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Intracellular proliferation of Legionella pneumophila within a vacuole in human alveolar macrophages is essential for manifestation of Legionnaires’ pneumonia. Intravacuolar growth of the pathogen is totally dependent on remodeling the L. pneumophila-containing vacuole (LCV) by the ER and on its evasion of the endosomal-lysosomal degradation pathway., Diversion of the Legionella pneumophila-containing vacuole (LCV) from the host endosomal-lysosomal degradation pathway is one of the main virulence features essential for manifestation of Legionnaires’ pneumonia. Many of the ∼350 Dot/Icm-injected effectors identified in L. pneumophila have been shown to interfere with various host pathways and processes, but no L. pneumophila effector has ever been identified to be indispensable for lysosomal evasion. While most single effector mutants of L. pneumophila do not exhibit a defective phenotype within macrophages, we show that the MavE effector is essential for intracellular growth of L. pneumophila in human monocyte-derived macrophages (hMDMs) and amoebae and for intrapulmonary proliferation in mice. The mavE null mutant fails to remodel the LCV with endoplasmic reticulum (ER)-derived vesicles and is trafficked to the lysosomes where it is degraded, similar to formalin-killed bacteria. During infection of hMDMs, the MavE effector localizes to the poles of the LCV membrane. The crystal structure of MavE, resolved to 1.8 Å, reveals a C-terminal transmembrane helix, three copies of tyrosine-based sorting motifs, and an NPxY eukaryotic motif, which binds phosphotyrosine-binding domains present on signaling and adaptor eukaryotic proteins. Two point mutations within the NPxY motif result in attenuation of L. pneumophila in both hMDMs and amoeba. The substitution defects of P78 and D64 are associated with failure of vacuoles harboring the mutant to be remodeled by the ER and results in fusion of the vacuole to the lysosomes leading to bacterial degradation. Therefore, the MavE effector of L. pneumophila is indispensable for phagosome biogenesis and lysosomal evasion.

Mycobacterium tuberculosis is the main etiological agent of tuberculosis. The Bacillus Calmette–Guerin (BCG) microbes that are primarily used as a vaccine against tuberculosis also constitute the dominant infection model for studying the interaction of mycobacteria with the host cell types. The majority of interaction experiments have been conducted using macrophages and monocytes as prototype phagocyte cell types. Here, we report that M. bovis BCG infects mouse primary B cells as well as human B cell line. The complement receptors, along with B cell receptors, are engaged in the process of bacterial entry into the host B cells. Once inside the B cells, the intracellular trafficking of BCG follows the complete endocytic pathway of the ingested particles, which is in contrast to the events taking place during ingestion of BCG by macrophages. In vivo infection of mice with M. bovis BCG activated peritoneal as well as splenic B cells to produce proinflammatory cytokines. This paper further supports the evidence that B cells are involved in a host's early interactions with intracellular bacterial pathogens and participate in the induction of innate defense responses.

Legionella pneumophila has co-evolved with amoebae, their natural hosts. Upon transmission to humans, the bacteria proliferate within alveolar macrophages causing pneumonia. Here, we show L. pneumophila injects the effector LamA, an amylase, into the cytosol of human macrophage (hMDMs) and amoebae to rapidly degrade glycogen to generate cytosolic hyper-glucose. In response, hMDMs shift their metabolism to aerobic glycolysis, which directly triggers an M1-like pro-inflammatory differentiation and nutritional innate immunity through enhanced tryptophan degradation. This leads to a modest restriction of bacterial proliferation in hMDMs. In contrast, LamA-mediated glycogenolysis in amoebae deprives the natural host from the main building blocks for synthesis of the cellulose-rich cyst wall, leading to subversion of amoeba encystation. This is non-permissive for bacterial proliferation. Therefore, LamA of L. pneumophila is an amoebae host-adapted effector that subverts encystation of the amoebae natural host, and the paradoxical hMDMs' pro-inflammatory response is likely an evolutionary accident.

