Legionella eukaryotic-like type IV substrates interfere with organelle trafficking

Legionella pneumophila, the causative agent of Legionnaires' disease, evades phago-lysosome fusion in mammalian and protozoan hosts to create a suitable niche for intracellular replication. To modulate vesicle trafficking pathways, L. pneumophila translocates effector proteins into eukaryotic cells through a Type IVB macro-molecular transport system called the Icm-Dot system. In this study, we employed a fluorescence-based translocation assay to show that 33 previously identified Legionella eukaryotic-like genes (leg) encode substrates of the Icm-Dot secretion system. To assess which of these proteins may contribute to the disruption of vesicle trafficking, we expressed each gene in yeast and looked for phenotypes related to vacuolar protein sorting. We found that LegC3-GFP and LegC7/YlfA-GFP caused the mis-secretion of CPY-Invertase, a fusion protein normally restricted to the yeast vacuole. We also found that LegC7/YlfA-GFP and its paralog LegC2/YlfB-GFP formed large structures around the yeast vacuole while LegC3-GFP localized to the plasma membrane and a fragmented vacuole. In mammalian cells, LegC2/YlfB-GFP and LegC7/YlfA-GFP were found within large structures that co-localized with anti-KDEL antibodies but excluded the lysosomal marker LAMP-1, similar to what is observed in Legionella-containing vacuoles. LegC3-GFP, in contrast, was observed as smaller structures which had no obvious co-localization with KDEL or LAMP-1. Finally, LegC3-GFP caused the accumulation of many endosome-like structures containing undigested material when expressed in the protozoan host Dictyostelium discoideum. Our results demonstrate that multiple Leg proteins are Icm/Dot-dependent substrates and that LegC3, LegC7/YlfA, and LegC2/YlfB may contribute to the intracellular trafficking of L. pneumophila by interfering with highly conserved pathways that modulate vesicle maturation.     

The perplexing functions and surprising origins of Legionella pneumophila type IV secretion effectors

Only a limited number of bacterial pathogens evade destruction by phagocytic cells such as macrophages. Legionella pneumophila is a Gram-negative γ-proteobacterial species that can infect and replicate in alveolar macrophages, causing Legionnaires' disease, a severe pneumonia. L. pneumophila uses a complex secretion system to inject host cells with effector proteins capable of disrupting or altering the host cell processes. The L. pneumophila effectors target multiple processes but are essentially aimed at modifying the properties of the L. pneumophila phagosome by altering vesicular trafficking, gradually creating a specialized vacuole in which the bacteria replicate robustly. In nature, L. pneumophila is thought to parasitize free-living protists, which may have selected for traits that promote virulence of L. pneumophila in humans. Indeed, many effector genes encode proteins with eukaryotic domains and are likely to be of protozoan origin. Sustained horizontal gene transfer events within the protozoan niche may have allowed L. pneumophila to become a professional parasite of phagocytes, simultaneously giving rise to its ability to infect macrophages, cells that constitute the first line of cellular defence against bacterial infections.     

A Legionella effector acquired from protozoa is involved in sphingolipids metabolism and is targeted to the host cell mitochondria

Legionella pneumophila infects alveolar macrophages and protozoa through establishment of an intracellular replication niche. This process is mediated by bacterial effectors translocated into the host cell via the Icm/Dot type IV secretion system. Most of the effectors identified so far are unique to L. pneumophila ; however, some of the effectors are homologous to eukaryotic proteins. We performed a distribution analysis of many known L. pneumophila effectors and found that several of them, mostly eukaryotic homologous proteins, are present in different Legionella species. In-depth analysis of LegS2, a L. pneumophila homologue of the highly conserved eukaryotic enzyme sphingosine-1-phosphate lyase (SPL), revealed that it was most likely acquired from a protozoan organism early during Legionella evolution. The LegS2 protein was found to translocate into host cells using a C-terminal translocation domain absent in its eukaryotic homologues. LegS2 was found to complement the sphingosine-sensitive phenotype of a Saccharomyces serevisia SPL-null mutant and this complementation depended on evolutionary conserved residues in the LegS2 catalytic domain. Interestingly, unlike the eukaryotic SPL that localizes to the endoplasmic reticulum, LegS2 was found to be targeted mainly to host cell mitochondria. Collectively, our results demonstrate the remarkable adaptations of a eukaryotic protein to the L. pneumophila pathogenesis system.     

