The Legionella effector RavD binds phosphatidylinositol-3-phosphate and helps suppress endolysosomal maturation of the Legionella-containing vacuole.

Upon phagocytosis into macrophages, the intracellular bacterial pathogen Legionella pneumophila secretes effector proteins that manipulate host cell components, enabling it to evade lysosomal degradation. However, the bacterial proteins involved in this evasion are incompletely characterized. Here we show that the L. pneumophila effector protein RavD targets host membrane compartments and contributes to the molecular mechanism the pathogen uses to prevent encounters with lysosomes. Protein-lipid binding assays revealed that RavD selectively binds phosphatidylinositol-3-phosphate (PI(3)P) in vitro We further determined that a C-terminal RavD region mediates the interaction with PI(3)P and that this interaction requires Arg-292. In transiently transfected mammalian cells, mCherry-RavD colocalized with the early endosome marker EGFP-Rab5 as well as the PI(3)P biosensor EGFP-2×FYVE. However, treatment with the phosphoinositide 3-kinase inhibitor wortmannin did not disrupt localization of mCherry-RavD to endosomal compartments, suggesting that RavD's interaction with PI(3)P is not necessary to anchor RavD to endosomal membranes. Using superresolution and immunogold transmission EM, we observed that, upon translocation into macrophages, RavD was retained onto the Legionella-containing vacuole and was also present on small vesicles adjacent to the vacuole. We also report that despite no detectable effects on intracellular growth of L. pneumophila within macrophages or amebae, the lack of RavD significantly increased the number of vacuoles that accumulate the late endosome/lysosome marker LAMP-1 during macrophage infection. Together, our findings suggest that, although not required for intracellular replication of L. pneumophila, RavD is a part of the molecular mechanism that steers the Legionella-containing vacuole away from endolysosomal maturation pathways.

The recycling endosome and bacterial pathogens.

Bacterial pathogens have developed a wide range of strategies to survive within human cells. A number of pathogens multiply in a vacuolar compartment, whereas others can rupture the vacuole and replicate in the host cytosol. A common theme among many bacterial pathogens is the use of specialised secretion systems to deliver effector proteins into the host cell. These effectors can manipulate the host's membrane trafficking pathways to remodel the vacuole into a replication-permissive niche and prevent degradation. As master regulators of eukaryotic membrane traffic, Rab GTPases are principal targets of bacterial effectors. This review highlights the manipulation of Rab GTPases that regulate host recycling endocytosis by several bacterial pathogens, including Chlamydia pneumoniae, Chlamydia trachomatis, Shigella flexneri, Salmonella enterica serovar Typhimurium, Uropathogenic Escherichia coli, and Legionella pneumophila. Recycling endocytosis plays key roles in a variety of cellular aspects such as nutrient uptake, immunity, cell division, migration, and adhesion. Though much remains to be understood about the molecular basis and the biological relevance of bacterial pathogens exploiting Rab GTPases, current knowledge supports the notion that endocytic recycling Rab GTPases are differentially targeted to avoid degradation and support bacterial replication. Thus, future studies of the interactions between bacterial pathogens and host endocytic recycling pathways are poised to deepen our understanding of bacterial survival strategies.

Legionella effector AnkX interacts with host nuclear protein PLEKHN1.

BACKGROUND: The intracellular bacterial pathogen Legionella pneumophila proliferates in human alveolar macrophages, resulting in a severe pneumonia termed Legionnaires' disease. Throughout the course of infection, L. pneumophila remains enclosed in a specialized membrane compartment that evades fusion with lysosomes. The pathogen delivers over 300 effector proteins into the host cell, altering host pathways in a manner that sets the stage for efficient pathogen replication. The L. pneumophila effector protein AnkX targets host Rab GTPases and functions in preventing fusion of the Legionella-containing vacuole with lysosomes. However, the current understanding of AnkX's interaction with host proteins and the means through which it exerts its cellular function is limited. RESULTS: Here, we investigated the protein interaction network of AnkX by using the nucleic acid programmable protein array (NAPPA), a high-density platform comprising 10,000 unique human ORFs. This approach facilitated the discovery of PLEKHN1 as a novel interaction partner of AnkX. We confirmed this interaction through multiple independent in vitro pull-down, co-immunoprecipitation, and cell-based assays. Structured illumination microscopy revealed that endogenous PLEKHN1 is found in the nucleus and on vesicular compartments, whereas ectopically produced AnkX co-localized with lipid rafts at the plasma membrane. In mammalian cells, HaloTag-AnkX co-localized with endogenous PLEKHN1 on vesicular compartments. A central fragment of AnkX (amino acids 491-809), containing eight ankyrin repeats, extensively co-localized with endogenous PLEKHN1, indicating that this region may harbor a new function. Further, we found that PLEKHN1 associated with multiple proteins involved in the inflammatory response. CONCLUSIONS: Altogether, our study provides evidence that in addition to Rab GTPases, the L. pneumophila effector AnkX targets nuclear host proteins and suggests that AnkX may have novel functions related to manipulating the inflammatory response.