Legionella pneumophila invades protozoa with an “accidental” ability to cause pneumonia upon transmission to humans. To support its nutrition during intracellular residence, L. pneumophila relies on host amino acids as the main source of carbon and energy to feed the TCA cycle. Despite the apparent lack of a requirement for glucose for L. pneumophila growth in vitro and intracellularly, the organism contains multiple amylases, which hydrolyze polysaccharides into glucose monomers. Here we describe one predicted putative amylase, LamB, which is uniquely present only in L. pneumophila and L. steigerwaltii among the ~60 species of Legionella. Our data show that LamB has a strong amylase activity, which is abolished upon substitutions of amino acids that are conserved in the catalytic pocket of amylases. Loss of LamB or expression of catalytically-inactive variants of LamB results in a severe growth defect of L. pneumophila in Acanthamoeba polyphaga and human monocytes-derived macrophages. Importantly, the lamB null mutant is severely attenuated in intra-pulmonary proliferation in the mouse model and is defective in dissemination to the liver and spleen. Our data show an essential role for LamB in intracellular replication of L. pneumophila in amoeba and human macrophages and in virulence in vivo.

Several bacterial pathogens interact with their host through protein secretion effectuated by a type VI secretion system (T6SS). Francisella tularensis is a highly pathogenic intracellular bacterium that causes the disease tularemia. Proteins encoded by the Francisella pathogenicity island (FPI), which constitute a type VI secretion system, are essential for the virulence of the bacterium and a key mechanism behind this is the escape from the phagosome followed by productive cytosolic replication. It has been shown that T6SS in Francisella is distinct since all putative substrates of F. tularensis T6SS, except for VgrG, are unique to the species. Many of the FPI proteins are secreted into the macrophage cytosol and this is dependent on the functional components of DotU, VgrG, IglC and IglG. In addition, PdpC seems to have a regulatory role for the expression of iglABCD. Since previous results showed peculiar phenotypes of the ΔpdpC and ΔiglG mutants in mouse macrophages, their unique behavior was characterized in human monocyte-derived macrophages (HMDM) in this study. Our results show that both ΔpdpC and ΔiglG mutants of the live vaccine strain (LVS) of F. tularensis did not replicate within HMDMs. The ΔpdpC mutant did not escape from the Francisella containing phagosome (FCP), neither caused cytopathogenicity in primary macrophages and was attenuated in a mouse model. Interestingly, the ΔiglG mutant escaped from the HMDMs FCP and also caused pathological changes in the spleen and liver tissues of intradermally infected C57BL/6 mice. The ΔiglG mutant, with its unique phenotype, is a potential vaccine candidate.

Nonpathogenic bacteria are taken up by host cells into vacuoles or phagosomes that are processed through the endocytic pathway, through which the vacuoles mature and fuse to the lysosomes, in which the bacteria are degraded. To avoid this fate within phagocytic cells, intracellular pathogens have evolved different strategies to survive and evade phagosome–lysosome fusion [1]. Understanding the mechanisms by which pathogens manipulate vesicle trafficking in different hosts is extremely important for understanding the ability of various pathogens to cause disease and is essential for designing novel and effective strategies for prevention and therapeutic intervention. Francisella tularensis is a gram-negative, highly infectious, facultative intracellular bacterium that causes the zoonotic disease tularemia. The genus Francisella contains five species: F. tularensis, F. philomiragia, F. hispaniensis, F. noatunensis, and F. novicida [2,3]. Two subspecies of F. tularensis, tularensis (Type A) and holarctica (Type B), cause most cases of the illness in humans. F. novicida causes disease only in immunocompromised persons, but is highly virulent in mice [3]. However, it is important to note that in comparison to F. tularensis subsp. tularensis and F. tularensis subsp. holarctica, F. novicida elicits a different immune response in the host [2]. Humans acquire infection by several routes, including direct contact with infected animals, ingestion of water or food contaminated by infected animals, exposure to infected arthropod vectors, or by inhalation of infective aerosols, resulting in pneumonic, oropharyngeal, glandular, ulceroglandular, or oculoglandular tularemia [4]. Considering the ease of dissemination and high infectivity, F. tularensis subsp. tularensis and F. tularensis subsp. holarctica have been classified by the Centers for Disease Control and Prevention (CDC) as Tier 1 select agents. The existence of Francisella in the environment is divided into two cycles: the terrestrial cycle and aquatic cycle [5]. Small rodents, hares, and arthropods play a major role in the terrestrial cycle, while rodents associated with water are important in the water cycle [4,5]. Organisms such as ticks, flies, and mosquitoes are considered vectors of tularemia transmission to mammals [6]. Although it causes disease in various animal species, no animal has been identified as a main reservoir of this pathogen. F. tularensis subsp. holarctica and F. novicida have a strong association with freshwater environments, free-living amoeba, and biofilms [7,8]. Since mosquito larvae can feed on aquatic protozoa, they may be infected with F. tularensis during development in their natural aquatic environment [7]. The effect of wars, natural disasters, climate change, and global warming will probably have an impact on increased incidences of tularemia [9].

