An ABC Transporter Containing a Forkhead-Associated Domain Interacts with a Serine-Threonine Protein Kinase and Is Required for Growth of My
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感染与免疫杂志 2005年第8期
Division of Mycobacterial Research
Division of Molecular Neuroendocrinology
Division of Protein Structure, National Institute for Medical Research, Mill Hill, London NW7 1AA, United Kingdom
ABSTRACT
Forkhead-associated (FHA) domains are modular phosphopeptide recognition motifs with a striking preference for phosphothreonine-containing epitopes. FHA domains have been best characterized in eukaryotic signaling pathways but have been identified in six proteins in Mycobacterium tuberculosis, the causative organism of tuberculosis. One of these, coded by gene Rv1747, is an ABC transporter and the only one to contain two such modules. A deletion mutant of Rv1747 is attenuated in a mouse intravenous injection model of tuberculosis where the bacterial load of the mutant is 10-fold lower than that of the wild type in both lungs and spleen. In addition, growth of the mutant in mouse bone marrow-derived macrophages and dendritic cells is significantly impaired. In contrast, growth of this mutant in vitro was indistinguishable from that of the wild type. The mutant phenotype was lost when the mutation was complemented by the wild-type allele, confirming that it was due to mutation of Rv1747. Using yeast two-hybrid analysis, we have shown that the Rv1747 protein interacts with the serine-threonine protein kinase PknF. This interaction appears to be phospho-dependent since it is abrogated in a kinase-dead mutant and by mutations in the presumed activation loop of PknF and in the first FHA domain of Rv1747. These results demonstrate that the protein coded by Rv1747 is required for normal virulent infection by M. tuberculosis in mice and, since it interacts with a serine-threonine protein kinase in a kinase-dependent manner, indicate that it forms part of an important phospho-dependent signaling pathway.
INTRODUCTION
Tuberculosis (TB) was declared a global emergency by the World Health Organization in 1993. Despite this, estimates suggested that 8.2 million new cases were still occurring worldwide in the year 2000, with 2 to 3 million people dying from the disease, and a third of the world's population are infected (39). There has been enormous progress in combating the disease, including the publication of the complete genome of its causative agent, the bacterium Mycobacterium tuberculosis (7). However, the situation has deteriorated in a number of regions due to synergy between TB and AIDS (21). The only vaccine for TB, Mycobacterium bovis BCG, developed in the 1920s, does not provide adequate levels of protection in Africa, India, and some parts of the United States (12, 13). Moreover, the rise in multidrug-resistant TB makes the identification of new drug targets and vaccines imperative (39).
The identification of virulence determinants is one approach to the development of novel disease intervention strategies that could be used to develop new therapeutic agents or attenuated vaccine strains. One likely class of proteins for the search for such virulence determinants is in regulatory proteins (see, for example, references 27, 35, 38, and 40). In particular, protein kinases have, perhaps surprisingly, emerged as major targets for pharmaceutical intervention over the last few years. M. tuberculosis faces complex environmental and metabolic choices as it moves from the environment to infection of macrophages, dormancy, and growth in the host. It is therefore expected to have an extensive repertoire of regulatory proteins with more than 100 predicted in the genome (7). Phosphorylation on serine, threonine, or tyrosine is the most common posttranslational modification utilized in cellular signaling processes in eukaryotes, where the interplay of protein kinases and phosphatases provides for rapid reversibility. In prokaryotes, phosphorylation-dependent signaling is manifested in the use of so-called two-component systems in which phosphate is transferred from a histidine on a sensor kinase to a glutamate or aspartate on a response regulator. M. tuberculosis has only 11 two-component networks compared to Bacillus subtilis and Escherichia coli, which each contain more than 30. Nevertheless, it does contain 11 putative Ser-Thr protein kinases. Interestingly, four of these genes are linked in the genome to genes coding for proteins that have the forkhead-associated (FHA) domain that fulfills the important role of modular phosphothreonine binding motif (48). Indeed, the presence of a FHA domain is now generally taken to indicate that the protein is likely to interact with a phosphorylated protein partner (10), although such interactions have been demonstrated in only a relatively limited cohort of biological systems.
The M. tuberculosis gene Rv1747 encodes a 92-kDa protein containing an ABC transporter domain and two FHA domains. Although its function is unknown, it has been suggested that this gene functions as an exporter (5). It is one of two genes in a putative operon (7) in which the first encodes a eukaryote-like serine/threonine protein kinase, Rv1746 (pknF) (see Fig. 1). Taken together, these observations suggest that Rv1747 is not only likely to function as a transporter with potential importance in virulence but also possibly to be a part of a complex signaling system involving a eukaryote-like STK pathway. Here we have investigated the role of Rv1747 in microbial pathogenesis and further examined the potential for regulatory interactions between the FHA domains of Rv1747 and PknF.
MATERIALS AND METHODS
Strains, growth conditions, and reagents. The wild-type strain used was M. tuberculosis H37Rv Paris (kindly provided by S. Cole). All strains were grown at 37°C in Dubos broth supplemented with 0.05% (vol/vol) Tween 80, 0.2% (vol/vol) glycerol, and 4% (vol/vol) Dubos medium albumin (Becton Dickinson), either at 100 ml in a Bellco roll-in incubator (2 rpm) or at 10 ml in static universals. Mutant strains of M. tuberculosis were plated onto Middlebrook 7H11 agar supplemented with 4% (vol/vol) Dubos medium albumin and, as noted, with hygromycin (100 μg ml–1), kanamycin (25 μg ml–1), X-Gal (5-bromo-4-chloro-3-indolyl--D-galactopyranoside; 50 μg ml–1), or sucrose (2% [wt/vol]). The PCR products were cloned by using a Zero Blunt TOPO PCR cloning kit (Invitrogen). Adult (8-week-old) female BALB/c mice were obtained from the specific-pathogen-free animal unit at National Institute for Medical Research.
Isolation of nucleic acids from mycobacteria. Extraction of genomic DNA used a modification of the method of Belisle et al. (3) adapted from Whipple et al. (47). For RNA extraction, rolling cultures of M. tuberculosis were harvested by centrifugation, and RNA was extracted by mechanical lysis using the Fast RNA Pro Blue kit (Q-BIOgene-ALEXIS). The RNA had DNA removed by DNase digestion.
Sequence analysis. Sequencing was carried out by using the BigDye terminator cycle sequencing kit on an ABI Prism 377 DNA analyzer, both from Applied Biosystems. The results were compared to the published M. tuberculosis sequence (7) by using the BLAST facility (http://genolist.pasteur.fr/TubercuList/). Sequencing to check the constructs for the Rv1747 knockout was carried out with the primers 5'-TTTGCACCTCGCGTTCT-3' and 5'-ACCAACACCACCATCTG-3' for the BamHI insert and the primers 5'-GTGATGCTGTCGAGCTT-3' and 5'-TTGAGCACCTTGTGTGT-3' for the NotI insert. The complement was also sequenced entirely.
