Role of Mycobacterium tuberculosis pknD in the Pathogenesis of central nervous system tuberculosis

<p>Abstract</p> <p>Background</p> <p>Central nervous system disease is the most serious form of tuberculosis, and is associated with high mortality and severe neurological sequelae. Though recent clinical reports suggest an association of distinct <it>Mycobacterium tuberculosis </it>strains with central nervous system disease, the microbial virulence factors required have not been described previously.</p> <p>Results</p> <p>We screened 398 unique <it>M. tuberculosis </it>mutants in guinea pigs to identify genes required for central nervous system tuberculosis. We found <it>M. tuberculosis pknD </it>(<it>Rv0931c</it>) to be required for central nervous system disease. These findings were central nervous system tissue-specific and were not observed in lung tissues. We demonstrated that <it>pknD </it>is required for invasion of brain endothelia (primary components of the blood-brain barrier protecting the central nervous system), but not macrophages, lung epithelia, or other endothelia. <it>M. tuberculosis pknD </it>encodes a "eukaryotic-like" serine-threonine protein kinase, with a predicted intracellular kinase and an extracellular (sensor) domain. Using confocal microscopy and flow cytometry we demonstrated that the <it>M. tuberculosis </it>PknD sensor is sufficient to trigger invasion of brain endothelia, a process which was neutralized by specific antiserum.</p> <p>Conclusions</p> <p>Our findings demonstrate a novel <it>in vivo </it>role for <it>M. tuberculosis pknD </it>and represent an important mechanism for bacterial invasion and virulence in central nervous system tuberculosis, a devastating and understudied disease primarily affecting young children.</p

A modified scoring system to describe gross pathology in the rabbit model of tuberculosis

<p>Abstract</p> <p>Background</p> <p>The rabbit model is an ideal means to study the pathogenesis of tuberculosis due to its semblance to the disease in humans. We have previously described the results using a bronchoscopic route of infection with live bacilli as a reliable means of generating lung cavities in sensitized rabbits. The role of sensitization in the development of disease outcomes has been well established in several animal models. We have described here the varying gross pathology that result from lack of sensitization with heat-killed <it>M. bovis </it>prior to high-dose bronchoscopic infection with live bacilli.</p> <p>Results</p> <p>Rabbits lacking sensitization did not generate lung cavities, but instead formed solely a tuberculoid pneumonia that replaced the normal lung parenchyma in the area of infection. Extrapulmonary dissemination was seen in approximately equal frequency and distribution in both rabbit populations. Notable differences include the lack of intestinal lesions in non-sensitized rabbits likely due to the lack of ingestion of expectorated bacilli from cavitary lesions. The experiment also employed a modified scoring system developed initially in the primate model of tuberculosis to allow for the quantification of findings observed at necropsy.</p> <p>Conclusions</p> <p>To date, no such scoring system has been employed in the rabbit model to describe gross pathology. The quantitative methodology would allow for rapid comparative analyses and standardization of thoracic and extrapulmonary pathology that could be evaluated for statistical significance. The aim is to use such a scoring system as the foundation for all future rabbit studies describing gross pathology at all stages in TB pathogenesis.</p

Alterations in Phospholipid Catabolism in Mycobacterium Tuberculosis LysX Mutant

Mycobacterium tuberculosis lysX mutant, defective for production of lysinylated phosphatidylglycerol, is sensitive to cationic antimicrobial peptides, is not proficient for proliferation in mice lungs, and exhibits altered membrane potential (Maloney et al., 2009). In the present study we show that a lysX complement strain expressing lysX from inducible tet promoter is proficient in restoring lysX phenotypes, confirming that the observed phenotypes are specific to lysX. To evaluate the correlation between changes in membrane potential and lysX activity, we visualized regions of cardiolipin (CL), one of the abundant phospholipids of mycobacteria, by staining with fluorescent dye 10-N-nonyl acridine orange and found that CL is localized as bright spots at septal regions and poles of actively dividing cells, but not in stationary phase cells. lysX mutants were elongated and showed more numerous and brighter CL staining at both mid cell and quarter cell septa, compared with wild type, indicating a defect in the cell division process. Evaluation of 14C-acetic acid incorporation into major phospholipids such as CL, phosphatidylethanolamine (PE), phosphatidylinositol (PI), and their degradation between lysX mutant and its parent revealed differences in the turnover of PE and PI. Our results favor a hypothesis that alterations in phospholipid metabolism could be contributing to changes in membrane potential, hence the observed phenotype of lysX mutant

Mycobacterium tuberculosis directs T helper 2 cell differentiation by inducing interleukin-1β production in dendritic cells

