AgResearch, Wallaceville Animal Research Centre, PO Box 40063, Upper Hutt, New Zealand
Correspondence
Desmond M. Collins
desmond.collins{at}agresearch.co.nz
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ABSTRACT |
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INTRODUCTION |
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Since the identification of the first genes involved in mycobacterial virulence (Collins et al., 1995; Wilson et al., 1995
), further development and improvement of molecular genetic techniques has enabled the discovery of an increasing number of such genes (de Mendonca-Lima et al., 2003
; Glickman & Jacobs, 2001
). While there is continuing discussion about what constitutes a true virulence gene (Barry, 2001
; Wassenaar & Gaastra, 2001
), it is undoubtedly helpful to know if inactivation of a gene affects the virulence of an organism in an animal model, as this potentially enables further study of the hostpathogen interactions which are affected directly or indirectly by that gene. In most cases, virulence of the parent and a mutant daughter strain has been determined in a single model of animal virulence, usually in mice or guinea pigs. This use of a single animal model reflects practicalities in terms of cost and time of using more than one animal host under biological containment for investigating a chronic disease, as well as an underlying assumption that the features of tuberculosis in different animal hosts are sufficiently similar that most virulence mechanisms will be the same in all hosts. Nevertheless, there is long-standing evidence that different species of the M. tuberculosis complex, as well as different strains within a species, have different pathogenicities for different animal hosts. For example, strains of M. tuberculosis are not as virulent as strains of M. bovis in rabbits but both species appear similarly virulent in guinea pigs (Dannenberg & Collins, 2001
); and South Indian strains of M. tuberculosis appear of similar virulence to other M. tuberculosis strains for humans but are less virulent in guinea pigs (Balasubramanian et al., 1992
). Even when a single animal host is used to determine the level of virulence, the result obtained depends on the particular indices that are measured. Growth rate in mice is often used to assess virulence of strains of the M. tuberculosis complex but this only indicates a subset of virulence mechanisms. Commonly used strains of M. tuberculosis and M. bovis grow at similar rates in mice and by that criterion would have similar virulence, but mice infected with M. bovis die much sooner that those infected with M. tuberculosis, so by that criterion M. bovis would be more virulent (North et al., 1999
). These different ways of assessing virulence have recently been emphasized by the production of a number of single-gene mutants of M. tuberculosis that have similar growth to their parents in animals but differ markedly when compared on the basis of animal survival (Kaushal et al., 2002
; Steyn et al., 2002
).
In earlier work (Collins et al., 1995), it was shown that M. bovis ATCC 35721 carries a point mutation in the principal sigma factor gene, sigA (designated rpoV in the earlier work), that causes attenuation of its virulence in guinea pigs when it is inoculated subcutaneously (subcutaneous guinea pig model). That work used an in vivo complementation approach in which ATCC 35721 was complemented with a wild-type sigA gene (to produce M. bovis WAg320) that restored virulence in this guinea pig model. Recently, it was found that the arginine to histidine change this mutation encoded at position 515 in SigA (SigA-R515H) reduced the interaction of this sigma factor with a secondary transcription factor WhiB3 (Steyn et al., 2002
). It was also shown that inactivation of whiB3 in M. tuberculosis H37Rv had no effect on its ability to grow and persist in mice infected intravenously or in a subcutaneous guinea pig model but mice infected with this mutant had significantly longer mean survival times than those infected with the parent H37Rv strain. In contrast, inactivation of whiB3 in M. bovis completely attenuated its growth in a subcutaneous guinea pig model. These results indicate a different role for some M. tuberculosis and M. bovis genes in pathogenesis generated in different animal models.
In New Zealand, it has been difficult to eradicate bovine tuberculosis from cattle in some parts of the country because of continual reinfection from tuberculous populations of the Australian brushtail possum (Trichosurus vulpecula), an animal introduced into New Zealand in the nineteenth century (de Lisle et al., 2001). In the course of tuberculosis studies on possums, we discovered that both M. bovis ATCC 35721 and M. bovis WAg320, its complement with a wild-type sigA gene, are attenuated when inoculated intratracheally into these animals. This result was in stark contrast to the situation in guinea pigs, where ATCC 35721 itself is attenuated but WAg320 has virulence approaching that of a wild-type strain. In this study, we describe this discovery, its use to identify an ABC transporter gene, phoT, and the virulence properties of phoT in different animal models of tuberculosis.
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METHODS |
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Knockout of phoT in M. bovis.
