Adaptive traits of cysts of the snow alga Sanguina nivaloides unveiled by 3D subcellular imaging.

Sanguina nivaloides is the main alga forming red snowfields in high mountains and Polar Regions. It is non-cultivable. Analysis of environmental samples by X-ray tomography, focused-ion-beam scanning-electron-microscopy, physicochemical and physiological characterization reveal adaptive traits accounting for algal capacity to reside in snow. Cysts populate liquid water at the periphery of ice, are photosynthetically active, can survive for months, and are sensitive to freezing. They harbor a wrinkled plasma membrane expanding the interface with environment. Ionomic analysis supports a cell efflux of K+, and assimilation of phosphorus. Glycerolipidomic analysis confirms a phosphate limitation. The chloroplast contains thylakoids oriented in all directions, fixes carbon in a central pyrenoid and produces starch in peripheral protuberances. Analysis of cells kept in the dark shows that starch is a short-term carbon storage. The biogenesis of cytosolic droplets shows that they are loaded with triacylglycerol and carotenoids for long-term carbon storage and protection against oxidative stress.

Genome-wide screen in human plasma identifies multifaceted complement evasion of Pseudomonas aeruginosa.

Pseudomonas aeruginosa, an opportunistic Gram-negative pathogen, is a leading cause of bacteremia with a high mortality rate. We recently reported that P. aeruginosa forms a persister-like sub-population of evaders in human plasma. Here, using a gain-of-function transposon sequencing (Tn-seq) screen in plasma, we identified and validated previously unknown factors affecting bacterial persistence in plasma. Among them, we identified a small periplasmic protein, named SrgA, whose expression leads to up to a 100-fold increase in resistance to killing. Additionally, mutants in pur and bio genes displayed higher tolerance and persistence, respectively. Analysis of several steps of the complement cascade and exposure to an outer-membrane-impermeable drug, nisin, suggested that the mutants impede membrane attack complex (MAC) activity per se. Electron microscopy combined with energy-dispersive X-ray spectroscopy (EDX) revealed the formation of polyphosphate (polyP) granules upon incubation in plasma of different size in purD and wild-type strains, implying the bacterial response to a stress signal. Indeed, inactivation of ppk genes encoding polyP-generating enzymes lead to significant elimination of persisting bacteria from plasma. Through this study, we shed light on a complex P. aeruginosa response to the plasma conditions and discovered the multifactorial origin of bacterial resilience to MAC-induced killing.

The AAA+ ATPase RavA and its binding partner ViaA modulate E. coli aminoglycoside sensitivity through interaction with the inner membrane.

Enteric bacteria have to adapt to environmental stresses in the human gastrointestinal tract such as acid and nutrient stress, oxygen limitation and exposure to antibiotics. Membrane lipid composition has recently emerged as a key factor for stress adaptation. The E. coli ravA-viaA operon is essential for aminoglycoside bactericidal activity under anaerobiosis but its mechanism of action is unclear. Here we characterise the VWA domain-protein ViaA and its interaction with the AAA+ ATPase RavA, and find that both proteins localise at the inner cell membrane. We demonstrate that RavA and ViaA target specific phospholipids and subsequently identify their lipid-binding sites. We further show that mutations abolishing interaction with lipids restore induced changes in cell membrane morphology and lipid composition. Finally we reveal that these mutations render E. coli gentamicin-resistant under fumarate respiration conditions. Our work thus uncovers a ravA-viaA-based pathway which is mobilised in response to aminoglycosides under anaerobiosis and engaged in cell membrane regulation.

Inflammasome activation by Pseudomonas aeruginosa's ExlA pore-forming toxin is detrimental for the host.

During acute Pseudomonas aeruginosa infection, the inflammatory response is essential for bacterial clearance. Neutrophil recruitment can be initiated following the assembly of an inflammasome within sentinel macrophages, leading to activation of caspase-1, which in turn triggers macrophage pyroptosis and IL-1ß/IL-18 maturation. Inflammasome formation can be induced by a number of bacterial determinants, including Type III secretion systems (T3SSs) or pore-forming toxins, or, alternatively, by lipopolysaccharide (LPS) via caspase-11 activation. Surprisingly, previous studies indicated that a T3SS-induced inflammasome increased pathogenicity in mouse models of P. aeruginosa infection. Here, we investigated the immune reaction of mice infected with a T3SS-negative P. aeruginosa strain (IHMA879472). Virulence of this strain relies on ExlA, a secreted pore-forming toxin. IHMA879472 promoted massive neutrophil infiltration in infected lungs, owing to efficient priming of toll-like receptors, and thus enhanced the expression of inflammatory proteins including pro-IL-1ß and TNF-α. However, mature-IL-1ß and IL-18 were undetectable in wild-type mice, suggesting that ExlA failed to effectively activate caspase-1. Nevertheless, caspase-1/11 deficiency improved survival following infection with IHMA879472, as previously described for T3SS+ bacteria. We conclude that the detrimental effect associated with the ExlA-induced inflammasome is probably not due to hyperinflammation, rather it stems from another inflammasome-dependent process.

