Stefan S. Weber

Legionella pneumophila Exploits PI(4)P to Anchor Secreted Effector Proteins to the Replicative Vacuole

The causative agent of Legionnaires disease, Legionella pneumophila, employs the intracellular multiplication (Icm)/defective organelle trafficking (Dot) type IV secretion system (T4SS) to upregulate phagocytosis and to establish a replicative vacuole in amoebae and macrophages. Legionella-containing vacuoles (LCVs) do not fuse with endosomes but recruit early secretory vesicles. Here we analyze the role of host cell phosphoinositide (PI) metabolism during uptake and intracellular replication of L. pneumophila. Genetic and pharmacological evidence suggests that class I phosphatidylinositol(3) kinases (PI3Ks) are dispensable for phagocytosis of wild-type L. pneumophila but inhibit intracellular replication of the bacteria and participate in the modulation of the LCV. Uptake and degradation of an icmT mutant strain lacking a functional Icm/Dot transporter was promoted by PI3Ks. We identified Icm/Dot–secreted proteins which specifically bind to phosphatidylinositol(4) phosphate (PI(4)P) in vitro and preferentially localize to LCVs in the absence of functional PI3Ks. PI(4)P was found to be present on LCVs using as a probe either an antibody against PI(4)P or the PH domain of the PI(4)P-binding protein FAPP1 (phosphatidylinositol(4) phosphate adaptor protein-1). Moreover, the presence of PI(4)P on LCVs required a functional Icm/Dot T4SS. Our results indicate that L. pneumophila modulates host cell PI metabolism and exploits the Golgi lipid second messenger PI(4)P to anchor secreted effector proteins to the LCV.

The Legionella pneumophila phosphatidylinositol-4 phosphate-binding type IV substrate SidC recruits endoplasmic reticulum vesicles to a replication-permissive vacuole

Legionella pneumophila, the causative agent of Legionnaires disease, uses the intracellular multiplication/defective organelle trafficking (Icm/Dot) type IV secretion system to establish within amoebae and macrophages an endoplasmic reticulum (ER)‐derived replication‐permissive compartment, the Legionella‐containing vacuole (LCV). The Icm/Dot substrate SidC and its paralogue SdcA anchor to LCVs via phosphatidylinositol‐4 phosphate [PtdIns(4)P]. Here we identify the unique 20u2003kDa PtdIns(4)P‐binding domain of SidC, which upon heterologous expression in Dictyostelium binds to LCVs and thus is useful as a PtdIns(4)P‐specific probe. LCVs harbouring L.u2003pneumophilaΔsidC‐sdcA mutant bacteria recruit ER and ER‐derived vesicles less efficiently and carry endosomal but not lysosomal markers. The phenotypes are complemented by supplying sidC on a plasmid. L.u2003pneumophilaΔsidC‐sdcA grows at wild‐type rate in calnexin‐negative LCVs, suggesting that communication with the ER is dispensable for establishing a replicative compartment. The amount of SidC and calnexin is directly proportional on isolated LCVs, and in a cell‐free system, the recruitment of calnexin‐positive vesicles to LCVs harbouring ΔsidC‐sdcA mutant bacteria is impaired. Beads coated with purified SidC or its 70u2003kDa N‐terminal fragment recruit ER vesicles in Dictyostelium and macrophage lysates. Our results establish SidC as an L.u2003pneumophila effector protein, which anchors to PtdIns(4)P on LCVs and recruits ER vesicles to a replication‐permissive vacuole.

The Legionella pneumophila response regulator LqsR promotes host cell interactions as an element of the virulence regulatory network controlled by RpoS and LetA

