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Background Frequent testing for Legionella concentration in water is required by most health risk monitoring organizations worldwide. Domestic hot water and cooling tower water networks must be regularly controlled to prevent Legionnaires’ disease, a potentially deadly lung infection. MICA Legionella is the fastest culture-based detection method for all serogroups of Legionella pneumophila, with automatic enumeration in 48 h and no need for confirmation. Objective This study compares the performance and robustness of MICA Legionella with the reference method ISO 11731:2017 for the enumeration of culturable L. pneumophila. Methods MICA Legionella and ISO 11731:2017 results were compared for domestic hot water and cooling tower water. Inclusivity and exclusivity were tested on reference and environmental strains. Ruggedness, lot-to-lot consistency, and stability of the reagents kit were also studied. Results Enumeration of L. pneumophila by MICA Legionella was statistically equivalent to ISO 11731:2017 in both matrixes. In cooling tower waters, MICA Legionella showed better sensitivity than ISO 11731:2017. It presented a 94% sensitivity and a 97% specificity. Conclusion MICA Legionella is a highly sensitive and specific method for culturable L. pneumophila enumeration. It presents, in 48 hours, equivalent or better results than ISO 11731:2017. Its protocol is robust to variations. Its reagents kit is stable for up to 18 months. Highlights MICA Legionella is a robust and reliable method for the enumeration of culturable L. pneumophila in domestic and cooling tower water. It reduces significantly the number of sample pretreatments required in ISO 11731:2017. Automatic identification and enumeration of L. pneumophila microcolonies eliminates the requirement to have skilled analysts and limits the results variability. It also greatly reduces the time to results to 48 h instead of 7–10 days with ISO 11731:2017 while providing statistically equivalent results.

The CD86 occupancy assay has been developed to measure the number of CD86 molecules unbound to belatacept, but its association with clinical outcomes has not been assessed yet. All kidney transplant patients switched to belatacept in our center between 2016 and 2018 were included. Blood samples were collected before each infusion for 1 year to assess CD86 occupancy by CD86 antibody cytometry staining on the surface of CD14

Salmonella enterica is an intracellular bacterial pathogen. The formation of its replication niche, which is composed of a vacuole associated with a network of membrane tubules, depends on the secretion of a set of bacterial effector proteins whose activities deeply modify the functions of the eukaryotic host cell. By recruiting and regulating the activity of the kinesin-1 molecular motor, Salmonella effectors PipB2 and SifA play an essential role in the formation of the bacterial compartments. In particular, they allow the formation of tubules from the vacuole and their extension along the microtubule cytoskeleton, and thus promote membrane exchanges and nutrient supply. We have developed in vitro and in cellulo assays to better understand the specific role played by these two effectors in the recruitment and regulation of kinesin-1. Our results reveal a specific interaction between the two effectors and indicate that, contrary to what studies on infected cells suggested, interaction with PipB2 is sufficient to relieve the autoinhibition of kinesin-1. Finally, they suggest the involvement of other Salmonella effectors in the control of the activity of this molecular motor. This article has an associated First Person interview with the first author of the paper.

Almost all living organisms use protein chaperones with a view to preventing proteins from misfolding or aggregation either spontaneously or during cellular stress. This work uses a reaction-diffusion stochastic model to describe the dynamic localization of the Hsp70 chaperone DnaK in Escherichia coli cells during transient proteotoxic collapse characterized by the accumulation of insoluble proteins. In the model, misfolded ('abnormal') proteins are produced during alcoholic stress and have the propensity to aggregate with a polymerization-like kinetics. When aggregates diffuse more slowly they grow larger. According to Michaelis-Menten-type kinetics, DnaK has the propensity to bind with misfolded proteins or aggregates in order to catalyse refolding. To match experimental fluorescence microscopy data showing clusters of DnaK-GFP localized in multiple foci, the model includes spatial zones with local reduced diffusion rates to generate spontaneous assemblies of DnaK called 'foci'. Numerical simulations of our model succeed in reproducing the kinetics of DnaK localization experimentally observed. DnaK starts from foci, moves to large aggregates during acute stress, resolves those aggregates during recovery and finally returns to its initial punctate localization pattern. Finally, we compare real biological events with hypothetical repartitions of the protein aggregates or DnaK. We then notice that DnaK action is more efficient on protein aggregates than on protein homogeneously distributed.

