Dot/Icm-Dependent Restriction of Legionella pneumophila within Neutrophils.

The Dot/Icm type IV secretion system (T4SS) of Legionella pneumophila is essential for lysosomal evasion and permissiveness of macrophages for intracellular proliferation of the pathogen. In contrast, we show that polymorphonuclear cells (PMNs) respond to a functional Dot/Icm system through rapid restriction of L. pneumophila. Specifically, we show that the L. pneumophila T4SS-injected amylase (LamA) effector catalyzes rapid glycogen degradation in the PMNs cytosol, leading to cytosolic hyperglucose. Neutrophils respond through immunometabolic reprogramming that includes upregulated aerobic glycolysis. The PMNs become activated with spatial generation of intracellular reactive oxygen species within the Legionella-containing phagosome (LCP) and fusion of specific and azurophilic granules to the LCP, leading to rapid restriction of L. pneumophila. We conclude that in contrast to macrophages, PMNs respond to a functional Dot/Icm system, and specifically to the effect of the injected amylase effector, through rapid engagement of major microbicidal processes and rapid restriction of the pathogen. IMPORTANCE Legionella pneumophila is commonly found in aquatic environments and resides within a wide variety of amoebal hosts. Upon aerosol transmission to humans, L. pneumophila invades and replicates with alveolar macrophages, causing pneumonia designated Legionnaires' disease. In addition to alveolar macrophages, neutrophils infiltrate into the lungs of infected patients. Unlike alveolar macrophages, neutrophils restrict and kill L. pneumophila, but the mechanisms were previously unclear. Here, we show that the pathogen secretes an amylase (LamA) enzyme that rapidly breakdowns glycogen stores within neutrophils, and this triggers increased glycolysis. Subsequently, the two major killing mechanisms of neutrophils, granule fusion and production of reactive oxygen species, are activated, resulting in rapid killing of L. pneumophila.

Increased Sensitivity of Amoeba-Grown Francisella Species to Disinfectants.

Francisella tularensis is a highly infectious, intracellular bacterium and it is the causative agent of tularemia. The bacterium has been isolated from more than 250 species, including protozoa. Previous studies have shown that the growth of Legionella pneumophila within the amoeba results in a dramatic increase in the resistance to disinfectants. Since Francisella persists in the environment for years, this study investigates whether Acanthamoeba castellanii-grown F. novicida exhibits an alteration in the resistance to disinfectants. The disinfectants used are didecyldimethylammonium chloride (DDAC) combined with isopropyl alcohol (D1), benzalkonium chloride combined with DDAC and formic acid (D2), and polyhexamethylene biguanide (PHMB, D3). The effect of disinfectants on the bacterial viability is determined by a colony-forming unit (CFU), by transmission electron microscopy (TEM), by fluorescence microscopy, and the damage of the bacterial membrane. Our data has shown that only a one-log10 loss in bacterial viability is exhibited upon treatment of agar-grown Francisella, while in amoeba-grown Francisella there was a three-log10 difference with D3. The D1 disinfectant sterilized the bacteria within 10 s. The treatment of agar-grown F. novicida with D2 reduces bacterial viability by seven-log10 within 10 s and 15 min, respectively. Surprisingly, the treatment of amoeba-grown F. novicida with D2 results in a total loss of bacterial viability. In conclusion, A. castellanii-grown F. novicida is more susceptible to many disinfectants.

Divergent evolution of Di-lysine ER retention vs. farnesylation motif-mediated anchoring of the AnkB virulence effector to the Legionella-containing vacuolar membrane.

Within macrophages and amoeba, the Legionella-containing vacuole (LCV) membrane is derived from the ER. The bona fide F-box AnkB effector protein of L. pneumophila strain AA100/130b is anchored to the cytosolic side of the LCV membrane through host-mediated farnesylation of its C-terminal eukaryotic "CaaX" motif. Here we show that the AnkB homologue of the Paris strain has a frame shift mutation that led to a loss of the CaaX motif and a concurrent generation of a unique C-terminal KNKYAP motif, which resembles the eukaryotic di-lysine ER-retention motif (KxKxx). Our phylogenetic analyses indicate that environmental isolates of L. pneumophila have a potential positive selection for the ER-retention KNKYAP motif. The AnkB-Paris effector is localized to the LCV membrane most likely through the ER-retention motif. Its ectopic expression in HEK293T cells localizes it to the perinuclear ER region and it trans-rescues the ankB mutant of strain AA100/130b in intra-vacuolar replication. The di-lysine ER retention motif of AnkB-Paris is indispensable for function; most likely as an ER retention motif that enables anchoring to the ER-derived LCV membrane. Our findings show divergent evolution of the ankB allele in exploiting either host farnesylation or the ER retention motif to be anchored into the LCV membrane.

