1 Departamento de Microbiologia, Imunologia e Parasitologia, Universidade Federal de São Paulo Escola Paulista de Medicina (UNIFESP-EPM), Rua Botucatu, 862 3° andar, 04023-062, São Paulo, Brazil
2 Diagnostic Laboratory of Infectious Diseases and Perinatal Screening, National Institute of Public Health and the Environment (RIVM), PO Box 1, 3720 BA, The Netherlands
Correspondence
S. C. Leão
sylvia{at}ecb.epm.br
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ABSTRACT |
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INTRODUCTION |
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M. tuberculosis plc genes present significant similarity with Pseudomonas aeruginosa genes plcH and plcN, which encode haemolytic and non-haemolytic phospholipase C enzymes, respectively (Leão et al., 1995; Ostroff et al., 1990
). The
-haemolysis exhibited by Escherichia coli extracts overexpressing recombinant PlcA suggests enzymic activity of M. tuberculosis Plc proteins (Leão et al., 1995
). Moreover, sphingomyelinase and phospholipase C activities were detected in cell extracts from M. tuberculosis and in Mycobacterium smegmatis overexpressing recombinant PlcA or PlcB (Johansen et al., 1996
). The product of the plcA gene was detected in cell extracts from M. tuberculosis grown in vitro and inside macrophages (Matsui et al., 2000
).
Bacterial phospholipase C enzymes are recognized as important virulence factors of P. aeruginosa (Berka et al., 1981), Bacillus cereus (Gilmore et al., 1989
), Clostridium perfringens (Logan et al., 1991
) and Listeria monocytogenes (Vasquez-Boland et al., 1992
), playing roles in intracellular survival, cytolysis and cell-to-cell spread (Titball, 1993
). The precise role of phospholipase C in the pathogenicity of M. tuberculosis is unknown. Recent contributions to this subject were the demonstration that triple plcABC and quadruple plcABCD M. tuberculosis mutants were attenuated in the late phase of mouse infection (Raynaud et al., 2002
), and the observation that a plc
ABC
tlyA M. tuberculosis mutant, lacking plcABC and the haemolysin gene, is more virulent in mice than strain H37Rv, resulting in a higher bacterial load in organs and diminished survival (Smith et al., 2002
). These results are not necessarily contradictory, since the strains and procedures used in both plc mutant studies were different. Nevertheless, both studies suggested participation of phospholipase C genes in the virulence of M. tuberculosis.
Genetic variations in plcABC and in plcD were observed within the M. tuberculosis complex. M. tuberculosis complex subspecies include M. tuberculosis, Mycobacterium africanum, M. bovis (along with the attenuated M. bovis BCG) and Mycobacterium microti. Recently proposed subspecies include Mycobacterium pinnipedii, the seal bacillus (Cousins et al., 2003), Mycobacterium tuberculosis subsp. Canettii (M. canettii) (Pfyffer et al., 1998
) and M. bovis subsp. caprae (Aranaz et al., 1999
; Niemann et al., 2002
). Comparative genomics of the complete DNA sequence of M. tuberculosis H37Rv and M. bovis BCG disclosed a total of 16 M. bovis BCG-specific deletions, or regions of difference (RD) (Mahairas et al., 1996
; Brosch et al., 1998
; Behr et al., 1999
; Gordon et al., 1999
). Hybridization experiments on a DNA microarray (Behr et al., 1999
) and using ordered bacterial artificial chromosome (BAC) libraries (Gordon et al., 1999
) revealed that an 8·9 kb fragment containing plcABC genes, RD5 deletion (Gordon et al., 1999
) or RD7 deletion (Behr et al., 1999
) is absent from M. bovis BCG relative to M. tuberculosis H37Rv. This region is also absent from virulent M. bovis (Behr et al., 1999
; Gordon et al., 1999
).
