Type III secretion proteins PcrV and PcrG from Pseudomonas aeruginosa form a 1:1 complex through high affinity interactions

BACKGROUND: Pseudomonas aeruginosa, an increasingly prevalent opportunistic pathogen, utilizes a type III secretion system for injection of toxins into host cells in order to initiate infection. A crucial component of this system is PcrV, which is essential for cytotoxicity and is found both within the bacterial cytoplasm and localized extracellularly, suggesting that it may play more than one role in Pseudomonas infectivity. LcrV, the homolog of PcrV in Yersinia, has been proposed to participate in effector secretion regulation by interacting with LcrG, which may act as a secretion blocker. Although PcrV also recognizes PcrG within the bacterial cytoplasm, the roles played by the two proteins in type III secretion in Pseudomonas may be different from the ones suggested for their Yersinia counterparts. RESULTS: In this work, we demonstrate by native mass spectrometry that PcrV and PcrG expressed and purified from E. coli form a 1:1 complex in vitro. Circular dichroism results indicate that PcrG is highly unstable in the absence of PcrV; in contrast, both PcrV alone and the PcrV:PcrG complex have high structural integrity. Surface plasmon resonance measurements show that PcrV interacts with PcrG with nanomolar affinity (15.6 nM) and rapid kinetics, an observation which is valid both for the full-length form of PcrG (residues 1–98) as well as a form which lacks the C-terminal 24 residues, which are predicted to have low secondary structure content. CONCLUSIONS: PcrV is a crucial component of the type III secretion system of Pseudomonas, but the way in which it participates in toxin secretion is not understood. Here we have characterized the interaction between PcrV and PcrG in vitro, and shown that PcrG is highly unstable. However, it associates readily with PcrV through a region located within its first 74 amino acids to form a high affinity complex. The fact that PcrV associates and dissociates quickly from an unstable molecule points to the transient nature of a PcrV:PcrG complex. These results are in agreement with analyses from pcrV deletion mutants which suggest that PcrV:PcrG may play a different role in effector secretion than the one described for the LcrV:LcrG complex in Yersinia

The demand for money in developing countries: Assessing the role of financial innovation

Traditional specifications of money demand have been commonly plagU4:!d by persistent overprediction, implausible parameter estimates, and highly autocorrelated errors. This paper argues that some of those problems stem from the failure to account for the impact of financial innovation. We estimate money demand for ten developing countries employing various proxies for the innovation process and provide an assessment of the relative importance of this variable. We find that financial innovation plays an important role in determining money demand and its fluctuations, and that the importance of this role increases with the rate of inflation.

A pan-European epidemiological study reveals honey bee colony survival depends on beekeeper education and disease control

Reports of honey bee population decline has spurred many national efforts to understand the extent of the problem and to identify causative or associated factors. However, our collective understanding of the factors has been hampered by a lack of joined up trans-national effort. Moreover, the impacts of beekeeper knowledge and beekeeping management practices have often been overlooked, despite honey bees being a managed pollinator. Here, we established a standardised active monitoring network for 5 798 apiaries over two consecutive years to quantify honey bee colony mortality across 17 European countries. Our data demonstrate that overwinter losses ranged between 2% and 32%, and that high summer losses were likely to follow high winter losses. Multivariate Poisson regression models revealed that hobbyist beekeepers with small apiaries and little experience in beekeeping had double the winter mortality rate when compared to professional beekeepers. Furthermore, honey bees kept by professional beekeepers never showed signs of disease, unlike apiaries from hobbyist beekeepers that had symptoms of bacterial infection and heavy Varroa infestation. Our data highlight beekeeper background and apicultural practices as major drivers of honey bee colony losses. The benefits of conducting trans-national monitoring schemes and improving beekeeper training are discussed

The evolving SARS-CoV-2 epidemic in Africa: Insights from rapidly expanding genomic surveillance

