SummaryThe Francisella tularensis subsp. novicida -containing phagosome (FCP) matures into a late endosome-like stage that acquires the late endosomal marker LAMP-2 but does not fuse to lysosomes, for the first few hours after bacterial entry. This modulation in phagosome biogenesis is followed by disruption of the phagosome and bacterial escape into the cytoplasm where they replicate. Here we examined the role of the Francisella pathogenicity island (FPI) protein IglC and its regulator MglA in the intracellular fate of F. tularensis subsp. novicida within human macrophages. We show that F. tularensis mglA and iglC mutant strains are defective for survival and replication within U937 macrophages and human monocytederived macrophages (hMDMs). The defect in intracellular replication of both mutants is associated with a defect in disruption of the phagosome and failure to escape into the cytoplasm. Approximately, 80-90% of the mglA and iglC mutants containing phagosomes acquire the late endosomal/lysosomal marker LAMP-2 similar to the wild-type (WT) strain. Phagosomes harbouring the mglA or iglC mutants acquire the lysosomal enzyme Cathepsin D, which is excluded from the phagosomes harbouring the WT strain. In hMDMs in which the lysosomes are preloaded with BSA-gold or Texas Red Ovalbumin, phagosomes harbouring the mglA or the iglC mutants acquire both lysosomal tracers. We conclude that the FPI protein IglC and its regulator MglA are essential for modulating phagosome biogenesis and subsequent bacterial escape into the cytoplasm. Therefore, acquisition of the FPI, within which iglC is contained, is essential for the pathogenic evolution of F. tularensis to evade lysosomal fusion within human macrophages and cause tularemia. This is the first example of specific virulence factors of F. tularensis that are essential for evasion of fusion of the FCP to lysosomes.

Farnesylation involves covalent linkage of eukaryotic proteins to a lipid moiety to anchor them into membranes, which is essential for the biological function of Ras and other proteins. A large cadre of bacterial effectors is injected into host cells by intravacuolar pathogens through elaborate type III–VII translocation machineries, and many of these effectors are incorporated into the pathogen-containing vacuolar membrane by unknown mechanisms. The Dot/Icm type IV secretion system of Legionella pneumophila injects into host cells the F-box effector Ankyrin B (AnkB), which functions as platforms for the docking of polyubiquitinated proteins to the Legionella-containing vacuole (LCV) to enable intravacuolar proliferation in macrophages and amoeba. We show that farnesylation of AnkB is indispensable for its anchoring to the cytosolic face of the LCV membrane, for its biological function within macrophages and Dictyostelium discoideum, and for intrapulmonary proliferation in mice. Remarkably, the protein farnesyltransferase, RCE-1 (Ras-converting enzyme-1), and isoprenyl cysteine carboxyl methyltransferase host farnesylation enzymes are recruited to the LCV in a Dot/Icm-dependent manner and are essential for the biological function of AnkB. In conclusion, this study shows novel localized recruitment of the host farnesylation machinery and its anchoring of an F-box effector to the LCV membrane, and this is essential for biological function in vitro and in vivo.

SummaryThe Dot/Icm type IV secretion system of Legionella pneumophila triggers robust activation of caspase-3 during early and exponential stages of proliferation within human macrophages, but apoptosis is delayed till late stages of infection, which is novel. As caspase-3 is the executioner of the cell, we tested the hypothesis that L. pneumophila triggers antiapoptotic signalling within the infected human macrophages to halt caspase-3 from dismantling the cells. Here we show that during early and exponential replication, L. pneumophila-infected human monocyte-derived macrophages (hMDMs) exhibit a remarkable resistance to induction of apoptosis, in a Dot/Icm-dependent manner. Microarray analyses and real-time PCR reveal that during exponential intracellular replication, L. pneumophila triggers upregulation of 12 anti-apoptotic genes that are linked to activation of the nuclear transcription factor kappa-B (NF-kB). Our data show that L. pneumophila induces a Dot/Icm-dependent sustained nuclear translocation of the p50 and p65 subunits of NF-kB during exponential intracellular replication. Bacterial entry is essential both for the anti-apoptotic phenotype of infected hMDMs and for nuclear translocation of the p65. Using p65 -/-and IKKa -/-b -/-double knockout mouse embryonic fibroblast cell lines, we show that nuclear translocation of NF-kB is required for the resistance of L. pneumophila-infected cells to apoptosis-inducing agents. In addition, the L. pneumophila-induced nuclear translocation of NF-kB requires the activity of IKKa and/or IKKb. We conclude that although the Dot/Icm secretion system of L. pneumophila elicits an early robust activation of caspase-3 in human macrophages, it triggers a strong anti-apoptotic signalling cascade mediated, at least in part by NF-kB, which renders the cells refractory to external potent apoptotic stimuli.

