ABSTRACT
Biofilms are known to be critical for Legionella settlement in engineered water systems and are often associated with Legionnaire's Disease events. One of the key features of biofilms is their heterogeneous three-dimensional structure which supports the establishment of microbial interactions and confers protection to microorganisms. This work addresses the impact of Legionella pneumophila colonization of a Pseudomonas fluorescens biofilm, as information about the interactions between Legionella and biofilm structures is scarce. It combines a set of meso- and microscale biofilm analyses (Optical Coherence Tomography, Episcopic Differential Interference Contrast coupled with Epifluorescence Microscopy and Confocal Laser Scanning Microscopy) with PNA-FISH labelled L. pneumophila to tackle the following questions: (a) does the biofilm structure change upon L. pneumophila biofilm colonization?; (b) what happens to L. pneumophila within the biofilm over time and (c) where is L. pneumophila preferentially located within the biofilm? Results showed that P. fluorescens structure did not significantly change upon L. pneumophila colonization, indicating the competitive advantage of the first colonizer. Imaging of PNA-labelled L. pneumophila showed that compared to standard culture recovery it colonized to a greater extent the 3-day-old P. fluorescens biofilms, presumably entering in VBNC state by the end of the experiment. L. pneumophila was mostly located in the bottom regions of the biofilm, which is consistent with the physiological requirements of both bacteria and confers enhanced Legionella protection against external aggressions. The present study provides an expedited methodological approach to address specific systematic laboratory studies concerning the interactions between L. pneumophila and biofilm structure that can provide, in the future, insights for public health Legionella management of water systems.
Subject(s)
Biofilms , Legionella pneumophila , Pseudomonas fluorescens , Biofilms/growth & development , Legionella pneumophila/physiology , Pseudomonas fluorescens/physiology , Legionella/physiology , Microscopy, Confocal , Tomography, Optical CoherenceABSTRACT
The causative agent of Legionnaires' disease, Legionella pneumophila, is an environmental bacterium, that replicates in macrophages, parasitizes amoeba, and forms biofilms. L. pneumophila employs the Legionella quorum sensing (Lqs) system and the transcription factor LvbR to control various bacterial traits, including virulence and biofilm architecture. LvbR negatively regulates the nitric oxide (NO) receptor Hnox1, linking quorum sensing to NO signaling. Here, we assessed the response of L. pneumophila to NO and investigated bacterial receptors underlying this process. Chemical NO donors, such as dipropylenetriamine (DPTA) NONOate and sodium nitroprusside (SNP), delayed and reduced the expression of the promoters for flagellin (PflaA) and the 6S small regulatory RNA (P6SRNA). Marker-less L. pneumophila mutant strains lacking individual (Hnox1, Hnox2, or NosP) or all three NO receptors (triple knockout, TKO) grew like the parental strain in media. However, in the TKO strain, the reduction of PflaA expression by DPTA NONOate was less pronounced, suggesting that the NO receptors are implicated in NO signaling. In the ΔnosP mutant, the lvbR promoter was upregulated, indicating that NosP negatively regulates LvbR. The single and triple NO receptor mutant strains were impaired for growth in phagocytes, and phenotypic heterogeneity of non-growing/growing bacteria in amoebae was regulated by the NO receptors. The single NO receptor and TKO mutant strains showed altered biofilm architecture and lack of response of biofilms to NO. In summary, we provide evidence that L. pneumophila regulates virulence, intracellular phenotypic heterogeneity, and biofilm formation through NO and three functionally non-redundant NO receptors, Hnox1, Hnox2, and NosP. IMPORTANCE: The highly reactive diatomic gas molecule nitric oxide (NO) is produced by eukaryotes and bacteria to promote short-range and transient signaling within and between neighboring cells. Despite its importance as an inter-kingdom and intra-bacterial signaling molecule, the bacterial response and the underlying components of the signaling pathways are poorly characterized. The environmental bacterium Legionella pneumophila forms biofilms and replicates in protozoan and mammalian phagocytes. L. pneumophila harbors three putative NO receptors, one of which crosstalks with the Legionella quorum sensing (Lqs)-LvbR network to regulate various bacterial traits, including virulence and biofilm architecture. In this study, we used pharmacological, genetic, and cell biological approaches to assess the response of L. pneumophila to NO and to demonstrate that the putative NO receptors are implicated in NO detection, bacterial replication in phagocytes, intracellular phenotypic heterogeneity, and biofilm formation.
