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Metabolic pathways are now considered as intrinsic virulence attributes of pathogenic bacteria and thus represent potential targets for antibacterial strategies. Here we focused on the role of the pentose phosphate pathway (PPP) and its connections with other metabolic pathways in the pathophysiology of Francisella novicida. The involvement of the PPP in the intracellular life cycle of Francisella was first demonstrated by studying PPP inactivating mutants. Indeed, we observed that inactivation of the tktA, rpiA or rpe genes severely impaired intramacrophage multiplication during the first 24 hours. However, time-lapse video microscopy demonstrated that rpiA and rpe mutants were able to resume late intracellular multiplication. To better understand the links between PPP and other metabolic networks in the bacterium, we also performed an extensive proteo-metabolomic analysis of these mutants. We show that the PPP constitutes a major bacterial metabolic hub with multiple connections to glycolysis, the tricarboxylic acid cycle and other pathways, such as fatty acid degradation and sulfur metabolism. Altogether our study highlights how PPP plays a key role in the pathogenesis and growth of Francisella in its intracellular niche., Competing Interests: No - The authors have declared that no competing interests exist.

One of the major challenges in Nile tilapia (Oreochromis niloticus L.) farming is the occurrence of bacterial infections, and the Francisella noatunensis subsp. orientalis (FNO) is an important pathogen that has emerged in last decades. Francisellosis outbreaks have been reported in the literature as occurring seasonally when water temperature is below 24 °C. The aim of this study was to quantify the median lethal doses (LD 50 ) of FNO in experimental challenges at 28 °C and 22 °C, and to investigate the impact of temperature changes in whole genome expression using microarray technology. The LD 50 for Nile tilapia at 28 °C was ∼10 5.7 , whereas at 22 °C, the LD 50 was ∼10 2.2 , showing that the decrease in temperature enhanced disease outcome. Out of 1917 genes screened, a total of 31 and 19 genes were down- and up-regulated at 22 °C, respectively. These genes were grouped by orthology into functional categories of: amino acid, inorganic ion, and carbohydrate transport and metabolism; transcription; and posttranslational modification, protein turnover, and chaperones. Expression of genes related to metabolism, oxidative stress, and thermal shock were regulated by temperature changes, reflecting an ability of FNO to adapt to the environment. Expression of virulence genes usually required for the Francisella genus was not changed between tested temperatures, including that of genes located on the Francisella Pathogenicity Island., (Copyright © 2019. Published by Elsevier Ltd.)

The Francisella genus comprises highly pathogenic bacteria that can cause fatal disease in their vertebrate and invertebrate hosts including humans. In general, Francisella growth depends on iron availability, hence, iron homeostasis must be tightly regulated during Francisella infection. We used the system of the professional phagocyte Dictyostelium and the fish pathogen F. noatunensis subsp. noatunensis ( F.n.n .) to investigate the role of the host cell iron transporters Nramp (natural resistance associated macrophage proteins) during Francisella infection. Like its mammalian ortholog, Dictyostelium Nramp1 transports iron from the phagosome into the cytosol, whereas the paralog NrampB is located on the contractile vacuole and controls, together with Nramp1, the cellular iron homeostasis. In Dictyostelium , Nramp1 localized to the F.n.n .-phagosome but disappeared from the compartment dependent on the presence of IglC, an established Francisella virulence factor. In the absence of Nramp transporters the bacteria translocated more efficiently from the phagosome into the host cell cytosol, its replicative niche. Increased escape rates coincided with increased proteolytic activity in bead-containing phagosomes indicating a role of the Nramp transporters for phagosomal maturation. In the nramp mutants, a higher bacterial load was observed in the replicative phase compared to wild-type host cells. Upon bacterial access to the cytosol of wt cells, mRNA levels of bacterial iron uptake factors were transiently upregulated. Decreased iron levels in the nramp mutants were compensated by a prolonged upregulation of the iron scavenging system. These results show that Nramps contribute to host cell immunity against Francisella infection by influencing the translocation efficiency from the phagosome to the cytosol but not by restricting access to nutritional iron in the cytosol.

