Critical Role of a Sheath Phosphorylation Site On the Assembly and Function of an Atypical Type VI Secretion System.

The bacterial pathogen Francisella tularensis possesses a noncanonical type VI secretion system (T6SS) that is required for phagosomal escape in infected macrophages. KCl stimulation has been previously used to trigger assembly and secretion of the T6SS in culture. By differential proteomics, we found here that the amounts of the T6SS proteins remained unchanged upon KCl stimulation, suggesting involvement of post-translational modifications in T6SS assembly. A phosphoproteomic analysis indeed identified a unique phosphorylation site on IglB, a key component of the T6SS sheath. Substitutions of Y139 with alanine or phosphomimetics prevented T6SS formation and abolished phagosomal escape whereas substitution with phenylalanine delayed but did not abolish phagosomal escape in J774-1 macrophages. Altogether our data demonstrated that the Y139 site of IglB plays a critical role in T6SS biogenesis, suggesting that sheath phosphorylation could participate to T6SS dynamics.Data are available via ProteomeXchange with identifier PXD013619; and on MS-Viewer, key lkaqkllxwx.

The metabolic enzyme fructose-1,6-bisphosphate aldolase acts as a transcriptional regulator in pathogenic Francisella.

The enzyme fructose-bisphosphate aldolase occupies a central position in glycolysis and gluconeogenesis pathways. Beyond its housekeeping role in metabolism, fructose-bisphosphate aldolase has been involved in additional functions and is considered as a potential target for drug development against pathogenic bacteria. Here, we address the role of fructose-bisphosphate aldolase in the bacterial pathogen Francisella novicida. We demonstrate that fructose-bisphosphate aldolase is important for bacterial multiplication in macrophages in the presence of gluconeogenic substrates. In addition, we unravel a direct role of this metabolic enzyme in transcription regulation of genes katG and rpoA, encoding catalase and an RNA polymerase subunit, respectively. We propose a model in which fructose-bisphosphate aldolase participates in the control of host redox homeostasis and the inflammatory immune response.The enzyme fructose-bisphosphate aldolase (FBA) plays central roles in glycolysis and gluconeogenesis. Here, Ziveri et al. show that FBA of the pathogen Francisella novicida acts, in addition, as a transcriptional regulator and is important for bacterial multiplication in macrophages.

Importance of Metabolic Adaptations in Francisella Pathogenesis.

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.

Role of Glycosylation/Deglycolysation Processes in Francisella tularensis Pathogenesis.

Francisella tularensis is able to invade, survive and replicate inside a variety of cell types. However, in vivo F. tularensis preferentially enters host macrophages where it rapidly escapes to the cytosol to avoid phagosomal stresses and to multiply to high numbers. We previously showed that human monocyte infection by F. tularensis LVS triggered deglycosylation of the glutamine transporter SLC1A5. However, this deglycosylation, specifically induced by Francisella infection, was not restricted to SLC1A5, suggesting that host protein deglycosylation processes in general might contribute to intracellular bacterial adaptation. Indeed, we later found that Francisella infection modulated the transcription of numerous glycosidase and glycosyltransferase genes in human macrophages and analysis of cell extracts revealed an important increase of N and O-protein glycosylation. In eukaryotic cells, glycosylation has significant effects on protein folding, conformation, distribution, stability, and activity and dysfunction of protein glycosylation may lead to development of diseases like cancer and pathogenesis of infectious diseases. Pathogenic bacteria have also evolved dedicated glycosylation machineries and have notably been shown to use these glycoconjugates as ligands to specifically interact with the host. In this review, we will focus on Francisella and summarize our current understanding of the importance of these post-translational modifications on its intracellular niche adaptation.

Host glycosylation pathways and the unfolded protein response contribute to the infection by Francisella.

