Triggering Ras signalling by intracellular Francisella tularensis through recruitment of PKCα and βI to the SOS2/GrB2 complex is essential for bacterial proliferation in the cytosol

Authors

  • Souhaila Al-Khodor,

    1. Department of Microbiology and Immunology, Room 413, College of Medicine
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    • Present address: National Institutes of Health, Bethesda, MD 20892, USA.

  • Yousef Abu Kwaik

    Corresponding author
    1. Department of Microbiology and Immunology, Room 413, College of Medicine
    2. Department of Biology, University of Louisville, Louisville, KY 40202, USA.
      E-mail: abukwaik@louisville.edu; Tel. (+1) 502 852 4117; Fax (+1) 502 852 7531.
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E-mail: abukwaik@louisville.edu; Tel. (+1) 502 852 4117; Fax (+1) 502 852 7531.

Summary

Intracellular proliferation of Francisella tularensis is essential for manifestation of the fatal disease tularaemia, and is classified as a category A bioterrorism agent. The F. tularensis-containing phagosome (FCP) matures into a late endosome-like phagosome with limited fusion to lysosomes, followed by rapid bacterial escape into the cytosol. The Francisella pathogenicity island (FPI) encodes a type VI-like secretion system, and the FPI-encoded IglC is essential for evasion of lysosomal fusion and phagosomal escape. Many host signalling events are likely to be modulated by F. tularensis to render the cell permissive for intracellular proliferation but they are not fully understood. Here we show that within 15 min of infection, intracellular F. tularensis ssp. novicida triggers IglC-dependent temporal activation of Ras, but attached extracellular bacteria fail to trigger Ras activation, which has never been shown for other intracellular pathogens. Intracellular F. tularensis ssp. novicida triggers activation of Ras through recruitment of PKCα and PKCβI to the SOS2/GrB2 complex. Silencing of SOS2, GrB2 and PKCα and PKCβI by RNAi has no effect on evasion of lysosomal fusion and bacterial escape into the cytosol but renders the cytosol non-permissive for replication of F. tularensis ssp. novicida. Since Ras activation promotes cell survival, we show that silencing of SOS2, GrB2 and PKCα and βI is associated with rapid early activation of caspase-3 within 8 h post infection. However, silencing of SOS2, GrB2 and PKCα and βI does not affect phosphorylation of Akt or Erk, indicating that activation of the PI3K/Akt and the Erk signalling cascade are independent of the F. tularensis-triggered Ras activation. We conclude that intracellular F. tularensis ssp. novicida triggers temporal and early activation of Ras through the SOS2/GrB2/PKCα/PKCβI quaternary complex. Temporal and rapid trigger of Ras signalling by intracellular F. tularensis is essential for intracellular bacterial proliferation within the cytosol, and this is associated with downregulation of early caspase-3 activation.

Introduction

Francisella tularensis is a Gram-negative facultative intracellular bacterium that can cause a fatal disease designated tularaemia in humans and animals (Pechous et al., 2009; Santic et al., 2010). Being easily disseminated and highly infectious, F. tularensis has been classified as a category A select agent (Darling et al., 2002). Four closely related subspecies of F. tularensis have been identified: tularensis, holarctica, mediasiatica and novicida (Pechous et al., 2009). Two subspecies of F. tularensis cause most human illness, subspecies tularensis and subspecies holarctica (Keim et al., 2007; Champion et al., 2009). F. tularensis ssp. novicida has low virulence for humans but shares a high degree of antigenic and genetic similarities with F. tularensis ssp. tularensis and ssp. holarctica (Forsman et al., 1994) and maintains high virulence in mice (Lauriano et al., 2004; Pammit et al., 2006). In addition, F. tularensis ssp. novicida has proven to be much more amenable to genetic manipulation than the other F. tularensis subspecies (Pammit et al., 2006).

Francisella tularensis enters the host cell by inducing pseudopod loops in macrophages forming the Francisella-containing phagosome (FCP) (Clemens et al., 2005; Santic et al., 2010). The FCP matures transiently to an acidified late endosome-like phagosome followed by bacterial escape into the cytosol by 30–60 min post infection (reviewed in Santic et al., 2010). Post phagosomal escape, the bacteria replicate in the cytosol and inducescaspase-3-mediated apoptosis of the host cell during late stages on infection in vitro and in the mice model in vivo (Lai et al., 2001; 2004; Rajaram et al., 2009; Wickstrum et al., 2009), but F. tularensis-infected macrophages are resistant to apoptosis during early stages of infection (Santic et al., 2009). Importantly, modulation of phagosome biogenesis and escape into the cytosol by F. tularensis ssp. novicida is indistinguishable from F. tularensis ssp. tularensis and ssp. holarctica (Clemens et al., 2004; Santic et al., 2005a).

While F. tularensis ssp. tularensis and holarctica have two identical copies of the FPI, F. tularensis ssp. novicida contains one single copy of the FPI (Gray et al., 2002; de Bruin et al., 2007; Santic et al., 2007). The FPI-encoded genes share homology with genes found in secretion loci of a number of bacteria, including the T6SS clusters of Vibrio cholerae and Pseudomonas aeruginosa, suggesting that the FPI encodes a T6SS-like apparatus (de Bruin et al., 2007; Barker et al., 2009). Inactivation of the FPI genes leads to decreased intramacrophage growth and decreased virulence (Gray et al., 2002; Lai et al., 2004; Tempel et al., 2006; Santic et al., 2007). The two FPI-encoded proteins VgrG and IglI are translocated into the cytosol of infected macrophages (Barker et al., 2009). Both proteins are required for evasion of lysosomal fusion and bacterial escape to the cytosol (Barker et al., 2009), and the secretion of IglI is T6SS-dependent (Barker et al., 2009). The FPI-encoded iglC gene is essential for evasion of lysosomal fusion and subsequent bacterial escape into the cytosol. Recent studies have shown that bacterial escape into the cytosol of human and arthropod-derived cells is complex and requires a large portion of the bacterial genome (Asare and Abu Kwaik, 2010; Asare et al., 2010). In addition, numerous Drosophila host factors have been identified to be essential for bacterial proliferation in arthropod-derived cells and many of these factors are conserved in mammalian cells (Akimana et al., 2010).