Microarrays. Microarrays of the M. tuberculosis genome were obtained from J. Hinds and P. Butcher, Bacterial Microarray Group, St. Georges Hospital Medical School, London, England. The arrays were scanned by using an Axon 4000A together with GenePix software.
Construction of Rv1747-null mutant. Plasmid constructs were made by inserting 2-kb regions of amplified H37Rv Paris DNA flanking each side of the knockout gene into the vector p2NIL (36) (See Fig. 1). The primers for the flanking regions were 5'-GGATCC-GTAACATCGCGCACGAATTG-3' and 5'-GGATCC-CAGCCGTTGCTTCTGCGAAT-3' (BamHI insert) and 5'-GCGGCCGCGTGACGTGCGACTGATTCTG-3' and 5'-GCGGCCGCTTGCGAGCAGGTGACACCTT-3' (NotI insert), both amplified with the enzyme Elongase (Promega). The hyg cassette was cut from pUC-HY vector (26, 41) by using KpnI and inserted between the 5' and 3' flanking regions. Finally, the sacB gene and the lacZ gene were inserted from the plasmid pGOAL17 (36) by using PacI. The plasmid constructs were pretreated with 100 mJ of UV light cm–2 (19) and used to electroporate M. tuberculosis (34). This was plated onto 7H11 plates supplemented with X-Gal, kanamycin, and hygromycin and left to grow for 3 weeks. Single-crossover events, seen as blue colonies, were streaked onto 7H11 supplemented with hygromycin and grown for a further 3 weeks before they were serially diluted and streaked onto 7H11 supplemented with sucrose and hygromycin. The resulting colonies were patch tested on 7H11 plates containing hygromycin, with X-Gal, or kanamycin to check for white, kanamycin-sensitive colonies, indicating a double-crossover event.
Check of Rv1747-null mutant. The Rv1747-null mutant was checked by PCR, Southern blotting, and genomic DNA microarray to ensure the gene had been knocked out. For PCR, the following primer pairs were used: 5'-GCGTTGACCTGCGTGTT-3' from M. tuberculosis 5' of the BamHI insert and 5'-CGTTCGAACGCGGCTAC-3' from the p2NIL multiple cloning site and 5'-GGTCAGCGAACCAATCA-3' from the p2NIL multiple cloning site and 5'-ACAGGACACCTGCTATG-3' from M. tuberculosis 3' of the NotI insert. The fragments were amplified at an annealing temperature of 55°C by using Hotstar Taq. For Southern blots genomic DNA was digested with the restriction enzyme AscI, and blots were probed with DNA from outside the knockout construct, one from pknE (derived by PCR with the primers 5'-GGATCCTTCAACGAACCCATCTGTCC-3' and 5'-TCGAATAGTTCTTGCTG-3') and the other from Rv1750c (fad1) (derived by using primers 5-CATAGCAGGTGTCCTGTT-3' and 5'-GGCTGAATTGGTGGCTG-3'). An AscI site is lost when the Rv1747 gene was deleted so that an 7-kb fragment that hybridized with the pknE probe and an 10-kb fragment that hybridized with the fad1 probe were replaced by an 15-kb fragment (data not shown). Genomic DNA was isolated and used to probe M. tuberculosis DNA microarrays as described previously (14).
Complementation of the Rv1747 mutation. The genes Rv1747, Rv1746, and 609 bp of Rv1745 (See Fig. 1) were amplified by PCR with the primers 5'-AAGCTTGCACGCCTTGAGGCGAATCT-3' (5' HindIII restriction site) and 5'-GAATTC-GTAACATCGCGCACGAATTG-3' (5' EcoRI restriction site) and the Expand Long Template PCR system. The PCR product was sequenced to check that there were no base changes. After restriction enzyme digestion, the PCR product was then cloned into the integrating vector pMV306 (43) and used to electroporate the M. tuberculosis Rv1747 knockout. This was plated onto 7H11 plates supplemented with kanamycin and hygromycin and left to grow for 3 weeks. The resulting colonies were harvested, the DNA was extracted, and the colonies were checked for complementation by PCR and microarrays.
In vitro growth determination. Wild-type H37Rv Paris and the Rv1747 knockout mutant and its complement strain were each initially removed from storage at –80°C, grown in 10 ml of Dubos medium as static cultures, and then at 37°C in a rolling incubator as described above. Once they each reached an optical density at 600 nm (OD600) of 0.5, 1 ml of each culture was subcultured to a fresh bottle of 100 ml of Dubos medium and incubation continued in the rolling incubator. Aliquots of 1 ml of each culture were removed for OD600 readings at 24-h intervals.
Growth of bacteria in mice. Stock cultures of M. tuberculosis H37Rv Paris, Rv1747 knockout, and Rv1747 complement were grown in 10 ml of Dubos medium standing cultures at 37°C for 14 days. The cultures were diluted in phosphate-buffered saline to an OD600 of 0.02. Infection was induced by injecting 0.2 ml of viable M. tuberculosis (5 x 105 CFU) into a lateral tail vein of 8-week-old adult female BALB/c mice. The infection was monitored by removing the lungs and spleens of infected mice at various intervals, homogenizing the tissues, and plating 10-fold dilutions to determine numbers of CFU of M. tuberculosis (45). The growth curves were compared by graph and statistical analysis. The results for each time point are the means of CFU determinations performed on organs from three mice, and the error bars show the standard deviations (see Fig. 3 and 4). The asterisk indicates that the result is statistically significantly different from that of the wild type by the two-tailed Student t test for groups of unequal variance (P < 0.01), as well as by single-factor analysis of variance (P < 0.01).
Growth of bacteria in mouse cell culture. Bone marrow cells were extracted from the hind legs of 8-week-old adult female BALB/C mice as described previously (44). The cells were resuspended in Iscove’s modified Dulbecco’s medium plus 5% fetal calf serum, 2 mM L-glutamine, and 80 μM -mercaptoethanol, to which was added, for macrophages, 10% by volume of supernatant from L929 cells that produce macrophage colony-stimulating factor or, for dendritic cells, 10% supernatant from X-63 cells transfected with granulocyte-macrophage colony-stimulating factor cDNA. The cells were plated in six-well plates and incubated at 37°C and 5% CO2 atmosphere for 24 h. At day 2 the cells were washed with fresh prewarmed medium, and nonadherent dendritic cells were plated in new six-well plates. Fresh medium was added to the plates containing the adherent bone marrow macrophage cells. The cells were cultured for a further 3 days, and at day 5 they were infected for 6 h at a cell/acid fast bacillus ratio of 2:1. After infection the medium was removed and replaced with fresh medium. The bone marrow macrophages and dendritic cells were lysed with 2% saponin and incubated for 1 h at 37°C. Growth was determined by viable counts on Middlebrook 7H11 agar plates containing Middlebrook OADC supplement at days 0 (6 h postinfection) and 4, 7, and 11 postinfection. The growth curves were compared by graph and statistical analysis. The results for each time point are the means of CFU determinations from at least three wells, and the error bars show the standard deviations. The asterisk indicates that the result is statistically significantly different from that of the wild type by the two-tailed Student t test for groups of unequal variance (P < 0.02), as well as by single-factor analysis of variance (P < 0.02).