Influences of Nanoparticles Characteristics on the Cellular Responses: The Example of Iron Oxide and Macrophages.

Iron oxide nanoparticles/microparticles are widely present in a variety of environments, e.g., as a byproduct of steel and iron degradation, as, for example, in railway brakes (e.g., metro station) or in welding fumes. As all particulate material, these metallic nanoparticles are taken up by macrophages, a cell type playing a key role in the innate immune response, including pathogen removal phagocytosis, secretion of free radical species such as nitric oxide or by controlling inflammation via cytokine release. In this paper, we evaluated how macrophages functions were altered by two iron based particles of different size (100 nm and 20 nm). We showed that at high, but subtoxic concentrations (1 mg/mL, large nanoparticles induced stronger perturbations in macrophages functions such as phagocytic capacity (tested with fluorescent latex microspheres) and the ability to respond to bacterial endotoxin lipopolysaccharide stimulus (LPS) in secreting nitric oxide and pro-cytokines (e.g., Interleukin-6 (IL-6) and Tumor Necrosis Factor (TNF)). These stronger effects may correlate with an observed stronger uptake of iron for the larger nanoparticles.

Canalicular domain structure and function in matrix-free hepatic spheroids.

Liver is pivotal in organism metabolism. This organ is receiving nutriments from the portal vein and then storing, metabolizing, distributing in the circulation or excreting excess and xenobiotics in bile. Liver architecture and hepatocyte polarization are crucial to achieve these functions. To study these mechanisms in details, relevant cell culture systems are required, which is not the case with standard 2D cell culture. Besides, primary hepatocytes rapidly de-differenciate making them inefficient in forming physiological system. Herein, we used an hepatoma-derived cell line to produce matrix-free hepatic spheroids and developed an integrated structural cell biology methodology by combining light sheet fluorescence microscopy and 3D electron microscopy to study their function and structure. Within these spheroids, hepatocytes polarize and organize to form bile canaliculi active for both organics and inorganics excretion. Besides, live imaging revealed the high dynamic of actin networks in basal membranes compared to their high stability in the apical pole that constitutes bile canaliculi. Finally, the first structure of active bile canaliculi was solved at nm resolution and showed the very high density of microvilli coming from all cells constituting the canaliculus. Therefore, this study is the first comprehensive and in-depth functional and structural study of bile canaliculi in a physiological-relevant context.

Pseudomonas aeruginosa Exolysin promotes bacterial growth in lungs, alveolar damage and bacterial dissemination.

Exolysin (ExlA) is a recently-identified pore-forming toxin secreted by a subset of Pseudomonas aeruginosa strains identified worldwide and devoid of Type III secretion system (T3SS), a major virulence factor. Here, we characterized at the ultrastructural level the lesions caused by an ExlA-secreting strain, CLJ1, in mouse infected lungs. CLJ1 induced necrotic lesions in pneumocytes and endothelial cells, resulting in alveolo-vascular barrier breakdown. Ectopic expression of ExlA in an exlA-negative strain induced similar tissue injuries. In addition, ExlA conferred on bacteria the capacity to proliferate in lungs and to disseminate in secondary organs, similar to bacteria possessing a functional T3SS. CLJ1 did not promote a strong neutrophil infiltration in the alveoli, owing to the weak pro-inflammatory cytokine reaction engendered by the strain. However, CLJ1 was rapidly eliminated from the blood in a bacteremia model, suggesting that it can be promptly phagocytosed by immune cells. Together, our study ascribes to ExlA-secreting bacteria the capacity to proliferate in the lung and to damage pulmonary tissues, thereby promoting metastatic infections, in absence of substantial immune response exacerbation.

The Use of Adenovirus Dodecahedron in the Delivery of an Enzymatic Activity in the Cell.

Penton-dodecahedron (Pt-Dd) derived from adenovirus type 3 is a symmetric complex of pentameric penton base plus fiber which can be produced in the baculovirus system at a high concentration. The size of Pt-Dd is smaller than the virus, but this virus-like particle (VLP) has the major proteins recognized by specific receptors on the surface of almost all types of cell. In this study, by direct observation with fluorescence microscopy on a fixed and living cell, the intracellular trafficking and localization of Pt-Dd labeled with fluorescence dyes in the cytoplasm of HeLa Tub-GFP showed a rapid internalization characteristic. Subsequently, the linkage of horseradish peroxidase (HRP) with Pt-Dd as the vector demonstrated an efficient system to deliver this enzyme into the cell without interfering its enzymatic activity as shown by biochemical and cellular experiments. These results were supported by additional studies using Bs-Dd or free form of the HRP used as the control. Overall, this study strengthens the potential role of Pt-Dd as an alternative vector for delivering therapeutic agents.