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PhoP, a key player in Mycobacterium tuberculosis virulence
Review
PhoP, a key player in Mycobacterium tuberculosis virulence Michelle Ryndak1, Shuishu Wang2 and Issar Smith1,3 1
Public Health Research Institute Center, University of Medicine and Dentistry of New Jersey, New Jersey Medical School, 225 Warren Street Newark, NJ 07103, USA 2 Department of Biochemistry, Uniformed Services University of Health Sciences, 4301 Jones Bridge Road, Bethesda, MD 20814, USA 3 Department of Medicine, University of Medicine and Dentistry of New Jersey, New Jersey Medical School, 225 Warren Street Newark, NJ 07103, USA
The Mycobacterium tuberculosis PhoPR two-component system is essential for virulence in animal models of tuberculosis. Recent articles have shown that among the reasons for the attenuation of the M. tuberculosis H37Ra strain is a mutation in the phoP gene that prevents the secretion of proteins that are important for virulence. There is a need for new anti-tubercular therapies because of the emergence of multi-drug-resistant M. tuberculosis strains and also the variable efficacy of the currently used bacille Calmette-Gue´rin vaccine. Because of its major role in M. tuberculosis pathogenicity, PhoP is a potential target candidate. This review summarizes our understanding of PhoPR’s role in virulence and discusses areas in which our knowledge is limited. Mycobacterium tuberculosis pathogenesis and PhoP Modern approaches to studying Mycobacterium tuberculosis (Mtb) virulence have greatly increased our knowledge of potential targets for new therapies for tuberculosis (TB). The PhoPR two-component system (2CS) has attracted attention in the past few years because Mtb phoP mutants are severely attenuated for growth in animal models. Recently, three important papers have appeared that address the role of PhoP in Mtb pathogenicity [1–3] and indicate how a mutation in PhoP can help to explain the loss of virulence in Mtb H37Ra, one of the first experimentally attenuated Mtb strains. These papers have provided the field with important information, but many questions must be answered before we fully understand the contribution of PhoPR to Mtb pathogenesis and how this knowledge can be used to create new anti-tubercular therapies. These issues are discussed in this review. The need for new anti-tubercular therapies TB has been a scourge of mankind throughout human history, yet it remains a major cause of mortality and morbidity. One-third of the world’s 6.67 billion population is infected with Mtb and, annually, there are nine million new cases and almost two million deaths from TB. The emergence of multi-drug-resistant TB and extensively drug-resistant TB has made traditional treatment ineffective in an alarmingly increasing number of cases [4], and Corresponding author: Smith, I. ([email protected])
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the AIDS epidemic has compounded the crisis by providing an immunocompromised population that is highly susceptible to TB [5]. A widely used vaccine, Mycobacterium bovis bacille Calmette-Gue´rin (BCG), was developed in the 1920s, but its efficacy is variable [6]. To overcome these problems, the World Health Organization (WHO) launched a new ‘Stop TB Strategy’ in 2006, and new diagnostics, drugs and vaccines are among its objectives. Basic research in these fields has focused on the identification and characterization of Mtb genes and mechanisms that could be exploited for the treatment and prevention of TB. Strategies to find new targets In the modern era, especially with the sequencing of the genomes of Mtb H37Rv and CDC1551 [7,8], random and directed mutagenesis has been used to identify Mtb genes and processes that are necessary for pathogenicity in tissue culture and animal models of TB (reviewed in Ref. [9]). Another strategy for the identification of virulence factors has been the comparative analyses of closely related virulent and attenuated strains of Mycobacteria. DNA hybridization studies comparing BCG and virulent M. bovis revealed several major differences, including RD1, a chromosomal segment that is present in virulent M. bovis and Mtb but missing from BCG. Introduction of this region into BCG increases its virulence [10], and the deletion of RD1 from Mtb leads to attenuation [11]. Similar comparisons were made between the virulent Mtb H37Rv and the attenuated Mtb H37Ra strains (reviewed in Ref. [12]). Both of these strains have the same ancestral parent, H37, which was isolated in 1905 from a pulmonary TB patient at the Trudeau Sanitorium, and researchers at Trudeau found that the virulence of this strain could be lessened by repeated passages (reviewed in Ref. [13]). Now, with the complete genome sequences of H37Ra, M. bovis and M. bovis BCG available (http://www.tbdb.org/), in addition to those of H37Rv and CDC1551, it is possible to discern DNA differences between these bacteria at the single-nucleotide level. The importance of PhoPR Recently, comparisons of the genome sequences of Mtb H37Rv and H37Ra revealed, among many differences, a single-nucleotide change in the phoP gene of the Mtb PhoPR 2CS [1–3,13]. 2CSs are highly conserved prokar-
0966-842X/$ – see front matter ß 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.tim.2008.08.006 Available online 3 October 2008
Review Box 1. Two-component systems Two-component systems (2CS) are highly conserved prokaryotic signal-transduction systems that in their simplest form consist of a sensor histidine kinase (HK) and an effector response regulator (RR). In response to a specific environmental signal (such as deprivation of carbon, nitrogen or magnesium, osmotic changes or many other stimuli), the HK phosphorylates itself and then transfers this phosphate to the RR, which becomes activated to perform its response function. In most cases, this is the modulation of gene expression, usually through DNA binding, which enables bacterial adaptation to the initial stimulus. Some pathogens use 2CSs to respond to host defense mechanisms, and they are often essential for virulence, with the Salmonella PhoPQ being the most widely studied example. The annotated genome of Mtb H37Rv lists the presence of 11 paired 2CS genes and individual genes for five RRs and two HKs. Many of these have been inactivated and have been shown to have various roles in Mtb physiology and, to a lesser extent, the virulence of this pathogen. PhoP, the RR member of the Mtb PhoPR 2CS and the subject of this review, plays an important part in Mtb pathogenicity. The annotated name, PhoP, is a misnomer because the PhoPR system does not respond to phosphate starvation. This confusing nomenclature is similar to the naming of the enteric bacterial PhoPQ, which responds to Mg2+ limitation and the presence of antibacterial peptides, not phosphate starvation. This problem of mistakes in annotation stems from the highly conserved nature of the 2CS proteins.
yotic signal transduction modules that in their simplest form consist of a sensor histidine kinase (HK) and an effector response regulator (RR) (Box 1). PhoP, the RR member of the Mtb PhoPR 2CS (Box 1), has an important role in Mtb virulence because phoP mutants are greatly
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attenuated for growth in macrophages and mice [14,15]. The nucleotide change in PhoP converts serine 219 (in H37Rv) to a leucine codon (in H37Ra) in the predicted DNA-recognition helix of PhoP (Figure 1) [16]. The H37Ra PhoP was unable to bind to phoP promoter sequences [1,2], unlike the H37Rv PhoP that binds to this region. Importantly, the introduction of the H37Rv phoP into H37Ra increases the ability of the strain to survive and grow in macrophages and in mice, although not to levels observed for H37Rv [3]. This is not surprising; earlier attempts to restore the virulence of H37Ra to the levels of H37Rv, using a cosmid library of H37Rv, were only partly successful [17], which indicates that multiple, unlinked changes were responsible for the attenuation of H37Ra. Comparative sequence analysis of the H37Rv and H37Ra genomes has shown the nature of these changes, many of which are in genes encoding transcriptional regulators, cell-envelope components and other factors that could also explain the loss of virulence of H37Ra [1,12,13]. The role of PhoPR in M. tuberculosis virulence Exactly how the PhoPR 2CS contributes to Mtb virulence has been an important question. Forty-four genes have reduced expression in the phoP mutant [15], and 19 of these are expressed at lower levels in H37Ra than in H37Rv [1]. More than half of the 44 genes are annotated to encode proteins involved in lipid metabolism and secretion, in addition to components of the Mtb cell envelope [15]. Among these are the pks2 and msl3 gene clusters, which encode enzymes for the synthesis and transport of sulfatides (SLs) and the acyl trehaloses (ATs), respectively [18,19]. These trehalose-containing complex lipids, found only in bacteria
Figure 1. The structure of the Mtb PhoP DNA-binding domain. (a) Ribbon diagram of the Mtb PhoP DNA-binding domain structure. The DNA-binding domain of PhoP has the typical fold of the winged helix–turn–helix DNA-binding domain [16]. A four-stranded antiparallel b-sheet at the N terminus is followed by helix a6, which is mostly buried and forms the hydrophobic core for the rest of the domain to pack. A long loop between a7 and a8 is partially disordered. This sequence is termed the transactivation loop because the corresponding regions in the Escherichia coli RRs OmpR [53] (which regulates expression of genes encoding outer-membrane proteins) and PhoB [54] (which controls the expression of genes for phosphate utilization) interact with components of the RNA polymerase. Helix a8 is the recognition helix, which is expected to have an important role in DNA-sequence recognition by binding in the major groove of DNA, as shown in panel (b). The C-terminal b-hairpin is called the wing of the winged helix–turn–helix structure. (b) Model of interactions between PhoP and DNA. This model is constructed on the basis of structural superposition of the recognition helix from the Mtb PhoP DNA-binding domain (Protein Data Bank code 2PMU) [16] with that of the PhoB-DNA complex (Protein Data Bank code 1GXP) [55]. The DNA shown (the magenta trace of the phosphate backbone) is the pho box DNA of the PhoB-DNA complex. Some residues of PhoP that have side chains that are likely to interact with DNA are labeled. Residue Ser219, which is found to be mutated into a leucine in PhoP of H37Ra [1–3,13], is at the middle of the recognition helix and is likely to have hydrogen bonds with DNA. Mutation of Ser219 into a bulkier hydrophobic leucine would lose hydrogen-bond interactions and introduce steric repulsions. Consequently, the DNA-binding affinity and specificity will be affected.