Legionella pneumophila is an opportunistic human pathogen that replicates within environmental amoebae including Acanthamoeba castellanii and Dictyostelium discoideum. The Icm/Dot type IV secretion system promotes phagocytosis and intracellular replication of L.u2003pneumophila in an endoplasmic reticulum‐derived ‘Legionella‐containing vacuole’ (LCV). L.u2003pneumophila adopts a biphasic life cycle consisting of a replicative growth phase and a transmissive (stationary) phase, the latter of which is characterized by the preferential expression of genes required for motility and virulence. A bioinformatic analysis of the L.u2003pneumophila genome revealed a gene cluster homologous to the Vibrio cholerae cqsAS genes, encoding a putative quorum sensing autoinducer synthase (lqsA) and a sensor kinase (lqsS), which flank a novel response regulator (lqsR). We report here that an L.u2003pneumophila lqsR deletion mutant grew in broth with the same rate as wild‐type bacteria, but entered the replicative growth phase earlier. Overexpression of lqsR led to an elongated morphology of the bacteria. The lqsR mutant strain was found to be more salt‐resistant and impaired for intracellular growth in A.u2003castellanii, D.u2003discoideum and macrophages, formation of the ER‐derived LCV and toxicity. Moreover, L.u2003pneumophila lacking LqsR, as well as strains lacking the stationary sigma factor RpoS or the two‐component response regulator LetA, were phagocytosed less efficiently by A.u2003castellanii, D.u2003discoideum or macrophages. The expression of lqsR was dependent on RpoS and, to a lesser extent, also on LetA. DNA microarray experiments revealed that lqsR regulates the expression of genes involved in virulence, motility and cell division, consistent with a role for LqsR in the transition from the replicative to the transmissive (virulent) phase. Our findings indicate that LqsR is a novel pleiotropic regulator involved in RpoS‐ and LetA‐controlled interactions of L.u2003pneumophila with phagocytes.

Pathogen trafficking pathways and host phosphoinositide metabolism

Phosphoinositide (PI) glycerolipids are key regulators of eukaryotic signal transduction, cytoskeleton architecture and membrane dynamics. The host cell PI metabolism is targeted by intracellular bacterial pathogens, which evolved intricate strategies to modulate uptake processes and vesicle trafficking pathways. Upon entering eukaryotic host cells, pathogenic bacteria replicate in distinct vacuoles or in the host cytoplasm. Vacuolar pathogens manipulate PI levels to mimic or modify membranes of subcellular compartments and thereby establish their replicative niche. Legionella pneumophila, Brucella abortus, Mycobacterium tuberculosis and Salmonella enterica translocate effector proteins into the host cell, some of which anchor to the vacuolar membrane via PIs or enzymatically turnover PIs. Cytoplasmic pathogens target PI metabolism at the plasma membrane, thus modulating their uptake and antiapoptotic signalling pathways. Employing this strategy, Shigella flexneri directly injects a PI‐modifying effector protein, while Listeria monocytogenes exploits PI metabolism indirectly by binding to transmembrane receptors. Thus, regardless of the intracellular lifestyle of the pathogen, PI metabolism is critically involved in the interactions with host cells.

The inositol polyphosphate 5‐phosphatase OCRL1 restricts intracellular growth of Legionella, localizes to the replicative vacuole and binds to the bacterial effector LpnE

Legionella pneumophila, the causative agent of Legionnaires disease, replicates within a specific vacuole in amoebae and macrophages. To form these ‘Legionella‐containing vacuoles’ (LCVs), the bacteria employ the Icm/Dot type IV secretion system and effector proteins, some of which anchor to the LCV membrane via the host glycolipid phosphatidylinositol 4‐phosphate [PtdIns(4)P]. Here we analysed the role of inositol polyphosphate 5‐phosphatases (IP5Ps) during L.u2003pneumophila infections. Bacterial replication and LCV formation occurred more efficiently in Dictyostelium discoideum amoebae lacking the IP5P Dd5P4, a homologue of human OCRL1 (Oculocerebrorenal syndrome of Lowe), implicated in retrograde endosome to Golgi trafficking. The phenotype was complemented by Dd5P4 but not the catalytically inactive 5‐phosphatase. Ectopically expressed Dd5P4 or OCRL1 localized to LCVs in D.u2003discoideum via an N‐terminal domain previously not implicated in membrane targeting, and OCRL1 was also identified on LCVs in macrophages. Dd5P4 was catalytically active on LCVs and accumulated on LCVs harbouring wild‐type but not ΔicmT mutant L.u2003pneumophila. The N‐terminal domain of OCRL1 bound L.u2003pneumophila LpnE, a Sel1‐like repeat protein involved in LCV formation, which localizes to LCVs and selectively binds PtdIns(3)P. Our results indicate that OCRL1 restricts intracellular growth of L.u2003pneumophila and binds to LCVs in association with LpnE.

Antibody-Fc receptor interactions in protection against intracellular pathogens.

Intracellular pathogen‐specific antibodies (Abs) can contribute to host protection by a number of different mechanisms. Ab opsonization of pathogens residing outside a host cell can prevent infection of target cells either via neutralization of the critical surface epitopes required for host cell entry, complement‐mediated degradation, or via subsequent intracellular degradation. In the case of intracellular localization, Abs can bind to infected cells and thus mark them for destruction by Fc receptor (FcR)‐bearing effector cells. This review focuses on the protective role of Abs against intracellular bacteria and parasites involving FcR interactions that modulate the intracellular trafficking of the pathogen, the ability of FcRs to interfere with the establishment of an intracellular replicative niche and the involvement of FcRs to modulate pathogen‐specific T‐cell responses.