Protein glycosylation processes play a crucial role in most physiological functions, including cell signalling, cellular differentiation and adhesion. We previously demonstrated that rapid deglycosylation of membrane proteins was specifically triggered after infection of human macrophages by the bacterial pathogen Francisella tularensis. Using a glycan processing gene microarray, we found here that Francisella infection modulated expression of numerous glycosidase and glycosyltransferase genes. Furthermore, analysis of cell extracts from infected macrophages by Lectin and Western blotting revealed an important increase of N- and O-protein glycosylation. We chose to focus in the present work on one of the O-glycosylated proteins identified by mass spectrometry, the multifunctional endoplasmic reticulum chaperone BiP (HSPA5/GRP78). We demonstrate that BiP expression is modulated upon Francisella infection and is required to support its intracellular multiplication. Moreover, we show that Francisella differentially modulates the BiP-dependent activation of three key proteins of the unfolded protein response (UPR), IRE1, PERK and ATF6. The effects exerted on human cells by Francisella may thus constitute a novel excample of UPR manipulation contributing to intracellular bacterial adaptation.

Gluconeogenesis, an essential metabolic pathway for pathogenic Francisella.

Intracellular multiplication and dissemination of the infectious bacterial pathogen Francisella tularensis implies the utilization of multiple host-derived nutrients. Here, we demonstrate that gluconeogenesis constitutes an essential metabolic pathway in Francisella pathogenesis. Indeed, inactivation of gene glpX, encoding the unique fructose 1,6-bisphosphatase of Francisella, severely impaired bacterial intracellular multiplication when cells were supplemented by gluconeogenic substrates such as glycerol or pyruvate. The ΔglpX mutant also showed a severe virulence defect in the mouse model, confirming the importance of this pathway during the in vivo life cycle of the pathogen. Isotopic profiling revealed the major role of the Embden-Meyerhof (glycolysis) pathway in glucose catabolism in Francisella and confirmed the importance of glpX in gluconeogenesis. Altogether, the data presented suggest that gluconeogenesis allows Francisella to cope with the limiting glucose availability it encounters during its infectious cycle by relying on host amino acids. Hence, targeting the gluconeogenic pathway might constitute an interesting therapeutic approach against this pathogen.

The complex amino acid diet of Francisella in infected macrophages.

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.

Importance of host cell arginine uptake in Francisella phagosomal escape and ribosomal protein amounts.

Upon entry into mammalian host cells, the pathogenic bacterium Francisella must import host cell arginine to multiply actively in the host cytoplasm. We identified and functionally characterized an arginine transporter (hereafter designated ArgP) whose inactivation considerably delayed bacterial phagosomal escape and intracellular multiplication. Intramacrophagic growth of the ΔargP mutant was fully restored upon supplementation of the growth medium with excess arginine, in both F. tularensis subsp. novicida and F. tularensis subsp. holarctica LVS, demonstrating the importance of arginine acquisition in these two subspecies. High-resolution mass spectrometry revealed that arginine limitation reduced the amount of most of the ribosomal proteins in the ΔargP mutant. In response to stresses such as nutritional limitation, repression of ribosomal protein synthesis has been observed in all kingdoms of life. Arginine availability may thus contribute to the sensing of the intracellular stage of the pathogen and to trigger phagosomal egress. All MS data have been deposited in the ProteomeXchange database with identifier PXD001584 (http://proteomecentral.proteomexchange.org/dataset/PXD001584).

Importance of branched-chain amino acid utilization in Francisella intracellular adaptation.