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Review closely related to Mtb (the Mtb complex), are major components of the Mtb cell envelope that is believed to protect against host defense mechanisms [20]. phoP mutants and H37Ra are missing these complex lipids [2,15,21]. However, Mtb strains with individual mutations in the msl3 or the pks2 gene clusters demonstrate essentially normal growth in mice [22–24]. The possibility that the absence of both the SLs and the ATs, as in the phoP mutants, would have a synergistic effect on Mtb virulence has been ruled out because a H37Rv strain with mutations in both pks2 and msl3 exhibited growth similar to wild type in mice [2]. Other components whose absence could explain the attenuation of the phoP mutant are the mycolyltransferase FbpA and the esterase LipF because fbpA and lipF mutants show severe reductions of bacterial growth in macrophages and mice [25,26]. This possibility is also not likely because expressing both lipF and fbpA from a PhoP-independent promoter in a phoP mutant did not restore the ability of this strain to grow at wild-type levels in macrophages (E. Dubnau and I. Smith, unpublished). Recent work has shown other possibilities for the attenuation of phoP mutants, i.e. a link between PhoP function and the secretion of ESAT-6 and CFP-10 [3]. ESAT-6 and CFP10 (also known as EsxA and EsxB, respectively) are secreted mycobacterial proteins that are immunodominant antigens in a majority of human TB cases [27], but it is not currently known what effect the secretion of these proteins has on the ability of Mtb to cause human TB or the severity of the disease. Interestingly, the genes encoding ESAT-6 and CFP10, and the system that secretes them, ESX-1, are located in an extended region encompassing the aforementioned virulence-related RD1. The presence of the ESX-1-secretion system promotes the uptake of mycobacteria into macrophages [28]. Other studies have reported that ESAT-6 secretion by ESX-1 causes apoptosis of a human macrophage cell line [29] and also leads to the secretion of proteins from the macrophage phagolysosome and the production of type-1 interferons [30]. In another pathogenic mycobacterial species, Mycobacterium marinum, secretion of ESAT-6 and CFP-10 is required for intra-macrophage growth of the bacterium and inhibition of phagosome maturation [31]. Genes within the extended RD1 are required for the cellto-cell spread of M. marinum in macrophage and lung epithelial monolayers [32]. ESX-1 also has a role in cellular processes that do not concern pathogenicity because it is involved in the regulation of DNA conjugation in the nonpathogen Mycobacterium smegmatis [33,34]. The link between PhoP and secretion of ESAT-6 and CFP-10 was demonstrated in several ways [3]. Splenocytes (primary splenic immune-system cells, including antigenpresenting cells and T cells) isolated from H37Ra-infected mice were drastically impaired in their ability to produce interferon-g in response to either ESAT-6 or CFP-10, compared to splenocytes from mice infected with H37Rv, and this defect was partially complemented by the introduction of the H37Rv phoP into H37Ra. These results indicated that mice infected with H37Ra were not exposed to ESAT-6 or CFP-10 and that the lack of functional PhoP in H37Ra was a likely reason. This was confirmed in other immunological experiments studying antigen presentation by dendritic cells isolated from similarly infected mice. 530
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Biochemical studies then showed that intracellular levels of ESAT-6 are similar in both H37Rv and H37Ra, but H37Ra is defective in ESAT-6 secretion, as are Mtb phoP mutants. The introduction of the H37Rv PhoP into H37Ra consequently restored ESAT-6 secretion. Other data indicate a relationship between PhoP and ESAT-6 secretion because the gene cluster Rv3616c–Rv3612c, which is required for ESAT-6 secretion [35], is downregulated in H37Ra [1,12] and in a phoP mutant of H37Rv [15]. In addition to ESAT-6 and CFP-10, ESX-1 is also known to secrete other proteins, such as EspA, a protein of unknown function that is encoded by Rv3616c [36]. The secretion of ESAT-6, CFP-10, EspA and other ESX-1 substrates is also co-dependent on the secretion of each substrate [36]. Therefore, the loss of ESAT-6 secretion in a phoP mutant is likely to result from a defect in EspA synthesis. Although the mechanism of PhoP regulation of Rv3616c is unknown, these new results establish a close relationship between PhoP and ESX-1-dependent secretion of ESAT-6, CFP-10 and other proteins that play a part in Mtb pathogenicity, at least in animal models. Important unanswered questions regarding PhoPR The preceding part of this review has demonstrated that PhoP is extremely important for Mtb pathogenicity and discussed its role in regulating a secretory system important for virulence. There are still some important questions about PhoP and its role in virulence, the genes it directly regulates, and how phoPR itself is regulated. The next part of this review highlights some of these areas where knowledge is lacking (Box 2). What other PhoP-controlled genes are important for virulence? The inability of a phoP mutant to secrete ESAT-6, CFP-10 and other ESX-1 substrates accounts for only some of its attenuation. Mtb strains that cannot secrete these two proteins because of mutations in RD1 genes or other loci show 1–2-log-order decreases in bacterial loads during mouse infections [37], and phoP mutants show a 4-logorder loss in similar experiments [14,15], as does H37Ra [2]. The absence of ESX-1 function and the missing SLs, ATs, FbpA and LipF, as well as other components in the phoP mutant could possibly explain the higher attenuation Box 2. Outstanding questions How does the Mtb PhoP control the expression of Rv3616c (espA) and other genes that are important for the secretion of ESX-1 substrates such as ESAT-6 and CFP-10? Is Mtb ESX-1 function important for the ability of the bacterium to infect humans and for the progression of TB? Which other genes regulated by PhoP are important for Mtb pathogenicity other than those involved in ESAT-6 and CFP-10 secretion, and which of these are direct targets for this RR? In addition, what is the role of PhoP in controlling ESX-1 function and DNA conjugation in M. smegmatis? How is PhoPR regulated (i.e. what external signals does the PhoPR system respond to and what are the structural features of PhoR that control this response)? Can the PhoPR 2CS be used as the target for new, effective antitubercular therapies?
Review of this strain. However, this would be difficult to test because PhoP upregulates 44 genes in H37Rv [15]. It would not be realistic to inactivate all or a large number of these genes in the same strain. There are also 70 genes that are more highly expressed in the H37Rv phoP mutant [15], and some of these could potentially negatively impact on Mtb virulence. For example, a Mtb strain lacking the heat-shock regulator HspR cannot repress genes that encode some heat-shock proteins [38], and the hspR mutant shows a 2-log decrease in bacterial load in the lungs of infected mice [39]. It is hypothesized that the higher levels of the heat-shock proteins in the hspR mutant during mouse infection cause a more efficient immuno-surveillance by the innate immune system of the host and an inhibition of Mtb growth. Suggestively, acr2 and htpG, both encoding heat-shock proteins, are expressed at higher levels in the phoP mutant [15], and acr2 is one of the heat-shock genes that is repressed by HspR [38]. Thus, the phoP mouse-attenuated virulence phenotype could also be due to the upregulated synthesis of Acr2 and HtpG, and possibly other proteins. It would be interesting to determine whether the artificially high expression of these heatshock genes could inhibit wild-type Mtb H37Rv growth during murine infections. Which genes are directly regulated by PhoP? PhoP regulates 114 genes, but it has only been reported to bind to the promoter of phoP [40]. Preliminary gel-retardation studies show that specific interactions occur between PhoP and the putative promoter regions of msl3, whiB6 and acr2 (J. Ngohang-Ndong et al., unpublished). msl3 is positively regulated by PhoP, and expression of whiB6 and acr2 is higher in the phoP mutant [15]. Clearly, much work must be performed to find other direct targets for PhoP that could explain its role in pathogenicity. For example, it will be important to test whether PhoP directly regulates Rv3616c (espA) by binding to its promoter. As discussed previously, this gene, encoding EspA that is required for ESAT-6 secretion, is not expressed in the phoP mutant. Rv3617 (ephA), the gene upstream of Rv3616c, is divergently transcribed and, thus, Rv3616c must have its own promoter. Additional PhoP target genes could be found by doing DNA-binding studies with promoter regions of additional genes that have been shown to be regulated by PhoP. More physiological answers could also be obtained by utilizing chromatin immunoprecipitation-type assays to find promoters that bind to PhoP in intact cells exposed to different conditions. When several more promoters have been shown to bind PhoP, it should be possible to define a consensus binding sequence that could be used to identify putative new genes that are directly regulated by PhoP. Because ESX-1 controls DNA conjugation in M. smegmatis [33,34], it also would be important to determine whether PhoP plays a direct part in this process. Which signals are sensed by the PhoPR 2CS? Little is known about the pathway(s) by which PhoP is activated (i.e. what signals are sensed by PhoR, postulated to be the cognate HK for PhoP; see the next section). An Mtb phoP mutant cannot grow in low magnesium con-
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ditions [15], similar to Salmonella phoPQ mutants. The Salmonella PhoPQ 2CS senses low Mg2+ levels and activates expression of genes encoding high-affinity Mg2+ transport systems [41]. However, transcriptional profiling studies with Mtb wild-type and phoP mutant strains growing in high and low Mg2+ show no differences in genes believed to be involved in magnesium transport [15]. In addition, Mg2+ starvation does not upregulate phoPR nor downstream genes that are ordinarily controlled by PhoP [15], as occurs in Salmonella, where a lack of Mg2+ [41] or the presence of antibacterial peptides (which remove Mg2+ from the sensory domain of PhoP [42]) induce phoPQ and the genes in its regulon. Why the Mtb phoP mutant requires high Mg2+ levels is not known, but this could be due to this metal stabilizing a structurally impaired cell envelope, as is observed in some cell-wall mutants in other bacteria [15]. To determine which environmental cue is inducing a PhoR response, bioinformatic analyses were made of the N terminus of PhoR, which has an external loop of 120 amino acids (residues 38–157) that is flanked by potential membrane-spanning domains (Figure 2). A computer search using the Quick Phyre program (http:// www.sbg.bio.ic.ac.uk/phyre/index.cgi) has shown that the most similar structure to the putative PhoR external loop is the middle b-domain of the Escherichia coli YggB, a membrane-spanning small ion sensor that responds to mechanical stress and forms a gatable pore [43]. This structural similarity is interesting because PhoP positively regulates genes for cell-surface components. In addition, phoP mutants show an altered cell envelope, missing several complex lipids and losing acid fast stainability, in addition to showing a different cell morphology [15,21]. It is interesting to speculate that PhoR could be sensing and responding to conditions such as alterations in the structure of the cell wall and/or envelope. There are many cases in which 2CSs are activated by cell-wall or envelope changes, for example, OmpR:EnvZ and CpxRA in E. coli and the Staphylococcus aureus LytRS [44]. Compared to the wild-type H37Rv parent, the phoP mutant is more sensitive to cloxacillin and vancomycin [15], antibiotics that disrupt bacterial cell walls [45]. In enterococci and streptomycetes, vancomycin induces the VanRS 2CS. VanRS then upregulates downstream genes necessary for vancomycin resistance [46,47]. Identification of the ligand(s) or stresses that regulate the activation of PhoR is an important step in characterizing this system. Most 2CS genes, including those of Mtb [48,49], are induced via an autocatalytic loop as the first phase of the response to a specific condition. Thus far, conditions have not been found in which the phoPR operon is upregulated. It would be important to know whether low levels of vancomycin or other cell-surface active antibiotics induce phoPR and downstream genes because this would strongly indicate that the phoPR locus is responding to cell-wall and/or membrane perturbation. Does PhoP interact exclusively with PhoR, and are they co-regulated? Generally, cognate HKs and RRs (i.e. those whose structural genes are closely linked and are usually co-tran531
Review
Figure 2. Model of the PhoR modular structure. The model is based on structural information of homologous HKs such as EnvZ of Escherichia coli [56] and HK835 of Thermotoga maritima [57]. PhoR has an extracytosolic sensor domain flanked by two transmembrane (TM) helices, followed by a HAMP linker domain (a domain commonly found in HKs, adenyl cyclases, methyl-accepting proteins and phosphatases), a dimerization-phosphorylation domain and an ATPase domain. PhoR functions as a dimer with one subunit phosphorylating the phosphoacceptor histidine of the other. The sensor, HAMP and dimerization domains form homodimers. The structure of the entire cytosolic portion of HK835 shows that the HAMP domain and the dimerization domain are involved in an extensive dimer interface [57]. A solution structure of a HAMP domain from Archaeoglobus fulgidus shows a dimer with a four-helical coiled coil [58]. The ATPase domain of HKs has a conserved structure of an a/b sandwich fold, exemplified by the structures of the ATPase domain of the HKs PrrB of Mtb [59], as well as EnvZ [60] and PhoQ [61] of E. coli.
