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Francisella tularensis is a highly infectious Gram-negative bacterium and the causative agent of the zoonotic disease tularemia. This bacterial pathogen can infect a broad variety of animal species and can be transmitted to humans in numerous ways with various clinical outcomes. Although, Francisella possesses the capacity to infect numerous mammalian cell types, the macrophage constitutes the main intracellular niche, used for in vivo bacterial dissemination. To survive and multiply within infected macrophages, Francisella must imperatively escape from the phagosomal compartment. In the cytosol, the bacterium needs to control the host innate immune response and adapt its metabolism to this nutrient-restricted niche. Our laboratory has shown that intracellular Francisella mainly relied on host amino acid as major gluconeogenic substrates and provided evidence that the host metabolism was also modified upon Francisella infection. We will review here our current understanding of how Francisella copes with the available nutrient sources provided by the host cell during the course of infection.

Genetic context in the vicinity of muf genes. Genetic context in the vicinity of muf genes detected in bacteriophage genomes (A) and bacterial chromosomes (typically prophages, B). The total number of genes encoding portal (in yellow), terminase large subunit (in blue), and small subunit (in purple) proteins according to their distances to muf genes (distance = 0) were reported (see Methods). 85% and 87% of muf genes were detected close to at least a portal-encoding gene or a terminase-encoding gene in bacteriophage genomes and bacterial chromosomes, respectively. The genetic context around muf genes was highly conserved in both datasets. (PDF 442 kb)

Pairwise comparison of the genetic organization of muf and maf genes. Top: the muf gene and the downstream ORF found in phage SM1 (NC_004996) infecting Streptococcus mitis. Bottom: the mafB toxin gene (encoding WP_003711327.1) and the cognate immunity gene mafI (encoding WP_002235294.1) found in Neisseria meningitidis NM3001 (assembly GCA_000293665.1). Nucleotide comparison was generated using BLASTn implemented in Easyfig 2.1. Gray vertical block indicates regions of shared similarity shaded according to BLASTn identity. The level of nucleotide identity is shown in the gradient scale. (PDF 74 kb)

Distribution of the length of the proteins encoded by genes annotated immediately downstream of muf genes. The association of small ORFs with MuF toxins compared to short MuFs is highly significant (two-tailed unpaired Student’s t test, p < 0.0001). The three categories correspond to the MuF protein architecture. Ct-tox MuF domains associated with a C-terminal extension with known toxin domains, Ct-unk MuF domains associated with a C-terminal extension with unknown domains, Short MuF domains without C-terminal extension. (PDF 70 kb)

Relationships between MuF families. Network association of the four HMM profiles of MuF proteins. Each node corresponds to a MuF HMM profile, and each edge width is proportional to the probability of homologous relationship computed by HHsearch for pairwise comparison of HMM profiles using HH-suite (see Methods). (PDF 381 kb)

Proportion of toxin domains in each MuF family and taxonomical clade. Distribution of MuF protein families in bacteriophages (A) and in bacterial chromosomes (B). The number of MuF proteins detected was reported (in orange) for each clade. Proportion of MuF domains associated with a C-terminal extension (Ct_ext) with known toxin domains (Ct_ext-tox, in red), with unknown domains (Ct_ext-unk, in gray) or without Ct_ext (Short, in blue) in bacteriophages (C) and in bacterial chromosomes (D). Only the number of MuF proteins with a known toxin domain, and only bacterial clades with at least four genomes sequenced were indicated (for simplicity). (PDF 832 kb)

International audience

Background Polymorphic toxins (PTs) are multi-domain bacterial exotoxins belonging to distinct families that share common features in terms of domain organization. PTs are found in all major bacterial clades, including many toxic effectors of type V and type VI secretion systems. PTs modulate the dynamics of microbial communities by killing or inhibiting the growth of bacterial competitors lacking protective immunity proteins. Results In this work, we identified a novel widespread family of PTs, named MuF toxins, which were exclusively encoded within temperate phages and their prophages. By analyzing the predicted proteomes of 1845 bacteriophages and 2464 bacterial genomes, we found that MuF-containing proteins were frequently part of the DNA packaging module of tailed phages. Interestingly, MuF toxins were abundant in the human gut microbiome. Conclusions Our results uncovered the presence of the MuF toxin family in the temperate phages of Firmicutes. The MuF toxin family is likely to play an important role in the ecology of the human microbiota where pathogens and commensal species belonging to the Firmicutes are abundant. We propose that MuF toxins could be delivered by phages into host bacteria and either influence the lysogeny decision or serve as bacterial weapons by inhibiting the growth of competing bacteria. Electronic supplementary material The online version of this article (doi:10.1186/s12915-017-0415-1) contains supplementary material, which is available to authorized users.

Francisella tularensis, the agent of the zoonotic disease tularemia, is a highly infectious bacterium for a large number of animal species and can be transmitted to humans by various means. The bacterium is able to infect a variety of cell types but replicates in mammalian hosts mainly in the cytosol of infected macrophages. In order to resist the stressful and nutrient-restricted intracellular environments, it encounters during its systemic dissemination, Francisella has developed dedicated stress resistance mechanisms and adapted its metabolic and nutritional needs. Recent data form our laboratory and from several other groups have shown that Francisella simultaneously relies on multiple host amino acid sources during its intracellular life cycle. This review will summarize how intracellular Francisella use different amino acid sources, and their role in phagosomal escape and/or cytosolic multiplication and systemic dissemination. We will first summarize the data that we have obtained on two amino acid transporters involved in Francisella phagosomal escape and cytosolic multiplication i.e., the glutamate transporter GadC and the asparagine transporter AnsP, respectively. The specific contribution of glutamate and asparagine to the physiology of the bacterium will be evoked. Then, we will discuss how Francisella has adapted to obtain and utilize host amino acid resources, and notably the contribution of host transporters and autophagy process in the establishment of a nutrient-replete intracellular niche.

International audience; 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. Francisella 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 Francisella. Amino acid transporters are likely to constitute underappreciated players in bacterial intracellular parasitism.

International audience; 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 as L. monocytogenes or an inert particle. Association of nucleolin with F. tularensis during infection continues intracellularly after endocytosis of the bacteria. The present work therefore unravels for the first time the presence of nucleolin in the phagosomal compartment of macrophages.