Systematic characterization of all Toxoplasma gondii TBC domain-containing proteins identifies an essential regulator of Rab2 in the secretory pathway.

Toxoplasma gondii resides in its intracellular niche by employing a series of specialized secretory organelles that play roles in invasion, host cell manipulation, and parasite replication. Rab GTPases are major regulators of the parasite's secretory traffic that function as nucleotide-dependent molecular switches to control vesicle trafficking. While many of the Rab proteins have been characterized in T. gondii, precisely how these Rabs are regulated remains poorly understood. To better understand the parasite's secretory traffic, we investigated the entire family of Tre2-Bub2-Cdc16 (TBC) domain-containing proteins, which are known to be involved in vesicle fusion and secretory protein trafficking. We first determined the localization of all 18 TBC domain-containing proteins to discrete regions of the secretory pathway or other vesicles in the parasite. Second, we use an auxin-inducible degron approach to demonstrate that the protozoan-specific TgTBC9 protein, which localizes to the endoplasmic reticulum (ER), is essential for parasite survival. Knockdown of TgTBC9 results in parasite growth arrest and affects the organization of the ER and mitochondrial morphology. TgTBC9 knockdown also results in the formation of large lipid droplets (LDs) and multi-membranous structures surrounded by ER membranes, further indicating a disruption of ER functions. We show that the conserved dual-finger active site in the TBC domain of the protein is critical for its GTPase-activating protein (GAP) function and that the Plasmodium falciparum orthologue of TgTBC9 can rescue the lethal knockdown. We additionally show by immunoprecipitation and yeast 2 hybrid analyses that TgTBC9 preferentially binds Rab2, indicating that the TBC9-Rab2 pair controls ER morphology and vesicular trafficking in the parasite. Together, these studies identify the first essential TBC protein described in any protozoan and provide new insight into intracellular vesicle trafficking in T. gondii.

Golgi associated RAB2 interactor protein family contributes to murine male fertility to various extents by assuring correct morphogenesis of sperm heads.

Sperm heads contain not only the nucleus but also the acrosome which is a distinctive cap-like structure located anterior to the nucleus and is derived from the Golgi apparatus. The Golgi Associated RAB2 Interactors (GARINs; also known as FAM71) protein family shows predominant expression in the testis and all possess a RAB2-binding domain which confers binding affinity to RAB2, a small GTPase that is responsible for membrane transport and vesicle trafficking. Our previous study showed that GARIN1A and GARIN1B are important for acrosome biogenesis and that GARIN1B is indispensable for male fertility in mice. Here, we generated KO mice of other Garins, namely Garin2, Garin3, Garin4, Garin5a, and Garin5b (Garin2-5b). Using computer-assisted morphological analysis, we found that the loss of each Garin2-5b resulted in aberrant sperm head morphogenesis. While the fertilities of Garin2-/- and Garin4-/- males are normal, Garin5a-/- and Garin5b-/- males are subfertile, and Garin3-/- males are infertile. Further analysis revealed that Garin3-/- males exhibited abnormal acrosomal morphology, but not as severely as Garin1b-/- males; instead, the amounts of membrane proteins, particularly ADAM family proteins, decreased in Garin3 KO spermatozoa. Moreover, only Garin4 KO mice exhibit vacuoles in the sperm head. These results indicate that GARINs assure correct head morphogenesis and some members of the GARIN family function distinctively in male fertility.

FAM71F1 binds to RAB2A and RAB2B and is essential for acrosome formation and male fertility in mice.

The acrosome is a cap-shaped, Golgi-derived membranous organelle that is located over the anterior of the sperm nucleus and highly conserved throughout evolution. Although morphological changes during acrosome biogenesis in spermatogenesis have been well described, the molecular mechanism underlying this process is still largely unknown. Family with sequence similarity 71, member F1 and F2 (FAM71F1 and FAM71F2) are testis-enriched proteins that contain a RAB2B-binding domain, a small GTPase involved in vesicle transport and membrane trafficking. Here, by generating mutant mice for each gene, we found that Fam71f1 is essential for male fertility. In Fam71f1-mutant mice, the acrosome was abnormally expanded at the round spermatid stage, likely because of enhanced vesicle trafficking. Mass spectrometry analysis after immunoprecipitation indicated that, in testes, FAM71F1 binds not only RAB2B, but also RAB2A. Further study suggested that FAM71F1 binds to the GTP-bound active form of RAB2A/B, but not the inactive form. These results indicate that a complex of FAM71F1 and active RAB2A/B suppresses excessive vesicle trafficking during acrosome formation.

