macrophage-pathogen interaction and pathogenesis is presented.     

Comparative genomics of metabolic pathways in Mycobacterium species: gene duplication, gene decay and lateral gene transfer

The genus Mycobacterium comprises significant pathogenic species that infect both humans and animals. One species within this genus, Mycobacterium tuberculosis, is the primary killer of humans resulting from bacterial infections. Five mycobacterial genomes belonging to four different species (M. tuberculosis, Mycobacterium bovis, Mycobacterium leprae and Mycobacterium avium ssp. paratuberculosis) have been sequenced to date and another 14 mycobacterial genomes are at various stages of completion. A comparative analysis of the gene products of key metabolic pathways revealed that the major differences among these species are in the gene products constituting the cell wall and the gene families encoding the acidic glycine-rich (PE/PPE/PGRS) proteins. Mycobacterium leprae has evolved by retaining a minimal gene set for most of the gene families, whereas M. avium ssp. paratuberculosis has acquired some of the virulence factors by lateral gene transfer.     

Development of a simple and low-cost real-time PCR method for the identification of commonly encountered mycobacteria in a high throughput laboratory

Aims:  To facilitate efficient identification of commonly encountered mycobacteria species ( Mycobacterium tuberculosis , Mycobacterium avium , Mycobacterium intracellulare , Mycobacterium fortuitum complex , Mycobacterium chelonae/abscessus , Mycobacterium kansasii , Mycobacterium gordonae ) in high throughput laboratories, a 16s rDNA sequence based real-time PCR assay was developed and evaluated.
Methods and Results:  Oligonucleotide primers and hybridization probes were designed based on sequence differences of the mycobacterial 16S rDNA gene. This assay was evaluated with 1649 suspected non-tuberculosis mycobacterial isolates. Apart from 3 out of 40  M. avium isolates that showed false signal with M. intracellulare specific probe, 100% specificity was obtained for all tested probes. Assay sensitivity varied from 88·9 to 100% depending on species. Average cost for obtaining a definite identification was only USD 1·1 with an average turn around time of less than 3 days.
Conclusions:  A rapid, simple and inexpensive real-time PCR assay was developed for the identification of common encountered mycobacteria in a high throughput laboratory setting.
Significance and Impact of the Study:  With this assay, more than 80% of the clinically isolated nontuberculous mycobacteria could be identified in a highly cost effective manner. This helped to save resources for other laboratory activities especially in high throughput mycobacterial laboratories.     

Mutation rates in the complex microsatellite MYCL1 and related simple repeats in cultured human cells

Microsatellite instability is a phenotype observed in tumors cells that have defects in DNA mismatch repair (MMR). Most markers used for detecting microsatellite instability are mono- and dinucleotide repeats, but one tetranucleotide repeat (MYCL1) has been reported to be useful for this purpose. The MYCL1 repeat is actually a complex repeat, made up of approximately 14 GAAA tetranucleotides plus various other GA-rich repeats. In order to determine the nature of the instability of the this sequence, we have used a frameshift-reversion assay in MMR-proficient and -deficient human cells to compare the mutation rates and the types of mutation of MYCL1 to those of the related simple repeats (GAAA)17, (GA)17, and (CA)17. We found that the complex repeat was the most stable of the repeats examined in cells deficient in MMR; the tetranucleotide was less stable, while the dinucleotides were the least stable. In MMR-proficient cells, the relative rates were reversed; the MYCL1 repeat was the least stable, the tetranucleotide was more stable, and the dinucleotides were the most stable. These results suggest that MYCL1 and the pure tetranucleotide have relatively low rates of errors during replication, but that the errors in these repeats are corrected less efficiently than those in the smaller repeats. Because of their high rate of instability in MMR-proficient cells, MYCL1 and other tetranucleotide repeats appear to lack specificity for detection of tumors with defective MMR.     

Evolutionary relationships among strains of Mycobacterium tuberculosis with few copies of IS6110

Molecular typing of Mycobacterium tuberculosis by using IS6110 shows low discrimination when there are fewer than five copies of the insertion sequence. Using a collection of such isolates from a study of the epidemiology of tuberculosis in London, we have shown a substantial degree of congruence between IS6110 patterns and both spoligotype and PGRS type. This indicates that the IS6110 types mainly represent distinct families of strains rather than arising through the convergent insertion of IS6110 into favored positions. This is supported by identification of the genomic sites of the insertion of IS6110 in these strains. The combined data enable identification of the putative evolutionary relationships of these strains, comprising three lineages broadly associated with patients born in South Asia (India and Pakistan), Africa, and Europe, respectively. These lineages appear to be quite distinct from M. tuberculosis isolates with multiple copies of IS6110.     