Primers designed to amplify internal RD5 portions were used in PCRs to evaluate the occurrence of RD5 deletion in M. tuberculosis clinical isolates and in other members of the complex. Primers PT1/PT2 were used to amplify a 396 bp region of mtp40, a fragment situated at the 3' end of plcA. This product was amplified from the reference strains M. tuberculosis H37Rv (TMC102) and M. tuberculosis H37Ra (ATCC 25177), but not from M. bovis (TMC410), M. bovis BCG Pasteur (TMC1011), M. microti (NCTC 8710) or M. africanum (ATCC 25240) (Del Portillo et al., 1991). The mtp40 product was amplified from 54·6 % of 105 M. africanum isolates (Viana-Niero et al., 2001
), and also from human, pig, llama and ferret M. microti isolates, but not from vole isolates or the hyrax strain (van Soolingen et al., 1998
). This product was detected in mycobacteria isolates from seals (Liébana et al., 1996
) and in M. canettii (Brosch et al., 2002
), but not in M. bovis subsp. caprae (Niemann et al., 2002
). Parsons et al. (2002)
studied 88 members of the M. tuberculosis complex, using a different pair of primers to amplify a 152 bp product of plcA. They concluded that RD5 is present in most, but not all, isolates of M. tuberculosis, M. africanum and M. microti, and consistently absent from M. bovis and M. bovis BCG isolates.
IS6110 is a repetitive element frequently used in the epidemiology of tuberculosis. Transposition of this element generates genome plasticity of members of the tubercle bacilli (Fang et al., 1999; Warren et al., 2000
). Genetic variations caused by IS6110 insertions at different points in plcABC and plcD were observed in M. tuberculosis clinical isolates (Vera-Cabrera et al., 1997
, 2001
; Warren et al., 2000
), indicating that these domains represent preferential regions for integration (Warren et al., 2000
). Considering the potential role that the plc genes may have in the virulence of the tubercle bacillus, the study of genetic variations affecting the four genes in isolates cultured from patients may provide insights into the significance of phospholipase C enzymes for the pathogenicity of the bacillus. Genetic polymorphisms affecting both plcABC and plcD loci in 25 representative M. tuberculosis clinical isolates, three M. africanum and four M. canettii strains are described here.
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METHODS |
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Cultures were grown on LöwensteinJensen (LJ) solid medium or in Middlebrook 7H9 (Difco) liquid medium supplemented with albumin, glucose and catalase (Difco) and kept frozen at 70 °C in aliquots of 7H9-ADC with 15 % (v/v) glycerol. DNA was prepared as described by van Embden et al. (1993).
PCR and sequencing.
For primer design, sequences for plcABC from M. tuberculosis H37Rv and plcD from M. tuberculosis CDC1551 were obtained from GenBank (http://www.ncbi.nlm.nih.gov/). Primers were designed using OLIGO (version 5.0; National Biosciences) and are depicted in Table 2. Each of the primer pairs AD/AR, BD/BR, CD/CR and DD/DR amplified an individual plc gene and amplification specificity was evaluated by restriction analysis of the amplified fragments (data not shown). Internal primers AD2/AR2, BD2/BR2, CD2/CR2 and DD3/DR3, PT1/PT2 primers specific for the mtp40 sequence, and IS1/IS2 primers from IS6110 and PPE1 from Rv2356c (H37Rv) were used to address specific sequencing issues (Table 2
). PCR amplicons were sequenced in an automated ABI Prism 377 sequencer (Perkin-Elmer) using the BigDye Terminator Cycle Sequencing Ready Reaction kit (Applied Biosystems). Sequences were compared using BLAST (http://www.ncbi.nlm.nih.gov/blast).
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Cloning and sequencing of a plcC-specific PstI restriction fragment.
