Etienne Richer

Polyphyletic origins of bacterial Nramp transporters

The redox-active metals iron and manganese are required for energy metabolism, protection against oxidative stress and defense against infections. In eukaryotes, both divalent metals are transported by Nramp transporters. The sequence of these transporters was remarkably conserved during evolution. Several bacterial Nramp homologs (MntH) are also proton-dependent manganese transporters. Here, we present phylogenetic evidence for the polyphyletic origins of three groups of MntH proteins and for possible Nramp horizontal gene transfer with eukaryotes. We propose that the evolution of the MntH/Nramp family is related to adaptation to oxidative environments, including those arising during infection of animals and plants.

Horizontal gene transfer of "prototype" Nramp in bacteria

Eukaryotic Nramp genes encode divalent metal ion permeases important for nutrition and resistance to microbial infection. Bacterial homologs encode proton-dependent transporters of manganese (MntH), and other divalent metal ions. Bacterial MntH were classified in three homology groups (A, B, C) and MntH C further subdivided in Cα, Cβ, Cγ. The proteins from C. tepidum (MntH B) and E. faecalis (MntH Cβ1, 2), divergent in sequence and hydropathy profile, conferred increased metal sensitivity when expressed in E. coli, suggesting conservation of divalent metal transport function in MntH B and C. Several genomic evidence suggest horizontal gene transfer (HGT) of mntH C genes: (i) The enterobacteria WigglesworthiamntH Cβ gene is linked to an Asnt-RNA, and its sequence most conserved with Gram positive bacteria homologs; (ii) all the Cβ genes identified in oral streptococcaceae are associated with different potentially mobile DNA elements; (iii) Lactococcuslactis and Burkholderiamallei genomes contain an mntH gene prematurely terminated and a novel full-length mntH C gene; (iv) remarkable sequence relatedness between the unicellular alga C. reinhardtii “prototype” Nramp and some MntH Cα (e.g., Nostoc spp., Listeria spp.) suggests HGT between Eukarya and Bacteria. Other “prototype” Nramp genes (intronless, encoding proteins strongly conserved with MntH A and B proteins) identified in invertebrates represent a possible source for transfer of Nramp genes toward opportunistic bacteria. This study demonstrates complex evolution of MntH in Bacteria. It is proposed that “prototype” Nramp are ancestors of bacterial MntH C proteins, which could facilitate bacterial infection.

N-Ethyl-N-Nitrosourea–Induced Mutation in Ubiquitin-Specific Peptidase 18 Causes Hyperactivation of IFN-αβ Signaling and Suppresses STAT4-Induced IFN-γ Production, Resulting in Increased Susceptibility to Salmonella Typhimurium

To deepen our knowledge of the natural host response to pathogens, our team undertook an in vivo screen of mutagenized 129S1 mice with Salmonella Typhimurium. One mutation affecting Salmonella susceptibility was mapped to a region of 1.3 Mb on chromosome 6 that contains 15 protein-coding genes. A missense mutation was identified in the Usp18 (ubiquitin-specific peptidase 18) gene. This mutation results in an increased inflammatory response (IL-6, type 1 IFN) to Salmonella and LPS challenge while paradoxically reducing IFN-γ production during bacterial infection. Increased STAT1 phosphorylation correlated with impaired STAT4 phosphorylation, resulting in overwhelming IL-6 secretion but reduced IFN-γ production during infection. The reduced IFN-γ levels, along with the increased inflammation, rationalize the S. Typhimurium susceptibility in terms of increased bacterial load in target organs and cytokine-induced septic shock and death.

Regulation of NRAMP1 gene expression by 1α,25‐dihydroxy‐vitamin D3 in HL‐60 phagocytes

The natural resistance‐associated macrophage protein 1 (Nramp1) is a proton‐dependent transporter of divalent metals. We studied NRAMP1 expression during HL‐60 differentiation induced by VD and VD agonists. NRAMP1 and CD14 gene expression differed in kinetics of induction, mRNA levels and stability, and response to VD combined with PMA, whereas a combination of VD and IFN‐γ induced similar up‐regulation. NRAMP1 protein expression paralleled the accumulation of mRNA and was localized in the phagosomal membrane after phagocytosis. A promoter construct extending 647 bp upstream of NRAMP1 ATG showed myeloid‐specific transcription in transient trasfection assays, which was up‐regulated by VD in HL‐60. In HL‐60 clones stably transfected with this construct, transcription was apparently induced through indirect VD genomic effects, and there was accordance between the levels of reporter transcription and endogenous NRAMP1 mRNA in response to VD but not to IFN‐γ. Thus, VD genomic effects stimulate NRAMP1 transcription and protein expression in maturing phagocytes.