Neisseria meningitidis is a bacterium responsible for severe sepsis and meningitis. Following type IV pilus-mediated adhesion to endothelial cells, bacteria proliferating on the cellular surface trigger a potent cellular response that enhances the ability of adhering bacteria to resist the mechanical forces generated by the blood flow. This response is characterized by the formation of numerous 100 nm wide membrane protrusions morphologically related to filopodia. Here, a high-resolution quantitative live-cell fluorescence microscopy procedure was designed and used to study this process. A farnesylated plasma membrane marker was first detected only a few seconds after bacterial contact, rapidly followed by actin cytoskeleton reorganization and bulk cytoplasm accumulation. The bacterial type IV pili-associated minor pilin PilV is necessary for the initiation of this cascade. Plasma membrane composition is a key factor as cholesterol depletion with methyl-β-cyclodextrin completely blocks the initiation of the cellular response. In contrast membrane deformation does not require the actin cytoskeleton. Strikingly, plasma membrane remodelling undermicrocolonies is also independent of common intracellular signalling pathways as cellular ATP depletion is not inhibitory. This study shows that bacteria-induced plasma membrane reorganization is a rapid event driven by a direct cross-talk between type IV pili and the plasma membrane rather than by the activation of an intracellular signalling pathway that would lead to actin remodelling.

