Repeats-in-Toxin (RTX) proteins of Gram-negative bacteria are excreted through the type I secretion system (T1SS) that recognizes non-cleavable C-terminal secretion signals. These are preceded by arrays of glycine and aspartate-rich nonapeptide repeats grouped by four to eight β strands into blocks that fold into calcium-binding parallel β-roll structures. The β-rolls are interspersed by linkers of variable length and sequence and the organization of multiple RTX repeat blocks within large RTX domains remains unknown. Here we examined the structure and function of the RTX domain of Bordetella pertussis adenylate cyclase toxin (CyaA) that is composed of five β-roll RTX blocks. We show that the non-folded RTX repeats maintain the stability of the CyaA polypeptide in the Ca2+-depleted bacterial cytosol and thereby enable its efficient translocation through the T1SS apparatus. The efficacy of secretion of truncated CyaA constructs was dictated by the number of retained RTX repeat blocks and depended on the presence of extracellular Ca2+ ions. We further describe the crystal structure of the RTX blocks IV-V of CyaA (CyaA1372-1681) that consists of a contiguous assembly of two β-rolls that differs substantially from the arrangement of the RTX blocks observed in RTX lipases or other RTX proteins. These results provide a novel structural insight into the architecture of the RTX domains of large RTX proteins and support the "push-ratchet" mechanism of the T1SS-mediated secretion of very large RTX proteins.

• MeSH
• adenylátcyklasový toxin chemie   genetika   metabolismus   MeSH
• bakteriální proteiny chemie   metabolismus   MeSH
• bakteriální toxiny chemie   genetika   metabolismus   MeSH
• Bordetella pertussis metabolismus   MeSH
• cytosol metabolismus   MeSH
• gramnegativní bakterie metabolismus   MeSH
• konformace proteinů MeSH
• sbalování proteinů MeSH
• sekreční systém typu I MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

In 2011-2012, a survey was performed in three regional hospitals in the Czech Republic to determine the incidence of Clostridium difficile infections (CDIs) and to characterize bacterial isolates. C. difficile isolates were characterized by PCR ribotyping, toxin genes detection, multiple-locus variable-number tandem-repeat analysis (MLVA), and antimicrobial susceptibility testing to fidaxomicin, vancomycin, metronidazole, clindamycin, LFF571, and moxifloxacin using agar dilution method. The incidence of CDI in three studied hospitals was 145, 146, and 24 cases per 100,000 inhabitants in 2011 and 177, 258, and 67 cases per 100,000 inhabitants in 2012. A total of 64 isolates of C. difficile was available for molecular typing and antimicrobial susceptibility testing. 60.9% of the isolates were classified as ribotype 176. All 41 isolates of ribotypes 176 and 078 were positive for the presence of binary toxin genes. Ribotype 176 also carried 18-bp deletion in the regulatory gene tcdC. Tested isolates of C. difficile were fully susceptible to vancomycin and metronidazole, whereas 65.1% of the isolates were resistant to moxifloxacin. MLVA results indicated that isolates from three different hospitals were genetically related, suggesting transmission between healthcare facilities.

• MeSH
• antibakteriální látky farmakologie   MeSH
• bakteriální toxiny analýza   genetika   MeSH
• Clostridioides difficile klasifikace   genetika   izolace a purifikace   MeSH
• diskové difúzní antimikrobiální testy MeSH
• incidence MeSH
• klostridiové infekce epidemiologie   mikrobiologie   MeSH
• lidé MeSH
• minisatelitní repetice MeSH
• molekulární typizace * MeSH
• nemocnice MeSH
• ribotypizace MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Geografické názvy
• Česká republika epidemiologie   MeSH

