Effector Contributions to Gβγ-mediated Signaling as Revealed by Muscarinic Potassium Channel Gating

Receptor-mediated activation of heterotrimeric G proteins leading to dissociation of the Gα subunit from Gβγ is a highly conserved signaling strategy used by numerous extracellular stimuli. Although Gβγ subunits regulate a variety of effectors, including kinases, cyclases, phospholipases, and ion channels (Clapham, D.E., and E.J. Neer. 1993. Nature (Lond.). 365:403–406), few tools exist for probing instantaneous Gβγ-effector interactions, and little is known about the kinetic contributions of effectors to the signaling process. In this study, we used the atrial muscarinic K⁺ channel, which is activated by direct interactions with Gβγ subunits (Logothetis, D.E., Y. Kurachi, J. Galper, E.J. Neer, and D.E. Clap. 1987. Nature (Lond.). 325:321–326; Wickman, K., J.A. Iniguez-Liuhi, P.A. Davenport, R. Taussig, G.B. Krapivinsky, M.E. Linder, A.G. Gilman, and D.E. Clapham. 1994. Nature (Lond.). 366: 654–663; Huang, C.-L., P.A. Slesinger, P.J. Casey, Y.N. Jan, and L.Y. Jan. 1995. Neuron. 15:1133–1143), as a sensitive reporter of the dynamics of Gβγ-effector interactions. Muscarinic K⁺ channels exhibit bursting behavior upon G protein activation, shifting between three distinct functional modes, characterized by the frequency of channel openings during individual bursts. Acetylcholine concentration (and by inference, the concentration of activated Gβγ) controls the fraction of time spent in each mode without changing either the burst duration or channel gating within individual modes. The picture which emerges is of a Gβγ effector with allosteric regulation and an intrinsic “off” switch which serves to limit its own activation. These two features combine to establish exquisite channel sensitivity to changes in Gβγ concentration, and may be indicative of the factors regulating other Gβγ-modulated effectors.     

Host–pathogen interaction between Phytophthora infestans and Solanum tuberosum following exposure to short and long daylight hours

The influence of short and long day length on the expression of qualitative and quantitative resistance to Phytophthora infestans in potato was studied. The incompatible interaction was tested for available set of isolates avirulent in greenhouse conditions to potato Black’s differentials possessing the genes: R2, R5, R6, R8, R9, R10, and standard potato cultivar Tarpan (no known R gene). The avirulent isolates either were completely avirulent regardless of plant growing conditions, or they infected leaflets of these differentials more frequently when plants were exposed previously to short day conditions than to long day conditions. This study highlights the importance of day length, among many other factors which are controlled, in testing the expression of the virulence of P. infestans isolates. In compatible interactions, when quantitative resistance was evaluated in differentials with gene R1, R3, R4, R7, R11, and potato cultivar Craigs Royal (no known R gene), stronger infection expressed by lesion growth rate, as well as stronger sporulation, were observed on potato leaflets of plants exposed to short day for 6–7 weeks before inoculation. The analysis of variance revealed a significant contribution to variation in lesion growth rate of day length, genotype, as well as day length by genotype interaction. Significant influence of isolate, and genotype, but not day length, on the expression of the incubation period was found. The results indicate the necessity of evaluating components of partial resistance present in potato lines used in breeding potato resistant to P. infestans in destined day length growing conditions.     

Bacillus bombysepticus α-Toxin Binding to G Protein-Coupled Receptor Kinase 2 Regulates cAMP/PKA Signaling Pathway to Induce Host Death