DNA from isolate 97-1177 was digested using PstI at 37 °C for 2 h, and subjected to electrophoresis in 0·8 % (w/v) agarose, along with the internal size marker. DNA was blotted onto a nylon membrane and hybridized with probes from plcC, IS6110 and the internal size marker, as described previously. The size of the fragment hybridizing with both the plcC and the IS6110 probe was calculated using GELCOMPAR II. Agarose gel slices around the selected band of PstI restriction fragment were excised from a second gel, purified using the Gel Extraction Kit (Qiagen) and ligated into PstI-digested dephosphorylated (CIAP; Invitrogen) pBluescript SK+ (Stratagene), using T4 DNA ligase (Invitrogen) at 16 °C, overnight. Ligated DNA was transformed into electrocompetent E. coli DH5 cells, which were plated onto LB-agar plates containing 100 µg ampicillin ml1, 0·25 mM IPTG and 1 mM X-Gal. Colony lifts were carried out on Whatman 541 filters and filters were hybridized with the plcC probe, labelled with [
-32P]dCTP with Ready-To-Go DNA Labelling Beads (Amersham), according to the protocol described by Maas (1983)
. Positive colonies visualized by exposure to autoradiographic film were confirmed by PCR with primers CD2/CR2 and by PstI digestion of the plasmid DNA. The recombinant plasmid, isolated using the Qiaprep Spin miniprep kit (Qiagen), was sequenced using M13 primers, as described previously.
RNA purification and RT-PCR.
Two-week-old cultures in Middlebrook 7H9 ADC medium were used for RNA isolation using FastRNA kit-BLUE (QBiogene) and a FastPrep Instrument (QBiogene), according to the manufacturer's instructions. DNA was removed by DNase I (Invitrogen). RT-PCR was performed with 1 µg RNA using the Access RT-PCR System (Promega) and internal primers specific for each plc gene. Amplicons were sequenced. Control of RT-PCR was performed using primers from atpB, a gene encoding a key component of the proton channel, which is transcribed during axenic growth and after infection of human macrophages (Graham & Clark-Curtiss, 1999).
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RESULTS |
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Instead of the normal mtp40 product of 396 bp, two isolates, 97-858 and 97-2433, produced amplicons of approximately 2 kb in size with primers PT1/PT2. plcC products larger than expected, of approximately 3 kb, were generated from isolates 97-742, 97-818 and 97-1505. Isolates 97-432 and 97-1389 produced plcD fragments of approximately 3 kb in size. In all cases, re-amplification of these amplicons using primers IS1/IS2 generated products of 123 bp, confirming the insertion of IS6110 elements in the mtp40 region of plcA, and in plcC and plcD, respectively.
To characterize the polymorphisms detected by PCR in the plcABC and plcD genomic regions of these selected isolates, plc-based fingerprints were obtained by the use of restriction endonuclease PvuII and specific DNA probes, homologous to the sequences of each of the four plc genes.
Southern hybridization and sequence analysis confirmed insertions and deletions
All isolates were subjected to Southern hybridization analysis. Polymorphisms in plc genes detected by PCR and hybridization were analysed by sequencing of selected amplicons.
Fig. 1 shows the physical map and PvuII restriction sites of plcABC obtained from analysis of M. tuberculosis H37Rv cosmid MTCY98 (GenBank accession no. Z83860) and plcD from M. tuberculosis CDC1551 cosmid MT1799 (GenBank accession no. AE007040). Fragments A, B, C and D were generated by PCR using primers AD/AR, BD/BR, CD/CR and DD/DR, respectively, and were used as probes in Southern blot hybridization (Fig. 1
). Information about PvuII fragment sizes was used to identify specific bands hybridizing with each probe. Empirical results showed that the size of the band located at the 5' region of plcA, calculated using GELCOMPAR II, was 2983 bp instead of 4657 bp, the expected size according to M. tuberculosis H37Rv genome data (Fig. 2
).
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In isolates 97-858 and 97-2433, Southern hybridization using probe A was consistent with the presence of a copy of IS6110 in the mtp40 region of plcA (Fig. 2). Sequence analysis of the amplicon from isolate 97-858 revealed the site of insertion at position 22 319, which is related to the sequence of cosmid MCTY98 (GenBank accession no. Z83860), and a direct repeat of three nucleotides TGT : TGT at the ends of the insertion element (Table 1
and Fig. 3
a). The exact insertion site in isolate 97-2433 was not determined.