Legionella Effector AnkX Disrupts Host Cell Endocytic Recycling in a Phosphocholination-Dependent Manner.

The facultative intracellular bacterium Legionella pneumophila proliferates within amoebae and human alveolar macrophages, and it is the causative agent of Legionnaires' disease, a life-threatening pneumonia. Within host cells, L. pneumophila establishes a replicative haven by delivering numerous effector proteins into the host cytosol, many of which target membrane trafficking by manipulating the function of Rab GTPases. The Legionella effector AnkX is a phosphocholine transferase that covalently modifies host Rab1 and Rab35. However, a detailed understanding of the biological consequence of Rab GTPase phosphocholination remains elusive. Here, we broaden the understanding of AnkX function by presenting three lines of evidence that it interferes with host endocytic recycling. First, using immunogold transmission electron microscopy, we determined that GFP-tagged AnkX ectopically produced in mammalian cells localizes at the plasma membrane and tubular membrane compartments, sites consistent with targeting the endocytic recycling pathway. Furthermore, the C-terminal region of AnkX was responsible for association with the plasma membrane, and we determined that this region was also able to bind the phosphoinositide lipids PI(3)P and PI(4)P in vitro. Second, we observed that mCherry-AnkX co-localized with Rab35, a regulator of recycling endocytosis and with major histocompatibility class I protein (MHC-I), a key immunoregulatory protein whose recycling from and back to the plasma membrane is Rab35-dependent. Third, we report that during infection of macrophages, AnkX is responsible for the disruption of endocytic recycling of transferrin, and AnkX's phosphocholination activity is critical for this function. These results support the hypothesis that AnkX targets endocytic recycling during host cell infection. Finally, we have demonstrated that the phosphocholination activity of AnkX is also critical for inhibiting fusion of the Legionella-containing vacuole (LCV) with lysosomes.

Host-pathogen interaction profiling using self-assembling human protein arrays.

Host-pathogen protein interactions are fundamental to every microbial infection, yet their identification has remained challenging due to the lack of simple detection tools that avoid abundance biases while providing an open format for experimental modifications. Here, we applied the Nucleic Acid-Programmable Protein Array and a HaloTag-Halo ligand detection system to determine the interaction network of Legionella pneumophila effectors (SidM and LidA) with 10 000 unique human proteins. We identified known targets of these L. pneumophila proteins and potentially novel interaction candidates. In addition, we applied our Click chemistry-based NAPPA platform to identify the substrates for SidM, an effector with an adenylyl transferase domain that catalyzes AMPylation (adenylylation), the covalent addition of adenosine monophosphate (AMP). We confirmed a subset of the novel SidM and LidA targets in independent in vitro pull-down and in vivo cell-based assays, and provided further insight into how these effectors may discriminate between different host Rab GTPases. Our method circumvents the purification of thousands of human and pathogen proteins, and does not require antibodies against or prelabeling of query proteins. This system is amenable to high-throughput analysis of effectors from a wide variety of human pathogens that may bind to and/or post-translationally modify targets within the human proteome.

Structural basis for Rab1 de-AMPylation by the Legionella pneumophila effector SidD.

The covalent attachment of adenosine monophosphate (AMP) to proteins, a process called AMPylation (adenylylation), has recently emerged as a novel theme in microbial pathogenesis. Although several AMPylating enzymes have been characterized, the only known virulence protein with de-AMPylation activity is SidD from the human pathogen Legionella pneumophila. SidD de-AMPylates mammalian Rab1, a small GTPase involved in secretory vesicle transport, thereby targeting the host protein for inactivation. The molecular mechanisms underlying Rab1 recognition and de-AMPylation by SidD are unclear. Here, we report the crystal structure of the catalytic region of SidD at 1.6 Å resolution. The structure reveals a phosphatase-like fold with additional structural elements not present in generic PP2C-type phosphatases. The catalytic pocket contains a binuclear metal-binding site characteristic of hydrolytic metalloenzymes, with strong dependency on magnesium ions. Subsequent docking and molecular dynamics simulations between SidD and Rab1 revealed the interface contacts and the energetic contribution of key residues to the interaction. In conjunction with an extensive structure-based mutational analysis, we provide in vivo and in vitro evidence for a remarkable adaptation of SidD to its host cell target Rab1 which explains how this effector confers specificity to the reaction it catalyses.

