Petros C. Karakousis

Dormancy Phenotype Displayed by Extracellular Mycobacterium tuberculosis within Artificial Granulomas in Mice

Mycobacterium tuberculosis residing within pulmonary granulomas and cavities represents an important reservoir of persistent organisms during human latent tuberculosis infection. We present a novel in vivo model of tuberculosis involving the encapsulation of bacilli in semidiffusible hollow fibers that are implanted subcutaneously into mice. Granulomatous lesions develop around these hollow fibers, and in this microenvironment, the organisms demonstrate an altered physiologic state characterized by stationary-state colony-forming unit counts and decreased metabolic activity. Moreover, these organisms show an antimicrobial susceptibility pattern similar to persistent bacilli in current models of tuberculosis chemotherapy in that they are more susceptible to the sterilizing drug, rifampin, than to the bactericidal drug isoniazid. We used this model of extracellular persistence within host granulomas to study both gene expression patterns and mutant survival patterns. Our results demonstrate induction of dosR (Rv3133c) and 20 other members of the DosR regulon believed to mediate the transition into dormancy, and that rel Mtb is required for Mycobacterium tuberculosis survival during extracellular persistence within host granulomas. Interestingly, the dormancy phenotype of extracellular M. tuberculosis within host granulomas appears to be immune mediated and interferon-γ dependent.

Mycobacterium avium complex in patients with HIV infection in the era of highly active antiretroviral therapy

Disseminated Mycobacterium avium complex (MAC) infection is a common complication of late-stage HIV-1 infection. Since the advent of highly active antiretroviral therapy (HAART), the rate of MAC infection has declined substantially, but patients with low CD4 cell counts remain at risk. Among patients in the Johns Hopkins cohort with advanced HIV disease, the proportion developing MAC has fallen from 16% before 1996 to 4% after 1996, with a current rate of less than 1% per year. Factors associated with developing MAC include younger age, no use of HAART, and enrollment before 1996. Prophylaxis with azithromycin or clarithromycin is recommended for all patients with CD4 counts less than 50 cells/mL. Optimum treatment for disseminated MAC includes clarithromycin and ethambutol, and another investigation suggests that the addition of rifabutin might reduce mortality. Both prophylaxis and treatment of disseminated MAC can be discontinued in patients who have responded to HAART, and specific guidelines for withdrawing treatment have been published. Although HAART has altered the frequency and outcome of MAC infection, it remains an important complication of AIDS.

Mycobacterium tuberculosis cell envelope lipids and the host immune response

., 1999). The organism is a slow-growing bacillusthat is transmitted by the respiratory route. Although it iscapable of causing disease in most organs, pulmonaryinvolvement is most common. Soon after infection, thebacilli are phagocytosed by alveolar macrophages andsurvive within early phagosomes. Innate immuneresponses directed by macrophages predominate early ininfection. Subsequent recruitment of dendritic cells leadsto cell-mediated responses involving CD4

Tuberculosis-associated haemophagocytic syndrome

Haemophagocytic syndrome is a disorder characterised by fevers, lymphadenopathy, hepatosplenomegaly, cytopenias, and hyperferritinaemia due to dysregulated activation and proliferation of macrophages, leading to uncontrolled phagocytosis of platelets, erythrocytes, lymphocytes, and their haematopoietic precursors throughout the reticuloendothelial system. Primary or familial haemophagocytic syndrome appears to have a genetic aetiology, whereas secondary haemophagocytic syndrome may be associated with malignancy, autoimmune disease, or infection. Epstein-Barr virus is the most common infectious aetiology implicated in haemophagocytic syndrome, but the syndrome has been associated with a variety of other viral, bacterial, and parasitic pathogens. We describe a case of haemophagocytic syndrome associated with disseminated Mycobacterium tuberculosis. We review all cases of M tuberculosis-associated haemophagocytic syndrome reported in the English language literature and discuss important issues pertaining to the epidemiology, diagnosis, and management of this disease.

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.

Phosphate Depletion: A Novel Trigger for Mycobacterium tuberculosis Persistence

Persistent Mycobacterium tuberculosis (MTB) likely encounters a phosphate-limited environment within macrophage phagosomes. We studied MTB growth, antibiotic susceptibility, and gene expression during phosphate limitation. With use of MTB mutants deficient in phosphate-related genes, we assessed bacillary survival under phosphate-limited conditions and in mouse and guinea pig lungs. Phosphate limitation restricted MTB growth in a dose-dependent manner, and phosphate-starved bacilli became phenotypically tolerant to isoniazid. The MTB genes ppk1 and relA were upregulated significantly after phosphate starvation, consistent with inorganic polyphosphate accumulation and MTB stringent response induction. The phosphate-specific transport operon pstS3-pstC2-pstA1 was induced during phosphate starvation and its expression was dependent on the 2-component regulatory system SenX3-RegX3. The MTB gene regX3 appears to be essential for bacillary survival during phosphate limitation and in mammalian lungs. Our data suggest that MTB encounters phosphate-limited conditions during mammalian lung infection and that expression of the phosphate starvation response (PSR) is important for MTB persistence.

