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

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

Mycobacterium tuberculosis TlyA protein negatively regulates T helper (Th) 1 and Th17 differentiation and promotes tuberculosis pathogenesis

Mycobacterium tuberculosis, the causative agent of tuberculosis, is an ancient pathogen and a major cause of death worldwide. Although various virulence factors of M. tuberculosis have been identified, its pathogenesis remains incompletely understood. TlyA is a virulence factor in several bacterial infections and is evolutionarily conserved in many Gram-positive bacteria, but its function in M. tuberculosis pathogenesis has not been elucidated. Here, we report that TlyA significantly contributes to the pathogenesis of M. tuberculosis. We show that a TlyA mutant M. tuberculosis strain induces increased IL-12 and reduced IL-1β and IL-10 cytokine responses, which sharply contrasts with the immune responses induced by wild type M. tuberculosis. Furthermore, compared with wild type M. tuberculosis, TlyA-deficient M. tuberculosis bacteria are more susceptible to autophagy in macrophages. Consequently, animals infected with the TlyA mutant M. tuberculosis organisms exhibited increased host-protective immune responses, reduced bacillary load, and increased survival compared with animals infected with wild type M. tuberculosis. Thus, M. tuberculosis employs TlyA as a host evasion factor, thereby contributing to its virulence

Heightened Vulnerability to MDR-TB Epidemics after Controlling Drug-Susceptible TB

Prior infection with one strain TB has been linked with diminished likelihood of re-infection by a new strain. This paper attempts to determine the role of declining prevalence of drug-susceptible TB in enabling future epidemics of MDR-TB.A computer simulation of MDR-TB epidemics was developed using an agent-based model platform programmed in NetLogo (See http://mdr.tbtools.org/). Eighty-one scenarios were created, varying levels of treatment quality, diagnostic accuracy, microbial fitness cost, and the degree of immunogenicity elicited by drug-susceptible TB. Outcome measures were the number of independent MDR-TB cases per trial and the proportion of trials resulting in MDR-TB epidemics for a 500 year period after drug therapy for TB is introduced.MDR-TB epidemics propagated more extensively after TB prevalence had fallen. At a case detection rate of 75%, improving therapeutic compliance from 50% to 75% can reduce the probability of an epidemic from 45% to 15%. Paradoxically, improving the case-detection rate from 50% to 75% when compliance with DOT is constant at 75% increases the probability of MDR-TB epidemics from 3% to 45%.The ability of MDR-TB to spread depends on the prevalence of drug-susceptible TB. Immunologic protection conferred by exposure to drug-susceptible TB can be a crucial factor that prevents MDR-TB epidemics when TB treatment is poor. Any single population that successfully reduces its burden of drug-susceptible TB will have reduced herd immunity to externally or internally introduced strains of MDR-TB and can experience heightened vulnerability to an epidemic. Since countries with good TB control may be more vulnerable, their self interest dictates greater promotion of case detection and DOTS implementation in countries with poor control to control their risk of MDR-TB

Statewide Molecular Epidemiology of Mycobacterium tuberculosis Transmission in a Moderate- to Low-Incidence State: Are Contact Investigations Enough?

To assess the circumstances of recent transmission of tuberculosis (TB) (progression to active disease <2 years after infection), we obtained DNA fingerprints for 1,172 (99%) of 1,179 Mycobacterium tuberculosis isolates collected from Maryland TB patients from 1996 to 2000. We also reviewed medical records and interviewed patients with genetically matching M. tuberculosis strains to identify epidemiologic links (cluster investigation). Traditional settings for transmission were defined as households or close relatives and friends; all other settings were considered nontraditional. Of 436 clustered patients, 114 had recently acquired TB. Cluster investigations were significantly more likely than contact investigations to identify patients who recently acquired TB in nontraditional settings (33/42 vs. 23/72, respectively; p<0.001). Transmission from a foreign-born person to a U.S.-born person was rare and occurred mainly in public settings. The time from symptom onset to diagnosis was twice as long for transmitters as for nontransmitters (16.8 vs. 8.5 weeks, respectively; p<0.01). Molecular epidemiologic studies showed that eliminating diagnostic delays can prevent TB transmission in nontraditional settings, which elude contact investigations