We did not test each strain for the percentage of bacteria expressing GFP, which could be a possible variable, as described in other pathogens[21]. co-localized with LAMP-1, a late endosomal marker. Co-immunoprecipation assays further exhibited GSK-5498A an conversation of nucleolin with LAMP-1. Co-localization of nucleolin with LVS was no longer detectable at 24 h when bacteria were multiplying in the cytoplasm. In contrast, with aniglCmutant of LVS, which remains trapped into the phagosomal compartment, or with inert particles, nucleolin/bacteria co-localization remained almost constant. == Conclusions/Significance == We herein confirm the importance of nucleolin expression for LVS binding and its specificity as nucleolin is not involved in binding of another intracellular pathogen asL. monocytogenesor an inert particle. Association of nucleolin withF. tularensisduring contamination continues intracellularly after endocytosis of the bacteria. The present work GSK-5498A therefore unravels for the first time the presence of nucleolin in the phagosomal compartment of macrophages. == Introduction == Francisella tularensisis a small non-motile Gram-negative bacterium that causes the zoonotic disease tularemia in a large number of animals, such as rabbits, hares, and small rodents[1].F. tularensisis also one of the most infectious human bacterial pathogens as ten bacteria can cause disease in humans[1],[2]. Humans acquire contamination by direct contact with sick animals, inhalation, ingestion of contaminated water or food, or by bites from ticks, mosquitoes or flies.F. tularensishas significant potential as an agent of bioterrorism due to its infectivity and capacity to infect in form of aerosols and its ability to cause illness and death[1]. Three subspecies (subsp) are pathogenic for humans:F. tularensissubsptularensis(type A strain),F. tularensissubspholarctica(type B strain) andF. tularensissubspmediasiatica. They differ in terms of their pathogenicity and geographic origin, but are phylogenetically very closely related. The fourth subsp,novicidaprovokes disease in mice, but is usually rarely pathogenic in humans.F. tularensislive vaccine strain (LVS) is an attenuated type B strain[3].F. tularensisis a highly virulent facultative intracellular bacterium, disseminating within host mononuclear phagocytes. After access into macrophages,F. tularensisinitially resides in a phagosomal compartment, whose maturation is usually then arrested. Bacterial escape into the cytoplasm, in the beginning reported to occur after 26 hours of contamination, has now been observed as early as 3060 min after phagocytosis[4]. Bacteria then replicate freely in the cytoplasm of the macrophages[3],[5]. Bacteria are ultimately released from infected cells after induction of apoptosis and pyropoptosis[6][8]. Among the mechanisms that mediate uptake ofF. tularensisby phagocytic cells, participation of C3[9], CR3[10], class A scavenger receptors[11]and mannose receptor[12], have been reported. More recently, we have shown that nucleolin, an eukaryotic protein able to traffic from your nucleus to the cell surface acted as a surface receptor forF. tularensisLVS on human monocyte-like THP-1 cells[13]. We also exhibited that this ligand for human nucleolin at the bacterial surface was the elongation factor Tu (EF-Tu) and that EF-Tu interacted specifically with the C-terminal RGG domain name of nucleolin. In the present work, we were interested in the fate of nucleolin afterF. tularensisLVS access in cells. We first confirmed by siRNA silencing experiments that expression of nucleolin was essential for binding and contamination by LVS of human monocyte/macrophage-type cells. Down-regulation of nucleolin expression had no effect on binding ofListeria GSK-5498A monocytogenesor inert particles to human cells. We then tracked nucleolin localization at different time points of contamination, by confocal microscopy analysis. We found that nucleolin co-localized with intracellular GSK-5498A bacteria at a high level in the phagosomal compartment. This co-localization strongly decreased when the bacteria reached the cytosol to multiply. == Results and Conversation == == Down-regulation of nucleolin expression decreases LVS binding and contamination == We have previously shown[13]that nucleolin, expressed on human cell surface, was involved in LVS contamination. To confirm that expression of nucleolin was essential for LVS binding on human cells, we performed silencing RNA experiments, using siRNA specifically knocking down nucleolin (Fig. 1). We controlled the specificity of the assay: i) by using a siRNA knocking down another eukaryotic protein, histone H1, which has been associated with many effects of PEPCK-C nucleolin[14],[15]; and ii) by monitoring access of either.