Knockout of the phoT gene from ATCC 35723 was performed in a similar way to that described previously (Wards et al., 2000). Briefly, a suicide plasmid was constructed by inserting a hygromycin resistance gene into the EcoRV site of phoT (Fig. 1
) in the 1973 bp insert from pUHA37. The phoT : : hygr fragment was transferred into pUHA9 to produce pUHA43, which was electroporated into ATCC 35723 using a high-efficiency electroporation technique (Wards & Collins, 1996
). Cells were plated onto medium containing hygromycin and resistant colonies were subcultured and their DNA was extracted (van Soolingen et al., 1991
). DNA from allelic-exchange mutants in which the phoT gene had been deleted was identified by restriction digestion and Southern blot hybridization and confirmed by PCR analysis using primers on each side of the phoT site into which the hygromycin resistance gene had been ligated.
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M. bovis culture in ciprofloxacin and low-phosphate medium.
For determination of the MIC of strains in ciprofloxacin, 10-fold serial dilutions of each strain were cultured in duplicate on supplemented Middlebrook 7H11 medium containing no ciprofloxacin and on the same medium containing doubling dilutions of 0·60·0375 µg ciprofloxacin ml-1. For culture in low-phosphate medium, approximately 3x106 c.f.u. of each strain in 50 µl Tween-albumin broth were inoculated into 5 ml Sauton medium containing serial dilutions of phosphate. The final concentration of phosphate for each culture was calculated taking into account the phosphate added in the 50 µl inoculum.
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RESULTS |
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Knockout of phoT in M. bovis
In order to determine the role of phoT in a virulent strain of M. bovis with a wild-type sigA genotype, a suicide plasmid approach was used to inactivate the phoT gene in M. bovis ATCC 35723 by allelic exchange. A Southern blot hybridization representing two successful knockout recombinants, a single homologous recombinant and three illegitimate recombinants is shown in Fig. 3(a). An agarose gel of PCR products from these knockouts in comparison to reference products is shown in Fig. 3(b)
. Compared to its moderately virulent parent, M. bovis ATCC 35723, the phoT knockout strain (WAg758) was found to have reduced virulence when assessed in the subcutaneous guinea pig model on the basis of spleen lesions (Table 2
) and confirmed by enumeration of c.f.u. in the spleen (Table 3
).
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WAg320, which contains sigA, was significantly more virulent than its parent, ATCC 35721, in both the subcutaneous and aerosol guinea pig models and in the lungs of mice but both strains were highly attenuated in possums. WAg320 was particularly virulent in the aerosol guinea pig model and the animals were sufficiently clinically affected after 29 days to be killed on ethical criteria. In contrast, WAg324, which contains phoT, was significantly more virulent than its parent in possums and in the subcutaneous guinea pig model but was significantly less virulent than its parent in the aerosol guinea pig model and of similar virulence to its parent in the mouse model. The phoT knockout WAg758 was significantly less virulent than its parent ATCC 35723 in all the animal models except mice, although even in mice the mean c.f.u. was 0·5 log lower.
In vitro growth of M. bovis strains in ciprofloxacin and low-phosphate medium
The growth of M. bovis strains in liquid medium containing various low concentrations of phosphate is given in Table 4. Growth was clearly associated with the presence of a wild-type sigA gene. In the absence of the wild-type version of sigA, phoT (WAg324) was unable to restore growth in low-phosphate medium. At the lowest phosphate concentration, strains with wild-type sigA and knockout of phoT (WAg320 and WAg758) did not grow, in contrast to ATCC 35723 with wild types of both sigA and phoT, which did grow. This indicates that in wild-type strains of M. bovis, phoT plays a role in acquiring phosphate when it is at very low concentrations. The MIC of ciprofloxacin for various M. bovis strains is also given in Table 4
. There was no difference between any of the strains except for WAg758, which had half the MIC of the other strains. This indicates that phoT may play a role in phosphate transport in ATCC 35723, as resistance to ciprofloxacin has been shown to be associated with phosphate transport in Mycobacterium smegmatis (Bhatt et al., 2000
).
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DISCUSSION |
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Work to this point had established that phoT was necessary to restore virulence to ATCC 35721 in the intratracheal possum model, that it also restored virulence in the subcutaneous guinea pig model and that ATCC 35721 has mutations in both its sigA and phoT genes. Clearly, ATCC 35721 is substantially different from a wild-type M. bovis strain and might even have further mutations perhaps of a compensatory nature, so it was desirable to confirm the virulence properties of phoT in a virulent M. bovis strain. In order to do this, the gene was inactivated by allelic exchange from the moderately virulent M. bovis strain, ATCC 35723, to produce WAg758. WAg758 was significantly less virulent than its parent in both the subcutaneous guinea pig and intratracheal possum models, establishing that phoT activity is necessary to maintain the virulence of ATCC 35723 in these models.