Regulation of neuromuscular junction organization by Rab2 and its effector ICA69 in Drosophila.

The mechanisms underlying synaptic differentiation, which involves neuronal membrane and cytoskeletal remodeling, are not completely understood. We performed a targeted RNAi-mediated screen of Drosophila BAR-domain proteins and identified islet cell autoantigen 69 kDa (ICA69) as one of the key regulators of morphological differentiation of the larval neuromuscular junction (NMJ). We show that Drosophila ICA69 colocalizes with α-Spectrin at the NMJ. The conserved N-BAR domain of ICA69 deforms liposomes in vitro Full-length ICA69 and the ICAC but not the N-BAR domain of ICA69 induce filopodia in cultured cells. Consistent with its cytoskeleton regulatory role, ICA69 mutants show reduced α-Spectrin immunoreactivity at the larval NMJ. Manipulating levels of ICA69 or its interactor PICK1 alters the synaptic level of ionotropic glutamate receptors (iGluRs). Moreover, reducing PICK1 or Rab2 levels phenocopies ICA69 mutation. Interestingly, Rab2 regulates not only synaptic iGluR but also ICA69 levels. Thus, our data suggest that: (1) ICA69 regulates NMJ organization through a pathway that involves PICK1 and Rab2, and (2) Rab2 functions genetically upstream of ICA69 and regulates NMJ organization and targeting/retention of iGluRs by regulating ICA69 levels.

Rap2B knockdown suppresses malignant progression of hepatocellular carcinoma by inactivating the PTEN/PI3K/Akt and ERK1/2 pathways.

Rap2B, belonging to the Ras superfamily of small guanosine triphosphate-binding proteins, is upregulated and contributes to the progression of several tumors by acting as an oncogene, including hepatocellular carcinoma (HCC). However, the mechanism underlying the functional roles of Rap2B in HCC remains unclear. In this study, the evaluation of Rap2B expression in HCC cells and tissues was achieved by qRT-PCR and western blot assays. The effects of Rap2B on the malignant biological behaviors in HCC were explored by means of MTT assay, flow cytometry analysis, and Transwell invasion assay, respectively. Protein levels of Ki67, matrix metalloproteinase (MMP)-2, MMP-9, and cleaved caspase-3, together with the alternations of the ERK1/2 and PTEN/PI3K/Akt pathways were qualified by western blot assay. Further verification of the Rap2B function on HCC tumorigenesis was attained by performing in vivo assays. We found that Rap2B levels were upregulated in HCC tissues and cells. Rap2B silencing led to a reduction of cell-proliferative and invasive abilities, and an increase of apoptosis in HCC cells. In addition, xenograft tumor assay demonstrated that Rap2B silencing repressed HCC xenograft tumor growth in vivo. In addition, we found that Rap2B knockdown significantly inhibited the ERK1/2 and PTEN/PI3K/Akt cascades in HCC cells and xenograft tumor tissues. Together, Rap2B knockdown inhibited HCC-malignant progression, which was involved in inhibiting the ERK1/2 and PTEN/PI3K/Akt pathways. Our findings contribute to understanding of the molecular mechanism of Rap2B in HCC progression.

The EARP Complex and Its Interactor EIPR-1 Are Required for Cargo Sorting to Dense-Core Vesicles.

The dense-core vesicle is a secretory organelle that mediates the regulated release of peptide hormones, growth factors, and biogenic amines. Dense-core vesicles originate from the trans-Golgi of neurons and neuroendocrine cells, but it is unclear how this specialized organelle is formed and acquires its specific cargos. To identify proteins that act in dense-core vesicle biogenesis, we performed a forward genetic screen in Caenorhabditis elegans for mutants defective in dense-core vesicle function. We previously reported the identification of two conserved proteins that interact with the small GTPase RAB-2 to control normal dense-core vesicle cargo-sorting. Here we identify several additional conserved factors important for dense-core vesicle cargo sorting: the WD40 domain protein EIPR-1 and the endosome-associated recycling protein (EARP) complex. By assaying behavior and the trafficking of dense-core vesicle cargos, we show that mutants that lack EIPR-1 or EARP have defects in dense-core vesicle cargo-sorting similar to those of mutants in the RAB-2 pathway. Genetic epistasis data indicate that RAB-2, EIPR-1 and EARP function in a common pathway. In addition, using a proteomic approach in rat insulinoma cells, we show that EIPR-1 physically interacts with the EARP complex. Our data suggest that EIPR-1 is a new interactor of the EARP complex and that dense-core vesicle cargo sorting depends on the EARP-dependent trafficking of cargo through an endosomal sorting compartment.