Most signalling events during infection by F. tularensis have been studied using F. tularensis ssp. novicida as a model system (Parsa et al., 2006; 2008; Rajaram et al., 2006; 2009; Butchar et al., 2008; Cremer et al., 2009a). Within the host cytosol, F. tularensis subvert host immune responses by negative regulation of TLR signalling (Telepnev et al., 2003), which results in dampening innate immunity (Butchar et al., 2008). A key element in the signalling pathways involved in transducing F. tularensis-initiated signals to cellular responses is the family of mitogen-activated protein (MAP) kinases such as p38, c-Jun N-terminal kinase (JNK) and Erk (Telepnev et al., 2005; Parsa et al., 2008). In addition to the MAP kinase pathway, the phosphoinositol 3 kinase (PI3K)/Akt pathway is also activated upon infection by F. tularensis resulting in a strong innate immune response (Parsa et al., 2006; 2008; Rajaram et al., 2006; Cremer et al., 2009b). Inhibition of PI3K/Akt results in suppression of F. tularensis ssp. novicida-induced innate immune responses (Parsa et al., 2006; Rajaram et al., 2006). The SH2 domain inositol phosphatase (SHIP) is phosphorylated upon infection by F. tularensis ssp. novicida, and negatively regulates PI3K activation resulting in dampening the innate immune response (Parsa et al., 2006; Rajaram et al., 2006). The SHIP-deficient macrophages display enhanced Akt activation upon infection by F. tularensis ssp. novicida, resulting in elevated PI3K-dependent activation and enhanced NF-κB-driven gene transcription (Parsa et al., 2006; Rajaram et al., 2006). In contrast, overexpression of the negative regulator SHIP results in decreased NF-κB activation and antagonizes the PI3K/Akt pathway (Parsa et al., 2006; Rajaram et al., 2006). In addition, a constitutive activation of Akt, or deletion of SHIP, promotes phagolysosomal fusion and limits bacterial burden in the host cytosol (Rajaram et al., 2009). Thus, signalling through the PI3K/Akt pathway by F. tularensis is required for evasion of lysosomal fusion and escape into the cytosol (Cremer et al., 2009b). It has been shown that Akt activation in macrophages occurs early during infection by F. tularensis ssp. novicida and decreases after 30 min, a pattern that is similar to the activation of Erk by F. tularensis (Rajaram et al., 2006). Activation of the Erk pathway is a critical event for immune cell activation, leading to transcriptional regulation of cytokine genes, translational regulation and other effector functions (Altman and Deckert, 1999; Ancrile et al., 2008).

Activation of Erk by F. tularensis may be dependent on the GTPase-activating protein (GAP) protein Ras, which triggers activation of the Raf-1/MEK1/ERK kinase cascade (Boguski and McCormick, 1993; Kawakami et al., 2003; Byun et al., 2009). Ras is at the point of convergence for many signalling pathways, and this activity is modulated by the guanine nucleotide binding (Plyte et al., 2000). Ras is activated transiently in response to a diverse array of extracellular signals such as growth factors, cytokines, hormones and neurotransmitters that stimulate cell surface receptors such as receptor tyrosine kinases (RTKs), non-receptor tyrosine kinase-associated receptors and G protein-coupled receptors (Satoh and Kaziro, 1992; Satoh et al., 1992). The best-characterized Ras-mediated signal transduction pathway involves the activation of the epidermal growth factor receptor (Egan et al., 1993; Warner et al., 1993). Upon ligand binding, the receptor undergoes autophosphorylation, which creates binding sites of the Shc and/or Grb2 adaptor proteins (Lowenstein et al., 1992; Rotin et al., 1992). GrB2 is stably associated with the son of sevenless homologue 2 (SOS2), which is the Ras guanine nucleotide exchange factors (GEF) (Campbell et al., 1998). The recruitment of the SOS2/GrB2 complex to Ras in the plasma membrane (Plyte et al., 2000) results in a temporal activation of Ras (Feig, 1994; Feig and Schaffhausen, 1994; Campbell et al., 1998). The recruitment of SOS2/GrB2 to Ras can occur directly or indirectly through the Shc adaptor protein (Lowenstein et al., 1992; Rotin et al., 1992). Shc, GrB2 and SOS2 provide the link between various activated cell surface receptors and Ras (Lowenstein et al., 1992; Rotin et al., 1992, Feig, 1994; Feig and Schaffhausen, 1994).

The PKC-dependent Syk signalling pathway also triggers the Ras/Erk pathway (Kawakami et al., 2003; Parsa et al., 2008). Upon receptor stimulation, the serine/threonine kinases PKCα and PKCβ1 recruit the SOS2/GrB2 complex to the vicinity of Ras, which facilitates the exchange of GTP for GDP on Ras (Kawakami et al., 2003). The active Ras-GTP stimulates four downstream effectors including Raf protein kinase, PI3K and the phospholipase-C (PLC) (Jasinski et al., 2008a). Each of these effectors regulates essential pathways of cell-cycle progression, survival and anti-apoptosis (Boguski and McCormick, 1993, Plyte et al., 2000; Kawakami et al., 2003; Honma et al., 2006; Jasinski et al., 2008a).

It is not known whether F. tularensis signals Ras activation within infected cells. In this article we show that F. tularensis ssp. novicida activates Ras in human cells during early stages of infection and that this activation depends on bacterial entry and on the FPI-encoded IglC protein. Both PKCα and βI isoforms are recruited to the SOS2/GrB2 complex resulting in the activation of Ras. Silencing of SOS2, GrB2 or PKCα and βI abrogates Ras activation by F. tularensis but has no effect on evasion of lysosomal fusion and bacterial escape into the cytosol and results in non-permissiveness of the cytosol for bacterial proliferation. The SOS2, GrB2 or PKCα and βI are essential for prevention of early activation of caspase-3 in F. tularensis-infected cells.

Results

F. tularensis activates Ras upon entry to the host cell

It has never been examined whether F. tularensis activates Ras during infection. Therefore, we determined whether F. tularensis triggers activation of Ras in HEK293T cells and human monocytes-derived macrophages (hMDMs). The cells were infected with F. tularensis ssp. novicida strain U112 for the indicated time points, and the iglC mutant which is defective in evasion of lysosomal fusion, phagosomal escape, was used as a negative control (Fig. 1). The post-nuclear supernatant (PNS) from infected cells was incubated with the Raf1 Ras-binding domain (RBD) agarose beads and the bound Ras proteins were analysed by Western blot using anti-Ras antibody that only detects active GTP-Ras. GTPγS was used as a positive control and the untreated cells were used as a second negative control. The data showed that by 5–15 min post infection of HEK293T cells and hMDMs, wild-type (WT) F. tularensis ssp. novicida triggered rapid Ras activation while the iglC mutant and the formalin-killed (FK) WT strain failed to activate Ras, similar to non-infected cells (Fig. 1B and D).

Figure 1.

Temporal Ras activation in F. tularensis-infected cells.
A. For Ras activation assay, the PNS from uninfected HEK293T cells or F. tularensis-infected cells and formalin-killed (FK) bacteria-infected cells were incubated with the Raf1 RBD (Ras-binding domain) agarose beads and the bound Ras proteins were analysed by Western blot using the Ras antibody using GTPγS as a positive control. Equal loading was verified by anti-actin antibody.
B. Kinetics of Ras activation during infection by F. tularensis. PNS from uninfected HEK293T cells or F. tularensis and iglC mutant-infected cells for 15 min, 2 h and 6 h were incubated with the Raf1 RBD (Ras-binding domain) agarose beads, processed and probed as described in (A).
C. Role of bacterial entry in Ras activation. HEK293T cells were pre-treated with 1 µg ml−1 Cytochalasin-D (CD) for 45 min prior to GTPγS treatment or infection for 15 min. The PNS from uninfected cells, and CD- or DMSO-treated cells were incubated with the Raf1 RBD (Ras-binding domain) agarose beads, processed and probed as described in (A).
D. Kinetics of Ras activation in hMDMs: PNS from uninfected hMDMs or F. tularensis-infected cells and iglC mutant-infected cells as well as CD-treated cells infected with the WT strain were incubated with the Raf1 RBD (Ras-binding domain) agarose beads and the bound Ras proteins were analysed by Western blot using the Ras antibody, using GTPγS as a positive control. Equal loading was verified by anti-actin antibody. This experiment was performed three times and the results are representative of one experiment.

