A Microscope Automated Fluidic System to Study Bacterial Processes in Real Time

Most time lapse microscopy experiments studying bacterial processes ie growth, progression through the cell cycle and motility have been performed on thin nutrient agar pads. An important limitation of this approach is that dynamic perturbations of the experimental conditions cannot be easily performed. In eukaryotic cell biology, fluidic approaches have been largely used to study the impact of rapid environmental perturbations on live cells and in real time. However, all these approaches are not easily applicable to bacterial cells because the substrata are in all cases specific and also because microfluidics nanotechnology requires a complex lithography for the study of micrometer sized bacterial cells. In fact, in many cases agar is the experimental solid substratum on which bacteria can move or even grow. For these reasons, we designed a novel hybrid micro fluidic device that combines a thin agar pad and a custom flow chamber. By studying several examples, we show that this system allows real time analysis of a broad array of biological processes such as growth, development and motility. Thus, the flow chamber system will be an essential tool to study any process that take place on an agar surface at the single cell level

Rules Governing Selective Protein Carbonylation

BACKGROUND:Carbonyl derivatives are mainly formed by direct metal-catalysed oxidation (MCO) attacks on the amino-acid side chains of proline, arginine, lysine and threonine residues. For reasons unknown, only some proteins are prone to carbonylation. METHODOLOGY/PRINCIPAL FINDINGS:we used mass spectrometry analysis to identify carbonylated sites in: BSA that had undergone in vitro MCO, and 23 carbonylated proteins in Escherichia coli. The presence of a carbonylated site rendered the neighbouring carbonylatable site more prone to carbonylation. Most carbonylated sites were present within hot spots of carbonylation. These observations led us to suggest rules for identifying sites more prone to carbonylation. We used these rules to design an in silico model (available at http://www.lcb.cnrs-mrs.fr/CSPD/), allowing an effective and accurate prediction of sites and of proteins more prone to carbonylation in the E. coli proteome. CONCLUSIONS/SIGNIFICANCE:We observed that proteins evolve to either selectively maintain or lose predicted hot spots of carbonylation depending on their biological function. As our predictive model also allows efficient detection of carbonylated proteins in Bacillus subtilis, we believe that our model may be extended to direct MCO attacks in all organisms

Mortality and pulmonary complications in patients undergoing surgery with perioperative SARS-CoV-2 infection: an international cohort study

Background: The impact of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) on postoperative recovery needs to be understood to inform clinical decision making during and after the COVID-19 pandemic. This study reports 30-day mortality and pulmonary complication rates in patients with perioperative SARS-CoV-2 infection. Methods: This international, multicentre, cohort study at 235 hospitals in 24 countries included all patients undergoing surgery who had SARS-CoV-2 infection confirmed within 7 days before or 30 days after surgery. The primary outcome measure was 30-day postoperative mortality and was assessed in all enrolled patients. The main secondary outcome measure was pulmonary complications, defined as pneumonia, acute respiratory distress syndrome, or unexpected postoperative ventilation. Findings: This analysis includes 1128 patients who had surgery between Jan 1 and March 31, 2020, of whom 835 (74·0%) had emergency surgery and 280 (24·8%) had elective surgery. SARS-CoV-2 infection was confirmed preoperatively in 294 (26·1%) patients. 30-day mortality was 23·8% (268 of 1128). Pulmonary complications occurred in 577 (51·2%) of 1128 patients; 30-day mortality in these patients was 38·0% (219 of 577), accounting for 81·7% (219 of 268) of all deaths. In adjusted analyses, 30-day mortality was associated with male sex (odds ratio 1·75 [95% CI 1·28–2·40], p\textless0·0001), age 70 years or older versus younger than 70 years (2·30 [1·65–3·22], p\textless0·0001), American Society of Anesthesiologists grades 3–5 versus grades 1–2 (2·35 [1·57–3·53], p\textless0·0001), malignant versus benign or obstetric diagnosis (1·55 [1·01–2·39], p=0·046), emergency versus elective surgery (1·67 [1·06–2·63], p=0·026), and major versus minor surgery (1·52 [1·01–2·31], p=0·047). Interpretation: Postoperative pulmonary complications occur in half of patients with perioperative SARS-CoV-2 infection and are associated with high mortality. Thresholds for surgery during the COVID-19 pandemic should be higher than during normal practice, particularly in men aged 70 years and older. Consideration should be given for postponing non-urgent procedures and promoting non-operative treatment to delay or avoid the need for surgery. Funding: National Institute for Health Research (NIHR), Association of Coloproctology of Great Britain and Ireland, Bowel and Cancer Research, Bowel Disease Research Foundation, Association of Upper Gastrointestinal Surgeons, British Association of Surgical Oncology, British Gynaecological Cancer Society, European Society of Coloproctology, NIHR Academy, Sarcoma UK, Vascular Society for Great Britain and Ireland, and Yorkshire Cancer Research