Mycobacterium tuberculosis (M. tb), the causative agent of tuberculosis (TB), resides and replicates within phagocytes and persists in susceptible hosts by modulating protective innate immune responses. Furthermore, M. tb promotes T helper 2(Th2) immune responses by altering the balance of T cell polarising cytokines in infected cells. However, cytokines that regulate Th2 cell differentiation during TB infection remain unknown. Here we show that IL-1β produced by phagocytes infected by virulent M. tb strain H37Rv directs Th2 cell differentiation. In sharp contrast, the vaccine strain BCG as well as RD-1 and ESAT-6 mutants of H37Rv failed to induce IL-1β and promote Th2 cell differentiation. Furthermore, ESAT-6 induced IL-1β production in dendritic cells, and CD4+ T cells co-cultured with infected DCs differentiated into Th2 cells. Taken together our findings indicate that IL-1β induced by RD-1/ESAT-6 plays an important role in the differentiation of Th2 cells, which in turn facilitates progression of TB by inhibiting host protective Th1 responses

Small molecule-directed immunotherapy against recurrent infection by Mycobacterium tuberculosis

Tuberculosis remains the biggest infectious threat to humanity with one-third of the population infected and 1.4 million deaths and 8.7 million new cases annually. Current tuberculosis therapy is lengthy and consists of multiple antimicrobials, which causes poor compliance and high treatment dropout, resulting in the development of drug-resistant variants of tuberculosis. Therefore, alternate methods to treat tuberculosis are urgently needed. Mycobacterium tuberculosis evades host immune responses by inducing T helper (Th)2 and regulatory T (Treg) cell responses, which diminish protective Th1 responses. Here, we show that animals (Stat-6−/−CD4-TGFβRIIDN mice) that are unable to generate both Th2 cells and Tregs are highly resistant to M. tuberculosis infection. Furthermore, simultaneous inhibition of these two subsets of Th cells by therapeutic compounds dramatically reduced bacterial burden in different organs. This treatment was associated with the generation of protective Th1 immune responses. As these therapeutic agents are not directed to the harbored organisms, they should avoid the risk of promoting the development of drug-resistant M. tuberculosis variants

Altered cellular infiltration and cytokine levels during early Mycobacterium tuberculosis sigC mutant infection are associated with late-stage disease attenuation and milder immunopathology in mice

<p>Abstract</p> <p>Background</p> <p>Mouse virulence assessments of certain <it>Mycobacterium tuberculosis </it>mutants have revealed an immunopathology defect in which high tissue CFU counts are observed but the tissue pathology and lethality are reduced. <it>M. tuberculosis </it>mutants which grow and persist in the mouse lungs, but have attenuated disease progression, have the immunopathology (<it>imp</it>) phenotype. The antigenic properties of these strains may alter the progression of disease due to a reduction in host immune cell recruitment to the lungs resulting in disease attenuation and prolonged host survival.</p> <p>Results</p> <p>In this study we focused on the mouse immune response to one such mutant; the <it>M. tuberculosis </it>Δ<it>sigC </it>mutant. Aerosol infection of DBA/2 and SCID mice with the <it>M. tuberculosis </it>Δ<it>sigC </it>mutant, complemented mutant and wild type strain showed proliferation of mutant bacilli in mouse lungs, but with decreased inflammation and mortality in DBA/2 mice. SCID mice shared the same phenotype as the DBA/2 mice in response to the Δ<it>sigC </it>mutant, however, they succumbed to the infection faster. Bronchoalveolar lavage (BAL) fluid analysis revealed elevated numbers of infiltrating neutrophils in the lungs of mice infected with wild type and complemented Δ<it>sigC </it>mutant strains but not in mice infected with the Δ<it>sigC </it>mutant. In addition, DBA/2 mice infected with the Δ<it>sigC </it>mutant had reduced levels of TNF-α, IL-1β, IL-6 and IFN-γ in the lungs. Similarly, there was a reduction in proinflammatory cytokines in the lungs of SCID mice. In contrast to the mouse model, the Δ<it>sigC </it>mutant had reduced initial growth in guinea pig lungs. A possible mechanism of attenuation in the Δ<it>sigC </it>mutant may be a reduction in neutrophilic-influx in the alveolar spaces of the lungs, and decreased proinflammatory cytokine secretion. In contrast to mouse data, the <it>M. tuberculosis </it>Δ<it>sigC </it>mutant proliferates slowly in guinea pig lungs, a setting characterized by caseating necrosis.</p> <p>Conclusion</p> <p>Our observations suggest that the immunopathology phenotype is associated with the inability to trigger a strong early immune response, resulting in disease attenuation. While macrophages and T cells have been shown to be important in containing <it>M. tuberculosis </it>disease our study has shown that neutrophils may also play an important role in the containment of this organism.</p