To study the kinetics of Ras activation during infection by F. tularensis ssp. novicida, we examined Ras activation at different time points of infection (Fig. 1B and D). The data showed that Ras activation was temporal and occurred within 5–15 min, but it disappeared by 2–6 h after infection (Fig. 1B and D). Importantly, the iglC mutant, and replication, did not trigger activation of Ras (Fig. 1B and D). Taken together, the data show that intracellular F. tularensis activates Ras temporally within 5–15 min of infection and the FPI-encoded IglC protein is essential for Ras activation.

It is possible that attached extracellular F. tularensis triggered Ras activation through a receptor-mediated binding and signalling (Keates et al., 2001). However, it is also possible that Ras activation is triggered by the intracellular bacteria. To determine whether bacterial entry was essential to trigger Ras activation, the HEK293T cells and hMDMs were pre-treated with Cytochalasin-D (CD) prior to infection to block bacterial entry to the host cell (Goddette and Frieden, 1986; Gavrilin et al., 2006). The data clearly showed that Ras was not activated by the attached extracellular bacteria in both cells (Fig. 1C and D), indicating that bacterial entry to the host cell is essential for triggering temporal activation of Ras. Taken together, we showed that Ras activation occurs in both hMDMs and HEK293T cells infected with the WT F. tularensis ssp. novicida at 5–15 min post infection and that bacterial entry is required for the Ras activation in an IglC-dependent manner. This is the first demonstration of intracellular triggering of Ras activation by an intracellular bacterial pathogen.

For the rest of this study we used HEK293T cells as a model system for infection due to the ease and efficiency of RNAi-mediated gene silencing, since, clearly, Ras was rapidly triggered by intracellular bacteria and IglC was required for this activation in both cells. In addition, we verified that evasion of lysosomal fusion by F. tularensis, bacterial escape into the cytosol, and intracellular replication within HEK293T cells is indistinguishable from hMDMs (data not shown).

Mechanism of Ras activation by F. tularensis

PKC has been described as a major activator of the Ras pathway (Robin et al., 2004). This activation can occur through phosphorylation of the PKC isoforms by Syk and their recruitment to the SOS2/GrB2 complex. To decipher whether the mechanism of the Ras activation pathway triggered by F. tularensis is PKC-dependent, we examined whether F. tularensis triggered activation of PKC and its recruitment to the SOS2/GrB2 complex. The HEK293T cells were infected with F. tularensis ssp. novicida for 15 min and Formalin-killed bacteria (FK) was used as a negative control. The PNS from both uninfected and infected cells were immunoprecipitated with the anti-SOS2, anti-GrB2, anti-PKCα or anti-PKC-β1 antibodies followed by immunoblotting. The constitutive interaction between SOS2 and GrB2 proteins was considered as a positive control. As expected, GrB2 co-immunoprecipitated with SOS2 in both live WT- and FK-infected cells (Fig. 2, lanes C and D). In control experiments, PNS from uninfected cells did not show any interaction between the PKC isoforms and the SOS2/GrB2 complex (Fig. 2, lane F). The PKCα and β1 isoforms interacted with the SOS2/GrB2 complex within 15 min of infection by live F. tularensis (Fig. 2, lane A1, lane B1). However, in FK bacteria-infected cells, the PKCα and β1 isoforms did not interact with the SOS2/GrB2 complex, indicating that only viable bacteria specifically triggered association of both PKC isoforms with the SOS2/GrB2 bi-molecular complex (Fig. 2, lane A2, lane B2). When the blots were re-probed with the anti-Shc antibody the data showed that Shc did not bind to the SOS2/GrB2 complex in live WT- and FK bacteria-infected cells (data not shown). Taken together, intracellular F. tularensis triggers temporal Ras activation within 15 min of infection via a Shc-independent mechanism that involves interaction of both PKCα and β1 isoforms with the SOS2/GrB2 bi-molecular complex.

Figure 2.

Recruitment of PKCα and βI isoforms to the SOS2/GrB2 complex during infection by F. tularensis. The PNS from WT strain or FK bacteria-infected HEK293T cells for 15 min were incubated with anti-PKC-α antibody (lanes A1 and A2), anti-PKC-βI antibody (lanes B1 and B2), anti-SOS2 antibody (lanes C1 and C2) or anti-GrB2 antibody (lanes D1 and D2). The PNS from uninfected cells (lane F) were used as a control. No antibody was added to the samples in lanes E1 and E2, as additional controls. After overnight incubation at 4°C, G protein-coupled beads were added to all samples, except the one in lane F. After serial washes, pulled-down proteins were analysed by Western blot with the respective antibodies as shown. Equal loading was verified by anti-actin antibody. This experiment was performed three times and the results are representative of one experiment.

Role of the SOS2/GrB2 complex and PKCα/β1 isoforms in F. tularensis-induced Ras activation

After stimulation of a receptor, PKCα/βI recruit the SOS2/GrB2 complex to the vicinity of Ras resulting in exchange of GDP to GTP, which activates Ras. To determine if both SOS2 and GrB2 were essential for Ras activation upon infection by F. tularensis, both factors were silenced separately by specific RNAi. Specificity of gene silencing was confirmed by Western blot (Fig. 3A). We utilized the HEK293T cells that exhibited efficient silencing. Cell viability was monitored by trypan blue staining, which showed no effect of silencing of SOS2 or GrB2 on cell viability, similar to the untreated cells or the RNAi control-treated cells. To determine whether Ras was activated in the SOS2 and GrB2-silenced cells, the silenced and control cells were infected by F. tularensis for 15 min. The PNS were incubated with the Raf1 RBD agarose beads and the bound Ras proteins were analysed by Western blot using the anti-Ras antibody that only detects active GTP-Ras. The GTPγS treatment was performed in the untreated cells and was used as a positive control for the Ras activation assay. Active Ras was detected in the F. tularensis-infected but not uninfected cells (Fig. 3B). No difference in the levels of Ras activation was observed between untreated and RNAi control-treated cells (data not shown). By 15 min post infection, Ras was not activated by F. tularensis in the SOS2 or GrB2-silenced cells compared with the non-silenced cells (Fig. 3B). We conclude that F. tularensis triggers Ras activation through the SOS2/GrB2 bi-molecular complex and that both SOS2 and GrB2 are essential for the temporal activation of Ras by intracellular F. tularensis.

Figure 3.

Role of PKCα/βI/SOS2/GrB2 complex in Ras activation during infection by F. tularensis.
A. Western blot analysis of SOS2, GrB2, PKCα and βI gene silencing in HEK293T cells. Untreated or RNAi-treated cells were lysed at 48 h after treatment by RNAi. The PNS were analysed by SDS-PAGE and immunoblotting with specific antibodies as shown. The blots were then stripped and re-probed with the anti-actin antibody for a loading control.
B. Role of SOS2/GrB2 complex and PKCα and βI isoforms in the Ras activation during infection by F. tularensis. Cells were either left untreated or treated with RNAi to silence SOS2, GrB2, PKCα and PKCβI using specific RNAi. Gene silencing was verified by Western blot. Both untreated and RNAi-treated HEK293T cells were infected with the WT strain for 15 min. The PNS were then processed for the Ras activation assay and bound proteins were analysed by Western blot using anti-Ras antibody (see legend 1 for details). GTPγS was used as positive control and was performed in the untreated cells. Equal loading was verified by anti-actin antibody. This experiment was performed three times and the results are representative of one experiment.

