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Title: Glutathione Provides a Source of Cysteine Essential for Intracellular Multiplication of Francisella tularensis}
Article:
Abstract:
Francisella tularensis is a highly infectious bacterium causing the zoonotic disease tularemia. Its ability to multiply and survive in macrophages is critical for its virulence. By screening a bank of HimarFT transposon mutants of the F. tularensis live vaccine strain( LVS) to isolate intracellular growth-deficient mutants, we selected one mutant in a gene encoding a putative γ-glutamyl transpeptidase( GGT). This gene( FTL_0766) was hence designated ggt. The mutant strain showed impaired intracellular multiplication and was strongly attenuated for virulence in mice. Here we present evidence that the GGT activity of F. tularensis allows utilization of glutathione( GSH, γ-glutamyl-cysteinyl-glycine) and γ-glutamyl-cysteine dipeptide as cysteine sources to ensure intracellular growth. This is the first demonstration of the essential role of a nutrient acquisition system in the intracellular multiplication of F. tularensis. GSH is the most abundant source of cysteine in the host cytosol. Thus, the capacity this intracellular bacterial pathogen has evolved to utilize the available GSH, as a source of cysteine in the host cytosol, constitutes a paradigm of bacteria–host adaptation.
Introduction:
Francisella tularensis is a Gram-negative bacterium capable of causing the disease tularemia in a large number of mammalian species. It is a highly infectious bacterium that can be transmitted to humans in numerous ways. Four different subspecies( subsp.) of F. tularensis that differ in virulence and geographic distribution exist. These are designated subsp. tularensis( type A), holarctica( type B), novicida and mediasiatica. F. tularensis subsp. tularensis is the most virulent subspecies causing a severe disease in humans, whereas F. tularensis subsp. holarctica causes a similar disease but of less severity[1]. Because if its high infectivity and lethality, F. tularensis is considered a potential bioterrorism agent[2]. F. tularensis is a facultative intracellular bacterium that infects and replicates mainly inside macrophages, but which can also infect hepatocytes, endothelial cells, epithelial cells, fibroblasts, chicken embryos, and amoebae[3]. Although the molecular mechanisms by which Francisella adapts to life inside host cells are not well understood, a number of genes that are necessary for Francisella to survive inside its host and cause disease are known[4]. These include genes located in the Francisella pathogenicity island( FPI)[5],[6],[7],[8],[9],[10],[11], the genes encoding the regulatory proteins MglA, SspA, and PmrA, which regulate expression of the FPI[5],[12],[13] and the genes responsible for lipopolysaccharide( LPS) production[14],[15],[16],[17]. Furthermore, several genome-scale screening approaches have been formerly utilized in F. tularensis subsp. novicida, tularensis, and holarctica, to identify additional genes that are important for replication inside macrophages or survival in mice[7],[18],[19],[20],[21]. In the present work, we designed an in vitro negative selection method, based on the use of a bacteriostatic antibiotic, to recover intracellular growth mutants directly from a pool of mutants. A similar approach has been reported earlier to select intracellular growth-deficient mutants of Listeria monocytogenes[22]. One of the mutants isolated by this procedure( see Table S1) showed a drastic intracellular growth defect. This mutant had an insertion into gene FTL_0766, encoding a putative gamma-glutamyl transpeptidase, GGT. We focus here on the role of this gene in the virulence of F. tularensis. GGT's are involved in hydrolysis of γ-glutamyl compounds such as glutathione( GSH). GSH is a non-ribosomal tripeptide( L-γ-L-glutamyl-L-cysteinyl-glycine) present in almost all eukaryotic cells and in some prokaryotes. In eukaryotes, GSH is essential for cell homeostasis and GSH-deficiency has been associated with various severe diseases[23]. In mammalian tissues, GSH is generally found in the cytosol at mM levels( 1–10 mM)[24]; and GSH biosynthesis and metabolism proceeds through the γ-glutamyl cycle[25]. In all living organisms, GGT catalyzes the first step in the degradation of GSH, which involves the cleavage and transfer of the γ-glutamyl moiety from GSH to an amino acid acceptor( or hydrolysis to release glutamate) and release of cysteinyl-glycine. The four subspecies of F. tularensis require cysteine for growth; and defined media, containing cysteine and other amino acids essential to support growth of F. tularensis strains, have been developed[26],[27]. In silico analysis of the F. tularensis SCHU S4 genome suggests that the specific requirement for cysteine is due to a nonfunctional pathway for sulfate assimilation, resulting from a pseudogene encoding adenylylsulfate kinase[27]. We demonstrate here that FTL_0766 of F. tularensis encodes a genuine GGT involved in the metabolism of γ-glutamyl-containing peptides. GGT allows the utilization of γ-glutamyl peptides as a source of cysteine during intracellular multiplication of the pathogen, and is thus critical for virulence.