Molecular mechanisms underlying bacterial persisters

All bacteria form persisters, cells that are multidrug tolerant and therefore able to survive antibiotic treatment. Due to the low frequencies of persisters in growing bacterial cultures and the complex underlying molecular mechanisms, the phenomenon has been challenging to study. However, recent technological advances in microfluidics and reporter genes have improved this scenario. Here, we summarize recent progress in the field, revealing the ubiquitous bacterial stress alarmone ppGpp as an emerging central regulator of multidrug tolerance and persistence, both in stochastically and environmentally induced persistence. In several different organisms, toxin-antitoxin modules function as effectors of ppGpp-induced persistence

Agrégation et carbonylation des protéines (deux facteurs impliqués dans le vieillissement cellulaire d'Escherichia coli)

Longtemps considéré comme une spécificité des organismes eucaryotes, le processus de vieillissement a été plus récemment observé chez des procaryotes comme Escherichia coli. L agrégation et les lésions oxydatives des protéines semblent être deux facteurs potentiellement présents et prépondérants au cours du processus de vieillissement. Notre étude a permis de mettre en évidence et de caractériser le rôle et les conséquences de ces deux paramètres sur la physiologie d E. coli. Nous avons pu montrer l existence d agrégats protéiques chez E. coli. La caractérisation biochimique de ces agrégats montre qu ils présentent un sur-enrichissement en protéines anormales suggérant qu ils puissent constituer un compartiment poubelle dans la cellule. L analyse des facteurs modulant la quantité de ces agrégats a révélé que le niveau de Formes Réactive de l Oxygène ainsi que celui de protéines non et/ou mal repliées jouait un rôle prépondérant. Le fait que les protéines carbonylées (modification oxydative irréversible) soient détectables uniquement dans un état agrégé nous a mené à utiliser ce marqueur dans l étude de la dynamique des agrégats. Nous avons pu montrer qu une fois agrégées les protéines carbonylées n étaient pas dégradées ni solubilisées suggérant que les agrégats protéiques constituent une zone de stockage permanente. Finalement nous avons montré que ces agrégats protéiques s accumulaient au cours du temps et qu ils représentaient un facteur de vieillissement aboutissant à la mort cellulaire. En conclusion ce travail de thèse a permis de mettre en évidence le rôle et l importance de l accumulation des agrégats protéiques et des lésions oxydatives protéiques au cours du vieillissement cellulaire chez E. coli.Longtemps considéré comme une spécificité des organismes eucaryotes, le processus de vieillissement a été plus récemment observé chez des procaryotes comme Escherichia coli. L agrégation et les lésions oxydatives des protéines semblent être deux facteurs potentiellement présents et prépondérants au cours du processus de vieillissement. Notre étude a permis de mettre en évidence et de caractériser le rôle et les conséquences de ces deux paramètres sur la physiologie d E. coli. Nous avons pu montrer l existence d agrégats protéiques chez E. coli. La caractérisation biochimique de ces agrégats montre qu ils présentent un sur-enrichissement en protéines anormales suggérant qu ils puissent constituer un compartiment poubelle dans la cellule. L analyse des facteurs modulant la quantité de ces agrégats a révélé que le niveau de Formes Réactive de l Oxygène ainsi que celui de protéines non et/ou mal repliées jouait un rôle prépondérant. Le fait que les protéines carbonylées (modification oxydative irréversible) soient détectables uniquement dans un état agrégé nous a mené à utiliser ce marqueur dans l étude de la dynamique des agrégats. Nous avons pu montrer qu une fois agrégées les protéines carbonylées n étaient pas dégradées ni solubilisées suggérant que les agrégats protéiques constituent une zone de stockage permanente. Finalement nous avons montré que ces agrégats protéiques s accumulaient au cours du temps et qu ils représentaient un facteur de vieillissement aboutissant à la mort cellulaire. En conclusion ce travail de thèse a permis de mettre en évidence le rôle et l importance de l accumulation des agrégats protéiques et des lésions oxydatives protéiques au cours du vieillissement cellulaire chez E. coli.AIX-MARSEILLE2-BU Sci.Luminy (130552106) / SudocSudocFranceF