Contribution of increased ISG15, ISGylation and deregulated type I IFN signaling in Usp18 mutant mice during the course of bacterial infections

Host genetics has a key role in susceptibility to Salmonella Typhimurium infection. We previously used N-ethyl-N-nitrosourea (ENU) mutagenesis to identify a loss-of-function mutation within the gene ubiquitin-specific peptidase 18 (Usp18Ity9), which confers increased susceptibility to Salmonella Typhimurium. USP18 functions to regulate type I interferon (IFN) signaling and as a protease to remove ISG15 from substrate proteins. Usp18Ity9 mice are susceptible to infection with Salmonella Typhimurium and have increased expression and function of ISG15, but Usp18Ity9 mice lacking Isg15 do not show improved survival with Salmonella challenge. Type I IFN signaling is increased in Usp18Ity9 mice and inhibition of type I IFN signaling is associated with improved survival in mutant mice. Hyperactivation of type I IFN signaling leads to increased IL-10, deregulated expression of autophagy markers and elevated interleukin (IL)-1β and IL-17. Furthermore, Usp18Ity9 mice are more susceptible to infection with Mycobacterium tuberculosis, have increased bacterial load in the lung and spleen, elevated inflammatory cytokines and more severe lung pathology. These findings demonstrate that regulation of type I IFN signaling is the predominant mechanism affecting the susceptibility of Usp18Ity9 mice to Salmonella infection and that hyperactivation of signaling leads to increased IL-10, deregulation of autophagic markers and increased proinflammatory cytokine production.

Transcription factors Sp1 and C/EBP regulate NRAMP1 gene expression

The natural resistance‐associated macrophage protein 1 (Nramp1), which belongs to a conserved family of membrane metal transporters, contributes to phagocyte‐autonomous antimicrobial defense mechanisms. Genetic polymorphisms in the human NRAMP1 gene predispose to susceptibility to infectious or inflammatory diseases. To characterize the transcriptional mechanisms controlling NRAMP1 expression, we previously showed that a 263 bp region upstream of the ATG drives basal promoter activity, and that a 325 bp region further upstream confers myeloid specificity and activation during differentiation of HL‐60 cells induced by vitamin D. Herein, the major transcription start site was mapped in the basal region by S1 protection assay, and two cis‐acting elements essential for myeloid transactivation were characterized by in vitro DNase footprinting, electrophoretic mobility shift experiments, in vivo transfection assays using linker‐mutated constructs, and chromatin immunoprecipitation assays in differentiated monocytic cells. One distal cis element binds Sp1 and is required for NRAMP1 myeloid regulation. Another site in the proximal region binds CCAAT enhancer binding proteins α or β and is crucial for transcription. This study implicates Sp1 and C/EBP factors in regulating the expression of the NRAMP1 gene in myeloid cells.

Suppression of Hepcidin Expression and Iron Overload Mediate Salmonella Susceptibility in Ankyrin 1 ENU-Induced Mutant

Salmonella, a ubiquitous Gram-negative intracellular bacterium, is a food borne pathogen that infects a broad range of hosts. Infection with Salmonella Typhimurium in mice is a broadly recognized experimental model resembling typhoid fever in humans. Using a N-ethyl-N-nitrosurea (ENU) mutagenesis recessive screen, we report the identification of Ity16 (Immunity to Typhimurium locus 16), a locus responsible for increased susceptibility to infection. The position of Ity16 was refined on chromosome 8 and a nonsense mutation was identified in the ankyrin 1 (Ank1) gene. ANK1 plays an important role in the formation and stabilization of the red cell cytoskeleton. The Ank1Ity16/Ity16 mutation causes severe hemolytic anemia in uninfected mice resulting in splenomegaly, hyperbilirubinemia, jaundice, extramedullary erythropoiesis and iron overload in liver and kidneys. Ank1Ity16/Ity16 mutant mice demonstrated low levels of hepcidin (Hamp) expression and significant increases in the expression of the growth differentiation factor 15 (Gdf15), erythropoietin (Epo) and heme oxygenase 1 (Hmox1) exacerbating extramedullary erythropoiesis, tissue iron deposition and splenomegaly. As the infection progresses in Ank1Ity16/Ity16, the anemia worsens and bacterial load were high in liver and kidneys compared to wild type mice. Heterozygous Ank1+/Ity16 mice were also more susceptible to Salmonella infection although to a lesser extent than Ank1Ity16/Ity16 and they did not inherently present anemia and splenomegaly. During infection, iron accumulated in the kidneys of Ank1+/Ity16 mice where bacterial loads were high compared to littermate controls. The critical role of HAMP in the host response to Salmonella infection was validated by showing increased susceptibility to infection in Hamp-deficient mice and significant survival benefits in Ank1 +/Ity16 heterozygous mice treated with HAMP peptide. This study illustrates that the regulation of Hamp and iron balance are crucial in the host response to Salmonella infection in Ank1 mutants.

