Asymmetric Structure of the Dimerization Domain of PhoR, a Sensor Kinase Important for the Virulence of Mycobacterium tuberculosis.

The PhoP-PhoR two-component system is essential for the virulence of Mycobacterium tuberculosis (Mtb) and therefore represents a potential target for developing novel antituberculosis therapies. However, little is known about the mechanism by which this two-component system regulates the virulence. In this study, we demonstrated that a phoR mutant Mtb strain has phenotypes similar to those of a phoP mutant, suggesting that PhoP and PhoR work in the same pathway to regulate Mtb virulence. We determined the structure of the dimerization and histidine phosphotransfer (DHp) domain of PhoR to a 1.9 Å resolution. The structure revealed that the DHp domain is a dimer. Each subunit consists of two antiparallel α helices connected by a loop of five residues. The two subunits of the dimer fold into a four-helical bundle with a continuous hydrophobic core. The topology of the four-helical bundle is identical to the histidine kinases that are known to have a cis-autophosphorylation mechanism, suggesting that PhoR is likely to autophosphorylate in cis. The dimer is asymmetric, with one subunit having a greater bending angle than the other at the highly conserved proline residue five-residues downstream of the phosphorylation site histidine. This structural asymmetry of the dimer suggests the flexibility of the PhoR DHp domain, which is likely to be important for the signal transduction mechanism in controlling the autophosphorylation and phosphotransfer reactions and communicating with the upstream structure.

Cholesterol is not an essential source of nutrition for Mycobacterium tuberculosis during infection.

Rv1106c (hsd; 3ß-hydroxysteroid dehydrogenase) is required by Mycobacterium tuberculosis for growth on cholesterol as a sole carbon source, whereas Rv3409c is not. Mutation of Rv1106c does not reduce Mycobacterium tuberculosis growth in infected macrophages or guinea pigs. We conclude that cholesterol is not required as a nutritional source during infection.

Immunogenicity and protection induced by a Mycobacterium tuberculosis sigE mutant in a BALB/c mouse model of progressive pulmonary tuberculosis.

Tuberculosis is still one of the main challenges to human global health, leading to about two million deaths every year. One of the reasons for its success is the lack of efficacy of the widely used vaccine Mycobacterium bovis BCG. In this article, we analyze the potential use of an attenuated mutant of Mycobacterium tuberculosis H37Rv lacking the sigma factor sigma(E) as a live vaccine. We have demonstrated that BALB/c mice infected by the intratracheal route with this mutant strain showed significantly higher survival rates and less tissue damage than animals infected with the parental or complemented mutant strain. Although animals infected with the sigE mutant had low bacillary loads, their lungs showed significantly higher production of the protective factors gamma interferon (IFN-gamma), tumor necrosis factor alpha (TNF-alpha), inducible nitric oxide synthase (iNOS), and beta-defensins than those of animals infected with the parental or complemented mutant strain. Moreover, we demonstrate that the sigE mutant, when inoculated subcutaneously, was more attenuated than BCG in immunodeficient nude mice, thus representing a good candidate for a novel attenuated live vaccine strain. Finally, when we used the sigE mutant as a subcutaneous vaccine, it was able to induce a higher level of protection than did BCG with both H37Rv and a highly virulent strain of M. tuberculosis (Beijing code 9501000). Taken together, our findings suggest that the sigE mutant is a very promising strain for the development of a new vaccine against tuberculosis.

A thiolase of Mycobacterium tuberculosis is required for virulence and production of androstenedione and androstadienedione from cholesterol.