The taming of a Rab GTPase by Legionella pneumophila.

Small GTPases of the Rab family represent an attractive target for microbial pathogens due to their role in controlling many aspects of intracellular cargo transport. Legionella pneumophila is an intravacuolar pathogen that survives inside host cells by manipulating protein trafficking pathways through a number of effector proteins secreted by the bacterium. These act as functional mimics of host proteins that modulate the activity of switch proteins such as guanosine triphosphatases (GTPases). L. pneumophila exploits the ER (endoplasmic reticulum)-to-Golgi vesicle transport pathway by modifying activity of Rab1, the GTPase regulating this pathway. This pathogen recruits Rab1 to the vacuole in which it resides, where effector proteins located on the surface of the vacuole regulate the activity status of Rab1 by mimicking the function of a guanine dissociation inhibitor (GDI) displacement factor, guanine exchange factor (GEF), or a GTPase-activating protein (GAP). In addition to these non-covalent modifications that alter the nucleotide binding state of Rab1, the bacterium also uses covalent modifications such as adenylylation (AMPylation) to control the dynamic of Rab1 on the Legionella-containing vacuole. Remarkably, AMPylation of Rab1 by SidM can be reversed by the L. pneumophila effector protein SidD, which exhibits de-AMPylation activity, demonstrating that L. pneumophila and related pathogens may utilize covalent modifications in order to transiently alter the activity of host proteins.

Legionella pneumophila LidA affects nucleotide binding and activity of the host GTPase Rab1.

Legionella pneumophila, the causative agent of a severe pneumonia known as Legionnaires' disease, intercepts material from host cell membrane transport pathways to create a specialized vacuolar compartment that supports bacterial replication. Delivery of bacterial effector proteins into the host cell requires the Dot/Icm type IV secretion system. Several effectors, including SidM, SidD, and LepB, were shown to target the early secretory pathway by manipulating the activity of the host GTPase Rab1. While the function of these effectors has been well characterized, the role of another Rab1-interacting protein from L. pneumophila, the effector protein LidA, is poorly understood. Here, we show that LidA binding to Rab1 stabilized the Rab1-guanosine nucleotide complex, protecting it from inactivation by GTPase-activating proteins (GAPs) and from nucleotide extraction. The protective effect of LidA on the Rab1-guanine nucleotide complex was concentration dependent, consistent with a 1:1 stoichiometry of the LidA-Rab1 complex. The central coiled-coil region of LidA was sufficient for Rab1 binding and to prevent GAP-mediated inactivation or nucleotide extraction from Rab1. In addition, the central region mediated binding to phosphatidylinositol 3-phosphate and other phosphoinositides. When bound to Rab1, LidA interfered with the covalent modification of Rab1 by phosphocholination or AMPylation, and it also blocked de-AMPylation of Rab1 by SidD and dephosphocholination by Lem3. Based on these findings, we propose a role for LidA in bridging the membrane of the Legionella-containing vacuole (LCV) with that of secretory transport vesicles surrounding the LCV.

De-AMPylation of the small GTPase Rab1 by the pathogen Legionella pneumophila.

The bacterial pathogen Legionella pneumophila exploits host cell vesicle transport by transiently manipulating the activity of the small guanosine triphosphatase (GTPase) Rab1. The effector protein SidM recruits Rab1 to the Legionella-containing vacuole (LCV), where it activates Rab1 and then AMPylates it by covalently adding adenosine monophosphate (AMP). L. pneumophila GTPase-activating protein LepB inactivates Rab1 before its removal from LCVs. Because LepB cannot bind AMPylated Rab1, the molecular events leading to Rab1 inactivation are unknown. We found that the effector protein SidD from L. pneumophila catalyzed AMP release from Rab1, generating de-AMPylated Rab1 accessible for inactivation by LepB. L. pneumophila mutants lacking SidD were defective for Rab1 removal from LCVs, identifying SidD as the missing link connecting the processes of early Rab1 accumulation and subsequent Rab1 removal during infection.

The sigE gene is required for normal expression of heterocyst-specific genes in Anabaena sp. strain PCC 7120.