The Francisella tularensis-containing phagosome (FCP) matures to a late-endosome-like phagosome prior to bacterial escape into the cytosols of macrophages, where bacterial proliferation occurs. Our data show that within the first 15 min after infection of primary human monocyte-derived macrophages (hMDMs), ϳ90% of the FCPs acquire the proton vacuolar ATPase (vATPase) pump and the lysomotropic dye LysoTracker, which concentrates in acidic compartments, similar to phagosomes harboring the Listeria monocytogenes control. The acquired proton vATPase pump and lysomotropic dye are gradually lost by 30 to 60 min postinfection, which coincides with bacterial escape into the cytosols of hMDMs. Colocalization of phagosomes harboring the iglD mutant with the vATPase pump and the LysoTracker dye was also transient, and the loss of colocalization was faster than that observed for the wild-type strain, which is consistent with the faster escape of the iglD mutant into the macrophage cytosol. In contrast, colocalization of both makers with phagosomes harboring the iglC mutant was persistent, which is consistent with fusion to the lysosomes and failure of the iglC mutant to escape into the macrophage cytosol. We have utilized a fluorescence microscopy-based phagosome integrity assay for differential labeling of vacuolar versus cytosolic bacteria, using antibacterial antibodies loaded into the cytosols of live hMDMs. We show that specific inhibition of the proton vATPase pump by bafilomycin A1 (BFA) blocks rapid bacterial escape into the cytosols of hMDMs, but 30% to 50% of the bacteria escape into the cytosol by 6 to 12 h after BFA treatment. The effect of BFA on the blocking of bacterial escape into the cytosol is completely reversible, as the bacteria escape after removal of BFA. We also show that the limited fusion of the FCP to lysosomes is not due to failure to recruit the late-endosomal fusion regulator Rab7. Therefore, within few minutes of its biogenesis, the FCP transiently acquires the proton vATPase pump to acidify the phagosome, and this transient acidification is essential for subsequent bacterial escape into the macrophage cytosol.

Amoebae are the natural hosts for Legionella pneumophila and play essential roles in bacterial ecology and infectivity to humans. When L. pneumophila colonizes an aquatic installation, it can persist for years despite repeated treatments with disinfectants. We hypothesized that freshwater amoebae play an important role in bacterial resistance to disinfectants, and in subsequent resuscitation of viable non-culturable (VNC) L. pneumophila that results in re-emergence of the disease-causing strain in the disinfected water source. Our work showed that in the absence of Acanthamoeba polyphaga, seven L. pneumophila strains became non-culturable after treatment by 256 p.p.m. of sodium hypochlorite (NaOCl). In contrast, intracellular L. pneumophila within A. polyphaga was resistant to 1024 p.p.m. of NaOCl. In addition, L. pneumophila-infected A. polyphaga exhibited increased resistance to NaOCl. When chlorine-sterilized water samples were co-cultured with A. polyphaga, the non-culturable L. pneumophila were resuscitated and proliferated robustly within A. polyphaga. Upon treatment by NaOCl, uninfected amoebae differentiated into cysts within 48 h. In contrast, L. pneumophila-infected A. polyphaga failed to differentiate into cysts, and L. pneumophila was never detected in cysts of A. polyphaga. We conclude that amoebic trophozoites protect intracellular L. pneumophila from eradication by NaOCl, and play an essential role in resuscitation of VNC L. pneumophila in NaOCl-disinfected water sources. Intracellular L. pneumophila within trophozoites of A. polyphaga block encystation of the amoebae, and the resistance of both organisms to NaOCl is enhanced. To ensure long-term eradication and complete loss of the VNC state of L. pneumophila, we recommend that Legionella-protozoa co-culture should be an important tool to ensure complete loss of the VNC state of L. pneumophila.

