Francisella tularensis regulates the expression of the amino acid transporter SLC1A5 in infected THP‐1 human monocytes

Francisella tularensis, a Gram‐negative bacterium that causes the disease tularemia in a large number of animal species, is thought to reside preferentially within macrophages in vivo. F. tularensis has developed mechanisms to rapidly escape from the phagosome into the cytoplasm of infected cells, a habitat with a rich supply of nutrients, ideal for multiplication. SLC1A5 is a neutral amino acid transporter expressed by human cells, which serves, along with SLC7A5 to equilibrate cytoplasmic amino acid pools. We herein analysed whether SLC1A5 was involved in F. tularensis intracellular multiplication. We demonstrate that expression of SLC1A5 is specifically upregulated by F. tularensis in infected THP‐1 human monocytes. Furthermore, we show that SLC1A5 downregulation decreases intracellular bacterial multiplication, supporting the involvement of SLC1A5 in F. tularensis infection. Notably, after entry of F. tularensis into cells and during the whole infection, the highly glycosylated form of SLC1A5 was deglycosylated only by bacteria capable of cytosolic multiplication. These data suggest that intracellular replication of F. tularensis depends on the function of host cell SLC1A5. Our results are the first, which show that Francisella intracellular multiplication in human monocyte cytoplasm is associated with a post‐translational modification of a eukaryotic amino acid transporter.     

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.     

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.     

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.     

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.     

Multiple MyD88-dependent responses contribute to pulmonary clearance of Legionella pneumophila

MyD88-dependent signalling is important for secretion of early inflammatory cytokines and host protection in response to Legionella pneumophila infection. Although toll-like receptor (TLR)2 contributes to MyD88-dependent clearance of L. pneumophila , TLR-independent functions of MyD88 could also be important. To determine why MyD88 is critical for host protection to L. pneumophila , the contribution of multiple TLRs and IL-18 receptor (IL-18R)-dependent interferon-gamma (IFN-γ) production in a mouse was examined. Mice deficient for TLR5 or TLR9, or deficient for TLR2 along with either TLR5 or TLR9, were competent for controlling bacterial replication and had no apparent defects in cytokine production compared with control mice. MyD88-dependent production of IFN-γ in the lung was mediated primarily by natural killer cells and required IL-18R signalling. Reducing IFN-γ levels did not greatly affect the kinetics of L. pneumophila replication or clearance in infected mice. Additionally, IFN-γ-deficient mice did not have a susceptibility phenotype as severe as the MyD88-deficient mice and were able to control a pulmonary infection by L. pneumophila . Thus, MyD88-dependent innate immune responses induced by L. pneumophila involve both TLR-dependent responses and IL-18R-dependent production of IFN-γ by natural killer cells, and these MyD88-dependent pathways can function independently to provide host protection against an intracellular pathogen.     

Legionella pneumophila requires polyamines for optimal intracellular growth

The Gram-negative intracellular pathogen Legionella pneumophila replicates in a membrane-bound compartment known as the Legionella-containing vacuole (LCV), into which it abundantly releases its chaperonin, HtpB. To determine whether HtpB remains within the LCV or reaches the host cell cytoplasm, we infected U937 human macrophages and CHO cells with L. pneumophila expressing a translocation reporter consisting of the Bordetella pertussisa denylate cyclase fused to HtpB. These infections led to increased cyclic AMP levels, suggesting that HtpB reaches the host cell cytoplasm. To identify potential functions of cytoplasmic HtpB, we expressed it in the yeast Saccharomyces cerevisiae, where HtpB induced pseudohyphal growth. A yeast-two-hybrid screen showed that HtpB interacted with S-adenosylmethionine decarboxylase (SAMDC), an essential yeast enzyme (encoded by SPE2) that is required for polyamine biosynthesis. Increasing the copy number of SPE2 induced pseudohyphal growth in S. cerevisiae; thus, we speculated that (i) HtpB induces pseudohyphal growth by activating polyamine synthesis and (ii) L. pneumophila may require exogenous polyamines for growth. A pharmacological inhibitor of SAMDC significantly reduced L. pneumophila replication in L929 mouse cells and U937 macrophages, whereas exogenously added polyamines moderately favored intracellular growth, confirming that polyamines and host SAMDC activity promote L. pneumophila proliferation. Bioinformatic analysis revealed that most known enzymes required for polyamine biosynthesis in bacteria (including SAMDC) are absent in L. pneumophila, further suggesting a need for exogenous polyamines. We hypothesize that HtpB may function to ensure a supply of polyamines in host cells, which are required for the optimal intracellular growth of L. pneumophila.     

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.     