The posttranslational Ca2+-dependent "clip-and-link" activity of large repeat-in-toxin (RTX) proteins starts by Ca2+-dependent structural rearrangement of a highly conserved self-processing module (SPM). Subsequently, an internal aspartate-proline (Asp-Pro) peptide bond at the N-terminal end of SPM breaks, and the liberated C-terminal aspartyl residue can react with a free ε-amino group of an adjacent lysine residue to form a new isopeptide bond. Here, we report a solution structure of the calcium-loaded SPM (Ca-SPM) derived from the FrpC protein of Neisseria meningitidis The Ca-SPM structure defines a unique protein architecture and provides structural insight into the autocatalytic cleavage of the Asp-Pro peptide bond through a "twisted-amide" activation. Furthermore, in-frame deletion of the SPM domain from the ApxIVA protein of Actinobacillus pleuropneumoniae attenuated the virulence of this porcine pathogen in a pig respiratory challenge model. We hypothesize that the Ca2+-dependent clip-and-link activity represents an unconventional strategy for Gram-negative pathogens to adhere to the host target cell surface.IMPORTANCE The Ca2+-dependent clip-and-link activity of large repeat-in-toxin (RTX) proteins is an exceptional posttranslational process in which an internal domain called a self-processing module (SPM) mediates Ca2+-dependent processing of a highly specific aspartate-proline (Asp-Pro) peptide bond and covalent linkage of the released aspartyl to an adjacent lysine residue through an isopeptide bond. Here, we report the solution structures of the Ca2+-loaded SPM (Ca-SPM) defining the mechanism of the autocatalytic cleavage of the Asp414-Pro415 peptide bond of the Neisseria meningitidis FrpC exoprotein. Moreover, deletion of the SPM domain in the ApxIVA protein, the FrpC homolog of Actinobacillus pleuropneumoniae, resulted in attenuation of virulence of the bacterium in a pig infection model, indicating that the Ca2+-dependent clip-and-link activity plays a role in the virulence of Gram-negative pathogens.

• MeSH
• Actinobacillus pleuropneumoniae chemie   patogenita   MeSH
• bakteriální proteiny chemie   genetika   MeSH
• bakteriální toxiny chemie   MeSH
• infekce bakteriemi rodu Actinobacillus veterinární   MeSH
• membránové proteiny chemie   MeSH
• Neisseria meningitidis chemie   MeSH
• posttranslační úpravy proteinů * MeSH
• prasata MeSH
• vápník metabolismus   MeSH
• virulence MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Despite more than 50 years of vaccination, pertussis is still an endemic disease, with regular epidemic outbreaks. With the exception of Poland, European countries have replaced whole-cell vaccines (WCVs) by acellular vaccines (ACVs) in the 1990s. Worldwide, antigenic divergence in vaccine antigens has been found between vaccine strains and circulating strains. In this work, 466 Bordetella pertussis isolates collected in the period 1998-2012 from 13 European countries were characterised by multi-locus antigen sequence typing (MAST) of the pertussis toxin promoter (ptxP) and of the genes coding for proteins used in the ACVs: pertussis toxin (Ptx), pertactin (Prn), type 2 fimbriae (Fim2) and type 3 fimbriae (Fim3). Isolates were further characterised by fimbrial serotyping, multi-locus variable-number tandem repeat analysis (MLVA) and pulsed-field gel electrophoresis (PFGE). The results showed a very similar B. pertussis population for 12 countries using ACVs, while Poland, which uses a WCV, was quite distinct, suggesting that ACVs and WCVs select for different B. pertussis populations. This study forms a baseline for future studies on the effect of vaccination programmes on B. pertussis populations.

• MeSH
• antigeny bakteriální genetika   MeSH
• Bordetella pertussis genetika   izolace a purifikace   klasifikace   MeSH
• genetická variace * MeSH
• lidé MeSH
• minisatelitní repetice MeSH
• molekulární epidemiologie MeSH
• multilokusová sekvenční typizace MeSH
• pertuse * epidemiologie   mikrobiologie   MeSH
• pertusový toxin genetika   MeSH
• promotorové oblasti (genetika) MeSH
• pulzní gelová elektroforéza MeSH
• sérotypizace MeSH
• Check Tag
• lidé MeSH
• Geografické názvy
• Evropa MeSH

PURPOSE: To characterise and compare twenty-eight Finnish Clostridium difficile RT027-like isolates, selected based on the presence of 18 bp deletion in the tcdC gene and toxin gene profile (A, B, binary), with eleven RT027 isolates from different Finnish geographical areas and time periods. METHODS: Twenty-eight C. difficile RT027-like isolates and 11 RT027 comparative strains were characterised by capillary-electrophoresis (CE) ribotyping, multi-locus variable tandem-repeats analysis (MLVA), multi-locus sequence typing (MLST), and sequencing of tcdC and gyrA gene fragments. Susceptibility to moxifloxacin was determined by E-test. RESULTS: Of 28 RT027-like isolates, seven RTs (016, 034, 075, 080, 153, 176 and 328), three WEBRIBO types (411, 475, AI-78) and three new profiles (F1-F3) were identified. MLVA revealed six clonal complexes (RTs 016, 027, 176 and F3). MLST showed eleven sequence types (1, 41, 47, 67, 95, 191,192, 223, 229, 264 and new ST). Twenty-two isolates (RTs 016, 080, 176, 328, F1, F2, F3 and WRTAI-78) carried Δ117 in the tcdC gene. Isolates of RTs 016, 027 and 176 were moxifloxacin resistant and harboured Thr82Ile in the GyrA. CONCLUSION: Our results show a high diversity within 28 Finnish RT027-like C. difficile isolates, with twelve CE-ribotyping profiles and eleven STs. MLVA revealed the regional spread of RTs 016, 027, 176 and F3. The presence of Δ117 in the tcdC gene in eight non-027 RTs highlights the importance of careful interpretation of the results from molecular systems targeting this site in the genome of C. difficile and the need of strain typing for epidemiological purposes.