Membrane skeletal association and post-translational allosteric regulation of Toxoplasma gondii GAPDH1.

When Toxoplasma gondii egresses from the host cell, glyceraldehyde-3-phosphate dehydrogenase 1 (GAPDH1), which is primary a glycolysis enzyme but actually a quintessential multifunctional protein, translocates to the unique cortical membrane skeleton. Here, we report the 2.25 Å resolution crystal structure of the GAPDH1 holoenzyme in a quaternary complex providing the basis for the molecular dissection of GAPDH1 structure-function relationships Knockdown of GAPDH1 expression and catalytic site disruption validate the essentiality of GAPDH1 in intracellular replication but we confirmed that glycolysis is not strictly essential. We identify, for the first time, S-loop phosphorylation as a novel, critical regulator of enzymatic activity that is consistent with the notion that the S-loop is critical for cofactor binding, allosteric activation and oligomerization. We show that neither enzymatic activity nor phosphorylation state correlate with the ability to translocate to the cortex. However, we demonstrate that association of GAPDH1 with the cortex is mediated by the N-terminus, likely palmitoylation. Overall, glycolysis and cortical translocation are functionally decoupled by post-translational modifications.

Huntingtin differentially regulates the axonal transport of a sub-set of Rab-containing vesicles in vivo.

Loss of huntingtin (HTT), the Huntington's disease (HD) protein, was previously shown to cause axonal transport defects. Within axons, HTT can associate with kinesin-1 and dynein motors either directly or via accessory proteins for bi-directional movement. However, the composition of the vesicle-motor complex that contains HTT during axonal transport is unknown. Here we analyze the in vivo movement of 16 Rab GTPases within Drosophila larval axons and show that HTT differentially influences the movement of a particular sub-set of these Rab-containing vesicles. While reduction of HTT perturbed the bi-directional motility of Rab3 and Rab19-containing vesicles, only the retrograde motility of Rab7-containing vesicles was disrupted with reduction of HTT. Interestingly, reduction of HTT stimulated the anterograde motility of Rab2-containing vesicles. Simultaneous dual-view imaging revealed that HTT and Rab2, 7 or 19 move together during axonal transport. Collectively, our findings indicate that HTT likely influences the motility of different Rab-containing vesicles and Rab-mediated functions. These findings have important implications for our understanding of the complex role HTT plays within neurons normally, which when disrupted may lead to neuronal death and disease.

Small GTPase Rab2B and Its Specific Binding Protein Golgi-associated Rab2B Interactor-like 4 (GARI-L4) Regulate Golgi Morphology.

Rab small GTPases are crucial regulators of the membrane traffic that maintains organelle identity and morphology. Several Rab isoforms are present in the Golgi, and it has been suggested that they regulate the compacted morphology of the Golgi in mammalian cells. However, the functional relationships among the Golgi-resident Rabs, e.g. whether they are functionally redundant or different, are poorly understood. In this study, we used specific siRNAs to perform genome-wide screening for human Rabs that are involved in Golgi morphology in HeLa-S3 cells. The results showed that knockdown of any one of the six Rab isoforms (Rab1A/1B/2A/2B/6B/8A) induced fragmentation of the Golgi in HeLa-S3 cells and that its phenotype was rescued by re-expression of their respective siRNA-resistant construct. We then performed systematic knockdown-rescue experiments in relation to each of the six Rabs. Interestingly, with the exception of the Rab8A knockdown, the Golgi fragmentation phenotype induced by knockdown of a single Rab isoform, e.g. Rab2B, was efficiently rescued by re-expression of its siRNA-resistant Rab alone, not by any of the other five Rabs, e.g. Rab2A, which is highly homologous to Rab2B, indicating that these Rab isoforms non-redundantly regulate Golgi morphology possibly through interaction with isoform-specific effector molecules. In addition, we identified Golgi-associated Rab2B interactor-like 4 (GARI-L4) as a novel Golgi-resident Rab2B-specific binding protein whose knockdown also induced fragmentation of the Golgi. Our findings suggest that the compacted Golgi morphology of mammalian cells is finely tuned by multiple sets of Rab (or Rab-effector complexes) that for the most part function independently.

