Sp1 phosphorylation regulates inducible expression of platelet-derived growth factor B-chain gene via atypical protein kinase C-ζ

Platelet-derived growth factor (PDGF) is a broadly expressed mitogenic and chemotactic factor with diverse roles in a number of physiologic and pathologic settings. The zinc finger transcription factors Sp1, Sp3 and Egr-1 bind to overlapping elements in the proximal PDGF B-chain promoter and activate transcription of this gene. The anthracycline nogalamycin has previously been reported to inhibit the capacity of Egr-1 to bind DNA in vitro. Here we used electrophoretic mobility shift assays to show that nogalamycin added to cells in culture did not alter the interaction of Egr-1 with the PDGF-B promoter. Instead, it enhanced the capacity of Sp1 to bind DNA. Nogalamycin increased PDGF-B mRNA expression at the level of transcription, which was abrogated by mutation of the Sp1 binding site in the PDGF-B promoter or overexpression of mutant Sp1. Rather than increasing total levels of Sp1, nogalamycin altered the phosphorylation state of the transcription factor. Overexpression of dominant-negative PKC-ζ blocked nogalamycin-inducible Sp1 phosphorylation and PDGF-B promoter-dependent expression. Nogalamycin stimulated the phosphorylation of PKC-ζ (on residue Thr410). These findings demonstrate for the first time that PKC-ζ and Sp1 phosphorylation mediate the inducible expression of this growth factor.

Crystal structure of cytochrome P450 14α-sterol demethylase (CYP51) from Mycobacterium tuberculosis in complex with azole inhibitors

Cytochrome P450 14α-sterol demethylases (CYP51) are essential enzymes in sterol biosynthesis in eukaryotes. CYP51 removes the 14α-methyl group from sterol precursors such as lanosterol, obtusifoliol, dihydrolanosterol, and 24(28)-methylene-24,25-dihydrolanosterol. Inhibitors of CYP51 include triazole antifungal agents fluconazole and itraconazole, drugs used in treatment of topical and systemic mycoses. The 2.1- and 2.2-Å crystal structures reported here for 4-phenylimidazole- and fluconazole-bound CYP51 from Mycobacterium tuberculosis (MTCYP51) are the first structures of an authentic P450 drug target. MTCYP51 exhibits the P450 fold with the exception of two striking differences—a bent I helix and an open conformation of BC loop—that define an active site-access channel running along the heme plane perpendicular to the direction observed for the substrate entry in P450BM3. Although a channel analogous to that in P450BM3 is evident also in MTCYP51, it is not open at the surface. The presence of two different channels, with one being open to the surface, suggests the possibility of conformationally regulated substrate-in/product-out openings in CYP51. Mapping mutations identified in Candida albicans azole-resistant isolates indicates that azole resistance in fungi develops in protein regions involved in orchestrating passage of CYP51 through different conformational stages along the catalytic cycle rather than in residues directly contacting fluconazole. These new structures provide a basis for rational design of new, more efficacious antifungal agents as well as insight into the molecular mechanism of P450 catalysis.

Virulence of a Mycobacterium tuberculosis clinical isolate in mice is determined by failure to induce Th1 type immunity and is associated with induction of IFN-α/β

To understand how virulent mycobacteria subvert host immunity and establish disease, we examined the differential response of mice to infection with various human outbreak Mycobacterium tuberculosis clinical isolates. One clinical isolate, HN878, was found to be hypervirulent, as demonstrated by unusually early death of infected immune-competent mice, compared with infection with other clinical isolates. The differential effect on survival required lymphocyte function because severe combined immunodeficiency (SCID) mice infected with HN878 or other clinical isolates all died at the same rate. The hypervirulence of HN878 was associated with failure to induce M. tuberculosis-specific proliferation and IFN-γ production by spleen and lymph node cells from infected mice. In addition, 2- to 4-fold lower levels of tumor necrosis factor-α (TNF-α), IL-6, IL-12, and IFN-γ mRNAs were observed in lungs of HN878-infected mice. IL-10, IL-4, and IL-5 mRNA levels were not significantly elevated in lungs of HN878 infected mice. In contrast, IFN-α mRNA levels were significantly higher in lungs of these mice. To further investigate the role of Type 1 IFNs, mice infected with HN878 were treated intranasally with purified IFN-α/β. The treatment resulted in increased lung bacillary loads and even further reduced survival. These results suggest that the hypervirulence of HN878 may be due to failure of this strain to stimulate Th1 type immunity. In addition, the lack of development of Th1 immunity in response to HN878 appears to be associated with increased induction of Type 1 IFNs.