Metabolic glycan labeling has recently emerged as a very powerful method for studying cell-surface glycans, which has applications that range from imaging glycans in living multicellular organisms, such as zebrafish or mice, to the identification of metastasis-associated cell-surface sialoglycoproteins. This strategy relies on the cellular biosynthetic machinery assimilating a modified monosaccharide that contains a bioorthogonal chemical reporter. The metabolic incorporation of this reporter into glycans can be further visualized by chemical ligation with a label, such as a fluorescent probe. Somewhat surprisingly, previous studies have mainly focused on the labeling of vertebrate glycans by using derivatives of common monosaccharides, such as Nacetyl neuraminic acid (or its N-acetylmannosamine precursor), N-acetylglucosamine, N-acetylgalactosamine, and fucose. In spite of a much higher degree of diversity in their monosaccharide building blocks as well as an essential role in bacterium–host interactions and bacterial virulence, bacterial polysaccharides have been poorly explored in terms of in vivo structural modifications. Bacteria are divided into Grampositive and Gram-negative bacteria. Whereas Gram-positive bacteria are surrounded by a peptidoglycan cell wall, Gramnegative bacteria are covered by a dense layer of lipopolysaccharides that are embedded in their outer membrane. These lipopolysaccharides are involved in the structural integrity of the cell and are often considered as determinants of pathogenicity. Although lipopolysaccharides appear to be an interesting target for specific and well-defined glycan metabolic labeling in Gram-negative bacteria, attempts to achieve this goal have been limited to the introduction of modified l-fucose derivatives into a customized, genetically engineered strain of Escherichia coli. Although it is a very interesting proof of concept, this l-fucose-based approach has some limitations as l-fucose is not generally present within the lipopolysaccharides of all Gram-negative bacteria, but is found in the O-antigens of specific strains. Secondly, free lfucose is not an intermediate in the normal E. coli “de novo” pathway and, therefore, should not be directly activable into a nucleotide-sugar donor without the introduction of an alternative pathway, known as the “salvage pathway”, into the organism of interest by genetic engineering (metabolic pathway engineering). Furthermore, once activated in the form of a modified guanosine-5’-diphosphate–fucose (GDP– Fuc), the l-fucose analogue might be transformed into a correspondingly modified GDP–mannose (GDP–Man) by the reverse de novo pathway, and potentially further metabolized into various other compounds, a process which could result in the chemical reporter being spread through other pathways of sugar metabolism or beyond. As a result of all of these limitations, and as our goal was labeling the lipopolysaccharides of bacteria with no genetic modification, we investigated whether another sugar could be used as a target for the metabolic modification of glycans. From all of the potential targets, 3-deoxy-d-mannooctulosonic acid (KDO) appears to be a very attractive candidate. Indeed, KDO is a specific and essential component of the inner core of lipopolysaccharides, and has long been considered as being present in the lipopolysaccharides of almost all Gram-negative species (as well as higher plants and algae), in which at least one residue is directly connected to lipid A (Scheme 1a). Because of its vital importance, KDO has been considered as a determinant for the characterization of Gram-negative bacteria, and the KDO pathway as a potential target for the development of new antibacterial compounds. In the KDO pathway (Scheme 1b), arabinose5-phosphate (arabinose-5-P) is condensed with phosphoenolpyruvate (PEP) to give KDO-8-phosphate (KDO-8-P), which is then transformed into free KDO, and further activated to form the cytidine monophosphate (CMP)–KDO donor prior to lipopolysaccharide elaboration. For all of these reasons, we hypothesized that the KDO pathway, as a lipopolysaccharidespecific pathway, may be tolerant enough to incorporate a modified analogue of KDO, such as 8-azido-8-deoxy-KDO (1, Scheme 2), into the core of E. coli lipopolysaccharides, and potentially other Gram-negative bacteria. Given the presence of free KDO as an intermediate in the pathway, we postulated that if the cell penetration of this analogue of KDO was sufficient, it could then be directly activated, partially replace endogenous KDO in lipopolysaccharides, and be detected on the cell surface by azide–alkyne click chemistry (Figure S1 in the Supporting Information). Moreover, modification of the C8-position of KDOwith a bioorthogonal azido group should prevent reverse metabolism by KDO-8-P [*] Dr. A. Dumont, Dr. S. Dukan Aix Marseille Universit , Laboratoire de Chimie Bact rienne (UMR 7283), Institut de Microbiologie de la M diterran e (IMM), CNRS, 31 Chemin Joseph Aiguier 13402 Marseille (France) E-mail: sdukan@imm.cnrs.fr

International audience; Legionella pneumophila is a pathogenic bacterium involved in regular outbreaks characterized by a relatively high fatality rate and an important societal impact. Frequent monitoring of the presence of this bacterium in environmental water samples is necessary to prevent these epidemic events, but the traditional culture-based detection and identification method requires up to 10 days. Reported herein is a method allowing identification of Legionella pneumophila by metabolic lipopolysaccharide labeling which targets, for the first time, a precursor to monosaccharides that are specifically present within the O-antigen of the bacterium. This new approach allows easy detection of living Legionella pneumophila, while other Legionella species are not labeled.

Neisseria meningitidis is a bacterium responsible for severe sepsis and meningitis. Following type IV pilus-mediated adhesion to endothelial cells, bacteria proliferating on the cellular surface trigger a potent cellular response that enhances the ability of adhering bacteria to resist the mechanical forces generated by the blood flow. This response is characterized by the formation of numerous 100 nm wide membrane protrusions morphologically related to filopodia. Here, a high-resolution quantitative live-cell fluorescence microscopy procedure was designed and used to study this process. A farnesylated plasma membrane marker was first detected only a few seconds after bacterial contact, rapidly followed by actin cytoskeleton reorganization and bulk cytoplasm accumulation. The bacterial type IV pili-associated minor pilin PilV is necessary for the initiation of this cascade. Plasma membrane composition is a key factor as cholesterol depletion with methyl-β-cyclodextrin completely blocks the initiation of the cellular response. In contrast membrane deformation does not require the actin cytoskeleton. Strikingly, plasma membrane remodelling undermicrocolonies is also independent of common intracellular signalling pathways as cellular ATP depletion is not inhibitory. This study shows that bacteria-induced plasma membrane reorganization is a rapid event driven by a direct cross-talk between type IV pili and the plasma membrane rather than by the activation of an intracellular signalling pathway that would lead to actin remodelling.