To decipher the role of PKCα/βI in the activation of Ras by F. tularensis, we silenced the PKC isoforms in HEK293T cells using specific RNAi. Cell viability was monitored by trypan blue staining, which showed no effect of silencing of either PKCα or βI on cell viability, similar to untreated cells or RNAi control-treated cells. Specificity of gene silencing was confirmed by Western blot (Fig. 3A). The PKCα- or βI-silenced cells were infected by F. tularensis for 15 min and PNS were prepared as described above. The data showed that silencing of both PKC isoforms abolished the activation of Ras by F. tularensis compared with the mock-treated or RNAi control-treated cells (Fig. 3B). We conclude that in addition to the SOS2/GrB2 bi-molecular complex, both PKCα and βI isoforms are essential for the temporal activation of Ras by intracellular F. tularensis.

SOS2, GrB2, PKCα and PKCβI are required for intracellular proliferation of F. tularensis

Since Ras was activated during early stages of infection by F. tularensis in an IglC-dependent mechanism and IglC is essential for intracellular proliferation, we examined whether the F. tularensis-induced Ras activation pathway is essential for bacterial replication. We examined the role of the SOS2/GrB2 complex and the PKC isoforms in intracellular replication of F. tularensis. The HEK293T cells were either left untreated or treated for 48 h with the RNAi-negative control or gene-specific RNAi. Moreover, to exclude non-specific effect of silencing, we silenced calpain-9 (Cap9), as a control molecule, and showed that Cap9 silencing does not affect bacterial replication (data not shown). Gene silencing was verified by Western blot (Fig. 3A). Untreated HEK293T cells and successfully silenced cells for each of the factors were infected by F. tularensis. The infection was carried our for 1 h using multiplicity of infection (moi) of 10 followed by 1 h of gentamicin treatment and further incubation for additional 6 h and 22 h for a total of 8 h and 24 h respectively. At each time point, cells were fixed and labelled with anti-F. tularensis antibodies and analysed by microscopy-based single cell analysis (Fig. 4). At 2 h post infection, there was no difference in the efficiency or frequency of bacterial internalization between the non-treated or the RNAi-treated cells (one bacterium per cell) indicating that bacterial uptake was not affected upon silencing of those factors, resulting in inhibition of Ras activation (Fig. 4A). At 8 h post infection, ∼80% of the untreated cells or cells transfected by the RNAi-negative control harboured 15–20 bacteria per cell (Fig. 4B). In contrast, 80–85% of the SOS2-silenced or GrB2-silenced cells did not show any bacterial proliferation at 8 h and 24 h post infection (Fig. 4B–D). Results obtained from the RNAi-negative control-treated cells were comparable to the untreated cells confirming the specificity of silencing (Fig. 4B). These data show that the SOS2 and GrB2 factors are not required for bacterial entry but are essential for intracellular replication of F. tularensis throughout the intracellular infection.

Figure 4.

Replication of F. tularensis within RNAi-silenced HEK293T cells.
A–C. Immunofluorescence images of HEK293T cells at 2 h after infection by F. tularensis. HEK293T cells were either left untreated (Un) or treated with the RNAi-negative control or the gene-specific RNAi for 48 h before infection, then infected with the WT strain U112 or formalin-killed (FK) bacteria at moi of 10 for 1 h, followed by gentamicin treatment for 1 h to kill extracellular bacteria. The cells were further incubated for a total of 8 h and 24 h. At all time points, cells were processed for confocal microscopy as described in Experimental procedures.
D. Quantitative analysis of the % of the infected cells harbouring replicating F. tularensis ssp. novicida in both untreated (UN) or RNAi-treated cells at 8 h post infection. The normal levels of replication within untreated HEK293T cells was > 15 bacteria per cell, and ∼25% of the cells are infected under our experimental conditions. At least 100 cells were analysed from two different coverslips. Error bars represent standard deviation. The P-value < 0.01 is considered significant and was represented by the asterisks. This experiment was performed three times and the results are representative of one experiment.

Silencing of either PKCα or PKCβI isoforms resulted in a dramatic loss of temporal activation of Ras by F. tularensis indicating an essential role of those isoforms in Ras activation (Fig. 3). To determine the role of PKCα and PKCβI isoforms in intracellular bacterial proliferation, both isoforms were silenced. The data showed that silencing of either isoforms did not affect the efficiency or frequency of bacterial internalization, and most of the infected cells harboured one bacterium at 2 h post infection. By 8–24 h post infection, silencing of either PKCα or PKCβI isoforms inhibited bacterial replication (Fig. 4B). The data showed that by 8 h or 24 h post infection, 70–80% of the PKCα- and PKCβI-silenced infected cells contained only one bacterium per cell (Fig. 4), indicating that PKCα and PKCβI are essential for permissiveness of the host cells for intracellular proliferation of F. tularensis. We conclude that components of the Ras activation pathway including the bi-molecular complex SOS2/GrB2 and both PKCα and PKCβI isoforms are dispensable for bacterial entry but are indispensable for intracellular proliferation of F. tularensis.

The role of SOS2, GrB2, PKCα and PKCβI in intracellular trafficking of F. tularensis

The FCP transiently matures to an acidified late endosome-like phagosome followed by bacterial escape into the cytosol by 30–60 min post infection (reviewed in Santic et al., 2010). However, the iglC mutant-containing phagosome fuses to the lysosomes and the mutant is defective in phagosomal escape into the cytosol (Santic et al., 2005a; 2007). The host factors involved in evasion of endocytic fusion and phagosomal escape of F. tularensis are not known. We postulated that non-permissiveness of the SOS2-, GrB2-, PKCα- and βI-silenced cells to bacterial replication might be due to alteration of biogenesis of the FCP. To test this hypothesis, we examined intracellular trafficking within untreated or SOS2, GrB2, PKCα and βI RNAi-treated cells and used the iglC mutant as a positive control.

Since the FCP transiently colocalizes with the LAMP2 late-endosomal marker for 15–30 min followed by a gradual loss of this colocalization by 1–2 h, we examined colocalization of the phagosomes with LAMP-2 and the lysosomal marker Cathepsin-D at 30 min and 2 h after infection (Fig. 5 and Fig. S1). At 30 min after infection, 75–80% of the iglC mutant-containing phagosomes colocalized with LAMP2 and Cathepsin-D respectively (Fig. 5B and Fig. S1A) (Santic et al., 2007). As expected, ∼45–48% of the WT strain containing phagosomes in untreated cells colocalized with LAMP2 at 30 min post infection (Fig. 5B). No significant difference (Student's t-test, > 0.1) in LAMP2 and Cathepsin-D colocalization was observed between the WT strain-containing phagosomes in untreated or RNAi-treated cells at 30 min post infection (Fig. 5B and Fig. S1).

Figure 5.

Evasion of lysosomal fusion by F. tularensis is independent of the SOS2/GrB2 complex and the PKCα and βI isoforms.
A. Representative confocal microscopy images of the late endosomal/lysosomal marker LAMP-2 colocalization with the phagosomes containing F. tularensis ssp. novicida. Untreated (UN) HEK293T cells or treated with the RNAi-negative control or the gene-specific RNAi for 48 h were infected with the WT strain U112 or formalin-killed (FK) bacteria using moi of 10 for 1 h followed by 1 h treatment with gentamicin to kill extracellular bacteria. The iglC mutant was used as a positive control. The white arrow points at colocalization of the iglC mutant-containing vacuole with the LAMP2 marker.
B and C. Quantitative analysis of colocalization of the WT strain-containing phagosomes with LAMP2 at 30 min (B) and 2 h (C) after infection. At least 100 cells were analysed from two coverslips. A P-value < 0.01 is considered significant and is represented by the asterisks. This experiment was performed three times and the results are representative of one experiment.