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Six isolates, 97-1289, 97-1177, 97-1200, 97-803, 97-488 and 97-1389, lacked plcA- and plcB-hybridizing bands. Southern hybridization with probe C disclosed shifts in the 2156 bp band. Amplicons of approximately 1 kb in size were produced using primers IS2/CR2. Sequence analysis of these amplicons revealed insertion of IS6110 elements in plcC at position 19 849 in four isolates, position 19 500 in one isolate and position 19 916 in one isolate (Table 1 and Fig. 3d, e, f
).
In four isolates, 97-66, 97-279, 97-464 and 97-1438, Southern hybridizations with probes A, B and C revealed a single plcC-hybridizing band of approximately 1·3 kb in size. Generation of a 450 bp product using primers CD2/CR2 and lack of hybridization of the plcC probe with the 574 bp restriction fragment confirmed that these isolates retain only the central part of plcC.
In isolates 97-432 and 97-1389, amplification using primers DD/DR resulted in a fragment of 3·0 kb in size. Hybridization using probe D was suggestive of the presence of an intervening sequence in plcD (Fig. 2). Sequence analysis of the amplicon from isolate 97-432 revealed the insertion of a copy of IS6110, with the right imperfect repeat inserted at position 2611 and the left repeat at position 2698 (relative to the sequence of cosmid MT1799; GenBank accession no. AE007040), and deletion of 87 bp from plcD. No direct repeats were detected at the ends of the IS element (Table 1
and Fig. 4
a).
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Polymorphisms in plc genes were characterized in a group of genetically related isolates
An interesting polymorphism in the plcABC locus was detected in a group of six genetically related isolates, 97-818, 97-1505, 97-803, 97-1289, 97-1177 and 97-1200. Analysis of the RFLP patterns generated upon hybridization with a probe complementary to the IS6110 element revealed similarities greater than 70 % in the IS6110-RFLP profiles (data not shown). All six isolates carried a copy of IS6110 in plcC at position 19 849. The orientation of the IS element was the same in all isolates. Four isolates, 97-803, 97-1289, 97-1177 and 97-1200, were mtp40-PCR-negative and presented deletions of plcA, plcB and part of plcC. The remaining two isolates retained the genes plcA and plcB. Polymorphism in the plcD genomic region was shared by all six isolates (Fig. 4b).
To understand the mechanism involved in the deletion of plc genes in this group of isolates, the sequences flanking the ends of the IS insertion were determined in isolate 97-1177. DNA was digested using PstI and hybridized in Southern blots, using probes complementary to plcC and IS6110 (data not shown). A 4·0 kb PstI DNA fragment, which hybridized with both probes, was cloned and sequenced. The right imperfect repeat of IS6110 was inserted in plcC at position 19 849, and the left repeat was inserted at position 28 509 from MTCY98 (Table 1 and Fig. 3b
). Sequence analysis confirmed the deletion of a segment of 8·66 kb in size, containing the 5' region of plcC and the complete genes plcB, plcA, PPE38 and PPE39, and its substitution by a copy of the IS6110 element. No direct repeats were detected at the ends of the IS element, suggesting that deletion occurred by homologous recombination between two copies of IS6110. Identical polymorphism was observed in the other three isolates and was corroborated by sequence analysis of a product of 2·0 kb, generated by amplification with primers PPE1 and CR2.
Isolates 97-818 and 97-1505, which carry the IS element in plcC without subsequent gene deletion, presented a second copy of IS6110 at position 28 509. Sequence analysis of amplicons obtained by PCR using primers PPE and IS1 confirmed that the second IS element, located between genes PPE39 and PPE40, has the same orientation of the IS6110 copy inserted in plcC (Fig. 3a). These results, in conjunction, represent strong evidence that the homologous recombination between these two IS6110 elements resulted in deletion of the chromosomal domain located between them.