Bacterial pathogens and their toxins target host receptors, leading to aberrant behavior or host death by changing signaling events through subversion of host intracellular cAMP level. This is an efficient and widespread mechanism of microbial pathogenesis. Previous studies describe toxins that increase cAMP in host cells, resulting in death through G protein-coupled receptor (GPCR) signaling pathways by influencing adenylyl cyclase or G protein activity. G protein-coupled receptor kinase 2 (GRK2) has a central role in regulation of GPCR desensitization. However, little information is available about the pathogenic mechanisms of toxins associated with GRK2. Here, we reported a new bacterial toxin-Bacillus bombysepticus (Bb) α-toxin that was lethal to host. We showed that Bb α-toxin interacted with BmGRK2. The data demonstrated that Bb α-toxin directly bound to BmGRK2 to promote death by affecting GPCR signaling pathways. This mechanism involved stimulation of G_αs, increase level of cAMP and activation of protein kinase A (PKA). Activated cAMP/PKA signal transduction altered downstream effectors that affected homeostasis and fundamental biological processes, disturbing the structural and functional integrity of cells, resulting in death. Preventing cAMP/PKA signaling transduction by inhibitions (NF449 or H-89) substantially reduced the pathogenicity of Bb α-toxin. The discovery of a toxin-induced host death specifically linked to GRK2 mediated signaling pathway suggested a new model for bacterial toxin action. Characterization of host genes whose expression and function are regulated by Bb α-toxin and GRK2 will offer a deeper understanding of the pathogenesis of infectious diseases caused by pathogens that elevate cAMP.     

Trichomonas vaginalis Exosomes Deliver Cargo to Host Cells and Mediate Host∶Parasite Interactions

Trichomonas vaginalis is a common sexually transmitted parasite that colonizes the human urogential tract where it remains extracellular and adheres to epithelial cells. Infections range from asymptomatic to highly inflammatory, depending on the host and the parasite strain. Here, we use a combination of methodologies including cell fractionation, immunofluorescence and electron microscopy, RNA, proteomic and cytokine analyses and cell adherence assays to examine pathogenic properties of T. vaginalis. We have found that T.vaginalis produces and secretes microvesicles with physical and biochemical properties similar to mammalian exosomes. The parasite-derived exosomes are characterized by the presence of RNA and core, conserved exosomal proteins as well as parasite-specific proteins. We demonstrate that T. vaginalis exosomes fuse with and deliver their contents to host cells and modulate host cell immune responses. Moreover, exosomes from highly adherent parasite strains increase the adherence of poorly adherent parasites to vaginal and prostate epithelial cells. In contrast, exosomes from poorly adherent strains had no measurable effect on parasite adherence. Exosomes from parasite strains that preferentially bind prostate cells increased binding of parasites to these cells relative to vaginal cells. In addition to establishing that parasite exosomes act to modulate host∶parasite interactions, these studies are the first to reveal a potential role for exosomes in promoting parasite∶parasite communication and host cell colonization.     

Staphylococcus epidermidis Antimicrobial δ-Toxin (Phenol-Soluble Modulin-γ) Cooperates with Host Antimicrobial Peptides to Kill Group A Streptococcus

Antimicrobial peptides play an important role in host defense against pathogens. Recently, phenol-soluble modulins (PSMs) from Staphylococcus epidermidis (S. epidermidis) were shown to interact with lipid membranes, form complexes, and exert antimicrobial activity. Based on the abundance and innocuity of the cutaneous resident S. epidermidis, we hypothesized that their PSMs contribute to host defense. Here we show that S. epidermidis δ-toxin (PSMγ) is normally present in the epidermis and sparsely in the dermis of human skin using immunohistochemistry. Synthetic δ-toxin interacted with neutrophil extracellular traps (NETs) and colocalized with cathelicidin while also inducing NET formation in human neutrophils. In antimicrobial assays against Group A Streptococcus (GAS), δ-toxin cooperated with CRAMP, hBD2, and hBD3. In whole blood, addition of δ-toxin exerted a bacteriostatic effect on GAS, and in NETs, δ-toxin increased their killing capacity against this pathogen. Coimmunoprecipitation and tryptophan spectroscopy demonstrated direct binding of δ-toxin to host antimicrobial peptides LL-37, CRAMP, hBD2, and hBD3. Finally, in a mouse wound model, GAS survival was reduced (along with Mip-2 cytokine levels) when the wounds were pretreated with δ-toxin. Thus, these data suggest that S. epidermidis–derived δ-toxin cooperates with the host-derived antimicrobial peptides in the innate immune system to reduce survival of an important human bacterial pathogen.     

