Macroautophagy is dispensable for intracellular replication of Legionella pneumophila in Dictyostelium discoideum

The Gram-negative bacterium Legionella pneumophila is a facultative intracellular pathogen of free-living amoebae and mammalian phagocytes. L. pneumophila is engulfed in phagosomes that initially avoid fusion with lysosomes. The phagosome associates with endoplasmic reticulum (ER) and mitochondria and eventually resembles ER. The morphological similarity of the replication vacuole to autophagosomes, and enhanced bacterial replication in response to macroautophagy-inducing starvation, led to the hypothesis that L. pneumophila infection requires macroautophagy. As L. pneumophila replicates in Dictyostelium discoideum, and macroautophagy genes have been identified and mutated in D. discoideum, we have taken a genetic and cell biological approach to evaluate the relationship between host macroautophagy and intracellular replication of L. pneumophila. Mutation of the apg1, apg5, apg6, apg7 and apg8 genes produced typical macroautophagy defects, including reduced bulk protein degradation and cell viability during starvation. We show that L. pneumophila replicates normally in D. discoideum macroautophagy mutants and produces replication vacuoles that are morphologically indistinguishable from those in wild-type D. discoideum. Furthermore, a green fluorescent protein (GFP)-tagged marker of autophagosomes, Apg8, does not systematically co-localize with DsRed-labelled L. pneumophila. We conclude that macroautophagy is dispensable for L. pneumophila intracellular replication in D. discoideum.     

The mechanism of killing and exiting the protozoan host Acanthamoeba polyphaga by Legionella pneumophila

The ability of Legionella pneumophila to cause legionnaires' disease is dependent on its capacity to replicate within cells in the alveolar spaces. The bacteria kill mammalian cells in two phases: induction of apoptosis during the early stages of infection, followed by an independent and rapid necrosis during later stages of the infection, mediated by a pore-forming activity. In the environment, L. pneumophila is a parasite of protozoa. The molecular mechanisms by which L. pneumophila kills the protozoan cells, after their exploitation for intracellular proliferation, are not known. In an effort to decipher these mechanisms, we have examined induction of both apoptosis and necrosis in the protozoan Acanthamoeba polyphaga upon infection by L. pneumophila. Our data show that, although A. polyphaga undergoes apoptosis following treatment with actinomycin D, L. pneumophila does not induce apoptosis in these cells. Instead, intracellular L. pneumophila induces necrotic death in A. polyphaga, which is mediated by the pore-forming activity. Mutants of L. pneumophila defective in expression of the pore-forming activity are indistinguishable from the parental strain in intracellular replication within A. polyphaga. The parental strain bacteria cause necrosis-mediated lysis of all the A. polyphaga cells within 48 h after infection, and all the intracellular bacteria are released into the tissue culture medium. In contrast, all cells infected by the mutants remain intact, and the intracellular bacteria are 'trapped' within A. polyphaga after the termination of intracellular replication. Failure to exit the host cell after termination of intracellular replication results in a gradual decline in the viability of the mutant strain bacteria within A. polyphaga starting 48h after infection. Our data show that the pore-forming activity of L. pneumophila is not required for intracellular bacterial replication within A. polyphaga but is required for killing and exiting the protozoan host upon termination of intracellular replication.     

Intracellular multiplication of Legionella pneumophila depends on host cell amino acid transporter SLC1A5

The infectious agent of Legionnaires' disease, Legionella (L) pneumophila, multiplies intracellularly in eukaryotic cells. This study has been performed to explore the nutrient requirements of L. pneumophila during intracellular replication. In human monocytes, bacterial replication rate was reduced by 76% in defined medium lacking L-cysteine, L-glutamine or L-serine. SLC1A5 (hATB(0,+)), a neutral amino acid transporter, was upregulated in the host cells after infection with L. pneumophila. Inhibition of SLC1A5 by BCH, a competitive inhibitor of amino acid uptake as well as siRNA silencing of the slc1a5 gene blocked intracellular multiplication of L. pneumophila without compromising viability of host cells. These observations suggest that replication of L. pneumophila depends on the function of host cell SLC1A5.     