scribed) exclusively interact with each other. One set of experiments indicated that Mtb PhoR and PhoP form a typical, exclusively interacting cognate pair because their structural genes are closely linked, and the macrophage attenuation phenotype of the H37Rv phoP mutant could only be complemented by phoPR and not phoP alone [15]. This indicated that the phoPR genes were cotranscribed and that the upstream phoP mutation prevented phoR expression. This inference has been confirmed directly by quantitative mRNA determinations, which showed that phoP and phoR are cotranscribed and that the phoP mutation in Mtb H37Rv abolishes phoR transcription (M. Ryndak et al., unpublished). However, the macrophage growth defect of a different phoP mutation in MT103, a recent Mtb clinical isolate, could be complemented by phoP alone [14], indicating that either the phoP mutation in this strain did not prevent phoR expression or that PhoP can function independently of PhoR. The essentiality of PhoR is an important point because it is relevant to the suitability of the PhoPR system as a potential target for anti532
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tubercular drugs and is discussed in the last section of this review. Biochemical experiments have shown that PhoR can phosphorylate PhoP [40] but, according to this work, phosphorylation is not required for PhoP binding to the PhoP promoter. However, another report presented the opposite result that PhoP will not bind to the phoP promoter unless it is phosphorylated [2]. The reasons for these disparate results are unknown, but the fact that phoR mutants have phenotypes that are similar to those of the phoP strain (M. Ryndak et al., unpublished) indicates that PhoR-dependent phosphorylation is important for PhoP function in physiological conditions. Recent published studies have provided a possible resolution of these conflicting reports about the effects of PhoP phosphorylation on its DNAbinding function. Although unphosphorylated PhoP does bind to oligonucleotide sequences derived from the phoP promoter, phosphorylation of the protein increases its binding affinity, according to DNase footprinting experiments [50]. This study also showed that phosphorylation of PhoP causes changes in its conformation and might facilitate dimerization of the protein. However, there is an important caveat in the interpretation of these experiments. In both this study [50] and a previous one from the same group, studying the interaction of PhoP with the phoP promoter [40], the PhoP protein from Mtb H37Ra was used. As discussed elsewhere in this review, the PhoP in H37Ra has an S219L mutation that prevents it from binding to DNA [1,2]. It is hoped that these carefully performed experiments measuring the effect of phosphorylation on PhoP binding to DNA will be repeated with the wild-type protein. Outside of the contradictory results on phoP and phoR co-transcription and the necessity of PhoP phosphorylation for its DNA binding, the only other report of how the phoPR operon is regulated comes from reporter-gene studies that indicate that PhoP negatively regulates phoP [40]. This is consistent with the observation made in this report that PhoP binds to a region that overlaps the putative RNApolymerase-binding site and transcriptional start site of the promoter directly upstream of phoPR. Although this conclusion can be questioned because the H37Ra PhoP was used for these studies, as discussed in the previous paragraph, preliminary studies in another group have shown that levels of phoP mRNA are significantly higher in the H37Rv phoP mutant than the wild-type parent, at least during exponential growth (M. Ryndak, et al., unpublished results). These results indicate that phoPR regulation is unlike the classical 2CS paradigm, discussed above, in which the RR is usually an activator of its own structural gene and that of the HK. It will be important to find conditions that induce phoPR expression because this information will enable more meaningful approaches to understand how this important 2CS is regulated. Concluding remarks and future directions There are still many questions about the role of PhoP in the virulence of Mtb (Box 2), but it is clear that this RR plays an important part in this process. Thus, an important question is, ‘How can our current knowledge concerning this protein enable its development as a target for new anti-
Review tubercular therapies?’ An Mtb phoP mutant strain is already being studied as a vaccine strain, and it shows much promise because it is more attenuated than the classical BCG vaccine strain – that is, it does not kill immunodeficient (SCID) mice, whereas BCG does – and the phoP mutant strain also confers protective immunity in mice and guinea pigs against subsequent virulent Mtb challenges [51,52]. These results are important because they indicate that the phoP vaccine strain can be further developed to be an effective live attenuated vaccine strain that could be used against immunocompromised individuals. Among the refinements would be the introduction of other mutations so that the vaccine strain could not revert to virulence. In addition, the fact that Mtb can grow in vitro without a functional PhoP and phoP mutants can survive for extended periods during infections indicates that it would be feasible to try to develop drugs that specifically target the PhoPR 2CS. In this regard, if PhoR is an obligatory phosphate-donating partner of PhoP, its loss should give an attenuation phenotype equivalent to that of a phoP mutation. PhoR is predicted to be an integral membrane protein and has an external domain that is presumably involved in sensing external signals (Figure 2). Antibiotics that interact with the external domains of PhoR and prevent its action might be better anti-tubercular agents than those that target PhoP because these anti-PhoR compounds will not have to be internalized by the bacteria. To provide information on whether PhoR is an obligatory kinase for PhoP, a phoR mutant has just been constructed in Mtb H37Rv, and preliminary results have shown that this strain shows phenotypes very similar to the phoP mutant (M. Ryndak et al., unpublished). It will be important to further characterize the phoR mutant and perform structure-function studies on the sensory domains of PhoR, which will enable rational drug design. It is hoped that this projected research will fulfill the promise of using the PhoPR 2CS as a target for new, effective anti-tubercular therapies. Note added in proof After this review was in press, two articles appeared that provide important information for the first two questions in Box 2. The first [62] demonstrates that the EspR protein, also secreted by the ESX-1 system, is a transcriptional activator of Rv3616c–Rv3614c, binding to the promoter region directly upstream of Rv3616c. It is not currently known how EspR activation of Rv3616c–3614c is related to PhoP’s positive regulation of this operon, but it is not through PhoP control of EspR synthesis because phoP mutants show normal expression of espR, according to the transcriptome experiments discussed in the review. Future experiments should clarify the nature of PhoP and EspR regulation of Rv3616c–Rv3614c. The second article [63] is based on previous studies in the Gambia that showed that people infected with Mycobacterium africanum (a member of the MTB complex that is localized to Western Africa and causes >50% of TB cases in this region) and their household contacts have T cells with an attenuated interferon-g response to ESAT-6 but not to other Mtb antigens [64]. This defective immune response occurs because these T cells are not exposed to ESAT-6 during infection, most likely because
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M. africanum does not secrete this protein. The recent 2008 paper [63] shows that M. tuberculosis and M. africanum are transmitted with equal efficiency from TB patients to household contacts, but the individuals exposed to M. africanum are significantly less likely to progress to active TB than those exposed to M. tuberculosis. This result is the first indication that ESX-1 function is important for the progression from initial infection to active human TB, but this conclusion requires direct evidence that M. africanum strains isolated from TB patients show defects in ESAT-6 secretion. Acknowledgements Work from the authors’ laboratories was supported by NIH grants RO1 GM079185 (S.W.) and AI065987 (I.S.).