Retromer and Rab2-dependent trafficking mediate PS1 degradation by proteasomes in endocytic disturbance.

Accumulating evidence suggests that endocytic pathway deficits are involved in Alzheimer's disease pathogenesis. Several reports show that endocytic disturbance affects ß-amyloid peptide (Aß) cleavage from ß-amyloid precursor protein (APP). Presenilin-1 (PS1) is the catalytic core of the γ-secretase complex required for Aß generation. Previously, we showed that aging induces endocytic disturbance, resulting in the accumulation of Aß and APP in enlarged endosomes. It remains unclear, however, whether PS1 localization and function are affected with endocytic disturbance. Here, we report that in endocytic disturbance, PS1 is transported from endosomes to ER/Golgi compartments via retromer trafficking, and that PS1 interacts with vacuolar protein sorting-associated protein 35 both in vitro and in vivo. Moreover, PS1 is degraded by proteasomes via a Rab2-dependent trafficking pathway, only during endocytic disturbance. These findings suggest that PS1 levels and localization in endosomes are regulated by retromer trafficking and ER-associated degradation system, even if endocytic disturbance significantly induces the endosomal accumulation of APP and ß-site APP-cleaving enzyme 1. Results of this study also suggest that retromer deficiency can affect PS1 localization in endosomes, where Aß cleavage mainly occurs, possibly leading to enhanced Aß pathology. We proposed the following mechanism for intracellular transport of presenilin-1 (PS1). When endosome/lysosome trafficking is disturbed, PS1 is transported from endosome to endoplasmic reticulum (ER)/Golgi compartments via retromer and Rab2-mediated trafficking, and then degraded by endoplasmic reticulum-associated degradation (ERAD). Perturbations in this trafficking can cause abnormal endosomal accumulation of PS1, and then may lead to exacerbated Aß pathology. Cover Image for this issue: doi: 10.1111/jnc.13318.

TBC-8, a putative RAB-2 GAP, regulates dense core vesicle maturation in Caenorhabditis elegans.

Dense core vesicles (DCVs) are thought to be generated at the late Golgi apparatus as immature DCVs, which subsequently undergo a maturation process through clathrin-mediated membrane remodeling events. This maturation process is required for efficient processing of neuropeptides within DCVs and for removal of factors that would otherwise interfere with DCV release. Previously, we have shown that the GTPase, RAB-2, and its effector, RIC-19, are involved in DCV maturation in Caenorhabditis elegans motoneurons. In rab-2 mutants, specific cargo is lost from maturing DCVs and missorted into the endosomal/lysosomal degradation route. Cargo loss could be prevented by blocking endosomal delivery. This suggests that RAB-2 is involved in retention of DCV components during the sorting process at the Golgi-endosomal interface. To understand how RAB-2 activity is regulated at the Golgi, we screened for RAB-2-specific GTPase activating proteins (GAPs). We identified a potential RAB-2 GAP, TBC-8, which is exclusively expressed in neurons and which, when depleted, shows similar DCV maturation defects as rab-2 mutants. We could demonstrate that RAB-2 binds to its putative GAP, TBC-8. Interestingly, TBC-8 also binds to the RAB-2 effector, RIC-19. This interaction appears to be conserved as TBC-8 also interacted with the human ortholog of RIC-19, ICA69. Therefore, we propose that a dynamic ON/OFF cycling of RAB-2 at the Golgi induced by the GAP/effector complex is required for proper DCV maturation.

The GTPase ARFRP1 controls the lipidation of chylomicrons in the Golgi of the intestinal epithelium.