Mycobacterium tuberculosis, the causative agent of tuberculosis, is an intracellular pathogen that shifts to a lipid-based metabolism in the host. Moreover, metabolism of the host lipid cholesterol plays an important role in M. tuberculosis infection. We used transcriptional profiling to identify genes transcriptionally regulated by cholesterol and KstR (Rv3574), a TetR-like repressor. The fadA5 (Rv3546) gene, annotated as a lipid-metabolizing thiolase, the expression of which is upregulated by cholesterol and repressed by KstR, was deleted in M. tuberculosis H37Rv. We demonstrated that fadA5 is required for utilization of cholesterol as a sole carbon source in vitro and for full virulence of M. tuberculosis in the chronic stage of mouse lung infection. Cholesterol is not toxic to the fadA5 mutant strain, and, therefore, toxicity does not account for its attenuation. We show that the wild-type strain, H37Rv, metabolizes cholesterol to androst-4-ene-3,17-dione (AD) and androsta-1,4-diene-3,17-dione (ADD) and exports these metabolites into the medium, whereas the fadA5 mutant strain is defective for this activity. We demonstrate that FadA5 catalyzes the thiolysis of acetoacetyl-coenzyme A (CoA). This catalytic activity is consistent with a beta-ketoacyl-CoA thiolase function in cholesterol beta-oxidation that is required for the production of androsterones. We conclude that the attenuated phenotype of the fadA5 mutant is a consequence of disrupted cholesterol metabolism that is essential only in the persistent stage of M. tuberculosis infection and may be caused by the inability to produce AD/ADD from cholesterol.

The Mycobacterium tuberculosis high-affinity iron importer, IrtA, contains an FAD-binding domain.

Iron is an essential nutrient not freely available to microorganisms infecting mammals. To overcome iron deficiency, bacteria have evolved various strategies including the synthesis and secretion of high-affinity iron chelators known as siderophores. The siderophores produced and secreted by Mycobacterium tuberculosis, exomycobactins, compete for iron with host iron-binding proteins and, together with the iron-regulated ABC transporter IrtAB, are required for the survival of M. tuberculosis in iron deficient conditions and for normal replication in macrophages and in mice. This study further characterizes the role of IrtAB in M. tuberculosis iron acquisition. Our results demonstrate a role for IrtAB in iron import and show that the amino terminus domain of IrtA is a flavin-adenine dinucleotide-binding domain essential for iron acquisition. These results suggest a model in which the amino terminus of IrtA functions to couple iron transport and assimilation.

Identification and functional characterization of the iron-dependent regulator (IdeR) of Mycobacterium avium subsp. paratuberculosis.

Mycobacterium avium subspecies paratuberculosis (MAP), the causative agent of Johne's disease in cattle and sheep, has unique iron requirements in that it is mycobactin-dependent for cultivation in vitro. The iron-dependent regulator (IdeR) is a well-characterized global regulator responsible for maintaining iron homeostasis in Mycobacterium tuberculosis (MTB). We identified an orthologous segment in the MAP genome, MAP2827, with >93 % amino acid identity to MTB IdeR. Electrophoretic mobility shift assays and DNase protection assays confirmed that MAP2827 binds the 19 bp consensus motif (iron box) on the MAP genome. Sequencing of MAP2827 from multiple isolates revealed a non-synonymous change (R91G) exclusive to sheep strains. Reporter gene assays and quantitative real-time RT-PCR assays in two diverse MAP strains and in an ideR deletion mutant of M. smegmatis (mc(2)155) suggested that both sheep MAP IdeR (sIdeR) and cattle MAP IdeR (cIdeR) repress mbtB transcription at high iron concentrations and relieve repression at low iron concentrations. On the other hand, bfrA (an iron storage gene) was upregulated by cIdeR when presented with MTB or the cattle MAP bfrA promoter, and was downregulated by sIdeR in the presence of MTB, or sheep or cattle MAP bfrA promoters, at high iron concentrations. The differential iron regulatory mechanisms between IdeR-regulated genes across strains may contribute to the differential growth or pathogenic characteristics of sheep and cattle MAP strains. Taken together, our study provides a possible reason for mycobactin dependency and suggests strong implications in the differential iron acquisition and storage mechanisms in MAP.

Cholesterol metabolism increases the metabolic pool of propionate in Mycobacterium tuberculosis.