Subject(s)
Bacterial Proteins , Biofilms , Gene Expression Regulation, Bacterial , Legionella pneumophila , Nitric Oxide , Signal Transduction , Biofilms/growth & development , Legionella pneumophila/genetics , Legionella pneumophila/pathogenicity , Legionella pneumophila/physiology , Legionella pneumophila/metabolism , Nitric Oxide/metabolism , Virulence , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Phenotype , Macrophages/microbiology , Quorum SensingABSTRACT
Phthalates constitute a family of anthropogenic chemicals developed to be used in the manufacture of plastics, solvents, and personal care products. Their dispersion and accumulation in many environments can occur at all stages of their use (from synthesis to recycling). However, many phthalates together with other accumulated engineered chemicals have been shown to interfere with hormone activities. These compounds are also in close contact with microorganisms that are free-living, in biofilms or in microbiota, within multicellular organisms. Herein, the activity of several phthalates and their substitutes were investigated on the opportunistic pathogen Legionella pneumophila, an aquatic microbe that can infect humans. Beside showing the toxicity of some phthalates, data suggested that Acetyl tributyl citrate (ATBC) and DBP (Di-n-butyl phthalate) at environmental doses (i.e. 10-6 M and 10-8 M) can modulate Legionella behavior in terms of motility, biofilm formation and response to antibiotics. A dose of 10-6 M mostly induced adverse effects for the bacteria, in contrast to a dose of 10-8 M. No perturbation of virulence towards Acanthamoeba castellanii was recorded. These behavioral alterations suggest that L. pneumophila is able to sense ATBC and DBP, in a cross-talk that either mimics the response to a native ligand, or dysregulates its physiology.
Subject(s)
Legionella pneumophila , Legionella , Phthalic Acids , Humans , Legionella pneumophila/physiology , Phthalic Acids/pharmacology , BiofilmsABSTRACT
The host type I interferon (IFN) response protects against Legionella pneumophila infections. Other bacterial pathogens inhibit type I IFN-mediated cell signaling; however, the interaction between this signaling pathway and L. pneumophila has not been well described. Here, we demonstrate that L. pneumophila inhibits the IFN-ß signaling pathway but does not inhibit IFN-γ-mediated cell signaling. The addition of IFN-ß to L. pneumophila-infected macrophages limited bacterial growth independently of NOS2 and reactive nitrogen species. The type IV secretion system of L. pneumophila is required to inhibit IFN-ß-mediated cell signaling. Finally, we show that the inhibition of the IFN-ß signaling pathway occurs downstream of STAT1 and STAT2 phosphorylation. In conclusion, our findings describe a novel host cell signaling pathway inhibited by L. pneumophila via its type IV secretion system.
Subject(s)
Interferon Type I , Legionella pneumophila , Legionnaires' Disease , Humans , Legionella pneumophila/physiology , Type IV Secretion Systems , Interferon-gamma/metabolism , Signal TransductionABSTRACT
Legionella pneumophila is an environmental bacterium, which replicates in amoeba but also in macrophages, and causes a life-threatening pneumonia called Legionnaires' disease. The opportunistic pathogen employs the α-hydroxy-ketone compound Legionella autoinducer-1 (LAI-1) for intraspecies and interkingdom signaling. LAI-1 is produced by the autoinducer synthase Legionella quorum sensing A (LqsA), but it is not known, how LAI-1 is released by the pathogen. Here, we use a Vibrio cholerae luminescence reporter strain and liquid chromatography-tandem mass spectrometry to detect bacteria-produced and synthetic LAI-1. Ectopic production of LqsA in Escherichia coli generated LAI-1, which partitions to outer membrane vesicles (OMVs) and increases OMV size. These E. coli OMVs trigger luminescence of the V. cholerae reporter strain and inhibit the migration of Dictyostelium discoideum amoeba. Overexpression of lqsA in L.pneumophila under the control of strong stationary phase promoters (PflaA or P6SRNA), but not under control of its endogenous promoter (PlqsA), produces LAI-1, which is detected in purified OMVs. These L. pneumophila OMVs trigger luminescence of the Vibrio reporter strain and inhibit D. discoideum migration. L. pneumophila OMVs are smaller upon overexpression of lqsA or upon addition of LAI-1 to growing bacteria, and therefore, LqsA affects OMV production. The overexpression of lqsA but not a catalytically inactive mutant promotes intracellular replication of L. pneumophila in macrophages, indicating that intracellularly produced LA1-1 modulates the interaction in favor of the pathogen. Taken together, we provide evidence that L. pneumophila LAI-1 is secreted through OMVs and promotes interbacterial communication and interactions with eukaryotic host cells.