Dictyostelium discoideum strains lacking the RtoA protein are defective for maturation of the Legionella pneumophila replication vacuole

To identify host proteins involved in Legionella pneumophila intracellular replication, the soil amoeba Dictyostelium discoideum was analysed. The absence of the amoebal RtoA protein is demonstrated here to depress L. pneumophila intracellular growth. Uptake of L. pneumophila into a D. discoideum rtoA(-) strain was marginally defective, but this effect was not sufficient to account for the defective intracellular growth of L. pneumophila. The rtoA mutant was also more resistant to high-multiplicity killing by the bacterium. A targeting assay testing the colocalization of L. pneumophila-containing vacuole with an endoplasmic reticulum/pre-Golgi intermediate compartment marker protein, GFP-HDEL, was used to analyse these defects. In parental D. discoideum, the L. pneumophila vacuole showed recruitment of GFP-HDEL within 40 min after introduction of bacteria to the amoebae. By 6 h after infection it was clear that the rtoA mutant acquired and retained the GFP-HDEL less efficiently than the parental strain, and that the mutant was defective for promoting the physical expansion of the membranous compartment surrounding the bacteria. Depressed intracellular growth of L. pneumophila in a D. discoideum rtoA(-) mutant therefore appeared to result from a lowered efficiency of vesicle trafficking events that are essential for the modification and expansion of the L. pneumophila-containing compartment.     

Genomic and proteomic analyses reveal multiple homologs of genes encoding enzymes of the methanol:coenzyme M methyltransferase system that are differentially expressed in methanol- and acetate-grown Methanosarcina thermophila

The opportunistic pathogen Legionella pneumophila, the etiologic agent of Legionnaires disease, is able to invade and multiply intracellularly in human macrophages. This process is controlled by several bacterial virulence factors. As recently demonstrated, one of these virulence factors, the macrophage infectivity potentiator (Mip) protein, is important for invasion and proper intracellular establishment of L. pneumophila in macrophages and protozoa. Knockout mutants devoid of a functional mip-gene enter host cells much less effectively but intracellular replication is not affected. Using a P(mip)-green fluorescent protein reporter construct in L. pneumophila substrain Corby, P(mip) was recently shown to be constitutively active in replicating bacteria. A stringent regulation during the infection process could not be observed, neither in intracellular nor in BYE broth-grown bacteria. For enhanced temporal and quantitative resolution, we examined the activity of mip on RNA level in order to detect short transient regulatory events. Our results show that P(mip) of L. pneumophila is temporarily repressed directly after invasion of the monocytic human cell line MonoMac 6 and regains activity after 24 h of intracellular replication.     

NAIP and Ipaf control Legionella pneumophila replication in human cells

In mice, different alleles of the mNAIP5 (murine neuronal apoptosis inhibitory protein-5)/mBirc1e gene determine whether macrophages restrict or support intracellular replication of Legionella pneumophila, and whether a mouse is resistant or (moderately) susceptible to Legionella infection. In the resistant mice strains, the nucleotide-binding oligomerization domain (Nod)-like receptor (NLR) family member mNAIP5/mBirc1e, as well as the NLR protein mIpaf (murine ICE protease-activating factor), are involved in recognition of Legionella flagellin and in restriction of bacterial replication. Human macrophages and lung epithelial cells support L. pneumophila growth, and humans can develop severe pneumonia (Legionnaires disease) after Legionella infection. The role of human orthologs to mNAIP5/mBirc1e and mIpaf in this bacterial infection has not been elucidated. Herein we demonstrate that flagellin-deficient L. pneumophila replicate more efficiently in human THP-1 macrophages, primary monocyte-derived macrophages, and alveolar macrophages, and in A549 lung epithelial cells compared with wild-type bacteria. Additionally, we note expression of the mNAIP5 ortholog hNAIP in all cell types examined, and expression of hIpaf in human macrophages. Gene silencing of hNAIP or hIpaf in macrophages or of hNAIP in lung epithelial cells leads to an enhanced bacterial growth, and overexpression of both molecules strongly reduces Legionella replication. In contrast to experiments with wild-type L. pneumophila, hNAIP or hIpaf knock-down affects the (enhanced) replication of flagellin-deficient Legionella only marginally. In conclusion, hNAIP and hIpaf mediate innate intracellular defense against flagellated Legionella in human cells.     