Co-ordination of Legionella pneumophila virulence with entry into stationary phase by ppGpp

Legionella pneumophila survives in aquatic environments, but replicates within amoebae or the alveolar macrophages of immunocompromised individuals. Here, the signal transduction pathway that co-ordinates L. pneumophila virulence expression in response to amino acid depletion was investigated. To facilitate kinetic and genetic studies, a phenotypic reporter of virulence was engineered by fusing flaA promoter sequences to a gene encoding green fluorescent protein. When subjected to amino acid depletion, L. pneumophila accumulated ppGpp and converted from a replicative to a virulent state, as judged by motility and sodium sensitivity. ppGpp appeared to initiate this response, as L. pneumophila induced to express the Escherichia coli RelA ppGpp synthetase independently of nutrient depletion accumulated ppGpp, exited the exponential growth phase and expressed flaAgfp, motility, sodium sensitivity, cytotoxicity and infectivity, five traits correlated with virulence. Although coincident with the stationary phase, L. pneumophila virulence expression appeared to require an additional factor: mutant Lp120 accumulated ppGpp and acquired two stationary phase traits but none of six virulence phenotypes analysed. We propose that, when nutrients are limiting, ppGpp acts as an alarmone, triggering the expression of multiple traits that enable L. pneumophila to escape its spent host, to survive and disperse in the environment and to re-establish a protected intracellular replication niche.     

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.     

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.     

Studies on the uptake and intracellular replication of Legionella pneumophila in protozoa and in macrophage-like cells

Abstract Legionella pneumophila strains isolated from different sources were tested for their host range in the protists Acanthamoeba castellanii, Hartmannella vermiformis and Entamoeba histolytica . It has been shown that A. castellanii and H. vermiformis but not E. histolytica support the intracellular replication of L. pneumophila . Furthermore it could be demonstrated that in vivo virulence in the guinea pig and the intracellular growth in U937 cells coincides with the capability to replicate intracellularly in A. castellanii at 37°C. The infectivity of L. pneumophila that had sustained a 48 hours nutrient deprivation was not significantly different from that of legionellae grown to log-phase on BCYE plates. In contrast the nutrient limitation on A. castellanii increased the amount of intracellular legionellae at the beginning of infection. An initial opsonin independent attachement stage of legionellae to U937 cells was demonstrated by scanning electron microscopy. In contrast, L. pneumophila's capability of stable or long term attachmennt to A. castellanii was shown to be inefficient.     

Role of protein kinase C-alpha in the control of infection by intracellular pathogens in macrophages.

The protein kinase C (PKC) family regulates macrophage function involved in host defense against infection. In this study, we investigated the role of macrophage PKC-alpha in the uptake and subsequent fate of Leishmania donovani promastigotes and Legionella pneumophila infections. To this end, we used clones of the murine macrophage cell line RAW 264.7 overexpressing a dominant-negative (DN) mutant of PKC-alpha. While phagocytosis of L. donovani promastigotes was not affected by DN PKC-alpha overexpression, their intracellular survival was enhanced by 10- to 20-fold at 48 h postinfection. Intracellular survival of a L. donovani mutant defective in lipophosphoglycan repeating units synthesis, which normally is rapidly degraded in phagolysosomes, was enhanced by 100-fold at 48 h postinfection. However, IFN-gamma-induced leishmanicidal activity was not affected by DN PKC-alpha overexpression. Similar to macrophages from genetically resistant C57BL/6 mice, control RAW 264.7 cells were not permissive for the intracellular replication of Legionella pneumophila. In contrast, DN PKC-alpha-overexpressing RAW 264.7 clones were phenotypically similar to macrophages from genetically susceptible A/J mice, as they allowed intracellular replication of L. pneumophila. Permissiveness to L. pneumophila was not the consequence of a general defect in the microbicidal capacities because killing of a temperature-sensitive mutant of Pseudomonas aeruginosa was normal in DN PKC-alpha-overexpressing RAW 264.7 clones. Collectively, these results support a role for PKC-alpha in the regulation of innate macrophage functions involved in the control of infection by intracellular parasites.     

Evidence for acquisition of Legionella type IV secretion substrates via interdomain horizontal gene transfer

Intracellular pathogens exploit host cell functions to create a replication niche inside eukaryotic cells. The causative agent of Legionnaires' disease, the gamma-proteobacterium Legionella pneumophila, resides and replicates within a modified vacuole of protozoan and mammalian cells. L. pneumophila translocates effector proteins into host cells through the Icm-Dot complex, a specialized type IVB secretion system that is required for intracellular growth. To find out if some effector proteins may have been acquired through interdomain horizontal gene transfer (HGT), we performed a bioinformatic screen that searched for eukaryotic motifs in all open reading frames of the L. pneumophila Philadelphia-1 genome. We found 44 uncharacterized genes with many distinct eukaryotic motifs. Most of these genes contain G+C biases compared to other L. pneumophila genes, supporting the theory that they were acquired through HGT. Furthermore, we found that several of them are expressed and up-regulated in stationary phase in an RpoS-dependent manner. In addition, at least seven of these gene products are translocated into host cells via the Icm-Dot complex, confirming their role in the intracellular environment. Reminiscent of the case with most Icm-Dot substrates, most of the strains containing mutations in these genes grew comparably to the parent strain intracellularly. Our findings suggest that in L. pneumophila, interdomain HGT may have been a major mechanism for the acquisition of determinants of infection.     