Rab2 Utilizes Glyceraldehyde-3-phosphate Dehydrogenase and Protein Kinase C�� to Associate with Microtubules and to Recruit Dynein

Rab2 requires glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and atypical protein kinase Cι (aPKCι) for retrograde vesicle formation from vesicular tubular clusters that sort secretory cargo from recycling proteins returned to the endoplasmic reticulum. However, the precise role of GAPDH and aPKCι in the early secretory pathway is unclear. GAPDH was the first glycolytic enzyme reported to co-purify with microtubules (MTs). Similarly, aPKC associates directly with MTs. To learn whether Rab2 also binds directly to MTs, a MT binding assay was performed. Purified Rab2 was found in a MT-enriched pellet only when both GAPDH and aPKCι were present, and Rab2-MT binding could be prevented by a recombinant fragment made to the Rab2 amino terminus (residues 2-70), which directly interacts with GAPDH and aPKCι. Because GAPDH binds to the carboxyl terminus of α-tubulin, we characterized the distribution of tyrosinated/detyrosinated α-tubulin that is recruited by Rab2 in a quantitative membrane binding assay. Rab2-treated membranes contained predominantly tyrosinated α-tubulin; however, aPKCι was the limiting and essential factor. Tyrosination/detyrosination influences MT motor protein binding; therefore, we determined whether Rab2 stimulated kinesin or dynein membrane binding. Although kinesin was not detected on membranes incubated with Rab2, dynein was recruited in a dose-dependent manner, and binding was aPKCι-dependent. These combined results suggest a mechanism by which Rab2 controls MT and motor recruitment to vesicular tubular clusters.The small GTPase Rab2 is essential for membrane trafficking in the early secretory pathway and associates with vesicular tubular clusters (VTCs)² located between the endoplasmic reticulum (ER) and the cis-Golgi compartment (1, 2). VTCs are pleomorphic structures that sort anterograde-directed cargo from recycling proteins and trafficking machinery retrieved to the ER (3-6). Rab2 bound to a VTC microdomain stimulates recruitment of soluble factors that results in the release of vesicles containing the recycling protein p53/p58 (7). In that regard, we have previously reported that glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and atypical PKC ι (aPKCι) are Rab2 effectors that interact directly with the Rab2 amino terminus and with each other (8, 9). Their interaction requires Src-dependent tyrosine phosphorylation of GAPDH and aPKCι (10). Moreover, GAPDH is a substrate for aPKCι (11). GAPDH catalytic activity is not required for ER to Golgi transport indicating that GAPDH provides a specific function essential for membrane trafficking from VTCs independent of glycolytic function (9). Indeed, phospho-GAPDH influences MT dynamics in the early secretory pathway (11).GAPDH was the first glycolytic enzyme reported to co-purify with microtubules (MTs) (12) and subsequently was shown to interact with the carboxyl terminus of α-tubulin (13). The binding of GAPDH to MTs promotes formation of cross-linked parallel MT arrays or bundles (14, 15). GAPDH has also been reported to possess membrane fusogenic activity, which is inhibited by tubulin (16). Similarly, aPKC associates directly with tubulin and promotes MT stability and MT remodeling at specific intracellular sites (17-21). It may not be coincidental that these two Rab2 effectors influence MT dynamics because recent studies indicate that the cytoskeleton plays a central role in the organization and operation of the secretory pathway (22).MTs are dynamic structures that grow or shrink by the addition or loss of α- and β-tubulin heterodimers from the ends of protofilaments (23). Their assembly and stability is regulated by a variety of proteins traditionally referred to as microtubule-associated proteins (MAPs). In addition to the multiple α/β isoforms that are present in eukaryotes, MTs undergo an assortment of post-translational modifications, including acetylation, glycylation, glutamylation, phosphorylation, palmitoylation, and detyrosination, which further contribute to their biochemical heterogeneity (24, 25). It has been proposed that these tubulin modifications regulate intracellular events by facilitating interaction with MAPs and with other specific effector proteins (24). For example, the reversible addition of tyrosine to the carboxyl terminus of α-tubulin regulates MT interaction with plus-end tracking proteins (+TIPs) containing the cytoskeleton-associated protein glycine-rich (CAP-Gly) motif and with dynein-dynactin (27-29). Additionally, MT motility and cargo transport rely on the cooperation of the motor proteins kinesin and dynein (30). Kinesin is a plus-end directed MT motor, whereas cytoplasmic dynein is a minus-end MT-based motor, and therefore the motors transport vesicular cargo toward the opposite end of a MT track (31).Although MT assembly does not appear to be directly regulated by small GTPases, Rab proteins provide a molecular link for vesicle movement along MTs to the appropriate target (22, 32-34). In this study, the potential interaction of Rab2 with MTs and motor proteins was characterized. We found that Rab2 does not bind directly to preassembled MTs but does associate when both GAPDH and aPKCι are present and bound to MTs. Moreover, the MTs predominantly contained tyrosinated α-tubulin (Tyr-tubulin) suggesting that a dynamic pool of MTs that differentially binds MAPs/effector proteins/motors associates with VTCs in response to Rab2. To that end, we determined that Rab2-promoted dynein/dynactin binding to membranes and that the recruitment required aPKCι.     