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.

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.

Legionella pneumophila utilizes the Dot/Icm type IV translocation system to proliferate within a vacuole in a wide variety of natural amoebal hosts and in alveolar macrophages of the human accidental host. Although L. pneumophila utilizes host amino acids as the main sources of carbon and energy, it is not known whether de novo synthesis of amino acids by intravacuolar L. pneumophila contributes to its nutrition. The aroB and aroE genes encode enzymes for the shikimate pathway that generates the aromatic amino acids Phe, Trp, and Tyr. Here we show the aroB and aroE mutants of L. pneumophila to be defective in growth in human monocyte-derived macrophages (hMDMs) but not in Acanthamoeba spp. The aroB and aroE mutants are severely attenuated in intrapulmonary proliferation in the A/J mouse model of Legionnaires' disease, and the defect is fully complemented by the respective wild-type alleles. The two mutants grow normally in rich media but do not grow in defined media lacking aromatic amino acids, and the growth defect is rescued by inclusion of the aromatic amino acids, which are essential for production of the pyomelanin pigment. Interestingly, supplementation of infected hMDMs with the three aromatic amino acids or with Trp alone rescues the intramacrophage defect of the aroE but not the aroB mutant. Therefore, the shikimate pathway of L. pneumophila is differentially required for optimal growth within human macrophages, which are auxotrophic for Trp and Phe, but is dispensable for growth within the Acanthamoeba spp. that synthesize the aromatic amino acids.

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/Icm-dependent 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.

Legionella pneumophila proliferates in environmental amoeba and human cells within the Legionella- containing vacuole (LCV). The exported AnkB F-box effector of L. pneumophila is anchored into the LCV membrane by host-mediated farnesylation. Here, we report that host proteasomal degradation of Lys(48)- linked polyubiquitinated proteins, assembled on the LCV by AnkB, generates amino acids required for intracellular bacterial proliferation. The severe defect of the ankB null mutant in proliferation within amoeba and human cells is rescued by supplementation of a mixture of amino acids or cysteine, serine, pyruvate, or citrate, similar to rescue by genetic complementation. Defect of the ankB mutant in intrapulmonary proliferation in mice is rescued upon injection of a mixture of amino acids or cysteine. Therefore, Legionella promotes eukaryotic proteasomal degradation to generate amino acids needed as carbon and energy sources for bacterial proliferation within evolutionarily distant hosts.

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.

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 9L10P 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-9L10P/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-9L10P/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., Author Summary Legionella pneumophila is abundantly found in the aquatic environment within various protozoa and can cause a severe pneumonia called Legionnaires' disease when it invades human macrophages in the lung. The ability of L. pneumophila to invade and proliferate within macrophages and protozoa is dependent on the translocation of specific proteins into the invaded cell via a specialized secretory device, and these proteins modulate various host cell processes. Of these translocated proteins, AnkB is indispensable for intracellular growth of L. pneumophila within macrophages and protozoa. Here we show that AnkB is essential for establishing a favorable intracellular replicative niche by promoting the decoration of the Legionella containing vacuole (LCV) with polyubiquitinated proteins. The AnkB effector achieves this by mimicking the action of host cell F-box proteins, a highly conserved component of the SCF ubiquitin ligase complex that is found in both unicellular organisms and mammalian cells. Our study provides new insights into the ability of intracellular pathogens to hijack evolutionarily conserved host cell processes through molecular mimicry to establish a favorable replicative niche within various hosts and to cause disease in mammals.

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.

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), ∼90% 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.