Intra-Vacuolar Proliferation of F. Novicida within H. Vermiformis.

Francisella tularensis is a gram negative facultative intracellular bacterium that causes the zoonotic disease tularemia. Free-living amebae, such as Acanthamoeba and Hartmannella, are environmental hosts of several intracellular pathogens. Epidemiology of F. tularensis in various parts of the world is associated with water-borne transmission, which includes mosquitoes and amebae as the potential host reservoirs of the bacteria in water resources. In vitro studies showed intracellular replication of F. tularensis within A. castellanii cells. Whether ameba is a biological reservoir for Francisella in the environment is not known. We used Hartmannella vermiformis as an amebal model system to study the intracellular life of F. novicida. For the first time we show that F. novicida survives and replicates within H. vermiformis. The iglC mutant strain of F. novicida is defective for survival and replication not only within A. castellanii but also in H. vermiformis cells. In contrast to mammalian cells, where bacteria replicate in the cytosol, F. novicida resides and replicates within membrane-bound vacuoles within the trophozoites of H. vermiformis. In contrast to the transient residence of F. novicida within acidic vacuoles prior to escaping to the cytosol of mammalian cells, F. novicida does not reside transiently or permanently in an acidic compartment within H. vermiformis when examined 30 min after initiation of the infection. We conclude that F. tularensis does not replicate within acidified vacuoles and does not escape into the cytosol of H. vermiformis. The Francisella pathogenicity island locus iglC is essential for intra-vacuolar proliferation of F. novicida within H. vermiformis. Our data show a distinct intracellular lifestyle for F. novicida within H. vermiformis compared to mammalian cells.

Molecular Characterization of Exploitation of the Polyubiquitination and Farnesylation Machineries of Dictyostelium Discoideum by the AnkB F-Box Effector of Legionella Pneumophila.

The Dot/Icm-translocated Ankyrin B (AnkB) F-box effector of Legionella pneumophila is essential for intra-vacuolar proliferation and functions as a platform for the docking of polyubiquitinated proteins to the Legionella-containing vacuole (LCV) within macrophages and ameba. Here we show that ectopically expressed AnkB in Dictyostelium discoideum is targeted to the plasma membrane where it recruits polyubiquitinated proteins and it trans-rescues the intracellular growth defect of the ankB null mutant, which has never been demonstrated for any effector in ameba. Using co-immunoprecipitation and bimolecular fluorescence complementation we show specific interaction of Skp1 of D. discoideum with the F-box domain of AnkB, which has never been demonstrated in ameba. We show that anchoring of AnkB to the cytosolic face of the LCV membrane in D. discoideum is mediated by the host farnesylation of the C-terminal eukaryotic CaaX motif of AnkB and is independent of the F-box and the two ANK domains, which has never been demonstrated in ameba. Importantly, the three host farnesylation enzymes farnesyl transferase, RCE-1, and isoprenyl cysteine carboxyl methyl transferase of D. discoideum are recruited to the LCV in a Dot/Icm-dependent manner, which has never been demonstrated in ameba. We conclude that the polyubiquitination and farnesylation enzymatic machineries of D. discoideum are recruited to the LCV in a Dot/Icm-dependent manner and the AnkB effector exploits the two evolutionarily conserved eukaryotic machineries to proliferate within ameba, similar to mammalian cells. We propose that L. pneumophila has acquired ankB through inter-kingdom horizontal gene transfer from primitive eukaryotes, which facilitated proliferation of L. pneumophila within human cells and the emergence of Legionnaires' disease.

Francisella-arthropod vector interaction and its role in patho-adaptation to infect mammals.

