Enterohemorrhagic E. coli Requires N-WASP for Efficient Type III Translocation but Not for EspFU-Mediated Actin Pedestal Formation

Upon infection of mammalian cells, enterohemorrhagic E. coli (EHEC) O157:H7 utilizes a type III secretion system to translocate the effectors Tir and EspFU (aka TccP) that trigger the formation of F-actin-rich ‘pedestals’ beneath bound bacteria. EspFU is localized to the plasma membrane by Tir and binds the nucleation-promoting factor N-WASP, which in turn activates the Arp2/3 actin assembly complex. Although N-WASP has been shown to be required for EHEC pedestal formation, the precise steps in the process that it influences have not been determined. We found that N-WASP and actin assembly promote EHEC-mediated translocation of Tir and EspFU into mammalian host cells. When we utilized the related pathogen enteropathogenic E. coli to enhance type III translocation of EHEC Tir and EspFU, we found surprisingly that actin pedestals were generated on N-WASP-deficient cells. Similar to pedestal formation on wild type cells, Tir and EspFU were the only bacterial effectors required for pedestal formation, and the EspFU sequences required to interact with N-WASP were found to also be essential to stimulate this alternate actin assembly pathway. In the absence of N-WASP, the Arp2/3 complex was both recruited to sites of bacterial attachment and required for actin assembly. Our results indicate that actin assembly facilitates type III translocation, and reveal that EspFU, presumably by recruiting an alternate host factor that can signal to the Arp2/3 complex, exhibits remarkable versatility in its strategies for stimulating actin polymerization

Etude fonctionnelle du facteur sigma principal de Bacteroides fragilis

Bacteroides fragilis est un germe anaérobie du tractus intestinal de l homme. Les promoteurs végétatifs de B. fragilis sont reconnus par un facteur sigma principal particulier (SigA), restreint aux phyla Bacteroidetes et Chlorobium. SigA possède un court segment basique, à la place de la région N-terminale 1.1 acide retrouvée chez tous les autres facteurs sigma principaux connus. Il contient des résidus "signature" dans les régions conservées 2 à 4, impliquées dans la reconnaissance des promoteurs et l interaction avec l ARN polymérase-cœur. Le segment basique de SigA conditionne le positionnement de l enzyme sur le promoteur et l étendue de la bulle de transcription. Le rôle de plusieurs résidus dans la formation du complexe ouvert de transcription et l interaction avec la séquence consensus -33 des promoteurs de B. fragilis est décrit. Cette thèse révèle l existence d un couple facteur sigma/promoteur unique au sein d un lignage majeur qui est apparu tôt dans l évolution bactérienne.PARIS-BIUSJ-Thèses (751052125) / SudocPARIS-BIUSJ-Physique recherche (751052113) / SudocSudocFranceF

The ability of an attaching and effacing pathogen to trigger localized actin assembly contributes to virulence by promoting mucosal attachment

Enterohaemorrhagic Escherichia coli (EHEC) colonizes the intestine and causes bloody diarrhoea and kidney failure by producing Shiga toxin. Upon binding intestinal cells, EHEC triggers a change in host cell shape, generating actin \u27pedestals\u27 beneath bound bacteria. To investigate the importance of pedestal formation to disease, we infected genetically engineered mice incapable of supporting pedestal formation by an EHEC-like mouse pathogen, or wild type mice with a mutant of that pathogen incapable of generating pedestals. We found that pedestal formation promotes attachment of bacteria to the intestinal mucosa and vastly increases the severity of Shiga toxin-mediated disease

Structural basis for complex formation between human IRSp53 and the translocated intimin receptor Tir of enterohemorrhagic E. coli.

Actin assembly beneath enterohemorrhagic E. coli (EHEC) attached to its host cell is triggered by the intracellular interaction of its translocated effector proteins Tir and EspF(U) with human IRSp53 family proteins and N-WASP. Here, we report the structure of the N-terminal I-BAR domain of IRSp53 in complex with a Tir-derived peptide, in which the homodimeric I-BAR domain binds two Tir molecules aligned in parallel. This arrangement provides a protein scaffold linking the bacterium to the host cell's actin polymerization machinery. The structure uncovers a specific peptide-binding site on the I-BAR surface, conserved between IRSp53 and IRTKS. The Tir Asn-Pro-Tyr (NPY) motif, essential for pedestal formation, is specifically recognized by this binding site. The site was confirmed by mutagenesis and in vivo-binding assays. It is possible that IRSp53 utilizes the NPY-binding site for additional interactions with as yet unknown partners within the host cell

Mechanical Thrombectomy for Acute Ischemic Stroke Amid the COVID-19 Outbreak

International audienceBackground and Purpose: The efficiency of prehospital care chain response and the adequacy of hospital resources are challenged amid the coronavirus disease 2019 (COVID-19) outbreak, with suspected consequences for patients with ischemic stroke eligible for mechanical thrombectomy (MT). Methods: We conducted a prospective national-level data collection of patients treated with MT, ranging 45 days across epidemic containment measures instatement, and of patients treated during the same calendar period in 2019. The primary end point was the variation of patients receiving MT during the epidemic period. Secondary end points included care delays between onset, imaging, and groin puncture. To analyze the primary end point, we used a Poisson regression model. We then analyzed the correlation between the number of MTs and the number of COVID-19 cases hospitalizations, using the Pearson correlation coefficient (compared with the null value). Results: A total of 1513 patients were included at 32 centers, in all French administrative regions. There was a 21% significant decrease (0.79; [95%CI, 0.76–0.82]; P <0.001) in MT case volumes during the epidemic period, and a significant increase in delays between imaging and groin puncture, overall (mean 144.9±SD 86.8 minutes versus 126.2±70.9; P <0.001 in 2019) and in transferred patients (mean 182.6±SD 82.0 minutes versus 153.25±67; P <0.001). After the instatement of strict epidemic mitigation measures, there was a significant negative correlation between the number of hospitalizations for COVID and the number of MT cases ( R 2 −0.51; P =0.04). Patients treated during the COVID outbreak were less likely to receive intravenous thrombolysis and to have unwitnessed strokes (both P <0.05). Conclusions: Our study showed a significant decrease in patients treated with MTs during the first stages of the COVID epidemic in France and alarming indicators of lengthened care delays. These findings prompt immediate consideration of local and regional stroke networks preparedness in the varying contexts of COVID-19 pandemic evolution