Two-component regulatory signal transduction systems are important elements of the adaptative response of prokaryotes to a variety of environmental stimuli. Disruption of PhoP-PhoR in Mycobacterium tuberculosisdramatically attenuates virulence, implying that this system directly and/or indirectly coordinates the expression of important virulence factors whose identity remains to be established. Interestingly, in knockingout the PhoP-PhoR two-component system in M. tuberculosis Mt103, dramatic changes in the colonial morphology, cording properties, and reactivity of the mutant strain to the basic dye neutral red, all intrinsic properties of tubercle bacilli known to correlate with virulence, were noted. Because deficiencies in the ability of the mutant to form serpentine cords and stain with the dye are likely the results of alterations of its cell envelope composition, we undertook to analyze the lipid content of phoP and phoP-phoR mutants constructed in two different strains of M. tuberculosis. Our results indicate that PhoP coordinately and positively regulates the synthesis of methyl-branched fatty acid-containing acyltrehaloses known to be restricted to pathogenic species of the M. tuberculosis complex, namely diacyltrehaloses, polyacyltrehaloses, and sulfolipids. Evidence is also provided that PhoP but not PhoR is required for the production of these lipids. This work represents an important step toward the functional characterization of PhoP-PhoR and the understanding of complex lipid synthesis in M. tuberculosis.Mycobacterium tuberculosis, the causative agent of tuberculosis in humans, is one of the leading causes of mortality due to a single infectious agent (1). In the tubercle bacillus as in other prokaryotes, two-component signal transduction systems are important elements of the adaptative response to a variety of stimuli (2). So far, of the 11 paired two-component systems, 5 unpaired response regulators and 2 unpaired protein sensors that M. tuberculosis possesses, the two-component system PhoP-PhoR is the one whose disruption was shown to affect the most dramatically the ability of M. tuberculosis to replicate in cellular and animal models (3). Interestingly, PhoP shows high similarity to the PhoP response regulator of Salmonella enterica serovar typhimurium, which senses Mg 2ϩ starvation and controls the expression of at least 40 genes, among which some encoding important virulence determinants (4). Further supporting the concept that PhoP is important for virulence and transmissibility of tubercle bacilli, a multidrug-resistant strain of Mycobacterium bovis (strain B) responsible for large tuberculosis outbreaks in Spain was found to carry an IS6110 insertion in the promoter region of phoP causing a strong up-regulation of the expression of this gene (5). To date, the stimuli sensed by the sensor histidine kinase PhoR and the genes controlled by the DNA-binding response regulator PhoP are not known. The identification of the environmental signals regulating PhoP-PhoR and the characterization of t...

SummaryMycobacterium tuberculosis and other pathogenic mycobacterial species produce large amounts of a glycogen-like a-glucan that represents the major polysaccharide of their outermost capsular layer. To determine the role of the surface-exposed glucan in the physiology and virulence of these bacteria, orthologues of the glg genes involved in the biosynthesis of glycogen in Escherichia coli were identified in M. tuberculosis H37Rv and inactivated by allelic replacement. Biochemical analyses of the mutants and complemented strains indicated that the synthesis of glucan and glycogen involves the a-1,4-glucosyltransferases Rv3032 and GlgA (Rv1212c), the ADP-glucose pyrophosphorylase GlgC (Rv1213) and the branching enzyme GlgB (Rv1326c). Disruption of glgC reduced by half the glucan and glycogen contents of M. tuberculosis, whereas the inactivation of glgA and Rv3032 affected the production of capsular glucan and glycogen, respectively. Attempts to disrupt Rv3032 in the glgA mutant were unsuccessful, suggesting that a functional copy of at least one of the two a-1,4-glucosyltransferases is required for growth. Importantly, the glgA mutant was impaired in its ability to persist in mice, suggesting a role for the capsular glucan in the persistence phase of infection. Unexpectedly, GlgB was found to be an essential enzyme.