At 2 h after infection, 65% of the WT strain-containing phagosomes did not colocalize with LAMP-2 or Cathepsin-D (Fig. 5C and Fig. S1), while ∼90% of the iglC mutant-containing phagosomes showed persistent colocalization with both markers, consistent with previous observation (Fig. 5C and Fig. S1) (Santic et al., 2005a). Silencing of SOS2, GrB2 or the two PKC isoforms did not have a significant effect on colocalization of the FCP with LAMP2 or Cathepsin-D, when compared with untreated cells or RNAi control-treated cells (Student's t-test, > 0.4) (Fig. 5 and Fig. S1). Taken together, neither the SOS2/GrB2 complex nor the PKCα/βI is essential for evasion of lysosomal fusion by F. tularensis. Therefore, activation of the Ras signalling by F. tularensis through recruitment of PKCα and βI to the SOS2/GrB2 complex is independent of modulation of phagosomal biogenesis and evasion of lysosomal fusion.

The SOS2/GrB2 complex and PKCα/βI isoforms are dispensable for escape of F. tularensis into the cytosol

Loss of the late endosomal marker LAMP-2 from the FCP by 30–60 min coincides with phagosomal escape of F. tularensis into the cytosol (Checroun et al., 2006; Santic et al., 2007; 2008; 2010; Wehrly et al., 2009). To study the role of the SOS2/GrB2 complex and PKCα/βI isoforms in bacterial escape, we utilized the fluorescence microscopy-based phagosomal integrity assay (Checroun et al., 2006; Santic et al., 2008). This assay allows differential labelling of bacteria that are cytosolic or within a compromised phagosomes versus those enclosed within intact phagosomes. After preferential permeabilization of the plasma membrane, an antibacterial antibody was loaded into the host cell cytosol to allow binding to the cytosolic bacteria, while bacteria within intact FCPs do not bind the antibody.

To test whether the Ras activation cascade is involved in bacterial escape to the cytosol, we silenced the major components of the Ras activation pathway including SOS2, GrB2, PKCα/βI using gene-specific RNAi, and examined the effect on phagosomal escape. Untreated HEK293T cells and successfully silenced cells for each of the factors were infected by F. tularensis for 1 h using moi of 10, followed by 1 h of gentamicin treatment, and then processed for the fluorescence-based phagosomal integrity assay (Checroun et al., 2006; Santic et al., 2008). The iglC mutant was used as a positive control for bacteria within intact FCPs (Santic et al., 2005b). The data showed that in the untreated HEK293T cells that were infected by the WT strain, > 80% of the WT bacteria were cytosolic or within disrupted phagosomes by 2 h post infection, as expected (Fig. 6). Similarly, > 80% of the intracellular bacteria were cytosolic in the SOS2-, GrB2-, PKCα- and βI-silenced cells. Consistent with previous findings, ∼80% of the iglC mutant control was within intact phagosomes (Fig. 6). These data show that the SOS2, GrB2, PKCα and PKCβII components of the Ras activation pathway play no detectable role in bacterial escape into the host cytosol. This indicates that escape of F. tularensis into the cytosol is independent of F. tularensis-triggered Ras activation.

Figure 6.

Escape of F. tularensis into the cytosol is independent of the SOS2/GrB2 complex and the PKCα and βI isoforms.
A. Representative confocal microscopy images of the WT F. tularensis within untreated (UN) or RNAi-treated cells to determine phagosomal escape using the fluorescence-based phagosomal integrity assay. This was determined by the ability of the intracellular bacteria to bind anti-F. tularensis antibody loaded into the host cell cytosol after preferential permeabilization of the plasma membrane. This is in contrast to bacteria found within intact vacuoles that are impermeable to the antibody. The iglC mutant was used as a negative control for bacteria trapped within intact phagosomes.
B. Quantification of the % of disrupted phagosomes (FCP) from untreated or RNAi-treated cells. At least 100 infected cells from two different coverslips. This experiment was performed twice and the results are representative of one experiment.

To determine whether silencing of SOS2, GrB2, PKCα and βI caused a re-entry of the bacteria into the endocytic pathway later in the infection (Checroun et al., 2006), LAMP-2 colocalization experiments were conducted at 12 and 24 h post infection. In the untreated cells and the RNAi-treated cells, F. tularensis did not re-enter LAMP-2-positive vacuoles at 12 h and 24 h post infection (data not shown). Taken together, we conclude that SOS2, GrB2, PKCα and PKCβI are dispensable for evasion of lysosomal fusion and escape of F. tularensis into the cytosol, but they are indispensable for bacterial proliferation in the cytosol.

Signalling of Ras through the SOS2/GrB2/PKCα/PKCβI quaternary complex protect F. tularensis-infected cells from early activation of caspase-3

It has been shown that intracellular proliferation of F. tularensis results in the activation of caspase-3-mediated apoptosis during late stages of infection in tissue culture and mice organs in vivo (Rajaram et al., 2009; Wickstrum et al., 2009), but F. tularensis-infected macrophages are resistant to apoptosis during early stages of infection (Santic et al., 2009). Ras is involved in promoting cell survival through blocking activation of caspase-3 (Jasinski et al., 2008b). Therefore, we postulated that the Ras activation pathway by intracellular F. tularensis contributed to survival of the infected cells by blocking early activation of the pro-apoptotic caspase-3.

To test our hypothesis, we determined the role of SOS2, GrB2, PKCα/βI in blocking early activation of caspase-3 in F. tularensis-infected cells. We silenced SOS2, GrB2, PKCα/βI in the HEK293T cells using gene-specific RNAi. Untreated cells and successfully silenced cells for each of the factors were infected by F. tularensis. The infection was carried out for 1 h using moi of 10, followed by 1 h of gentamicin treatment, and incubated for additional 6 and 22 h for a total of 8 and 24 h. The cells were labelled with anti-active caspase-3 antibody and analysed by confocal microscopy. Staurosporine (STS, 1 µM), which triggers caspase-3-mediated apoptotic death in the HEK293T cells (Nakamura et al., 2007), was used as a positive control (Fig. 7B). DMSO-treated cells either uninfected or infected by F. tularensis were used as a negative control. Caspase-3 activation was not detected in mock-treated cells, but an increase of 10- to 12-fold in the number of cells exhibiting caspase-3 activation was observed among the uninfected cells after STS treatment compared with the DMSO-treated cells (Fig. 7B and C). The data showed that infection of cells silenced for SOS2, GrB2, PKCα and βI with F. tularensis resulted in early caspase-3 activation by 8 h post infection, where four- to eightfold increase in the number of cells exhibiting caspase-3 activation was detected in the RNAi-treated cells compared with the untreated or RNAi control-treated cells (Fig. 7C) (Student's t-test, < 0.01). Importantly, silencing of each of the four factors did not trigger early caspase-3 activation in uninfected cells (Fig. 7). A 12- to 14-fold increase in the number of cells exhibiting capsase-3 activation was also observed at 24 h post infection in the cells where expression of SOS2, GrB2, PKCα and βI was knocked down compared with untreated cells or RNAi control-treated cells (Fig. 7A and C). Therefore, early activation of caspase-3 is specifically triggered by 8 h post infection by the WT strain of F. tularensis in the cells silenced for SOS2, GrB2 or PKCα and βI (Fig. 7). We conclude that SOS2, GrB2, PKCα and βI are crucial for protecting F. tularensis-infected cells from early activation of caspase-3 within F. tularensis-infected cells.