Francisella tularensis is a Gram-negative, intracellular, zoonotic bacterium, and is the causative agent of tularemia with a broad host range. Arthropods such as ticks, mosquitoes, and flies maintain F. tularensis in nature by transmitting the bacteria among small mammals. While the tick is largely believed to be a biological vector of F. tularensis, transmission by mosquitoes and flies is largely believed to be mechanical on the mouthpart through interrupted feedings. However, the mechanism of infection of the vectors by F. tularensis is not well understood. Since F. tularensis has not been localized in the salivary gland of the primary human biting ticks, it is thought that bacterial transmission by ticks is through mechanical inoculation of tick feces containing F. tularensis into the skin wound. Drosophila melanogaster is an established good arthropod model for arthropod vectors of tularemia, where F. tularensis infects hemocytes, and is found in hemolymph, as seen in ticks. In addition, phagosome biogenesis and robust intracellular proliferation of F. tularensis in arthropod-derived cells are similar to that in mammalian macrophages. Furthermore, bacterial factors required for infectivity of mammals are often required for infectivity of the fly by F. tularensis. Several host factors that contribute to F. tularensis intracellular pathogenesis in D. melanogaster have been identified, and F. tularensis targets some of the evolutionarily conserved eukaryotic processes to enable intracellular survival and proliferation in evolutionarily distant hosts.

Exploitation of host cell biology and evasion of immunity by francisella tularensis.

Francisella tularensis is an intracellular bacterium that infects humans and many small mammals. During infection, F. tularensis replicates predominantly in macrophages but also proliferate in other cell types. Entry into host cells is mediate by various receptors. Complement-opsonized F. tularensis enters into macrophages by looping phagocytosis. Uptake is mediated in part by Syk, which may activate actin rearrangement in the phagocytic cup resulting in the engulfment of F. tularensis in a lipid raft rich phagosome. Inside the host cells, F. tularensis resides transiently in an acidified late endosome-like compartment before disruption of the phagosomal membrane and escape into the cytosol, where bacterial proliferation occurs. Modulation of phagosome biogenesis and escape into the cytosol is mediated by the Francisella pathogenicity island-encoded type VI-like secretion system. Whilst inside the phagosome, F. tularensis temporarily induce proinflammatory cytokines in PI3K/Akt-dependent manner, which is counteracted by the induction of SHIP that negatively regulates PI3K/Akt activation and promotes bacterial escape into the cytosol. Interestingly, F. tularensis subverts CD4 T cells-mediated killing by inhibiting antigen presentation by activated macrophages through ubiquitin-dependent degradation of MHC II molecules on activated macrophages. In the cytosol, F. tularensis is recognized by the host cell inflammasome, which is down-regulated by F. tularensis that also inhibits caspase-1 and ASC activity. During late stages of intracellular proliferation, caspase-3 is activated but apoptosis is delayed through activation of NF-κB and Ras, which ensures cell viability.

Host-mediated post-translational prenylation of novel dot/icm-translocated effectors of legionella pneumophila.

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.

Exploitation of Host Polyubiquitination Machinery through Molecular Mimicry by Eukaryotic-Like Bacterial F-Box Effectors.

Microbial pathogens have evolved exquisite mechanisms to interfere and intercept host biological processes, often through molecular mimicry of specific host proteins. Ubiquitination is a highly conserved eukaryotic post-translational modification essential in determining protein fate, and is often hijacked by pathogenic bacteria. The conserved SKP1/CUL1/F-box (SCF) E3 ubiquitin ligase complex plays a key role in ubiquitination of proteins in eukaryotic cells. The F-box protein component of the SCF complex provides specificity to ubiquitination by binding to specific cellular proteins, targeting them to be ubiquitinated by the SCF complex. The bacterial pathogens. Legionella pneumophila, Agrobacterium tumefaciens, and Ralstonia solanacearum utilize type III or IV translocation systems to inject into the host cell eukaryotic-like F-box effectors that interact with the host SKP1 component of the SCF complex to trigger ubiquitination of specific host cells targets, which is essential to promote proliferation of these pathogens. Our bioinformatic analyses have identified at least 74 genes encoding putative F-box proteins belonging to 22 other bacterial species, including human pathogens, plant pathogens, and amebal endosymbionts. Therefore, subversion of the host ubiquitination machinery by bacterial F-box proteins may be a widespread strategy amongst pathogenic bacteria. The findings that bacterial F-box proteins harbor Ankyrin repeats as protein-protein interaction domains, which are present in F-box proteins of primitive but not higher eukaryotes, suggest acquisition of many bacterial F-box proteins from primitive eukaryotic hosts rather than the mammalian host.