Host cell processes that influence the intracellular survival of Legionella pneumophila

Key to the pathogenesis of intracellular pathogens is their ability to manipulate host cell processes, permitting the establishment of an intracellular replicative niche. In turn, the host cell deploys defence mechanisms that limit intracellular infection. The bacterial pathogen Legionella pneumophila, the aetiological agent of Legionnaire's Disease, has evolved virulence mechanisms that allow it to replicate within protozoa, its natural host. Many of these tactics also enable L. pneumophila's survival and replication inside macrophages within a membrane-bound compartment known as the Legionella-containing vacuole. One of the virulence factors indispensable for L. pneumophila's intracellular survival is a type IV secretion system, which translocates a large repertoire of bacterial effectors into the host cell. These effectors modulate multiple host cell processes and in particular, redirect trafficking of the L. pneumophila phagosome and mediate its conversion into an ER-derived organelle competent for intracellular bacterial replication. In this review, we discuss how L. pneumophila manipulates host cells, as well as host cell processes that either facilitate or impede its intracellular survival.     

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.     

Legionella pneumophila CsrA is a pivotal repressor of transmission traits and activator of replication

Legionella pneumophila can replicate inside amoebae and also alveolar macrophages to cause Legionnaires' Disease in susceptible hosts. When nutrients become limiting, a stringent-like response coordinates the differentiation of L. pneumophila to a transmissive form, a process mediated by the two-component system LetA/S and the sigma factors RpoS and FliA. Here we demonstrate that the broadly conserved RNA binding protein CsrA is a global repressor of L. pneumophila transmission phenotypes and an essential activator of intracellular replication. By analysing csrA expression and the phenotypes of csrA single and double mutants and a strain that expresses csrA constitutively, we demonstrate that, during replication in broth, CsrA represses every post-exponential phase phenotype examined, including cell shape shortening, motility, pigmentation, stress resistance, sodium sensitivity, cytotoxicity and efficient macrophage infection. At the transition to the post-exponential phase, LetA/S relieves CsrA repression to induce transmission phenotypes by both FliA-dependent and -independent pathways. For L. pneumophila to avoid lysosomal degradation in macrophages, CsrA repression must be relieved by LetA/S before phagocytosis; conversely, before intracellular bacteria can replicate, CsrA repression must be restored. The reciprocal regulation of replication and transmission exemplified by CsrA likely enhances the fitness of microbes faced with fluctuating environments.     

Interferon-gamma reverses the evasion of Birc1e/Naip5 gene mediated murine macrophage immunity by Legionella pneumophila mutant lacking flagellin

Legionella pneumophila is the etiologic agent of Legionnaires' disease. This bacterium contains a single monopolar flagellum, of which the FlaA subunit is a major protein constituent. The murine macrophage resistance against this bacterium is controlled by the Birc1e/Naip5 gene, which belongs to the NOD family. We evaluated the intracellular growth of the flaA mutant bacteria as well as another aflagellated fliA mutant, within bone marrow-derived macrophages from mice with an intact (C57BL/6, BALB/c) or mutated (A/J) Birc1e/Naip5 gene. The flaA mutant L. pneumophila multiplied within C57BL/6 and BALB/c macrophages while the wild-type strain did not. Cell viability was not impaired until 3 days after infection when the flaA mutant bacteria replicated 10(2-3)-fold in macrophages, implying that L. pneumophila inhibited host cell death during the early phase of intracellular replication. The addition of recombinant interferon-gamma (IFN-gamma) to the infected macrophages restricted replication of the flaA mutant within macrophages; these treated cells also showed enhanced nitric oxide production, although inhibition of nitric oxide production did not affect the IFN-gamma induced inhibition of Legionella replication. These findings suggested that IFN-gamma activated macrophages to restrict the intracellular growth of the L. pneumophila flaA mutant by a NO independent pathway.     