The receptor-like protein kinase PknB from Mycobacterium tuberculosis is encoded by the distal gene in a highly conserved operon, present in all actinobacteria, that may control cell shape and cell division. Genes coding for a PknB-like protein kinase are also found in many more distantly related gram-positive bacteria. Here, we report that the pknB gene can be disrupted by allelic replacement in M. tuberculosis and the saprophyte Mycobacterium smegmatis only in the presence of a second functional copy of the gene. We also demonstrate that eukaryotic Ser/Thr protein kinase inhibitors, which inactivate PknB in vitro with a 50% inhibitory concentration in the submicromolar range, are able to kill M. tuberculosis H37Rv, M. smegmatis mc 2 155, and Mycobacterium aurum A؉ with MICs in the micromolar range. Furthermore, significantly higher concentrations of these compounds are required to inhibit growth of M. smegmatis strains overexpressing PknB, suggesting that this protein kinase is the molecular target. These findings demonstrate that the Ser/Thr protein kinase PknB is essential for sustaining mycobacterial growth and support the development of protein kinase inhibitors as new potential antituberculosis drugs.Mycobacterium tuberculosis is the causative agent of tuberculosis, a major world health problem that is responsible for the deaths of over two million people every year. A better understanding of the biology of the tubercle bacillus, with the goal of unveiling and validating new therapeutic targets, is an imperative requirement for improving the control and treatment of tuberculosis. The study of signaling elements, in particular Ser/Thr protein kinases (STPKs), is of outstanding interest in this context, given their likely important roles in mycobacterial physiology and virulence, as well as the available expertise on the design of specific inhibitors for eukaryotic STPKs, which currently represent one of the most actively studied groups of drug targets.Signal transduction in prokaryotes is conducted primarily by two-component regulatory systems, basically consisting of a sensor histidine kinase and a response regulator (45). The M. tuberculosis genome encodes 11 complete two-component systems (11,19), several of which contribute to the virulence of M. tuberculosis (33,34,36,38,49), but only one system (MtrA, MtrB) was found to be essential for cell growth (48). The relatively low number of two-component systems is offset by alternative signal transduction mechanisms involving Ser/Thr phosphorylation (2) that are generally less common in bacteria than in eukaryotes (26). M. tuberculosis has genes for one phospho-Ser/Thr phosphatase (pstP) and as many as 11 STPKs (pknA to pknL) (11). In mycobacteria with larger genomes, such as Mycobacterium marinum or Mycobacterium smegmatis, STPKs outnumber two-component systems, suggesting that the bulk of signal transduction is via Ser/Thr (de)phosphorylation.Paradoxically, most of these STPKs do not appear to control essential physiological processes. Mycobacterium lepra...

Similarities between Mycobacterium tuberculosis phoP-phoR mutants and the attenuated laboratory strain M. tuberculosis H37Ra in terms of morphological and cytochemical properties, lipid content, gene expression and virulence attenuation prompted us to analyze the functionality of this two-component regulator in the latter strain. Sequence analysis revealed a base substitution resulting in a one-amino-acid change in the likely DNA-binding region of PhoP in H37Ra relative to H37Rv. Using gel-shift assays, we show that this mutation abrogates the ability of the H37Ra PhoP protein to bind to a 40-bp segment of its own promoter. Consistent with this result, the phoP gene from H37Rv but not that from H37Ra was able to restore the synthesis of sulfolipids, diacyltrehaloses and polyacyltrehaloses in an isogenic phoP-phoR knock-out mutant of M. tuberculosis Moreover, complementation of H37Ra with phoP from H37Rv fully restored sulfolipid, diacyltrehalose and polyacyltrehalose synthesis, clearly indicating that the lack of production of these lipids in H37Ra is solely due to the point mutation in phoP. Using a pks2-3/4 knock-out mutant of M. tuberculosis H37Rv, evidence is further provided that the above-mentioned polyketide-derived acyltrehaloses do not significantly contribute to the virulence of the tubercle bacillus in a mouse model of infection. Reasons for the attenuation of H37Ra thus most likely stand in other virulence factors, many of which are expected to belong to the PhoP regulon and another of which, unrelated to PhoP, appears to be the lack of production of phthiocerol dimycocerosates in this strain.H37Rv and H37Ra are two variants of an Mycobacterium tuberculosis strain named H37 that was originally isolated from the sputum of a tuberculosis patient in 1905. Serial passaging of H37 through different media led to the dissociation of this isolate into two variants, a virulent one known as H37Rv and an avirulent one known as H37Ra, which also differ in their colonial morphology and cording properties (27,39). With the goal of identifying the molecular determinants underlying the virulence attenuation of H37Ra, numerous approaches including genetic complementation of H37Ra with H37Rv genomic DNA (29), gene expression profiling (15, 28, 32), subtractive RNA hybridization (22) and comparative genomics, proteomics and lipidomics (4,7,10,18,26) have been undertaken. Although these studies have led to the identification of a number of genes or gene products the expression of which differs between H37Rv and H37Ra, it is still at present unclear to what extent these products account for the virulence attenuation of H37Ra. Furthermore, as attempts to restore the virulence of the H37Ra strain through genetic complementation with H37Rv DNA have so far yielded negative results (4, 29), it has been concluded that the virulence attenuation of H37Ra was certainly the result of multiple mutations and/or rearrangements affecting multiple chromosomal loci.With the recent study by different groups of the two-component regulator...

Mycobacterium tuberculosis PknB is an essential receptor-like protein kinase involved in cell growth control. Here, we demonstrate that mitoxantrone, an anthraquinone derivative used in cancer therapy, is a PknB inhibitor capable of preventing mycobacterial growth. The structure of the complex reveals that mitoxantrone partially occupies the adeninebinding pocket in PknB, providing a framework for the design of compounds with potential therapeutic applications. PknB crystallizes as a 'back-to-back' homodimer identical to those observed in other structures of PknB in complex with ATP analogs. This organization resembles that of the RNA-dependent protein kinase PKR, suggesting a mechanism for kinase activation in mycobacteria.