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.

SummaryIntracellular bacterial pathogens generally express chaperones such as Hsp100s during multiplication in host cells, allowing them to survive potentially hostile conditions. Francisella tularensis is a highly infectious bacterium causing the zoonotic disease tularaemia. The ability of F. tularensis to multiply and survive in macrophages is considered essential for its virulence. Although previous mutant screens in Francisella have identified the Hsp100 chaperone ClpB as important for intracellular survival, no detailed study has been performed. We demonstrate here that ClpB of F. tularensis live vaccine strain (LVS) is important for resistance to cellular stress. Promoter analysis shows that the transcriptional start is preceded by a s 32-like promoter sequence and we demonstrate that expression of clpB is induced by heat shock. This indicates that expression of clpB is dependent on the heat-shock response mediated by s 32, the only alternative s-factor present in Francisella. Our studies demonstrate that ClpB contributes to intracellular multiplication in vitro, but is not essential. However, ClpB is absolutely required for Francisella to replicate in target organs and induce disease in mice. Proteomic analysis of membrane-enriched fractions shows that five proteins are recovered at lower levels in the mutant strain. The crucial role of ClpB for in vivo persistence of Francisella may be linked to its assumed function in reactivation of aggregated proteins under in vivo stress conditions.

Francisella tularensis is a highly infectious pathogen that infects animals and humans, causing tularemia. The ability to replicate within macrophages is central for virulence and relies on expression of genes located in the Francisella pathogenicity island (FPI), as well as expression of other genes. Regulation of FPI-encoded virulence gene expression in F. tularensis involves at least four regulatory proteins and is not fully understood. Here we studied the RNA-binding protein Hfq in F. tularensis and particularly the role that it plays as a global regulator of gene expression in stress tolerance and pathogenesis. We demonstrate that Hfq promotes resistance to several cellular stresses (including osmotic and membrane stresses). Furthermore, we show that Hfq is important for the ability of the F. tularensis vaccine strain LVS to induce disease and persist in organs of infected mice. We also demonstrate that Hfq is important for stress tolerance and full virulence in a virulent clinical isolate of F. tularensis, FSC200. Finally, microarray analyses revealed that Hfq regulates expression of numerous genes, including genes located in the FPI. Strikingly, Hfq negatively regulates only one of two divergently expressed putative operons in the FPI, in contrast to the other known regulators, which regulate the entire FPI. Hfq thus appears to be a new pleiotropic regulator of virulence in F. tularensis, acting mostly as a repressor, in contrast to the other regulators identified so far. Moreover, the results obtained suggest a novel regulatory mechanism for a subset of FPI genes.Regulation of gene expression by noncoding or small RNAs (sRNAs) is prevalent in both prokaryotes and eukaryotes, and recent studies have identified numerous sRNAs in different organisms. Most sRNAs encoded by bacterial chromosomes act by base pairing with mRNA targets that are encoded in trans, and these sRNAs commonly require Hfq in order to function (47). Hfq is a bacterial RNA-binding protein that was initially recognized as a host factor for replication of the Q␤ RNA phage in Escherichia coli (17). The Hfq protein is very abundant, and it belongs to the eukaryotic and archaeal families of Sm and Sm-like proteins, respectively, that form homohexameric structures (for reviews, see references 5 and 53). The importance of Hfq became clear when an E. coli hfq null mutant was created. This mutant had pleiotropic phenotypes, such as a decreased growth rate, increased sensitivity to cellular stresses, and increased cell length (51). Hfq is a posttranscriptional regulator that binds sRNAs and mRNA and facilitates RNA-RNA interaction (1,18,23,32,35,55). For the most part, sRNA-mRNA interactions result in mRNA degradation and/or inhibition of translation, but they can also increase translation (for reviews, see references 1, 20, and 48). As might be expected from the pleiotropic phenotypes of an hfq mutant, deletion of hfq has been correlated with consider-

HIV-specific CD8 + T cells play a major role in the control of virus during HIV primary infection (PI) but do not completely prevent viral replication. We used IFN-γ enzyme-linked immunospot assay and intracellular staining to characterize the ex vivo CD8 + T-cell responses to a large variety of HIV epitopic peptides in 24 subjects with early HIV PI. We observed HIV-specific responses in 71% of subjects. Gag and Nef peptides were more frequently recognized than Env and Pol peptides. The number of peptides recognized was low (median 2, range 0-6). In contrast, a much broader response was observed in 30 asymptomatic subjects with chronic infection: all were responders with a median of 5 peptides recognized (range 1-13). The frequency of HIV-specific CD8 + T cells among PBMC for a given peptide was of the same order of magnitude in both groups. The proportion of HIV-specific CD8 + CD28 -terminally differentiated T cells was much lower in PI than at the chronic stage of infection. The weakness of the immune response during HIV PI could partially account for the failure to control HIV. These findings have potential importance for defining immunotherapeutic strategies and establishing the goals for effective vaccination.

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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-yetunidentified receptor for F. tularensis internalization has been suggested.

The Gram-positive human pathogen Staphylococcus aureus is a leading cause of severe bacterial infections. Recent studies have shown that various cell types could readily internalize S. aureus and infected cells have been proposed to serve as vehicle for the systemic dissemination of the pathogen. Here we focused on the intracellular behavior of the Community-Associated Methicillin-Resistant S. aureus strain USA300. Supporting earlier observations, we found that wild-type S. aureus strain USA300 persisted for longer period within endothelial cells than within macrophages and that a mutant displaying the small colony variant phenotype (ΔhemDBL) had increased intracellular persistence. Time-lapse microscopy revealed that initial persistence of wild-type bacteria in endothelial cells corresponded to distinct single cell events, ranging from active intracellular bacterial proliferation, leading to cell lysis, to non-replicating bacterial persistence even 1 week after infection. In sharp contrast, ΔhemDBL mutant bacteria were essentially non-replicating up to 10 days after infection. These findings suggest that internalization of S. aureus in endothelial cells triggers its persistence and support the notion that endothelial cells might constitute an intracellular persistence niche responsible for reported relapse of infection after antibiotic therapy.

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.