Iron- and Hepcidin-Independent Downregulation of the Iron Exporter Ferroportin in Macrophages during Salmonella Infection

Retention of iron in tissue macrophages via upregulation of hepcidin (HAMP) and downregulation of the iron exporter ferroportin (FPN) is thought to participate in the establishment of anemia of inflammation after infection. However, an upregulation of FPN has been proposed to limit macrophages iron access to intracellular pathogens. Therefore, we studied the iron homeostasis and in particular the regulation of FPN after infection with Salmonella enterica serovar Typhimurium in mice presenting tissue macrophages with high iron (AcB61), basal iron (A/J and wild-type mice), or low iron (Hamp knock out, Hamp−/−) levels. The presence of iron in AcB61 macrophages due to extravascular hemolysis and strong erythrophagocytosis activity favored the proliferation of Salmonella in the spleen and liver with a concomitant decrease of FPN protein expression. Despite systemic iron overload, no or slight increase in Salmonella burden was observed in Hamp−/− mice compared to controls. Importantly, FPN expression at both mRNA and protein levels was strongly decreased during Salmonella infection in Hamp−/− mice. The repression of Fpn mRNA was also observed in Salmonella-infected cultured macrophages. In addition, the downregulation of FPN was associated with decreased iron stores in both the liver and spleen in infected mice. Our findings show that during Salmonella infection, FPN is repressed through an iron and hepcidin-independent mechanism. Such regulation likely provides the cellular iron indispensable for the growth of Salmonella inside the macrophages.

Impact of Usp18 and IFN signaling in Salmonella -induced typhlitis

In humans, Salmonella infection causes two major clinical diseases, typhoid fever and a self-limiting gastro-enteritidis. Salmonella transmission occurs by the fecal–oral route and the interactions between the bacteria and the digestive tract epithelium are central to the outcome of the infection. Using a mouse model of typhoid fever, we previously identified a mutation in USP18 affecting type I interferon (IFN) signaling resulting in increased susceptibility to systemic Salmonella infection. In this study, we demonstrate the effects of this mutation during the early response to Salmonella using a model of typhlitis. Mutant Usp18 mice showed a minimal inflammatory response early after Salmonella Typhimurium infection that was associated with low pathologic scores and low IFN-γ production. This resulted in an increased interaction of Salmonella with the cecal epithelium and earlier systemic dissemination of the bacteria. The global transcriptional signature in the cecum of mouse during Salmonella infection showed normal expression of tissue specific genes and upregulation of type I IFN pathway in mutant mice. In control mice, there was a significant over-representation of genes involved in cellular recruitment and antibacterial activity paralleling the histopathological features. These results show the impact of USP18 in the development of Salmonella-induced typhlitis.

Chemical mutagenesis: a new strategy against the global threat of infectious diseases

The perpetual evolution of drug-resistant microbes, the overwhelming burden of acquired immune suppression due to HIV, the emergence or re-emergence of various pathogens (West Nile virus, pandemic influenza, Creutzfeld-Jacob disease), and increased fears of bioterrorism has drawn a great deal of new attention to infectious diseases. The pathogenesis of infection is characterized by complex interactions of potentially virulent microorganisms with host genetic and acquired factors. Chemical mutagenesis of the mouse genome provides a robust method to unravel this challenging problem. To deepen our understanding of the natural host response to pathogens, our team and others are interrogating the mouse genome to define genes that are crucial to the defense against infectious diseases (pathogen recognition, viral defense, bacterial defense, prion infection). In this review we highlight the current progress of these efforts and propose a toolbox for other groups that are interested in this endeavor.