• MeSH
• ADP-ribosatransferasy genetika   MeSH
• antibakteriální látky farmakologie   MeSH
• bakteriální léková rezistence MeSH
• bakteriální proteiny genetika   MeSH
• bakteriální toxiny analýza   genetika   MeSH
• Clostridioides difficile klasifikace   účinky léků   genetika   izolace a purifikace   MeSH
• DNA bakterií analýza   MeSH
• DNA gyráza genetika   MeSH
• dospělí MeSH
• enterotoxiny genetika   MeSH
• fluorochinolony farmakologie   MeSH
• genotyp MeSH
• klostridiové infekce epidemiologie   mikrobiologie   MeSH
• lidé středního věku MeSH
• lidé MeSH
• mikrobiální testy citlivosti MeSH
• minisatelitní repetice genetika   MeSH
• mladý dospělý MeSH
• molekulární epidemiologie MeSH
• moxifloxacin MeSH
• multilokusová sekvenční typizace metody   MeSH
• polymerázová řetězová reakce metody   MeSH
• represorové proteiny genetika   MeSH
• ribotypizace metody   MeSH
• sekvence nukleotidů MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• techniky typizace bakterií * MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mladý dospělý MeSH
• mužské pohlaví MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• Geografické názvy
• Finsko MeSH

Repeated injection cycles with abobotulinumtoxinA, a botulinum toxin type A, are recommended in current clinical guidelines as a treatment option for adults with upper limb spastic paresis. However, the magnitude of the maximal therapeutic effect of repeated abobotulinumtoxinA treatment across different efficacy parameters and the number of injection cycles required to reach maximal effect remain to be elucidated. Here, we present a post hoc exploratory analysis of a randomized, double-blind, placebo-controlled trial (12-24 weeks; NCT01313299) and open-label extension study (up to 12 months; NCT0131331), in patients aged 18-80 years with hemiparesis for ≥6 months after stroke/traumatic brain injury. Two inferential methods were used to assess the changes in efficacy parameters after repeat abobotulinumtoxinA treatment cycles: Mixed Model Repeated Measures analysis and Non-Linear Random Coefficients analysis. Using the latter model, the expected maximal effect size (not placebo-controlled) and the number of treatment cycles to reach 90% of this maximal effect were estimated. Treatment responses in terms of passive and perceived parameters (i.e. modified Ashworth scale in primary target muscle group, disability assessment scale for principal target for treatment or limb position, and angle of catch at fast speed) were estimated to reach near-maximal effect in two to three cycles. Near-maximal treatment effect for active parameters (i.e. active range of motion against the resistance of extrinsic finger flexors and active function, assessed by the Modified Frenchay Scale) was estimated to be reached one to two cycles later. In contrast to most parameters, active function showed greater improvements at Week 12 (estimated maximal change from baseline-modified Frenchay Scale overall score: +0.8 (95% confidence interval, 0.6; 1.0) than at Week 4 (+0.6 [95% confidence interval, 0.4; 0.8]). Overall, the analyses suggest that repeated treatment cycles with abobotulinumtoxinA in patients chronically affected with upper limb spastic paresis allow them to relearn how to use the affected arm with now looser antagonists. Future studies should assess active parameters as primary outcome measures over repeated treatment cycles, and assess efficacy at the 12-week time-point of each cycle, as the benefits of abobotulinumtoxinA may be underestimated in the studies of insufficient duration. Abbreviated summary In this post hoc analysis of repeated abobotulinumtoxinA injection cycles in upper limb spastic paresis, Gracies et al. used statistical modelling to elucidate the maximal therapeutic effect of abobotulinumtoxinA. Notably, the number of injections required to reach this maximal effect was higher for active (e.g. active function) compared with passive (e.g. tone) parameters.