Efficient intracellular multiplication of Legionella pneumophila in human monocytes requires functional host cell L-type calcium channels

The infectious agent of Legionnaires' disease, Legionella pneumophila, multiplies intracellularly in a variety of eukaryotic cells. Genistein, a tyrosine kinase inhibitor, has been shown to block intracellular replication of L. pneumophila without harming the infected host cell. The present study has been performed to investigate the underlying mechanism. We demonstrate that inhibition of intracellular bacterial growth by genistein is not mediated by its protein tyrosine kinase-modulating effect but by inhibition of L-type calcium channels of the infected host cell.     

Alveolar macrophage activation in experimental legionellosis.

Legionella pneumophila is a facultative intracellular parasite of alveolar macrophages. In vitro studies have shown that lymphokine-activated mononuclear phagocytes inhibit intracellular replication of L. pneumophila. To determine if recovery from legionellosis is associated with activation of alveolar macrophages in vivo to resist L. pneumophila, we studied an animal model of Legionnaires' disease. Rats were exposed to aerosolized L. pneumophila and alveolar macrophages were harvested during the recovery phase of infection. We compared these alveolar exudate macrophages with normal resident alveolar macrophages for the capacity to support or inhibit the intracellular growth of L. pneumophila. We also measured Ia expression as a marker of immunologic activation, and studied binding of bacteria, superoxide release, and the expression of transferrin receptors as potential mechanisms of resistance to L. pneumophila. For perspective on the specificity of these responses, we also studied alveolar exudate cells elicited by inhalation of heat-killed L. pneumophila, live Listeria monocytogenes, and live Escherichia coli. We found that alveolar exudate macrophages elicited by live L. pneumophila, but not heat-killed L. pneumophila, resisted the intracellular growth of L. pneumophila. Exudate macrophages in resolving legionellosis exhibited increased Ia expression, diminished superoxide production, and downregulation of transferrin receptors. Binding of L. pneumophila to exudate macrophages was indistinguishable from that to resident macrophages in the presence of normal serum, and augmented in the presence of immune serum. Alveolar exudate macrophages elicited by E. coli also inhibited growth of L. pneumophila, and exhibited a modest increase in Ia expression without change in transferrin receptors. Exudate cells induced by L. monocytogenes exhibited up-regulation of Ia without diminution of superoxide release. Alveolar cells harvested after inhalation of heat-killed L. pneumophila did not differ from resident alveolar macrophages in the expression of surface markers. These findings suggest that alveolar macrophages are immunologically activated in vivo to serve as effector cells in resolving legionellosis, and that live bacteria are required to induce this expression of immunity. The mechanism of resistance to parasitism by L. pneumophila may entail restriction of the intracellular availability of iron, but does not involve diminished bacterial binding or an augmented respiratory burst.     

Type I IFN protects permissive macrophages from Legionella pneumophila infection through an IFN-gamma-independent pathway

