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.

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.

MitoNEET-dependent formation of intermitochondrial junctions.

MitoNEET (mNEET) is a dimeric mitochondrial outer membrane protein implicated in many facets of human pathophysiology, notably diabetes and cancer, but its molecular function remains poorly characterized. In this study, we generated and analyzed mNEET KO cells and found that in these cells the mitochondrial network was disturbed. Analysis of 3D-EM reconstructions and of thin sections revealed that genetic inactivation of mNEET did not affect the size of mitochondria but that the frequency of intermitochondrial junctions was reduced. Loss of mNEET decreased cellular respiration, because of a reduction in the total cellular mitochondrial volume, suggesting that intermitochondrial contacts stabilize individual mitochondria. Reexpression of mNEET in mNEET KO cells restored the WT morphology of the mitochondrial network, and reexpression of a mutant mNEET resistant to oxidative stress increased in addition the resistance of the mitochondrial network to H2O2-induced fragmentation. Finally, overexpression of mNEET increased strongly intermitochondrial contacts and resulted in the clustering of mitochondria. Our results suggest that mNEET plays a specific role in the formation of intermitochondrial junctions and thus participates in the adaptation of cells to physiological changes and to the control of mitochondrial homeostasis.

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.

L-OPA1 regulates mitoflash biogenesis independently from membrane fusion.

Mitochondrial flashes mediated by optic atrophy 1 (OPA1) fusion protein are bioenergetic responses to stochastic drops in mitochondrial membrane potential (Δψm) whose origin is unclear. Using structurally distinct genetically encoded pH-sensitive probes, we confirm that flashes are matrix alkalinization transients, thereby establishing the pH nature of these events, which we renamed "mitopHlashes". Probes located in cristae or intermembrane space as verified by electron microscopy do not report pH changes during Δψm drops or respiratory chain inhibition. Opa1 ablation does not alter Δψm fluctuations but drastically decreases the efficiency of mitopHlash/Δψm coupling, which is restored by re-expressing fusion-deficient OPA1K301A and preserved in cells lacking the outer-membrane fusion proteins MFN1/2 or the OPA1 proteases OMA1 and YME1L, indicating that mitochondrial membrane fusion and OPA1 proteolytic processing are dispensable. pH/Δψm uncoupling occurs early during staurosporine-induced apoptosis and is mitigated by OPA1 overexpression, suggesting that OPA1 maintains mitopHlash competence during stress conditions. We propose that OPA1 stabilizes respiratory chain supercomplexes in a conformation that enables respiring mitochondria to compensate a drop in Δψm by an explosive matrix pH flash.

Identification of the epidermal growth factor receptor as the receptor for Salmonella Rck-dependent invasion.

The Salmonella Rck outer membrane protein binds to the cell surface, which leads to bacterial internalization via a Zipper mechanism. This invasion process requires induction of cellular signals, including phosphorylation of tyrosine proteins, and activation of c-Src and PI3K, which arises as a result of an interaction with a host cell surface receptor. In this study, epidermal growth factor receptor (EGFR) was identified as the cell signaling receptor required for Rck-mediated adhesion and internalization. First, Rck-mediated adhesion and internalization were shown to be altered when EGFR expression and activity were modulated. Then, immunoprecipitations were performed to demonstrate the Rck-EGFR interaction. Furthermore, surface plasmon resonance biosensor and homogeneous time-resolved fluorescence technologies were used to demonstrate the direct interaction of Rck with the extracellular domain of human EGFR. Finally, our study strongly suggests a noncompetitive binding of Rck and EGF to EGFR. Overall, these results demonstrate that Rck is able to bind to EGFR and thereby establish a tight adherence to provide a signaling cascade, which leads to internalization of Rck-expressing bacteria.-Wiedemann, A., Mijouin, L., Ayoub, M. A., Barilleau, E., Canepa, S., Teixeira-Gomes, A. P., Le Vern, Y., Rosselin, M., Reiter, E., Velge, P. Identification of the epidermal growth factor receptor as the receptor for Salmonella Rck-dependent invasion.

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

The 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 signaling cascade remains unclear. The present study showed that using Rck-coated beads or Rck-overexpressing Escherichia 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 signaling 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.

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

The 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.

Heterogeneity of type III secretion system (T3SS)-1-independent entry mechanisms used by Salmonella Enteritidis to invade different cell types.

Salmonella causes a wide range of diseases from acute gastroenteritis to systemic typhoid fever, depending on the host. To invade non-phagocytic cells, Salmonella has developed different mechanisms. The main invasion system requires a type III secretion system (T3SS) known as T3SS-1, which promotes a Trigger entry mechanism. However, other invasion factors have recently been described in Salmonella, including Rck and PagN, which were not expressed under our bacterial culture conditions. Based on these observations, we used adhesion and invasion assays to analyse the respective roles of Salmonella Enteritidis T3SS-1-dependent and -independent invasion processes at different times of infection. Diverse cell lines and cell types were tested, including endothelial, epithelial and fibroblast cells. We demonstrated that cell susceptibility to the T3SS-1-independent entry differs by a factor of nine between the most and the least permissive cell lines tested. In addition, using scanning electron and confocal microscopy, we showed that T3SS-1-independent entry into cells was characterized by a Trigger-like alteration, as for the T3SS-1-dependent entry, and also by Zipper-like cellular alteration. Our results demonstrate for what is believed to be the first time that Salmonella can induce Trigger-like entry independently of T3SS-1 and can induce Zipper-like entry independently of Rck. Overall, these data open new avenues for discovering new invasion mechanisms in Salmonella.

Rck of Salmonella enterica, subspecies enterica serovar enteritidis, mediates zipper-like internalization.

Salmonella can invade non-phagocytic cells through its type III secretion system (T3SS-1), which induces a Trigger entry process. This study showed that Salmonella enterica, subspecies enterica serovar Enteritidis can also invade cells via the Rck outer membrane protein. Rck was necessary and sufficient to enable non-invasive E. coli and Rck-coated beads to adhere to and invade different cells. Internalization analysis of latex beads coated with different Rck peptides showed that the peptide containing amino acids 140-150 promoted adhesion, whereas amino acids between 150 and 159 modulated invasion. Expression of dominant-negative derivatives and use of specific inhibitors demonstrated the crucial role of small GTPases Rac1 and Cdc42 in activating the Arp2/3 complex to trigger formation of actin-rich accumulation, leading to Rck-dependent internalization. Finally, scanning and transmission electron microscopy with Rck-coated beads and E. coli expressing Rck revealed microvillus-like extensions that formed a Zipper-like structure, engulfing the adherent beads and bacteria. Overall, our results provide new insights into the Salmonella T3SS-independent invasion mechanisms and strongly suggest that Rck induces a Zipper-like entry mechanism. Consequently, Salmonella seems to be the first bacterium found to be able to induce both Zipper and Trigger mechanisms to invade host cells.