Phytophthora ×stagnum nothosp. nov., a New Hybrid from Irrigation Reservoirs at Ornamental Plant Nurseries in Virginia

A novel Phytophthora species was frequently recovered from irrigation reservoirs at several ornamental plant production facilities in eastern Virginia. Initial sequencing of the internal transcribed spacer (ITS) region of this species generated unreadable sequences due to continual polymorphic positions. Cloning and sequencing the ITS region as well as sequencing the mitochondrially encoded cytochrome c oxidase 1 and beta-tubulin genes revealed that it is a hybrid between P. taxon PgChlamydo as its paternal parent and an unknown species genetically close to P. mississippiae as its maternal parent. This hybrid has some diagnostic morphological features of P. taxon PgChlamydo and P. mississippiae. It produces catenulate hyphal swellings, characteristic of P. mississippiae, and chlamydospores, typical of P. taxon PgChlamydo. It also produces both ornamented and relatively smooth-walled oogonia. Ornamented oogonia are another important diagnostic character of P. mississippiae. The relatively smooth-walled oogonia may be indicative of oogonial character of P. taxon PgChlamydo. The new hybrid is described here as Phytophthora ×stagnum.     

Strain-Transcendent Immune Response to Recombinant Var2CSA DBL5-ε Domain Block P. falciparum Adhesion to Placenta-Derived BeWo Cells under Flow Conditions

Background

Pregnancy-associated malaria (PAM) is a serious consequence of the adhesion to the placental receptor chondroitin sulfate A (CSA) of Plasmodium falciparum-infected erythrocytes (PE) expressing the large cysteine-rich multi-domain protein var2CSA. Women become resistant to PAM, and develop strain-transcending immunity against CSA-binding parasites. The identification of var2CSA regions that could elicit broadly neutralizing and adhesion-blocking antibodies is a key step for the design of prophylactic vaccine strategies.

Methodology

Escherichia coli expressed var2CSA DBL domains were refolded and purified prior to immunization of mice and a goat. Protein-G-purified antibodies were tested for their ability to block FCR3^CSA-infected erythrocytes binding to placental (BeWo) and monkey brain endothelial (ScC2) cell lines using a flow cytoadhesion inhibition assay mimicking closely the physiological conditions present in the placenta at shear stress of 0.05 Pa. DBL5-ε, DBL6-ε and DBL5-6-ε induced cross-reactive antibodies using Alum and Freund as adjuvants, which blocked cytoadhesion at values ranging between 40 to 96% at 0.5 mg IgG per ml. Importantly, antibodies raised against recombinant DBL5-ε from 3 distinct parasites genotypes (HB3, Dd2 and 7G8) showed strain-transcending inhibition ranging from 38 to 64% for the heterologuous FCR3^CSA.