Analysis of transcription of plc genes
We also studied possible disturbances caused by insertions of IS6110 elements in the transcription of plc genes in selected isolates. Disruption of plcA or plcC by an IS6110 element did not impair transcription of adjacent plc genes in isolates 97-858 and 97-818, respectively (Fig. 5). Transcription of plcD was confirmed by RT-PCR in isolates with an intact plcD gene, such as M. bovis BCG Pasteur and isolate 97-279 (Fig. 5
). Products of plc genes were not detected by RT-PCR in isolates 97-1289, 97-1177, 97-1200, 97-803 and 97-1389 (not shown).
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DISCUSSION |
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To examine polymorphisms in plc genes in members of the M. tuberculosis complex that cause disease in humans, we analysed 25 selected M. tuberculosis clinical isolates and well-characterized isolates from M. africanum and M. canettii. This analysis revealed the presence of insertions inside the coding regions of plcA, plcC and plcD in M. tuberculosis isolates. No polymorphisms in the plcABC region were detected in M. africanum and M. canettii, but the small number of isolates analysed so far does not allow a definite conclusion. The finding of partial plcD deletion in one M. africanum isolate suggests that this region is also prone to polymorphisms in this subspecies.
The original report on IS6110 distribution in the genome of M. tuberculosis H37Rv indicated that insertions occurred preferentially in non-coding regions (Philipp et al., 1996). A recent analysis of data collated from various sources demonstrated that 57 of the 95 analysed insertions (60 %) occurred within coding regions (Sampson et al., 2001
). The impact of these insertions on the phenotype is unknown, as most of the disrupted genes are multicopy genes present in the genome. Disruption of individual members in a family of genes may have limited impact on phenotype, depending on the activity of the protein encoded.
Insertions of IS6110 elements were detected in plcA and plcC. RT-PCR analysis provided evidence that disruption of either of these genes does not have a significant polar effect on transcription from neighbouring plc genes. Raynaud et al. (2002) obtained similar results using mutants that harboured transposon insertions in plcA, plcB or plcC.
In this work, discrete IS6110 insertions in plcC at positions 19 849 and 19 668 were detected. Insertion events at positions 19 589, 19 645 and 19 848, located in plcC, were observed in a previous study including 11 isolates (Vera-Cabrera et al., 2001). These findings indicate that this domain is a preferential integration region, defined as a chromosomal domain of <500 bp, where several points of IS6110 insertion have been identified in different clinical isolates (Warren et al., 2000
).
A preferential insertion site was also detected in plcD. In 10 isolates, insertion of a copy of IS6110 was detected at position 2611, relative to M. tuberculosis CDC1551 cosmid MT1799 (GenBank accession no. AE007040). Insertions at position 2611 were reported by other authors in clones ISL540.9 and ISL0480.11 (GenBank accession nos AF126473 and AF077945, respectively) (Sampson et al., 1999), confirming that this site represents a hot spot for integration. Ho et al. (2000)
studied 22 non-related clinical isolates and found 18 discrete IS6110 insertion sites in plcD and adjacent genes. Lari et al. (2001)
detected an IS6110 insertion in plcD in one isolate and deletions of the entire RvD2 region in 15 out of 45 clinical M. tuberculosis isolates.
Detailed analysis of six genetically related isolates disclosed a genetic mechanism of deletion of a fragment located between two IS6110 elements. Deletion was accompanied by loss of the characteristic three- or four-nucleotide direct repeat flanking IS6110 elements. The observed deletion is most likely explained by the homologous recombination mechanism described by Fang et al. (1999), and this explanation agrees with findings of other authors (Ho et al., 2000
).
Fang et al. (1999) reported deletions in a preferential locus for IS6110 insertion, named ipl, in clinical M. tuberculosis isolates, and attributed these deletions to homologous recombination between two IS6110 elements. This hypothesis was based on the assumption that a recent related ancestor had two IS6110 insertions, with the same orientation, in this locus. Homologous recombination of these two IS6110 sequences would have led to deletion of the DNA segment between them. To our knowledge, in this study, for the first time this hypothesis was substantiated by the finding of clinical isolates that are genetically related as determined by IS6110 typing and that could represent evolutionary intermediates.