Temporal and spatial trigger of post-exponential virulence-associated regulatory cascades by Legionella pneumophila after bacterial escape into the host cell cytosol.

During late stages of infection and prior to lysis of the infected macrophages or amoeba, the Legionella pneumophila-containing phagosome becomes disrupted, followed by bacterial escape into the host cell cytosol, where the last few rounds of bacterial proliferation occur prior to lysis of the plasma membrane. This coincides with growth transition into the post-exponential (PE) phase, which is controlled by regulatory cascades including RpoS and the LetA/S two-component regulator. Whether the temporal expression of flagella by the regulatory cascades at the PE phase is exhibited within the phagosome or after bacterial escape into the host cell cytosol is not known. We have utilized fluorescence microscopy-based phagosome integrity assay to differentiate between vacuolar and cytosolic bacteria/or bacteria within disrupted phagosomes. Our data show that during late stages of infection, expression of FlaA is triggered after bacterial escape into the macrophage cytosol and the peak of FlaA expression is delayed for few hours after cytosolic residence of the bacteria. Importantly, bacterial escape into the host cell cytosol is independent of flagella, RpoS and the two-component regulator LetA/S, which are all triggered by L. pneumophila upon growth transition into the PE phase. Disruption of the phagosome and bacterial escape into the cytosol of macrophages is independent of the bacterial pore-forming activity, and occurs prior to the induction of apoptosis during late stages of infection. We conclude that the temporal and spatial engagement of virulence-associated regulatory cascades by L. pneumophila at the PE phase is temporally and spatially triggered after phagosomal escape and bacterial residence in the host cell cytosol.

Regulation of apoptosis and anti-apoptosis signalling by Francisella tularensis.

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-kappaB) is not known. Whether escape of the bacteria into the cytosol is sufficient and/or essential for activation of NF-kappaB is not known. Our results show that F. tularensis subsp. novicida induces sustained nuclear translocation of NF-kappaB at early time points after infection of human monocytes derived macrophages (hMDMs). The sustained nuclear translocation of NF-kappaB is defective in the iglC mutant that fails to escape into the cytosol of macrophages. Nuclear translocation of NF-kappaB by the wild type strain is abolished upon treatment with the NF-kappaB 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-kappaB.

Molecular characterization of the Dot/Icm-translocated AnkH and AnkJ eukaryotic-like effectors of Legionella pneumophila.

Although most Dot/Icm-translocated effectors of Legionella pneumophila are not required for intracellular proliferation, the eukaryotic-like ankyrin effectors, AnkH and AnkJ are required for intracellular proliferation. In this report, we show that the IcmSW chaperones are essential for translocation of AnkJ but not AnkH. The 10 C-terminal residues and the ANK domains of AnkH and AnkJ are required for translocation. Our data indicate that the two ANK domains of AnkH are critical domains required for the function of the effector in intracellular replication of L. pneumophila. The ankH and ankJ mutants are severely defective in intrapulmonary proliferation in mice. Expression of AnkH and AnkJ fusions within HEK293 cells show a punctuate distribution in the cytosol but no association with endocytic vesicles, the Golgi apparatus or the endoplasmic reticulum. Interestingly, the defect in intracellular proliferation of the ankH or ankJ mutants is rescued in HEK293 cells expressing the respective protein. We conclude that AnkH and AnkJ are effectors translocated by the Dot/Icm system by distinct mechanisms and modulate distinct cytosolic processes in the host cell.

Molecular mimicry by an F-box effector of Legionella pneumophila hijacks a conserved polyubiquitination machinery within macrophages and protozoa.