Discussion:
Screenings of banks of mutants have been previously reported in F. tularensis[7],[18],[19],[20],[21],[45]. A number of genes were repeatedly found by these different approaches, in particular genes involved in purine/pyrimidine synthesis. Notably, three of these studies identified the ggt gene[19],[21],[45] but its properties were not further characterized. GGT shares some sequence similarity with the CapD enzyme responsible for covalent anchoring of the B. anthracis capsule. The existence of a capsule surrounding F. tularensis has been evoked repeatedly but never persuasively demonstrated experimentally. Therefore, we evaluated the impact of ggt inactivation on serum resistance of LVS as the putative Francisella capsule confers serum resistance[46]. The ggt mutant showed a serum resistance comparable to that of wild-type LVS, demonstrating that GGT is not involved in serum resistance. Inactivation of ggt had a drastic effect on the ability of LVS to multiply in eukaryotic cells. Strikingly, we showed that the intracellular growth defect of the ggt mutant could be eliminated by providing external cysteine to the culture medium, establishing a direct link between GGT activity and cysteine availability. Also, the capacity of the ggt mutant to cause disease in mice was severely decreased, further demonstrating the critical role of GGT in pathogenesis. The involvement of GGT in the utilization of glutathione or other γ-L-glutamyl peptides has been demonstrated in both prokaryotes and eukaryotes[38],[47],[48],[49],[50]. While some bacterial species have the capacity to directly import GSH from the growth medium and degrade it into glutamate and cysteine in their cytoplasm[51], species such as H. pylori cannot transport this substrate and must secrete or export GGT to degrade GSH extracellularly. GGT of H. pylori has been described as a virulence factor essential for the establishment of the infection in the mouse model[52],[53],[54] and as an apoptosis-inducing protein[55]. Furthermore, a role for GGT in the metabolism of glutamine and glutathione has been recently established, providing information on the role of the enzyme in the physiopathology of this extracellular bacterial pathogen[56]. We did not detect any GGT activity in culture supernatants of LVS under the conditions we tested, suggesting that GGT-mediated degradation of γ-glutamyl compounds occurs in the bacterial cytoplasm. Future work will be required to address the molecular mechanisms of uptake of these compounds in F. tularensis. Our results show that both GSH and γ-glutamyl-cysteine can functionally replace cysteine to support growth of Francisella in broth, and that GGT activity is required for this, by cleaving the γ-glutamyl-cysteine linkage. We also showed that F. tularensis GGT could functionally complement an E. coli GGT-deficient strain. The F. tularensis GGT was able to cleave the γ-Glu bond of the dipeptide γ-Glu-Leu, allowing, like the genuine E. coli enzyme, the utilization of the dipeptide as a source of amino acid( providing leucine to supplement the leucine auxothrophy of the strain). Finally, a metabolic labeling assay confirmed that GSH utilization, as a source of cysteine, specifically requires the action of the ggt gene product. Furthermore, the use of radiolabeled-GSH, carrying the 35S isotope on the cysteine side chain, demonstrated that cysteine from GSH is indeed incorporated into bacterial proteins during growth. The infectious cycle of F. tularensis is essentially intracellular. Since the severe intracellular growth defect of the ggt mutant could be completely relieved by the addition of free cysteine to the cell culture medium, it seems most likely that GGT provides a source of cysteine to cytosolic bacteria by degrading internalized γ-glutamyl-cysteine peptides. GSH is the most abundant non-protein thiol in mammalian cells and the prevalent low-molecular weight peptide in eukaryotic cells, attaining concentrations of 1 to 10 mM in many different cell types, most of which( 85–90%) is present in the cytosol. Biosynthesis of GSH is dependent on the availability of the amino acid precursors glutamate, glycine, and cysteine. The intracellular pool of cysteine is relatively small( 0. 10–0. 25 mM) and cysteine is generally the limiting amino acid for GSH synthesis( the other two precursors, glycine and glutamate, are found in considerable higher intracellular concentration). It is estimated that at the low levels of γ-glutamyl-cysteine normally present, 95% is converted to GSH( see for reviews[23],[57],[58] and references therein). We propose a model for intracellular cysteine acquisition by F. tularensis( Figure 8), in which intracellular F. tularensis uses γ-glutamyl-cysteine dipeptide and GSH as the primary sources of cysteine. The ggt mutant, unable to cleave the γ-glutamyl-cysteine bond, does not obtain sufficient free cysteine( or cystine) from its surroundings to support growth. Therefore, only the addition of mM amounts of free cysteine compensates its enzymatic deficiency. Utilization of cytosolic GSH and/or cysteine-containing dipeptides as a source of cysteine might be used by other intracellular bacterial pathogens. More generally, acquisition of nutrients should be considered as an integral part of the pathogenicity of intracellular bacteria. This concept is comforted by recent transcriptomic analyses, which revealed the upregulation of many bacterial metabolic and transport functions in infected cells( see for examples[59],[60],[61],[62]). A remarkable example of the interplay between intracellular pathogens and their host has been provided with L. monocytogenes. Indeed, it has been shown that a listerial membrane transporter, Hpt, was essential for acquisition of phosphorylated hexoses within the cystosol of infected cells and was, thus, of major importance for bacterial replication[63],[64]. Clearly, intracellular bacterial pathogens have adapted a fine balance between their metabolic needs and the nutrients available( nature and concentration), to multiply efficiently and to avoid a premature death of the host cell that would lead to an aborted infection.
The role of nutrient acquisition systems in survival and multiplication of intracellular bacterial pathogens within infected cells is yet poorly understood . The data presented here suggest that Francisella tularensis , a highly infectious facultative intracellular bacterium , is capable of utilizing glutathione ( GSH ) and γ–glutamyl-cysteine peptides present in the cytosol of infected host cells . An in vitro negative selection method , based on the use of a bacteriostatic antibiotic , to recover intracellular growth mutants directly from a pool of mutants , allowed us to select one mutant in a gene encoding a γ-glutamyl transpeptidase ( GGT ) . The mutant strain showed impaired intracellular multiplication and was strongly attenuated for virulence in mice . The cleavage of these cysteine-containing peptides by GGT activity provides thus the essential source of cysteine required for intracellular multiplication . The capacity F . tularensis has evolved to utilize GSH , the most abundant source of cysteine in the host cytosol , constitutes a model of bacterial adaptation to intracellular lifestyle .