Exploitation of evolutionarily conserved amoeba and mammalian processes by Legionella

Legionella pneumophila proliferates within various protists and metazoan cells, where a cadre of ～300 effectors is injected into the host cell by the defect in organelle trafficking/intracellular multiplication (Dot/Icm) type IVB translocation system. Interkingdom horizontal gene transfer of genes of protists and their subsequent convergent evolution to become translocated effectors has probably enabled L. pneumophila to adapt to the intracellular life within various protists and metazoan cells through exploitation of evolutionarily eukaryotic processes, such as endoplasmic reticulum-to-Golgi vesicle traffic, phosphoinositol metabolism, AMPylation, deAMPylation, prenylation, polyubiquitination, proteasomal degradation and cytosolic amino- and oligo-peptidases. This is highlighted by the ankyrin B (AnkB) F-box effector that exploits multiple conserved eukaryotic machineries to generate high levels of free amino acids as sources of carbon and energy essential for intracellular proliferation in protists and metazoan cells and for manifestation of pulmonary disease in mammals.     

An ancient horizontal gene transfer between mosquito and the endosymbiotic bacterium Wolbachia pipientis

The extent and biological relevance of horizontal gene transfer (HGT) in eukaryotic evolution remain highly controversial. Recent studies have demonstrated frequent and large-scale HGT from endosymbiotic bacteria to their hosts, but the great majority of these transferred genes rapidly become nonfunctional in the recipient genome. Here, we investigate an ancient HGT between a host metazoan and an endosymbiotic bacterium, Wolbachia pipientis. The transferred gene has so far been found only in mosquitoes and Wolbachia. In mosquitoes, it is a member of a gene family encoding candidate receptors required for malaria sporozoite invasion of the mosquito salivary gland. The gene copy in Wolbachia has substantially diverged in sequence from the mosquito homolog, is evolving under purifying selection, and is expressed, suggesting that this gene is also functional in the bacterial genome. Several lines of evidence indicate that the gene may have been transferred from eukaryotic host to bacterial endosymbiont. Regardless of the direction of transfer, however, these results demonstrate that interdomain HGT may give rise to functional, persistent, and possibly evolutionarily significant new genes.     

Diverse mechanisms of metaeffector activity in an intracellular bacterial pathogen,Legionella pneumophila

Pathogens deliver complex arsenals of translocated effector proteins to host cells during infection, but the extent to which these proteins are regulated once inside the eukaryotic cell remains poorly defined. Among all bacterial pathogens, Legionella pneumophila maintains the largest known set of translocated substrates, delivering over 300 proteins to the host cell via its Type IVB, Icm/Dot translocation system. Backed by a few notable examples of effector–effector regulation in L. pneumophila, we sought to define the extent of this phenomenon through a systematic analysis of effector–effector functional interaction. We used Saccharomyces cerevisiae, an established proxy for the eukaryotic host, to query > 108,000 pairwise genetic interactions between two compatible expression libraries of ~330 L. pneumophila‐translocated substrates. While capturing all known examples of effector–effector suppression, we identify fourteen novel translocated substrates that suppress the activity of other bacterial effectors and one pair with synergistic activities. In at least nine instances, this regulation is direct—a hallmark of an emerging class of proteins called metaeffectors, or “effectors of effectors”. Through detailed structural and functional analysis, we show that metaeffector activity derives from a diverse range of mechanisms, shapes evolution, and can be used to reveal important aspects of each cognate effector's function. Metaeffectors, along with other, indirect, forms of effector–effector modulation, may be a common feature of many intracellular pathogens—with unrealized potential to inform our understanding of how pathogens regulate their interactions with the host cell.     

The Legionella pneumophila IcmSW complex interacts with multiple Dot/Icm effectors to facilitate type IV translocation

Many gram-negative pathogens use a type IV secretion system (T4SS) to deliver effector proteins into eukaryotic host cells. The fidelity of protein translocation depends on the efficient recognition of effector proteins by the T4SS. Legionella pneumophila delivers a large number of effector proteins into eukaryotic cells using the Dot/Icm T4SS. How the Dot/Icm system is able to recognize and control the delivery of effectors is poorly understood. Recent studies suggest that the IcmS and IcmW proteins interact to form a stable complex that facilitates translocation of effector proteins by the Dot/Icm system by an unknown mechanism. Here we demonstrate that the IcmSW complex is necessary for the productive translocation of multiple Dot/Icm effector proteins. Effector proteins that were able to bind IcmSW in vitro required icmS and icmW for efficient translocation into eukaryotic cells during L. pneumophila infection. We identified regions in the effector protein SidG involved in icmSW-dependent translocation. Although the full-length SidG protein was translocated by an icmSW-dependent mechanism, deletion of amino terminal regions in the SidG protein resulted in icmSW-independent translocation, indicating that the IcmSW complex is not contributing directly to recognition of effector proteins by the Dot/Icm system. Biochemical and genetic studies showed that the IcmSW complex interacts with a central region of the SidG protein. The IcmSW interaction resulted in a conformational change in the SidG protein as determined by differences in protease sensitivity in vitro. These data suggest that IcmSW binding to effectors could enhance effector protein delivery by mediating a conformational change that facilitates T4SS recognition of a translocation domain located in the carboxyl region of the effector protein.     