The ability of phoT to restore virulence to ATCC 35721 for both the subcutaneous guinea pig and intratracheal possum models contrasts with the ability of sigA to restore virulence to ATCC 35721 only for the subcutaneous guinea pig model. This raised the question of whether the difference between the models with respect to sigA was due to differences in the host response to infection of guinea pigs and possums or was related to the mode of administration. It was possible that sigA might be sufficient for restoring virulence to ATCC 35721 when the strain is inoculated subcutaneously but not when animals are infected via the lungs. To investigate this possibility and also the effect of the presence or absence of phoT, the same group of five strains was inoculated into two additional animal models: aerosol models of the mouse and guinea pig. An important result from this work was the finding that WAg320 (ATCC 35721 complemented with sigA) was significantly more virulent than ATCC 35721 in both the subcutaneous and aerosol guinea pig models as well as in the mouse model. The original finding that WAg320 is virulent in guinea pigs (Collins et al., 1995) therefore reflects a general host susceptibility of guinea pigs to this strain that is not dependent on route of administration. The corollary of this is that because WAg320 is not virulent when administered into the lungs of possums, there is a clear difference in susceptibility to this strain between possums and both guinea pigs and mice. At the molecular level, it has now been shown that the mutation in SigA-R515H in ATCC 35721 affects its binding to an accessory transcription factor, WhiB3, which is thought to regulate genes that influence the immune response of the host (Steyn et al., 2002
). If this is the case, then regulation of these genes by WhiB3 does not appear to be important for influencing the immune response of possums.
Inoculation of ATCC 35723 and its phoT knockout strain (WAg758) in four different models gave consistent results. In three of the models, WAg758 was significantly less virulent than its parent while in the mouse model the trend was in the same direction but the difference was not significant with the small number of animals used. Clearly, phoT is important for the virulence of M. bovis in a range of animals. ATCC 35721 recombinants complemented with phoT were also found to be virulent when inoculated in the subcutaneous guinea pig model irrespective of the presence of the wild-type allele of the principal sigma factor gene, sigA. In our earlier work in which sigA was identified in a similar in vivo complementation approach to that used here for phoT, we analysed the predominant virulence-restoring clone from the library which represented 80 % of the recombinant M. bovis isolates recovered from guinea pigs (Collins et al., 1995). In light of the present finding that phoT also restores virulence to ATCC 35721 in the subcutaneous guinea pig model, we would now conclude that some of the remaining 20 % of virulence-restoring recombinants from the first study probably contained cosmids incorporating the phoT locus. The most likely explanation for their unequal representation in the earlier study is the under-representation of cosmids containing phoT in the library used in that study. Another possible explanation is that phoT restores ATCC 35721 (WAg324) to a slightly lower degree of virulence than does sigA (WAg320) and is outcompeted by WAg320 when both are inoculated together. However, far greater numbers of animals per group would be needed than those used throughout this study in order to determine if there is a significant degree of difference in virulence between these two strains.
Sequencing of a PCR product made from genomic DNA of M. bovis BCG showed that one of the frameshift mutations in the phoT of ATCC 35721 was also present in BCG. It is now well established (Wards et al., 2000; Lewis et al., 2003
) that the absence of the RD1 region from BCG contributes greatly to its attenuation and that much of this attenuation can be accounted for by the loss of the esat-6 gene (Wards et al., 2000
), one of the nine genes deleted from BCG in the RD1 region. The identification of a frameshift mutation causing inactivation of the phoT gene, which has virulence properties and is very distant from RD1 on the chromosome, indicates that phoT may also be contributing to the attenuation of BCG.