Functional dissection of the PE domain responsible for translocation of PE_PGRS33 across the mycobacterial cell wall

PE are peculiar exported mycobacterial proteins over-represented in pathogenic mycobacterial species. They are characterized by an N-terminal domain of about 110 amino acids (PE domain) which has been demonstrated to be responsible for their export and localization. In this paper, we characterize the PE domain of PE_PGRS33 (PE(Rv1818c)), one of the best characterized PE proteins. We constructed several mutated proteins in which portions of the PE domain were deleted or subjected to defined mutations. These proteins were expressed in different mycobacterial species and their localization was characterized. We confirmed that the PE domain is essential for PE_PGRS33 surface localization, and demonstrated that a PE domain lacking its first 30 amino acids loses its function. However, single amino acid substitutions in two regions extremely well conserved within the N-terminal domain of all PE proteins had some effect on the stability of PE_PGRS33, but not on its localization. Using Mycobacterium marinum we could show that the type VII secretion system ESX-5 is essential for PE_PGRS33 export. Moreover, in M. marinum, but not in Mycobacterium bovis BCG and in Mycobacterium tuberculosis, the PE domain of PE_PGRS33 is processed and secreted into the culture medium when expressed in the absence of the PGRS domain. Finally, using chimeric proteins in which different portions of the PE(Rv1818c) domain were fused to the N-terminus of the green fluorescent protein, we could hypothesize that the first 30 amino acids of the PE domain contain a sequence that allows protein translocation.     

Isolation and analysis of IS6120, a new insertion sequence from Mycobacterium smegmatis

Insertion sequence IS6120 from Mycobacterium smegmatis was identified by its ability to transpose into different sites in the lambda repressor gene, cl857, carried on an Escherichia coli/mycobacteria shuttle plasmid. IS6120 is a novel 1.5 kb insertion sequence, which has 24-bp imperfect terminal inverted repeats and generates 9-bp duplications of the target DNA following insertion. IS6120 is present in at least three copies in M. smegmatis but was not found in other species, including Mycobacterium tuberculosis. Nucleotide sequence analysis revealed that IS6120 contains two open reading frames, one of which encodes a putative transposase with similarities to those found in IS256 from Staphylococcus aureus, IST2 from Thiobacillus ferrooxidans, and ISRm3 from Rhizobium meliloti. The fact that IS6120 does not recognize a consensus target sequence for insertion and has no homologous sequences in the other strains studied makes IS6120 useful for transposon mutagenesis in mycobacteria.     

Modulation of C-to-T mutation by recombination-independent pairing of closely positioned DNA repeats

Repeat-induced point mutation is a genetic process that creates cytosine-to-thymine (C-to-T) transitions in duplicated genomic sequences in fungi. Repeat-induced point mutation detects duplications (irrespective of their origin, specific sequence, coding capacity, and genomic positions) by a recombination-independent mechanism that likely matches intact DNA double helices directly, without relying on the annealing of complementary single strands. In the fungus Neurospora crassa, closely positioned repeats can induce mutation of the adjoining nonrepetitive regions. This process is related to heterochromatin assembly and requires the cytosine methyltransferase DIM-2. Using DIM-2-dependent mutation as a readout of homologous pairing, we find that GC-rich repeats produce a much stronger response than AT-rich repeats, independently of their intrinsic propensity to become mutated. We also report that direct repeats trigger much stronger DIM-2-dependent mutation than inverted repeats. These results can be rationalized in the light of a recently proposed model of homologous DNA pairing, in which DNA double helices associate by forming sequence-specific quadruplex-based contacts with a concomitant release of supercoiling. A similar process featuring pairing-induced supercoiling may initiate epigenetic silencing of repetitive DNA in other organisms, including humans.     

Complete nucleotide sequence of pAL5000, a plasmid from Mycobacterium fortuitum

We have determined the complete nucleotide sequence of pAL5000, a plasmid from Mycobacterium fortuitum; the plasmid contains 4837 bp with 65% G + C. Five open reading frames (ORF1 to ORF5) have been identified. A number of sequences corresponding to palindromes, repeats, a helix-turn-helix motif, a signal sequence and repetitive amino acid motifs can be identified. This sequence should facilitate the construction of vectors based on pAL5000 for transfer and expression studies in mycobacteria.     

Sequence-dependent effect of interruptions on microsatellite mutation rate in mismatch repair-deficient human cells

Although microsatellite mutation rates generally increase with increasing length of the repeat tract, interruptions in a microsatellite may stabilize it. We have performed a direct analysis of the effect of microsatellite interruptions on mutation rate and spectrum in cultured mammalian cells. Two mononucleotide sequences (G₁₇ and A₁₇) and a dinucleotide [(CA)₁₇] were compared with interrupted repeats of the same size and with sequences of 8 repeat units. MMR-deficient (MMR⁻) cells were used for these studies to eliminate effects of this repair process. Mutation rates were determined by fluctuation analysis on cells containing a microsatellite sequence at the 5′ end of an antibiotic-resistance gene; the vector carrying this sequence was integrated in the genome of the cells. In general, interrupted sequences had lower mutation rates than perfect ones of the same size, but the magnitude of the difference was dependent upon the sequence of the interrupting base(s). Some interrupted repeats had mutation rates that were lower than those of perfect sequences of the same length but similar to those of half the length. This suggests that interrupting bases effectively divide microsatellites into smaller repeat runs with mutational characteristics different from those of the corresponding full-length microsatellite. We conclude that interruptions decrease microsatellite mutation rate and influence the spectrum of frameshift mutations. The sequence of the interrupting base(s) determines the magnitude of the effect on mutation rate.