Effector proteins translocated by Legionella pneumophila: strength in numbers

The Gram-negative bacterium Legionella pneumophila is a parasite of eukaryotic cells. It has evolved to survive and replicate in a wide range of protozoan hosts and can also infect human alveolar macrophages as an opportunistic pathogen. Crucially for the infection process, L. pneumophila uses a type IV secretion system called Dot/Icm to translocate bacterial proteins into host cells. In recent years a large number of Dot/Icm-translocated proteins have been identified. The study of these proteins, referred to as effectors, is providing valuable insight into the mechanism by which an intracellular pathogen can manipulate eukaryotic cellular processes to traffic and replicate in host cells.     

Modulation of phosphoinositide metabolism by pathogenic bacteria

Phosphoinositide metabolism plays a pivotal role in the regulation of receptor-mediated signal transduction, actin remodelling and membrane dynamics. Phosphoinositides co-ordinate these processes by recruiting protein effectors to distinct cellular membranes in a time- and organelle-dependent manner. Intracellular bacterial pathogens interfere with phosphoinositide metabolism to direct their entry into eukaryotic cells, form replication-permissive vacuoles, modulate apoptosis, or trigger fluid secretion. Gram-negative pathogens such as Legionella pneumophila, Shigella flexneri, or Salmonella enterica employ secretion systems to invade host cells by 'pathogen-triggered phagocytosis' and thereby bypass a requirement for phosphatidylinositol 3-kinases [PI(3)Ks]. Contrarily, 'receptor-mediated phagocytosis' of Yersinia spp., Listeria monocytogenes and other pathogenic bacteria depends on PI(3)Ks. Secreted effector proteins have been found to directly bind to and modify host cell phosphoinositides, thus modulating phagocytosis and intracellular survival of the pathogens. These effectors include L. pneumophila proteins that specifically attach to phosphatidylinositol 4-phosphate [PI(4)P] on the Legionella-containing vacuole, and phosphoinositide phosphatases produced by S. flexneri, S. enterica or Mycobacterium tuberculosis. This review covers current knowledge about subversion of host cell phosphoinositide metabolism by intracellular bacterial pathogens with an emphasis on recently identified secreted effector proteins directly engaging phosphoinositides.     

The Coxiella burnetii Dot/Icm system delivers a unique repertoire of type IV effectors into host cells and is required for intracellular replication

Coxiella burnetii, the causative agent of human Q fever, is an intracellular pathogen that replicates in an acidified vacuole derived from the host lysosomal network. This pathogen encodes a Dot/Icm type IV secretion system that delivers bacterial proteins called effectors to the host cytosol. To identify new effector proteins, the functionally analogous Legionella pneumophila Dot/Icm system was used in a genetic screen to identify fragments of C. burnetii genomic DNA that when fused to an adenylate cyclase reporter were capable of directing Dot/Icm-dependent translocation of the fusion protein into mammalian host cells. This screen identified Dot/Icm effectors that were proteins unique to C. burnetii, having no overall sequence homology with L. pneumophila Dot/Icm effectors. A comparison of C. burnetii genome sequences from different isolates revealed diversity in the size and distribution of the genes encoding many of these effectors. Studies examining the localization and function of effectors in eukaryotic cells provided evidence that several of these proteins have an affinity for specific host organelles and can disrupt cellular functions. The identification of a transposon insertion mutation that disrupts the dot/icm locus was used to validate that this apparatus was essential for translocation of effectors. Importantly, this C. burnetii Dot/Icm-deficient mutant was found to be defective for intracellular replication. Thus, these data indicate that C. burnetii encodes a unique subset of bacterial effector proteins translocated into host cells by the Dot/Icm apparatus, and that the cumulative activities exerted by these effectors enables C. burnetii to successfully establish a niche inside mammalian cells that supports intracellular replication.     