References 1 Lee, J.S. et al. (2008) Mutation in the transcriptional regulator PhoP contributes to avirulence of Mycobacterium tuberculosis H37Ra strain. Cell Host Microbe 3, 97–103 2 Chesne-Seck, M.L. et al. (2008) A point mutation in the two-component regulator PhoP–PhoR accounts for the absence of polyketide-derived acyltrehaloses but not that of phthiocerol dimycocerosates in Mycobacterium tuberculosis H37Ra. J. Bacteriol. 190, 1329–1334 3 Frigui, W. et al. (2008) Control of M. tuberculosis ESAT-6 secretion and specific T cell recognition by PhoP. PLoS Pathog. 4, e33 4 World Health Organization (2008) Global Tuberculosis Control: Surveillance, Planning, Financing. WHO 5 Gandhi, N.R. et al. (2006) Extensively drug-resistant tuberculosis as a cause of death in patients co-infected with tuberculosis and HIV in a rural area of South Africa. Lancet 368, 1575–1580 6 Andersen, P. and Doherty, T.M. (2005) The success and failure of BCG – implications for a novel tuberculosis vaccine. Nat. Rev. Microbiol. 3, 656–662 7 Cole, S.T. et al. (1998) Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence. Nature 393, 537–544 8 Fleischmann, R.D. et al. (2002) Whole-genome comparison of Mycobacterium tuberculosis clinical and laboratory strains. J. Bacteriol. 184, 5479–5490 9 Smith, I. (2003) Mycobacterium tuberculosis pathogenesis and molecular determinants of virulence. Clin. Microbiol. Rev. 16, 463–496 10 Pym, A.S. et al. (2002) Loss of RD1 contributed to the attenuation of the live tuberculosis vaccines Mycobacterium bovis BCG and Mycobacterium microti. Mol. Microbiol. 46, 709–717 11 Lewis, K.N. et al. (2003) Deletion of RD1 from Mycobacterium tuberculosis mimics bacille Calmette-Gue´rin attenuation. J. Infect. Dis. 187, 117–123 12 Gao, Q. et al. (2004) Comparative expression studies of a complex phenotype: cord formation in Mycobacterium tuberculosis. Tuberculosis (Edinb.) 84, 188–196 13 Zheng, H. et al. (2008) Genetic basis of virulence attenuation revealed by comparative genomic analysis of Mycobacterium tuberculosis strain H37Ra versus H37Rv. Plos One 3, e2375 14 Perez, E. et al. (2001) An essential role for phoP in Mycobacterium tuberculosis virulence. Mol. Microbiol. 41, 179–187 15 Walters, S.B. et al. (2006) The Mycobacterium tuberculosis PhoPR twocomponent system regulates genes essential for virulence and complex lipid biosynthesis. Mol. Microbiol. 60, 312–330 16 Wang, S. et al. (2007) Structure of the DNA-binding domain of the response regulator PhoP from Mycobacterium tuberculosis. Biochemistry 46, 14751–14761 17 Pascopella, L. et al. (1994) Use of in vivo complementation in Mycobacerium tuberculosis to identify a genomic fragment associated with virulence. Infect. Immun. 62, 1313–1319 18 Sirakova, T.D. et al. (2001) The Mycobacterium tuberculosis pks2 gene encodes the synthase for the hepta- and octamethyl-branched fatty acids required for sulfolipid synthesis. J. Biol. Chem. 276, 16833–16839 19 Dubey, V.S. et al. (2002) Disruption of msl3 abolishes the synthesis of mycolipanoic and mycolipenic acids required for polyacyltrehalose synthesis in Mycobacterium tuberculosis H37Rv and causes cell aggregation. Mol. Microbiol. 45, 1451–1459 533
Review 20 Daffe, M. and Etienne, G. (1999) The capsule of Mycobacterium tuberculosis and its implications for pathogenicity. Tuber. Lung Dis. 79, 153–169 21 Gonzalo Asensio, J. et al. (2006) The virulence-associated twocomponent PhoP–PhoR system controls the biosynthesis of polyketide-derived lipids in Mycobacterium tuberculosis. J. Biol. Chem. 281, 1313–1316 22 Rousseau, C. et al. (2003) Deficiency in mycolipenate- and mycosanoate-derived acyltrehaloses enhances early interactions of Mycobacterium tuberculosis with host cells. Cell. Microbiol. 5, 405–415 23 Converse, S.E. et al. (2003) MmpL8 is required for sulfolipid-1 biosynthesis and Mycobacterium tuberculosis virulence. Proc. Natl. Acad. Sci. U. S. A. 100, 6121–6126 24 Domenech, P. et al. (2004) The role of MmpL8 in sulfatide biogenesis and virulence of Mycobacterium tuberculosis. J. Biol. Chem. 279, 21257–21265 25 Camacho, L.R. et al. (1999) Identification of a virulence gene cluster of Mycobacterium tuberculosis by signature-tagged transposon mutagenesis. Mol. Microbiol. 34, 257–267 26 Copenhaver, R.H. et al. (2004) A mutant of Mycobacterium tuberculosis H37Rv that lacks expression of antigen 85A is attenuated in mice but retains vaccinogenic potential. Infect. Immun. 72, 7084–7095 27 Brodin, P. et al. (2004) ESAT-6 proteins: protective antigens and virulence factors? Trends Microbiol. 12, 500–508 28 Brodin, P. et al. (2006) Dissection of ESAT-6 system 1 of Mycobacterium tuberculosis and impact on immunogenicity and virulence. Infect. Immun. 74, 88–98 29 Derrick, S.C. and Morris, S.L. (2007) The ESAT6 protein of Mycobacterium tuberculosis induces apoptosis of macrophages by activating caspase production. Cell. Microbiol. 9, 1547–1555 30 Stanley, S.A. et al. (2007) The type I IFN response to infection with Mycobacterium tuberculosis requires ESX-1-mediated secretion and contributes to pathogenesis. J. Immunol. 178, 3143–3152 31 Xu, J. et al. (2007) A unique Mycobacterium ESX-1 protein co-secretes with CFP-10/ESAT-6 and is necessary for inhibiting phagosome maturation. Mol. Microbiol. 66, 787–800 32 Gao, L.Y. et al. (2004) A mycobacterial virulence gene cluster extending RD1 is required for cytolysis, bacterial spreading and ESAT-6 secretion. Mol. Microbiol. 53, 1677–1693 33 Flint, J.L. et al. (2004) The RD1 virulence locus of Mycobacterium tuberculosis regulates DNA transfer in Mycobacterium smegmatis. Proc. Natl. Acad. Sci. U. S. A. 101, 12598–12603 34 Coros, A. et al. (2008) The specialized secretory apparatus ESX-1 is essential for DNA transfer in Mycobacterium smegmatis. Mol. Microbiol. 69, 794–808 35 MacGurn, J.A. et al. (2005) A non-RD1 gene cluster is required for Snm secretion in Mycobacterium tuberculosis. Mol. Microbiol. 57, 1653– 1663 36 Fortune, S.M. et al. (2005) Mutually dependent secretion of proteins required for mycobacterial virulence. Proc. Natl. Acad. Sci. U. S. A. 102, 10676–10681 37 Guinn, K.M. et al. (2004) Individual RD1-region genes are required for export of ESAT-6/CFP-10 and for virulence of Mycobacterium tuberculosis. Mol. Microbiol. 51, 359–370 38 Stewart, G.R. et al. (2002) Dissection of the heat-shock response in Mycobacterium tuberculosis using mutants and microarrays. Microbiology 148, 3129–3138 39 Stewart, G.R. et al. (2001) Overexpression of heat-shock proteins reduces survival of Mycobacterium tuberculosis in the chronic phase of infection. Nat. Med. 7, 732–737 40 Gupta, S. et al. (2006) Transcriptional autoregulation by Mycobacterium tuberculosis PhoP involves recognition of novel direct repeat sequences in the regulatory region of the promoter. FEBS Lett. 580, 5328–5338 41 Chamnongpol, S. et al. (2003) Mg2+ sensing by the Mg2+ sensor PhoQ of Salmonella enterica. J. Mol. Biol. 325, 795–807
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Trends in Microbiology Vol.16 No.11 42 Bader, M.W. et al. (2005) Recognition of antimicrobial peptides by a bacterial sensor kinase. Cell 122, 461–472 43 Koprowski, P. and Kubalski, A. (2003) C termini of the Escherichia coli mechanosensitive ion channel (MscS) move apart upon the channel opening. J. Biol. Chem. 278, 11237–11245 44 Mascher, T. (2006) Intramembrane-sensing histidine kinases: a new family of cell envelope stress sensors in Firmicutes bacteria. FEMS Microbiol. Lett. 264, 133–144 45 Nagarajan, R. (1991) Antibacterial activities and modes of action of vancomycin and related glycopeptides. Antimicrob. Agents Chemother. 35, 605–609 46 Arthur, M. et al. (1992) The VanS–VanR two-component regulatory system controls synthesis of depsipeptide peptidoglycan precursors in Enterococcus faecium BM4147. J. Bacteriol. 174, 2582–2591 47 Hutchings, M.I. et al. (2006) The vancomycin resistance VanRS twocomponent signal transduction system of Streptomyces coelicolor. Mol. Microbiol. 59, 923–935 48 Park, H.D. et al. (2003) Rv3133c/dosR is a transcription factor that mediates the hypoxic response of Mycobacterium tuberculosis. Mol. Microbiol. 48, 833–843 49 He, H. et al. (2006) MprAB is a stress-responsive two-component system that directly regulates expression of sigma factors SigB and SigE in Mycobacterium tuberculosis. J. Bacteriol. 188, 2134–2143 50 Sinha, A. et al. (2008) PhoP-PhoP interaction at adjacent PhoP binding sites is influenced by protein phosphorylation. J. Bacteriol. 190, 1317– 1328 51 Martin, C. et al. (2006) The live Mycobacterium tuberculosis phoP mutant strain is more attenuated than BCG and confers protective immunity against tuberculosis in mice and guinea pigs. Vaccine 24, 3408–3419 52 Aguilar, D. et al. (2007) Immunological responses and protective immunity against tuberculosis conferred by vaccination of Balb/C mice with the attenuated Mycobacterium tuberculosis ( phoP) SO2 strain. Clin. Exp. Immunol. 147, 330–338 53 Martinez-Hackert, E. and Stock, A.M. (1997) Structural relationships in the OmpR family of winged-helix transcription factors. J. Mol. Biol. 269, 301–312 54 Makino, K. et al. (1996) DNA binding of PhoB and its interaction with RNA polymerase. J. Mol. Biol. 259, 15–26 55 Blanco, A.G. et al. (2002) Tandem DNA recognition by PhoB, a twocomponent signal transduction transcriptional activator. Structure 10, 701–713 56 Zhu, Y. and Inouye, M. (2004) The HAMP linker in histidine kinase dimeric receptors is critical for symmetric transmembrane signal transduction. J. Biol. Chem. 279, 48152–48158 57 Marina, A. et al. (2005) Structure of the entire cytoplasmic portion of a sensor histidine-kinase protein. EMBO J. 24, 4247–4259 58 Hulko, M. et al. (2006) The HAMP domain structure implies helix rotation in transmembrane signaling. Cell 126, 929–940 59 Nowak, E. et al. (2006) Structural and functional aspects of the sensor histidine kinase PrrB from Mycobacterium tuberculosis. Structure 14, 275–285 60 Tanaka, T. et al. (1998) NMR structure of the histidine kinase domain of the E. coli osmosensor EnvZ. Nature 396, 88–92 61 Marina, A. et al. (2001) Structural and mutational analysis of the PhoQ histidine kinase catalytic domain. Insight into the reaction mechanism. J. Biol. Chem. 276, 41182–41190 62 Ragahavan, S. et al. (2008) Secreted transcription factor controls Mycobacterium tuberculosis virulence. Nature 454, 717–721 63 de Jong, B.C. et al. (2008) Progression to active tuberculosis, but not transmission, varies by Mycobacterium tuberculosis lineage in The Gambia. J. Infect. Dis. 198, 1037–1043 64 de Jong, B.C. et al. (2006) Mycobacterium africanum elicits an attenuated T cell response to early secreted antigenic target, 6 kda, in patients with tuberculosis and their household contacts. J. Infect. Dis. 193, 1279–1286