The Legionella pneumophila -containing phagosome evades endocytic fusion and intercepts endoplasmic reticulum (ER)-to-Golgi vesicle traffic, which is believed to be mediated by the Dot/Icm type IV secretion system. Although phagosomes harboring dot/icm mutants are thought to mature through the endosomal-lysosomal pathway, colocalization studies with lysosomal markers have reported contradictory results. In addition, phagosomes harboring the dot/icm mutants do not interact with endocytosed materials, which is inconsistent with maturation of the phagosomes in the endosomal-lysosomal pathway. Using multiple strategies, we show that the dot/icm mutants defective in the Dot/Icm structural apparatus are unable to maintain the integrity of their phagosomes and escape into the cytoplasm within minutes of entry into various mammalian and protozoan cells in a process independent of the type II secretion system. In contrast, mutants defective in cytoplasmic chaperones of Dot/Icm effectors and rpoS , letA/S , and letE regulatory mutants are all localized within intact phagosomes. Importantly, non- dot/icm L. pneumophila mutants whose phagosomes acquire late endosomal-lysosomal markers are all located within intact phagosomes. Using high-resolution electron microscopy, we show that phagosomes harboring the dot/icm transporter mutants do not fuse to lysosomes but are free in the cytoplasm. Inhibition of ER-to-Golgi vesicle traffic by brefeldin A does not affect the integrity of the phagosomes harboring the parental strain of L. pneumophila . We conclude that the Dot/Icm transporter is involved in maintaining the integrity of the L. pneumophila phagosome, independent of interception of ER-to-Golgi vesicle traffic, which is a novel function of type IV secretion systems.

Francisella tularensis is an intracellular bacterial pathogen, and is a category A bioterrorism agent. Within quiescent human macrophages, the F. tularensis pathogenicity island (FPI) is essential for bacterial growth within quiescent macrophages. The F. tularensis-containing phagosome matures to a late endosome-like stage that does not fuse to lysosomes for 1–8 h, followed by gradual bacterial escape into the macrophage cytosol. Here we show that the FPI protein IglD is essential for intracellular replication in primary human monocyte-derived macrophages (hMDMs). While the parental strain replicates robustly in pulmonary, hepatic and splenic tissues of BALB/c mice associated with severe immunopathologies, the isogenic iglD mutant is severely defective. Within hMDMs, the iglD mutant-containing phagosomes mature to either a late endosome-like phagosome, similar to the parental strain, or to a phagolysosome, similar to phagosomes harbouring the iglC mutant control. Despite heterogeneity and alterations in phagosome biogenesis, the iglD mutant bacteria escape into the cytosol faster than the parental strain within hMDMs and pulmonary cells of BALB/c mice. Co-infections of hMDMs with the wild-type strain and the iglD mutant, or super-infection of iglD mutantinfected hMDMs with the wild-type strain show that the mutant strain replicates robustly within the cytosol of hMDMs coinhabited by the wild strain. However, when the wild-type strain-infected hMDMs are super-infected by the iglD mutant, the mutant fails to replicate in the cytosol of communal macrophages. This is the first demonstration of a F. tularensis novel protein essential for proliferation in the macrophage cytosol. Our data indicate that F. tularensis transduces signals to the macrophage cytosol to remodel it into a proliferative niche, and IglD is essential for transduction of these signals.