Yeast two-hybrid analysis. The pknF and Rv1747 genes were amplified by PCR with the proofreading polymerase Pfu turbo (Stratagene), and template chromosomal DNA from M. tuberculosis H37Rv. For pknF the amplification primers were 5'-CTGCAGCATGCCGCTCGCGGAAGGTT-3' (forward primer) and 5'-CTGCAGCGGCCAGCCGTTGCTTCTGC-3' (reverse primer), both carrying a PstI site at the 5' end. For Rv1747, the primers were 5'-GGATCCGTGCCGATGAGCCAACCAGC-3' and 5'-GGATCCGCACGCCTTGAGGCGAATCT-3', both carrying a 5' BamHI site. The restriction sites were used to clone the genes into the yeast two-hybrid Gal4 activation domain vectors pGAD-C3 and pGAD-C1, respectively, and the Gal4-binding domain vectors pGBD-C3 and pGBD-C1, respectively (22). Constructs were confirmed by sequencing. Saccharomyces cerevisiae Y187 (MAT ura3-52 his3-200 ade2-101 trp1-901 leu2-3,112 gal4 met– gal80 URA3::GAL1UAS-GAL1TATA-lacZ) (17) was cotransformed with Gal4 activation domain and Gal4-binding domain vectors according to the LiAc TRAFO method (16) and plated onto minimal Difco plates lacking Leu and Trp for the selection of transformants. Protein interactions were measured by assaying for lacZ expression by using ONPG (o-nitrophenyl--D-galactopyranoside) as a substrate (28) and are expressed as U/min/mg of protein. All pknF and Rv1747 Gal4 constructs were cotransformed with either empty pGAD or pGBD vectors to eliminate false positives.
Site-directed mutagenesis. Site-directed mutagenesis was carried out according to the Stratagene QuickChange XL site-directed mutagenesis manual, using SoloPack Gold Supercompetent E. coli for transformation. Primers used for mutagenesis are described in Table 1. A mutation was introduced into the PknF active site, where Lys-41 was substituted by Ala. Mutation of the activation loop of PknF was created by individual substitution of Thr-173, -175, and -178 with alanines. Rv1747 was mutated in FHA domain-1, in which Ser-47 was substituted by Ala, and a second mutant was created in which Ser-248 in FHA domain-2 was also substituted by Ala. The presence of the desired mutations was confirmed by sequencing.
RESULTS
Construction of a Rv1747 knockout. The serine-threonine protein kinase pknF is adjacent to and probably in the same operon as Rv1747, an ABC transporter protein with two FHA domains (Fig. 1). Most of the coding region of gene Rv1747 was successfully deleted from M. tuberculosis H37Rv by using homologous recombination, and this was confirmed by PCR, Southern blotting, and DNA microarray analyses (see Materials and Methods). A complement strain was constructed by using the region of DNA including the promoter, Rv1746, and Rv1747 to demonstrate that phenotypic effects were due to deletion of Rv1747 and not to downstream polar effects of the mutation on Rv1748. Microarray experiments with DNA from the complement strain showed that the Rv1747 gene had indeed been replaced and that there was an additional Rv1746 gene (data not shown).
Growth of the Rv1747 mutant is normal in vitro but attenuated in vivo. Growth, as measured by OD600 readings, of the Rv1747 mutant and its complement compared to growth of the H37Rv wild type in 100 ml of Dubos rolling culture was not statistically different (Fig. 2). However, mouse infection with the Rv1747 mutant resulted in severely impaired growth of the mutant compared to the wild-type H37Rv. This was demonstrated in both the lungs (by Student t test, P = 6.4 x 10–4) and spleens (P = 3.5 x 10–4) of mice (Fig. 3). The mutant was also significantly different compared to the complement (P = 1.4 x 10–3 and 4.4 x 10–5 in lungs and spleens, respectively). The mutant strain did, however, persist within the animals. In mouse bone marrow-derived macrophages, growth of the Rv1747 knockout was impaired compared to both the H37Rv wild type and the Rv1747 complement as compared by Student t test (Fig. 4, P = 4.5 x 10–4 and 3.9 x 10–4, respectively). In bone marrow-derived dendritic cells the Rv1747 complement did not grow as well as the H37Rv wild type, but the difference was not significantly different (see Fig. 4), whereas the Rv1747 mutant was again significantly impaired in growth compared to the H37Rv wild type as determined by Student t test (P = 0.019). The mutant was also significantly different compared to the complement (P = 0.034).
Interaction between PknF and Rv1747 is phospho and FHA domain dependent. Using yeast two-hybrid analysis, a strong interaction was observed between PknF and Rv1747 (Fig. 5, bar 5). This interaction was abolished upon mutation of Lys-41 to Ala in the active site of PknF (23) (Fig. 5, bar 8), strongly suggesting that the interaction between PknF and Rv1747 is phosphorylation dependent.
Interaction of PknF with Rv1747 was abolished by mutation to Ala of Thr-173 in the activation loop of PknF (Fig. 5, bar 9). Mutation of Thr-175 or Thr-178 (Fig. 5, bars 10 and 11) reduced the interaction by <3-fold, showing that all three threonines are involved in the interaction but that only Thr-173 is essential for recognition by the FHA-containing Rv1747.
The X-ray structure of FHA1 of Rad53p in complex with a phospho-threonine peptide has indicated that there are six highly conserved residues in the FHA domain (11). Five are located around the peptide-binding site, three of which make interactions with the peptide. Of these three, only two, Arg-70 and Ser-85, bind directly to the pThr residue itself (Fig. 6). We have therefore mutated the equivalent serine residues in the two FHA domains of Rv1747, Ser-47 in FHA-1, and Ser-248 in FHA-2. The interaction of Rv1747 with PknF was reduced by >99% by substitution of Ser-47 by Ala in FHA domain 1 of Rv1747 (Fig. 5, bar 6) and was reduced by 60% by the mutation of Ser-248 to Ala in FHA-2 (Fig. 5, bar 7), suggesting that both FHA domains are involved in the interaction with PknF, but only FHA-1 is essential for this process. These data complement and extend previous observations that the level of phosphorylation of Rv1747 by PknF is dependent on both FHA domains (31). A weak but measurable interaction was noted when Rv1747 was present in both the activation and binding domains of Gal4 showing that this protein interacts with itself (Fig. 5, bar 4). This is consistent with the fact that ABC transporters are generally active as dimers or higher oligomers. Nevertheless, this weak interaction was not affected by either of the mutations in the Rv1747 FHA domains (data not shown).