Mycobacterium tuberculosis can metabolize cholesterol to both acetate and propionate. The mass of isolated phthiocerol dimycoserate, a methyl-branched fatty acylated polyketide, was used as a reporter for intracellular propionate metabolic flux. When M. tuberculosis is grown using cholesterol as the only source of carbon, a 42 amu increase in average phthiocerol dimycoserate molecular weight is observed, consistent with the cellular pool of propionate and, thus, methylmalonyl CoA increasing upon cholesterol metabolism. In contrast, no shift in phthiocerol dimycoserate molecular weight is observed upon supplementation of medium containing glycerol and glucose with cholesterol. We conclude that cholesterol is a significant source of propionate only in the absence of sugar carbon sources.

PhoP, a key player in Mycobacterium tuberculosis virulence.

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-Guérin 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.

Protein dynamics in iron-starved Mycobacterium tuberculosis revealed by turnover and abundance measurement using hybrid-linear ion trap-Fourier transform mass spectrometry.

To study the proteome response of Mycobacterium tuberculosis H37Rv to a change in iron level, iron-starved late-log-phase cells were diluted in fresh low- and high-iron media containing [ (15)N]-labeled asparagine as the sole nitrogen source for labeling the proteins synthesized upon dilution. We determined the relative protein abundance and protein turnover in M. tuberculosis H37Rv under these two conditions. For measurements, we used a high-resolution hybrid-linear ion trap-Fourier transform mass spectrometer coupled with nanoliquid chromatography separation. While relative protein abundance analysis shows that only 5 proteins were upregulated by high iron, 24 proteins had elevated protein turnover for the cells in the high-iron medium. This suggests that protein turnover is a sensitive parameter to assess the proteome dynamics. Cluster analysis was used to explore the interconnection of protein abundance and turnover, revealing coordination of the cellular processes of protein synthesis, degradation, and secretion that determine the abundance and allocation of a protein in the cytosol and the extracellular matrix of the cells. Further potential utility of the approach is discussed.

Mycobacterium tuberculosis sigma factor E regulon modulates the host inflammatory response.

Mycobacterium tuberculosis survives in macrophages and usually subverts the bactericidal mechanisms of these phagocytes. The understanding of this host-pathogen interaction is relevant for the development of new treatments for tuberculosis. The adaptation of M. tuberculosis to intracellular life depends on its ability to regulate the expression of its genes. Sigma factors are important bacterial transcription activators that bind to the RNA polymerase and give it promoter specificity. Sigma factor E (SigE) controls the expression of genes that are essential for virulence. We have identified the SigE regulon during infection of macrophages, and we analyzed the impact of this regulon on the transcriptional response of phagocytes. Our results indicate that SigE regulates the expression of genes involved in the maintenance of M. tuberculosis cell envelope integrity and function during macrophage infection. Analysis of the phagocytes' transcriptional response indicates that the SigE regulon is involved in the modulation of the inflammatory response.

Global transcriptional profile of Mycobacterium tuberculosis during THP-1 human macrophage infection.

During lung infection, Mycobacterium tuberculosis resides in macrophages and subverts the bactericidal mechanisms of these professional phagocytes. Comprehension of this host-pathogen relationship is fundamental for the development of new therapies to cure and prevent tuberculosis. In this work, we analyzed the transcriptional profile of M. tuberculosis infecting human macrophage-like THP-1 cells in order to identify putative bacterial pathogenic factors that can be relevant for the intracellular survival of M. tuberculosis. We compared the gene expression profile of M. tuberculosis H37Rv after 4 h and 24 h of infection of human macrophage-like THP-1 cells with the gene expression profile of the strain growing exponentially in broth cultures. We found 585 genes expressed differentially by intracellular M. tuberculosis. An analysis of the gene expression profile of M. tuberculosis inside THP-1 cells suggests the perturbation of the cell envelope as a major intracellular stress inside THP-1 macrophages.

Structure of the DNA-binding domain of the response regulator PhoP from Mycobacterium tuberculosis.