Legionella longbeachae belongs to the family Legionellaceae, which causes a severe and fatal pneumonia known as Legionnaires' disease. In the United States, more than 90% of cases of Legionnaires' disease are caused by Legionella pneumophila (6). Interestingly, the most predominant species responsible for Legionnaires' disease in Western Australia is L. longbeachae (15). In addition, infection due to L. longbeachae has been reported in New Zealand, Germany, Japan, Denmark, Sweden, Canada, and The Netherlands. Unlike L. pneumophila, which inhabits mostly aquatic environments, L. longbeachae is commonly isolated from moist potting soil (29). In aquatic environments, amoeba serves as a reservoir for the amplification and dissemination of L. pneumophila and is considered the natural host for the bacterium (34). In addition, amoeba has been shown to resuscitate viable nonculturable L. pneumophila after disinfection by biocides, which may account for the reemergence of Legionella in water systems after disinfection (24). L. pneumophila replicates in alveolar macrophages, which is necessary for the manifestation of Legionnaires' disease. After phagocytosis, L. pneumophila is localized in a unique phagosome that is isolated from the endocytic pathway (26, 41, 43). The L. pneumophila-containing phagosome excludes endocytic markers, including the lysosome-associated membrane glycoproteins lysosome-associated membrane protein 1 (LAMP-1) and LAMP-2 as well as the lysosomal acid protease cathepsin D (10). While the L. pneumophila-containing phagosome does not interact with the dynamic endocytic traffic, the L. longbeachae-containing phagosome interacts with the endocytic traffic and its biogenesis exhibits some maturation within the endocytic pathway (5). Recent studies have shown that within human macrophages, the L. longbeachae-containing phagosome is trafficked into a nonacidified late endosome-like phagosome that acquires the LAMPs and the mannose-6-phosphate receptor late endosomal markers but excludes the vacuolar ATPase proton pump and lysosomal markers (5). In addition, the L. longbeachae-containing phagosome is remodeled by the rough endoplasmic reticulum and bacterial replication occurs within the rough endoplasmic reticulum-remodeled late endosome-like phagosomes (5). Thus, there is a divergence in the mechanisms of pathogenesis of L. longbeachae and L. pneumophila in human macrophages (5). Further studies are needed to dissect further the host-parasite interaction of L. longbeachae, which is lagging behind that of most other intracellular pathogens, including the closely related species L. pneumophila. Many intracellular pathogens, including L. pneumophila, have been shown to modulate the intrinsic and extrinsic apoptotic pathways of apoptosis that converge on the activation of caspase 3, resulting in apoptosis/programmed cell death (20). L. pneumophila induces the activation of caspase 3 in human macrophages during early stages of infection, which is thought to be essential for evasion of vesicle traffic, since inhibition of caspase 3 in human macrophages results in fusion of the phagosomes to lysosomes (18, 35). The activation of caspase 3 and the subsequent isolation of the phagosome from the endocytic pathway are mediated by the Dot/Icm type IV secretion system (47). Although caspase 3 is induced robustly during early stages of infection in human macrophages, apoptosis is not triggered until late stages of infection, concomitant with the termination of intracellular replication (2, 3, 35). The delay in apoptosis is associated with the induction of antiapoptotic signaling through the activation of NF-κB-dependent and -independent pathways (3, 30). In contrast, caspase 3 is not activated and is not required for the intracellular infection of mouse-derived macrophages (36, 45). Whether L. longbeachae also triggers caspase 3 and subsequent apoptosis in human macrophages is not known. Among inbred mouse strains, A/J is the only inbred mouse strain susceptible to infection by L. pneumophila, while all the other strains are resistant (31). In contrast, many inbred strains of mice are susceptible to infection by many Legionella species (31). Only one study of permissiveness of mouse macrophages in vitro to L. longbeachae has been reported using a single isolate and indicated that the isolate replicates in both A/J and C57BL/6 thioglycolate-elicited mouse peritoneal macrophages, but whether the growth kinetics are similar to those of L. pneumophila is not known (27). Whether L. longbeachae can replicate in mouse lungs in vivo and whether mice are a suitable animal model for L. longbeachae are not known. The genetic susceptibility of mice has been attributed to a polymorphism in the neuronal apoptosis inhibitory protein 5 (naip5)-birc1e gene (13). At least eight murine homologues of naip genes have been identified (25), and naip5 has been identified as the gene responsible for the differential susceptibilities of A/J mice to L. pneumophila infection (13). The family of Naips is expressed abundantly in macrophage-rich tissues in mice, and their collective expression is increased after phagocytosis by murine macrophages (14), but whether Naip5 is one of the induced Naips is not known. The differential susceptibilities of different inbred mouse strains to infection by L. pneumophila are due to the rapid activation of caspase 1 in C57BL/6 versus A/J mice, resulting in early macrophage pyropoptosis-mediated cell death in C57BL/6 mice (36, 45). The L. pneumophila product that is responsible for the activation of caspase 1 is flagellin, but it is not known how Naip5 contributes to the process (36). Whether L. pneumophila triggers caspase 1 activation in human macrophages is not known, and whether L. longbeachae is capable of activating caspase 1 in mouse or human macrophages is also not known. Some Naips have been shown to possess antiapoptotic activity (40) due to inhibition of caspase 3, caspase 7, and caspase 9 (17). The role of Naip5 in the activation of caspase 3 and apoptosis has not been determined, although it has been shown that the differential susceptibilities of mice to L. pneumophila are not related to the activation of caspase 3 (36, 45). Here, we show that polymorphism of the naip5 allele does not play a role in the susceptibility of inbred mouse strains to infection by L. longbeachae. Both in vitro and in vivo studies show that L. longbeachae replicates efficiently in bone marrow-derived macrophages and in the lungs of A/J, C57BL/6, and BALB/c mice. In addition, we show that the induction of naip5 transcription in both L. pneumophila- and L. longbeachae-infected A/J mouse macrophages is less compared to that in C57BL/6 mice. We show that unlike what was observed with L. pneumophila, caspase 3 activation and late-stage apoptosis are triggered only at very low levels in both mouse and human macrophages infected by L. longbeachae. Flagellated L. longbeachae does not trigger caspase 1-mediated pyropoptosis in mouse macrophages, which correlates with the lack of detectable pore-forming activity in this species. Neither species activates caspase 1 in human macrophages.