Stochastic induction of persister cells by HipA through (p)ppGpp-mediated activation of mRNA endonucleases

The model organism Escherichia coli codes for at least 11 type II toxin–antitoxin (TA) modules, all implicated in bacterial persistence (multidrug tolerance). Ten of these encode messenger RNA endonucleases (mRNases) inhibiting translation by catalytic degradation of mRNA, and the 11th module, hipBA, encodes HipA (high persister protein A) kinase, which inhibits glutamyl tRNA synthetase (GltX). In turn, inhibition of GltX inhibits translation and induces the stringent response and persistence. Previously, we presented strong support for a model proposing (p)ppGpp (guanosine tetra and penta-phosphate) as the master regulator of persistence. Stochastic variation of [(p)ppGpp] in single cells induced TA-encoded mRNases via a pathway involving polyphosphate and Lon protease. Polyphosphate activated Lon to degrade all known type II antitoxins of E. coli. In turn, the activated mRNases induced persistence and multidrug tolerance. However, even though it was known that activation of HipA stimulated (p)ppGpp synthesis, our model did not explain how hipBA induced persistence. Here we show that, in support of and consistent with our initial model, HipA-induced persistence depends not only on (p)ppGpp but also on the 10 mRNase-encoding TA modules, Lon protease, and polyphosphate. Importantly, observations with single cells convincingly show that the high level of (p)ppGpp caused by activation of HipA does not induce persistence in the absence of TA-encoded mRNases. Thus, slow growth per se does not induce persistence in the absence of TA-encoded toxins, placing these genes as central effectors of bacterial persistence

Regulation of ytfK by cAMP-CRP Contributes to SpoT-Dependent Accumulation of (p)ppGpp in Response to Carbon Starvation YtfK Responds to Glucose Exhaustion

International audienceGuanosine penta- or tetraphosphate (known as (p)ppGpp) serves as second messenger to respond to nutrient downshift and other environmental stresses, a phenomenon called stringent response. Accumulation of (p)ppGpp promotes the coordinated inhibition of macromolecule synthesis, as well as the activation of stress response pathways to cope and adapt to harmful conditions. In Escherichia coli , the (p)ppGpp level is tightly regulated by two enzymes, the (p)ppGpp synthetase RelA and the bifunctional synthetase/hydrolase SpoT. We recently identified the small protein YtfK as a key regulator of SpoT-mediated activation of stringent response in E. coli . Here, we further characterized the regulation of ytfK . We observed that ytfK is subjected to catabolite repression and is positively regulated by the cyclic AMP (cAMP)-cAMP receptor protein (CRP) complex. Importantly, YtfK contributes to SpoT-dependent accumulation of (p)ppGpp and cell survival in response to glucose starvation. Therefore, regulation of ytfK by the cAMP-CRP appears important to adjust (p)ppGpp level and coordinate cellular metabolism in response to glucose availability

Protein Aggregates: an Aging Factor Involved in Cell Death▿

In a previous study, we demonstrated the presence of protein aggregates in an exponentially grown Escherichia coli culture. In light of these observations, protein aggregates could be considered damage to cells that is able to pass from one generation to the next. Based on the assumption that the amount of aggregate protein could represent an aging factor, we monitored this amount in a bacterial culture during senescence. In doing so, we observed (i) a significant increase in the amount of aggregate protein over time, (ii) a proportional relationship between the amount of aggregate protein and the level of dead cells, (iii) a larger amount in dead cells than in culturable cells, (iv) a heterogeneous distribution of different amounts within a homogenous population of culturable cells entering stasis, and (v) that the initial amount of aggregate protein within a culturable population conditioned the death rate of the culture. Together, the results presented in this study suggest that protein aggregates indeed represent one aging factor leading to bacterial cell death