Legionella pneumophila is an intracellular pathogen whose replication in macrophages is mainly controlled by IFN-gamma. Freshly isolated peritoneal macrophages elicited in vivo with thioglycolate (TG) from A/J mice are highly permissive to L. pneumophila growth in vitro, while TG-elicited macrophages from CD1 mice are resistant. In this study, we show that when CD1 TG-macrophages are cultured for 7 days, they become permissive to Legionella infection. We demonstrate that treatment with type I IFN (IFN-alphabeta) totally inhibits the growth of L. pneumophila in both freshly isolated A/J and in vitro-aged CD1 TG-macrophages. IFN-alphabeta protective effect on permissive macrophages was comparable to that induced by IFN-gamma. Even low doses of either IFN-alpha or IFN-beta alone were effective in inhibiting L. pneumophila multiplication in macrophage cultures. Notably, treatment of resistant, freshly isolated CD1 TG-macrophages with Ab to mouse IFN-alphabeta significantly enhanced their susceptibility to Legionella infection in vitro, thus implying a role of endogenous IFN-alphabeta in mediating the natural resistance of macrophages to L. pneumophila infection. Finally, addition of anti-IFN-gamma-neutralizing Ab did not restore Legionella growth in IFN-alpha- or IFN-beta-treated A/J or CD1 permissive macrophages, indicating that IFN-alphabeta effect was not mediated by IFN-gamma. This observation was further confirmed by the finding that IFN-alphabeta was effective in inhibiting L. pneumophila replication in macrophages from IFN-gamma receptor-deficient mice. Taken together, our results provide the first evidence for a role of IFN-alphabeta in the control of L. pneumophila infection in mouse models of susceptible macrophages and suggest the existence of different pathways for the control of intracellular bacteria in macrophages.     

Early trafficking and intracellular replication of Legionella longbeachaea within an ER-derived late endosome-like phagosome

Legionella pneumophila is the predominant cause of Legionnaires' disease in the USA and Europe in contrast to Legionella longbeachaea, which is the leading cause of the disease in Western Australia. The ability of L. pneumophila to replicate intracellularly is triggered at the post-exponential phase along with expression of other virulence traits, such as motility. We show that while motility of L. longbeachaea is triggered upon growth transition into post-exponential phase, its ability to proliferate intracellularly is totally independent of the bacterial growth phase. Within macrophages, L. pneumophila replicates in a phagosome that excludes early and late endocytic markers and is surrounded by the rough endoplasmic reticulum (RER). In contrast, the L. longbeachaea phagosome colocalizes with the early endosomal marker early endosomal antigen 1 (EEA1) and the late endosomal markers lysosomal associated membrane glycoprotein 2 (LAMP-2) and mannose 6-phosphate receptor (M6PR), and is surrounded by the RER. The L. longbeachaea phagosome does not colocalize with the vacuolar ATPase (vATPase) proton pump, and the lysosomal luminal protease Cathepsin D, or the lysosomal tracer Texas red Ovalbumin (TROV). Intracellular proliferation of L. longbeachaea occurs in LAMP-2-positive phagosomes that are remodelled by the RER. Despite their distinct trafficking, both L. longbeachaea and L. pneumophila can replicate in communal phagosomes whose biogenesis is predominantly modulated by L. longbeachaea into LAMP-2-positive phagosomes. In addition, the L. pneumophila dotA mutant is rescued for intracellular replication if it co-inhabits the phagosome with L. longbeachaea. During late stages of infection, L. longbeachaea escape into the cytoplasm, prior to lysis of the macrophage, similar to L. pneumophila. We conclude that the L. longbeachaea phagosome matures to a non-acidified late endosome-like stage that is remodelled by the RER, indicating an idiosyncratic trafficking of L. longbeachaea compared with other intracellular pathogens, and a divergence in its intracellular lifestyle from L. pneumophila. In addition, re-routing biogenesis of the L. pneumophila phagosome into a late endosome controlled by L. longbeachaea has no effect on intracellular replication.     

Legionella pneumophila Philadelphia-1 tatB and tatC affect intracellular replication and biofilm formation

Legionella pneumophila is a facultative intracellular human pathogen and an important cause of Legionnaires' disease, a severe form of pneumonia. Recently, we showed the presence of a putative twin-arginine translocation (Tat) pathway in L. pneumophila Philadelphia-1. This secretion pathway is used to transport completely folded proteins across the cytoplasmic membrane. The importance of the Tat pathway in L. pneumophila was investigated by constructing a tatB and a tatC mutant. Functionality of the Tat pathway was shown using a proven heterologous Tat substrate. It was shown that tatB and tatC are involved in intracellular replication in Acanthamoeba castellanii and differentiated U937 cells, and in biofilm forming ability. A putative Legionella Tat substrate was identified via 2D gel electrophoresis.     