Figure 7.

Early activation of caspase-3 in the SOS2-, GrB2-, PKCα- and βI-silenced cells upon infection by F. tularensis.
A. Representative confocal microscopy images of the caspase-3 activation after infection with the F. tularensis WT strain. HEK293T cells were either left untreated of treated with the SOS2, GrB2, PKCα and βI respective RNAi. After verifying specific gene silencing, cells were infected with the WT strain U112 using moi of 10 for 1 h followed by 1 h treatment with gentamicin to kill extracellular bacteria. Cells were further incubated for a total of 24 h. Caspase-3 activation was detected by anti-active caspase-3 antibody at 24 h post-infection.
B. Controls for caspase-3 activation: Staurosporine (STS)-treated cells were used as a positive control and DMSO-treated cells were used as a negative control. Caspase-3 activation was detected by anti-active caspase-3 antibody at 24 h post infection, compared with Staurosporine (STS)-treated cells were used as a positive control and DMSO-treated cells used as a negative control.
C. Quantitative analysis of caspase-3 activation at 8 and 24 h post infection. At least 100 cells were analysed from two different coverslips. The levels of caspase-3 activation are presented as ratio of number of cells with active caspase-3 in RNAi-treated cells that were infected by F. tularensis, versus uninfected cells treated with the same RNAi. This experiment was performed twice and the results are representative of one experiment.

SOS2, GrB2, PKCα and βI are not required for activation of Akt or Erk by F. tularensis

It has been shown that F. tularensis induces Akt and Erk phosphorylation that peaks during the first few minutes of infection and Akt phosphorylation occurs downstream of the PI3K pathway (Rajaram et al., 2006). It is also possible the Erk phosphorylation occurs downstream of Ras activation by F. tularensis. Moreover, class I PI3K contain RBDs and can also be activated through association with active Ras. To determine if the Ras activation pathway interacted with the PI3K pathway or Erk upon infection by F. tularensis, we analysed Akt and Erk phosphorylation upon silencing the major players of the Ras activation pathway including SOS2, GrB2 and both PKC isoforms. Untreated and RNAi-treated cells were infected with F. tularensis and verification of the specific gene silencing was accomplished by Western blot. The cells were lysed 5–30 min after infection and protein lysates were analysed by Western blotting with the anti-pAkt or anti-pErk antibody (Fig. 8 and data not shown). To ensure equal protein loading, the membranes were re-probed with the anti-Akt and Anti-Erk antibody. The results showed that infection of untreated cells by F. tularensis induced robust phosphorylation of Akt and Erk (Fig. 8 and data not shown). Silencing of SOS2, GrB2, PKCα and βI did not have any detectable effect on the phosphorylation of Akt or Erk by 5–30 min after infection (Fig. 8 and data not shown). This indicates the activation of the PI3K/Akt pathway and Erk activation are independent of Ras activation by F. tularensis. Independence of F. tularensis-mediated Ras activation from F. tularensis-mediated activation of the PI3k/Akt pathway is consistent with the role of the PI3K/Akt pathway in evasion of lysosomal fusion and phagosomal escape of F. tularensis (Rajaram et al., 2009), while Ras signalling by F. tularensis through SOS2, GrB2, PKCα and βI is independent of evasion of lysosomal fusion and phagosomal escape.

Figure 8.

Triggering the PI3K and the Ras activation pathways by F. tularensis is independent. Untreated or RNAi-treated HEK293T cells were infected with the WT strain F. tularensis ssp. novicida for 5 min and compared with uninfected cells. Phosphorylation of Akt was detected using anti-pAkt antibody. The same membrane was re-probed with anti-Akt antibody to verify equal loading.

Discussion

Early phases of the interaction of F. tularensis with the host and modulating the signalling cascades are thought to be key events in subsequent intracellular proliferation (Butchar et al., 2008; Santic et al., 2010). However, the signalling steps that are triggered during this crucial stage of initiating the infection are largely unknown. Many studies have demonstrated the role of the PI3K/Akt/SHIP/MIR-155 in signalling during infection by F. tularensis (Parsa et al., 2006; 2008; Rajaram et al., 2006; 2009). The receptor-mediated stimulation of Ras and subsequent activation of its downstream effector molecules is critical in the biological response to numerous signals (Meadows et al., 2001), but Ras activation has never been shown to be triggered intracellularly by any intracellular bacterial pathogen. Whether Ras is activated by intracellular F. tularensis has not been reported yet. In this article we describe for the first time the mechanism of Ras activation by intracellular F. tularensis ssp. novicida and its indispensable role in intracellular bacterial proliferation, which has never been shown for any intracellular pathogen.

We show a SOS2/GrB2/PKCα- and PKCβI-dependent signalling pathway is temporally triggered within 15 min of infection by F. tularensis, leading to the activation of Ras in F. tularensis-infected cells. Fifteen minutes after infection, Ras activation peaks then declines, indicating that the temporal Ras activation occurs rapidly during early stages of F. tularensis–host cell interaction. Activation of Ras within gastric cells has been shown to be triggered by Helicobacter pylori, which binds the EGF receptor leading to the activation of Ras (Keates et al., 2001). Moreover, Internalin B (InlB), a surface protein of Listeria monocytogenes binds the c-Met receptor leading to Ras activation, which is essential for uptake of L. monocytogenes, and for activation of the PI3K/Akt signalling pathway (Mansell et al., 2001). In contrast, we show that entry of F. tularensis into the host cell is essential to trigger Ras activation and that bacterial attachment to the host cell membrane is not sufficient to trigger Ras activation. Moreover, Ras signalling by F. tularensis is independent of the PI3k/Akt signalling cascade, which is triggered by attached extracellular F. tularensis (Jasinski et al., 2008b). Importantly, entry of F. tularensis ssp. novicida into the host cell is independent of the SOS2, GrB2, or PKCα and βI isoforms factors and Ras activation. We conclude that the temporal activation of Ras by F. tularensis ssp. novicida is not surface receptor-mediated; but is triggered by intracellular bacteria and is essential for intracellular bacterial proliferation, which is novel.

A F. tularensis-secreted effector is likely responsible of the activation of Ras. This may be mediated by interacting with some components of the Ras activation pathway such as SOS2 or GrB2. The FPI encodes a type T6SS-like apparatus that plays an essential role in evasion of lysosomal fusion and bacterial escape into the cytosol (Broms et al., 2009; Santic et al., 2010). The IglC protein is encoded by the FPI and in addition to its role in evasion of lysosomal fusion and escape into the cytosol, we show that the IglC protein plays an essential role in triggering Ras activation. Bacterial effectors of various pathogens can interfere with the Ras signalling pathways through distinct mechanisms (Chen et al., 1999; Mimuro et al., 2002). For example, the H. pylori CagA secreted effector interacts with GrB2, which results in the activation of the Ras signalling cascade (Mimuro et al., 2002). Similarly, Salmonella typhimurium activates JNK, a downstream effector of Ras signalling, which is mediated by the effector protein SopE (Chen et al., 1999). Our data clearly show that activation of Ras by F. tularensis is IglC-dependent. However, it is still to be determined whether F. tularensis translocates IglC that interacts directly or indirectly with a component of the Ras activation pathway. This mechanism of Ras activation occurs at the early stages of infection of both primary macrophages and the HEK293T cell line. Activation of Ras does not occur if bacterial entry to the cell is blocked, and occurs earlier (∼5 min) than bacterial escape to the cytosol (30–60 min) (Santic et al., 2010). These observations exclude the possibility that bacterial escape into the cytosol is required is for Ras activation.

