The Different Strategies Used by Salmonella to Invade Host Cells

Chapitre 17International audienc

Identification and role of the T3SS-1 independant invasion mechanism in salmonella enteritidis

Le principal système d’invasion chez Salmonella requiert un système de sécrétion de type III(T3SS-1) qui induit un mécanisme d’entrée de type Trigger. Cependant, d’autres invasine sont été décrites chez Salmonella mais leurs rôles dans la virulence restent peu connus. Nous avons ainsi caractérisé le rôle de Rck en tant qu’invasine chez Salmonella Enteritidis, et démontré par différentes approches que Rck induit un mécanisme d’invasion de type Zipper.L'étude de la cascade de signalisation cellulaire induite par Rck a permis de suggérer un modèle d'internalisation impliquant l’actine, le complexe Arp2/3, les GTPases Rac et Cdc42,Akt, la PI3K de classe I et Src. L'analyse du pouvoir d'invasion d'un mutant de S. Enteritidis cultivé dans des conditions où il est incapable d'exprimer les facteurs d'invasion connus : T3SS-1, Rck et PagN montre l’existence d'autre facteurs d’entrée encore non identifiés chez Salmonella qui semblent induire une invasion de type Zipper et de type Trigger. L’ensemble de ces données font de Salmonella, la première bactérie capable d’envahir les cellules soit via un mécanisme de type Trigger, dépendant au moins du T3SS-1, soit via un mécanisme de type Zipper, dépendant au moins de Rck et révèlent la complexité des mécanismes d’invasion de Salmonella.The main invasion system of Salmonella requires a type III secretion system (T3SS-1) which promotes a Trigger entry mechanism. However, other invasins were described in Salmonella but their roles in virulence remain unclear. We have shown that Salmonella Enteritidis caninvade cells via the Rck outer membrane protein and we have demonstrated by different approaches that Rck mediates a Zipper entry process. Characterisation of the cellular transduction pathway induced by Rck enable us to propose a model of internalisation involving actin, Arp2/3, Rac and Cdc42, Akt, class I PI3K and Src. Finally, the invasion ability of a S. Enteritidis mutant grown under culture conditions that did not allow the expression of any identified invasion factors (T3SS-1, Rck and PagN) provides evidences for still non-characterised Salmonella invasion factors which seem to induce both Zipper and Trigger mechanisms. Overall, our data indicate that Salmonella is the first bacterium found tobe able to invade cells by both a Trigger mechanism at least mediated by the T3SS-1 and a Zipper entry process at least mediated by Rck. Study of the T3SS-1-independent invasion systems could bring to a better understanding of Salmonella pathogenicity, particularly in regard to the different diseases induced by Salmonella and to its great diversity of hosts

Redox Control of Mitochondrial Calcium Uptake

Dong et al. (2017) establish how the mitochondrial Ca2+ uniporter (MCU) integrates Ca2+ and oxidative stress signals by identifying a cysteine residue that controls MCU channel activity, a mechanism causing mitochondrial Ca2+ overload and cell death during oxidative stress

Identification et rôle des mécanismes d'invasion cellulaire indépendants du T3SS-1 chez Salmonella Enteritidis