Francisella tularensis is a facultative intracellular bacterium that causes the deadly disease tularemia. Most evidence suggests that Francisella is not well recognized by the innate immune system that normally leads to cytokine expression and cell death. In previous work, we identified new bacterial factors that were hyper-cytotoxic to macrophages. Four of the identified hyper-cytotoxic strains (lpcC, manB, manC, and kdtA) had an impaired lipopolysaccharide (LPS) synthesis and produced an exposed lipid A lacking the O-antigen. These mutants were not only hyper-cytotoxic but also were phagocytosed at much higher rates compared with the wild type parent strain. To elucidate the cellular signaling underlying this enhanced phagocytosis and cell death, we performed a large-scale comparative phosphoproteomic analysis of cells infected with wild-type and delta-lpcC F. novicida. Our data suggest that not only actin but also intermediate filaments and microtubules are important for F. novicida entry into the host cells. In addition, we observed differential phosphorylation of tristetraprolin, a key component of the mRNA-degrading machinery that controls the expression of a variety of genes including many cytokines. Infection with the delta-lpcC mutant induced the hyper-phosphorylation and inhibition of tristetraprolin, leading to the production of cytokines such as IL-1beta and TNF-alpha that may kill the host cells by triggering apoptosis. Together, our data provide new insights for Francisella invasion and a post-transcriptional mechanism that prevents the expression of host immune response factors that control infection by this pathogen.

Francisella tularensis is a gram-negative bacterium that is highly virulent in humans, causing the disease tularemia. F. novicida is closely related to F. tularensis and exhibits high virulence in mice, but it is avirulent in healthy humans. An F. novicida-specific gene cluster (FTN0451 to FTN0456) encodes two proteins with diguanylate cyclase (DGC) and phosphodiesterase (PDE) domains that modulate the synthesis and degradation of cyclic di-GMP (cdGMP). No DGC- or PDE-encoding protein genes are present in the F. tularensis genome. F. novicida strains lacking either the two DGC/PDE genes (cdgA and cdgB) or the entire gene cluster (strain KKF457) are defective for biofilm formation. In addition, expression of CdgB or a heterologous DGC in strain KKF457 stimulated F. novicida biofilms, even in a strain lacking the biofilm regulator QseB. Genetic evidence suggests that CdgA is predominantly a PDE, while CdgB is predominantly a DGC. The F. novicida qseB strain showed reduced cdgA and cdgB transcript levels, demonstrating an F. novicida biofilm signaling cascade that controls cdGMP levels. Interestingly, KKF457 with elevated cdGMP levels exhibited a decrease in intramacrophage replication and virulence in mice, as well as increased growth yields and biofilm formation in vitro. Microarray analyses revealed that cdGMP stimulated the transcription of a chitinase (ChiB) known to contribute to biofilm formation. Our results indicate that elevated cdGMP in F. novicida stimulates biofilm formation and inhibits virulence. We suggest that differences in human virulence between F. novicida and F. tularensis may be due in part to the absence of cdGMP signaling in F. tularensis.

Modification of specific Gram-negative bacterial cell envelope components, such as capsule, O-antigen and lipid A, are often essential for the successful establishment of infection. Francisella species express lipid A molecules with unique characteristics involved in circumventing host defences, which significantly contribute to their virulence. In this study, we show that NaxD, a member of the highly conserved YdjC superfamily, is a deacetylase required for an important modification of the outer membrane component lipid A in Francisella. Mass spectrometry analysis revealed that NaxD is essential for the modification of a lipid A phosphate with galactosamine in Francisella novicida, a model organism for the study of highly virulent Francisella tularensis. Significantly, enzymatic assays confirmed that this protein is necessary for deacetylation of its substrate. In addition, NaxD was involved in resistance to the antimicrobial peptide polymyxin B and critical for replication in macrophages and in vivo virulence. Importantly, this protein is also required for lipid A modification in F. tularensis as well as Bordetella bronchiseptica. Since NaxD homologues are conserved among many Gram-negative pathogens, this work has broad implications for our understanding of host subversion mechanisms of other virulent bacteria., (© 2012 Blackwell Publishing Ltd.)