• Publikační typ
• časopisecké články MeSH

Cíle: Botulinum toxin A (BoNT-A) umožňuje cílenou terapii spasticity a stal se běžným prostředkem léčby u pacientů s dětskou mozkovou obrnou (DMO). Nicméně, informace o efektu opakovaného podávání BoNT-A a dlouhodobé výsledky zatím chybí. Naší hypotézou bylo, že děti léčené víceetážovou opakovanou aplikací vysokodávkovaného BoNT-A nezhorší svůj chůzový stereotyp po dobu minimálně čtyř let. Materiál a metodika: Osmnáct pacientů s diplegickou, spastickou formou DMO, Gross Motor Function Classification System I–III a průměrným věkem 6 let a 9 měsíců bylo léčeno pomocí konceptu integrované léčby BoNT-A po minimální dobu 4 let. Jejich chůzový stereotyp byl opakovaně měřen pomocí kinematické a kinetické analýzy chůze a jako hlavní výsledný parametr byl zvolen Gait Deviation Index (GDI). Výsledky: Průměrná doba sledování činila 5 let a 9 měsíců (SD 1 rok a 5 měsíců). Každému z dětí byl aplikován BoNT-A průměrně 1,02krát (SD 0,37) ročně. Celkový GDI zůstal nezměněn po celou dobu sledování, nicméně některé kinematické a kinetické parametry poukazují na změnu chůzového stereotypu během léčby. Nejlepších výsledků bylo dosaženo v oblasti hlezna, kdežto hybnost kyčelního kloubu se i přes léčbu zhoršovala. Závěr: Studie prokazuje, že pomocí integrovaného přístupu k aplikaci BoNT-A můžeme dlouhodobě zabránit deterioraci chůzového stereotypu u dětí se spastickou formou DMO. Integrovaný přístup má však své limity při léčbě flexorů kyčelního kloubu a hamstringů.

Aims: Botulinum toxin type A (BoNT-A) offers a targeted form of therapy to reduce spasticity and has become a standard form of treatment in children with cerebral palsy (CP). However, the effects of repeated BoNT-A treatment over a longer periods of time are not well known. We hypothesized that children treated with multi-level, high-dose, repeated BoNT-A applications would not deteriorate in their gait function over a minimum period of four years. Materials and methodology: Gait in 18 children with spastic diplegic CP, mean age 6 years 9 months, Gross Motor Function Classification System I–III, treated according to the concept of integrated BoNT-A application over a minimum of four years was evaluated using the kinematic and kinetic gait analysis. The main outcome measure was the Gait Deviation Index (GDI). Results: The mean follow-up time was 5 years and 9 months (SD 1 year 5 months). Each child received a mean of 1.02 (SD 0.37) applications of BoNT-A per year. The gait function assessed by GDI remained unchanged. However, gait kinematic and kinetic parameters revealed some intra-individual changes and documented an evolution of gait pattern. The majority of improvements occurred at the level of ankle, while hip function was deteriora­ting despite the therapy. Conclusions: The study demonstrated that integrated multi­level BoNT-A treatment can be effective in maintaining the same level of walking in children with spastic diplegic cerebral palsy over a period of more than five years. The described integrated approach has limitations in the management of hip flexor and hamstrings tightness.

• Klíčová slova
• dětská mozková obrna,
• MeSH
• botulotoxiny typ A * aplikace a dávkování   terapeutické užití   MeSH
• chůze (způsob) * fyziologie   účinky léků   MeSH
• dítě MeSH
• dlouhodobá péče MeSH
• injekce intramuskulární MeSH
• kosterní svaly patofyziologie   účinky léků   MeSH
• lidé MeSH
• mozková obrna * farmakoterapie   MeSH
• předškolní dítě MeSH
• retrospektivní studie MeSH
• rozsah kloubních pohybů účinky léků   MeSH
• rozvrh dávkování léků MeSH
• svalový tonus účinky léků   MeSH
• výsledek terapie MeSH
• vývojová kyčelní dysplazie komplikace   terapie   MeSH
• vztah mezi dávkou a účinkem léčiva MeSH
• Check Tag
• dítě MeSH
• lidé MeSH
• mužské pohlaví MeSH
• předškolní dítě MeSH
• ženské pohlaví MeSH