The ability of Legionella pneumophila to proliferate within various protozoa in the aquatic environment and in macrophages indicates a remarkable evolution and microbial exploitation of evolutionarily conserved eukaryotic processes. Ankyrin B (AnkB) of L. pneumophila is a non-canonical F-box-containing protein, and is the only known Dot/Icm-translocated effector of L. pneumophila essential for intra-vacuolar proliferation within both macrophages and protozoan hosts. We show that the F-box domain of AnkB and the (9)L(10)P conserved residues are essential for intracellular bacterial proliferation and for rapid acquisition of polyubiquitinated proteins by the Legionella-containing vacuole (LCV) within macrophages, Dictyostelium discoideum, and Acanthamoeba. Interestingly, translocation of AnkB and recruitment of polyubiquitinated proteins in macrophages and Acanthamoeba is rapidly triggered by extracellular bacteria within 5 min of bacterial attachment. Ectopically expressed AnkB within mammalian cells is localized to the periphery of the cell where it co-localizes with host SKP1 and recruits polyubiquitinated proteins, which results in restoration of intracellular growth to the ankB mutant similar to the parental strain. While an ectopically expressed AnkB-(9)L(10)P/AA variant is localized to the cell periphery, it does not recruit polyubiquitinated proteins and fails to trans-rescue the ankB mutant intracellular growth defect. Direct in vivo interaction of AnkB but not the AnkB-(9)L(10)P/AA variant with the host SKP1 is demonstrated. Importantly, RNAi-mediated silencing of expression of SKP1 renders the cells non-permissive for intracellular proliferation of L. pneumophila. The role of AnkB in exploitation of the polyubiquitination machinery is essential for intrapulmonary bacterial proliferation in the mouse model of Legionnaires' disease. Therefore, AnkB exhibits a novel molecular and functional mimicry of eukaryotic F-box proteins that exploits conserved polyubiquitination machinery for intracellular proliferation within evolutionarily distant hosts.

Intracellular fate of Francisella tularensis within arthropod-derived cells.

Since transmission of Francisella tularensis into the mammalian host occurs via arthropod vectors such as ticks, mosquitoes, horseflies and deerflies, recent studies have established Drosophila melanogaster as an arthropod vector model system. Nothing is known about the intracellular fate of F. tularensis within arthropod-derived cells, and the role of this host-parasite adaptation in the evolution of this pathogen to infect mammals. In this report, we explored intracellular trafficking of F. tularensis ssp. novicida in D. melanogaster-derived S2 cells. First, we show that similar to the F. tularensis ssp. holarctica-derived LVS strain, F. tularensis ssp. novicida is highly infectious, replicates exponentially within S2 cells and within adult flies, and is fatal to adult fruit flies in a dose-dependent manner, while the iglC, iglD and mglA mutants are defective. Using electron and fluorescence microscopy-based phagosome integrity assays, we show that the wild-type strain escapes into the cytosol of S2 cells within 30-60 min post infection and by 6 h, 90% were cytosolic. In contrast, approximately 40-50% of the iglC and iglD mutants escape into the cytosol by 6 h while the other subpopulation becomes enclosed within multilamellar vesicles (MLVs). Pre-treatment of S2 cells with the autophagy inhibitor methyl adenine blocks formation of the MLVs and all the vacuolar subpopulation of the iglC and iglD mutant bacteria become enclosed within single membrane-surrounded vacuoles. Endocytic trafficking studies of F. tularensis within S2 cells show transient colocalization of the bacterial phagosome with D. melanogaster LAMP2-GFP fusion but not with lysosomes pre-loaded with fluorescent dextran. Our data show that MLVs harbouring the iglC mutant acquire Lamp2 and dextran while MLVs harbouring the iglD mutant exclude these late endosomal and lysosomal markers. Our data indicate crucial differences in the role of the pathogenicity island-encoded proteins in modulating intracellular trafficking within human macrophages and arthropod vector-derived cells.

Role for the Ankyrin eukaryotic-like genes of Legionella pneumophila in parasitism of protozoan hosts and human macrophages.