DISCUSSION
Reversible phosphorylation is a ubiquitous mechanism of signaling transduction. In eukaryotes, proteins are phosphorylated on either tyrosine residues or serine/threonine residues by two distinct but structurally related families of kinases. In contrast, prokaryotes utilize the phosphorylation of histidine residues and subsequent phosphoryl transfer to aspartate residues in so-called two-component phospho-relay systems. Somewhat surprisingly, the genome sequence of M. tuberculosis revealed not only a number of putative two-component histidine kinases but also a complement of 11 eukaryote-like serine-threonine kinases, together with a number of phosphatases (7). It has been postulated that these kinases are likely to play regulatory roles (1), and some of them have been investigated biochemically (2, 23, 29, 30, 37) and structurally (32, 49). Potential regulatory functions have been investigated in some cases (for a review, see reference 33). For example, PknA has been implicated in the regulation of morphological changes associated with cell division (6), PknH is differentially expressed under stress conditions (42), and PknG is linked to cellular glutamate levels (8) and is secreted within macrophages and inhibits phagosome-lysosome fusion (46). Inactivation of PknG in M. bovis BCG led to lysosomal localization and mycobacterial cell death due to the loss of inhibition of phagosome-lysosome fusion (46).
The FHA domain is a modular phosphopeptide recognition motif (9) showing a striking specificity for phosphothreonine-containing epitopes (11). FHA domains have been best studied in their roles in the eukaryotic DNA damage response, in particular, in the context of the Rad53/Chk2 family of checkpoint kinases. Of particular relevance is the observation that these molecules encode modular proteins, each containing an STK domain together with one or more FHA domains. In this context, the FHA domains seem to play a variety of regulatory roles in kinase activation and subcellular localization. However, relatively little is known about how prokaryotic FHA domains function. Notably, four M. tuberculosis STK-encoding genes are linked to genes coding for proteins containing the FHA phosphopeptide recognition domain suggesting, by analogy to Chk2 and Rad53, that they may play a direct role in regulating STK activity. Support for this notion has emerged from studies of PknH, which has been shown to phosphorylate the FHA-containing EmbR protein, an activity that is abolished by mutations in the FHA domain of EmbR that would be predicted to compromise pThr-binding (30).
In the present study we have demonstrated the interaction of PknF with the product of Rv1747. We have corroborated this result by mutating both the PknF kinase and Rv1747. Thus, this interaction was abrogated by mutation of Lys-41, which is known to abolish the kinase activity of PknF (23), providing evidence that the interaction between PknF and Rv1747 is phosphorylation dependent. It was also abolished by mutation of Thr-173 in the activation loop of PknF but reduced only threefold by mutation of Thr-175 or Thr-178. Thr-173 and Thr-175 are homologous with residues Thr-171 and Thr-173 in PknB, identified as autophosphorylation sites in PknB (4). The presence of pThr in FHA domain binding sites has been shown to be essential for interaction (11), suggesting that Thr-173 may be the phosphorylation site of PknF that is recognized by the Rv1747 FHA domains. Alternatively, since mutation of Thr-173 in the activation loop could, potentially, prevent autophosphorylation at other sites in PknF, confident assignment of the actual FHA binding site will require further investigation, and these experiments are in progress. Lastly, the protein-protein interaction was disrupted by mutation of Ser-47 of Rv1747, which is highly conserved in FHA domains and directly interacts with the phosphothreonine residue in both Rad53 FHA-1 and the FHA domain of Chk2 (11, 24). In contrast, binding was reduced by <3-fold upon mutation of Ser-248 in the second putative FHA domain. These results suggest that phospho-dependent interactions between PknF and the FHA domain of Rv1747 form part of a regulatory cascade that involves phosphorylation by PknF that is directed predominantly by binding of FHA-1 to a pThr-containing autophosphorylation site on PknF, possibly located within the kinase activation loop itself.
Rv1747 codes for an ABC-transporter protein. ATP binding cassette (ABC) transport systems occur in all cells of all living species (18) and in M. tuberculosis genes encoding these proteins occupy 2.5% of the genome (5). They are involved in the active transport of solutes across cellular membranes, including the uptake of a large variety of essential nutrients and the secretion of virulence factors, proteases, and toxins. The ABC transporter gene Rv1747 contains only one membrane-spanning domain and one nucleotide binding domain and so is expected to dimerize to function as a transporter (5). There is presently no information as to what substrate it transports. Similarity has been found with the White protein from Drosophila melanogaster, a permease necessary for the transport of pigment precursors into pigment cells responsible for eye color, and with NodI from Rhizobium and Bradyrhizobium strains, a protein implicated in the nodulation process by the export of a polysaccharide (5). It has been suggested that the substance transported may be a lipooligosaccharide (http://genolist.pasteur.fr/TubercuList/) and that the Rv1747 product possibly functions as an exporter protein (5). Among eukaryotic ABC transporters with known structures, Rv1747 is most closely related at the sequence level to TAP (transporter associated with antigen processing; PDB ID 1JJ7, E = 7e-15) (25) and CTFR (cystic fibrosis transmembrane conductance channel; PDB ID 1R10, E = 4e-10) (15), which are both regulated by phosphorylation.
The phenotype of the Rv1747 knockout demonstrated here is the first reported mutation in an FHA domain-containing protein of M. tuberculosis and is intriguing in its very marked reduction in growth in macrophages and in mice. Thus, the mutant exhibited greatly reduced bacterial loads in the mouse lungs and spleens compared to infection by the wild type and therefore falls within the growth in vivo (giv) type of mutant defined by Hingley-Wilson et al. (20). This ABC transporter system therefore appears to be very important for the normal growth of the bacterium during the multiplication phase in macrophages and in mice but did not appear necessary for the persistence phase of growth. Clearly, whether the protein involved is importing or exporting a substrate, its absence results in failure of the Mycobacterium to thrive in mice but makes no difference to its growth in vitro. The fact that Rv1747 interacts with the PknF kinase suggests that its activity is modulated in response to unknown signals that are potentially detected by pknF through its extracellular C-terminal region/domain and transduced into the intracellular milieu via the activity of its STK domain.
ACKNOWLEDGMENTS
We thank Vangelis Stavropoulos and John Brennan for valuable help with the infection experiments. We thank Tanya Parish for providing plasmids p2NIL and pGOAL. We acknowledge BμG@S (the Bacterial Microarray Group at St. George's Hospital Medical School) and especially Jason Hinds and Philip Butcher for the supply of the M. tuberculosis microarray and advice, and The Wellcome Trust for funding the multicollaborative microbial pathogen microarray facility under its Functional Genomics Resources Initiative.