PhoP, a key player in Mycobacterium tuberculosis virulence Michelle Ryndak1, Shuishu Wang2 and Issar Smith1,3 1
Public Health Research Institute Center, University of Medicine and Dentistry of New Jersey, New Jersey Medical School, 225 Warren Street Newark, NJ 07103, USA 2 Department of Biochemistry, Uniformed Services University of Health Sciences, 4301 Jones Bridge Road, Bethesda, MD 20814, USA 3 Department of Medicine, University of Medicine and Dentistry of New Jersey, New Jersey Medical School, 225 Warren Street Newark, NJ 07103, USA
The Mycobacterium tuberculosis PhoPR two-component system is essential for virulence in animal models of tuberculosis. Recent articles have shown that among the reasons for the attenuation of the M. tuberculosis H37Ra strain is a mutation in the phoP gene that prevents the secretion of proteins that are important for virulence. There is a need for new anti-tubercular therapies because of the emergence of multi-drug-resistant M. tuberculosis strains and also the variable efficacy of the currently used bacille Calmette-Gue´rin vaccine. Because of its major role in M. tuberculosis pathogenicity, PhoP is a potential target candidate. This review summarizes our understanding of PhoPR’s role in virulence and discusses areas in which our knowledge is limited. Mycobacterium tuberculosis pathogenesis and PhoP Modern approaches to studying Mycobacterium tuberculosis (Mtb) virulence have greatly increased our knowledge of potential targets for new therapies for tuberculosis (TB). The PhoPR two-component system (2CS) has attracted attention in the past few years because Mtb phoP mutants are severely attenuated for growth in animal models. Recently, three important papers have appeared that address the role of PhoP in Mtb pathogenicity [1–3] and indicate how a mutation in PhoP can help to explain the loss of virulence in Mtb H37Ra, one of the first experimentally attenuated Mtb strains. These papers have provided the field with important information, but many questions must be answered before we fully understand the contribution of PhoPR to Mtb pathogenesis and how this knowledge can be used to create new anti-tubercular therapies. These issues are discussed in this review. The need for new anti-tubercular therapies TB has been a scourge of mankind throughout human history, yet it remains a major cause of mortality and morbidity. One-third of the world’s 6.67 billion population is infected with Mtb and, annually, there are nine million new cases and almost two million deaths from TB. The emergence of multi-drug-resistant TB and extensively drug-resistant TB has made traditional treatment ineffective in an alarmingly increasing number of cases [4], and Corresponding author: Smith, I. ([email protected])
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the AIDS epidemic has compounded the crisis by providing an immunocompromised population that is highly susceptible to TB [5]. A widely used vaccine, Mycobacterium bovis bacille Calmette-Gue´rin (BCG), was developed in the 1920s, but its efficacy is variable [6]. To overcome these problems, the World Health Organization (WHO) launched a new ‘Stop TB Strategy’ in 2006, and new diagnostics, drugs and vaccines are among its objectives. Basic research in these fields has focused on the identification and characterization of Mtb genes and mechanisms that could be exploited for the treatment and prevention of TB. Strategies to find new targets In the modern era, especially with the sequencing of the genomes of Mtb H37Rv and CDC1551 [7,8], random and directed mutagenesis has been used to identify Mtb genes and processes that are necessary for pathogenicity in tissue culture and animal models of TB (reviewed in Ref. [9]). Another strategy for the identification of virulence factors has been the comparative analyses of closely related virulent and attenuated strains of Mycobacteria. DNA hybridization studies comparing BCG and virulent M. bovis revealed several major differences, including RD1, a chromosomal segment that is present in virulent M. bovis and Mtb but missing from BCG. Introduction of this region into BCG increases its virulence [10], and the deletion of RD1 from Mtb leads to attenuation [11]. Similar comparisons were made between the virulent Mtb H37Rv and the attenuated Mtb H37Ra strains (reviewed in Ref. [12]). Both of these strains have the same ancestral parent, H37, which was isolated in 1905 from a pulmonary TB patient at the Trudeau Sanitorium, and researchers at Trudeau found that the virulence of this strain could be lessened by repeated passages (reviewed in Ref. [13]). Now, with the complete genome sequences of H37Ra, M. bovis and M. bovis BCG available (http://www.tbdb.org/), in addition to those of H37Rv and CDC1551, it is possible to discern DNA differences between these bacteria at the single-nucleotide level. The importance of PhoPR Recently, comparisons of the genome sequences of Mtb H37Rv and H37Ra revealed, among many differences, a single-nucleotide change in the phoP gene of the Mtb PhoPR 2CS [1–3,13]. 2CSs are highly conserved prokar-
0966-842X/$ – see front matter ß 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.tim.2008.08.006 Available online 3 October 2008
Review Box 1. Two-component systems Two-component systems (2CS) are highly conserved prokaryotic signal-transduction systems that in their simplest form consist of a sensor histidine kinase (HK) and an effector response regulator (RR). In response to a specific environmental signal (such as deprivation of carbon, nitrogen or magnesium, osmotic changes or many other stimuli), the HK phosphorylates itself and then transfers this phosphate to the RR, which becomes activated to perform its response function. In most cases, this is the modulation of gene expression, usually through DNA binding, which enables bacterial adaptation to the initial stimulus. Some pathogens use 2CSs to respond to host defense mechanisms, and they are often essential for virulence, with the Salmonella PhoPQ being the most widely studied example. The annotated genome of Mtb H37Rv lists the presence of 11 paired 2CS genes and individual genes for five RRs and two HKs. Many of these have been inactivated and have been shown to have various roles in Mtb physiology and, to a lesser extent, the virulence of this pathogen. PhoP, the RR member of the Mtb PhoPR 2CS and the subject of this review, plays an important part in Mtb pathogenicity. The annotated name, PhoP, is a misnomer because the PhoPR system does not respond to phosphate starvation. This confusing nomenclature is similar to the naming of the enteric bacterial PhoPQ, which responds to Mg2+ limitation and the presence of antibacterial peptides, not phosphate starvation. This problem of mistakes in annotation stems from the highly conserved nature of the 2CS proteins.
yotic signal transduction modules that in their simplest form consist of a sensor histidine kinase (HK) and an effector response regulator (RR) (Box 1). PhoP, the RR member of the Mtb PhoPR 2CS (Box 1), has an important role in Mtb virulence because phoP mutants are greatly
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attenuated for growth in macrophages and mice [14,15]. The nucleotide change in PhoP converts serine 219 (in H37Rv) to a leucine codon (in H37Ra) in the predicted DNA-recognition helix of PhoP (Figure 1) [16]. The H37Ra PhoP was unable to bind to phoP promoter sequences [1,2], unlike the H37Rv PhoP that binds to this region. Importantly, the introduction of the H37Rv phoP into H37Ra increases the ability of the strain to survive and grow in macrophages and in mice, although not to levels observed for H37Rv [3]. This is not surprising; earlier attempts to restore the virulence of H37Ra to the levels of H37Rv, using a cosmid library of H37Rv, were only partly successful [17], which indicates that multiple, unlinked changes were responsible for the attenuation of H37Ra. Comparative sequence analysis of the H37Rv and H37Ra genomes has shown the nature of these changes, many of which are in genes encoding transcriptional regulators, cell-envelope components and other factors that could also explain the loss of virulence of H37Ra [1,12,13]. The role of PhoPR in M. tuberculosis virulence Exactly how the PhoPR 2CS contributes to Mtb virulence has been an important question. Forty-four genes have reduced expression in the phoP mutant [15], and 19 of these are expressed at lower levels in H37Ra than in H37Rv [1]. More than half of the 44 genes are annotated to encode proteins involved in lipid metabolism and secretion, in addition to components of the Mtb cell envelope [15]. Among these are the pks2 and msl3 gene clusters, which encode enzymes for the synthesis and transport of sulfatides (SLs) and the acyl trehaloses (ATs), respectively [18,19]. These trehalose-containing complex lipids, found only in bacteria
Figure 1. The structure of the Mtb PhoP DNA-binding domain. (a) Ribbon diagram of the Mtb PhoP DNA-binding domain structure. The DNA-binding domain of PhoP has the typical fold of the winged helix–turn–helix DNA-binding domain [16]. A four-stranded antiparallel b-sheet at the N terminus is followed by helix a6, which is mostly buried and forms the hydrophobic core for the rest of the domain to pack. A long loop between a7 and a8 is partially disordered. This sequence is termed the transactivation loop because the corresponding regions in the Escherichia coli RRs OmpR [53] (which regulates expression of genes encoding outer-membrane proteins) and PhoB [54] (which controls the expression of genes for phosphate utilization) interact with components of the RNA polymerase. Helix a8 is the recognition helix, which is expected to have an important role in DNA-sequence recognition by binding in the major groove of DNA, as shown in panel (b). The C-terminal b-hairpin is called the wing of the winged helix–turn–helix structure. (b) Model of interactions between PhoP and DNA. This model is constructed on the basis of structural superposition of the recognition helix from the Mtb PhoP DNA-binding domain (Protein Data Bank code 2PMU) [16] with that of the PhoB-DNA complex (Protein Data Bank code 1GXP) [55]. The DNA shown (the magenta trace of the phosphate backbone) is the pho box DNA of the PhoB-DNA complex. Some residues of PhoP that have side chains that are likely to interact with DNA are labeled. Residue Ser219, which is found to be mutated into a leucine in PhoP of H37Ra [1–3,13], is at the middle of the recognition helix and is likely to have hydrogen bonds with DNA. Mutation of Ser219 into a bulkier hydrophobic leucine would lose hydrogen-bond interactions and introduce steric repulsions. Consequently, the DNA-binding affinity and specificity will be affected.