Subject(s)
Legionella pneumophila , Quorum Sensing , Humans , Bacterial Proteins/genetics , Dictyostelium , Escherichia coli , Legionella , Legionella pneumophila/physiology , Legionnaires' Disease/microbiologyABSTRACT
Legionella pneumophila grows within membrane-bound vacuoles in alveolar macrophages during human disease. Pathogen manipulation of the host cell is driven by bacterial proteins translocated through a type IV secretion system (T4SS). Although host protein synthesis during infection is arrested by the action of several of these translocated effectors, translation of a subset of host proteins predicted to restrict the pathogen is maintained. To identify the spectrum of host proteins selectively synthesized after L. pneumophila challenge, macrophages infected with the pathogen were allowed to incorporate the amino acid analog azidohomoalanine (AHA) during a 2-h time window, and newly synthesized macrophage proteins were isolated by orthogonal chemistry followed by mass spectrometry. Among the proteins isolated were interferon-stimulated genes as well as proteins translated from highly abundant transcripts. Surprisingly, a large number of the identified proteins were from low-abundance transcripts. These proteins were predicted to be among the most efficiently translated per unit transcript in the cell based on ribosome profiling data sets. To determine if high ribosome loading was a consequence of efficient translation initiation, the 5' untranslated regions (5' UTR) of transcripts having the highest and lowest predicted loading levels were inserted upstream of a reporter, and translation efficiency was determined in response to L. pneumophila challenge. The efficiency of reporter expression largely correlated with predicted ribosome loading and lack of secondary structure. Therefore, determinants in the 5' UTR allow selected host cell transcripts to overcome a pathogen-driven translation blockade.
Subject(s)
Legionella pneumophila , Humans , Legionella pneumophila/physiology , 5' Untranslated Regions , RNA, Messenger/genetics , RNA, Messenger/metabolism , Host-Pathogen Interactions/genetics , Vacuoles/microbiology , Bacterial Proteins/genetics , Bacterial Proteins/metabolismABSTRACT
Previously, we demonstrated that Cas2 encoded within the CRISPR-Cas locus of Legionella pneumophila strain 130b promotes the ability of the Legionella pathogen to infect amoebal hosts. Given that L. pneumophila Cas2 has RNase activity, we posited that the cytoplasmic protein is regulating the expression of another Legionella gene(s) that fosters intracellular infection. Proteomics revealed 10 proteins at diminished levels in the cas2 mutant, and reverse transcription-quantitative (qRT-PCR) confirmed the reduced expression of a gene encoding putative small heat shock protein C2 (HspC2), among several others. As predicted, the gene was expressed more highly at 37°C to 50°C than that at 30°C, and an hspC2 mutant, but not its complemented derivative, displayed ~100-fold reduced CFU following heat shock at 55°C. Compatible with the effect of Cas2 on hspC2 expression, strains lacking Cas2 also had impaired thermal tolerance. The hspC2 mutant, like the cas2 mutant before it, was greatly impaired for infection of Acanthamoeba castellanii, a frequent host for legionellae in waters. HspC2 and Cas2 were not required for entry into these host cells but promoted the replicative phase of intracellular infection. Finally, the hspC2 mutant exhibited an additional defect during the infection of macrophages, which are the primary host for legionellae during lung infection. In summary, hspC2 is upregulated by the presence of Cas2, and HspC2 uniquely promotes both L. pneumophila extracellular survival at high temperatures and infection of amoebal and human host cells. To our knowledge, these findings also represent the first genetic proof linking Cas2 to thermotolerance, expanding the repertoire of noncanonical functions associated with CRISPR-Cas proteins.
Subject(s)
Acanthamoeba castellanii , Heat-Shock Proteins, Small , Legionella pneumophila , Humans , Legionella pneumophila/physiology , Heat-Shock Proteins, Small/metabolism , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Ribonucleases/metabolismABSTRACT
Background: The intracellular pathogen Legionella pneumophila (L. pneumophila) is a causative agent of pneumonia and does great harm to human health. These bacteria are phagocytosed by alveolar macrophages and survive to replicate within the macrophages. Despite macrophage infectivity potentiator (MIP) protein serving as an essential virulence factor during the invasion process of L. pneumophila, the regulatory mechanism of MIP protein in the process of bacterial infection to host cells is not yet completely understood. This research thus aims to explore the interaction between MIP and macrophage phagocytosis. Methods: Through the experiment of the co-culture of RAW264.7 macrophages with different concentrations of MIP, the chemotactic activity of macrophages was detected and the phagocytosis was determined by a neutral red uptake assay. The expression of long noncoding RNA (lncRNA) GAS5, microRNA-21 (miR-21), and suppressor of cytokine signaling (SOCS)6 was determined by qRT-PCR. Target genes were detected by dual luciferase assay. Results: MIP could reduce the phagocytosis and improve the chemotaxis of RAW264.7 macrophages. The expression of both lncRNA GAS5 and SOCS6 was increased whereas the expression of miR-21 was decreased when macrophages were treated with MIP. Dual luciferase assay revealed that lncRNA GAS5 could interact with miR-21, and SOCS6 served as the target of miR-21. After GAS5 overexpression, the phagocytosis of RAW264.7 treated with MIP was increased whereas the chemotaxis was decreased. In contrast, the opposite results were found in RAW264.7 following GAS5 interference. Conclusions: The present results revealed that MIP could influence RAW264.7 macrophages on phagocytic and chemotactic activities through the axis of lncRNA GAS5/miR-21/SOCS6.
Subject(s)
Legionella pneumophila , MicroRNAs , RNA, Long Noncoding , Chemotaxis , Humans , Legionella pneumophila/physiology , Macrophages/metabolism , MicroRNAs/genetics , MicroRNAs/metabolism , Phagocytosis , RNA, Long Noncoding/genetics , RNA, Long Noncoding/metabolism , Suppressor of Cytokine Signaling Proteins/genetics , Suppressor of Cytokine Signaling Proteins/metabolismABSTRACT
The environmental bacterium Legionella pneumophila is an intracellular pathogen of various protozoan hosts and able to cause Legionnaires' disease, a severe pneumonia in humans. By encoding a wide selection of virulence factors, the infectious agent possesses several strategies to manipulate its host cells and evade immune detection. In the present study, we demonstrate that the L. pneumophila zinc metalloprotease ProA functions as a modulator of flagellin-mediated TLR5 stimulation and subsequent activation of the pro-inflammatory NF-κB pathway. We found ProA to be capable of directly degrading immunogenic FlaA monomers but not the polymeric form of bacterial flagella. These results indicate a role of the protease in antagonizing immune stimulation, which was further substantiated in HEK-BlueTM hTLR5 Detection assays. Addition of purified proteins, bacterial suspensions of L. pneumophila mutant strains as well as supernatants of human lung tissue explant infection to this reporter cell line demonstrated that ProA specifically decreases the TLR5 response via FlaA degradation. Conclusively, the zinc metalloprotease ProA serves as a powerful regulator of exogenous flagellin and presumably creates an important advantage for L. pneumophila proliferation in mammalian hosts by promoting immune evasion.
Subject(s)
Legionella pneumophila , Legionnaires' Disease , Animals , Flagellin , Humans , Legionella pneumophila/physiology , Legionnaires' Disease/microbiology , Mammals , Metalloproteases , Toll-Like Receptor 5/genetics , Zinc/pharmacologyABSTRACT
Acanthamoeba spp. feeds on bacteria, fungi, and algae to obtain nutrients from the environment. However, several pathogens can survive and multiply in Acanthamoeba. Mechanisms necessary for the survival and proliferation of microorganisms in Acanthamoeba remain unclear. The object of this study was to identify effective factors for the survival of microorganisms in Acanthamoeba. Differentially expressed genes (DEGs) in A. castellanii infected by Legionella pneumophila or Escherichia coli were identified based on mRNA sequencing. A total of 2342 and 1878 DEGs were identified in Acanthamoeba with L. pneumophila and E. coli, respectively. Among these DEGs, 502 were up-regulated and 116 were down-regulated in Acanthamoeba infected by L. pneumophila compared to those in Acanthamoeba feed on E. coli. Gene ontology analysis showed that the genes encoded small GTPase-mediated signal transduction proteins in the biological process domain, intracellular proteins in the cellular component domain, and ATP binding proteins in the molecular function domain were up-regulated while integral components of membrane proteins in the cellular component domain were down-regulated in Acanthamoeba infected by Legionella compared to those in Acanthamoeba feed on E. coli. During endosymbiosis with Legionella, Acanthamoeba showed various changes in the expression of genes supposed to be involved in phagosomal maturation. Acanthamoeba infected by Legionella also showed high expression levels of aminotransferase, methyltransferase, and cysteine proteinase but low expression levels of RNA pseudouridine synthase superfamily protein and 2OG-Fe(II) oxygenase superfamily. These results provide directions for further research to understand the survival strategy of L. pneumophila in A. castellanii.
Subject(s)
Acanthamoeba/genetics , Acanthamoeba/microbiology , Escherichia coli/physiology , Gene Expression , Legionella pneumophila/physiology , Down-Regulation , Phagocytosis/physiology , RNA, Protozoan/chemistry , Real-Time Polymerase Chain Reaction , Sequence Analysis, RNA , Symbiosis/genetics , Up-RegulationABSTRACT
Legionella pneumophila is an intracellular pathogen that can cause Legionnaire's disease by invading alveolar epithelial cells and macrophages. The major outer membrane protein (MOMP) plays an important role in the interaction between bacteria and host cells. However, the role of MOMP in the process of L. pneumophila invasion of macrophages and its working mechanism remain unknown. We aimed to explore the effects of MOMP on phagocytosis and chemotaxis of RAW 264.7 macrophages. The chemotactic activity, toxicity, and phagocytosis of RAW 264.7 cocultured with different concentrations of MOMP were determined by Transwell, CCK-8, and neutral red uptake assays, respectively. Target genes were detected by double-luciferase and pull down assays. qRT-PCR and Western blot were performed to analyze the expression of several important proteins involved in the immune response pathway, including coronin-1, interleukins (IL-10), forkhead transcription factor 1 (FOXO1), nucleotide-binding oligomerization domain protein (NOD) 1, NOD2, and receptor-interacting protein (RIP) 2. After coculturing with MOMP, cytological observation indicated a decrease of phagocytosis and a marked increase of chemotaxis in RAW 264.7 macrophages. The phagocytosis degree of RAW 264.7 macrophage varied with the concentration gradient of MOMP in a time-dependent manner. MOMP could increase the expression levels of MCP-1, IL-10, NOD2, and RIP2 and decrease the expression levels of FOXO1 and coronin-1 in cell culture supernatants. In addition, we found that FOXO1 could promote its transcription by binding to the promoter of coronin-1. The results of the present study suggested that MOMP could inhibit phagocytosis and facilitate chemotaxis of RAW 264.7 macrophage, which might be associated with the FOXO1/coronin-1 axis.
Subject(s)
Bacterial Proteins/metabolism , Forkhead Box Protein O1/metabolism , Legionella pneumophila/physiology , Legionnaires' Disease/immunology , Macrophages/immunology , Microfilament Proteins/metabolism , Porins/metabolism , Animals , Bacterial Proteins/immunology , Chemokine CCL2/metabolism , Chemotaxis , Gene Expression Regulation , Interleukin-10/metabolism , Mice , Microfilament Proteins/genetics , Phagocytosis , Porins/immunology , RAW 264.7 Cells , Signal TransductionABSTRACT
Legionella pneumophila is a facultative intracellular pathogen that uses the Dot/Icm Type IV secretion system (T4SS) to translocate many effectors into its host and establish a safe, replicative lifestyle. The bacteria, once phagocytosed, reside in a vacuolar structure known as the Legionella-containing vacuole (LCV) within the host cells and rapidly subvert organelle trafficking events, block inflammatory responses, hijack the host ubiquitination system, and abolish apoptotic signaling. This arsenal of translocated effectors can manipulate the host factors in a multitude of different ways. These proteins also contribute to bacterial virulence by positively or negatively regulating the activity of one another. Such effector-effector interactions, direct and indirect, provide the delicate balance required to maintain cellular homeostasis while establishing itself within the host. This review summarizes the recent progress in our knowledge of the structure-function relationship and biochemical mechanisms of select effector pairs from Legionella that work in opposition to one another, while highlighting the diversity of biochemical means adopted by this intracellular pathogen to establish a replicative niche within host cells.