This study was supported by the Medical Research Council.
Dedicated to the memory of M. Joseph Colston, who passed away on 20 February 2003.
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Division of Molecular Neuroendocrinology
Division of Protein Structure, National Institute for Medical Research, Mill Hill, London NW7 1AA, United Kingdom
ABSTRACT
Forkhead-associated (FHA) domains are modular phosphopeptide recognition motifs with a striking preference for phosphothreonine-containing epitopes. FHA domains have been best characterized in eukaryotic signaling pathways but have been identified in six proteins in Mycobacterium tuberculosis, the causative organism of tuberculosis. One of these, coded by gene Rv1747, is an ABC transporter and the only one to contain two such modules. A deletion mutant of Rv1747 is attenuated in a mouse intravenous injection model of tuberculosis where the bacterial load of the mutant is 10-fold lower than that of the wild type in both lungs and spleen. In addition, growth of the mutant in mouse bone marrow-derived macrophages and dendritic cells is significantly impaired. In contrast, growth of this mutant in vitro was indistinguishable from that of the wild type. The mutant phenotype was lost when the mutation was complemented by the wild-type allele, confirming that it was due to mutation of Rv1747. Using yeast two-hybrid analysis, we have shown that the Rv1747 protein interacts with the serine-threonine protein kinase PknF. This interaction appears to be phospho-dependent since it is abrogated in a kinase-dead mutant and by mutations in the presumed activation loop of PknF and in the first FHA domain of Rv1747. These results demonstrate that the protein coded by Rv1747 is required for normal virulent infection by M. tuberculosis in mice and, since it interacts with a serine-threonine protein kinase in a kinase-dependent manner, indicate that it forms part of an important phospho-dependent signaling pathway.
INTRODUCTION
Tuberculosis (TB) was declared a global emergency by the World Health Organization in 1993. Despite this, estimates suggested that 8.2 million new cases were still occurring worldwide in the year 2000, with 2 to 3 million people dying from the disease, and a third of the world's population are infected (39). There has been enormous progress in combating the disease, including the publication of the complete genome of its causative agent, the bacterium Mycobacterium tuberculosis (7). However, the situation has deteriorated in a number of regions due to synergy between TB and AIDS (21). The only vaccine for TB, Mycobacterium bovis BCG, developed in the 1920s, does not provide adequate levels of protection in Africa, India, and some parts of the United States (12, 13). Moreover, the rise in multidrug-resistant TB makes the identification of new drug targets and vaccines imperative (39).
The identification of virulence determinants is one approach to the development of novel disease intervention strategies that could be used to develop new therapeutic agents or attenuated vaccine strains. One likely class of proteins for the search for such virulence determinants is in regulatory proteins (see, for example, references 27, 35, 38, and 40). In particular, protein kinases have, perhaps surprisingly, emerged as major targets for pharmaceutical intervention over the last few years. M. tuberculosis faces complex environmental and metabolic choices as it moves from the environment to infection of macrophages, dormancy, and growth in the host. It is therefore expected to have an extensive repertoire of regulatory proteins with more than 100 predicted in the genome (7). Phosphorylation on serine, threonine, or tyrosine is the most common posttranslational modification utilized in cellular signaling processes in eukaryotes, where the interplay of protein kinases and phosphatases provides for rapid reversibility. In prokaryotes, phosphorylation-dependent signaling is manifested in the use of so-called two-component systems in which phosphate is transferred from a histidine on a sensor kinase to a glutamate or aspartate on a response regulator. M. tuberculosis has only 11 two-component networks compared to Bacillus subtilis and Escherichia coli, which each contain more than 30. Nevertheless, it does contain 11 putative Ser-Thr protein kinases. Interestingly, four of these genes are linked in the genome to genes coding for proteins that have the forkhead-associated (FHA) domain that fulfills the important role of modular phosphothreonine binding motif (48). Indeed, the presence of a FHA domain is now generally taken to indicate that the protein is likely to interact with a phosphorylated protein partner (10), although such interactions have been demonstrated in only a relatively limited cohort of biological systems.
The M. tuberculosis gene Rv1747 encodes a 92-kDa protein containing an ABC transporter domain and two FHA domains. Although its function is unknown, it has been suggested that this gene functions as an exporter (5). It is one of two genes in a putative operon (7) in which the first encodes a eukaryote-like serine/threonine protein kinase, Rv1746 (pknF) (see Fig. 1). Taken together, these observations suggest that Rv1747 is not only likely to function as a transporter with potential importance in virulence but also possibly to be a part of a complex signaling system involving a eukaryote-like STK pathway. Here we have investigated the role of Rv1747 in microbial pathogenesis and further examined the potential for regulatory interactions between the FHA domains of Rv1747 and PknF.
MATERIALS AND METHODS
Strains, growth conditions, and reagents. The wild-type strain used was M. tuberculosis H37Rv Paris (kindly provided by S. Cole). All strains were grown at 37°C in Dubos broth supplemented with 0.05% (vol/vol) Tween 80, 0.2% (vol/vol) glycerol, and 4% (vol/vol) Dubos medium albumin (Becton Dickinson), either at 100 ml in a Bellco roll-in incubator (2 rpm) or at 10 ml in static universals. Mutant strains of M. tuberculosis were plated onto Middlebrook 7H11 agar supplemented with 4% (vol/vol) Dubos medium albumin and, as noted, with hygromycin (100 μg ml–1), kanamycin (25 μg ml–1), X-Gal (5-bromo-4-chloro-3-indolyl--D-galactopyranoside; 50 μg ml–1), or sucrose (2% [wt/vol]). The PCR products were cloned by using a Zero Blunt TOPO PCR cloning kit (Invitrogen). Adult (8-week-old) female BALB/c mice were obtained from the specific-pathogen-free animal unit at National Institute for Medical Research.
Isolation of nucleic acids from mycobacteria. Extraction of genomic DNA used a modification of the method of Belisle et al. (3) adapted from Whipple et al. (47). For RNA extraction, rolling cultures of M. tuberculosis were harvested by centrifugation, and RNA was extracted by mechanical lysis using the Fast RNA Pro Blue kit (Q-BIOgene-ALEXIS). The RNA had DNA removed by DNase digestion.
Sequence analysis. Sequencing was carried out by using the BigDye terminator cycle sequencing kit on an ABI Prism 377 DNA analyzer, both from Applied Biosystems. The results were compared to the published M. tuberculosis sequence (7) by using the BLAST facility (http://genolist.pasteur.fr/TubercuList/). Sequencing to check the constructs for the Rv1747 knockout was carried out with the primers 5'-TTTGCACCTCGCGTTCT-3' and 5'-ACCAACACCACCATCTG-3' for the BamHI insert and the primers 5'-GTGATGCTGTCGAGCTT-3' and 5'-TTGAGCACCTTGTGTGT-3' for the NotI insert. The complement was also sequenced entirely.