The PhoP-PhoR two-component signaling system from Mycobacterium tuberculosis is essential for the virulence of the tubercle bacillus. The response regulator, PhoP, regulates expression of over 110 genes. In order to elucidate the regulatory mechanism of PhoP, we determined the crystal structure of its DNA-binding domain (PhoPC). PhoPC exhibits a typical fold of the winged helix-turn-helix subfamily of response regulators. The structure starts with a four-stranded antiparallel beta-sheet, followed by a three-helical bundle of alpha-helices, and then a C-terminal beta-hairpin, which together with a short beta-strand between the first and second helices forms a three-stranded antiparallel beta-sheet. Structural elements are packed through a hydrophobic core, with the first helix providing a scaffold for the rest of the domain to pack. The second and third helices and the long, flexible loop between them form the helix-turn-helix motif, with the third helix being the recognition helix. The C-terminal beta-hairpin turn forms the wing motif. The molecular surfaces around the recognition helix and the wing residues show strong positive electrostatic potential, consistent with their roles in DNA binding and nucleotide sequence recognition. The crystal packing of PhoPC gives a hexamer ring, with neighboring molecules interacting in a head-to-tail fashion. This packing interface suggests that PhoPC could bind DNA in a tandem association. However, this mode of DNA binding is likely to be nonspecific because the recognition helix is partially blocked and would be prevented from inserting into the major groove of DNA. Detailed structural analysis and implications with respect to DNA binding are discussed.

Molecular basis of the defective heat stress response in Mycobacterium leprae.

Mycobacterium leprae, a major human pathogen, grows poorly at 37 degrees C. The basis for its inability to survive at elevated temperatures was investigated. We determined that M. leprae lacks a protective heat shock response as a result of the lack of transcriptional induction of the alternative sigma factor genes sigE and sigB and the major heat shock operons, HSP70 and HSP60, even though heat shock promoters and regulatory circuits for these genes appear to be intact. M. leprae sigE was found to be capable of complementing the defective heat shock response of mycobacterial sigE knockout mutants only in the presence of a functional mycobacterial sigH, which orchestrates the mycobacterial heat shock response. Since the sigH of M. leprae is a pseudogene, these data support the conclusion that a key aspect of the defective heat shock response in M. leprae is the absence of a functional sigH. In addition, 68% of the genes induced during heat shock in M. tuberculosis were shown to be either absent from the M. leprae genome or were present as pseudogenes. Among these is the hsp/acr2 gene, whose product is essential for M. tuberculosis survival during heat shock. Taken together, these results suggest that the reduced ability of M. leprae to survive at elevated temperatures results from the lack of a functional transcriptional response to heat shock and the absence of a full repertoire of heat stress response genes, including sigH.

Rv1106c from Mycobacterium tuberculosis is a 3beta-hydroxysteroid dehydrogenase.

New approaches are required to combat Mycobacterium tuberculosis (Mtb), especially the multi-drug resistant and extremely drug resistant organisms (MDR-TB and XDR-TB). There are many reports that mycobacteria oxidize 3beta-hydroxysterols to 3-ketosteroids, but the enzymes responsible for this activity have not been identified in mycobacterial species. In this work, the Rv1106c gene that is annotated as a 3beta-hydroxysteroid dehydrogenase in Mtb has been cloned and heterologously expressed. The purified enzyme was kinetically characterized and found to have a pH optimum between 8.5 and 9.5. The enzyme, which is a member of the short chain dehydrogenase superfamily, uses NAD+ as a cofactor and oxidizes cholesterol, pregnenolone, and dehydroepiandrosterone to their respective 3-keto-4-ene products. The enzyme forms a ternary complex with NAD+ binding before the sterol. The enzyme shows no substrate preference for dehydroepiandrosterone versus pregnenolone with second-order rate constants (kcat/Km) of 3.2 +/- 0.4 and 3.9 +/- 0.9 microM-1 min-1, respectively, at pH 8.5, 150 mM NaCl, 30 mM MgCl2, and saturating NAD+. Trilostane is a competitive inhibitor of dehydroepiandrosterone with a Ki of 197 +/- 8 microM. The expression of the 3beta-hydroxysteroid dehydrogenase in Mtb is intracellular. Disruption of the 3beta-hydroxysteroid dehydrogenase gene in Mtb abrogates mycobacterial cholesterol oxidation activity. These data are consistent with the Rv1106c gene being the one responsible for 3beta-hydroxysterol oxidation in Mtb.