International audience; The Gram-negative bacterium Neisseria meningitidis asymptomatically colonizes the throat of 10 to 30% of the human population, but throat colonization can also act as the port of entry to the blood (septicemia) and then the brain (meningitis). Colonization is mediated by filamentous organelles referred to as type IV pili, which allow the formation of bacterial aggregates associated with host cells. We found that proliferation of N. meningitidis in contact with host cells increased the transcription of a bacterial gene encoding a transferase that adds phosphoglycerol onto type IV pili. This unusual posttranslational modification specifically released type IV pili-dependent contacts between bacteria. In turn, this regulated detachment process allowed propagation of the bacterium to new colonization sites and also migration across the epithelium, a prerequisite for dissemination and invasive disease.

International audience; In Salmonella-infected cells, the bacterial effector SifA forms a functional complex with the eukaryotic protein SKIP (SifA and kinesin-interacting protein). The lack of either partner has important consequences on the intracellular fate and on the virulence of this pathogen. In addition to SifA, SKIP binds the microtubule-based motor kinesin-1. Yet the absence of SifA or SKIP results in an unusual accumulation of kinesin-1 on the bacterial vacuolar membrane. To understand this apparent contradiction, we investigated the interaction between SKIP and kinesin-1 and the function of this complex. We show that the C-terminal RUN (RPIP8, UNC-14 and NESCA) domain of SKIP interacted specifically with the tetratricopeptide repeat (TPR) domain of the kinesin light chain. Overexpression of SKIP induced a microtubule- and kinesin-1-dependent anterograde movement of late endosomal/lysosomal compartments. In infected cells, SifA contributed to the fission of vesicles from the bacterial vacuole and the SifA/SKIP complex was required for the formation and/or the anterograde transport of kinesin-1-enriched vesicles. These observations reflect the role of SKIP as a linker and/or an activator for kinesin-1.

International audience; SifA is a Salmonella effector that is translocated into infected cells by the pathogenicity island 2-encoded type three secretion system. SifA is a critical virulence factor. Previous studies demonstrated that, upon translocation, SifA binds the pleckstrin homology motif of the eukaryotic host protein SKIP. In turn, the SifA-SKIP complex regulates the mobilization of the molecular motor kinesin-1 on the bacterial vacuole. SifA exhibits multiple domains containing functional motifs. Here we performed a molecular dissection and a mutational study of SifA to evaluate the relative contribution of the different domains to SifA functions. Biochemical and crystallographic analysis confirmed that the N-terminal domain of SifA is sufficient to interact with the PH domain of SKIP, forming a 1:1 complex with a micromolar dissociation constant. Mutation of the tryptophan (W) residue in the WxxxE motif, which has been proposed to mimic active form of GTPase, deeply affected the stability and the translocation of SifA while mutations of the glutamic residue (E) had no functional impact. A SifA L130D mutant that does not bind SKIP showed a sifA-like phenotype both in infected cells and in the mouse model of infection. We concluded that the WxxxE motif is essential for maintaining the tertiary structure of SifA, the functions of which require the interaction with the eukaryotic protein SKIP.

Salmonella enterica is an intracellular bacterial pathogen that causes gastroenteritis and typhoid fever. Inside host cells, the bacterium is enclosed in a membrane bound compartment, the Salmonella-containing vacuole (SCV). Intracellular replication of Salmonella requires the translocation of effector proteins into the host cytosol. The SifA effector protein is important for the membrane stability of the SCV. Recently, we have shown that the Salmonella sifA- mutant presents on its vacuole an important accumulation of kinesin-1, a molecular motor involved in the plus-end-directed transport of various organelles. Kinesin-1 is not recruited on SCVs of mutants that do not translocate effector proteins. This indicates that SifA is a negative regulator of the recruitment of this molecular motor and reveals the existence of another effector that recruits kinesin-1. This chapter describes techniques that are used to screen by immunofluorescence microscopy the accumulation of kinesin-1 on strains of Salmonella carrying multiple mutations.