Microarrays. Microarrays of the M. tuberculosis genome were obtained from J. Hinds and P. Butcher, Bacterial Microarray Group, St. Georges Hospital Medical School, London, England. The arrays were scanned by using an Axon 4000A together with GenePix software.
Construction of Rv1747-null mutant. Plasmid constructs were made by inserting 2-kb regions of amplified H37Rv Paris DNA flanking each side of the knockout gene into the vector p2NIL (36) (See Fig. 1). The primers for the flanking regions were 5'-GGATCC-GTAACATCGCGCACGAATTG-3' and 5'-GGATCC-CAGCCGTTGCTTCTGCGAAT-3' (BamHI insert) and 5'-GCGGCCGCGTGACGTGCGACTGATTCTG-3' and 5'-GCGGCCGCTTGCGAGCAGGTGACACCTT-3' (NotI insert), both amplified with the enzyme Elongase (Promega). The hyg cassette was cut from pUC-HY vector (26, 41) by using KpnI and inserted between the 5' and 3' flanking regions. Finally, the sacB gene and the lacZ gene were inserted from the plasmid pGOAL17 (36) by using PacI. The plasmid constructs were pretreated with 100 mJ of UV light cm–2 (19) and used to electroporate M. tuberculosis (34). This was plated onto 7H11 plates supplemented with X-Gal, kanamycin, and hygromycin and left to grow for 3 weeks. Single-crossover events, seen as blue colonies, were streaked onto 7H11 supplemented with hygromycin and grown for a further 3 weeks before they were serially diluted and streaked onto 7H11 supplemented with sucrose and hygromycin. The resulting colonies were patch tested on 7H11 plates containing hygromycin, with X-Gal, or kanamycin to check for white, kanamycin-sensitive colonies, indicating a double-crossover event.
Check of Rv1747-null mutant. The Rv1747-null mutant was checked by PCR, Southern blotting, and genomic DNA microarray to ensure the gene had been knocked out. For PCR, the following primer pairs were used: 5'-GCGTTGACCTGCGTGTT-3' from M. tuberculosis 5' of the BamHI insert and 5'-CGTTCGAACGCGGCTAC-3' from the p2NIL multiple cloning site and 5'-GGTCAGCGAACCAATCA-3' from the p2NIL multiple cloning site and 5'-ACAGGACACCTGCTATG-3' from M. tuberculosis 3' of the NotI insert. The fragments were amplified at an annealing temperature of 55°C by using Hotstar Taq. For Southern blots genomic DNA was digested with the restriction enzyme AscI, and blots were probed with DNA from outside the knockout construct, one from pknE (derived by PCR with the primers 5'-GGATCCTTCAACGAACCCATCTGTCC-3' and 5'-TCGAATAGTTCTTGCTG-3') and the other from Rv1750c (fad1) (derived by using primers 5-CATAGCAGGTGTCCTGTT-3' and 5'-GGCTGAATTGGTGGCTG-3'). An AscI site is lost when the Rv1747 gene was deleted so that an 7-kb fragment that hybridized with the pknE probe and an 10-kb fragment that hybridized with the fad1 probe were replaced by an 15-kb fragment (data not shown). Genomic DNA was isolated and used to probe M. tuberculosis DNA microarrays as described previously (14).
Complementation of the Rv1747 mutation. The genes Rv1747, Rv1746, and 609 bp of Rv1745 (See Fig. 1) were amplified by PCR with the primers 5'-AAGCTTGCACGCCTTGAGGCGAATCT-3' (5' HindIII restriction site) and 5'-GAATTC-GTAACATCGCGCACGAATTG-3' (5' EcoRI restriction site) and the Expand Long Template PCR system. The PCR product was sequenced to check that there were no base changes. After restriction enzyme digestion, the PCR product was then cloned into the integrating vector pMV306 (43) and used to electroporate the M. tuberculosis Rv1747 knockout. This was plated onto 7H11 plates supplemented with kanamycin and hygromycin and left to grow for 3 weeks. The resulting colonies were harvested, the DNA was extracted, and the colonies were checked for complementation by PCR and microarrays.
In vitro growth determination. Wild-type H37Rv Paris and the Rv1747 knockout mutant and its complement strain were each initially removed from storage at –80°C, grown in 10 ml of Dubos medium as static cultures, and then at 37°C in a rolling incubator as described above. Once they each reached an optical density at 600 nm (OD600) of 0.5, 1 ml of each culture was subcultured to a fresh bottle of 100 ml of Dubos medium and incubation continued in the rolling incubator. Aliquots of 1 ml of each culture were removed for OD600 readings at 24-h intervals.
Growth of bacteria in mice. Stock cultures of M. tuberculosis H37Rv Paris, Rv1747 knockout, and Rv1747 complement were grown in 10 ml of Dubos medium standing cultures at 37°C for 14 days. The cultures were diluted in phosphate-buffered saline to an OD600 of 0.02. Infection was induced by injecting 0.2 ml of viable M. tuberculosis (5 x 105 CFU) into a lateral tail vein of 8-week-old adult female BALB/c mice. The infection was monitored by removing the lungs and spleens of infected mice at various intervals, homogenizing the tissues, and plating 10-fold dilutions to determine numbers of CFU of M. tuberculosis (45). The growth curves were compared by graph and statistical analysis. The results for each time point are the means of CFU determinations performed on organs from three mice, and the error bars show the standard deviations (see Fig. 3 and 4). The asterisk indicates that the result is statistically significantly different from that of the wild type by the two-tailed Student t test for groups of unequal variance (P < 0.01), as well as by single-factor analysis of variance (P < 0.01).
Growth of bacteria in mouse cell culture. Bone marrow cells were extracted from the hind legs of 8-week-old adult female BALB/C mice as described previously (44). The cells were resuspended in Iscove’s modified Dulbecco’s medium plus 5% fetal calf serum, 2 mM L-glutamine, and 80 μM -mercaptoethanol, to which was added, for macrophages, 10% by volume of supernatant from L929 cells that produce macrophage colony-stimulating factor or, for dendritic cells, 10% supernatant from X-63 cells transfected with granulocyte-macrophage colony-stimulating factor cDNA. The cells were plated in six-well plates and incubated at 37°C and 5% CO2 atmosphere for 24 h. At day 2 the cells were washed with fresh prewarmed medium, and nonadherent dendritic cells were plated in new six-well plates. Fresh medium was added to the plates containing the adherent bone marrow macrophage cells. The cells were cultured for a further 3 days, and at day 5 they were infected for 6 h at a cell/acid fast bacillus ratio of 2:1. After infection the medium was removed and replaced with fresh medium. The bone marrow macrophages and dendritic cells were lysed with 2% saponin and incubated for 1 h at 37°C. Growth was determined by viable counts on Middlebrook 7H11 agar plates containing Middlebrook OADC supplement at days 0 (6 h postinfection) and 4, 7, and 11 postinfection. The growth curves were compared by graph and statistical analysis. The results for each time point are the means of CFU determinations from at least three wells, and the error bars show the standard deviations. The asterisk indicates that the result is statistically significantly different from that of the wild type by the two-tailed Student t test for groups of unequal variance (P < 0.02), as well as by single-factor analysis of variance (P < 0.02).