Differential gene expression between Mycobacterium bovis and Mycobacterium tuberculosis.

The high sequence identity among the Mycobacterium bovis and Mycobacterium tuberculosis genomes contrasts with the physiological differences reported between these pathogens, suggesting that variations in gene expression may be involved. In this study, microarray hybridization was used to compare the total transcriptome of M. bovis and M. tuberculosis, during the exponential phase of growth. Differential expression was detected in 258 genes, representing a 6% of the total genome. Variable genes were grouped according to functional categories. The main variations were found in genes encoding proteins involved in intermediary metabolism and respiration, cell wall processes, and hypothetical proteins. It is noteworthy that, compared to M. tuberculosis, the expression of a higher number of transcriptional regulators were detected in M. bovis. Likewise, in M. tuberculosis we found a higher expression of the PE/PPE genes, some of which code for cell wall related proteins. Also, in both pathogens we detected the expression of a number of genes not annotated in the M. tuberculosis H37Rv or M. bovis 2122 genomes, but annotated in the M. tuberculosis CDC1551 genome. Our results provide new evidence concerning differences in gene expression between both pathogens, and confirm previous hypotheses inferred from genome comparisons and proteome analysis. This study may shed some new light on our understanding of the mechanisms relating to differences in gene expression and pathogenicity in mycobacteria.

Global analysis of the Mycobacterium tuberculosis Zur (FurB) regulon.

The proteins belonging to the Fur family are global regulators of gene expression involved in the response to several environmental stresses and to the maintenance of divalent cation homeostasis. The Mycobacterium tuberculosis genome encodes two Fur-like proteins, FurA and a protein formerly annotated FurB. Since in this paper we show that it represents a zinc uptake regulator, we refer to it as Zur. The gene encoding Zur is found in an operon together with the gene encoding a second transcriptional regulator (Rv2358). In a previous work we demonstrated that Rv2358 is responsible for the zinc-dependent repression of the Rv2358-zur operon, favoring the hypothesis that these genes represent key regulators of zinc homeostasis. In this study we generated a zur mutant in M. tuberculosis, examined its phenotype, and characterized the Zur regulon by DNA microarray analysis. Thirty-two genes, presumably organized in 16 operons, were found to be upregulated in the zur mutant. Twenty-four of them belonged to eight putative transcriptional units preceded by a conserved 26-bp palindrome. Electrophoretic mobility shift experiments demonstrated that Zur binds to this palindrome in a zinc-dependent manner, suggesting its direct regulation of these genes. The proteins encoded by Zur-regulated genes include a group of ribosomal proteins, three putative metal transporters, the proteins belonging to early secretory antigen target 6 (ESAT-6) cluster 3, and three additional proteins belonging to the ESAT-6/culture filtrate protein 10 (CFP-10) family known to contain immunodominant epitopes in the T-cell response to M. tuberculosis infection.

Infection of human dendritic cells with a Mycobacterium tuberculosis sigE mutant stimulates production of high levels of interleukin-10 but low levels of CXCL10: impact on the T-cell response.