Understanding the mechanisms of Salmonella virulence is an important challenge. The capacity of this intracellular bacterial pathogen to cause diseases depends on the expression of virulence factors including the second type III secretion system (TTSS-2), which is used to translocate into the eukaryotic cytosol a set of effector proteins that divert the biology of the host cell and shape the bacterial replicative niche. Yet little is known about the eukaryotic functions affected by individual Salmonella effectors. Here we report that the TTSS-2 effector PipB2 interacts with the kinesin light chain, a subunit of the kinesin-1 motor complex that drives anterograde transport along microtubules. Translocation of PipB2 is both necessary and sufficient for the recruitment of kinesin-1 to the membrane of the Salmonella -containing vacuole. In vivo , PipB2 contributes to the attenuation of Salmonella mutant strains in mice. Taken together, our data indicate that the TTSS-2-mediated fine-tuning of kinesin-1 activity associated with the bacterial vacuole is crucial for the virulence of Salmonella .

La maladie athéromateuse rénovasculaire est responsable d’une morbi-mortalité élevée. Plusieurs essais randomisés ont conclu à l’absence de bénéfice de la revascularisation percutanée en comparaison au traitement médical optimisé. Cependant ces études ne prenaient pas en compte les présentations cliniques à haut-risque regroupant hypertension artérielle (HTA) résistante, œdème aigu pulmonaire (OAP) flash ou insuffisance rénale rapidement progressive. Ainsi, nous avons évalué l’intérêt de l’angioplastie plus particulièrement dans cette population en contexte de sténoses bilatérales des artères rénales sévères.ObjectifsNous avons réalisé une étude observationnelle rétrospective au CHU de Rouen sur 83 patients, entre le 01/01/1996 et le 31/12/2017 inclus, atteints de sténose athéromateuse des artères rénales (SAAR) ≥ 50% bilatérales ou unilatérales sur rein unique (40% des SAAR ≥ 80%). L’objectif principal de l’étude était d’analyser à la fois pour les présentations à haut-risque (n=53, 64%) regroupant HTA résistantes (n=29), OAP flashs (n=11), HTA résistantes avec insuffisance rénale aiguë (IRA) associée (n=9) et IRA isolée (n=4) ; et à bas-risque (n=30, 36%), l’impact de l’angioplastie sur la mortalité, l’évolution vers l’insuffisance rénale chronique (IRC) terminale et la survenue d’évènements cardiovasculaires (ECV) en comparaison au traitement médical. Secondairement, nous avons évalué l’intérêt de l’angioplastie sur le profil tensionnel et la fonction rénale.RésultatsAu total 54 patients ont été revascularisés dont 66% dans le groupe haut-risque. Au cours d’un suivi médian de 52 mois, 60% des patients haut-risque sont décédés, 25% ont évolué vers l’IRC terminale et enfin plus de 40% ont subi au moins un ECV. Nous mettons en évidence, dans ce groupe, un bénéfice de l’angioplastie à la fois sur la mortalité et la survie rénale en cas de SAAR ≥ 80% (respectivement p=0,01 et p=0,02). Nous montrons également un bénéfice de l’angioplastie sur l’amélioration de la PA systolique (groupe haut-risque) à 6 et 12 mois avec respectivement une baisse de 9 mmHg (p=0,01) et 10 mmHg (p=0,005). L’interprétation de nos résultats est limitée par l’aspect rétrospectif de l’étude et ses biais, le faible effectif et le manque de puissance de notre analyse statistique.ConclusionCette étude tend à montrer un bénéfice supérieur de l’angioplastie comparativement au traitement médical, dans la prise en charge des patients à haut-risque en contexte de MARV sévère, en cohérence avec les publications récentes.