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Review closely related to Mtb (the Mtb complex), are major components of the Mtb cell envelope that is believed to protect against host defense mechanisms [20]. phoP mutants and H37Ra are missing these complex lipids [2,15,21]. However, Mtb strains with individual mutations in the msl3 or the pks2 gene clusters demonstrate essentially normal growth in mice [22–24]. The possibility that the absence of both the SLs and the ATs, as in the phoP mutants, would have a synergistic effect on Mtb virulence has been ruled out because a H37Rv strain with mutations in both pks2 and msl3 exhibited growth similar to wild type in mice [2]. Other components whose absence could explain the attenuation of the phoP mutant are the mycolyltransferase FbpA and the esterase LipF because fbpA and lipF mutants show severe reductions of bacterial growth in macrophages and mice [25,26]. This possibility is also not likely because expressing both lipF and fbpA from a PhoP-independent promoter in a phoP mutant did not restore the ability of this strain to grow at wild-type levels in macrophages (E. Dubnau and I. Smith, unpublished). Recent work has shown other possibilities for the attenuation of phoP mutants, i.e. a link between PhoP function and the secretion of ESAT-6 and CFP-10 [3]. ESAT-6 and CFP10 (also known as EsxA and EsxB, respectively) are secreted mycobacterial proteins that are immunodominant antigens in a majority of human TB cases [27], but it is not currently known what effect the secretion of these proteins has on the ability of Mtb to cause human TB or the severity of the disease. Interestingly, the genes encoding ESAT-6 and CFP10, and the system that secretes them, ESX-1, are located in an extended region encompassing the aforementioned virulence-related RD1. The presence of the ESX-1-secretion system promotes the uptake of mycobacteria into macrophages [28]. Other studies have reported that ESAT-6 secretion by ESX-1 causes apoptosis of a human macrophage cell line [29] and also leads to the secretion of proteins from the macrophage phagolysosome and the production of type-1 interferons [30]. In another pathogenic mycobacterial species, Mycobacterium marinum, secretion of ESAT-6 and CFP-10 is required for intra-macrophage growth of the bacterium and inhibition of phagosome maturation [31]. Genes within the extended RD1 are required for the cellto-cell spread of M. marinum in macrophage and lung epithelial monolayers [32]. ESX-1 also has a role in cellular processes that do not concern pathogenicity because it is involved in the regulation of DNA conjugation in the nonpathogen Mycobacterium smegmatis [33,34]. The link between PhoP and secretion of ESAT-6 and CFP-10 was demonstrated in several ways [3]. Splenocytes (primary splenic immune-system cells, including antigenpresenting cells and T cells) isolated from H37Ra-infected mice were drastically impaired in their ability to produce interferon-g in response to either ESAT-6 or CFP-10, compared to splenocytes from mice infected with H37Rv, and this defect was partially complemented by the introduction of the H37Rv phoP into H37Ra. These results indicated that mice infected with H37Ra were not exposed to ESAT-6 or CFP-10 and that the lack of functional PhoP in H37Ra was a likely reason. This was confirmed in other immunological experiments studying antigen presentation by dendritic cells isolated from similarly infected mice. 530
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Biochemical studies then showed that intracellular levels of ESAT-6 are similar in both H37Rv and H37Ra, but H37Ra is defective in ESAT-6 secretion, as are Mtb phoP mutants. The introduction of the H37Rv PhoP into H37Ra consequently restored ESAT-6 secretion. Other data indicate a relationship between PhoP and ESAT-6 secretion because the gene cluster Rv3616c–Rv3612c, which is required for ESAT-6 secretion [35], is downregulated in H37Ra [1,12] and in a phoP mutant of H37Rv [15]. In addition to ESAT-6 and CFP-10, ESX-1 is also known to secrete other proteins, such as EspA, a protein of unknown function that is encoded by Rv3616c [36]. The secretion of ESAT-6, CFP-10, EspA and other ESX-1 substrates is also co-dependent on the secretion of each substrate [36]. Therefore, the loss of ESAT-6 secretion in a phoP mutant is likely to result from a defect in EspA synthesis. Although the mechanism of PhoP regulation of Rv3616c is unknown, these new results establish a close relationship between PhoP and ESX-1-dependent secretion of ESAT-6, CFP-10 and other proteins that play a part in Mtb pathogenicity, at least in animal models. Important unanswered questions regarding PhoPR The preceding part of this review has demonstrated that PhoP is extremely important for Mtb pathogenicity and discussed its role in regulating a secretory system important for virulence. There are still some important questions about PhoP and its role in virulence, the genes it directly regulates, and how phoPR itself is regulated. The next part of this review highlights some of these areas where knowledge is lacking (Box 2). What other PhoP-controlled genes are important for virulence? The inability of a phoP mutant to secrete ESAT-6, CFP-10 and other ESX-1 substrates accounts for only some of its attenuation. Mtb strains that cannot secrete these two proteins because of mutations in RD1 genes or other loci show 1–2-log-order decreases in bacterial loads during mouse infections [37], and phoP mutants show a 4-logorder loss in similar experiments [14,15], as does H37Ra [2]. The absence of ESX-1 function and the missing SLs, ATs, FbpA and LipF, as well as other components in the phoP mutant could possibly explain the higher attenuation Box 2. Outstanding questions How does the Mtb PhoP control the expression of Rv3616c (espA) and other genes that are important for the secretion of ESX-1 substrates such as ESAT-6 and CFP-10? Is Mtb ESX-1 function important for the ability of the bacterium to infect humans and for the progression of TB? Which other genes regulated by PhoP are important for Mtb pathogenicity other than those involved in ESAT-6 and CFP-10 secretion, and which of these are direct targets for this RR? In addition, what is the role of PhoP in controlling ESX-1 function and DNA conjugation in M. smegmatis? How is PhoPR regulated (i.e. what external signals does the PhoPR system respond to and what are the structural features of PhoR that control this response)? Can the PhoPR 2CS be used as the target for new, effective antitubercular therapies?
Review of this strain. However, this would be difficult to test because PhoP upregulates 44 genes in H37Rv [15]. It would not be realistic to inactivate all or a large number of these genes in the same strain. There are also 70 genes that are more highly expressed in the H37Rv phoP mutant [15], and some of these could potentially negatively impact on Mtb virulence. For example, a Mtb strain lacking the heat-shock regulator HspR cannot repress genes that encode some heat-shock proteins [38], and the hspR mutant shows a 2-log decrease in bacterial load in the lungs of infected mice [39]. It is hypothesized that the higher levels of the heat-shock proteins in the hspR mutant during mouse infection cause a more efficient immuno-surveillance by the innate immune system of the host and an inhibition of Mtb growth. Suggestively, acr2 and htpG, both encoding heat-shock proteins, are expressed at higher levels in the phoP mutant [15], and acr2 is one of the heat-shock genes that is repressed by HspR [38]. Thus, the phoP mouse-attenuated virulence phenotype could also be due to the upregulated synthesis of Acr2 and HtpG, and possibly other proteins. It would be interesting to determine whether the artificially high expression of these heatshock genes could inhibit wild-type Mtb H37Rv growth during murine infections. Which genes are directly regulated by PhoP? PhoP regulates 114 genes, but it has only been reported to bind to the promoter of phoP [40]. Preliminary gel-retardation studies show that specific interactions occur between PhoP and the putative promoter regions of msl3, whiB6 and acr2 (J. Ngohang-Ndong et al., unpublished). msl3 is positively regulated by PhoP, and expression of whiB6 and acr2 is higher in the phoP mutant [15]. Clearly, much work must be performed to find other direct targets for PhoP that could explain its role in pathogenicity. For example, it will be important to test whether PhoP directly regulates Rv3616c (espA) by binding to its promoter. As discussed previously, this gene, encoding EspA that is required for ESAT-6 secretion, is not expressed in the phoP mutant. Rv3617 (ephA), the gene upstream of Rv3616c, is divergently transcribed and, thus, Rv3616c must have its own promoter. Additional PhoP target genes could be found by doing DNA-binding studies with promoter regions of additional genes that have been shown to be regulated by PhoP. More physiological answers could also be obtained by utilizing chromatin immunoprecipitation-type assays to find promoters that bind to PhoP in intact cells exposed to different conditions. When several more promoters have been shown to bind PhoP, it should be possible to define a consensus binding sequence that could be used to identify putative new genes that are directly regulated by PhoP. Because ESX-1 controls DNA conjugation in M. smegmatis [33,34], it also would be important to determine whether PhoP plays a direct part in this process. Which signals are sensed by the PhoPR 2CS? Little is known about the pathway(s) by which PhoP is activated (i.e. what signals are sensed by PhoR, postulated to be the cognate HK for PhoP; see the next section). An Mtb phoP mutant cannot grow in low magnesium con-
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ditions [15], similar to Salmonella phoPQ mutants. The Salmonella PhoPQ 2CS senses low Mg2+ levels and activates expression of genes encoding high-affinity Mg2+ transport systems [41]. However, transcriptional profiling studies with Mtb wild-type and phoP mutant strains growing in high and low Mg2+ show no differences in genes believed to be involved in magnesium transport [15]. In addition, Mg2+ starvation does not upregulate phoPR nor downstream genes that are ordinarily controlled by PhoP [15], as occurs in Salmonella, where a lack of Mg2+ [41] or the presence of antibacterial peptides (which remove Mg2+ from the sensory domain of PhoP [42]) induce phoPQ and the genes in its regulon. Why the Mtb phoP mutant requires high Mg2+ levels is not known, but this could be due to this metal stabilizing a structurally impaired cell envelope, as is observed in some cell-wall mutants in other bacteria [15]. To determine which environmental cue is inducing a PhoR response, bioinformatic analyses were made of the N terminus of PhoR, which has an external loop of 120 amino acids (residues 38–157) that is flanked by potential membrane-spanning domains (Figure 2). A computer search using the Quick Phyre program (http:// www.sbg.bio.ic.ac.uk/phyre/index.cgi) has shown that the most similar structure to the putative PhoR external loop is the middle b-domain of the Escherichia coli YggB, a membrane-spanning small ion sensor that responds to mechanical stress and forms a gatable pore [43]. This structural similarity is interesting because PhoP positively regulates genes for cell-surface components. In addition, phoP mutants show an altered cell envelope, missing several complex lipids and losing acid fast stainability, in addition to showing a different cell morphology [15,21]. It is interesting to speculate that PhoR could be sensing and responding to conditions such as alterations in the structure of the cell wall and/or envelope. There are many cases in which 2CSs are activated by cell-wall or envelope changes, for example, OmpR:EnvZ and CpxRA in E. coli and the Staphylococcus aureus LytRS [44]. Compared to the wild-type H37Rv parent, the phoP mutant is more sensitive to cloxacillin and vancomycin [15], antibiotics that disrupt bacterial cell walls [45]. In enterococci and streptomycetes, vancomycin induces the VanRS 2CS. VanRS then upregulates downstream genes necessary for vancomycin resistance [46,47]. Identification of the ligand(s) or stresses that regulate the activation of PhoR is an important step in characterizing this system. Most 2CS genes, including those of Mtb [48,49], are induced via an autocatalytic loop as the first phase of the response to a specific condition. Thus far, conditions have not been found in which the phoPR operon is upregulated. It would be important to know whether low levels of vancomycin or other cell-surface active antibiotics induce phoPR and downstream genes because this would strongly indicate that the phoPR locus is responding to cell-wall and/or membrane perturbation. Does PhoP interact exclusively with PhoR, and are they co-regulated? Generally, cognate HKs and RRs (i.e. those whose structural genes are closely linked and are usually co-tran531
Review
Figure 2. Model of the PhoR modular structure. The model is based on structural information of homologous HKs such as EnvZ of Escherichia coli [56] and HK835 of Thermotoga maritima [57]. PhoR has an extracytosolic sensor domain flanked by two transmembrane (TM) helices, followed by a HAMP linker domain (a domain commonly found in HKs, adenyl cyclases, methyl-accepting proteins and phosphatases), a dimerization-phosphorylation domain and an ATPase domain. PhoR functions as a dimer with one subunit phosphorylating the phosphoacceptor histidine of the other. The sensor, HAMP and dimerization domains form homodimers. The structure of the entire cytosolic portion of HK835 shows that the HAMP domain and the dimerization domain are involved in an extensive dimer interface [57]. A solution structure of a HAMP domain from Archaeoglobus fulgidus shows a dimer with a four-helical coiled coil [58]. The ATPase domain of HKs has a conserved structure of an a/b sandwich fold, exemplified by the structures of the ATPase domain of the HKs PrrB of Mtb [59], as well as EnvZ [60] and PhoQ [61] of E. coli.