INTRODUCTION Investment in Africa over the past year with regard to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) sequencing has led to a massive increase in the number of sequences, which, to date, exceeds 100,000 sequences generated to track the pandemic on the continent. These sequences have profoundly affected how public health officials in Africa have navigated the COVID-19 pandemic. RATIONALE We demonstrate how the first 100,000 SARS-CoV-2 sequences from Africa have helped monitor the epidemic on the continent, how genomic surveillance expanded over the course of the pandemic, and how we adapted our sequencing methods to deal with an evolving virus. Finally, we also examine how viral lineages have spread across the continent in a phylogeographic framework to gain insights into the underlying temporal and spatial transmission dynamics for several variants of concern (VOCs). RESULTS Our results indicate that the number of countries in Africa that can sequence the virus within their own borders is growing and that this is coupled with a shorter turnaround time from the time of sampling to sequence submission. Ongoing evolution necessitated the continual updating of primer sets, and, as a result, eight primer sets were designed in tandem with viral evolution and used to ensure effective sequencing of the virus. The pandemic unfolded through multiple waves of infection that were each driven by distinct genetic lineages, with B.1-like ancestral strains associated with the first pandemic wave of infections in 2020. Successive waves on the continent were fueled by different VOCs, with Alpha and Beta cocirculating in distinct spatial patterns during the second wave and Delta and Omicron affecting the whole continent during the third and fourth waves, respectively. Phylogeographic reconstruction points toward distinct differences in viral importation and exportation patterns associated with the Alpha, Beta, Delta, and Omicron variants and subvariants, when considering both Africa versus the rest of the world and viral dissemination within the continent. Our epidemiological and phylogenetic inferences therefore underscore the heterogeneous nature of the pandemic on the continent and highlight key insights and challenges, for instance, recognizing the limitations of low testing proportions. We also highlight the early warning capacity that genomic surveillance in Africa has had for the rest of the world with the detection of new lineages and variants, the most recent being the characterization of various Omicron subvariants. CONCLUSION Sustained investment for diagnostics and genomic surveillance in Africa is needed as the virus continues to evolve. This is important not only to help combat SARS-CoV-2 on the continent but also because it can be used as a platform to help address the many emerging and reemerging infectious disease threats in Africa. In particular, capacity building for local sequencing within countries or within the continent should be prioritized because this is generally associated with shorter turnaround times, providing the most benefit to local public health authorities tasked with pandemic response and mitigation and allowing for the fastest reaction to localized outbreaks. These investments are crucial for pandemic preparedness and response and will serve the health of the continent well into the 21st century

PscF is a major component of the Pseudomonas aeruginosa type III secretion needle.

International audiencePseudomonas aeruginosa, a Gram-negative opportunistic pathogen, translocates exoenzymes (Exo) directly into the eukaryotic cell cytoplasm. This is accomplished by a type III secretion/translocation machinery. Here, we show that the P. aeruginosa type III secretory needle structure is composed essentially of PscF, a protein required for secretion and P. aeruginosa cytotoxicity. Partially purified needles, detached from the bacterial surface, are 60-80 nm in length and 7 nm in width, resembling needles from Yersinia spp.. YscF of Yersinia enterocolitica was able to functionally complement the pscF deletion, but required 11 P. aeruginosa-specific amino acids at the N-terminus for its function

The V Antigen of Pseudomonas aeruginosa Is Required for Assembly of the Functional PopB/PopD Translocation Pore in Host Cell Membranes

Pseudomonas aeruginosa efficiently intoxicates eukaryotic cells through the activity of the type III secretion-translocation system (TTSS). Gene deletions within the translocation operon pcrGVH-popBD abolish pore-forming activity of P. aeruginosa strains with macrophages and TTSS-dependent hemolysis. Here we investigated the requirements for PcrV, PopB, and PopD in pore formation by analyzing specific mutants using red blood cells (RBCs) and fibroblasts expressing green fluorescent protein fused to actin. Simultaneous secretion of three proteins, PopB, PopD, and PcrV, was required to achieve wild-type hemolysis and effector translocation. Deletion of pcrV in a cytotoxic strain did not affect secretion of PopB and PopD but abolished hemolytic activity and translocation of effectors into fibroblasts. Notably, the PcrV-deficient mutant was not capable of inserting PopD into host cell membranes, whereas PopB and PopD, but not PcrV, were readily found within membranes of wild-type-infected RBCs. Immunoprecipitation experiments performed by using a liposome model of pore assembly revealed a direct interaction between PopD and PopB but not between PopD and PcrV. Consequently, PcrV is necessary for the functional assembly of the PopB/D translocon complex but does not interact directly with pore-forming Pop proteins