• Klíčová slova
• neurogenní měchýř, inkontinence,
• MeSH
• botulotoxiny typ A aplikace a dávkování   škodlivé účinky   terapeutické užití   MeSH
• dítě MeSH
• endoskopie metody   využití   MeSH
• financování organizované MeSH
• hyperaktivní močový měchýř diagnóza   farmakoterapie   MeSH
• inkontinence moči farmakoterapie   MeSH
• lidé MeSH
• následné studie MeSH
• neurogenní močový měchýř diagnóza   farmakoterapie   MeSH
• statistika jako téma MeSH
• urodynamika MeSH
• výsledky a postupy - zhodnocení (zdravotní péče) MeSH
• způsoby aplikace léků MeSH
• Check Tag
• dítě MeSH
• lidé MeSH

This study aimed to implement a toxigenic culture as an optional third diagnostic step for glutamate dehydrogenase (GDH)-positive and toxin A/B-negative diarrheal stool samples into a diagnostic algorithm for Clostridioides (Clostridium) difficile infection (CDI), and to characterise C. difficile isolates for epidemiological purposes. During the 5-month study, 481 diarrhoeal stool samples from three Slovak hospitals were investigated and 66 non-duplicated GDH-positive stool samples were found. Of them, 36 were also toxin A/B-positive. Twenty-three GDH-positive and toxin A/B-negative stool samples were shown subsequently to be positive following toxigenic culture (TC). Molecular characterisation of C. difficile isolates showed the predominance of PCR ribotype (RT) 001 (n = 37, 56.1%) and the occurrence of RT 176 (n = 3, 4.5%). C. difficile RT 001 isolates clustered to eight clonal complexes (CCs) using multiple-locus variable-number tandem repeats analysis (MLVA). Interestingly, one third of RT 001 isolates clustering in these CCs were cultured from toxin A/B-negative stool samples. Our observations highlight the need of use multiple step diagnostic algorithm in CDI diagnosis in order to detect all CDI cases and to avoid the spread of C. difficile in healthcare settings.

• MeSH
• algoritmy MeSH
• bakteriální proteiny analýza   MeSH
• Clostridioides difficile klasifikace   izolace a purifikace   MeSH
• diagnostické techniky molekulární MeSH
• enterotoxiny analýza   nedostatek   MeSH
• feces mikrobiologie   MeSH
• glutamátdehydrogenasa analýza   MeSH
• klostridiové infekce diagnóza   epidemiologie   mikrobiologie   MeSH
• lidé MeSH
• minisatelitní repetice genetika   MeSH
• molekulární typizace * MeSH
• multilokusová sekvenční typizace MeSH
• nemocnice MeSH
• polymerázová řetězová reakce * MeSH
• průjem diagnóza   epidemiologie   mikrobiologie   MeSH
• ribotypizace metody   MeSH
• shluková analýza MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• multicentrická studie MeSH
• Geografické názvy
• Slovenská republika MeSH

OBJECTIVE: To determine electrical changes in the heart in a chronic, nonstatus model of epilepsy. METHODS: Electrocorticography (ECoG) and electrocardiography (ECG) of nine animals (five made epileptic by intrahippocampal injection of tetanus neurotoxin (TeNT) and four controls), are monitored continuously by radiotelemetry for up to 7 weeks. RESULTS: Epileptic animals develop a median of 168 seizures, with postictal tachycardias reaching a mean of 487 beats/min and lasting a mean of 661 seconds. Ictal changes in heart rate include tachycardia and in the case of convulsive seizures, bradyarrhythmias resembling Mobitz type 1 second-degree atrioventricular block; notably the P-R interval increased before block. Postictally, the amplitude of T wave increases. Interictally, QT dependence on RR is modest and conventional QT corrections prove ineffective. Interictal QT intervals, measured at a heart rate of 400 bpm, increased from 65 to 75 ms, an increase dependent on seizure incidence over the preceding 10-14 days. SIGNIFICANCE: Repeated seizures induce a sustained tachycardia and increase in QT interval of the ECG and evoke arrhythmias including periods of atrioventricular block during Racine type 4 and 5 seizures. These changes in cardiac function may predispose to development in fatal arrhythmias and sudden death in humans with epilepsy.

• MeSH
• bradykardie etiologie   MeSH
• elektrokardiografie MeSH
• elektrokortikografie MeSH
• krysa rodu rattus MeSH
• náhlá neočekávaná smrt při epilepsii etiologie   MeSH
• neurotoxiny toxicita   MeSH
• potkani Wistar MeSH
• tachykardie etiologie   MeSH
• tetanový toxin toxicita   MeSH
• záchvaty chemicky indukované   komplikace   patofyziologie   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH