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Mitigation of Expression of Virulence Genes in Legionella pneumophila Internalized in the Free-Living Amoeba Willaertia magna C2c Maky
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Abstract
Legionella pneumophila is a human pathogen responsible for a severe form of pneumonia named Legionnaire disease. Its natural habitat is aquatic environments, being in a free state or intracellular parasites of free-living amoebae, such as Acanthamoeba castellanii. This pathogen is able to replicate within some amoebae. Willaertia magna C2c Maky, a non-pathogenic amoeba, was previously demonstrated to resist to L. pneumophila and even to be able to eliminate the L. pneumophila strains Philadelphia, Lens, and Paris. Here, we studied the induction of seven virulence genes of three L. pneumophila strains (Paris, Philadelphia, and Lens) within W. magna C2c Maky in comparison within A. castellanii and with the gene expression level of L. pneumophila strains alone used as controls. We defined a gene expression-based virulence index to compare easily and without bias the transcript levels in different conditions and demonstrated that W. magna C2c Maky did not increase the virulence of L. pneumophila strains in contrast to A. castellanii. These results confirmed the non-permissiveness of W. magna C2c Maky toward L. pneumophila strains.
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Secreted Bacterial Effectors That Inhibit Host Protein Synthesis Are Critical for Induction of the Innate Immune Response to Virulent Legionella pneumophila
Mary Fontana, Simran Banga, Kevin C. Barry … (2011)
Introduction In metazoans, the innate immune system senses infection through the use of germline-encoded pattern recognition receptors (PRRs) that detect pathogen-associated molecular patterns (PAMPs), such as lipopolysaccharide or flagellin [1]. PAMPs are conserved molecules that are found on non-pathogenic and pathogenic microbes alike, and consequently, even commensal microbes are capable of activating PRRs [2]. Thus, it has been proposed that additional innate immune mechanisms may exist to discriminate between pathogens and non-pathogens [3], [4]. In plants, selective recognition of pathogens is accomplished by detection of the enzymatic activities of “effector” molecules that are delivered specifically by pathogens into host cells. Typically, the effector is an enzyme that disrupts host cell signaling pathways to the benefit of the pathogen. Host sensors monitoring or “guarding” the integrity of the signaling pathway are able to detect the pathogen-induced disruption and initiate a protective response. This mode of innate recognition is termed “effector-triggered immunity” [5] and represents a significant component of the plant innate immune response. It has been suggested that innate recognition of pathogen-encoded activities, which have been termed “patterns of pathogenesis” in metazoans [3], could act in concert with PRRs to distinguish pathogens from non-pathogens, leading to qualitatively distinct responses that are commensurate with the potential threat. However, few if any examples of “patterns of pathogenesis” have been shown to elicit innate responses in metazoans. The gram negative bacterial pathogen Legionella pneumophila provides an excellent model to address whether metazoans respond to pathogen-encoded activities in addition to PAMPs. L. pneumophila replicates in the environment within amoebae [6], but can also replicate within alveolar macrophages in the mammalian lung [7], where it causes a severe inflammatory pneumonia called Legionnaires' Disease [6]. Because its evolution has occurred primarily or exclusively in amoebae, L. pneumophila appears not to have evolved significant immune-evasive mechanisms. Indeed, most healthy individuals mount a robust protective inflammatory response to L. pneumophila, resulting from engagement of multiple redundant innate immune pathways [8]. We hypothesize, therefore, that as a naïve pathogen, L. pneumophila may reveal novel innate immune responses that better adapted pathogens may evade or disable [9]. In host cells, L. pneumophila multiplies within a specialized replicative vacuole, the formation of which is orchestrated by bacterial effector proteins translocated into the host cytosol via the Dot/Icm type IV secretion system [10]. In addition to its essential roles in bacterial replication and virulence, the Dot/Icm system also translocates bacterial PAMPs, such as flagellin, nucleic acids, or fragments of peptidoglycan, that activate cytosolic immunosurveillance pathways [8], [11], [12], [13], [14], [15], [16]. There are also recent suggestions in the literature that Dot/Icm+ L. pneumophila may stimulate additional, uncharacterized immunosurveillance pathways [8], [17]. Overall, the molecular basis of the host response to Dot/Icm+ L. pneumophila remains poorly understood. Here we show that macrophages infected with virulent L. pneumophila make a unique transcriptional response to a bacterial activity that disrupts a vital host process. We show that this robust transcriptional response requires the Dot/Icm system, and cannot be explained solely by known PAMP-sensing pathways. Instead, we provide evidence that the response requires the enzymatic activity of five secreted bacterial effectors that inhibit host protein synthesis. Effector-dependent inhibition of protein synthesis synergized with PRR signaling to elicit the full transcriptional response to L. pneumophila. The response to the bacterial effectors could be recapitulated through the use of pharmacological agents or toxins that inhibit host translation, administered in conjunction with a PRR agonist. Thus, our results provide a striking example of a host response that is shaped not only by PAMPs but also by a complementary “effector-triggered” mechanism that represents a novel mode of immune responsiveness in metazoans. Results Induction of an ‘effector-triggered’ transcriptional signature in macrophages infected with virulent L. pneumophila We initially sought to identify host responses that discriminate between pathogenic and non-pathogenic bacteria. Our strategy was to compare the host response to wildtype virulent L. pneumophila with the host response to an avirulent L. pneumophila mutant, ΔdotA. ΔdotA mutants lack a functional Dot/Icm secretion system, and thus fail to translocate effectors into the host cytosol, but they nevertheless express the normal complement of PAMPs that engage Toll-like receptor pathways. We performed transcriptional profiling experiments on macrophages infected with either wildtype L. pneumophila or the avirulent ΔdotA mutant. In the microarray experiments, Caspase-1−/− macrophages were used to eliminate flagellin-dependent macrophage death, which would otherwise differ between wildtype and ΔdotA infections [12], [14], [16], but our results were later validated with wildtype macrophages (see below). RNA was collected from macrophages at a timepoint when there were similar numbers of bacteria in both wildtype-infected and ΔdotA-infected macrophages. Microarray analysis revealed 166 genes that were differentially induced >2-fold in a manner dependent on type IV secretion (Figure 1A and Table S1). The induction of some of the Dot/Icm-dependent genes, e.g. Ifnb, could be explained by cytosolic sensing pathways that have been previously characterized [11], [13], [18]. However, much of the response to Dot/Icm+ bacteria did not appear to be accounted for by host pathways known to recognize L. pneumophila. For reasons discussed below, we refer specifically to this unexplained Dot/Icm-dependent transcriptional signature as the ‘effector-triggered response,’ or ETR. 10.1371/journal.ppat.1001289.g001 Figure 1 A unique transcriptional response in macrophages infected with virulent L. pneumophila. (A) Caspase-1−/− macrophages were infected for 6 h with the specified strains of L. pneumophila. RNA was amplified and hybridized to MEEBO microarrays. Black and red dots, genes exhibiting greater than 2-fold difference in induction between wildtype (WT) and mutant. Red dots indicate labeled genes. Data shown are the average of two experiments. (B) B6 macrophages were infected for 6 h with the specified strains of L. pneumophila. Levels of the indicated transcripts were measured by quantitative RT-PCR. (C) Mice were infected intranasally with 2×106 L. pneumophila and bronchoalveolar lavage was performed 24 h post infection. Host cells recovered from bronchoalveolar lavage fluid (BALF) were counted with a hemocytometer. A portion of each sample was plated on BCYE plates to enumerate cfu. (D) Macrophages were infected for 6 h with L. pneumophila. Levels of the indicated transcripts were measured by quantitative RT-PCR. N.S., not significant. Data shown are representative of two (a, d) or at least three (B, C) experiments (mean ± sd in b, d). *, p 1000-fold more by pathogenic wildtype L. pneumophila as compared to the ΔdotA mutant (Figure 1B). In subsequent experiments we focused on these three genes, as they provided a sensitive readout of the ETR. To assess whether the ETR might be important during L. pneumophila infection in vivo, we infected B6 and Il23a −/− mice intranasally with L. pneumophila. Il23a −/− mice displayed a significant defect in host cell recruitment to the lungs 24 hours after infection (Figure 1C), consistent with the known role of IL-23 in neutrophil recruitment to sites of infection [19]. The phenotype of Il23a −/− mice was not due to decreased bacterial burden in these mice (Figure 1C). Thus at least one transcriptional target of the ETR plays a role in the host response, though there are clearly numerous redundant pathways that recognize L. pneumophila in vivo [8]. Known innate immune pathways are not sufficient to induce the full ‘effector-triggered response’ In order to identify the host pathway(s) responsible for induction of the ETR, we first examined innate immune pathways known to recognize L. pneumophila. Induction of the representative genes Il23a, Csf2, and Gem did not require the previously described Naip5/Nlrc4 flagellin-sensing pathway [20], as infection with a flagellin-deficient mutant (ΔflaA) also induced robust expression of these genes (Figure 1A, B and Table S2). Moreover, Il23a, Csf2 and Gem were strongly (>1000-fold) induced in the absence of the Mavs/Irf3/Irf7 signaling axis shown previously to respond to L. pneumophila [11], [13], [18] (Figure 1D, and data not shown). As suggested by previous transcriptional profiling experiments [17], we confirmed that Myd88 −/−and Rip2 −/−macrophages, which are defective in TLR and Nod1/Nod2 signaling, respectively, strongly upregulated Il23a and Gem following infection with wildtype L. pneumophila (Figure 2A). Induction of Il23a was abrogated in Myd88 −/− Rip2 −/− and Myd88 −/− Nod1 −/− Nod2 −/− macrophages; however, these macrophages still robustly induced Gem (Figure 2A, and data not shown). These data indicate that TLR/Nod signaling is necessary for induction of some, but not all, genes in the ETR. Furthermore, the intact induction of Gem in Myd88 −/− Nod1 −/− Nod2 −/− macrophages implies the existence of an additional pathway. 10.1371/journal.ppat.1001289.g002 Figure 2 MyD88 and Nod signaling alone do not account for the unique response to virulent L. pneumophila, which can be recapitulated by ER stress inducers that also inhibit translation. In all panels, the indicated transcripts were measured by quantitative RT-PCR. (A) Macrophages were infected with ΔflaA L. pneumophila for 6 h. (B) Macrophages were infected with L. pneumophila or were treated with Pam3CSK4 (10 ng/mL) and/or transfected with MDP (10 µg/mL) for 6 h. (C) B6 macrophages were infected with L. pneumophila, wildtype L. monocytogenes or the avirulent L. monocytogenes Δhly mutant for 4 h. (D) B6 macrophages were infected with the indicated strains of L. pneumophila for 6 h. **, p 50 genes, including Il23a, Gem, and Csf2, that constitute an ‘effector-triggered’ response. We propose that at least some of these genes are superinduced upon the sustained activation of transcription factors such as NF-κB, although it is important to emphasize that the host response to protein synthesis inhibition is complex and likely involves other pathways as well, such as MAP kinase activation (data not shown). Interestingly, we observed that not all NF-κB-dependent target genes are superinduced by translation inhibition. For example, Nfkbia (encoding the IκB protein) was not superinduced in wildtype L. pneumophila infection (Figure 5F). This selective superinduction of certain target genes may be significant, since it allows the host to respond to a pathogen-dependent stress by altering not only the magnitude but also the composition of the transcriptional response. Moreover, if IκB were superinduced, this would presumably act to reverse or prevent sustained NF-κB signaling, resulting in little net gain. The mechanism by which prolonged NF-κB signaling may preferentially enhance transcription of the specific subset of effector-triggered genes is not yet clear. However, recent studies have shown that the chromatin context for several of these genes (e.g., Il23a, Csf2) is in a relatively ‘closed’ conformation [34], [35]. This may render the genes refractory to strong transcriptional induction under a normal TLR stimulus, but enable them to become highly induced upon prolonged NF-κB activation. It is interesting to note that genes such as Il23a and Csf2 are classified as ‘primary’ response genes [34], [35] simply because they are inducible in the presence of cycloheximide. What is not often discussed is the possibility, demonstrated here, that inhibition of protein synthesis by cycloheximide is a key stimulus that induces transcription of these genes. The consequences of the host response to translation inhibition are likely to be difficult to measure in the context of a microbial infection in vivo. Presumably, most pathogens that disrupt host translation derive benefit from this activity, perhaps by increasing availability of amino acid nutrients or by dampening production of the host response. These benefits may be offset by an enhanced host response to translation inhibition itself. It is possible that the robust innate immune response to translation inhibition serves primarily to compensate for the decrease in translation, resulting in little net change in the output of the immune response. Accordingly, the lack of an apparent phenotype during in vivo infection with Δ5 may reflect the sum of multiple positive and negative effects that result from translation inhibition. Additionally, as suggested by our data (Figure S4) the response to L. pneumophila in vivo may involve non-macrophage cell types in which translation inhibition does not play a crucial role. While PRR-based sensing of microbial molecules is certainly a fundamental mode of innate immune recognition, it is not clear how PRRs alone might be able to distinguish pathogens from non-pathogens, and thereby mount responses commensurate with the potential threat. Our results demonstrate that pathogen-mediated interference with a key host process (i.e., host protein synthesis), in concert with PRR signaling, results in an immune response that is qualitatively distinct from the response to an avirulent microbe. Although induction of some genes in the ETR (e.g., Gem) occurs in response to inhibition of protein synthesis alone, much of the ETR is due to the combined effects of PAMP recognition and effector-dependent inhibition of protein synthesis. A requirement for two signals might be rationalized by the fact that the ETR includes potent inflammatory cytokines such as GM-CSF or IL-23, which can drive pathological inflammation [36] and autoimmunity [37] if expressed inappropriately. Restricting production of potentially dangerous cytokines to instances where a pathogenic microbe is present may be a strategy by which hosts avoid self-damage unless necessary for self-defense. Thus, we propose that the host response to a harmful pathogen-encoded activity may represent a general mechanism by which the immune systems of metazoans distinguish pathogens from non-pathogens. Materials and Methods Ethics statement This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Animal Care and Use Committee at the University of California, Berkeley (Protocol number R301-0311BCR). Mice and cell culture Macrophages were derived from the bone marrow of the following mouse strains: C57BL/6J (Jackson Labs), A20 −/− (A. Ma, UCSF), Caspase-1 −/− (M. Starnbach, Harvard Medical School), Mavs −/− (Z. Chen, University of Texas SW), Irf3/Irf7 −/− (K. Fitzgerald, U. Mass Medical School), Myd88 −/− (G. Barton, UC Berkeley), Rip2 −/− (M. Kelliher, U. Mass Medical School), Myd88 −/− Rip2 −/− (C. Roy, Yale University), and Myd88 −/− Nod1 −/− Nod2 −/− (generated from crosses at UC Berkeley). Il23a−/− mice were from N. Ghilardi (Genentech). Macrophages were derived from bone marrow by 8d culture in RPMI supplemented with 10% serum, 100 µM streptomycin, 100 U/mL penicillin, 2 mM L-glutamine, and 10% supernatant from 3T3-M-CSF cells, with feeding on day 5. Dendritic cells were derived from B6 bone marrow by 6d culture in RPMI supplemented with 10% serum, 100 µM streptomycin, 100 U/mL penicillin, 2 mM glutamine, and recombinant GM-CSF (1:1000, PeproTech). Dictyostelium discoideum amoebae were cultured at 21°C in HL-5 medium (0.056 M glucose, 0.5% yeast extract, 0.5% proteose peptone, 0.5% thiotone, 2.5 mM Na2HPO4, 2.5 mM KH2PO4, pH 6.9). Bacterial strains The L. pneumophila wildtype strain LP02 is a streptomycin-resistant thymidine auxotroph derived from L. pneumophila LP01. The ΔdotA, ΔflaA, ΔicmS and ΔicmW mutants have been described [14], [15]. Mutants lacking one or more effectors were generated from LP02 by sequential in-frame deletion using the suicide plasmid pSR47S as described [24]. Sequences of primers used for constructing deletion plasmids are listed in Table S3. Mutants were complemented with the indicated effectors expressed from the L. pneumophila sidF promoter in the plasmid pJB908, which encodes thymidine synthetase as a selectable marker. L. monocytogenes strain 10403S and the isogenic Δhly mutant have been described [21]. Microarrays Macrophage RNA from 1.5×106 cells (6 well dishes) was isolated using the Ambion RNAqueous Kit (Applied Biosystems) and amplified with the Ambion Amino Allyl MessageAmp II aRNA Amplification Kit (Applied Biosystems) according to the manufacturer's protocol. Microarrays were performed as described [38]. Briefly, spotted microarrays utilizing the MEEBO 70-mer oligonucleotide set (Illumina) were printed at the UCSF Center for Advanced Technology. Microarray probes were generated by coupling amplified RNA to Cy dyes. After hybridization, arrays were washed, scanned on a GenePix 4000B Scanner (Molecular Devices), and gridded using SpotReader software (Niles Scientific). Analysis was performed using the GenePix Pro 6 and Acuity 4 software packages (Molecular Devices). Two independent experiments were performed. Microarray data have been deposited in the Gene Expression Omnibus database (http://www.ncbi.nlm.nih.gov/geo/) under the accession number GSE26491. Infection and stimulation Macrophages were plated in 6 well dishes at a density of 1.5×106 cells per well and infected at an MOI of 1 by centrifugation for 10 min at 400× g, or were treated with puromycin, thapsigargin, tunicamycin, cycloheximide (all Sigma), Exotoxin A (List Biological Labs), transfected synthetic muramyl-dipeptide (MDP) (CalBiochem), or a synthetic bacterial lipopeptide (Pam3CSK4) (Invivogen). Dendritic cells were plated at a density of 106 cells per well and infected at an MOI of 2 as described above. Lipofectamine 2000 (Invitrogen) was used for transfections. Bruceantin was the kind gift of S. Starck and N. Shastri (UC Berkeley), who obtained it from the National Cancer Institute, NIH (Open Repository NSC165563). A fusion of diphtheria toxin to the lethal factor translocation signal (LFn-DT) was the gift of B. Krantz (UC Berkeley) and was delivered to cells via the pore formed by anthrax protective antigen (PA) as described [39]. Quantitative RT-PCR Macrophage RNA was harvested 4-6 hours post infection, as indicated, and isolated with the RNeasy kit (Qiagen) according to the manufacturer's protocol. RNA samples were treated with RQ1 DNase (Promega) prior to reverse transcription with Superscript III (Invitrogen). cDNA reactions were primed with poly dT for measurement of mature transcripts, and with random hexamers (Invitrogen) for measurement of unspliced transcripts. Quantitative PCR was performed as described [13] using the Step One Plus RT PCR System (Applied Biosystems) with Platinum Taq DNA polymerase (Invitrogen) and EvaGreen (Biotium). Transcript levels were normalized to Rps17. Primer sequences are listed in Table S5. mRNA stabilization assay Macrophages were infected in 6-well dishes at an MOI of 1, as described above. The transcription inhibitor Actinomycin D (10 µg/mL, Sigma) was added 4 hours post infection. RNA was harvested at successive timepoints and levels of indicated transcripts were assessed by quantitative RT-PCR. In vivo experiments Age- and sex-matched B6 or Il23a−/− mice were anesthetized with ketamine and infected intranasally with 2×106 LP01 in 20 µL PBS essentially as described [13], or were treated with ExoA or Pam3CSK4 in 25 µL PBS. Bronchoalveolar lavage was performed 24 hours post infection by introducing 800 µL PBS into the trachea with a catheter (BD Angiocath 18 g, 1.3×48 mm). Lavage fluid was analyzed by ELISA. Total host cells in the lavage were counted on a hemocytometer. For RT-PCR experiments, all lavage samples receiving identical treatments were pooled, and RNA was isolated from the pooled cells using the RNeasy Kit as described above. FACS analysis of lavage samples labeled with anti-GR-1-PeCy7 and anti-Ly6G-PE (eBioscience) indicated that most cells in lavage were neutrophils. CFU were enumerated by hypotonic lysis of host cells in the lavage followed by plating on CBYE plates. Western blots Macrophages were plated in 6 well dishes at a density of 2×106 cells per well and infected at an MOI of 2. For whole cell extract, cells were lysed in RIPA buffer supplemented with 2 mM NaVO3, 1 mM PMSF, 1 mM DTT, and 1 X Complete Protease Inhibitor Cocktail (Roche). For nuclear translocation experiments, nuclear and cytosolic fractions were obtained using the NE-PER kit (Pierce) according to the manufacturer's protocol. Protein levels were normalized using the micro-BCA kit (Pierce) and then separated on 10% NuPAGE bis-tris gels (Invitrogen). Proteins were transferred to PVDF membranes and immunoblotted with antibodies to IκBα, NF-κB p65, lamin-B or β-actin (all Santa Cruz). ELISA Macrophages were plated in 24 well dishes at a density of 5×105 cells per well and infected at an MOI of 1. After 24 h, supernatants were collected, sterile-filtered, and analyzed by ELISA using paired GM-CSF antibodies (eBioscience). For quantification of intracellular GM-CSF, ELISAs were performed using cytoplasmic extract of macrophages infected for 6 h with the indicated strains. Levels of GM-CSF were normalized to total protein concentration. Recombinant GM-CSF (eBioscience) was used as a standard. Growth in bone marrow derived macrophages Intracellular bacterial growth of wildtype and mutant L. pneumophila was evaluated in A/J macrophages as described [24]. Growth in amoebae D. discoideum was plated into 24-well plates at a density of 5×105 cells per well in MB medium (modified HL-5 medium, without glucose and with 20 mM MES buffer) three hours before infection with the indicated L. pneumophila strains at an MOI of 0.05. The plates were spun at 1000 rpm for 5 minutes and incubated at 25°C. After two hours, wells were washed 3X with PBS to synchronize the infection. At successive time points, infected cells were lysed with 0.2% saponin and bacterial growth was determined by plating on growth medium. Protein synthesis assay 2×106 macrophages were seeded in 6-well plates and infected with bacterial strains at an MOI of 2. After 2.5 h, the infected cells were incubated with 1 µCi 35S-methionine (Perkin Elmer) in RPMI-met (Invitrogen). After chase-labeling for an hour, the cells were washed 3× with PBS, lysed with 0.1% SDS and precipitated with TCA [24]. The protein precipitates were filtered onto 0.45 mm Millipore membranes and washed twice with PBS. Retained 35S was determined by a liquid scintillation counter. Cytotoxicity assay Macrophages were plated in 96 well dishes at a density of 5×104 cells per well and infected at an MOI of 1. At successive timepoints, Neutral Red (Sigma) was added to a final concentration of 1% and incubated for 1 h. Cells were then washed with PBS, photographed, and counted [14]. Supporting Information Figure S1 Genetic maps of the five deleted effectors. Numbers refer to the nucleotide position in the published L. pneumophila LP01 genome (GenBank Accession #AE017354). (0.67 MB TIF) Click here for additional data file. Figure S2 New transcription and mRNA stabilization of ETR target genes. (A) After a 6h infection in B6 macrophages, de novo transcription of the indicated genes was measured by quantitative RT-PCR with primers that specifically targeted the pre-spliced mRNA. (B) To assess RNA stability, the transcription inhibitor Actinomycin D (10μg/mL) was added to macrophages 4h post infection. RNA was collected at successive timepoints, and transcripts were measured by quantitative RT-PCR. Results are representative of two to three experiments (mean ± sd). (0.56 MB TIF) Click here for additional data file. Figure S3 Cytotoxicity assay and measurement of intracellular GM-CSF in macrophages infected with ΔflaA or Δ5ΔflaA L. pneumophila. (A) B6 macrophages were infected at an MOI of 1. At indicated timepoints, the number of surviving cells was determined by Neutral Red assay. Bacteria lacking flagellin were used to avoid caspase-1-dependent cell death. (B) Intracellular GM-CSF levels were measured by performing ELISA on cytoplasmic extracts of macrophages infected for 6h with the indicated strains. Results are representative of two experiments (mean ± sd in A). (0.60 MB TIF) Click here for additional data file. Figure S4 Induction of Il23a, Gem, and Csf2 in dendritic cells occurs independently of Type IV secretion. B6 bone marrow derived dendritic cells were infected with the indicated strains at an MOI of 2. After 6h, RNA was harvested and transcripts were measured by quantitative RT-PCR. Results are representative of two experiments (mean ± sd). (0.52 MB TIF) Click here for additional data file. Figure S5 In vivo induction of Csf2 and Gem by translation inhibition. Quantitative RT-PCR measurement of Csf2 and Gem expression in bronchoalveolar lavage cells collected from mice 24h after intranasal treatment with ExoA and/or Pam3CSK4. Results are representative of two experiments (mean ± sd). (0.47 MB TIF) Click here for additional data file. Table S1 Genes induced or repressed twofold or more in caspase-1−/− macrophages infected with wildtype or ΔdotA L. pneumophila. (0.31 MB XLS) Click here for additional data file. Table S2 Genes induced or repressed twofold or more in caspase-1−/− macrophages infected with wildtype or ΔflaA L. pneumophila. (0.32 MB XLS) Click here for additional data file. Table S3 Deleted gene information and deletion primers for the Δ5 strain. (0.04 MB DOC) Click here for additional data file. Table S4 Genes induced or repressed twofold or more in caspase-1−/− macrophages infected with wildtype or Δ5 L. pneumophila. (0.43 MB XLS) Click here for additional data file. Table S5 Quantitative RT-PCR primer sequences used in this study. (0.04 MB DOC) Click here for additional data file.
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- Abstract: found
- Article: not found
Legionella pneumophila inhibits macrophage apoptosis by targeting pro-death members of the Bcl2 protein family.
Ping Gao, Zhao-Qing Luo, Lingling Chen … (2007)
To establish a vacuole that supports bacterial replication, Legionella pneumophila translocates a large number of bacterial proteins into host cells via the Dot/Icm type IV secretion system. Functions of most of these translocated proteins are unknown, but recent investigations suggest their roles in modulating diverse host processes such as vesicle trafficking, autophagy, ubiquitination, and apoptosis. Cells infected by L. pneumophila exhibited resistance to apoptotic stimuli, but the bacterial protein directly involved in this process remained elusive. We show here that SidF, one substrate of the Dot/Icm transporter, is involved in the inhibition of infected cells from undergoing apoptosis to allow maximal bacterial multiplication. Permissive macrophages harboring a replicating sidF mutant are more apoptotic and more sensitive to staurosporine-induced cell death. Furthermore, cells expressing SidF are resistant to apoptosis stimuli. SidF contributes to apoptosis resistance in L. pneumophila-infected cells by specifically interacting with and neutralizing the effects of BNIP3 and Bcl-rambo, two proapoptotic members of Bcl2 protein family. Thus, inhibiting the functions of host pro-death proteins by translocated effectors constitutes a mechanism for L. pneumophila to protect host cells from apoptosis.
- Record: found
- Abstract: found
- Article: not found
Growth of Legionella pneumophila in Acanthamoeba castellanii enhances invasion.
L. Tompkins, Vincent J Cirillo, S Falkow (1994)
Legionella pneumophila is considered to be a facultative intracellular parasite. Therefore, the ability of these bacteria to enter, i.e., invade, eukaryotic cells is expected to be a key pathogenic determinant. We compared the invasive ability of bacteria grown under standard laboratory conditions with that of bacteria grown in Acanthamoeba castellanii, one of the protozoan species that serves as a natural host for L. pneumophila in the environment. Amoeba-grown L. pneumophila cells were found to be at least 100-fold more invasive for epithelial cells and 10-fold more invasive for macrophages and A. castellanii than were L. pneumophila cells grown on agar. Comparison of agar- and amoeba-grown L. pneumophila cells by light and electron microscopy demonstrated dramatic differences in the morphology and structure of the bacteria. Analyses of protein expression in the two strains of bacteria suggest that these phenotypic differences may be due to the expression of new proteins in amoeba-grown L. pneumophila cells. In addition, the amoeba-grown bacteria were found to enter macrophages via coiling phagocytosis at a higher frequency than agar-grown bacteria did. Replication of L. pneumophila in protozoans present in domestic water supplies may be necessary to produce bacteria that are competent to enter mammalian cells and produce human disease.
Author and article information
Journal
Journal ID (nlm-ta): Pathogens
Journal ID (iso-abbrev): Pathogens
Journal ID (publisher-id): pathogens
Title:
Pathogens
Publisher:
MDPI
ISSN
(Electronic):
2076-0817
Publication date
(Electronic):
05
June
2020
Publication date Collection:
June
2020
Volume: 9
Issue: 6
Electronic Location Identifier: 447
Affiliations
[2
]Lyon Neuroscience Research Center CRNL UMR5292 U1028, University of Lyon, Univ Lyon
1, CNRS, Inserm, 69500 Bron, France;
jacques.bodennec@
123456univ-lyon1.fr
(J.B.);
laurent.bezin@
123456univ-lyon1.fr
(L.B.)
Author notes
Author information
Sandrine Demanèche
https://orcid.org/0000-0002-7430-9209
Article
Publisher ID: pathogens-09-00447DOI: 10.3390/pathogens9060447
PMC ID: 7350332
PubMed ID: 32517040
SO-VID: 5b97345c-d0a1-42a0-a47a-2d79b78306ff
Copyright © © 2020 by the authors.
License:
Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license ( http://creativecommons.org/licenses/by/4.0/).
History
Date
received
: 14
May
2020
Date
accepted
: 03
June
2020
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Subject:
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