Yeast two-hybrid analysis. The pknF and Rv1747 genes were amplified by PCR with the proofreading polymerase Pfu turbo (Stratagene), and template chromosomal DNA from M. tuberculosis H37Rv. For pknF the amplification primers were 5'-CTGCAGCATGCCGCTCGCGGAAGGTT-3' (forward primer) and 5'-CTGCAGCGGCCAGCCGTTGCTTCTGC-3' (reverse primer), both carrying a PstI site at the 5' end. For Rv1747, the primers were 5'-GGATCCGTGCCGATGAGCCAACCAGC-3' and 5'-GGATCCGCACGCCTTGAGGCGAATCT-3', both carrying a 5' BamHI site. The restriction sites were used to clone the genes into the yeast two-hybrid Gal4 activation domain vectors pGAD-C3 and pGAD-C1, respectively, and the Gal4-binding domain vectors pGBD-C3 and pGBD-C1, respectively (22). Constructs were confirmed by sequencing. Saccharomyces cerevisiae Y187 (MAT ura3-52 his3-200 ade2-101 trp1-901 leu2-3,112 gal4 met– gal80 URA3::GAL1UAS-GAL1TATA-lacZ) (17) was cotransformed with Gal4 activation domain and Gal4-binding domain vectors according to the LiAc TRAFO method (16) and plated onto minimal Difco plates lacking Leu and Trp for the selection of transformants. Protein interactions were measured by assaying for lacZ expression by using ONPG (o-nitrophenyl--D-galactopyranoside) as a substrate (28) and are expressed as U/min/mg of protein. All pknF and Rv1747 Gal4 constructs were cotransformed with either empty pGAD or pGBD vectors to eliminate false positives.
Site-directed mutagenesis. Site-directed mutagenesis was carried out according to the Stratagene QuickChange XL site-directed mutagenesis manual, using SoloPack Gold Supercompetent E. coli for transformation. Primers used for mutagenesis are described in Table 1. A mutation was introduced into the PknF active site, where Lys-41 was substituted by Ala. Mutation of the activation loop of PknF was created by individual substitution of Thr-173, -175, and -178 with alanines. Rv1747 was mutated in FHA domain-1, in which Ser-47 was substituted by Ala, and a second mutant was created in which Ser-248 in FHA domain-2 was also substituted by Ala. The presence of the desired mutations was confirmed by sequencing.
RESULTS
Construction of a Rv1747 knockout. The serine-threonine protein kinase pknF is adjacent to and probably in the same operon as Rv1747, an ABC transporter protein with two FHA domains (Fig. 1). Most of the coding region of gene Rv1747 was successfully deleted from M. tuberculosis H37Rv by using homologous recombination, and this was confirmed by PCR, Southern blotting, and DNA microarray analyses (see Materials and Methods). A complement strain was constructed by using the region of DNA including the promoter, Rv1746, and Rv1747 to demonstrate that phenotypic effects were due to deletion of Rv1747 and not to downstream polar effects of the mutation on Rv1748. Microarray experiments with DNA from the complement strain showed that the Rv1747 gene had indeed been replaced and that there was an additional Rv1746 gene (data not shown).
Growth of the Rv1747 mutant is normal in vitro but attenuated in vivo. Growth, as measured by OD600 readings, of the Rv1747 mutant and its complement compared to growth of the H37Rv wild type in 100 ml of Dubos rolling culture was not statistically different (Fig. 2). However, mouse infection with the Rv1747 mutant resulted in severely impaired growth of the mutant compared to the wild-type H37Rv. This was demonstrated in both the lungs (by Student t test, P = 6.4 x 10–4) and spleens (P = 3.5 x 10–4) of mice (Fig. 3). The mutant was also significantly different compared to the complement (P = 1.4 x 10–3 and 4.4 x 10–5 in lungs and spleens, respectively). The mutant strain did, however, persist within the animals. In mouse bone marrow-derived macrophages, growth of the Rv1747 knockout was impaired compared to both the H37Rv wild type and the Rv1747 complement as compared by Student t test (Fig. 4, P = 4.5 x 10–4 and 3.9 x 10–4, respectively). In bone marrow-derived dendritic cells the Rv1747 complement did not grow as well as the H37Rv wild type, but the difference was not significantly different (see Fig. 4), whereas the Rv1747 mutant was again significantly impaired in growth compared to the H37Rv wild type as determined by Student t test (P = 0.019). The mutant was also significantly different compared to the complement (P = 0.034).
Interaction between PknF and Rv1747 is phospho and FHA domain dependent. Using yeast two-hybrid analysis, a strong interaction was observed between PknF and Rv1747 (Fig. 5, bar 5). This interaction was abolished upon mutation of Lys-41 to Ala in the active site of PknF (23) (Fig. 5, bar 8), strongly suggesting that the interaction between PknF and Rv1747 is phosphorylation dependent.
Interaction of PknF with Rv1747 was abolished by mutation to Ala of Thr-173 in the activation loop of PknF (Fig. 5, bar 9). Mutation of Thr-175 or Thr-178 (Fig. 5, bars 10 and 11) reduced the interaction by <3-fold, showing that all three threonines are involved in the interaction but that only Thr-173 is essential for recognition by the FHA-containing Rv1747.
The X-ray structure of FHA1 of Rad53p in complex with a phospho-threonine peptide has indicated that there are six highly conserved residues in the FHA domain (11). Five are located around the peptide-binding site, three of which make interactions with the peptide. Of these three, only two, Arg-70 and Ser-85, bind directly to the pThr residue itself (Fig. 6). We have therefore mutated the equivalent serine residues in the two FHA domains of Rv1747, Ser-47 in FHA-1, and Ser-248 in FHA-2. The interaction of Rv1747 with PknF was reduced by >99% by substitution of Ser-47 by Ala in FHA domain 1 of Rv1747 (Fig. 5, bar 6) and was reduced by 60% by the mutation of Ser-248 to Ala in FHA-2 (Fig. 5, bar 7), suggesting that both FHA domains are involved in the interaction with PknF, but only FHA-1 is essential for this process. These data complement and extend previous observations that the level of phosphorylation of Rv1747 by PknF is dependent on both FHA domains (31). A weak but measurable interaction was noted when Rv1747 was present in both the activation and binding domains of Gal4 showing that this protein interacts with itself (Fig. 5, bar 4). This is consistent with the fact that ABC transporters are generally active as dimers or higher oligomers. Nevertheless, this weak interaction was not affected by either of the mutations in the Rv1747 FHA domains (data not shown).