scribed) exclusively interact with each other. One set of experiments indicated that Mtb PhoR and PhoP form a typical, exclusively interacting cognate pair because their structural genes are closely linked, and the macrophage attenuation phenotype of the H37Rv phoP mutant could only be complemented by phoPR and not phoP alone [15]. This indicated that the phoPR genes were cotranscribed and that the upstream phoP mutation prevented phoR expression. This inference has been confirmed directly by quantitative mRNA determinations, which showed that phoP and phoR are cotranscribed and that the phoP mutation in Mtb H37Rv abolishes phoR transcription (M. Ryndak et al., unpublished). However, the macrophage growth defect of a different phoP mutation in MT103, a recent Mtb clinical isolate, could be complemented by phoP alone [14], indicating that either the phoP mutation in this strain did not prevent phoR expression or that PhoP can function independently of PhoR. The essentiality of PhoR is an important point because it is relevant to the suitability of the PhoPR system as a potential target for anti532
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tubercular drugs and is discussed in the last section of this review. Biochemical experiments have shown that PhoR can phosphorylate PhoP [40] but, according to this work, phosphorylation is not required for PhoP binding to the PhoP promoter. However, another report presented the opposite result that PhoP will not bind to the phoP promoter unless it is phosphorylated [2]. The reasons for these disparate results are unknown, but the fact that phoR mutants have phenotypes that are similar to those of the phoP strain (M. Ryndak et al., unpublished) indicates that PhoR-dependent phosphorylation is important for PhoP function in physiological conditions. Recent published studies have provided a possible resolution of these conflicting reports about the effects of PhoP phosphorylation on its DNAbinding function. Although unphosphorylated PhoP does bind to oligonucleotide sequences derived from the phoP promoter, phosphorylation of the protein increases its binding affinity, according to DNase footprinting experiments [50]. This study also showed that phosphorylation of PhoP causes changes in its conformation and might facilitate dimerization of the protein. However, there is an important caveat in the interpretation of these experiments. In both this study [50] and a previous one from the same group, studying the interaction of PhoP with the phoP promoter [40], the PhoP protein from Mtb H37Ra was used. As discussed elsewhere in this review, the PhoP in H37Ra has an S219L mutation that prevents it from binding to DNA [1,2]. It is hoped that these carefully performed experiments measuring the effect of phosphorylation on PhoP binding to DNA will be repeated with the wild-type protein. Outside of the contradictory results on phoP and phoR co-transcription and the necessity of PhoP phosphorylation for its DNA binding, the only other report of how the phoPR operon is regulated comes from reporter-gene studies that indicate that PhoP negatively regulates phoP [40]. This is consistent with the observation made in this report that PhoP binds to a region that overlaps the putative RNApolymerase-binding site and transcriptional start site of the promoter directly upstream of phoPR. Although this conclusion can be questioned because the H37Ra PhoP was used for these studies, as discussed in the previous paragraph, preliminary studies in another group have shown that levels of phoP mRNA are significantly higher in the H37Rv phoP mutant than the wild-type parent, at least during exponential growth (M. Ryndak, et al., unpublished results). These results indicate that phoPR regulation is unlike the classical 2CS paradigm, discussed above, in which the RR is usually an activator of its own structural gene and that of the HK. It will be important to find conditions that induce phoPR expression because this information will enable more meaningful approaches to understand how this important 2CS is regulated. Concluding remarks and future directions There are still many questions about the role of PhoP in the virulence of Mtb (Box 2), but it is clear that this RR plays an important part in this process. Thus, an important question is, ‘How can our current knowledge concerning this protein enable its development as a target for new anti-
Review tubercular therapies?’ An Mtb phoP mutant strain is already being studied as a vaccine strain, and it shows much promise because it is more attenuated than the classical BCG vaccine strain – that is, it does not kill immunodeficient (SCID) mice, whereas BCG does – and the phoP mutant strain also confers protective immunity in mice and guinea pigs against subsequent virulent Mtb challenges [51,52]. These results are important because they indicate that the phoP vaccine strain can be further developed to be an effective live attenuated vaccine strain that could be used against immunocompromised individuals. Among the refinements would be the introduction of other mutations so that the vaccine strain could not revert to virulence. In addition, the fact that Mtb can grow in vitro without a functional PhoP and phoP mutants can survive for extended periods during infections indicates that it would be feasible to try to develop drugs that specifically target the PhoPR 2CS. In this regard, if PhoR is an obligatory phosphate-donating partner of PhoP, its loss should give an attenuation phenotype equivalent to that of a phoP mutation. PhoR is predicted to be an integral membrane protein and has an external domain that is presumably involved in sensing external signals (Figure 2). Antibiotics that interact with the external domains of PhoR and prevent its action might be better anti-tubercular agents than those that target PhoP because these anti-PhoR compounds will not have to be internalized by the bacteria. To provide information on whether PhoR is an obligatory kinase for PhoP, a phoR mutant has just been constructed in Mtb H37Rv, and preliminary results have shown that this strain shows phenotypes very similar to the phoP mutant (M. Ryndak et al., unpublished). It will be important to further characterize the phoR mutant and perform structure-function studies on the sensory domains of PhoR, which will enable rational drug design. It is hoped that this projected research will fulfill the promise of using the PhoPR 2CS as a target for new, effective anti-tubercular therapies. Note added in proof After this review was in press, two articles appeared that provide important information for the first two questions in Box 2. The first [62] demonstrates that the EspR protein, also secreted by the ESX-1 system, is a transcriptional activator of Rv3616c–Rv3614c, binding to the promoter region directly upstream of Rv3616c. It is not currently known how EspR activation of Rv3616c–3614c is related to PhoP’s positive regulation of this operon, but it is not through PhoP control of EspR synthesis because phoP mutants show normal expression of espR, according to the transcriptome experiments discussed in the review. Future experiments should clarify the nature of PhoP and EspR regulation of Rv3616c–Rv3614c. The second article [63] is based on previous studies in the Gambia that showed that people infected with Mycobacterium africanum (a member of the MTB complex that is localized to Western Africa and causes >50% of TB cases in this region) and their household contacts have T cells with an attenuated interferon-g response to ESAT-6 but not to other Mtb antigens [64]. This defective immune response occurs because these T cells are not exposed to ESAT-6 during infection, most likely because
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M. africanum does not secrete this protein. The recent 2008 paper [63] shows that M. tuberculosis and M. africanum are transmitted with equal efficiency from TB patients to household contacts, but the individuals exposed to M. africanum are significantly less likely to progress to active TB than those exposed to M. tuberculosis. This result is the first indication that ESX-1 function is important for the progression from initial infection to active human TB, but this conclusion requires direct evidence that M. africanum strains isolated from TB patients show defects in ESAT-6 secretion. Acknowledgements Work from the authors’ laboratories was supported by NIH grants RO1 GM079185 (S.W.) and AI065987 (I.S.).