Intracellular bacterial pathogens have adapted their metabolism to optimally utilize the nutrients available in infected host cells. We recently reported the identification of an asparagine transporter required specifically for cytosolic multiplication of Francisella. In the present work, we characterized a new member of the major super family (MSF) of transporters, involved in isoleucine uptake. We show that this transporter (here designated IleP) plays a critical role in intracellular metabolic adaptation of Francisella. Inactivation of IleP severely impaired intracellular F. tularensis subsp. novicida multiplication in all cell types tested and reduced bacterial virulence in the mouse model. To further establish the importance of the ileP gene in F. tularensis pathogenesis, we constructed a chromosomal deletion mutant of ileP (ΔFTL_1803) in the F. tularensis subsp. holarctica live vaccine strain (LVS). Inactivation of IleP in the F. tularensis LVS provoked comparable intracellular growth defects, confirming the critical role of this transporter in isoleucine uptake. The data presented establish, for the first time, the importance of isoleucine utilization for efficient phagosomal escape and cytosolic multiplication of Francisella and suggest that virulent F. tularensis subspecies have lost their branched-chain amino acid biosynthetic pathways and rely exclusively on dedicated uptake systems. This loss of function is likely to reflect an evolution toward a predominantly intracellular life style of the pathogen. Amino acid transporters should be thus considered major players in the adaptation of intracellular pathogens.

Detection of the interaction between host and bacterial proteins: eukaryotic nucleolin interacts with Francisella elongation factor Tu.

Dissecting the interaction between bacterial and host proteins is fundamental in understanding pathogenesis. It is also very helpful for exploring new therapeutic approaches, either preventive or curative. Here, we describe different techniques, which allowed us to detect new molecules involved in the binding and infection of the bacterium Francisella tularensis, on human cells. This facultative intracellular pathogen is the causative agent of tularemia and is considered as a bio-threatening agent. The privileged host cells are monocytes and macrophages. We used both "in vitro" and "in vivo" experiments to explore the modulation of F. tularensis infection and thereafter determine a bacterial ligand and its host receptor molecule.

Glutamate utilization couples oxidative stress defense and the tricarboxylic acid cycle in Francisella phagosomal escape.

Intracellular bacterial pathogens have developed a variety of strategies to avoid degradation by the host innate immune defense mechanisms triggered upon phagocytocis. Upon infection of mammalian host cells, the intracellular pathogen Francisella replicates exclusively in the cytosolic compartment. Hence, its ability to escape rapidly from the phagosomal compartment is critical for its pathogenicity. Here, we show for the first time that a glutamate transporter of Francisella (here designated GadC) is critical for oxidative stress defense in the phagosome, thus impairing intra-macrophage multiplication and virulence in the mouse model. The gadC mutant failed to efficiently neutralize the production of reactive oxygen species. Remarkably, virulence of the gadC mutant was partially restored in mice defective in NADPH oxidase activity. The data presented highlight links between glutamate uptake, oxidative stress defense, the tricarboxylic acid cycle and phagosomal escape. This is the first report establishing the role of an amino acid transporter in the early stage of the Francisella intracellular lifecycle.

Asparagine assimilation is critical for intracellular replication and dissemination of Francisella.

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.

Francisella tularensis intracellular survival: to eat or to die.

Francisella tularensis is a highly infectious facultative intracellular bacterium causing the zoonotic disease tularemia. Numerous attributes required for F. tularensis intracellular multiplication have been identified recently. However, the mechanisms by which the majority of them interfere with the infected host are still poorly understood. The following review summarizes our current knowledge on the different steps of Francisella intramacrophagic life cycle and expands on the importance of nutrient acquisition.

Proteins involved in Francisella tularensis survival and replication inside macrophages.

Francisella tularensis, the etiological agent of tularemia, is a member of the γ-proteobacteria class of Gram-negative bacteria. This highly virulent bacterium can infect a large range of mammalian species and has been recognized as a human pathogen for a century. F. tularensis is able to survive in vitro in a variety of cell types. In vivo, the bacterium replicates mainly in infected macrophages, using the cytoplasmic compartment as a replicative niche. To successfully adapt to this stressful environment, F. tularensis must simultaneously: produce and regulate the expression of a series of dedicated virulence factors; adapt its metabolic needs to the nutritional context of the host cytosol; and control the innate immune cytosolic surveillance pathways to avoid premature cell death. We will focus here on the secretion or release of bacterial proteins in the host, as well as on the envelope proteins, involved in bacterial survival inside macrophages.