Conclusions

Using single and double DBL domains from var2CSA and Alum as adjuvant, we identified recombinant subunits inducing an immune response in experimental animals which is able to block efficiently parasite adhesion in a flow cytoadhesion assay that mimics closely the erythrocyte flow in the placenta. These subunits show promising features for inclusion into a vaccine aiming to protect against PAM.     

Unlipidated Outer Membrane Protein Omp16 (U-Omp16) from Brucella spp. as Nasal Adjuvant Induces a Th1 Immune Response and Modulates the Th2 Allergic Response to Cow’s Milk Proteins

The discovery of novel mucosal adjuvants will help to develop new formulations to control infectious and allergic diseases. In this work we demonstrate that U-Omp16 from Brucella spp. delivered by the nasal route (i.n.) induced an inflammatory immune response in bronchoalveolar lavage (BAL) and lung tissues. Nasal co-administration of U-Omp16 with the model antigen (Ag) ovalbumin (OVA) increased the amount of Ag in lung tissues and induced OVA-specific systemic IgG and T helper (Th) 1 immune responses. The usefulness of U-Omp16 was also assessed in a mouse model of food allergy. U-Omp16 i.n. administration during sensitization ameliorated the hypersensitivity responses of sensitized mice upon oral exposure to Cow’s Milk Protein (CMP), decreased clinical signs, reduced anti-CMP IgE serum antibodies and modulated the Th2 response in favor of Th1 immunity. Thus, U-Omp16 could be used as a broad Th1 mucosal adjuvant for different Ag formulations.     

Wolbachia Age-Sex-Specific Density in Aedes albopictus: A Host Evolutionary Response to Cytoplasmic Incompatibility?

Background

Wolbachia bacteria have invaded many arthropod species by inducing Cytoplasmic Incompatibility (CI). These symbionts represent fascinating objects of study for evolutionary biologists, but also powerful potential biocontrol agents. Here, we assess the density dynamics of Wolbachia infections in males and females of the mosquito Aedes albopitcus, an important vector of human pathogens, and interpret the results within an evolutionary framework.

Methodology/Principal Findings

Wolbachia densities were measured in natural populations and in age controlled mosquitoes using quantitative PCR. We show that the density dynamics of the wAlbA Wolbachia strain infecting Aedes albopictus drastically differ between males and females, with a very rapid decay of infection in males only.

Conclusions/Significance

Theory predicts that Wolbachia and its hosts should cooperate to improve the transmission of infection to offspring, because only infected eggs are protected from the effects of CI. However, incompatible matings effectively lower the fertility of infected males, so that selection acting on the host genome should tend to reduce the expression of CI in males, for example, by reducing infection density in males before sexual maturation. The rapid decay of one Wolbachia infection in Aedes albopictus males, but not in females, is consistent with this prediction. We suggest that the commonly observed reduction in CI intensity with male age reflects a similar evolutionary process. Our results also highlight the importance of monitoring infection density dynamics in both males and females to assess the efficiency of Wolbachia-based control strategies.     

Shigella IpaH0722 E3 Ubiquitin Ligase Effector Targets TRAF2 to Inhibit PKC–NF-κB Activity in Invaded Epithelial Cells

NF-κB plays a central role in modulating innate immune responses to bacterial infections. Therefore, many bacterial pathogens deploy multiple mechanisms to counteract NF-κB activation. The invasion of and subsequent replication of Shigella within epithelial cells is recognized by various pathogen recognition receptors as pathogen-associated molecular patterns. These receptors trigger innate defense mechanisms via the activation of the NF-κB signaling pathway. Here, we show the inhibition of the NF-κB activation by the delivery of the IpaH E3 ubiquitin ligase family member IpaH0722 using Shigella''s type III secretion system. IpaH0722 dampens the acute inflammatory response by preferentially inhibiting the PKC-mediated activation of NF-κB by ubiquitinating TRAF2, a molecule downstream of PKC, and by promoting its proteasome-dependent degradation.