A novel sigma factor reveals a unique regulon controlling cell-specific recombination in Mycoplasma genitalium

The Mycoplasma genitalium MG428 protein shows homology to members of the sigma-70 family of sigma factors. Herein, we found that MG428 activates transcription of recA, ruvA and ruvB as well as several genes with unknown function. Deletion of MG_428 or some of the up-regulated unknown genes led to severe recombination defects. Single cell analyses revealed that activation of the MG428-regulon is a rare event under laboratory growth conditions. A conserved sequence with sigma-70 promoter architecture (TTGTCA-N-ATTWAT) was identified in the upstream region of all of the MG428-regulated genes or operons. Primer extension analyses demonstrated that transcription initiates immediately downstream of this sigma70-type promoter in a MG428-dependent manner. Furthermore, mutagenesis of the conserved −10 and −35 elements corroborated the requirement of these regions for promoter function. Therefore, a new mycoplasma promoter directs transcription of a unique recombination regulon. Additionally, MG428 was found to interact with the RNAP core enzyme, reinforcing the predicted role of this protein as an alternative sigma factor. Finally, our results indicate that MG428 contributes to the generation of genetic diversity in this model organism. Since recombination is an important mechanism to generate antigenic variation, MG428 emerges as a novel factor contributing to M. genitalium virulence

Heterogeneous catalysts with programmable topologies generated by reticulation of organocatalysts into metal-organic frameworks : The case of squaramide

A well-established strategy to synthesize heterogeneous, metal-organic framework (MOF) catalysts that exhibit nanoconfinement effects, and specific pores with highly-localized catalytic sites, is to use organic linkers containing organocatalytic centers. Here, we report that by combining this linker approach with reticular chemistry, and exploiting three-dimensioanl (3D) MOF-structural data from the Cambridge Structural Database, we have designed four heterogeneous MOF-based catalysts for standard organic transformations. These programmable MOFs are isoreticular versions of pcu IRMOF-16, fcu UiO-68 and pillared-pcu SNU-8X, the three most common topologies of MOFs built from the organic linker p,p'-terphenyldicarboxylic acid (tpdc). To synthesize the four squaramide-based MOFs, we designed and synthesized a linker, 4,4'-((3,4-dioxocyclobut-1-ene-1,2-diyl)bis(azanedyil))dibenzoic acid (Sq_tpdc), which is identical in directionality and length to tpdc but which contains organocatalytic squaramide centers. Squaramides were chosen because their immobilization into a framework enhances its reactivity and stability while avoiding any self-quenching phenomena. Therefore, the four MOFs share the same organocatalytic squaramide moiety, but confine it within distinct pore environments. We then evaluated these MOFs as heterogeneous H-bonding catalysts in organic transformations: a Friedel-Crafts alkylation and an epoxide ring-opening. Some of them exhibited good performance in both reactions but all showed distinct catalytic profiles that reflect their structural differences

Engineering <i>Mycoplasma pneumoniae</i> to bypass the association with Guillain-Barré syndrome

A non-pathogenic Mycoplasma pneumoniae-based chassis is leading the development of live biotherapeutic products (LBPs) for respiratory diseases. However, reports connecting Guillain-Barré syndrome (GBS) cases to prior M. pneumoniae infections represent a concern for exploiting such a chassis. Galactolipids, especially galactocerebroside (GalCer), are considered the most likely M. pneumoniae antigens triggering autoimmune responses associated with GBS development. In this work, we generated different strains lacking genes involved in galactolipids biosynthesis. Glycolipid profiling of the strains demonstrated that some mutants show a complete lack of galactolipids. Cross-reactivity assays with sera from GBS patients with prior M. pneumoniae infection showed that certain engineered strains exhibit reduced antibody recognition. However, correlation analyses of these results with the glycolipid profile of the engineered strains suggest that other factors different from GalCer contribute to sera recognition, including total ceramide levels, dihexosylceramide (DHCer), and diglycosyldiacylglycerol (DGDAG). Finally, we discuss the best candidate strains as potential GBS-free Mycoplasma chassis.</p