Regulation of the Mycobacterium tuberculosis hypoxic response gene encoding α-crystallin

Unlike many pathogens that are overtly toxic to their hosts, the primary virulence determinant of Mycobacterium tuberculosis appears to be its ability to persist for years or decades within humans in a clinically latent state. Since early in the 20th century latency has been linked to hypoxic conditions within the host, but the response of M. tuberculosis to a hypoxic signal remains poorly characterized. The M. tuberculosis α-crystallin (acr) gene is powerfully and rapidly induced at reduced oxygen tensions, providing us with a means to identify regulators of the hypoxic response. Using a whole genome microarray, we identified >100 genes whose expression is rapidly altered by defined hypoxic conditions. Numerous genes involved in biosynthesis and aerobic metabolism are repressed, whereas a high proportion of the induced genes have no known function. Among the induced genes is an apparent operon that includes the putative two-component response regulator pair Rv3133c/Rv3132c. When we interrupted expression of this operon by targeted disruption of the upstream gene Rv3134c, the hypoxic regulation of acr was eliminated. These results suggest a possible role for Rv3132c/3133c/3134c in mycobacterial latency.

Chemokine receptor 2 serves an early and essential role in resistance to Mycobacterium tuberculosis

Although the protective cellular immune response to Mycobacterium tuberculosis requires recruitment of macrophages and T lymphocytes to the site of infection, the signals that regulate this trafficking have not been defined. We investigated the role of C-C chemokine receptor 2 (CCR2)-dependent cell recruitment in the protective response to M. tuberculosis. CCR2−/− mice died early after infection and had 100-fold more bacteria in their lungs than did CCR2+/+ mice. CCR2−/− mice exhibited an early defect in macrophage recruitment to the lung and a later defect in recruitment of dendritic cells and T cells to the lung. CCR2−/− mice also had fewer macrophages and dendritic cells recruited to the mediastinal lymph node (MLN) after infection. T cell migration through the MLN was similar in CCR2−/− and CCR2+/+ mice. However, T cell priming was delayed in the MLNs of the CCR2−/− mice, and fewer CD4+ and CD8+ T cells primed to produce IFN-γ accumulated in the lungs of the CCR2−/− mice. These data demonstrate that cellular responses mediated by activation of CCR2 are essential in the initial immune response and control of infection with M. tuberculosis.

Major histocompatibility class I presentation of soluble antigen facilitated by Mycobacterium tuberculosis infection.

Cell-mediated immune responses are essential for protection against many intracellular pathogens. For Mycobacterium tuberculosis (MTB), protection requires the activity of T cells that recognize antigens presented in the context of both major histocompatibility complex (MHC) class II and I molecules. Since MHC class I presentation generally requires antigen to be localized to the cytoplasmic compartment of antigen-presenting cells, it remains unclear how pathogens that reside primarily within endocytic vesicles of infected macrophages, such as MTB, can elicit specific MHC class I-restricted T cells. A mechanism is described for virulent MTB that allows soluble antigens ordinarily unable to enter the cytoplasm, such as ovalbumin, to be presented through the MHC class I pathway to T cells. The mechanism is selective for MHC class I presentation, since MTB infection inhibited MHC class II presentation of ovalbumin. The MHC class I presentation requires the tubercle bacilli to be viable, and it is dependent upon the transporter associated with antigen processing (TAP), which translocates antigenic peptides from the cytoplasm into the endoplasmic reticulum. The process is mimicked by Listeria monocytogenes and soluble listeriolysin, a pore-forming hemolysin derived from it, suggesting that virulent MTB may have evolved a comparable mechanism that allows molecules in a vacuolar compartment to enter the cytoplasmic presentation pathway for the generation of protective MHC class I-restricted T cells.

The embAB genes of Mycobacterium avium encode an arabinosyl transferase involved in cell wall arabinan biosynthesis that is the target for the antimycobacterial drug ethambutol.

The antimycobacterial compound ethambutol [Emb; dextro-2,2'-(ethylenediimino)-di-1-butanol] is used to treat tuberculosis as well as disseminated infections caused by Mycobacterium avium. The critical target for Emb lies in the pathway for the biosynthesis of cell wall arabinogalactan, but the molecular mechanisms for drug action and resistance are unknown. The cellular target for Emb was sought using drug resistance, via target overexpression by a plasmid vector, as a selection tool. This strategy led to the cloning of the M. avium emb region which rendered the otherwise susceptible Mycobacterium smegmatis host resistant to Emb. This region contains three complete open reading frames (ORFs), embR, embA, and embB. The translationally coupled embA and embB genes are necessary and sufficient for an Emb-resistant phenotype which depends on gene copy number, and their putative novel membrane proteins are homologous to each other. The predicted protein encoded by embR, which is related to known transcriptional activators from Streptomyces, is expendable for the phenotypic expression of Emb resistance, but an intact divergent promoter region between embR and embAB is required. An Emb-sensitive cell-free assay for arabinan biosynthesis shows that overexpression of embAB is associated with high-level Emb-resistant arabinosyl transferase activity, and that embR appears to modulate the in vitro level of this activity. These data suggest that embAB encode the drug target of Emb, the arabinosyl transferase responsible for the polymerization of arabinose into the arabinan of arabinogalactan, and that overproduction of this Emb-sensitive target leads to Emb resistance.