Subject(s)
Host-Pathogen Interactions , Legionella pneumophila/physiology , Legionnaires' Disease/metabolism , Animals , Bacterial Proteins/metabolism , Homeostasis , Humans , Inflammation/metabolism , Inflammation/microbiology , Inflammation/pathology , Legionnaires' Disease/microbiology , Legionnaires' Disease/pathology , Type IV Secretion Systems/metabolism , Ubiquitination , Vacuoles/metabolism , Vacuoles/microbiology , Vacuoles/pathologyABSTRACT
Legionella pneumophila is the causative agent of Legionnaires' disease and is capable of replicating inside phagocytic cells, such as mammalian macrophages. The Dot/Icm type IV secretion system is a L. pneumophila virulence factor that is essential for successful intracellular replication. During infection, L. pneumophila builds a replication-permissive vacuole by recruiting multiple host molecules and hijacking host cellular signaling pathways, a process mediated by the coordinated functions of multiple Dot/Icm effector proteins. RavY is a predicted Dot/Icm effector protein found to be important for optimal L. pneumophila replication inside host cells. Here, we demonstrate that RavY is a Dot/Icm-translocated effector protein that is dispensable for axenic replication of L. pneumophila but critical for optimal intracellular replication of the bacteria. RavY is not required for avoidance of endosomal maturation, and RavY does not contribute to the recruitment of host molecules found on replication-permissive vacuoles, such as ubiquitin, RAB1a, and RTN4. Vacuoles containing L. pneumophila ravY mutants promote intracellular survival but limit replication. The replication defect of the L. pneumophila ravY mutant was complemented when the mutant was in the same vacuole as wild-type L. pneumophila. Thus, RavY is an effector that is essential for promoting intracellular replication of L. pneumophila once the specialized vacuole has been established.
Subject(s)
Host-Pathogen Interactions , Legionella pneumophila/physiology , Legionnaires' Disease/microbiology , Vacuoles/microbiology , Virulence Factors/genetics , Phagocytosis , Phagosomes/microbiology , Type IV Secretion Systems/metabolism , VirulenceABSTRACT
Legionella pneumophila cell surface hydrophobicity and charge are important determinants of their mobility and persistence in engineered water systems (EWS). These surface properties may differ depending on the growth phase of L. pneumophila resulting in variable adhesion and persistence within EWS. We describe the growth-dependent variations in L. pneumophila cell surface hydrophobicity and surface charge using the microbial adhesion to hydrocarbon assay and microelectrophoresis, respectively, and their role in cell adhesion to stainless steel using a quartz crystal microbalance with dissipation (QCM-D) monitoring instrument. We observed a steady increase in L. pneumophila hydrophobicity during their lifecycle in culture media. Cell surfaces of stationary phase L. pneumophila were significantly more hydrophobic than their lag and midexponential counterparts. No significant changes in L. pneumophila cell surface charge were noted. Morphology of L. pneumophila remained relatively constant throughout their lifecycle. In the QCM-D study, lag and exponential phase L. pneumophila weakly adhered to stainless steel surfaces resulting in viscoelastic layers. In contrast, stationary phase bacteria were tightly and irreversibly bound to the surfaces, forming rigid layers. Our results suggest that the stationary phase of L. pneumophila would highly favour their adhesion to plumbing surfaces and persistence in EWS.
Subject(s)
Bacterial Adhesion , Legionella pneumophila/physiology , Quartz Crystal Microbalance Techniques , Stainless Steel , Hydrophobic and Hydrophilic Interactions , Legionella pneumophila/ultrastructure , Microscopy, Electron, Scanning , Surface PropertiesABSTRACT
During infection, intracellular bacterial pathogens translocate a variety of effectors into host cells that modify host membrane trafficking for their benefit. We found a self-organizing system consisting of a bacterial phosphoinositide kinase and its opposing phosphatase that formed spatiotemporal patterns, including traveling waves, to remodel host cellular membranes. The Legionella effector MavQ, a phosphatidylinositol (PI) 3-kinase, was targeted to the endoplasmic reticulum (ER). MavQ and the Legionella PI 3-phosphatase SidP, even in the absence of other bacterial components, drove rapid PI 3-phosphate turnover on the ER and spontaneously formed traveling waves that spread along ER subdomains inducing vesicle and tubule budding. Thus, bacteria can exploit a self-organizing membrane-targeting mechanism to hijack host cellular structures for survival.