According to Sampson et al. (2003), preferential integration of IS6110 elements apparently triggers IS6110-mediated deletion events, as observed in the DR region and in a 20 kb hypervariable region (Ho et al., 2000
). Admitting that plcC and PPE genes are preferential insertion regions (Sampson et al., 1999
; Warren et al., 2000
), it could be speculated that recombination between two insertion sequences, one in a plc gene and the other in the vicinity of a neighbouring PPE gene, may have resulted in additional IS6110-mediated deletion events in plc genes, identified in this work.
Proximity and relative orientation of IS6110 elements are expected to influence recombination events within preferential integration regions, but other as-yet-unknown factors, such as IS6110 transposition activity, may also play a role.
Analysis of polymorphisms in the plcDcutinase region of H37 strains provides some insights. The genomes of M. tuberculosis H37Rv, M. tuberculosis H37Ra and M. tuberculosis CDC1551 contain a copy of IS6110 in front of the cutinase gene, in the same position and with the same orientation (Fig. 4). In H37Ra, a second copy of IS6110 is present in plcD. In M. tuberculosis H37Rv, a 7·9 kb fragment comprising the segment between position 2856 in plcD and the cutinase gene was deleted, leaving one copy of IS6110. The absence of flanking direct repeat elements strongly suggests that homologous recombination between two copies of IS6110 caused the deletion event (fragment RvD2). Therefore, the presence of two insertion elements, one in plcD and the other in the cutinase gene, triggered deletion in H37Rv but not in H37Ra, suggesting that IS6110 activity is in some way more pronounced in Rv than in Ra. Differences in IS6110 transposition activity between these two strains were confirmed by analysis of IS6110-RFLP patterns from 18 ATCC H37 variants (Bifani et al., 2000
). Nine distinct but similar patterns were detected in 15 H37Rv isolates and a single pattern was detected in three H37Ra isolates.
In five isolates, insertions and/or deletions occurred simultaneously in all four plc genes. Even with extensive deletions in plcABC genes, a truncated fragment of the plcC gene was still detected by Southern blot hybridization. Transcription of this truncated plcC gene was not detected by RT-PCR. Since this is the first description of gene interruptions affecting expression of all members in the same gene family, implications on the fitness of these organisms are unknown at this time. Nevertheless, the fact that these strains were recovered from patients with active tuberculosis demonstrates that they retain the ability to cause disease.
Despite the mobile quality of the IS6110 element, its instability is not sufficiently high to support short-term variations in the plc genes. It is also very unlikely that the variability in the plc genes could be influenced by culture procedures. First of all, cultures are not frequently re-cultured after primary isolation. Strains with the same genotypes often have the same plc gene composition. In fact, the half-life of IS6110 RFLP is between 3 and 5 years (De Boer et al., 1999). This includes transpositions in all genomic regions. Alterations of the plc genes due to transposition of IS6110 would be much less frequent.
Insertions and deletions were identified in 84 % of the M. tuberculosis isolates studied and in one M. africanum isolate, with effects ranging from disruption of individual open reading frames to loss of complete genes. Insertions in nearby locations indicate that these genomic regions may be particularly susceptible to insertion events. These findings suggest that insertions of IS6110 in plc loci result in a biological advantage for the bacteria, which is maintained during subsequent strain diversification. Deletions may be subjected to positive selection when the bacteria no longer require the removed genes. The question whether interruptions of these genes by insertion or deletion may have important effects on the biological properties of clinical isolates still deserves an answer. Differences in the virulence and phospholipase C activity of these isolates are currently under evaluation.
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ACKNOWLEDGEMENTS |
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Received 19 September 2003;
revised 3 November 2003;
accepted 4 November 2003.
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