The small GTP-binding proteins of the Ras superfamily are master controllers of cell physiology, and their functions range from cell cycle progression, cell morphology, actin rearrangement, endocytosis to intracellular trafficking of vesicles and organelles (Jasinski et al., 2008a,b). Moreover, the Ras activation pathway is involved in promoting survival and suppressing cell death (Jasinski et al., 2008a,b). Cell morphology and actin rearrangement are not affected upon knocking down the Ras activation pathway, which excludes both processes from being involved in Ras signalling by F. tularensis. We have tested two possibilities to unravel the role of Ras activation by F. tularensis in bacterial proliferation within the cytosol. First, our data confirm that the SOS2/GrB2/PKCα/PKCβI quaternary complex does not play roles in evasion of lysosomal fusion or in escape of F. tularensis into the host cytosol. Second, since Ras is essential for cell survival and anti-apoptosis (Jasinski et al., 2008a), we show that an early activation of caspase-3 is exhibited in infected cells in which expression of SOS2, GrB2, PKCα or PKCβI has been knocked down. This indicates that temporal and early triggering of the Ras activation pathway by F. tularensis plays a role in promoting cell survival during early stages of infection through prevention of caspase-3 activation, which is the executioner of apoptosis (Lai et al., 2001). We conclude that the temporal early activation of Ras by F. tularensis ssp. novicida is essential for intracellular proliferation of F. tularensis ssp. novicida and this is mediated, at least in part, by inhibition of early activation of caspase-3 in the infected cell. The time difference between early Ras activation and its effect in protecting cells from early caspase-3 activation may be due to a time-dependent event that may be triggered upon Ras activation. This may involve activation of other cellular pathways required to block caspase-3 activation, such as triggering expression of anti-apoptotic genes (Santic et al., 2009).

The PI3K/Akt signalling pathway is rapidly activated upon infection by F. tularensis, leading to phosphorylation of Akt, which is downregulated by SHIP (Parsa et al., 2006; 2008; Butchar et al., 2007; 2008; Cremer et al., 2009b; 2010; Rajaram et al., 2009). Infection by L. monocytogenes triggers the PI3K/Akt signalling pathway via the surface protein InlB leading to Ras activation, which is required for bacterial entry (Mansell et al., 2001). However, based on four lines of evidence, our data exclude the role of Ras activation in triggering the PI3K/Akt pathway by F. tularensis and show that the role of the Ras activation pathway in bacterial replication is independent of the PI3K/Akt pathway. First, triggering the PI3k/Akt by F. tularensis is mediated by attached extracellular bacteria, while Ras signalling is not triggered by attached extracellular bacteria (Mansell et al., 2001). Second, constitutive expression of Akt or deletion of SHIP promotes phagolysosomal fusion and prevents bacterial escape into the cytosol (Mansell et al., 2001). In contrast, triggering the Ras signalling pathway by F. tularensis does not play any detectable role in evasion of lysosomal fusion or bacterial escape to the cytosol. Third, the role of SHIP in the response of infected cells to F. tularensis is independent of Erk and Ras (Rajaram et al., 2009). Fourth, silencing SOS2, GrB2, PKCα and PKCβI by RNAi blocks activation of Ras by F. tularensis and inhibits intracellular bacterial proliferation, but has no effect on phosphorylation of Akt or Erk. Therefore, triggering the PI3K/Akt signalling pathway or Erk is independent of Ras activation by F. tularensis.

In conclusion, our data clearly demonstrate that upon entry into the host cell, intracellular F. tularensis triggers recruitment of the PKCα and PKCβI isoforms to the SOS2/GrB2 complex resulting in activation of the Ras pathway (Fig. 9), which is a novel strategy of triggering Ras signalling by an intracellular pathogen. The SOS2/GrB2/PKCα/PKCβI quaternary complex is essential for activation of the Ras pathway by F. tularensis, which is essential for bacterial proliferation within the cytosol associated with blocking early activation of the pro-apoptotic caspase-3.

Figure 9.

A model for the Ras activation pathway by intracellular F. tularensis. Within few minutes after bacterial entry into the host cell and biogenesis of the phagosome, F. tularensis ssp. novicida triggers activation of Ras. PKCα and βI isoforms in the cytosol are recruited to the plasma membrane where they bind to the SOS2/GrB2 complex which in turn activates Ras to the GTP-bound form. Activation of Ras through SOS2/GrB2/PKCα-βI is essential for bacterial proliferation. The Ras activation pathway by F. tularensis ssp. novicida inhibits early activation of caspase-3 in the infected cells. The Ras activation pathway is independent of evasion of lysosomal fusion and bacterial escape to the cytosol. The major PI3K/Akt and the Ras activation signalling pathways are independently triggered by F. tularensis ssp. novicida.

Experimental procedures

Bacterial strains and cells

The F. tularensis ssp. novicida strain U112 and its isogenic mutant iglC have been described previously (Lauriano et al., 2003). The bacterial strain was grown for 2 days on tryptic soy agar (TSA) supplemented with 0.1% cysteine and 10 µg ml−1 kanamycin was supplemented for the mutant. Isolation and preparation of the hMDMs from peripheral blood of volunteers was carried out as previously described (Al-Khodor et al., 2008). The HEK293T cells were maintained as we described previously (Price et al., 2009).

Silencing of SOS2, GrB2 and PCK isoforms in HEK293T cells

One day before transfection, 3 ml of 5 × 105 cells per ml of HEK293T were seeded in each well of the six-well plates and 1 ml of the same cell density was seeded on glass coverslip in 24-well plates. In both cases, the wells were treated with 0.2 mg ml−1 poly-l lysine for 30 min at room temperature to increase cell attachment. The SOS2, GrB2, PKCα, βI isoforms RNAi was purchased from Santa Cruz (Santa Cruz, CA). A scrambled RNAi was used as our negative control for silencing. At the time of the transfection, the cells were washed once with the transfection media and processed as recommended by the manufacturers. After 6 h of incubation at 37°C in a CO2 incubator, the growth medium containing 2× FBS was added and cells were incubated for additional 18 h. The media were then aspirated and new media containing 1× FBS were added to the cells and incubated for additional 24 h.

Cells in the six-well plates were than washed in 1 × PBS and lysed using the mammalian protein extraction reagent M-Per (Pierce-Thermo Scientific, Rockford, IL) supplemented with one tablet of the complete mini protease inhibitors EDTA-free cocktail (Roche, Indianapolis, IN). The PNS were boiled using 1× reducing buffer (Thermo Scientific, Waltham, MA), separated by SDS-PAGE and transferred onto PVDF membranes (Millipore, Billerica, MA). Western blotting was performed using 1:200 dilution of rabbit polyclonal anti-PKC-α, anti-PKC-βI, anti-SOS2 and anti-GrB2 antisera (Santa Cruz, Santa Cruz, CA), rabbit polyclonal anti-Erk1/2 antiserum (Abacam, Cambridge, MA) that detects total Erk, anti-P42/44 that detected phosphorylated Erk (Cell Signaling, Danvers, MA). Polyclonal rabbit anti-actin antiserum was used at 1:100 dilution (Sigma, St Louis, MO).