Subject(s)
Bacterial Proteins/metabolism , Endoplasmic Reticulum/metabolism , Intracellular Membranes/metabolism , Legionella pneumophila/physiology , Phosphatidylinositol 3-Kinase/metabolism , Phosphatidylinositol Phosphates/metabolism , Animals , Bacterial Proteins/chemistry , COS Cells , Chlorocebus aethiops , Endoplasmic Reticulum/ultrastructure , Feedback, Physiological , HeLa Cells , Host-Pathogen Interactions , Humans , Intracellular Membranes/ultrastructure , Legionella pneumophila/enzymology , Legionella pneumophila/genetics , Legionella pneumophila/growth & development , Mice , Mutation , Phosphatidylinositol 3-Kinase/chemistry , Phosphatidylinositol Phosphates/chemistry , Phosphoric Monoester Hydrolases/metabolism , Protein Domains , RAW 264.7 CellsABSTRACT
Legionella pneumophila is an opportunistic pathogen that survives and proliferates within protists such as Acanthamoeba spp. in environment. However, intracellular pathogenic endosymbiosis and its implications within Acanthamoeba spp. remain poorly understood. In this study, RNA sequencing analysis was used to investigate transcriptional changes in A. castellanii in response to L. pneumophila infection. Based on RNA sequencing data, we identified 1,211 upregulated genes and 1,131 downregulated genes in A. castellanii infected with L. pneumophila for 12 hr. After 24 hr, 1,321 upregulated genes and 1,379 downregulated genes were identified. Gene ontology (GO) analysis revealed that L. pneumophila endosymbiosis enhanced hydrolase activity, catalytic activity, and DNA binding while reducing oxidoreductase activity in the molecular function (MF) domain. In particular, multiple genes associated with the GO term 'integral component of membrane' were downregulated during endosymbiosis. The endosymbiont also induced differential expression of various methyltransferases and acetyltransferases in A. castellanii. Findings herein are may significantly contribute to understanding endosymbiosis of L. pneumophila within A. castellanii.
Subject(s)
Acanthamoeba castellanii/genetics , Acanthamoeba castellanii/microbiology , Genes, Protozoan/genetics , Legionella pneumophila/physiology , Symbiosis/genetics , Transcriptome/genetics , Acanthamoeba castellanii/enzymology , Acetyltransferases/genetics , Acetyltransferases/metabolism , Catalysis , Gene Ontology , Hydrolases/metabolism , Legionella pneumophila/pathogenicity , Methyltransferases/genetics , Methyltransferases/metabolism , Oxidoreductases/metabolism , Sequence Analysis, RNA , Transcription, GeneticABSTRACT
In large-building water systems, Legionella pneumophila is exposed to common environmental stressors such as copper. The aim of this study was to evaluate the susceptibility to copper of L. pneumophila isolates recovered from various sites: two clinical and seven environmental isolates from hot water system biofilm and water and from cooling tower water. After a 1-week acclimation in simulated drinking water, strains were exposed to various copper concentrations (0.8 to 5 mg/liter) for over 672 h. Complete loss of culturability was observed for three isolates following copper exposure to 5 mg/liter for 672 h. Two sequence type 1427 (ST1427)-like isolates were highly sensitive to copper, while the other two, isolated from biofilm samples, maintained higher culturability. The expression of the copper resistance gene copA evaluated by reverse transcription-quantitative PCR (RT-qPCR) was significantly higher for the biofilm isolates. All four ST1427-like isolates were recovered from the same water system during an outbreak. Whole-genome sequencing results confirmed that the four isolates are very close phylogenetically, differing by only 29 single nucleotide polymorphisms, suggesting in situ adaptation to microenvironmental conditions, possibly due to epigenetic regulation. These results indicate that the immediate environment within a building water distribution system influences the tolerance of L. pneumophila to copper. Increased contact of L. pneumophila biofilm strains with copper piping or copper alloys in the heat exchanger might lead to local adaptation. The phenotypic differences observed between water and biofilm isolates from the hot water system of a health care facility warrants further investigation to assess the relevance of evaluating disinfection performances based on water sampling alone.IMPORTANCELegionella pneumophila is a pathogen indigenous to natural and large building water systems in the bulk and the biofilm phases. The immediate environment within a system can impact the tolerance of L. pneumophila to environmental stressors, including copper. In health care facilities, copper levels in water can vary, depending on water quality, plumbing materials, and age. This study evaluated the impact of the isolation site (water versus biofilm, hot water system versus cooling tower) within building water systems. Closely related strains isolated from a health care facility hot water system exhibited variable tolerance to copper stress, shown by differential expression of copA, with biofilm isolates displaying highest expression and tolerance. Relying on the detection of L. pneumophila in water samples following exposure to environmental stressors such as copper may underestimate the prevalence of L. pneumophila, leading to inappropriate risk management strategies and increasing the risk of exposure for vulnerable patients.
