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.

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.

The urease locus of Mycobacterium tuberculosis and its utilization for the demonstration of allelic exchange in Mycobacterium bovis bacillus Calmette-Guérin.

The ureABC genes of Mycobacterium tuberculosis were cloned. By using a set of degenerate primers corresponding to a conserved region of the urease enzyme (EC 3.5.1.5), a fragment of the expected size was amplified by PCR and was used to screen a M. tuberculosis cosmid library. Three open reading frames with extensive similarity to the urease genes from other organisms were found. The locus was mapped on the chromosome, using an ordered M. tuberculosis cosmid library. A suicide vector containing a ureC gene disrupted by a kanamycin marker (aph) was used to construct a urease-negative Mycobacterium bovis bacillus Calmette-Guérin mutant by allelic exchange involving replacement of the ureC gene with the aph::ureC construct. To our knowledge, allelic exchange has not been reported previously in the slow-growing mycobacteria. Homologous recombination will be an invaluable genetic tool for deciphering the mechanisms of tuberculosis pathogenesis, a disease that causes 3 x 10(6) deaths a year worldwide.

Mutation of the principal sigma factor causes loss of virulence in a strain of the Mycobacterium tuberculosis complex.

Tuberculosis continues to be responsible for the deaths of millions of people, yet the virulence factors of the causative pathogens remain unknown. Genetic complementation experiments with strains of the Mycobacterium tuberculosis complex have identified a gene from a virulent strain that restores virulence to an attenuated strain. The gene, designated rpoV, has a high degree of homology with principal transcription or sigma factors from other bacteria, particularly Mycobacterium smegmatis and Streptomyces griseus. The homologous rpoV gene of the attenuated strain has a point mutation causing an arginine-->histidine change in a domain known to interact with promoters. To our knowledge, association of loss of bacterial virulence with a mutation in the principal sigma factor has not been previously reported. The results indicate either that tuberculosis organisms have an alternative principal sigma factor that promotes virulence genes or, more probably, that this particular mutant principal sigma factor is unable to promote expression of one or more genes required for virulence. Study of genes and proteins differentially regulated by the mutant transcription factor should facilitate identification of further virulence factors.

Surfactant protein a promotes attachment of Mycobacterium tuberculosis to alveolar macrophages during infection with human immunodeficiency virus.

The incidence of tuberculosis is increasing on a global scale, in part due to its strong association with human immunodeficiency virus (HIV) infection. Attachment of Mycobacterium tuberculosis to its host cell, the alveolar macrophage (AM), is an important early step in the pathogenesis of infection. Bronchoalveolar lavage of HIV-infected individuals demonstrated the presence of a factor which significantly enhances the attachment of tubercle bacilli to AMs 3-fold relative to a normal control population. This factor is surfactant protein A (SP-A). SP-A levels are increased in the lungs of HIV-infected individuals. SP-A levels and attachment of M. tuberculosis to AMs inversely correlate with peripheral blood CD4 lymphocyte counts. Elevated concentrations of SP-A during the progression of HIV infection may represent an important nonimmune risk factor for acquiring tuberculosis, even before significant depletion of CD4 lymphocytes in the peripheral blood occurs.

Iron acquisition by Mycobacterium tuberculosis: isolation and characterization of a family of iron-binding exochelins.

Mycobacterium tuberculosis, the primary agent of tuberculosis, must acquire iron from the host to cause infection. To do so, it releases high-affinity iron-binding siderophores called exochelins. Exochelins are thought to transfer iron to another type of high-affinity iron-binding molecule in the bacterial cell wall, mycobactins, for subsequent utilization by the bacterium. In this paper, we describe the purification of exochelins of M. tuberculosis and their characterization by mass spectrometry. Exochelins comprise a family of molecules whose most abundant species range in mass from 744 to 800 Da in the neutral Fe(3+)-loaded state. The molecules form two 14-Da-increment series, one saturated and the other unsaturated, with the increments reflecting different numbers of CH2 groups on a side chain. These series further subdivide into serine- or threonine-containing species. The virulent M. tuberculosis Erdman strain and the avirulent M. tuberculosis H37Ra strain produce a similar set of exochelins. Based on a comparison of their tandem mass spectra, exochelins share a common core structure with mycobactins. However, exochelins are smaller than mycobactins due to a shorter alkyl side chain, and the side chain of exochelins terminates in a methyl ester. These differences render exochelins more polar than the lipophilic mycobactins and hence soluble in the aqueous extracellular milieu of the bacterium in which they bind iron in the host.