RNA interference analysis of Legionella in Drosophila cells: exploitation of early secretory apparatus dynamics

Legionella pneumophila translocates multiple bacterial effector proteins into host cells to direct formation of a replication vacuole for the bacterium. The emerging consensus is that formation of this compartment involves recruitment of membrane material that traffics between the endoplasmic reticulum (ER) and Golgi. To investigate this model, a targeted approach was used to knock down expression of proteins involved in membrane trafficking, using RNA interference in Drosophila cells. Surprisingly, few single knockdowns of ER-Golgi transport proteins decreased L. pneumophila replication. By analyzing double-stranded RNAs in pairs, combinations were identified that together caused defects in intracellular replication, consistent with the model that membrane traffic funnels into the replication vacuole from multiple sources. In particular, simultaneous depletion of the intermediate compartment and Golgi-tethering factor transport protein particle together with the ER SNARE protein Sec22 reduced replication efficiency, indicating that introduction of lesions at distinct sites in the secretory system reduces replication efficiency. In contrast to knockdowns in secretory traffic, which required multiple simultaneous hits, knockdown of single cytosolic components of ER-associated degradation, including Cdc48/p97 and associated cofactors, was sufficient to inhibit intracellular replication. The requirement for the Cdc48/p97 complex was conserved in mammalian cells, in which replication vacuoles showed intense recruitment of ubiquitinated proteins, the preferred substrates of Cdc48/p97. This complex promoted dislocation of both ubiquitinated proteins and bacterial effectors from the replication vacuole, consistent with the model that maintenance of high-level replication requires surveillance of the vacuole surface. This work demonstrates that L. pneumophila has the ability to gain access to multiple sites in the secretory system and provides the first evidence for a role of the Cdc48/p97 complex in promoting intracellular replication of pathogens and maintenance of replication vacuoles.     

Restriction of Legionella pneumophila growth in macrophages requires the concerted action of cytokine and Naip5/Ipaf signalling pathways

Macrophages from the C57BL/6 (B6) mouse strain restrict intracellular growth of Legionella pneumophila, whereas A/J macrophages are highly permissive. The mechanism by which B6 macrophages restrict Legionella growth remains poorly understood, but is known to require the cytosolic microbe sensors Naip5 (Birc1e) and Ipaf. We hypothesized that Naip5 and Ipaf may act in partnership with other antimicrobial signalling pathways in macrophages. Indeed, we found that macrophages lacking either tumour necrosis factor (TNF)-alpha or type I interferon (IFN) signalling are permissive for growth of L. pneumophila, even in the presence of functional Naip5 and Ipaf alleles. Similarly, macrophages lacking Naip5 and/or Ipaf signalling were permissive even though we found that Naip5 or Ipaf were not required for induction of TNF-alpha and type I IFN. Therefore, our data suggest that the mechanism by which B6 macrophages restrict intracellular replication of L. pneumophila is more complex than previously appreciated, and involves the concerted action of cytokine and intracellular microbe sensor signalling pathways.     

Legionella phagosomes intercept vesicular traffic from endoplasmic reticulum exit sites

It is unknown how Legionella pneumophila cells escape the degradative lysosomal pathway after phagocytosis by macrophages and replicate in an organelle derived from the endoplasmic reticulum. Here we show that, after internalization, L. pneumophila-containing phagosomes recruit early secretory vesicles. Once L. pneumophila phagosomes have intercepted early secretory vesicles they begin to acquire proteins residing in transitional and rough endoplasmic reticulum. The functions of Sar1 and ADP-ribosylation factor-1 are important for biogenesis of the L. pneumophila replicative organelle. These data indicate that L. pneumophila intercepts vesicular traffic from endoplasmic-reticulum exit sites to create an organelle that permits intracellular replication and prevents destruction by the host cell.