Subject(s)
Copper/toxicity , Drinking Water/microbiology , Hospitals , Legionella pneumophila , Water Supply , Adaptation, Physiological , Biofilms/drug effects , Drug Tolerance/genetics , Genome, Bacterial , Legionella pneumophila/drug effects , Legionella pneumophila/genetics , Legionella pneumophila/isolation & purification , Legionella pneumophila/physiology , PhylogenyABSTRACT
Dictyostelium discoideum Sey1 is the single ortholog of mammalian atlastin 1-3 (ATL1-3), which are large homodimeric GTPases mediating homotypic fusion of endoplasmic reticulum (ER) tubules. In this study, we generated a D. discoideum mutant strain lacking the sey1 gene and found that amoebae deleted for sey1 are enlarged, but grow and develop similarly to the parental strain. The ∆sey1 mutant amoebae showed an altered ER architecture, and the tubular ER network was partially disrupted without any major consequences for other organelles or the architecture of the secretory and endocytic pathways. Macropinocytic and phagocytic functions were preserved; however, the mutant amoebae exhibited cumulative defects in lysosomal enzymes exocytosis, intracellular proteolysis, and cell motility, resulting in impaired growth on bacterial lawns. Moreover, ∆sey1 mutant cells showed a constitutive activation of the unfolded protein response pathway (UPR), but they still readily adapted to moderate levels of ER stress, while unable to cope with prolonged stress. In D. discoideum ∆sey1 the formation of the ER-associated compartment harbouring the bacterial pathogen Legionella pneumophila was also impaired. In the mutant amoebae, the ER was less efficiently recruited to the "Legionella-containing vacuole" (LCV), the expansion of the pathogen vacuole was inhibited at early stages of infection and intracellular bacterial growth was reduced. In summary, our study establishes a role of D. discoideum Sey1 in ER architecture, proteolysis, cell motility and intracellular replication of L. pneumophila.
Subject(s)
Dictyostelium/physiology , Endoplasmic Reticulum/ultrastructure , GTP Phosphohydrolases/metabolism , Legionella pneumophila/physiology , Protozoan Proteins/metabolism , Vacuoles/microbiology , Dictyostelium/growth & development , Dictyostelium/microbiology , Dictyostelium/ultrastructure , Endoplasmic Reticulum/metabolism , Endoplasmic Reticulum Stress , Endoplasmic Reticulum, Rough/microbiology , Endoplasmic Reticulum, Rough/physiology , GTP Phosphohydrolases/genetics , Homeostasis , Host-Pathogen Interactions , Legionella pneumophila/growth & development , Movement , Muramidase/metabolism , Phosphatidylinositol Phosphates/metabolism , Protozoan Proteins/genetics , Vacuoles/physiologyABSTRACT
The ubiquitin pathway is highly conserved across the eukaryotic domain of life and plays an essential role in a plethora of cellular processes. It is not surprising that many intracellular bacterial pathogens often target the essential host ubiquitin pathway. The intracellular bacterial pathogen Legionella pneumophila injects into the host cell cytosol multiple classes of classical and novel ubiquitin-modifying enzymes that modulate diverse ubiquitin-related processes in the host cell. Most of these pathogen-injected proteins, designated as effectors, mimic known E3-ubiquitin ligases through harboring F-box or U-box domains. The classical F-box effector, AnkB targets host proteins for K48-linked polyubiquitination, which leads to excessive proteasomal degradation that is required to generate adequate supplies of amino acids for metabolism of the pathogen. In contrast, the SidC and SdcA effectors share no structural similarity to known eukaryotic ligases despite having E3-ubiquitin ligase activity, suggesting that the number of E3-ligases in eukaryotes is under-represented. L. pneumophila also injects into the host many novel ubiquitin-modifying enzymes, which are the SidE family of effectors that catalyze phosphoribosyl-ubiquitination of serine residue of target proteins, independently of the canonical E1-2-3 enzymatic cascade. Interestingly, the environmental bacterium, L. pneumophila, has evolved within a diverse range of amoebal species, which serve as the natural hosts, while accidental transmission through contaminated aerosols can cause pneumonia in humans. Therefore, it is likely that the novel ubiquitin-modifying enzymes of L. pneumophila were acquired by the pathogen through interkingdom gene transfer from the diverse natural amoebal hosts. Furthermore, conservation of the ubiquitin pathway across eukaryotes has enabled these novel ubiquitin-modifying enzymes to function similarly in mammalian cells. Studies on the biological functions of these effectors are likely to reveal further novel ubiquitin biology and shed further lights on the evolution of ubiquitin.