Mycobacterium tuberculosis nuoG Is a Virulence Gene That Inhibits Apoptosis of Infected Host Cells

The survival and persistence of Mycobacterium tuberculosis depends on its capacity to manipulate multiple host defense pathways, including the ability to actively inhibit the death by apoptosis of infected host cells. The genetic basis for this anti-apoptotic activity and its implication for mycobacterial virulence have not been demonstrated or elucidated. Using a novel gain-of-function genetic screen, we demonstrated that inhibition of infection-induced apoptosis of macrophages is controlled by multiple genetic loci in M. tuberculosis. Characterization of one of these loci in detail revealed that the anti-apoptosis activity was attributable to the type I NADH-dehydrogenase of M. tuberculosis, and was mainly due to the subunit of this multicomponent complex encoded by the nuoG gene. Expression of M. tuberculosis nuoG in nonpathogenic mycobacteria endowed them with the ability to inhibit apoptosis of infected human or mouse macrophages, and increased their virulence in a SCID mouse model. Conversely, deletion of nuoG in M. tuberculosis ablated its ability to inhibit macrophage apoptosis and significantly reduced its virulence in mice. These results identify a key component of the genetic basis for an important virulence trait of M. tuberculosis and support a direct causal relationship between virulence of pathogenic mycobacteria and their ability to inhibit macrophage apoptosis

Phase I Study Assessing the Pharmacokinetic Profile, Safety, and Tolerability of a Single Dose of Ceftazidime-Avibactam in Hospitalized Pediatric Patients.

This study aimed to investigate the pharmacokinetics (PK), safety, and tolerability of a single dose of ceftazidime-avibactam in pediatric patients. A phase I, multicenter, open-label PK study was conducted in pediatric patients hospitalized with an infection and receiving systemic antibiotic therapy. Patients were enrolled into four age cohorts (cohort 1, ≥12 to &lt;18 years; cohort 2, ≥6 to &lt;12 years; cohort 3, ≥2 to &lt;6 years; cohort 4, ≥3 months to &lt;2 years). Patients received a single 2-h intravenous infusion of ceftazidime-avibactam (cohort 1, 2,000 to 500 mg; cohort 2, 2,000 to 500 mg [≥40 kg] or 50 to 12.5 mg/kg [&lt;40 kg]; cohorts 3 and 4, 50 to 12.5 mg/kg). Blood samples were collected to describe individual PK characteristics for ceftazidime and avibactam. Population PK modeling was used to describe characteristics of ceftazidime and avibactam PK across all age groups. Safety and tolerability were assessed. Thirty-two patients received study drug. Mean plasma concentration-time curves, geometric mean maximum concentration (Cmax), and area under the concentration-time curve from time zero to infinity (AUC0-∞) were similar across all cohorts for both drugs. Six patients (18.8%) reported an adverse event, all mild or moderate in intensity. No deaths or serious adverse events occurred. The single-dose PK of ceftazidime and avibactam were comparable between each of the 4 age cohorts investigated and were broadly similar to those previously observed in adults. No new safety concerns were identified. (This study has been registered at ClinicalTrials.gov under registration no. NCT01893346.)

Bacterial Thymidine Kinase as a Non-Invasive Imaging Reporter for Mycobacterium tuberculosis in Live Animals

Bacteria can be selectively imaged in experimentally-infected animals using exogenously administered 1-(2'deoxy-2'-fluoro-beta-D-arabinofuranosyl)-5-[(125)I]-iodouracil ([(125)I]-FIAU), a nucleoside analog substrate for bacterial thymidine kinase (TK). Our goal was to use this reporter and develop non-invasive methods to detect and localize Mycobacterium tuberculosis.We engineered a M. tuberculosis strain with chromosomally integrated bacterial TK under the control of hsp60 -- a strong constitutive mycobacterial promoter. [(125)I]FIAU uptake, antimicrobial susceptibilities and in vivo growth characteristics were evaluated for this strain. Using single photon emission computed tomography (SPECT), M. tuberculosis P(hsp60) TK strain was evaluated in experimentally-infected BALB/c and C3HeB/FeJ mice using the thigh inoculation or low-dose aerosol infection models. M. tuberculosis P(hsp60) TK strain actively accumulated [(125)I]FIAU in vitro. Growth characteristics of the TK strain and susceptibility to common anti-tuberculous drugs were similar to the wild-type parent strain. M. tuberculosis P(hsp60) TK strain was stable in vivo and SPECT imaging could detect and localize this strain in both animal models tested.We have developed a novel tool for non-invasive assessment of M. tuberculosis in live experimentally-infected animals. This tool will allow real-time pathogenesis studies in animal models of TB and has the potential to simplify preclinical studies and accelerate TB research

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 relMtb 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