Ras activation assay

One week before infection hMDMs from volunteers' blood were seeded in an ultra low attachment plate, then cells were detached after 4 days and 3 ml of 5 × 105 cells per well hMDMs were seeded in six-well plates. One day before infection, 3 ml of 5 × 105 HEK293T cells ml−1 were seeded in six-well platespre-treated with 0.2 mg ml−1 poly-l lysine for 30 min at room temperature in order to increase cell attachment.

Both untreated and RNAi-treated cells were infected with the F. tularensis ssp. novicida strain U112 or its iglC isogenic mutant for the indicated time points using moi of 10. When the infection was performed longer than 1 h, 50 µg ml−1 gentamicin was added to the cells for 1 h in order to kill extracellular bacteria. Where indicated, cells were pre-treated with 1 µg ml−1 Cytochalasin-D or 0.1% DMSO for 45 min at 37°C prior to infection. For Formalin-killed bacteria, the bacteria were killed by exposure to 3.7% formalin for 30 min at room temperature prior to infection. After the indicated times of infection, the medium was carefully removed and cells were rinsed once with ice-cold PBS. Both uninfected and infected cells, mock-treated cells or cells treated with RNAi were washed with ice-cold 1× PBS then lysed with 1× lysis/assay buffer (Ras activation assay from Cell Biolabs, San Diego, CA). To detect Ras activation, the agarose beads coated with Raf1 Ras-binding domain (RBD) were added to the PNS incubated at 4°C for 1 h with gentle mixing. Beads were then washed and after boiling the beads, the proteins were eluted and subjected to Western blot probed with the anti-Ras mouse monoclonal antibody (1:1000) (Cell Biolabs). The GTPγS was used as a positive control.

Co-immunoprecipitation

A total of 3 × 106 HEK293T cells in 3 ml were seeded in six-well plates and infected by the WT strain U112 or the formalin-killed (FK) F. tularensis for 15 min using moi of 10. Cells were then washed in 1 × PBS and lysed using the mammalian protein extraction reagent M-Per (Pierce-Thermo Scientific, Rockford, IL). Cell lysates were centrifuged for 10 min at 13 000 g and the supernatant was collected and treated with one tablet of the complete mini EDTA-free protease inhibitor cocktail (Roche, Indianapolis, IN). Approximately, 1 ml of the PNS (250 µg proteins) of both WT and FK-infected cells were incubated overnight at 4°C with 20 µl (equivalent to 4 µg) of rabbit anti-PKC-α, rabbit anti-PKC-βI, rabbit anti-SOS2 or rabbit anti-GrB2 antisera (Santa Cruz Biotechnology, Santa Cruz, CA). The PNS from uninfected cells incubated without antibodies were used as negative control. A total of 100 µl of immobilized G protein resin slurry (Pierce-Thermo Scientific, Rockford, IL) was added to the PNS and incubated with gentle end-to-end mixing for additional 2 h at room temperature. Five hundred microlitres of Tris buffer saline was added to the mixture and the beads were recovered and washed three times followed by centrifugation at 2500 g for 2–3 min. After the final wash, the complexes were processed for Western blot. The immunoprecipitated proteins were examined by immunoblotting with the rabbit polyclonal anti-PKC-α, anti-PKC-βI, anti-SOS2, anti-GrB2 and anti-Shc antisera (Santa Cruz) and re-probed with the mouse monoclonal anti-actin antibody.

Confocal laser scanning microscopy

After confirming specific gene silencing by Western blot, cell viability was determined by trypan blue exclusion. Untreated 5 × 105 HEK293T cells and successfully silenced cells seeded on coverslips in 24-well plates, cells were infected with F. tularensis ssp. novicida for 1 h (unless otherwise indicated) using moi of 10. After infection, the cells were washed three times with warm culture medium, and 50 µg ml−1 gentamicin was added for additional 1 h to kill extracellular bacteria. After three washes with DMEM, infected cells were incubated for the indicated time points after infection. The infected cells were fixed and permeabilized with ice-cold methanol for 5 min, then blocked in 3% BSA for 1 h. To label bacteria, goat anti-F. tularensis antibody (1:4000) was used followed by the anti-goat secondary antibody conjugated to Alexa fluor-488 (Molecular Probes Invitrogen, Carlsbad, CA). The anti-LAMP-2 (H4B4) 1:2000 mouse monoclonal antibody (developed by J.T. August and J.E.K. Hildreth) was obtained from the Developmental Studies Hybridoma Bank (University of Iowa, IA), mAbs anti-Cathepsin-D (1:200) was obtained from BD transduction (San Jose, CA). Anti-mouse secondary antibodies conjugated to Alexa fluor-555 were obtained from Molecular Probes. Colocalization of the FCP with LAMP-2 and Cathepsin-D was performed as we described previously (Santic et al., 2005a; 2008).

To detect activation of caspase-3 in the infected cells, anti-active caspase-3 rabbit polyclonal antiserum (1:200) (Cell Signaling, Danvers, MA) was used, followed by the donkey anti-rabbit secondary antibody conjugated to Alexa fluor-555 (Molecular Probes Invitrogen, Carlsbad, CA). As a negative control, uninfected HEK293T cells were mock-treated with 0.1% DMSO. As a positive control for caspase-3 activation, HEK293T cells were pre-treated with 1 µM Staurosporine (Sigma) for 18 h, as previously described (Nakamura et al., 2007). After fixation, cells were labelled and processed for confocal microscopy as we described previously (Santic et al., 2005b). The images were captured using the Fluoview FV-1000 confocal microscope and presented in the figures as a single z section after being processed using the Adobe illustrator software.

Quantification of bacterial escape into the cytosol

To quantify escape of F. tularensis from its phagosome into the host cytosol, we performed the fluorescence-based integrity assay, as described previously (Checroun et al., 2006; Santic et al., 2008). Briefly, untreated or RNAi-treated HEK293T cells in 24-well plate were infected with the WT strain or the iglC mutant. At 2 h after infection, cells were washed once with KHM buffer (110 mM potassium acetate/20 mM Hepes/2 mM MgCl2, pH 7.3), and their plasma membranes were selectively permeabilized and processed as described previously (Checroun et al., 2006; Santic et al., 2008). The images were captured using the Fluoview FV-1000 confocal microscope, and presented in the figures as a single z section after being processed using the Adobe illustrator software.

Detection of Akt phosphorylation

To study phosphorylation of Akt during infection by F. tularensis in the RNAi-silenced cells, untreated or RNAi-treated HEK293T cells were infected with an moi of 100 for 5 min. Uninfected and infected cells were lysed in M-Per buffer treated with the Complete EDTA-free protease inhibitor cocktail. PNS were processed by Western blot using rabbit polyclonal anti-pAkt antiserum (1:1000) (Cell Signaling, Danvers, MA). To ensure equal loading, the membrane was re-probed with the rabbit polyclonal anti-Akt antibody (1:200) (Santa Cruz, Santa Cruz, CA).

Data and statistical analyses

All experiments have been performed three times, the standard deviation was calculated, and statistically significant difference is indicated by asterisk in the bar graphs in the figures. Statistical analyses were performed using the two-tail Student's t-test and GraphPad Prism-5 software.

Acknowledgements

Y.A.K. is supported by Public Health Service Awards R01AI43965 and R01AI069321 from NIAID and by the commonwealth of Kentucky Research Challenge Trust Fund.

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