DISCUSSION
Reversible phosphorylation is a ubiquitous mechanism of signaling transduction. In eukaryotes, proteins are phosphorylated on either tyrosine residues or serine/threonine residues by two distinct but structurally related families of kinases. In contrast, prokaryotes utilize the phosphorylation of histidine residues and subsequent phosphoryl transfer to aspartate residues in so-called two-component phospho-relay systems. Somewhat surprisingly, the genome sequence of M. tuberculosis revealed not only a number of putative two-component histidine kinases but also a complement of 11 eukaryote-like serine-threonine kinases, together with a number of phosphatases (7). It has been postulated that these kinases are likely to play regulatory roles (1), and some of them have been investigated biochemically (2, 23, 29, 30, 37) and structurally (32, 49). Potential regulatory functions have been investigated in some cases (for a review, see reference 33). For example, PknA has been implicated in the regulation of morphological changes associated with cell division (6), PknH is differentially expressed under stress conditions (42), and PknG is linked to cellular glutamate levels (8) and is secreted within macrophages and inhibits phagosome-lysosome fusion (46). Inactivation of PknG in M. bovis BCG led to lysosomal localization and mycobacterial cell death due to the loss of inhibition of phagosome-lysosome fusion (46).
The FHA domain is a modular phosphopeptide recognition motif (9) showing a striking specificity for phosphothreonine-containing epitopes (11). FHA domains have been best studied in their roles in the eukaryotic DNA damage response, in particular, in the context of the Rad53/Chk2 family of checkpoint kinases. Of particular relevance is the observation that these molecules encode modular proteins, each containing an STK domain together with one or more FHA domains. In this context, the FHA domains seem to play a variety of regulatory roles in kinase activation and subcellular localization. However, relatively little is known about how prokaryotic FHA domains function. Notably, four M. tuberculosis STK-encoding genes are linked to genes coding for proteins containing the FHA phosphopeptide recognition domain suggesting, by analogy to Chk2 and Rad53, that they may play a direct role in regulating STK activity. Support for this notion has emerged from studies of PknH, which has been shown to phosphorylate the FHA-containing EmbR protein, an activity that is abolished by mutations in the FHA domain of EmbR that would be predicted to compromise pThr-binding (30).
In the present study we have demonstrated the interaction of PknF with the product of Rv1747. We have corroborated this result by mutating both the PknF kinase and Rv1747. Thus, this interaction was abrogated by mutation of Lys-41, which is known to abolish the kinase activity of PknF (23), providing evidence that the interaction between PknF and Rv1747 is phosphorylation dependent. It was also abolished by mutation of Thr-173 in the activation loop of PknF but reduced only threefold by mutation of Thr-175 or Thr-178. Thr-173 and Thr-175 are homologous with residues Thr-171 and Thr-173 in PknB, identified as autophosphorylation sites in PknB (4). The presence of pThr in FHA domain binding sites has been shown to be essential for interaction (11), suggesting that Thr-173 may be the phosphorylation site of PknF that is recognized by the Rv1747 FHA domains. Alternatively, since mutation of Thr-173 in the activation loop could, potentially, prevent autophosphorylation at other sites in PknF, confident assignment of the actual FHA binding site will require further investigation, and these experiments are in progress. Lastly, the protein-protein interaction was disrupted by mutation of Ser-47 of Rv1747, which is highly conserved in FHA domains and directly interacts with the phosphothreonine residue in both Rad53 FHA-1 and the FHA domain of Chk2 (11, 24). In contrast, binding was reduced by <3-fold upon mutation of Ser-248 in the second putative FHA domain. These results suggest that phospho-dependent interactions between PknF and the FHA domain of Rv1747 form part of a regulatory cascade that involves phosphorylation by PknF that is directed predominantly by binding of FHA-1 to a pThr-containing autophosphorylation site on PknF, possibly located within the kinase activation loop itself.
Rv1747 codes for an ABC-transporter protein. ATP binding cassette (ABC) transport systems occur in all cells of all living species (18) and in M. tuberculosis genes encoding these proteins occupy 2.5% of the genome (5). They are involved in the active transport of solutes across cellular membranes, including the uptake of a large variety of essential nutrients and the secretion of virulence factors, proteases, and toxins. The ABC transporter gene Rv1747 contains only one membrane-spanning domain and one nucleotide binding domain and so is expected to dimerize to function as a transporter (5). There is presently no information as to what substrate it transports. Similarity has been found with the White protein from Drosophila melanogaster, a permease necessary for the transport of pigment precursors into pigment cells responsible for eye color, and with NodI from Rhizobium and Bradyrhizobium strains, a protein implicated in the nodulation process by the export of a polysaccharide (5). It has been suggested that the substance transported may be a lipooligosaccharide (http://genolist.pasteur.fr/TubercuList/) and that the Rv1747 product possibly functions as an exporter protein (5). Among eukaryotic ABC transporters with known structures, Rv1747 is most closely related at the sequence level to TAP (transporter associated with antigen processing; PDB ID 1JJ7, E = 7e-15) (25) and CTFR (cystic fibrosis transmembrane conductance channel; PDB ID 1R10, E = 4e-10) (15), which are both regulated by phosphorylation.
The phenotype of the Rv1747 knockout demonstrated here is the first reported mutation in an FHA domain-containing protein of M. tuberculosis and is intriguing in its very marked reduction in growth in macrophages and in mice. Thus, the mutant exhibited greatly reduced bacterial loads in the mouse lungs and spleens compared to infection by the wild type and therefore falls within the growth in vivo (giv) type of mutant defined by Hingley-Wilson et al. (20). This ABC transporter system therefore appears to be very important for the normal growth of the bacterium during the multiplication phase in macrophages and in mice but did not appear necessary for the persistence phase of growth. Clearly, whether the protein involved is importing or exporting a substrate, its absence results in failure of the Mycobacterium to thrive in mice but makes no difference to its growth in vitro. The fact that Rv1747 interacts with the PknF kinase suggests that its activity is modulated in response to unknown signals that are potentially detected by pknF through its extracellular C-terminal region/domain and transduced into the intracellular milieu via the activity of its STK domain.
ACKNOWLEDGMENTS
We thank Vangelis Stavropoulos and John Brennan for valuable help with the infection experiments. We thank Tanya Parish for providing plasmids p2NIL and pGOAL. We acknowledge BμG@S (the Bacterial Microarray Group at St. George's Hospital Medical School) and especially Jason Hinds and Philip Butcher for the supply of the M. tuberculosis microarray and advice, and The Wellcome Trust for funding the multicollaborative microbial pathogen microarray facility under its Functional Genomics Resources Initiative.
This study was supported by the Medical Research Council.
Dedicated to the memory of M. Joseph Colston, who passed away on 20 February 2003.
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