Comparative DNA analysis of M. bovis PhoT shows that it is essentially identical to M. tuberculosis PhoT and encodes an ATP-binding cassette (ABC) protein with a probable role in phosphate transport (Cole et al., 1998). Bacteria generally have an active phosphate transport system that includes two membrane-spanning proteins, a substrate-binding protein and an ABC protein designated PstB which hydrolyses ATP and provides the energy required for transport (van Veen, 1997
; Linton & Higgins, 1998
). Normally, these proteins are in a single operon or at the same locus on the genome. The M. tuberculosis complex is unusual in having three putative pst operons that between them contain three phosphate-binding proteins and four membrane-spanning proteins (Braibant et al., 2000
) and it is thought that these multiple copies might enable subtle biochemical adaptations of these organisms to different phosphate-limiting conditions during the infectious cycle (Sarin et al., 2001
). pstB is the only gene encoding an ABC protein in these operons, but phoT, which is 130 kb from pstB on the chromosome, is also an ABC protein gene and PhoT has higher homology to PstB in some other organisms (http://genolist.pasteur.fr/TubercuList) than does PstB of M. tuberculosis. In particular, PhoT has very high homology to what appear to be PstB-encoding genes surrounded by other phosphate transport genes in the as yet unannotated, unfinished genome sequences of both M. smegmatis and M. avium (http://www.ncbi.nlm.nih.gov/sutils/genom_table.cgi?). Comparison of the two M. smegmatis sequences in TIGR_1772 and AAC15686 to each other and to the PstB proteins in other organisms indicates that they are almost certainly from the same gene but that the earlier sequence (AAC15686) is translated from a gene sequence that has a small number of sequencing errors (comparisons not shown). To investigate whether PhoT has a role in phosphate transport, the five strains of this study were cultured in decreasing concentrations of phosphate. Results of this work showed that growth at lowered phosphate concentrations required the presence of the wild-type principal sigma factor SigA but that, even with this factor present, growth did not occur at the lowest phosphate concentration tested without the presence of an active PhoT. Recently, it was shown that disruption of the pst operon in M. smegmatis reduced its phosphate uptake ability and doubled its sensitivity to the fluoroquinolone ciprofloxacin (Bhatt et al., 2000
). This group also showed that resistance to ciprofloxacin involved an efflux system (Banerjee et al., 2000
). Testing of the sensitivity of the strains used in this study to ciprofloxacin similarly showed that inactivation of phoT in ATCC 35723 doubled its sensitivity as well as reducing its ability to scavenge phosphate. Bhatt et al. (2000)
concluded that there is strong evidence that pstB plays a role in phosphate importing as well as having a second role in exporting. In this study, PhoT also appears to have two different functions: a phosphate transporting activity at low phosphate levels requiring SigA and a virulence role in the presence of either SigA or, for some animal models, also SigA-R515H. While the ciprofloxacin results and analogy to the M. smegmatis system suggest that this virulence role may involve the export of one or more substances, this remains to be determined. At this stage, these conclusions on the role of PhoT in M. bovis must be accepted with caution as the determination of growth in low phosphate concentrations measures an in vitro phenotype and virulence is an in vivo phenotype. In better-studied pathogenic bacteria, it has been shown that uptake of phosphate has its own regulatory system and that the relationship of this regulation with virulence is only partly understood (Rao et al., 2003
; von Kruger et al., 1999
). An additional complicating factor in interpreting these results in the broader context of other species of the M. tuberculosis complex is the very recent finding that pstB in M. bovis, including the strains used in this study, has a frameshift mutation that would be expected to affect the expression of a functional PstB. Since PstB and PhoT may have functionally overlapping roles and pstB is not frameshifted in M. tuberculosis, it is possible that the effects on either virulence or phosphate scavenging that occur when phoT is inactivated in M. bovis may not occur in M. tuberculosis.
Virulence of pathogenic organisms is a complex phenotype that depends on the function of many genes in the pathogen as well as on a multitude of responses by the infected host. In the case of tuberculosis, the importance of the disease has encouraged the development and use of many different animal models. No single model exactly replicates the situation in humans or in important economic species such as cattle, but different models have advantages for different studies. Mouse models have been especially useful in immunological studies of tuberculosis but gave less clear-cut results for the strains used in this study than did the other models. In particular, ATCC 35721 grew to higher numbers in mice than did ATCC 35723, even though this latter strain is much more virulent that ATCC 35721 in guinea pigs and possums. These results emphasize the caution that should be exercised in using a single animal model to study gene effects on a phenotype as complex as virulence even when isogenic strains are being used. In this study, the susceptibility of different animals to different strains of M. bovis enabled the discovery of the virulence role of phoT, the likely separation of this role from its phosphate-scavenging ability, and the particular importance of this gene for the virulence of M. bovis in a natural wildlife host. These discoveries illustrate the advantages of using different animal models as tools for the molecular biological investigation of tuberculosis virulence.
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ACKNOWLEDGEMENTS |
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Received 8 May 2003;
revised 15 July 2003;
accepted 28 July 2003.
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