The ability of the intracellular bacterium Legionella pneumophila to cause disease is totally dependent on its ability to modulate the biogenesis of its phagosome and to replicate within alveolar cells. Upon invasion, L. pneumophila activates caspase-3 in macrophages, monocytes, and alveolar epithelial cells in a Dot/Icmdependent manner that is independent of the extrinsic or intrinsic pathway of apoptosis, suggesting a novel mechanism of caspase-3 activation by this intracellular pathogen. We have shown that the inhibition of caspase-3 prior to infection results in altered biogenesis of the L. pneumophila-containing phagosome and in an inhibition of intracellular replication. In this report, we show that the preactivation of caspase-3 prior to infection does not rescue the intracellular replication of L. pneumophila icmS, icmR, and icmQ mutant strains. Interestingly, preactivation of caspase-3 through the intrinsic and extrinsic pathways of apoptosis in both human and mouse macrophages inhibits intracellular replication of the parental stain of L. pneumophila. Using single-cell analysis, we show that intracellular L. pneumophila induces a robust activation of caspase-3 during exponential replication. Surprisingly, despite this robust activation of caspase-3 in the infected cell, the host cell does not undergo apoptosis until late stages of infection. In sharp contrast, the activation of caspase-3 by apoptosis-inducing agents occurs concomitantly with the apoptotic death of all cells that exhibit caspase-3 activation. It is only at a later stage of infection, and concomitant with the termination of intracellular replication, that the L. pneumophila-infected cells undergo apoptotic death. We conclude that although a robust activation of caspase-3 is exhibited throughout the exponential intracellular replication of L. pneumophila, apoptotic cell death is not executed until late stages of the infection, concomitant with the termination of intracellular replication.

The Dot/Icm type IV translocated Ankyrin B (AnkB) effector of Legionella pneumophila is modified by the host prenylation machinery that anchors it into the outer leaflet of the Legionella-containing vacuole (LCV), which is essential for biological function of the effector in vitro and in vivo. Prenylation involves the covalent linkage of an isoprenoid lipid moiety to a C-terminal CaaX motif in eukaryotic proteins enabling their anchoring into membranes. We show here that the LCV harboring an ankB null mutant is decorated with prenylated proteins in a Dot/Icm-dependent manner, indicating that other LCV membrane-anchored proteins are prenylated. In silico analyses of four sequenced L. pneumophila genomes revealed the presence of eleven other genes that encode proteins with a C-terminal eukaryotic CaaX prenylation motif. Of these eleven designated Prenylated effectors of Legionella (Pel), seven are also found in L. pneumophila AA100. We show that six L. pneumophila AA100 Pel proteins exhibit distinct cellular localization when ectopically expressed in mammalian cells and this is dependent on action of the host prenylation machinery and the conserved cysteine residue of the CaaX motif. Although inhibition of the host prenylation machinery completely blocks intra-vacuolar proliferation of L. pneumophila, it only had a modest effect on intracellular trafficking of the LCV. Five of the Pel proteins are injected into human macrophages by the Dot/Icm type IV translocation system of L. pneumophila. Taken together, the Pel proteins are novel Dot/Icm-translocated effectors of L. pneumophila that are post-translationally modified by the host prenylation machinery, which enables their anchoring into cellular membranes, and the prenylated effectors contribute to evasion of lysosomal fusion by the LCV.

Francisella tularensis induces apoptosis within macrophages but the temporal and spatial modulation through activation of caspase-1, caspase-3, and the anti-apoptosis nuclear transcription factor B (NF-κB) is not known. Whether escape of the bacteria into the cytosol is sufficient and/or essential for activation of NF-κB is not known. Our results show that F. tularensis subsp. novicida induces sustained nuclear translocation of NF-κB at early time points after infection of human monocytes derived macrophages (hMDMs). The sustained nuclear translocation of NF-κB is defective in the iglC mutant that fails to escape into the cytosol of macrophages. Nuclear translocation of NF-κB by the wild type strain is abolished upon treatment with the NF-κB inhibitor caffein acid phenyl ester. While the wild type strain triggers caspase-3 and caspase-1 activation by 6 h post-infection the iglC mutant is defective in triggering both caspases. In hMDMs treated with the apoptosis-inducing agent, staurosporin, there is an induction of cell death in the iglC mutant-infected macrophages despite reduced frequency of caspase-1 and caspase-3 activity. The wt-infected macrophages are resistant to cell death-induced agent. We conclude that although caspase-1 and capsase-3 are triggered within F. tularensis-infected hMDMs during early stages of infection, cell death is delayed, which is correlated with simultaneous activation of NF-κB.