References 1 Lee, J.S. et al. (2008) Mutation in the transcriptional regulator PhoP contributes to avirulence of Mycobacterium tuberculosis H37Ra strain. Cell Host Microbe 3, 97–103 2 Chesne-Seck, M.L. et al. (2008) A point mutation in the two-component regulator PhoP–PhoR accounts for the absence of polyketide-derived acyltrehaloses but not that of phthiocerol dimycocerosates in Mycobacterium tuberculosis H37Ra. J. Bacteriol. 190, 1329–1334 3 Frigui, W. et al. (2008) Control of M. tuberculosis ESAT-6 secretion and specific T cell recognition by PhoP. PLoS Pathog. 4, e33 4 World Health Organization (2008) Global Tuberculosis Control: Surveillance, Planning, Financing. WHO 5 Gandhi, N.R. et al. (2006) Extensively drug-resistant tuberculosis as a cause of death in patients co-infected with tuberculosis and HIV in a rural area of South Africa. Lancet 368, 1575–1580 6 Andersen, P. and Doherty, T.M. (2005) The success and failure of BCG – implications for a novel tuberculosis vaccine. Nat. Rev. Microbiol. 3, 656–662 7 Cole, S.T. et al. (1998) Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence. Nature 393, 537–544 8 Fleischmann, R.D. et al. (2002) Whole-genome comparison of Mycobacterium tuberculosis clinical and laboratory strains. J. Bacteriol. 184, 5479–5490 9 Smith, I. (2003) Mycobacterium tuberculosis pathogenesis and molecular determinants of virulence. Clin. Microbiol. Rev. 16, 463–496 10 Pym, A.S. et al. (2002) Loss of RD1 contributed to the attenuation of the live tuberculosis vaccines Mycobacterium bovis BCG and Mycobacterium microti. Mol. Microbiol. 46, 709–717 11 Lewis, K.N. et al. (2003) Deletion of RD1 from Mycobacterium tuberculosis mimics bacille Calmette-Gue´rin attenuation. J. Infect. Dis. 187, 117–123 12 Gao, Q. et al. (2004) Comparative expression studies of a complex phenotype: cord formation in Mycobacterium tuberculosis. Tuberculosis (Edinb.) 84, 188–196 13 Zheng, H. et al. (2008) Genetic basis of virulence attenuation revealed by comparative genomic analysis of Mycobacterium tuberculosis strain H37Ra versus H37Rv. Plos One 3, e2375 14 Perez, E. et al. (2001) An essential role for phoP in Mycobacterium tuberculosis virulence. Mol. Microbiol. 41, 179–187 15 Walters, S.B. et al. (2006) The Mycobacterium tuberculosis PhoPR twocomponent system regulates genes essential for virulence and complex lipid biosynthesis. Mol. Microbiol. 60, 312–330 16 Wang, S. et al. (2007) Structure of the DNA-binding domain of the response regulator PhoP from Mycobacterium tuberculosis. Biochemistry 46, 14751–14761 17 Pascopella, L. et al. (1994) Use of in vivo complementation in Mycobacerium tuberculosis to identify a genomic fragment associated with virulence. Infect. Immun. 62, 1313–1319 18 Sirakova, T.D. et al. (2001) The Mycobacterium tuberculosis pks2 gene encodes the synthase for the hepta- and octamethyl-branched fatty acids required for sulfolipid synthesis. J. Biol. Chem. 276, 16833–16839 19 Dubey, V.S. et al. (2002) Disruption of msl3 abolishes the synthesis of mycolipanoic and mycolipenic acids required for polyacyltrehalose synthesis in Mycobacterium tuberculosis H37Rv and causes cell aggregation. Mol. Microbiol. 45, 1451–1459 533
Review 20 Daffe, M. and Etienne, G. (1999) The capsule of Mycobacterium tuberculosis and its implications for pathogenicity. Tuber. Lung Dis. 79, 153–169 21 Gonzalo Asensio, J. et al. (2006) The virulence-associated twocomponent PhoP–PhoR system controls the biosynthesis of polyketide-derived lipids in Mycobacterium tuberculosis. J. Biol. Chem. 281, 1313–1316 22 Rousseau, C. et al. (2003) Deficiency in mycolipenate- and mycosanoate-derived acyltrehaloses enhances early interactions of Mycobacterium tuberculosis with host cells. Cell. Microbiol. 5, 405–415 23 Converse, S.E. et al. (2003) MmpL8 is required for sulfolipid-1 biosynthesis and Mycobacterium tuberculosis virulence. Proc. Natl. Acad. Sci. U. S. A. 100, 6121–6126 24 Domenech, P. et al. (2004) The role of MmpL8 in sulfatide biogenesis and virulence of Mycobacterium tuberculosis. J. Biol. Chem. 279, 21257–21265 25 Camacho, L.R. et al. (1999) Identification of a virulence gene cluster of Mycobacterium tuberculosis by signature-tagged transposon mutagenesis. Mol. Microbiol. 34, 257–267 26 Copenhaver, R.H. et al. (2004) A mutant of Mycobacterium tuberculosis H37Rv that lacks expression of antigen 85A is attenuated in mice but retains vaccinogenic potential. Infect. Immun. 72, 7084–7095 27 Brodin, P. et al. (2004) ESAT-6 proteins: protective antigens and virulence factors? Trends Microbiol. 12, 500–508 28 Brodin, P. et al. (2006) Dissection of ESAT-6 system 1 of Mycobacterium tuberculosis and impact on immunogenicity and virulence. Infect. Immun. 74, 88–98 29 Derrick, S.C. and Morris, S.L. (2007) The ESAT6 protein of Mycobacterium tuberculosis induces apoptosis of macrophages by activating caspase production. Cell. Microbiol. 9, 1547–1555 30 Stanley, S.A. et al. (2007) The type I IFN response to infection with Mycobacterium tuberculosis requires ESX-1-mediated secretion and contributes to pathogenesis. J. Immunol. 178, 3143–3152 31 Xu, J. et al. (2007) A unique Mycobacterium ESX-1 protein co-secretes with CFP-10/ESAT-6 and is necessary for inhibiting phagosome maturation. Mol. Microbiol. 66, 787–800 32 Gao, L.Y. et al. (2004) A mycobacterial virulence gene cluster extending RD1 is required for cytolysis, bacterial spreading and ESAT-6 secretion. Mol. Microbiol. 53, 1677–1693 33 Flint, J.L. et al. (2004) The RD1 virulence locus of Mycobacterium tuberculosis regulates DNA transfer in Mycobacterium smegmatis. Proc. Natl. Acad. Sci. U. S. A. 101, 12598–12603 34 Coros, A. et al. (2008) The specialized secretory apparatus ESX-1 is essential for DNA transfer in Mycobacterium smegmatis. Mol. Microbiol. 69, 794–808 35 MacGurn, J.A. et al. (2005) A non-RD1 gene cluster is required for Snm secretion in Mycobacterium tuberculosis. Mol. Microbiol. 57, 1653– 1663 36 Fortune, S.M. et al. (2005) Mutually dependent secretion of proteins required for mycobacterial virulence. Proc. Natl. Acad. Sci. U. S. A. 102, 10676–10681 37 Guinn, K.M. et al. (2004) Individual RD1-region genes are required for export of ESAT-6/CFP-10 and for virulence of Mycobacterium tuberculosis. Mol. Microbiol. 51, 359–370 38 Stewart, G.R. et al. (2002) Dissection of the heat-shock response in Mycobacterium tuberculosis using mutants and microarrays. Microbiology 148, 3129–3138 39 Stewart, G.R. et al. (2001) Overexpression of heat-shock proteins reduces survival of Mycobacterium tuberculosis in the chronic phase of infection. Nat. Med. 7, 732–737 40 Gupta, S. et al. (2006) Transcriptional autoregulation by Mycobacterium tuberculosis PhoP involves recognition of novel direct repeat sequences in the regulatory region of the promoter. FEBS Lett. 580, 5328–5338 41 Chamnongpol, S. et al. (2003) Mg2+ sensing by the Mg2+ sensor PhoQ of Salmonella enterica. J. Mol. Biol. 325, 795–807
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Trends in Microbiology Vol.16 No.11 42 Bader, M.W. et al. (2005) Recognition of antimicrobial peptides by a bacterial sensor kinase. Cell 122, 461–472 43 Koprowski, P. and Kubalski, A. (2003) C termini of the Escherichia coli mechanosensitive ion channel (MscS) move apart upon the channel opening. J. Biol. Chem. 278, 11237–11245 44 Mascher, T. (2006) Intramembrane-sensing histidine kinases: a new family of cell envelope stress sensors in Firmicutes bacteria. FEMS Microbiol. Lett. 264, 133–144 45 Nagarajan, R. (1991) Antibacterial activities and modes of action of vancomycin and related glycopeptides. Antimicrob. Agents Chemother. 35, 605–609 46 Arthur, M. et al. (1992) The VanS–VanR two-component regulatory system controls synthesis of depsipeptide peptidoglycan precursors in Enterococcus faecium BM4147. J. Bacteriol. 174, 2582–2591 47 Hutchings, M.I. et al. (2006) The vancomycin resistance VanRS twocomponent signal transduction system of Streptomyces coelicolor. Mol. Microbiol. 59, 923–935 48 Park, H.D. et al. (2003) Rv3133c/dosR is a transcription factor that mediates the hypoxic response of Mycobacterium tuberculosis. Mol. Microbiol. 48, 833–843 49 He, H. et al. (2006) MprAB is a stress-responsive two-component system that directly regulates expression of sigma factors SigB and SigE in Mycobacterium tuberculosis. J. Bacteriol. 188, 2134–2143 50 Sinha, A. et al. (2008) PhoP-PhoP interaction at adjacent PhoP binding sites is influenced by protein phosphorylation. J. Bacteriol. 190, 1317– 1328 51 Martin, C. et al. (2006) The live Mycobacterium tuberculosis phoP mutant strain is more attenuated than BCG and confers protective immunity against tuberculosis in mice and guinea pigs. Vaccine 24, 3408–3419 52 Aguilar, D. et al. (2007) Immunological responses and protective immunity against tuberculosis conferred by vaccination of Balb/C mice with the attenuated Mycobacterium tuberculosis ( phoP) SO2 strain. Clin. Exp. Immunol. 147, 330–338 53 Martinez-Hackert, E. and Stock, A.M. (1997) Structural relationships in the OmpR family of winged-helix transcription factors. J. Mol. Biol. 269, 301–312 54 Makino, K. et al. (1996) DNA binding of PhoB and its interaction with RNA polymerase. J. Mol. Biol. 259, 15–26 55 Blanco, A.G. et al. (2002) Tandem DNA recognition by PhoB, a twocomponent signal transduction transcriptional activator. Structure 10, 701–713 56 Zhu, Y. and Inouye, M. (2004) The HAMP linker in histidine kinase dimeric receptors is critical for symmetric transmembrane signal transduction. J. Biol. Chem. 279, 48152–48158 57 Marina, A. et al. (2005) Structure of the entire cytoplasmic portion of a sensor histidine-kinase protein. EMBO J. 24, 4247–4259 58 Hulko, M. et al. (2006) The HAMP domain structure implies helix rotation in transmembrane signaling. Cell 126, 929–940 59 Nowak, E. et al. (2006) Structural and functional aspects of the sensor histidine kinase PrrB from Mycobacterium tuberculosis. Structure 14, 275–285 60 Tanaka, T. et al. (1998) NMR structure of the histidine kinase domain of the E. coli osmosensor EnvZ. Nature 396, 88–92 61 Marina, A. et al. (2001) Structural and mutational analysis of the PhoQ histidine kinase catalytic domain. Insight into the reaction mechanism. J. Biol. Chem. 276, 41182–41190 62 Ragahavan, S. et al. (2008) Secreted transcription factor controls Mycobacterium tuberculosis virulence. Nature 454, 717–721 63 de Jong, B.C. et al. (2008) Progression to active tuberculosis, but not transmission, varies by Mycobacterium tuberculosis lineage in The Gambia. J. Infect. Dis. 198, 1037–1043 64 de Jong, B.C. et al. (2006) Mycobacterium africanum elicits an attenuated T cell response to early secreted antigenic target, 6 kda, in patients with tuberculosis and their household contacts. J. Infect. Dis. 193, 1279–1286