Paul J. Converse

Studies of a Ring-Cleaving Dioxygenase Illuminate the Role of Cholesterol Metabolism in the Pathogenesis of Mycobacterium tuberculosis

Mycobacterium tuberculosis, the etiological agent of TB, possesses a cholesterol catabolic pathway implicated in pathogenesis. This pathway includes an iron-dependent extradiol dioxygenase, HsaC, that cleaves catechols. Immuno-compromised mice infected with a ΔhsaC mutant of M. tuberculosis H37Rv survived 50% longer than mice infected with the wild-type strain. In guinea pigs, the mutant disseminated more slowly to the spleen, persisted less successfully in the lung, and caused little pathology. These data establish that, while cholesterol metabolism by M. tuberculosis appears to be most important during the chronic stage of infection, it begins much earlier and may contribute to the pathogens dissemination within the host. Purified HsaC efficiently cleaved the catecholic cholesterol metabolite, DHSA (3,4-dihydroxy-9,10-seconandrost-1,3,5(10)-triene-9,17-dione; k cat/K m = 14.4±0.5 µM−1 s−1), and was inactivated by a halogenated substrate analogue (partition coefficient<50). Remarkably, cholesterol caused loss of viability in the ΔhsaC mutant, consistent with catechol toxicity. Structures of HsaC:DHSA binary complexes at 2.1 Å revealed two catechol-binding modes: bidentate binding to the active site iron, as has been reported in similar enzymes, and, unexpectedly, monodentate binding. The position of the bicyclo-alkanone moiety of DHSA was very similar in the two binding modes, suggesting that this interaction is a determinant in the initial substrate-binding event. These data provide insights into the binding of catechols by extradiol dioxygenases and facilitate inhibitor design.

Selective Killing of Nonreplicating Mycobacteria

Antibiotics are typically more effective against replicating rather than nonreplicating bacteria. However, a major need in global health is to eradicate persistent or nonreplicating subpopulations of bacteria such as Mycobacterium tuberculosis (Mtb). Hence, identifying chemical inhibitors that selectively kill bacteria that are not replicating is of practical importance. To address this, we screened for inhibitors of dihydrolipoamide acyltransferase (DlaT), an enzyme required by Mtb to cause tuberculosis in guinea pigs and used by the bacterium to resist nitric oxide-derived reactive nitrogen intermediates, a stress encountered in the host. Chemical screening for inhibitors of Mtb DlaT identified select rhodanines as compounds that almost exclusively kill nonreplicating mycobacteria in synergy with products of host immunity, such as nitric oxide and hypoxia, and are effective on bacteria within macrophages, a cellular reservoir for latent Mtb. Compounds that kill nonreplicating pathogens in cooperation with host immunity could complement the conventional chemotherapy of infectious disease.

Mycobacterium tuberculosis ECF sigma factor sigC is required for lethality in mice and for the conditional expression of a defined gene set

Bacterial alternative RNA polymerase sigma factors are key global adaptive response regulators with a likely role in Mycobacterium tuberculosis pathogenesis. We constructed a mutant lacking the sigma factor gene, sigC, by allelic exchange, in the virulent CDC1551 strain of M. tuberculosis and compared the resulting mutant with the isogenic wild‐type strain and complemented mutant strain. In vitro, compared to the wild‐type and complemented strains, the mutant was found to have similar ability to survive in both murine bone marrow‐derived macrophages and activated J774 macrophages. In time‐to‐death experiments in the mouse model, the ΔsigC mutant was significantly attenuated, causing no death in infected mice whereas the wild‐type and complemented strains caused 100% mortality within 235 days after aerosol infection with a median time to death of 170 days. Mouse organ bacterial burdens indicated that the mutant proliferated and persisted at the same level as the wild‐type and complemented strains in lung tissue and was able to persist in mice without causing death for > 300 days. A complete genomic microarray study demonstrated that SigC modulates the expression of several key virulence‐associated genes including hspX, senX3 and mtrA, encoding the α‐crystallin homologue, a two‐component sensor kinase and a two‐component response regulator respectively. Altered expression of a subset of these genes was confirmed by quantitative RT‐PCR analysis. Analysis of genes modulated by SigC also revealed a putative consensus DNA recognition sequence for SigC of SSSAAT–N16−20–CGTSSS (S = C or G). Promoter recognition for one of these genes was confirmed by in vitro transcription analysis after purification of recombinant SigC and reconstitution of an EσC RNA polymerase holoenzyme. These data indicate that the M. tuberculosis transcription factor SigC governs expression of an important M. tuberculosis regulon and is essential for lethality in mice, but is not required for bacterial survival in this species. These observations place the ΔsigC mutant in a class of M. tuberculosis mutants which persist in tissues but are attenuated in their ability to elicit lethal immunopathology.