Targeted replacement of the mycocerosic acid synthase gene in Mycobacterium bovis BCG produces a mutant that lacks mycosides.

A single gene (mas) encodes the multifunctional enzyme that catalyzes the synthesis of very long chain multiple methyl branched fatty acids called mycocerosic acids that are present only in slow-growing pathogenic mycobacteria and are thought to be important for pathogenesis. To achieve a targeted disruption of mas, an internal 2-kb segment of this gene was replaced with approximately the same size hygromycin-resistance gene (hyg), such that hyg was flanked by 4.7- and 1.4-kb segments of mas. Transformation of Mycobacterium bovis BCG with this construct in a plasmid that cannot replicate in mycobacteria yielded hygromycin-resistant transformants. Screening of 38 such transformants by PCR revealed several transformants representing homologous recombination with single crossover and one with double crossover. With primers representing the hyg termini and those representing the mycobacterial genome segments outside that used to make the transformation construct, the double-crossover mutant yielded PCR products expected from either side of hyg. Gene replacement was further confirmed by the absence of the vector and the 2-kb segment of mas replaced by hyg from the genome of the mutant. Thin-layer and radio-gas chromatographic analyses of the lipids derived from [1-14C]propionate showed that the mutant was incapable of synthesizing mycocerosic acids and mycosides. Thus, homologous recombination with double crossover was achieved in a slow-growing mycobacterium with an intron-containing RecA. The resulting mas-disrupted mutant should allow testing of the postulated roles of mycosides in pathogenesis.

A stationary-phase stress-response sigma factor from Mycobacterium tuberculosis.

Alternative RNA polymerase sigma factors are a common means of coordinating gene regulation in bacteria. Using PCR amplification with degenerate primers, we identified and cloned a sigma factor gene, sigF, from Mycobacterium tuberculosis. The deduced protein encoded by sigF shows significant similarity to SigF sporulation sigma factors from Streptomyces coelicolor and Bacillus subtilis and to SigB, a stress-response sigma factor, from B. subtilis. Southern blot surveys with a sigF-specific probe identified cross-hybridizing bands in other slow-growing mycobacteria, Mycobacterium bovis bacille Calmette-Guérin (BCG) and Mycobacterium avium, but not in the rapid-growers Mycobacterium smegmatis or Mycobacterium abscessus. RNase protection assays revealed that M. tuberculosis sigF mRNA is not present during exponential-phase growth in M. bovis BCG cultures but is strongly induced during stationary phase, nitrogen depletion, and cold shock. Weak expression of M. tuberculosis sigF was also detected during late-exponential phase, oxidative stress, anaerobiasis, and alcohol shock. The specific expression of M. tuberculosis sigF during stress or stationary phase suggests that it may play a role in the ability of tubercle bacilli to adapt to host defenses and persist during human infection.

An integrated map of the genome of the tubercle bacillus, Mycobacterium tuberculosis H37Rv, and comparison with Mycobacterium leprae.

An integrated map of the genome of the tubercle bacillus, Mycobacterium tuberculosis, was constructed by using a twin-pronged approach. Pulsed-field gel electrophoretic analysis enabled cleavage sites for Asn I and Dra I to be positioned on the 4.4-Mb circular chromosome, while, in parallel, clones from two cosmid libraries were ordered into contigs by means of fingerprinting and hybridization mapping. The resultant contig map was readily correlated with the physical map of the genome via the landmarked restriction sites. Over 165 genes and markers were localized on the integrated map, thus enabling comparisons with the leprosy bacillus, Mycobacterium leprae, to be undertaken. Mycobacterial genomes appear to have evolved as mosaic structures since extended segments with conserved gene order and organization are interspersed with different flanking regions. Repetitive sequences and insertion elements are highly abundant in M. tuberculosis, but the distribution of IS6110 is apparently nonrandom.

Efflux pump of the proton antiporter family confers low-level fluoroquinolone resistance in Mycobacterium smegmatis.

Due to the resurgence of tuberculosis and the emergence of multidrug-resistant strains, fluoroquinolones (FQ) are being used in selected tuberculosis patients, but FQ-resistant strains of Mycobacterium tuberculosis have rapidly begun to appear. The mechanisms involved in FQ resistance need to be elucidated if the effectiveness of this class of antibiotics is to be improved and prolonged. By using the rapid-growing Mycobacterium smegmatis as a model genetic system, a gene was selected that confers low-level FQ resistance when present on a multicopy plasmid. This gene, lfrA, encodes a putative membrane efflux pump of the major facilitator family, which appears to recognize the hydrophilic FQ, ethidium bromide, acridine, and some quaternary ammonium compounds. It is homologous to qacA from Staphylococcus aureus, tcmA, of Streptomyces glaucescens, and actII and mmr, both from Streptomyces coelicoler. Increased expression of lfrA augments the appearance of subsequent mutations to higher-level FQ resistance.