The Dot/Icm system of Legionella pneumophila triggers activation of caspase-3 during early stages of infection of human macrophages, but apoptosis is delayed until late stages of infection. During early stages of infection of mouse macrophages, the organism triggers rapid caspase-1-mediated cytotoxicity, which is mediated by bacterial flagellin. However, it is not known whether caspase-1 is triggered by L. pneumophila in human macrophages or whether caspase-3 is activated in permissive or nonpermissive mouse macrophages. Using single-cell analyses, we show that the wild-type strain of L. pneumophila does not trigger caspase-1 activation throughout the intracellular infection of human monocyte-derived macrophages (hMDMs), even when the flagellated bacteria escape into the cytoplasm during late stages. Using single-cell analyses, we show that the Dot/Icm system of L. pneumophila triggers caspase-3 but not caspase-1 within permissive A/J mouse bone marrow-derived primary macrophages by 2 to 8 h, but apoptosis is delayed until late stages of infection. While L. pneumophila triggers a Dot/Icm-dependent activation of caspase-1 in nonpermissive BALB/c mouse-derived macrophages, caspase-3 is not activated at any stage of infection. We show that robust intrapulmonary replication of the wild-type strain of L. pneumophila in susceptible A/J mice is associated with late-stage Dot/Icm-dependent pulmonary apoptosis and alveolar inflammation. In the lungs of nonpermissive BALB/c mice, L. pneumophila does not replicate and does not trigger pulmonary apoptosis or alveolar inflammation. Thus, similar to hMDMs, L. pneumophila does not trigger caspase-1 but triggers caspase-3 activation during early and exponential replication in permissive A/J mouse-derived macrophages, and apoptosis is delayed until late stages of infection. The Dot/Icm type IV secretion system is essential for pulmonary apoptosis in the genetically susceptible A/J mice.

Francisella tularensis is a highly infectious intracellular bacterium that causes fulminating disease and is a potential bioweapon. Although entry of the bacteria into macrophages is mediated by novel asymmetric, spacious pseudopod loops, the nascent phagosome becomes tight fitting within seconds of formation. Biogenesis of the Francisella-containing phagosome (FCP) is arrested for 2– 4 h at a unique stage within the endosomal– lysosomal degradation pathway, followed by gradual bacterial escape into the cytosol, where the microbe proliferates. By contrast, other intracellular pathogens either proliferate within an idiosyncratic phagosome or escape within minutes into the cytoplasm to avoid degradation. Thus, trafficking of the FCP defies the dogma of classification of intracellular pathogens into vacuolar or cytosolic. The Francisella pathogenicity island and its transcriptional regulator MglA are essential for arresting biogenesis of the FCP. Despite sophisticated microbial strategies to arrest phagosome biogenesis within quiescent macrophages, trafficking of F. tularensis and other intracellular pathogens within interferon-g-activated macrophages is similar, in that the bacterial phagosomes fuse to lysosomes. The potential use of F. tularensis as a bioweapon has generated interest in the study of its molecular pathogenesis to identify targets for therapy, vaccination and rapid diagnosis.

Legionella pneumophilais an intracellular pathogen that modulates the biogenesis of its phagosome to evade endocytic vesicle traffic. TheLegionella-containing phagosome (LCP) does not acquire any endocytic markers and is remodeled by the endoplasmic reticulum during early stages. Here we show that intracellular replication ofL. pneumophilais inhibited in gamma interferon (IFN-γ)-activated, bone marrow-derived mouse macrophages and IFN-γ-activated, human monocyte-derived macrophages in a dose-dependent manner. This inhibition of intracellular replication is associated with the maturation of the LCP into a phagolysosome, as documented by the acquisition of LAMP-2, cathepsin D, and lysosomal tracer Texas Red ovalbumin, and with the failure of the LCP to be remodeled by the rough endoplasmic reticulum. We conclude that IFN-γ-activated macrophages override the ability ofL. pneumophilato evade endocytic fusion and that the LCP is processed through the “default” endosomal-lysosomal degradation pathway.