Le principal système d invasion chez Salmonella requiert un système de sécrétion de type III(T3SS-1) qui induit un mécanisme d entrée de type Trigger. Cependant, d autres invasine sont été décrites chez Salmonella mais leurs rôles dans la virulence restent peu connus. Nous avons ainsi caractérisé le rôle de Rck en tant qu invasine chez Salmonella Enteritidis, et démontré par différentes approches que Rck induit un mécanisme d invasion de type Zipper.L'étude de la cascade de signalisation cellulaire induite par Rck a permis de suggérer un modèle d'internalisation impliquant l actine, le complexe Arp2/3, les GTPases Rac et Cdc42,Akt, la PI3K de classe I et Src. L'analyse du pouvoir d'invasion d'un mutant de S. Enteritidis cultivé dans des conditions où il est incapable d'exprimer les facteurs d'invasion connus : T3SS-1, Rck et PagN montre l existence d'autre facteurs d entrée encore non identifiés chez Salmonella qui semblent induire une invasion de type Zipper et de type Trigger. L ensemble de ces données font de Salmonella, la première bactérie capable d envahir les cellules soit via un mécanisme de type Trigger, dépendant au moins du T3SS-1, soit via un mécanisme de type Zipper, dépendant au moins de Rck et révèlent la complexité des mécanismes d invasion de Salmonella.The main invasion system of Salmonella requires a type III secretion system (T3SS-1) which promotes a Trigger entry mechanism. However, other invasins were described in Salmonella but their roles in virulence remain unclear. We have shown that Salmonella Enteritidis caninvade cells via the Rck outer membrane protein and we have demonstrated by different approaches that Rck mediates a Zipper entry process. Characterisation of the cellular transduction pathway induced by Rck enable us to propose a model of internalisation involving actin, Arp2/3, Rac and Cdc42, Akt, class I PI3K and Src. Finally, the invasion ability of a S. Enteritidis mutant grown under culture conditions that did not allow the expression of any identified invasion factors (T3SS-1, Rck and PagN) provides evidences for still non-characterised Salmonella invasion factors which seem to induce both Zipper and Trigger mechanisms. Overall, our data indicate that Salmonella is the first bacterium found tobe able to invade cells by both a Trigger mechanism at least mediated by the T3SS-1 and a Zipper entry process at least mediated by Rck. Study of the T3SS-1-independent invasion systems could bring to a better understanding of Salmonella pathogenicity, particularly in regard to the different diseases induced by Salmonella and to its great diversity of hosts.TOURS-Bibl.électronique (372610011) / SudocSudocFranceF

Ultrastructural Characterization of Flashing Mitochondria

Mitochondria undergo spontaneous transient elevations in matrix pH associated with drops in mitochondrial membrane potential. These mitopHlashes require a functional respiratory chain and the profusion protein optic atrophy 1, but their mechanistic basis is unclear. To gain insight on the origin of these dynamic events, we resolved the ultrastructure of flashing mitochondria by correlative light and electron microscopy. HeLa cells expressing the matrix-targeted pH probe mitoSypHer were screened for mitopHlashes and fixed immediately after the occurrence of a flashing event. The cells were then processed for imaging by serial block face scanning electron microscopy using a focused ion beam to generate ~1,200 slices of 10 nm thickness from a 28 μm × 15 μm cellular volume. Correlation of live/fixed fluorescence and electron microscopy images allowed the unambiguous identification of flashing and nonflashing mitochondria. Three-dimensional reconstruction and surface mapping revealed that each tomogram contained two flashing mitochondria of unequal sizes, one being much larger than the average mitochondrial volume. Flashing mitochondria were 10-fold larger than silent mitochondria but with a surface to volume ratio and a cristae volume similar to nonflashing mitochondria. Flashing mitochondria were connected by tubular structures, formed more membrane contact sites, and a constriction was observed at a junction between a flashing mitochondrion and a nonflashing mitochondrion. These data indicate that flashing mitochondria are structurally preserved and bioenergetically competent but form numerous membrane contact sites and are connected by tubular structures, consistent with our earlier suggestion that mitopHlashes might be triggered by the opening of fusion pores between contiguous mitochondria

Performance Evaluation of the Quantamatrix QMAC-dRAST System for Rapid Antibiotic Susceptibility Testing Directly from Blood Cultures

