Identification and in vitro Analysis of the GatD/MurT Enzyme-Complex Catalyzing Lipid II Amidation in Staphylococcus aureus

The peptidoglycan of Staphylococcus aureus is characterized by a high degree of crosslinking and almost completely lacks free carboxyl groups, due to amidation of the D-glutamic acid in the stem peptide. Amidation of peptidoglycan has been proposed to play a decisive role in polymerization of cell wall building blocks, correlating with the crosslinking of neighboring peptidoglycan stem peptides. Mutants with a reduced degree of amidation are less viable and show increased susceptibility to methicillin. We identified the enzymes catalyzing the formation of D-glutamine in position 2 of the stem peptide. We provide biochemical evidence that the reaction is catalyzed by a glutamine amidotransferase-like protein and a Mur ligase homologue, encoded by SA1707 and SA1708, respectively. Both proteins, for which we propose the designation GatD and MurT, are required for amidation and appear to form a physically stable bi-enzyme complex. To investigate the reaction in vitro we purified recombinant GatD and MurT His-tag fusion proteins and their potential substrates, i.e. UDP-MurNAc-pentapeptide, as well as the membrane-bound cell wall precursors lipid I, lipid II and lipid II-Gly5. In vitro amidation occurred with all bactoprenol-bound intermediates, suggesting that in vivo lipid II and/or lipid II-Gly5 may be substrates for GatD/MurT. Inactivation of the GatD active site abolished lipid II amidation. Both, murT and gatD are organized in an operon and are essential genes of S. aureus. BLAST analysis revealed the presence of homologous transcriptional units in a number of gram-positive pathogens, e.g. Mycobacterium tuberculosis, Streptococcus pneumonia and Clostridium perfringens, all known to have a D-iso-glutamine containing PG. A less negatively charged PG reduces susceptibility towards defensins and may play a general role in innate immune signaling

Predictors of the development of central post stroke pain after stroke in the somatosensory system

