A ß-Lactamase based reporter system for ESX dependent protein translocation in mycobacteria.

Protein secretion is essential for all bacteria in order to interact with their environment. Mycobacterium tuberculosis depends on protein secretion to subvert host immune response mechanisms. Both the general secretion system (Sec) and the twin-arginine translocation system (Tat) are functional in mycobacteria. Furthermore, a novel type of protein translocation system named ESX has been identified. In the genome of M. tuberculosis five paralogous ESX regions (ESX-1 to ESX-5) have been found. Several components of the ESX translocation apparatus have been identified over the last ten years. The ESX regions are composed of a basic set of genes for the translocation machinery and the main substrate - a heterodimer. The best studied of these heterodimers is EsxA (ESAT-6)/EsxB (CFP-10), which has been shown to be exported by ESX-1. EsxA/B is heavily involved in virulence of M. tuberculosis. EsxG/H is exported by ESX-3 and seems to be involved in an essential iron-uptake mechanism in M. tuberculosis. These findings make ESX-3 components high profile drug targets. Until now, reporter systems for determination of ESX protein translocation have not been developed. In order to create such a reporter system, a truncated ß-lactamase ('bla TEM-1) was fused to the N-terminus of EsxB, EsxG and EsxU, respectively. These constructs have then been tested in a ß-lactamase (BlaS) deletion strain of Mycobacterium smegmatis. M. smegmatis ΔblaS is highly susceptible to ampicillin. An ampicillin resistant phenotype was conferred by translocation of Bla TEM-1-Esx fusion proteins into the periplasm. BlaTEM-1-Esx fusion proteins were not found in the culture filtrate suggesting that plasma membrane translocation and outer membrane translocation are two distinct steps in ESX secretion. Thus we have developed a powerful tool to dissect the molecular mechanisms of ESX dependent protein translocation and to screen for novel components of the ESX systems on a large scale.

[Evaluation of an inpatient parent-child-rehabilitation program: symptoms of psychological distress and quality of life]. / Evaluation einer stationären Eltern-Kind-Rehabilitation: psychische Symptombelastung und Lebensqualität.

OBJECTIVE: To evaluate the short- and medium-term effectiveness of an inpatient parent-child rehabilitation program in reducing symptoms of psychological distress and in increasing the quality of life of parents and children. METHODS: Psychological problems and quality of life of parents and children were assessed 4 weeks before admission, at admission and discharge from the program, and 3 months post discharge using standardized questionnaires. The sample consisted of 256 parents, parent ratings of 397 children and self reports of 124 school-aged children. RESULTS: Compared to the waiting period prior to admission significant decreases in psychological problems as well as improvements in quality of life were found for both, parents and children. These effects proved to be stable over the follow-up period. CONCLUSIONS: Inpatient parent-child rehabilitation programs are effective in improving the psychological well-being and quality of life of parents and children.

Deletion of dop in Mycobacterium smegmatis abolishes pupylation of protein substrates in vivo.

Proteasome-bearing bacteria make use of a ubiquitin-like modification pathway to target proteins for proteasomal turnover. In a process termed pupylation, proteasomal substrates are covalently modified with the small protein Pup that serves as a degradation signal. Pup is attached to substrate proteins by action of PafA. Prior to its attachment, Pup needs to undergo deamidation at its C-terminal residue, converting glutamine to glutamate. This step is catalysed in vitro by Dop. In order to characterize Dop activity in vivo, we generated a dop deletion mutant in Mycobacterium smegmatis. In the Deltadop strain, pupylation is severely impaired and the steady-state levels of two known proteasomal substrates are drastically increased. Pupylation can be re-established by complementing the mutant with either DopWt or a Pup variant carrying a glutamate at its ultimate C-terminal position (PupGGE). Our data show that Pup is deamidated by Dop in vivo and that likely Dop alone is responsible for this activity. Furthermore, we demonstrate that a putative N-terminal ATP-binding motif is crucial for catalysis, as a single point mutation (E10A) in this motif abolishes Dop activity both in vivo and in vitro.