Francisella tularensis regulates the expression of the amino acid transporter SLC1A5 in infected THP-1 human monocytes.

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.

Identification of a putative chaperone involved in stress resistance and virulence in Francisella tularensis.

Francisella tularensis is a highly infectious bacterium causing the zoonotic disease tularemia. This facultative intracellular bacterium replicates in vivo mainly inside macrophages and therefore has developed strategies to resist this stressful environment. Here, we identified a novel genetic locus that is important for stress resistance and intracellular survival of F. tularensis. In silico and transcriptional analyses suggest that this locus (genes FTL_0200 to FTL_0209 in the live vaccine strain [LVS]) constitutes an operon controlled by the alternative sigma factor σ³². The first gene, FTL_0200, encodes a putative AAA+ ATPase of the MoxR subfamily. Insertion mutagenesis into genes FTL_0200, FTL_0205, and FTL_0206 revealed a role for the locus in both intracellular multiplication and in vivo survival of F. tularensis. Deletion of gene FTL_0200 led to a mutant bacterium with increased vulnerability to various stress conditions, including oxidative and pH stresses. Proteomic analyses revealed a pleiotropic impact of the ΔFTL_0200 deletion, supporting a role as a chaperone for FTL_0200. This is the first report of a role for a MoxR family member in bacterial pathogenesis. This class of proteins is remarkably conserved among pathogenic species and may thus constitute a novel player in bacterial virulence.

Nucleolin, a shuttle protein promoting infection of human monocytes by Francisella tularensis.

BACKGROUND: Francisella tularensis is a highly virulent facultative intracellular bacterium, disseminating in vivo mainly within host mononuclear phagocytes. After entry into macrophages, F. tularensis initially resides in a phagosomal compartment, whose maturation is then arrested. Bacteria escape rapidly into the cytoplasm, where they replicate freely. We recently demonstrated that nucleolin, an eukaryotic protein able to traffic from the nucleus to the cell surface, acted as a surface receptor for F. tularensis LVS on human monocyte-like THP-1 cells. METHODOLOGY/PRINCIPAL FINDINGS: Here, we followed the fate of nucleolin once F. tularensis has been endocytosed. We first confirmed by siRNA silencing experiments that expression of nucleolin protein was essential for binding of LVS on human macrophage-type THP-1 cells. We then showed that nucleolin co-localized with intracellular bacteria in the phagosomal compartment. Strikingly, in that compartment, nucleolin also co-localized with LAMP-1, a late endosomal marker. Co-immunoprecipation assays further demonstrated an interaction 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 an iglC mutant 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 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.

Loops and networks in control of Francisella tularensis virulence.

Francisella tularensis is a highly infectious, Gram-negative bacterium responsible for the disease tularemia in a broad variety of animals, including humans. F. tularensis intracellular multiplication occurs mainly in macrophages. However, F. tularensis is able to infect many other cell types, including other phagocytic (dendritic cells, polymorphonuclear leukocytes) and nonphagocytic (alveolar epithelial cells, hepatocytes, endothelial cells and fibroblasts) cells. The ability of professional phagocytic cells to engulf and kill microbes is an essential component of innate defense. The ability of F. tularensis to impair phagocyte function and survive in the cytosol of infected cells thus constitutes a central aspect of its virulence. The F. tularensis intracellular lifecycle relies on the tightly regulated expression of a series of genes. The unraveling secrets of the regulatory cascades governing the regulation of virulence of F. tularensis will be discussed along with future challenges yet to be solved.

Pivotal role of the Francisella tularensis heat-shock sigma factor RpoH.