Current Incidence and risk factors of fecal incontinence after acute stroke affecting functionally independent people

Background: Previously published retrospective series show a high prevalence of fecal incontinence (FI) in stroke patients. We aimed to analyze in a prospective series the current incidence of FI in acute stroke in functionally independent patients and its evolution over time and the patient characteristics associated with the appearance of FI in acute stroke. Methods: We included consecutive patients with acute stroke admitted in our stroke unit who fulfilled the following inclusion criteria: a first episode of stroke, aged >18 years, with no previous functional dependency [modified Rankin Scale (mRS) = 2] and without previous known FI. FI was assessed by a multidisciplinary trained team using dedicated questionnaires at 72 ± 24 h (acute phase) and at 90 ± 15 days (chronic phase). Demographic, medical history, clinical and stroke features, mortality, and mRS at 7 days were collected. Results: Three hundred fifty-nine (48.3%) of 749 patients (mean age 65.9 ± 10, 64% male, 84.1% ischemic) fulfilled the inclusion criteria and were prospectively included during a 20-month period. FI was identified in 23 patients (6.4%) at 72 ± 24 h and in 7 (1.9%) at 90 days ± 15 days after stroke onset. FI was more frequent in hemorrhagic strokes (18 vs. 5%, p 0.007) and in more severe strokes [median National Institute of Health Stroke Scale (NIHSS) 18 (14–22) vs. 5 (3–13), p < 0.0001]. No differences were found regarding age, sex, vascular risk factors, or other comorbidities, or affected hemisphere. Patients with NIHSS =12 (AUC 0.81, 95% CI 0.71 to 0.89) had a 17-fold increase for the risk of FI (OR 16.9, IC 95% 4.7–60.1) adjusted for covariates.Peer ReviewedPostprint (published version

Engineering Mycoplasma pneumoniae to bypass the association with Guillain-Barré syndrome

A non-pathogenic Mycoplasma pneumoniae-based chassis is leading the development of live biotherapeutic products (LBPs) for respiratory diseases. However, reports connecting Guillain-Barré syndrome (GBS) cases to prior M. pneumoniae infections represent a concern for exploiting such a chassis. Galactolipids, especially galactocerebroside (GalCer), are considered the most likely M. pneumoniae antigens triggering autoimmune responses associated with GBS development. In this work, we generated different strains lacking genes involved in galactolipids biosynthesis. Glycolipid profiling of the strains demonstrated that some mutants show a complete lack of galactolipids. Cross-reactivity assays with sera from GBS patients with prior M. pneumoniae infection showed that certain engineered strains exhibit reduced antibody recognition. However, correlation analyses of these results with the glycolipid profile of the engineered strains suggest that other factors different from GalCer contribute to sera recognition, including total ceramide levels, dihexosylceramide (DHCer), and diglycosyldiacylglycerol (DGDAG). Finally, we discuss the best candidate strains as potential GBS-free Mycoplasma chassis.This project has received funding from the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation programme ERC LUNG-BIOREPAIR (101020135). We also acknowledge the support of the Spanish Ministry of Science and Innovation through the Plan Nacional PID2021-122341NB-I00 and the Centro de Excelencia Severo Ochoa (CEX2020-001049-S, MCIN/AEI /10.13039/501100011033), the Generalitat de Catalunya through the CERCA programme, the Center for Industrial Technology Development (CDTI) through the Neotec programme (SNEO 20211019) and to the EMBL partnership. C.P.-L. acknowledges the support of ‘Programa Torres Quevedo’ grant [PTQ2020-011048] funded by MCIN/AEI/10.13039/501100011033; European Union ‘NextGenerationEU/PRTR’. The proteomics analyses were performed in the CRG/UPF Proteomics Unit which is part of the Spanish National Infrastructure for Omics Technologies (ICTS OmicsTech). We thank T. Hoogenboezem and C. Gago da Graça (Department of Pediatrics, Erasmus MC–Sophia Children's Hospital, University Medical Centre, Rotterdam, The Netherlands) for excellent technical assistance.Peer reviewe