Objectives: Rapid antibiotic susceptibility testing (AST) for positive blood cultures can improve patient clinical outcomes if the time to an effective antimicrobial therapy is shortened. In this study, we tested the Quantamatrix dRAST system (QMAC-dRAST), a rapid AST system based on time-lapse microscopic imagery of bacterial colony formation in agarose. Methods: Evaluation of the QMAC-dRAST was performed from 250 monobacterial blood cultures including 130 Enterobacterales, 20 non-fermentative Gram-negative bacteria, 69 staphylococci and 31 enterococci. Blood cultures were recovered from anonymous patients or from spiking experiments to enrich our study with bacterial species and resistant strains. Categorical agreement (CA), minor errors (me), major errors (ME) and very major errors (VME) were calculated in comparison to the results obtained from the BD Phoenix™ M50. Discrepancies between the Phoenix™ M50 and QMAC-dRAST results were investigated using the gradient strip method. The repeatability and reproducibility performance of the QMAC-dRAST was assessed for 16 strains, each strain being tested five times from a spiked blood culture. Results: The overall CAs for Enterobacterales, non-fermentative Gram-negative bacteria, staphylococci and enterococci were 95.1%, 91.2%, 93.4% and 94.5%, respectively. The VME percentage was below 4% for all the groups except for staphylococci, which showed a VME rate of 7%. The median time to result was 6.7 h (range: 4.7–7.9). Repeatability and reproducibility assays showed a high reliability of AST results with best and worst ratios of 98.8% and 99.6% and 95.0% and 98.3%, respectively. Conclusions: The QMAC-dRAST is a fast and reliable system to determine AST directly from monobacterial blood cultures with a major TAT reduction compared to conventional AST testing.</jats:p

Involvement of c-Src tyrosine kinase upstream of class I phosphatidylinositol (PI) 3-kinases in <em>Salmonella</em> Enteritidis Rck protein-mediated invasion

International audienceThe Salmonella outer membrane protein Rck mediates a Zipper entry mechanism controlled by tyrosine phosphorylation and class I phosphatidylinositol 3-kinase (PI 3-kinase). However, the underlying mechanism leading to this signalling cascade remains unclear. The present study showed that using Rck-coated beads or Rck-overexpressing E. coli, Rck-mediated actin polymerization and invasion were blocked by PP2, a Src-family tyrosine kinase inhibitor. In addition, phosphorylation of Src-family kinases significantly increased after stimulation with Rck. The specific contribution of c-Src, one member of the Src-family kinases, was demonstrated using c-Src-deficient fibroblasts or c-Src siRNA transfected epithelial cells. We also observed that Rck-mediated internalization led to the formation of a complex between c-Src and at least one tyrosine phosphorylated protein. Furthermore, our results revealed that the c-Src signal molecule was upstream of PI 3-kinase during the Rck-mediated signalling pathway, as Rck-mediated PI 3-kinase activation was blocked by PP2, and PI 3-kinase inhibitor had no effect on the Src phosphorylation. These results demonstrate the involvement of c-Src upstream of the PI 3-kinase in the Zipper entry process mediated by Rck

The T3SS-1 : not the only way for Salmonella to invade non phagocytic cells

International audienc

Multiplicity of Salmonella invasion mechanism

National audienc

<em>Salmonella enteritidis</em> Rck-mediated invasion requires activation of Rac1, which is dependent on the class I PI 3-kinases-Akt signaling pathway

International audienceThe Salmonella outer membrane protein Rck mediates a Zipper-like entry mechanism controlled by Rac, the Arp2/3 complex, and actin polymerization. However, little is known about the early steps leading to Rac activation and Rck-mediated internalization. The use of pharmacological inhibitors or PI 3-kinase dominant-negative mutant induced more than 80% less invasion without affecting attachment. Moreover, Rck-mediated internalization caused an increase in the association of p85 with at least one tyrosine-phosphorylated protein, indicating that class I PI 3-kinase activity was stimulated. We also report that this PI 3-kinase activity is essential for Rac1 activation. However, Rac recruitment at the Rck-mediated entry site was independent of its activation. Using a pharmacological approach or Akt-knockout cells, we also demonstrated that Akt was phosphorylated in response to Rck-mediated internalization as demonstrated by immunoblotting analysis and that all three Akt isoforms were required during this process. Overall, our results describe a signaling pathway involving tyrosine phosphorylation, class I PI 3-kinase, Akt activation, and Rac activation, leading to Rck-dependent Zipper entry. The specificity of this signaling pathway with regard to that of the type 3 secretion system, which is the other invasion process of Salmonella, is discussed