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In order to develop a successful infectious cycle, intracellular bacterial pathogens must be able to adapt their metabolism to optimally utilize the nutrients available in the cellular compartments and tissues where they reside. F rancisella tularensis, the agent of the zoonotic disease tularaemia, is a highly infectious bacterium for a large number of animal species. This bacterium replicates in its mammalian hosts mainly in the cytosol of infected macrophages. We report here the identification of a novel amino acid transporter of the major facilitator superfamily of secondary transporters that is required for bacterial intracellular multiplication and systemic dissemination. We show that inactivation of this transporter does not affect phagosomal escape but prevents multiplication in the cytosol of all cell types tested. Remarkably, the intracellular growth defect of the mutant was fully and specifically reversed by addition of asparagine or asparagine-containing dipeptides as well as by simultaneous addition of aspartic acid and ammonium. Importantly, bacterial virulence was also restored in vivo, in the mouse model, by asparagine supplementation. This work unravels thus, for the first time, the importance of asparagine for cytosolicmultiplication of F rancisella. Amino acid transporters are likely to constitute underappreciated players in bacterial intracellular parasitism. [ABSTRACT FROM AUTHOR]

Francisella tularensis, a Gram-negative bacterium that causes the disease tularemia in a large number of animal species, is thought to reside preferentially within macrophages in vivo. F. tularensis has developed mechanisms to rapidly escape from the phagosome into the cytoplasm of infected cells, a habitat with a rich supply of nutrients, ideal for multiplication. SLC1A5 is a neutral amino acid transporter expressed by human cells, which serves, along with SLC7A5 to equilibrate cytoplasmic amino acid pools. We herein analysed whether SLC1A5 was involved in F. tularensis intracellular multiplication. We demonstrate that expression of SLC1A5 is specifically upregulated by F. tularensis in infected THP-1 human monocytes. Furthermore, we show that SLC1A5 downregulation decreases intracellular bacterial multiplication, supporting the involvement of SLC1A5 in F. tularensis infection. Notably, after entry of F. tularensis into cells and during the whole infection, the highly glycosylated form of SLC1A5 was deglycosylated only by bacteria capable of cytosolic multiplication. These data suggest that intracellular replication of F. tularensis depends on the function of host cell SLC1A5. Our results are the first, which show that Francisella intracellular multiplication in human monocyte cytoplasm is associated with a post-translational modification of a eukaryotic amino acid transporter. [ABSTRACT FROM AUTHOR]