Identification of Protective B Cell Antigens of Legionella pneumophila

Abs confer protection from secondary infection with Legionella pneumophila, the causative agent of a severe form of pneumonia known as Legionnaires’ disease. In this study, we demonstrate that Ab-mediated protection is effective across L. pneumophila serogroups, suggesting that Abs specific for conserved protein Ags are sufficient to mediate this protective effect. We used two independent methods to identify immunogenic L. pneumophila protein Ags, namely, the screening of a λ phage library representing the complete L. pneumophila genome and two-dimensional gel electrophoresis combined with Western blot analysis and protein spot identification by mass spectrometry. A total of 30 novel L. pneumophila B cell Ags were identified, the majority of which are located in or associated with the bacterial membrane, where they are accessible for Abs and, therefore, likely to be relevant for Ab-mediated protection against L. pneumophila. Selected B cell Ags were recombinantly expressed and tested in a vaccination protocol. Mice immunized with either single-protein Ags or an Ag combination showed reduced bacterial titers in bronchoalveolar lavage and lung after L. pneumophila challenge. To determine the clinical relevance of these findings, we tested Legionnaires’ disease patient sera for reactivity with the identified L. pneumophila Ags. The recognized Ags were indeed conserved across host species, because Abs specific for all three selected Ags could be detected in patient sera, rendering the identified protein Ags potential vaccine candidates.

Dissecting the Contribution of IgG Subclasses in Restricting Airway Infection with Legionella pneumophila

Abs are able to mediate local protection from pulmonary infection with Legionella pneumophila, the causative agent of a severe form of pneumonia known as Legionnaires’ disease. L. pneumophila is able to infect alveolar macrophages in the lung and replicates intracellularly in a vacuolar compartment with endoplasmic reticulum–like characteristics. However, Abs opsonize the bacteria and confer an FcR-mediated signal to phagocytic host cells that vetoes the bacterial evasion strategies, thereby efficiently targeting the bacteria to intracellular lysosomal degradation. In this study we analyzed the prevalence of pathogen-specific IgG subclasses present in immunized mice and found that the presence of IgG2c and IgG3 correlated with reduced bacterial titers after intranasal infection. We then isolated different IgG subclasses and compared their differential prophylactic potential in restricting airway L. pneumophila replication. We found that all IgG subclasses were effective in restricting pulmonary airway infection in mice when administered at high and equivalent doses. However, at limiting Ab concentrations we found a superior role of IgG2c in restricting L. pneumophila replication in a prophylactic setting. Furthermore, we assessed the therapeutic efficacy of administering an mAb during an established infection and found that bacterial titers could be reduced very efficiently with such a treatment. Thus, we propose the therapeutic use of Abs for the treatment of intracellular bacterial infections in situations where antibiotics might be ineffective.

Assessment of legionella-specific immunity in mice.

Legionella pneumophila is the causative agent of the potentially fatal Legionnaires disease in humans. Mice have proved to be valuable model organisms to study the pathogenesis of this intracellular bacterium, as well as immune responses against it. In this chapter we describe a selection of mouse infection protocols to study the innate and adaptive immune responses raised after an infection with Legionella. Included are protocols for systemic and pulmonary infections, surgical collection of organs as well as determination of cell composition, cytokines, and antibody titers therein. Furthermore, we describe an immunohistology protocol to analyze lung tissue sections by fluorescence microscopy.

Antibody-Dependent Cellular Phagocytosis and Its Impact on Pathogen Control

In humans and mice there exist a multitude of Fcγ receptors (FcγRs) with different affinities and specificities for the different IgG subclasses. Engagement of FcRs on cells usually results in the initiation of an activating or inhibitory signaling cascade that can lead to multiple effector functions, including phagocytosis of opsonized pathogens or immune complexes. In this chapter, we discuss phagocytic cell types and their respective FcγRs that they utilize to phagocytize IgG-coated particles/pathogens. Furthermore, we discuss downstream signaling mechanisms and resulting effects of FcγR-mediated phagocytosis. Special emphasis is given to the role of FcR-mediated phagocytosis in phagocytes for pathogen uptake, subsequent intracellular localization, and pathogen control.