The Mycobacterium tuberculosis genome encodes 13 sigma factors. We have previously shown that mutations in some of these transcriptional activators render M. tuberculosis sensitive to various environmental stresses and can attenuate the virulence phenotype. In this work, we focused on extracytoplasmic factor sigmaE and studied the effects induced by the deletion of its structural gene (sigE) in the infection of human monocyte-derived dendritic cells (MDDC). We found that the wild-type M. tuberculosis strain (H37Rv), the sigE mutant (ST28), and the complemented strain (ST29) were able to infect dendritic cells (DC) to similar extents, although at 4 days postinfection a reduced ability to grow inside MDDC was observed for the sigE mutant ST28. After mycobacterium capture, the majority of MDDC underwent full maturation and expressed both inflammatory cytokines, such as tumor necrosis factor alpha, and the regulatory cytokines interleukin-12 (IL-12), IL-18, and beta interferon (IFN-beta). Conversely, a higher level of production of IL-10 was observed in ST28-infected MDDC compared to H37Rv- or ST29-infected cell results. However, in spite of the presence of IL-10, supernatants from ST28-infected DC induced IFN-gamma production by T cells similarly to those from H37Rv-infected DC culture. On the other hand, IL-10 impaired CXCL10 production in sigE mutant-infected DC and, indeed, its neutralization restored CXCL10 secretion. In line with these results, supernatants from ST28-infected cells showed a decreased capability to recruit CXCR3+ CD4+ T cells compared to those obtained from H37Rv-infected DC culture. Thus, our findings suggest that the sigE mutant-induced secretion of IL-10 inhibits CXCL10 expression and, in turn, the recruitment of activated-effector cells involved in the formation of granulomas.

The Mycobacterium tuberculosis PhoPR two-component system regulates genes essential for virulence and complex lipid biosynthesis.

Two-component signal transduction systems (2-CS) play an important role in bacterial pathogenesis. In the work presented here, we have studied the effects of a mutation in the Mycobacterium tuberculosis (Mtb) PhoPR 2-CS on the pathogenicity, physiology and global gene expression of this bacterial pathogen. Disruption of PhoPR causes a marked attenuation of growth in macrophages and mice and prevents growth in low-Mg2+ media. The inability to grow in THP-1 macrophages can be partially overcome by the addition of excess Mg2+ during infection. Global transcription assays demonstrate PhoP is a positive transcriptional regulator of several genes, but do not support the hypothesis that the Mtb PhoPR system is sensing Mg2+ starvation, as is the case with the Salmonella typhimurium PhoPQ 2-CS. The genes that were positively regulated include those found in the pks2 and the msl3 gene clusters that encode enzymes for the biosynthesis of sulphatides and diacyltrehalose and polyacyltrehalose respectively. Complementary biochemical studies, in agreement with recent results from another group, indicate that these complex lipids are also absent from the phoP mutant, and the lack of these components in its cell envelope may indirectly cause the mutant's high-Mg2+ growth requirement. The experiments reported here provide functional evidence for the PhoPR 2-CS involvement in Mtb pathogenesis, and they suggest that a major reason for the attenuation observed in the phoP mutant is the absence of certain complex lipids that are known to be important for virulence.

Posttranslational regulation of Mycobacterium tuberculosis extracytoplasmic-function sigma factor sigma L and roles in virulence and in global regulation of gene expression.

In this report, we demonstrate that SigL is posttranslationally regulated by a specific anti-sigma factor, RslA, and contributes to the expression of at least 28 genes. Several of these genes could mediate important cell envelope-related processes. Importantly, a sigL-rslA mutant strain was significantly attenuated in a mouse model of infection.

Identification of an ABC transporter required for iron acquisition and virulence in Mycobacterium tuberculosis.

Iron availability affects the course of tuberculosis infection, and the ability to acquire this metal is known to be essential for replication of Mycobacterium tuberculosis in human macrophages. M. tuberculosis overcomes iron deficiency by producing siderophores. The relevance of siderophore synthesis for iron acquisition by M. tuberculosis has been demonstrated, but the molecules involved in iron uptake are currently unknown. We have identified two genes (irtA and irtB) encoding an ABC transporter similar to the YbtPQ system involved in iron transport in Yersinia pestis. Inactivation of the irtAB system decreases the ability of M. tuberculosis to survive iron-deficient conditions. IrtA and -B do not participate in siderophore synthesis or secretion but are required for efficient utilization of iron from Fe-carboxymycobactin, as well as replication of M. tuberculosis in human macrophages and in mouse lungs. We postulate that IrtAB is a transporter of Fe-carboxymycobactin. The irtAB genes are located in a chromosomal region previously shown to contain genes regulated by iron and the major iron regulator IdeR. Taken together, our results and previous observations made by other groups regarding two other genes in this region indicate that this gene cluster is dedicated to siderophore synthesis and transport in M. tuberculosis.