Francisella tularensis is a highly infectious pathogen that infects animals and humans to cause the disease tularemia. The primary targets of this bacterium are macrophages, in which it replicates in the cytoplasm after escaping the initial phagosomal compartment. The ability to replicate within macrophages relies on the tightly regulated expression of a series of genes. One of the most commonly used means of coordinating the regulation of multiple genes in bacteria consists of the association of dedicated alternative sigma factors with the core of the RNA polymerase (RNAP). In silico analysis of the F. tularensis LVS genome led us to identify, in addition to the genes encoding the RNAP core (comprising the alpha1, alpha2, beta, beta' and omega subunits), one gene (designated rpoD) encoding the major sigma factor sigma(70), and a unique gene (FTL_0851) encoding a putative alternative sigma factor homologue of the sigma(32) heat-shock family (designated rpoH). Hence, F. tularensis represents one of the minority of bacterial species that possess only one or no alternative sigma factor in addition to the main factor sigma(70). In the present work, we show that FTL_0851 encodes a genuine sigma(32) factor. Transcriptomic analyses of the F. tularensis LVS heat-stress response allowed the identification of a series of orthologues of known heat-shock genes (including those for Hsp40, GroEL, GroES, DnaK, DnaJ, GrpE, ClpB and ClpP) and a number of genes implicated in Francisella virulence. A bioinformatic analysis was used to identify genes preceded by a putative sigma(32)-binding site, revealing both similarities to and differences from RpoH-mediated gene expression in Escherichia coli. Our results suggest that RpoH is an essential protein of F. tularensis, and positively regulates a subset of genes involved in the heat-shock response.

A novel receptor - ligand pathway for entry of Francisella tularensis in monocyte-like THP-1 cells: interaction between surface nucleolin and bacterial elongation factor Tu.

BACKGROUND: Francisella tularensis, the causative agent of tularemia, is one of the most infectious human bacterial pathogens. It is phagocytosed by immune cells, such as monocytes and macrophages. The precise mechanisms that initiate bacterial uptake have not yet been elucidated. Participation of C3, CR3, class A scavenger receptors and mannose receptor in bacterial uptake have been already reported. However, contribution of an additional, as-yet-unidentified receptor for F. tularensis internalization has been suggested. RESULTS: We show here that cell-surface expressed nucleolin is a receptor for Francisella tularensis Live Vaccine Strain (LVS) and promotes LVS binding and infection of human monocyte-like THP-1 cells. The HB-19 pseudopeptide that binds specifically carboxy-terminal RGG domain of nucleolin inhibits LVS binding and infection of monocyte-like THP-1 cells. In a pull-down assay, elongation factor Tu (EF-Tu), a GTP-binding protein involved in protein translation, usually found in cytoplasm, was recovered among LVS bacterial membrane proteins bound on RGG domain of nucleolin. A specific polyclonal murine antibody was raised against recombinant LVS EF-Tu. By fluorescence and electron microscopy experiments, we found that a fraction of EF-Tu could be detected at the bacterial surface. Anti-EF-Tu antibodies reduced LVS binding to monocyte-like THP-1 cells and impaired infection, even in absence of complement and complement receptors. Interaction between EF-Tu and nucleolin was illustrated by two different pull-down assays using recombinant EF-Tu proteins and either RGG domain of nucleolin or cell solubilized nucleolin. DISCUSSION: Altogether, our results demonstrate that the interaction between surface nucleolin and its bacterial ligand EF-Tu plays an important role in Francisella tularensis adhesion and entry process and may therefore facilitate invasion of host tissues. Since phagosomal escape and intra-cytosolic multiplication of LVS in infected monocytes are very similar to those of human pathogenic F. tularensis ssp tularensis, the mechanism of entry into monocyte-like THP-1 cells, involving interaction between EF-Tu and nucleolin, might be similar in the two subspecies. Thus, the use of either nucleolin-specific pseudopeptide HB-19 or recombinant EF-Tu could provide attractive therapeutic approaches for modulating F. tularensis infection.