Phagocytosis of Erythrocytes from Gaucher Patients Induces Phenotypic Modifications in Macrophages, Driving Them toward Gaucher Cells.

Gaucher disease (GD) is caused by glucocerebrosidase deficiency leading to the accumulation of sphingolipids in macrophages named "Gaucher's Cells". These cells are characterized by deregulated expression of cell surface markers, abnormal secretion of inflammatory cytokines, and iron sequestration. These cells are known to infiltrate tissues resulting in hematological manifestations, splenomegaly, and bone diseases. We have already demonstrated that Gaucher red blood cells exhibit altered properties suggesting their key role in GD clinical manifestations. We hypothesized that Gaucher's erythrocytes could be prone to premature destruction by macrophages contributing to the formation of altered macrophages and Gaucher-like cells. We conducted in vitro experiments of erythrophagocytosis using erythrocytes from Gaucher's patients or healthy donors. Our results showed an enhanced erythrophagocytosis of Gaucher red blood cells compared to healthy red blood cells, which is related to erythrocyte sphingolipids overload and reduced deformability. Importantly, we showed elevated expression of the antigen-presenting molecules CD1d and MHC-II and of the iron-regulator hepcidin in macrophages, as well as enhanced secretion of the pro-inflammatory cytokine IL-1ß after phagocytosis of GD erythrocytes. These results strongly suggested that erythrophagocytosis in GD contribute to phenotypic modifications in macrophages. This present study shows that erythrocytes-macrophages interactions may be crucial in GD pathophysiology and pathogenesis.

Effects of sphingolipids overload on red blood cell properties in Gaucher disease.

Gaucher disease (GD) is a genetic disease with mutations in the GBA gene that encodes glucocerebrosidase causing complications such as anaemia and bone disease. GD is characterized by accumulation of the sphingolipids (SL) glucosylceramide (GL1), glucosylsphingosine (Lyso-GL1), sphingosine (Sph) and sphingosine-1-phosphate (S1P). These SL are increased in the plasma of GD patients and the associated complications have been attributed to the accumulation of lipids in macrophages. Our recent findings indicated that red blood cells (RBCs) and erythroid progenitors may play an important role in GD pathophysiology. RBCs abnormalities and dyserythropoiesis have been observed in GD patients. Moreover, we showed higher SL levels in the plasma and in RBCs from untreated GD patients compared with controls. In this study, we quantified SL in 16 untreated GD patients and 15 patients treated with enzyme replacement therapy. Our results showed that the treatment significantly decreases SL levels in the plasma and RBCs. The increased SL content in RBCs correlates with abnormal RBC properties and with markers of disease activity. Because RBCs lack glucocerebrosidase activity, we investigated how lipid overload could occur in these cells. Our results suggested that SL overload in RBCs occurs both during erythropoiesis and during its circulation in the plasma.

Diversity of the auxotrophic requirements in natural isolates of Escherichia coli.

Isolates of Escherichia coli, except Shigella, are generally prototrophic; they do not require any growth factors to grow in mineral medium. However, a nicotinic acid requirement is common among B2 phylogroup STc95 O18 E. coli clone strains. Nicotinic acid is a precursor of nicotinamide adenine dinucleotide (NAD), an essential molecule that plays central role in cellular metabolism. The defect in NAD synthesis of these strains is due to alterations in de novo biosynthesis pathway nadB gene. Here, by studying growth on minimal medium with glycolytic (glucose) or gluconeogenic (pyruvate or succinate) substrates as the carbon supply in a large panel of E. coli natural isolates representative of the species diversity, we identify an absolute nicotinic acid requirement in non-STc95 strains due in one case to a nadA inactivation. The growth on glucose medium of some extraintestinal pathogenic E. coli strains belonging to various non-O18 B2 phylogroup STc95 clones is restored either by aspartate or nicotinate, demonstrating that the nicotinic acid requirement can also be due to an intracellular aspartate depletion. The auxotrophic requirements depend on the carbon source available in the environment. Moreover, some strains prototrophic in glucose medium become auxotrophic in succinate medium, and conversely, some strainsauxotrophic in glucose medium become prototrophic in succinate medium. Finally, a partial depletion of intracellular aspartate can be observed in some prototrophic strains belonging to various phylogroups. The observed more or less significant depletion according to isolates may be due to differences in tricarboxylic acid cycle enzyme activities. These metabolic defects could be involved in the adaptation of E. coli to its various niches.

High Recombinant Frequency in Extraintestinal Pathogenic Escherichia coli Strains.

Homologous recombination promotes genetic diversity by facilitating the integration of foreign DNA and intrachromosomal gene shuffling. It has been hypothesized that if recombination is variable among strains, selection should favor higher recombination rates among pathogens, as they face additional selection pressures from host defenses. To test this hypothesis we have developed a plasmid-based method for estimating the rate of recombination independently of other factors such as DNA transfer, selective processes, and mutational interference. Our results with 160 human commensal and extraintestinal pathogenic Escherichia coli (ExPEC) isolates show that the recombinant frequencies are extremely diverse (ranging 9 orders of magnitude) and plastic (they are profoundly affected by growth in urine, a condition commonly encountered by ExPEC). We find that the frequency of recombination is biased by strain lifestyle, as ExPEC isolates display strikingly higher recombination rates than their commensal counterparts. Furthermore, the presence of virulence factors is positively associated with higher recombination frequencies. These results suggest selection for high homologous recombination capacity, which may result in a higher evolvability for pathogens compared with commensals.