Role of the dosR-dosS Two-Component Regulatory System in Mycobacterium tuberculosis Virulence in Three Animal Models†

ABSTRACT The Mycobacterium tuberculosis dosR gene (Rv3133c) is part of an operon, Rv3134c-Rv3132c, and encodes a response regulator that has been shown to be upregulated by hypoxia and other in vitro stress conditions and may be important for bacterial survival within granulomatous lesions found in tuberculosis. DosR is activated in response to hypoxia and nitric oxide by DosS (Rv3132c) or DosT (Rv2027c). We compared the virulence levels of an M. tuberculosis dosR-dosS deletion mutant (ΔdosR-dosS [ΔdosR-S]), a dosR-complemented strain, and wild-type H37Rv in rabbits, guinea pigs, and mice infected by the aerosol route and in a mouse hollow-fiber model that may mimic in vivo granulomatous conditions. In the mouse and the guinea pig models, the ΔdosR-S mutant exhibited a growth defect. In the rabbit, the ΔdosR-S mutant did not replicate more than the wild type. In the hollow-fiber model, the mutant phenotype was not different from that of the wild-type strain. Our analyses reveal that the dosR and dosS genes are required for full virulence and that there may be differences in the patterns of attenuation of this mutant between the animal models studied.

An in vitro predictive test for graft versus host disease in patients with genotypic HLA-identical bone marrow transplants

Acute graft versus host disease remains a major cause of morbidity and mortality in allogeneic bone marrow transplantation. To date, no clinically useful test has been reported that will predict the occurrence of graft versus host disease in genotypic HLA-identical donor-recipient pairs. We have developed a skin-explant model using donor lymphocytes that have been sensitized against recipient lymphocytes in vitro and cocultured with the recipients skin. Histologic changes compatible with acute graft versus host disease are found in the positive explants. To date 32 patients have been tested in a prospective manner. Among the 18 recipient-donor pairs that were positive, 16 patients were found to have histologic Grade 2 or higher graft versus host disease of the skin on biopsy. Among the 14 negative pairs, only 3 patients had histologic Grade 2 or higher graft versus host disease of the skin on biopsy. Thus, the model has a sensitivity of 84 per cent and a specificity of 85 per cent, and is a significant predictor of the histologic occurrence of graft versus host disease (P less than 0.0005 by chi-square test). The test may be useful in the selection of donors for bone marrow transplantation and in the planning of prophylaxis against graft versus host disease.

Attenuation of Late-Stage Disease in Mice Infected by the Mycobacterium tuberculosis Mutant Lacking the SigF Alternate Sigma Factor and Identification of SigF-Dependent Genes by Microarray Analysis

ABSTRACT The Mycobacterium tuberculosis alternate sigma factor, SigF, is expressed during stationary growth phase and under stress conditions in vitro. To better understand the function of SigF we studied the phenotype of the M. tuberculosis ΔsigF mutant in vivo during mouse infection, tested the mutant as a vaccine in rabbits, and evaluated the mutants microarray expression profile in comparison with the wild type. In mice the growth rates of theΔ sigF mutant and wild-type strains were nearly identical during the first 8 weeks after infection. At 8 weeks, theΔ sigF mutant persisted in the lung, while the wild type continued growing through 20 weeks. Histopathological analysis showed that both wild-type and mutant strains had similar degrees of interstitial and granulomatous inflammation during the first 12 weeks of infection. However, from 12 to 20 weeks the mutant strain showed smaller and fewer lesions and less inflammation in the lungs and spleen. Intradermal vaccination of rabbits with the M. tuberculosis ΔsigF strain, followed by aerosol challenge, resulted in fewer tubercles than did intradermal M. bovis BCG vaccination. Complete genomic microarray analysis revealed that 187 genes were relatively underexpressed in the absence of SigF in early stationary phase, 277 in late stationary phase, and only 38 genes in exponential growth phase. Numerous regulatory genes and those involved in cell envelope synthesis were down-regulated in the absence of SigF; moreover, the ΔsigF mutant strain lacked neutral red staining, suggesting a reduction in the expression of envelope-associated sulfolipids. Examination of 5′-untranslated sequences among the downregulated genes revealed multiple instances of a putative SigF consensus recognition sequence: GGTTTCX18GGGTAT. These results indicate that in the mouse the M. tuberculosis ΔsigF mutant strain persists in the lung but at lower bacterial burdens than wild type and is attenuated by histopathologic assessment. Microarray analysis has identified SigF-dependent genes and a putative SigF consensus recognition site.

Comparison of a Tuberculin Interferon-γ Assay with the Tuberculin Skin Test in High-Risk Adults: Effect of Human Immunodeficiency Virus Infection

A novel, whole blood interferon-gamma (IFN-gamma) assay was evaluated to determine its suitability for detecting Mycobacterium tuberculosis exposure in intravenous drug users with or without human immunodeficiency virus (HIV) infection. Whole heparinized blood was incubated overnight in separate wells with tuberculin purified protein derivative (PPD), saline, and mitogen controls. Levels of IFN-gamma in plasma supernatants were determined by rapid ELISA. Participants were then administered the tuberculin skin test (TST) and tested for cutaneous anergy. The whole blood IFN-gamma test agreed (89%-100%) with a positive TST in both HIV-seropositive and -seronegative subjects, but reactivity to PPD was more detectable by the whole blood assay among those with negative TSTs or anergy. TST induration diameter and IFN-gamma responses were correlated (Spearmans p = .45, P = .0001), but both responses were blunted by HIV infection. In summary, tuberculin reactivity appears to be more detectable by the whole blood IFN-gamma assay than by TST, and the assay requires no return visit for test reading.