Paysage et contrat territorial d'exploitation : Entre adequation difficile et emergence contrariee. Cas du departement de la Dordogne

Available from INIST (FR), Document Supply Service, under shelf-number : GR 1906 / INIST-CNRS - Institut de l'Information Scientifique et TechniqueSIGLEFRFranc

The PscE-PscF-PscG complex controls type III secretion needle biogenesis in Pseudomonas aeruginosa.

International audienceType III secretion (T3S) systems play key roles in pathogenicity of many Gram-negative bacteria and are employed to inject toxins directly into the cytoplasm of target cells. They are composed of over 20 different proteins that associate into a basal structure that traverses both inner and outer bacterial membranes and a hollow, needle-like structure through which toxins travel. The PscF protein is the main component of the Pseudomonas aeruginosa T3S needle. Here we demonstrate that PscF, when purified on its own, is able to form needle-like fibers of 8 nm in width and >1 microm in length. In addition, we demonstrate for the first time that the T3S needle subunit requires two cytoplasmic partners, PscE and PscG, in P. aeruginosa, which trap PscF in a ternary, 1:1:1 complex, thus blocking it in a monomeric state. Knock-out mutants deficient in PscE and PscG are non-cytotoxic, lack PscF, and are unable to export PscF encoded extrachromosomally. Temperature-scanning circular dichroism measurements show that the PscE-PscF-PscG complex is thermally stable and displays a cooperative unfolding/refolding pattern. Thus, PscE and PscG prevent PscF from polymerizing prematurely in the P. aeruginosa cytoplasm and keep it in a secretion prone conformation, strategies which may be shared by other pathogens that employ the T3S system for infection

Que serait un dispositif hospitalier adéquat au soutien de la transition chez les patients alcooliques ? :

Partant des nombreux ponts possibles entre une clinique de la transition adolescentaire et les impasses des personnes dépendantes à l’alcool, nous avons mis en avant la difficulté pour ces personnes de vivre les divers moments de transition en dehors d’un vécu de rupture et de fragilisation identitaire. Toute séparation ramène à une perte réelle de l’autre et, en miroir, de soi. Comment un cadre thérapeutique peut tenter de soutenir un certain travail autour de ces « passages » ? Pour ce faire, nous avons exploré le concept de transitionnalité, de préoccupation maternelle primaire, d’accrochage à un objet réel de dépendance afin d’aboutir sur les processus de séparation et d’individuation. Nous évoquons comment les différentes étapes et approches spécifiques d’un dispositif peuvent être autant d’occasions d’éprouver ces enjeux. La reprise dans les espaces de soin, de parole et de lien à l’autre réhumanisant (soignants et non-soignants) devrait rendre possible une transformation du patient lui permettant de renouer avec ses parts « capables », ouvrant la porte à d’autres investissements du côté d’une liberté nouvelle.[What would a successful hospital alcohol dependency program for patients in transition look like ?] Taking as a starting point the many possible connections between a teenage transition clinic and the roadblocks encountered in alcohol dependency, we stress the difficulty for anyone going through multiple moments of transition outside of the experience of fragile identity and breakdown. Each separation is a real loss of the other, and a part of oneself. How does a therapeutic framework attempt to work around these episodes? In an attempt to answer this question, we have explored the concepts of transitionality and primary maternal preoccupation, of transitional attachment, in order to get to grips with the processes of separation and individuation. We discuss how the different stages and specific approaches of a program can provide an opportunity to appreciate these challenges. Reestablishing speech and connecting to the other in caring spaces rehumanizes health professionals and nonhealth professionals alike, and should encourage patients to make changes enabling them to rediscover their inner strengths, also paving the way for them to reclaim their freedom. © 2021 Boeck Universite. All rights reserved