Legionella pneumophila is a ubiquitous organism in the aquatic environment where it is capable of invasion and intracellular proliferation within various protozoan species and is also capable of causing pneumonia in humans. In silico analysis showed that the three sequenced L. pneumophila genomes each contained a common multigene family of 11 ankyrin (ank) genes encoding proteins with approximately 30-35 amino acid tandem Ankyrin repeats that are involved in protein-protein interactions in eukaryotic cells. To examine whether the ank genes are involved in tropism of protozoan hosts, we have constructed isogenic mutants of L. pneumophila in ten of the ank genes. Among the mutants, the DeltaankH and DeltaankJ mutants exhibit significant defects in robust intracellular replication within A. polyphaga, Hartmanella vermiformis and Tetrahymena pyriformis. A similar defect is also exhibited in human macrophages. Most of the ank genes are upregulated by L. pneumophila upon growth transition into the post-exponential phase in vitro and within Acanthamoeba polyphaga, and this upregulation is mediated, at least in part, by RpoS. Single-cell analyses have shown that upon co-infection of the wild-type strain with the ankH or ankJ mutant, the replication defect of the mutant is rescued within communal phagosomes harbouring the wild-type strain, similar to dot/icm mutants. Therefore, at least two of the L. pneumophila eukaryotic-like Ank proteins play a role in intracellular replication of L. pneumophila within amoeba, ciliated protozoa and human macrophages. The Ank proteins may not be involved in host tropism in the aquatic environment. Many of the L. pneumophila eukaryotic-like ank genes are triggered upon growth transition into post-exponential phase in vitro as well as within A. polyphaga. Our data suggest a role for AnkH and AnkJ in modulation of phagosome biogenesis by L. pneumophila independent of evasion of lysosomal fusion and recruitment of the rough endoplasmic reticulum.

Anti-apoptotic signalling by the Dot/Icm secretion system of L. pneumophila.

The 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 anti-apoptotic 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-kappaB). Our data show that L. pneumophila induces a Dot/Icm-dependent sustained nuclear translocation of the p50 and p65 subunits of NF-kappaB 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 IKKalpha-/- beta-/- double knockout mouse embryonic fibroblast cell lines, we show that nuclear translocation of NF-kappaB is required for the resistance of L. pneumophila-infected cells to apoptosis-inducing agents. In addition, the L. pneumophila-induced nuclear translocation of NF-kappaB requires the activity of IKKalpha and/or IKKbeta. 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-kappaB, which renders the cells refractory to external potent apoptotic stimuli.

The Francisella tularensis pathogenicity island protein IglC and its regulator MglA are essential for modulating phagosome biogenesis and subsequent bacterial escape into the cytoplasm.

The 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 monocyte-derived 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.

Structure-function analysis of the C-terminus of IcmT of Legionella pneumophila in pore formation-mediated egress from macrophages.

We have recently shown an essential role of the 32 amino acids C-terminus domain of IcmT of Legionella pneumophila in bacterial egress from macrophages. Mutants expressing an IcmT protein with a truncation in the C-terminus, replicate intracellularly but are defective in pore formation-mediated egress. The C-terminus domain of IcmT is the only hydrophilic domain of IcmT that is predicted to be in the cytoplasm while the rest of the protein is in the cytoplasmic membrane. In order to characterize the structure-function of the C-terminus of IcmT in the pore-forming activity and bacterial egress, we constructed 10 icmT missense mutant alleles differing by a single amino acid in the C-terminus of icmT and introduced them into the null icmT mutant. The H58Q, W69L, R71I, R79I and R86I icmT mutant alleles showed significantly lower pore-forming activity as measured by hemolysis of sRBC. The Y59S, R68L and S77L mutant alleles showed significantly lower cytopathogenicity to U937 macrophages. All 10 mutant alleles enabled the icmT null mutant to replicate intracellularly as efficiently as icmT null mutant harboring the wild-type icmT. Seven of the icmT alleles enabled the icmT null mutant to egress from infected macrophages as efficiently as icmT null mutant harboring the wild-type icmT. The other 3 substitutions conferred a partial defect in hemolysis and two of them also conferred a defect in egress from macrophages. Thus, two amino acid residues in the C-terminus of IcmT are required for both pore formation and bacterial egress. However, certain single amino acid substitutions in the C-terminus reduce the pore-forming activity when tested in vitro, but may or may not have a detectable effect on egress of L. pneumophila from U937 macrophages.