An epidemic of tuberculosis with a high rate of tuberculin anergy among a population previously unexposed to tuberculosis, the Yanomami Indians of the Brazilian Amazon

A survey of an emerging tuberculosis epidemic among the Yanomami Indians of the Amazonian rain forest provided a unique opportunity to study the impact of tuberculosis on a population isolated from contact with the tubercle bacillus for millennia until the mid-1960s. Within the Yanomami population, an extraordinary high prevalence of active tuberculosis (6.4% of 625 individuals clinically examined) was observed, indicating a high susceptibility to disease, even among bacille Calmette–Guérin-vaccinated individuals. Observational studies on cell-mediated and humoral immune responses of the Yanomami Indians compared with contemporary residents of the region suggest profound differences in immunological responsiveness to Mycobacterium tuberculosis infection. Among the Yanomami, a very high prevalence of tuberculin skin test anergy was found. Of patients with active tuberculosis, 46% had purified protein derivative of tuberculosis reactions <10 mm; similarly 58% of recent bacillus Calmette–Guérin vaccines exhibited skin test reactions <5 mm. The Yanomami also had higher titers of antibodies against M. tuberculosis glycolipid antigens (>70%) than the control subjects comprised of Brazilians of European descent (14%). The antibodies were mostly of the IgM isotype. Among the tuberculosis patients who also produced IgG antibodies, the titers of IgG4 were significantly higher among the Yanomami than in the control population. Although it was not possible to analyze T-cell responses or patterns of lymphokine production in vitro because of the remoteness of the villages from laboratory facilities, the results suggest that the first encounter of the Yanomami Indian population with tuberculosis engenders a diminished cell-mediated immune response and an increased production antibody responses, relative to other populations with extensive previous contact with the pathogen. These findings suggest that tuberculosis may represent a powerful selective pressure on human evolution that over centuries has shaped the nature of human immune responses to infection.

Identification of nitric oxide synthase as a protective locus against tuberculosis

Mutagenesis of the host immune system has helped identify response pathways necessary to combat tuberculosis. Several such pathways may function as activators of a common protective gene: inducible nitric oxide synthase (NOS2). Here we provide direct evidence for this gene controlling primary Mycobacterium tuberculosis infection using mice homozygous for a disrupted NOS2 allele. NOS2−/− mice proved highly susceptible, resembling wild-type littermates immunosuppressed by high-dose glucocorticoids, and allowed Mycobacterium tuberculosis to replicate faster in the lungs than reported for other gene-deficient hosts. Susceptibility appeared to be independent of the only known naturally inherited antimicrobial locus, NRAMP1. Progression of chronic tuberculosis in wild-type mice was accelerated by specifically inhibiting NOS2 via administration of N6-(1-iminoethyl)-l-lysine. Together these findings identify NOS2 as a critical host gene for tuberculostasis.

Effects of protein calorie malnutrition on tuberculosis in mice

Infectious diseases and malnutrition represent major burdens afflicting millions of people in developing countries. Both conditions affect individuals in industrialized nations, particularly the aged, the HIV-infected, and people with chronic diseases. While malnutrition is known to induce a state of immunodeficiency, the mechanisms responsible for compromised antimicrobial resistance in malnourished hosts remain obscure. In the present study, mice fed a 2% protein diet and developing protein calorie malnutrition, in contrast to well-nourished controls receiving a 20% protein diet, rapidly succumbed to infection with Mycobacterium tuberculosis. Malnourished mice exhibited a tissue-specific diminution in the expression of interferon γ, tumor necrosis factor α, and the inducible form of nitric oxide synthase in the lungs, but not the liver. The expression of these molecules critical to the production of mycobactericidal nitrogen oxides was depressed in malnourished animals in the lungs specifically at early times (<14 days) after infection. At later times, levels of expression became comparable to those in well-nourished controls, although the bacillary burden in the malnourished animals continued to rise. Nevertheless, urinary and serum nitrate contents, an index of total nitric oxide (NO) production in vivo, were not detectably diminished in malnourished, mycobacteria-infected mice. In contrast to the selective and early reduction of lymphokines and the inducible form of nitric oxide synthase in the lung, a marked diminution of the granulomatous reaction was observed in malnourished mice throughout the entire course of infection in all tissues examined (lungs, liver, and spleen). Remarkably, the progressively fatal course of tuberculosis observed in the malnourished mice could be reversed by restoring a full protein (20%) diet. The results indicate that protein calorie malnutrition selectively compromises several components of the cellular immune response that are important for containing and restricting tuberculous infection, and suggest that malnutrition-induced susceptibility to some infectious diseases can be reversed or ameliorated by nutritional intervention.