The filamentous cyanobacterium Anabaena (Nostoc) sp. strain PCC 7120 produces specialized cells for nitrogen fixation called heterocysts. Previous work showed that the group 2 sigma factor sigE (alr4249; previously called sigF) is upregulated in differentiating heterocysts 16 h after nitrogen step-down. We now show that the sigE gene is required for normal heterocyst development and normal expression levels of several heterocyst-specific genes. Mobility shift assays showed that the transcription factor NtcA binds to sites in the upstream region of sigE and that this binding is enhanced by 2-oxoglutarate (2-OG). Deletions of the region containing the NtcA binding sites in P(sigE)-gfp reporter plasmids showed that the sites contribute to normal developmental regulation but are not essential for upregulation in heterocysts. Northern RNA blot analysis of nifH mRNA revealed delayed and reduced transcript levels during heterocyst differentiation in a sigE mutant background. Quantitative reverse transcription-PCR (qRT-PCR) analyses of the sigE mutant showed lower levels of transcripts for nifH, fdxH, and hglE2 but normal levels for hupL. We developed a P(nifHD)-gfp reporter construct that showed strong heterocyst-specific expression. Time-lapse microscopy of the P(nifHD)-gfp reporter in a sigE mutant background showed delayed development and undetectable green fluorescent protein (GFP) fluorescence. Overexpression of sigE caused accelerated heterocyst development, an increased heterocyst frequency, and premature expression of GFP fluorescence from the P(nifHD)-gfp reporter.

Anabaena sp. strain PCC 7120 conR contains a LytR-CpsA-Psr domain, is developmentally regulated, and is essential for diazotrophic growth and heterocyst morphogenesis.

The conR (all0187) gene of the filamentous cyanobacterium Anabaena (Nostoc) sp. strain PCC 7120 is predicted to be part of a family of proteins that contain the LytR-CpsA-Psr domain associated with septum formation and cell wall maintenance. The conR gene was originally misannotated as a transcription regulator. Northern RNA blot analysis showed that conR expression was upregulated 8 h after nitrogen step-down. Fluorescence microscopy of a P(conR)-gfp reporter strain revealed increased GFP fluorescence in proheterocysts and heterocysts beginning 9 h after nitrogen step-down. Insertional inactivation of conR caused a septum-formation defect of vegetative cells grown in nitrate-containing medium. In nitrate-free medium, mutant filaments formed abnormally long heterocysts and were defective for diazotrophic growth. Septum formation between heterocysts and adjacent vegetative cells was abnormal, often with one or both poles of the heterocysts appearing partially open. In a conR mutant, expression of nifH was delayed after nitrogen step-down and nitrogenase activity was approximately 70 % of wild-type activity, indicating that heterocysts of the conR mutant strain are partially functional. We hypothesize that the diazotrophic growth defect is caused by an inability of the heterocysts to transport fixed nitrogen to the neighbouring vegetative cells.

The Anabaena sp. strain PCC 7120 gene all2874 encodes a diguanylate cyclase and is required for normal heterocyst development under high-light growth conditions.

The genome of the heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120 harbors 14 genes containing a GGDEF diguanylate cyclase domain. We found that inactivation of one of these genes, all2874, caused abnormal heterocyst development. The all2874 mutant showed a pronounced reduction in heterocyst frequency during diazotrophic growth and reduced vegetative cell size compared to the wild type. The severity of the mutant phenotype varied with light intensity; at high light intensity, the mutant phenotype was accentuated, whereas at low light intensity the phenotype was similar to wild type. Under high-light growth conditions, the initial heterocyst frequency and pattern for the all2874 mutant were normal, but within 4 days following nitrogen step-down, many intervals between heterocysts increased to as many as 200 vegetative cells, whereas in the wild type the intervals were less than 25 vegetative cells. Filaments containing these unusually long vegetative cell intervals between heterocysts also contained intervals of normal length. An all2874 mutant strain carrying a P(patS)-gfp transcriptional reporter fusion failed to show normal upregulation of the reporter, which indicates that the decrease in heterocyst frequency is due to an early block in differentiation before induction of the patS gene, which in the wild type takes place 8 h after nitrogen step-down. Genetic epistasis experiments suggest that All2874 acts upstream of the master regulator HetR in differentiating cells. We also showed that purified All2874 functions as a diguanylate cyclase in vitro. We hypothesize that All2874 is required for the normal regulation of heterocyst frequency under high-light growth conditions.