Manipulation of host vesicular trafficking and innate immune defence by Legionella Dot/Icm effectors

Legionella pneumophila, the causative agent of Legionnaires' disease, infects and replicates in macrophages and amoebas. Following internalization, L. pneumophila resides in a vacuole structure called Legionella-containing vacuole (LCV). The LCV escapes from the endocytic maturation process and avoids fusion with the lysosome, a hallmark of Legionella pathogenesis. Interference with the secretory vesicle transport and avoiding lysosomal targeting render the LCV permissive for L. pneumophila intracellular replication. Central to L. pneumophila pathogenesis is a defect in the organelle trafficking/intracellular multiplication (Dot/Icm) type IV secretion system that translocates a large number of effector proteins into host cells. Many of the Dot/Icm effectors employ diverse and sophisticated biochemical strategies to manipulate the host vesicular transport system, playing an important role in LCV biogenesis and trafficking. Similar to other bacterial pathogens, L. pneumophila also delivers effector proteins to modulate or counteract host innate immune defence pathways such as the NF-κB and apoptotic signalling. This review summarizes the known functions and mechanisms of Dot/Icm effectors that target host membrane trafficking and innate immune defence pathways.     

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

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

嗜肺军团菌效应蛋白质介导的新型泛素化过程

泛素化是真核细胞特有的蛋白质翻译后修饰方式,调节真核细胞内多种重要生理过程,例如蛋白质稳态、细胞周期、免疫反应、DNA修复以及囊泡转运等。鉴于泛素化对于生命活动的重要性,病原菌在与宿主细胞的长期进化过程中衍生出一系列针对宿主泛素化过程的效应蛋白质,调控宿主体内泛素化过程,从而构建有利于病原菌自身生长繁殖的内环境。嗜肺军团菌是一种革兰氏阴性菌,是军团菌肺炎的致病菌,能够引起发热和肺部感染,重型病死率高达15%~30%。Dot/Icm Ⅳ型分泌系统是嗜肺军团菌侵染过程中最主要的毒力系统。在侵染宿主细胞的过程中,嗜肺军团菌利用该分泌系统,分泌超过330种效应蛋白质,协助细菌在宿主胞内生存、增殖和逃逸。多种嗜肺军团菌效应蛋白质通过直接或者间接的方式对宿主泛素化过程进行调控。近年的研究发现,多种效应蛋白质可以介导不同于真核生物经典泛素化的新型泛素化过程。本文介绍了嗜肺军团菌效应蛋白质介导的新型泛素化过程的最新研究进展,为理解泛素化过程在嗜肺军团菌致病过程中的重要作用提供参考依据。     

A Dot/Icm-translocated ankyrin protein of Legionella pneumophila is required for intracellular proliferation within human macrophages and protozoa

The Dot/Icm type IV secretion system of Legionella pneumophila translocates numerous bacterial effectors into the host cell and is essential for bacterial proliferation within macrophages and protozoa. We have recently shown that L. pneumophila strain AA100/130b harbours 11 genes encoding eukaryotic-like ankyrin (Ank) proteins, a family of proteins involved in various essential eukaryotic cellular processes. In contrast to most Dot/Icm-exported substrates, which have little or no detectable role in intracellular proliferation, a mutation in ankB results in a severe growth defect in intracellular replication within human monocyte-derived macrophages (hMDMs), U937 macrophages and Acanthamoeba polyphaga. Single cell analyses of coinfections of hMDMs have shown that the intracellular growth defect of the ankB mutant is totally rescued in cis within communal phagosomes harbouring the wild type strain. Interestingly, distinct from dot/icm structural mutants, the ankB mutant is also rescued in trans within cells harbouring the wild type strain in a different phagosome, indicating that AnkB is a trans-acting secreted effector. Using adenylate cyclase fusions to AnkB, we show that AnkB is translocated into the host cell via the Dot/Icm secretion system in an IcmSW-dependent manner and that the last three C-terminal amino acid residues are essential for translocation. Distinct from the dot/icm structural mutants, the ankB mutant-containing phagosomes exclude late endosomal and lysosomal markers and their phagosomes are remodelled by the rough endoplasmic reticulum. We show that at the postexponential phase of growth, the LetA/S and PmrA/B Two Component Systems confer a positive regulation on expression of the ankB gene, whereas RpoS, LetE and RelA suppress its expression. Our data show that the eukaryotic-like AnkB protein is a Dot/Icm-exported effector that plays a major role in intracellular replication of L. pneumophila within macrophages and protozoa, and its expression is temporally controlled by regulators of the postexponential phase of growth.     