Strain FhSp1T, isolated from human blood in Spain in 2003, was studied for its taxonomic allocation. By 16S rRNA and recA gene sequencing, the strain was shown to belong to the genus Francisella. In the 16S rRNA gene sequence, Francisella sp. FhSp1T shared similarity of more than 99% with strains of Francisella tularensis subspecies and Francisella novicida U112T, 98% with Francisella piscicida GM2212T and 98.4% with Francisella philomiragia ATCC 25015T. In the recA gene sequence, Francisella sp. FhSp1T exhibited 91.6-91.7% similarity to strains of F. tularensis subspecies, 91.2% to F. novicida U112T and 84% to F. philomiragia ATCC 25017. The genus affiliation was supported by a quinone system typical of Francisella (Q-8 as the major component), a complex polar lipid profile similar to that of F. tularensis with the major components diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylcholine and an unknown aminophospholipid (APL4) and a fatty acid profile consisting mainly of C10:0 (17.2%), C14:0 (11.2%), C16:0 (13.1%), C18:0 3-OH (14.2%) and C18:1omega9c (7.1%). DNA-DNA hybridization, which showed unambiguously that FhSp1T represents a novel species, and the results of biochemical tests allowed genotypic and phenotypic differentiation of the isolate from all hitherto-described Francisella species. A multiplex PCR developed in the course of this study discriminated FhSp1T from representatives of all other Francisella species and subspecies, clades A.I and A.II of F. tularensis subsp. tularensis and F. tularensis subsp. holarctica biovar japonica and also between these representatives of the genus. Therefore, we propose the name Francisella hispaniensis sp. nov., with the type strain FhSp1T (=FnSp1T =FSC454T =F62T =DSM 22475T =CCUG 58020T). Furthermore, we formally propose the transfer of the species Francisella novicida to the species Francisella tularensis as Francisella tularensis subsp. novicida comb. nov. (type strain ATCC 15482T =CCUG 33449T =CIP 56.12T). We also present an emended description of the genus Francisella.

Francisella tularensis requires iron (Fe) for growth, but the biologic sources of Fe for this organism are largely unknown. We found that Francisella sp. growing in broth culture or within human macrophages can acquire Fe from the two major host Fe-binding proteins, lactoferrin (Lf) and transferrin (Tf). Fe acquisition is a potential target for novel therapies. Gallium (Ga) is a transition metal that interferes with cellular Fe metabolism by competing with Fe for uptake/utilization. Growth of either F. tularensis live vaccine strain (LVS) or Francisella novicida was inhibited by >or=2 microM Ga chelated to Tf or Lf, with GaLf being somewhat more potent. Francisella spp. express two Fe-containing antioxidant enzymes, catalase (KatG) and Fe cofactored superoxide dismutase (FeSOD). Growth of LVS with 10 muM GaTf or GaLf led to a dramatic decrease in bacterial catalase activity and in FeSOD activity that was associated with an increased susceptibility to H(2)O(2). Ga also protected mice from intranasal challenge with F. novicida. Whereas 100% of the F. novicida-infected mice died by day 9, 75% of the mice receiving Ga continued to survive to at least day 15. Thus, a single intranasal dose of Ga followed by daily intraperitoneal Ga at a dose tolerated by the animals resulted in prolonged survival. These data support the potential utility of Ga as a therapy for F. tularensis infection of the lung.

Several genes contained in the Francisella pathogenicity island (FPI) encode proteins needed for intracellular growth and virulence of Francisella tularensis. The pdpA gene is the first cistron in the larger of the two operons found in the FPI. In this work we studied the intracellular growth phenotype of a Francisella novicida mutant in the pdpA gene. The DeltapdpA strain was capable of a small amount of intracellular replication but, unlike wild-type F. novicida, remained associated with the lysosomal marker LAMP-1, suggesting that PdpA is necessary for progression from the early phagosome phase of infection. Strains with in cis complementation of the DeltapdpA lesion showed a restoration of intracellular growth to wild-type levels. Infection of macrophages with the DeltapdpA mutant generated a host-cell mRNA profile distinct from that generated by infection with wild-type F. novicida. The transcriptional response of the host macrophage indicates that PdpA functions directly or indirectly to suppress macrophage ability to signal via growth factors, cytokines and adhesion ligands.

Francisella tularensis is a highly virulent, intracellular pathogen that causes the disease tularaemia. A research surrogate for F. tularensis is Francisella novicida, which causes a tularaemia-like disease in mice, grows similarly in macrophages, and yet is unable to cause disease in humans. Both Francisella species contain a cluster of genes referred to as the Francisella pathogenicity island (FPI). Pathogenicity determinant protein A (PdpA), encoded by the pdpA gene, is located within the FPI and has been associated with the virulence of Francisella species. In this work we examined the properties of PdpA protein expression and localization as well as the phenotype of a F. novicida pdpA deletion mutant. Monoclonal antibody detection of PdpA showed that it is a soluble protein that is upregulated in iron-limiting conditions and undetectable in an mglA or mglB mutant background. Deletion of pdpA resulted in a strain that was highly attenuated for virulence in chicken embryos and mice.