Dose-Ranging Comparison of Rifampin and Rifapentine in Two Pathologically Distinct Murine Models of Tuberculosis

ABSTRACT In previous experiments, replacing the 10-mg/kg of body weight daily dose of rifampin with 7.5 to 10 mg/kg of rifapentine in combinations containing isoniazid and pyrazinamide reduced the duration of treatment needed to cure tuberculosis in BALB/c mice by approximately 50% due to rifapentines more potent activity and greater drug exposures obtained. In the present study, we performed dose-ranging comparisons of the bactericidal and sterilizing activities of rifampin and rifapentine, alone and in combination with isoniazid and pyrazinamide with or without ethambutol, in BALB/c mice and in C3HeB/FeJ mice, which develop necrotic lung granulomas after infection with Mycobacterium tuberculosis. Each rifamycin demonstrated a significant increase in sterilizing activity with increasing dose. Rifapentine was roughly 4 times more potent in both mouse strains. These results reinforce the rationale for ongoing clinical trials to ascertain the highest well-tolerated doses of rifampin and rifapentine. This study also provides an important benchmark for the efficacy of the first-line regimen in C3HeB/FeJ mice, a strain in which the lung lesions observed after M. tuberculosis infection may better represent the pathology of human tuberculosis.

Exosomes Isolated from Mycobacteria-Infected Mice or Cultured Macrophages Can Recruit and Activate Immune Cells In Vitro and In Vivo

More than 2 billion people are infected with Mycobacterium. tuberculosis; however, only 5–10% of those infected will develop active disease. Recent data suggest that containment is controlled locally at the level of the granuloma and that granuloma architecture may differ even within a single infected individual. Formation of a granuloma likely requires exposure to mycobacterial components released from infected macrophages, but the mechanism of their release is still unclear. We hypothesize that exosomes, which are small membrane vesicles containing mycobacterial components released from infected macrophages, could promote cellular recruitment during granuloma formation. In support of this hypothesis, we found that C57BL/6 mouse-derived bone marrow macrophages treated with exosomes released from M. tuberculosis-infected RAW264.7 cells secrete significant levels of chemokines and can induce migration of CFSE-labeled macrophages and splenocytes. Exosomes isolated from the serum of M. bovis bacillus Calmette-Guérin–infected mice could also stimulate macrophage production of chemokines and cytokines ex vivo, but the level and type differed during the course of a 60-d infection. Of interest, the exosome concentration in serum correlated strongly with mouse bacterial load, suggesting some role in immune regulation. Finally, hollow fiber-based experiments indicated that macrophages treated with exosomes released from M. tuberculosis-infected cells could promote macrophage recruitment in vivo. Exosomes injected intranasally could also recruit CD11b+ cells into the lung. Overall, our study suggests that exosomes may play an important role in recruiting and regulating host cells during an M. tuberculosis infection.

Latent Tuberculosis Infection: Myths, Models, and Molecular Mechanisms

SUMMARY The aim of this review is to present the current state of knowledge on human latent tuberculosis infection (LTBI) based on clinical studies and observations, as well as experimental in vitro and animal models. Several key terms are defined, including “latency,” “persistence,” “dormancy,” and “antibiotic tolerance.” Dogmas prevalent in the field are critically examined based on available clinical and experimental data, including the long-held beliefs that infection is either latent or active, that LTBI represents a small population of nonreplicating, “dormant” bacilli, and that caseous granulomas are the haven for LTBI. The role of host factors, such as CD4+ and CD8+ T cells, T regulatory cells, tumor necrosis factor alpha (TNF-α), and gamma interferon (IFN-γ), in controlling TB infection is discussed. We also highlight microbial regulatory and metabolic pathways implicated in bacillary growth restriction and antibiotic tolerance under various physiologically relevant conditions. Finally, we pose several clinically important questions, which remain unanswered and will serve to stimulate future research on LTBI.

Metronidazole Lacks Activity against Mycobacterium tuberculosis in an In Vivo Hypoxic Granuloma Model of Latency

During human latent tuberculosis (TB) infection, dormant bacilli putatively reside within the hypoxic environment of caseating lung granulomas. The anaerobic drug metronidazole has antituberculous activity under hypoxic conditions in vitro but lacks activity against murine TB. In the present study, we used the hypoxia marker pimonidazole to demonstrate the presence of hypoxia in a novel in vivo granuloma model of Mycobacterium tuberculosis latency. We also used a high-throughput, microarray-based technique to identify mycobacterial genes essential to hypoxia and showed that this in vivo model correctly identified 51% of hypoxia-attenuated mutants, a significantly larger percentage than that identified by the mouse (29%) and guinea pig (29%) aerosol models of TB. Although isoniazid showed activity during the first 28 days of therapy and rifampin was active against dormant bacilli after the establishment of hypoxia, metronidazole showed no antituberculous activity in this in vivo hypoxic granuloma model of M. tuberculosis dormancy.