The uptake and processing of dietary lipids by the small intestine is a multistep process that involves several steps including vesicular and protein transport. The GTPase ADP-ribosylation factor-related protein 1 (ARFRP1) controls the ARF-like 1 (ARL1)-mediated Golgi recruitment of GRIP domain proteins which in turn bind several Rab-GTPases. Here, we describe the essential role of ARFRP1 and its interaction with Rab2 in the assembly and lipidation of chylomicrons in the intestinal epithelium. Mice lacking Arfrp1 specifically in the intestine (Arfrp1(vil-/-)) exhibit an early post-natal growth retardation with reduced plasma triacylglycerol and free fatty acid concentrations. Arfrp1(vil-/-) enterocytes as well as Arfrp1 mRNA depleted Caco-2 cells absorbed fatty acids normally but secreted chylomicrons with a markedly reduced triacylglycerol content. In addition, the release of apolipoprotein A-I (ApoA-I) was dramatically decreased, and ApoA-I accumulated in the Arfrp1(vil-/-) epithelium, where it predominantly co-localized with Rab2. The release of chylomicrons from Caco-2 was markedly reduced after the suppression of Rab2, ARL1 and Golgin-245. Thus, the GTPase ARFRP1 and its downstream proteins are required for the lipidation of chylo-microns and the assembly of ApoA-I to these particles in the Golgi of intestinal epithelial cells.

Identifying Rab2 Protein as a Key Interactor of Centrin1 Essential for Leishmania donovani Growth.

Previously, we have demonstrated that deletion of a growth-regulating gene (LdCen1) in the Leishmania donovani parasite (LdCen1-/-) attenuated the parasite's intracellular amastigote growth but not the growth of extracellular promastigotes. LdCen1-/- parasites were found to be safe and efficacious against homologous and heterologous Leishmania species as a vaccine candidate in animal models. The reason for the differential growth of LdCen1-/- between the two stages of the parasite needed investigation. Here, we report that LdCen1 interacts with a novel Ras-associated binding protein in L. donovani (LdRab2) to compensate for the growth of LdCen1-/- promastigotes. LdRab2 was isolated by protein pull-down from the parasite lysate, followed by nano-LC-MS/MS identification. The RAB domain sequence and the functional binding partners of the LdRab2 protein were predicted via Search Tool for the Retrieval of Interacting Proteins (STRING) analysis. The closeness of the LdRab2 protein to other reported centrin-binding proteins with different functions in other organisms was analyzed via phylogenetic analysis. Furthermore, in vitro and in silico analyses revealed that LdRab2 also interacts with other L. donovani centrins 3-5. Since centrin is a calcium-binding protein, we further investigated calcium-based interactions and found that the binding of LdRab2 to LdCen1 and LdCen4 is calcium-independent, whereas the interactions with LdCen3 and LdCen5 are calcium-dependent. The colocalization of LdCen1 and LdRab2 at the cellular basal-body region by immunofluorescence supports their possible functional association. The elevated expression of the LdRab2 protein in the mutant promastigotes suggested a probable role in compensating for the promastigote growth of this mutant strain, probably in association with other parasite centrins.

Biophysical Evidence for the Amyloid Formation of a Recombinant Rab2 Isoform of Leishmania donovani.

BACKGROUND: The most fatal form of Visceral leishmaniasis or kala-azar is caused by the intracellular protozoan parasite Leishmania donovani. The life cycle and the infection pathway of the parasite are regulated by the small GTPase family of Rab proteins. The involvement of Rab proteins in neurodegenerative amyloidosis is implicated in protein misfolding, secretion abnormalities and dysregulation. The inter and intra-cellular shuttlings of Rab proteins are proposed to be aggregation-prone. However, the biophysical unfolding and aggregation of protozoan Rab proteins is limited. Understanding the aggregation mechanisms of Rab protein will determine their physical impact on the disease pathogenesis and individual health. OBJECTIVE: This work investigates the acidic pH-induced unfolding and aggregation of a recombinant Rab2 protein from L. donovani (rLdRab2) using multi-spectroscopic probes. METHODS: The acidic unfolding of rLdRab2 is characterised by intrinsic fluorescence and ANS assay, while aggregation is determined by Thioflavin-T and 90° light scattering assay. Circular dichroism determined the secondary structure of monomers and aggregates. The aggregate morphology was imaged by transmission electron microscopy. RESULTS: rLdRab2 was modelled to be a Rab2 isoform with loose globular packing. The acidinduced unfolding of the protein is a plausible non-two-state process. At pH 2.0, a partially folded intermediate (PFI) state characterised by ~ 30% structural loss and exposed hydrophobic core was found to accumulate. The PFI state slowly converted into well-developed protofibrils at high protein concentrations demonstrating its amyloidogenic nature. The native state of the protein was also observed to be aggregation-prone at high protein concentrations. However, it formed amorphous aggregation instead of fibrils. CONCLUSION: To our knowledge, this is the first study to report in vitro amyloid-like behaviour of Rab proteins in L donovani. This study provides a novel opportunity to understand the complete biophysical characteristics of Rab2 protein of the lower eukaryote, L. donovani.

Molecular structure of the Brucella abortus metalloprotein RicA, a Rab2-binding virulence effector.

The Gram-negative intracellular pathogen Brucella abortus is the causative agent of brucellosis, which is among the most common zoonoses globally. The B. abortus RicA protein binds the host-expressed guanosine nucleotide-binding protein, Rab2, and modulates B. abortus infection biology. We have solved the first X-ray crystal structure of RicA to 2.7 Å resolution and have quantified the affinity of RicA binding to human Rab2 in its GDP-bound and nucleotide-free forms. RicA adopts a classic γ-carbonic anhydrase (γ-CA) fold containing a left-handed ß-helix followed by a C-terminal α-helix. Two homotrimers of RicA occupy the crystallographic asymmetric unit. Though no zinc was included in the purification or crystallization buffers, zinc is contained within the RicA crystals, as demonstrated by X-ray fluorescence spectroscopy. Electron density for a Zn(2+) ion coordinated by three histidine residues is evident in the putative active site of RicA. However, purified RicA preparations do not exhibit carbonic anhydrase activity, suggesting that Zn(2+) may not be the physiologically relevant metal cofactor or that RicA is not a bona fide carbonic anhydrase enzyme. Isothermal titration calorimetry (ITC) measurements of purified RicA binding to purified human Rab2 and GDP-Rab2 revealed similar equilibrium affinities (Kd ≈ 35 and 40 µM, respectively). This study thus defines RicA as a Zn(2+)-binding γ-carbonic anhydrase-like protein that binds the human membrane fusion/trafficking protein Rab2 with low micromolar affinity in vitro. These results support a model in which γ-CA family proteins may evolve unique cellular functions while retaining many of the structural hallmarks of archetypal γ-CA enzymes.

Small GTPases and Brucella entry into the endoplasmic reticulum.

A key determinant for intracellular pathogenic bacteria to ensure their virulence within host cells is their ability to bypass the endocytic pathway and to reach a safe niche of replication. In the case of Brucella, the bacterium targets the ER (endoplasmic reticulum) to create a replicating niche called the BCV (Brucella-containing vacuole). The ER is a suitable strategic place for pathogenic Brucella. Indeed, bacteria can be hidden from host cell defences to persist within the host, and they can take advantage of the membrane reservoir delivered by the ER to replicate. Interaction with the ER leads to the presence on the BCV of the GAPDH (glyceraldehyde-3-phosphate dehydrogenase) and the small GTPase Rab2 known to be located on secretory vesicles that traffic between the ER and the Golgi apparatus. GAPDH and the small GTPase Rab2 controls Brucella replication at late times post-infection. A specific interaction between the human small GTPase Rab2 and a Brucella spp. protein named RicA was identified. Altered kinetics of intracellular trafficking and faster proliferation of the Brucella abortus ΔricA mutant was observed compared with the wild-type strain. RicA is the first reported effector with a proposed function for B. abortus.

Structural analysis of Brucella abortus RicA substitutions that do not impair interaction with human Rab2 GTPase.

BACKGROUND: Protein-protein interactions are at the basis of many cellular processes, and they are also involved in the interaction between pathogens and their host(s). Many intracellular pathogenic bacteria translocate proteins called effectors into the cytoplasm of the infected host cell, and these effectors can interact with one or several host protein(s). An effector named RicA was recently reported in Brucella abortus to specifically interact with human Rab2 and to affect intracellular trafficking of this pathogen. RESULTS: In order to identify regions of the RicA protein involved in the interaction with Rab2, RicA was subjected to extensive random mutagenesis using error prone polymerase chain reaction. The resulting allele library was selected by the yeast two-hybrid assay for Rab2-interacting clones that were isolated and sequenced, following the "absence of interference" approach. A tridimensional model of RicA structure was used to position the substitutions that did not affect RicA-Rab2 interaction, giving a "negative image" of the putative interaction region. Since RicA is a bacterial conserved protein, RicA homologs were also tested against Rab2 in a yeast two-hybrid assay, and the C. crescentus homolog of RicA was found to interact with human Rab2. Analysis of the RicA structural model suggested that regions involved in the folding of the "beta helix" or an exposed loop with the IGFP sequence could also be involved in the interaction with Rab2. Extensive mutagenesis of the IGFP loop suggested that loss of interaction with Rab2 was correlated with insolubility of the mutated RicA, showing that "absence of interference" approach also generates surfaces that could be necessary for folding. CONCLUSION: Extensive analysis of substitutions in RicA unveiled two structural elements on the surface of RicA, the most exposed ß-sheet and the IGFP loop, which could be involved in the interaction with Rab2 and protein folding. Our analysis of mutants in the IGFP loop suggests that, at least for some mono-domain proteins such as RicA, protein interaction analysis using allele libraries could be complicated by the dual effect of many substitutions affecting both folding and protein-protein interaction.

Identification of a Brucella spp. secreted effector specifically interacting with human small GTPase Rab2.

Bacteria of the Brucella genus are facultative intracellular class III pathogens. These bacteria are able to control the intracellular trafficking of their vacuole, presumably by the use of yet unknown translocated effectors. To identify such effectors, we used a high-throughput yeast two-hybrid screen to identify interactions between putative human phagosomal proteins and predicted Brucella spp. proteins. We identified a specific interaction between the human small GTPase Rab2 and a Brucella spp. protein named RicA. This interaction was confirmed by GST-pull-down with the GDP-bound form of Rab2. A TEM-ß-lactamase-RicA fusion was translocated from Brucella abortus to RAW264.7 macrophages during infection. This translocation was not detectable in a strain deleted for the virB operon, coding for the type IV secretion system. However, RicA secretion in a bacteriological culture was still observed in a ΔvirB mutant. In HeLa cells, a ΔricA mutant recruits less GTP-locked myc-Rab2 on its Brucella-containing vacuoles, compared with the wild-type strain. We observed altered kinetics of intracellular trafficking and faster proliferation of the B. abortusΔricA mutant in HeLa cells, compared with the wild-type control. Altogether, the data reported here suggest RicA as the first reported effector with a proposed function for B. abortus.

Targeting of host Rab GTPase function by the intravacuolar pathogen Legionella pneumophila.

The intracellular pathogen Legionella pneumophila replicates in a vacuole that recruits material from the host cell endoplasmic reticulum (ER). Biogenesis of this unique vacuole depends on the bacterial Dot/Icm type IV secretion system that translocates proteins across host cell membranes. Here, we show that two translocated substrates, SidM and LidA, target host cell Rab1, a small GTPase regulating ER-to-Golgi traffic. SidM is a guanosine nucleotide exchange factor for Rab1 that recruits Rab1 to Legionella-containing vacuoles, a process that is enhanced by LidA. Expression of sidM in mammalian cells interferes with the secretory pathway and causes Golgi fragmentation. Consistent with a collaborative relationship between the two proteins, immobilized SidM and LidA synergize to promote Rab1-dependent binding of early secretory vesicles. These results indicate that proteins translocated into the host cell by the intravacuolar pathogen L. pneumophila are able to recapitulate events involved in host secretory trafficking.

The glyceraldehyde-3-phosphate dehydrogenase and the small GTPase Rab 2 are crucial for Brucella replication.

The intracellular pathogen Brucella abortus survives and replicates inside host cells within an endoplasmic reticulum (ER)-derived replicative organelle named the "Brucella-containing vacuole" (BCV). Here, we developed a subcellular fractionation method to isolate BCVs and characterize for the first time the protein composition of its replicative niche. After identification of BCV membrane proteins by 2 dimensional (2D) gel electrophoresis and mass spectrometry, we focused on two eukaryotic proteins: the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and the small GTPase Rab 2 recruited to the vacuolar membrane of Brucella. These proteins were previously described to localize on vesicular and tubular clusters (VTC) and to regulate the VTC membrane traffic between the endoplasmic reticulum (ER) and the Golgi. Inhibition of either GAPDH or Rab 2 expression by small interfering RNA strongly inhibited B. abortus replication. Consistent with this result, inhibition of other partners of GAPDH and Rab 2, such as COPI and PKC iota, reduced B. abortus replication. Furthermore, blockage of Rab 2 GTPase in a GDP-locked form also inhibited B. abortus replication. Bacteria did not fuse with the ER and instead remained in lysosomal-associated membrane vacuoles. These results reveal an essential role for GAPDH and the small GTPase Rab 2 in B. abortus virulence within host cells.