Legionella metaeffector exploits host proteasome to temporally regulate cognate effector

Pathogen-associated secretion systems translocate numerous effector proteins into eukaryotic host cells to coordinate cellular processes important for infection. Spatiotemporal regulation is therefore important for modulating distinct activities of effectors at different stages of infection. Here we provide the first evidence of "metaeffector," a designation for an effector protein that regulates the function of another effector within the host cell. Legionella LubX protein functions as an E3 ubiquitin ligase that hijacks the host proteasome to specifically target the bacterial effector protein SidH for degradation. Delayed delivery of LubX to the host cytoplasm leads to the shutdown of SidH within the host cells at later stages of infection. This demonstrates a sophisticated level of coevolution between eukaryotic cells and L. pneumophila involving an effector that functions as a key regulator to temporally coordinate the function of a cognate effector protein.     

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.     

一个细菌的致命之旅——嗜肺军团菌分泌系统及效应蛋白的研究进展

嗜肺军团菌是引起军团菌肺炎以及庞蒂亚克热的革兰氏阴性胞内病原细菌,嗜肺军团菌侵染宿主的主要特点是可以通过其IVB型毒力分泌系统,向宿主细胞内分泌超过150种的底物效应蛋白。通过这些效应蛋白的作用,嗜肺军团菌能够调整宿主细胞的胞内运输途径,改变内外环境来伪装自己的吞噬泡,干扰宿主的细胞周期,抑制宿主细胞的凋亡,从而有效逃避宿主细胞的防御功能,创造出理想的胞内增殖环境。最后,效应蛋白还可以帮助军团菌从宿主细胞中逃逸。目前,嗜肺军团菌已经成为"病原菌-宿主相互作用"的重要研究模型,其毒力分泌系统及其底物效应蛋白的功能也成为细胞微生物学的研究热点。对嗜肺军团菌分泌系统及效应蛋白的研究不仅能够帮助阐明病原细菌的致病机理,还有助于推动对宿主免疫机制的更深层次的研究。文章主要针对嗜肺军团菌的毒力分泌系统,尤其是IVB型分泌系统的结构和功能,以及底物效应蛋白的研究进展进行了综述,向读者展示出一个小小的细菌所拥有的那令人惊叹的、如此狡猾的生存策略和它精致的杀伤武器。     

Pore-forming activity is not sufficient for Legionella pneumophila phagosome trafficking and intracellular growth

Bacterial pathogens often subvert eukaryotic cellular processes in order to establish a replicative niche and evade host immunity. Inhibition of phagosome lysosome fusion is a strategy used by several intracellular bacteria that grow within mammalian cells. It was shown recently that Legionella pneumophila possesses a cytolytic activity that results from the insertion of pores in the macrophage membrane upon contact, and that this activity requires the dot/icm gene products, which are necessary for intracellular growth and phagosome trafficking. Other bacteria that inhibit phagosome lysosome fusion, such as Mycobacterium tuberculosis , demonstrate similar cytolytic activities, which suggests that formation of pores in the phagosome membrane may account for the defects observed in phagosome trafficking. In this study, we identify a new class of L. pneumophila mutant that retains the pore-forming activity found in virulent bacteria, but is defective in phagosome lysosome fusion inhibition and intracellular growth. These data indicate that cytolytic activity is not sufficient for L. pneumophila -induced alterations in phagosome trafficking. Rather, the pore may be a vehicle that facilitates delivery of bacterial-derived effector molecules to the host cell cytoplasm.     

Presence in Legionella pneumophila of a mammalian-like mitochondrial permeability transition pore?

The genome of Legionella pneumophila reveals the presence of a large number of genes coding for eukaryotic-like proteins. By using database searches and homology investigations, we identified three proteins in L. pneumophila whose sequences share similarities with that of eukaryotic polypeptides (lpg0211, lpg1974 and lpg1982). In eukaryotes, the corresponding proteins (PBR, peripheral benzodiazepine receptor; VDAC, voltage-dependant anion channel; and CypD, cyclophilin D) participate in the formation of the mammalian mitochondrial permeability transition pore (MPTP), a complex involved in cell apoptosis. Intriguingly, the presence of these proteins has never been reported in the same bacterium and constitutes, up to now, a unique feature of L. pneumophila. In Legionella, we hypothesize that these proteins are recruited in a multiprotein complex close to the MPTP that may regulate intracellular survival and/or proliferation.