Einleitung: Zentrale neuropathische Schmerzen nach Schlaganfall (central post stroke pain, CPSP) entwickeln sich bei bis zu 12 % aller Schlaganfallpatienten und beeinträchtigen die Lebensqualität erheblich. Bislang ist unzureichend bekannt, welche Patienten einen CPSP entwickeln. Um zu untersuchen, inwiefern sich Patienten mit CPSP von schmerzfreien Patienten unterscheiden, wurde eine prospektive Beobachtungsstudie durchgeführt. Ziel war es, mögliche Prädiktoren für die Entstehung von CPSP zu erfassen. So könnten perspektivisch für Patienten mit hohem Risiko gezielt Präventionen entwickelt werden. Methoden: Patienten mit akutem Schlaganfall im somatosensorischen System wurden longitudinal mit Hilfe von klinisch-neurologischer Untersuchung, Quantitativer Sensorischer Testung (QST) und Magnetresonanztomographie über einen Zeitraum von sechs Monaten auf die Entstehung eines CPSP untersucht. Mit Hilfe der QST wurden somatosensorische Profile auf der zur zentralen Läsion kontralateralen, sowie ipsilateralen Körperseite analysiert. Verglichen wurden Patienten, die im Verlauf einen CPSP entwickelten, mit Patienten, die keine Schmerzen entwickelten. Ergebnisse: Von 78 Schlaganfallpatienten entwickelten 26 im Verlauf einen CPSP, während 52 Patienten keinen CPSP entwickelten. Patienten mit CPSP waren häufiger weiblich (p=0,014) und zeigten bereits vor Schmerzentwicklung eine schwerere neurologische Beeinträchtigung (p=0,001), schlechtere Schlafqualität (p=0,037) und körperliche Lebensqualität (p=0,034) als Patienten ohne CPSP. In der QST zeigten CPSP-Patienten auf der kontralateralen Seite der Läsion vor Schmerzbeginn eine signifikante Reduktion der Thermästhesie im Vergleich zu schmerzfreien Patienten. Prädiktive Faktoren für die Entstehung von CPSP waren ein weibliches Geschlecht (p=0,025) und eine schwerere neurologische Beeinträchtigung (p=0,007), während eine verstärkte Kälteschmerz- (p=0,012) und Vibrationswahrnehmung (p=0,015) mit einer Risikoreduktion einen CPSP zu entwickeln einherging. Schlussfolgerung: Die frühzeitige Identifikation von Prädiktoren für die Entstehung von CPSP im Akutstadium nach Schlaganfall, wie sie in dieser Arbeit erstmalig untersucht wurde, erleichtert die Entscheidungsfindung für die Einleitung einer prophylaktischen Behandlung erheblich und kann damit beitragen eine Chronifizierung von Schmerzen zu verhindern.Objective: Central post stroke pain (CPSP) develops in up to 12 % of all stroke patients and significantly affects the quality of life. So far, it is insufficiently known which stroke patients develop a CPSP. In order to investigate how patients who develop CPSP differ from patients who remain pain-free and whether there are predictive factors before pain development, a prospective observational study was carried out. The aim was to identify possible predictors for the development of CPSP. In perspective we intend to establish prophylactic treatment of pain to avoid pain chronification. Methods: Patients with acute stroke in the somatosensory system were examined longitudinally over a period of six months for the development of a CPSP using clinical neurological examination, standardized Quantitative Sensory Testing (QST) and magnetic resonance imaging. Using QST, somatosensory profiles contralateral and ipsilateral to the central lesion were analyzed. Patients who developed a CPSP during the observation period were compared to patients who did not develop pain (non-pain sensory stroke, NPSS). Results: Out of 78 patients with sensory stroke, 26 developed a CPSP, while 52 patients did not develop pain. Patients with CPSP were more often female (p=0,014) and showed more severe neurological impairment (p=0,001), poorer quality of sleep (p=0,037) and physical quality of life (p=0,034) even before pain development than patients with NPSS. Regarding QST, CPSP patients showed a significant reduction of thermesthesia on the contralateral side of the lesion compared to NPSS patients even before the onset of pain. Predictive factors for the development of CPSP were a female gender (p=0,025) and a more severe neurological impairment (p=0,007), while an improved perception of cold pain (p=0,012) and vibration (p=0,015) was associated with a reduction in the risk of de-veloping a CPSP. Conclusion: The early evaluation of predictors for the development of CPSP in the acute post-stroke stage, as investigated for the first time in this work, may help to better target prophylactic treatment and may thus help prevent pain from becoming chronic

MuSK-antibodies are associated with worse outcome in myasthenic crisis requiring mechanical ventilation

Myasthenic crisis (MC) is a life-threatening condition for patients with myasthenia gravis (MG). Muscle-specific kinase-antibodies (MuSK-ABs) are detected in ~ 6% of MG, but data on outcome of MuSK-MCs are still lacking. We made a subgroup analysis of patients who presented with MC with either acetylcholine-receptor-antibody positive MG (AchR-MG) or MuSK-MG between 2006 and 2015 in a retrospective German multicenter study. We identified 19 MuSK-AB associated MCs in 15 patients and 161 MCs in 144 patients with AchR-ABs only. In contrast to patients with AchR-AB, MuSK-AB patients were more often female (p = 0.05, OR = 2.74) and classified as Myasthenia Gravis Foundation of America-class IV before crisis (p = 0.04, OR = 3.25). MuSK-AB patients suffer more often from multiple chronic disease (p = 0.016, OR = 4.87) and were treated more invasively in terms of plasma exchanging therapies (not significant). The number of days of mechanical ventilation (MV) (43.0 ± 53.1 vs. 17.4 ± 18; p < 0.0001), days on an intensive care unit (ICU) (45.3 ± 49.5 vs. 21.2 ± 19.7; p < 0.0001), and hospital-length of stay (LOS) (55.9 ± 47.6 vs. 28.8 ± 20.9 days; p < 0.0001) were significantly increased in MuSK-MC. Remarkable is that these changes were mainly due to patients with MusK-ABs only, whereas patients' outcome with both antibodies was similar to AchR-MCs. Furthermore, our data showed a shortened duration of MV after treatment with plasma exchanging therapies compared to treatment with intravenous immunoglobulin in MuSK-MCs. We conclude that MuSK-AB-status is associated with a longer need of MV, ICU-LOS, and hospital-LOS in MC, and therefore recommend early initiation of a disease-specific therapy