Deletion of Mycobacterium tuberculosis sigma factor E results in delayed time to death with bacterial persistence in the lungs of aerosol-infected mice.

ABSTRACT The stress-induced extracytoplasmic sigma factor E (SigE) of Mycobacterium tuberculosis shows increased expression after heat shock, sodium dodecyl sulfate treatment, and oxidative stress, as well as after phagocytosis in macrophages. We report that deletion of sigE results in delayed lethality in mice without a significant reduction of bacterial numbers in lungs.

Assessment of Clofazimine Activity in a Second-Line Regimen for Tuberculosis in Mice

RATIONALE Although observational studies suggest that clofazimine-containing regimens are highly active against drug-resistant tuberculosis, the contribution of clofazimine for the treatment of this disease has never been systematically evaluated. OBJECTIVES Our goal was to directly compare the activity of a standard second-line drug regimen with or without the addition of clofazimine in a mouse model of multidrug-resistant tuberculosis. Our comparative outcomes included time to culture conversion in the mouse lungs and the percentage of relapses after treatment cessation. METHODS Mice were aerosol-infected with an isoniazid-resistant (as a surrogate of multidrug-resistant) strain of Mycobacterium tuberculosis. Treatment, which was administered for 5 to 9 months, was initiated 2 weeks after infection and comprised the following second-line regimen: daily (5 d/wk) moxifloxacin, ethambutol, and pyrazinamide, supplemented with amikacin during the first 2 months. One-half of the mice also received daily clofazimine. The decline in lung bacterial load was assessed monthly using charcoal-containing agar to reduce clofazimine carryover. Relapse was assessed 6 months after treatment cessation. MEASUREMENTS AND MAIN RESULTS After 2 months, the bacillary load in lungs was reduced from 9.74 log10 at baseline to 3.61 and 4.68 in mice treated with or without clofazimine, respectively (P < 0.001). Mice treated with clofazimine were culture-negative after 5 months, whereas all mice treated without clofazimine remained heavily culture-positive for the entire 9 months of the study. The relapse rate was 7% among mice treated with clofazimine for 8 to 9 months. CONCLUSIONS The clofazimine contribution was substantial in these experimental conditions.

Clofazimine shortens the duration of the first-line treatment regimen for experimental chemotherapy of tuberculosis

Significance The infectious disease tuberculosis (TB) is a major public health problem that affects millions of people worldwide. TB treatment consists of a multidrug regimen that needs to be taken for a minimum of 6 mo, and lack of adherence to this regimen is associated with treatment failure and emergence of drug resistance. In a mouse model of TB chemotherapy, we have found that inclusion of the antileprosy drug clofazimine in the first-line regimen for TB reduces the duration of treatment necessary to achieve relapse-free cure from 6 mo to 3 mo. Our data suggest that clofazimine, a drug already known to be safe for long-term administration to patients with leprosy, has the potential to significantly shorten the duration of TB treatment. A key drug for the treatment of leprosy, clofazimine has recently been associated with highly effective and significantly shortened regimens for the treatment of multidrug-resistant tuberculosis (TB). Consequently, we hypothesized that clofazimine may also shorten the duration of treatment for drug-susceptible TB. We conducted a controlled trial in the mouse model of TB chemotherapy comparing the activity of the 6-mo standard regimen for TB treatment, i.e., 2 mo of daily rifampin, isoniazid, pyrazinamide, and ethambutol followed by 4 mo of rifampin and isoniazid, with a 4-mo clofazimine-containing regimen: 2 mo of daily rifampin, isoniazid, pyrazinamide, and clofazimine followed by 2 mo of rifampin, isoniazid, and clofazimine. Treatment efficacy was assessed on the basis of Mycobacterium tuberculosis colony counts in the lungs and spleens during treatment and on the proportion of mice with culture-positive relapse 6 mo after treatment cessation. No additive effect of clofazimine was observed after the first week of treatment, but, by the second week of treatment, the colony counts were significantly lower in the clofazimine-treated mice than in the mice receiving the standard regimen. Lung culture conversion was obtained after 3 and 5 mo in mice treated with the clofazimine-containing and standard regimens, respectively, and relapse-free cure was obtained after 3 and 6 mo of treatment with the clofazimine-containing and standard regimens, respectively. Thus, clofazimine is a promising anti-TB drug with the potential to shorten the duration of TB chemotherapy by at least half (3 mo vs. 6 mo) in the mouse model of TB.