Balamuthia mandrillaris, free-living ameba and opportunistic agent of encephalitis, is a potential host for Legionella pneumophila bacteria

Balamuthia mandrillaris is a free-living ameba and an opportunistic agent of granulomatous encephalitis in humans and other mammalian species. Other free-living amebas, such as Acanthamoeba and Hartmannella, can provide a niche for intracellular survival of bacteria, including the causative agent of Legionnaires' disease, Legionella pneumophila. Infection of amebas by L. pneumophila enhances the bacterial infectivity for mammalian cells and lung tissues. Likewise, the pathogenicity of amebas may be enhanced when they host bacteria. So far, the colonization of B. mandrillaris by bacteria has not been convincingly shown. In this study, we investigated whether this ameba could host L. pneumophila bacteria. Our experiments showed that L. pneumophila could initiate uptake by B. mandrillaris and could replicate within the ameba about 4 to 5 log cycles from 24 to 72 h after infection. On the other hand, a dotA mutant, known to be unable to propagate in Acanthamoeba castellanii, also did not replicate within B. mandrillaris. Approaching completion of the intracellular cycle, L. pneumophila wild-type bacteria were able to destroy their ameboid hosts. Observations by light microscopy paralleled our quantitative data and revealed the rounding, collapse, clumping, and complete destruction of the infected amebas. Electron microscopic studies unveiled the replication of the bacteria in a compartment surrounded by a structure resembling rough endoplasmic reticulum. The course of intracellular infection, the degree of bacterial multiplication, and the ultrastructural features of a L. pneumophila-infected B. mandrillaris ameba resembled those described for other amebas hosting Legionella bacteria. We hence speculate that B. mandrillaris might serve as a host for bacteria in its natural environment.     

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.     

The Legionella pneumophila EnhC Protein Interferes with Immunostimulatory Muramyl Peptide Production to Evade Innate Immunity

Successful pathogens have evolved to evade innate immune recognition of microbial molecules by pattern recognition receptors (PRR), which control microbial growth in host tissues. Upon Legionella pneumophila infection of macrophages, the cytosolic PRR Nod1 recognizes anhydro-disaccharide-tetrapeptide (anhDSTP) generated by soluble lytic transglycosylase (SltL), the predominant bacterial peptidoglycan degrading enzyme, to activate NF-κB-dependent innate immune responses. We show that L.?pneumophila periplasmic protein EnhC, which is uniquely required for bacterial replication within macrophages, interferes with SltL to lower anhDSTP production. L.?pneumophila mutant strains lacking EnhC (ΔenhC) increase Nod1-dependent NF-κB activation in host cells, while reducing SltL activity in?a ΔenhC strain restores intracellular bacterial growth. Further, L.?pneumophila ΔenhC is specifically rescued in Nod1- but not Nod2-deficient macrophages, arguing that EnhC facilitates evasion from Nod1 recognition. These results indicate that?a bacterial pathogen regulates peptidoglycan degradation to control the production of PRR ligands and evade innate immune recognition.     

The Lly protein protects Legionella pneumophila from light but does not directly influence its intracellular survival in Hartmannella vermiformis.

The lly locus (legiolysin) mediates the browning of the culture medium of Legionella pneumophila in the late stationary growth phase, presumably as a result of synthesis of homogentisic acid. Mutagenesis of the lly gene of the L. pneumophila Philadelphia I derivative JR32 did not affect intracellular replication in the natural host Hartmannella vermiformis. The Lly-negative mutant, however, showed a markedly decreased resistance to ordinary light. The cloned lly gene conferred an increased resistance to light in recombinant L. pneumophila and Escherichia coli K-12, indicating a contribution of the Lly protein to ecological adaptation of Legionella species.     

Live Legionella pneumophila induces MUC5AC production by airway epithelial cells independently of intracellular invasion

The airway epithelium is the initial barrier against airborne pathogens, and it plays many roles in host airway defense. Legionella pneumophila is an intracellular pathogen that causes rapidly advancing pneumonia and is sometimes life-threatening. Here, we evaluated the role of the airway epithelial cells in the defense against L.?pneumophila by examining mucus production in vitro. The production of MUC5AC, a major mucin protein, was not induced by formalin- or ultraviolet-killed L.?pneumophila, but it was induced by live L.?pneumophila. Similarly, nuclear factor-kappaB (NF-κB) was activated only by live L.?pneumophila. Inhibitors of ERK and JNK, but not p38, dose-dependently inhibited the induction of MUC5AC by live L.?pneumophila. Inhibition of intracellular invasion by cytochalasin D did not affect MUC5AC production. Taken together, the results suggest that live L.?pneumophila induces MUC5AC production via the ERK-JNK and NF-κB pathways without internalization of bacteria and that the airway epithelium produces mucin as part of the immune response against L.?pneumophila.