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Journal articlePinzan CF, Sardinha-Silva A, Almeida F, et al., 2015,
Vaccination with Recombinant Microneme Proteins Confers Protection against Experimental Toxoplasmosis in Mice
, PLOS One, Vol: 10, ISSN: 1932-6203Toxoplasmosis, a zoonotic disease caused by Toxoplasma gondii, is an important publichealth problem and veterinary concern. Although there is no vaccine for human toxoplasmosis,many attempts have been made to develop one. Promising vaccine candidates utilizeproteins, or their genes, from microneme organelle of T. gondii that are involved in theinitial stages of host cell invasion by the parasite. In the present study, we used differentrecombinant microneme proteins (TgMIC1, TgMIC4, or TgMIC6) or combinations of theseproteins (TgMIC1-4 and TgMIC1-4-6) to evaluate the immune response and protectionagainst experimental toxoplasmosis in C57BL/6 mice. Vaccination with recombinantTgMIC1, TgMIC4, or TgMIC6 alone conferred partial protection, as demonstrated byreduced brain cyst burden and mortality rates after challenge. Immunization with TgMIC1-4or TgMIC1-4-6 vaccines provided the most effective protection, since 70% and 80% ofmice, respectively, survived to the acute phase of infection. In addition, these vaccinatedmice, in comparison to non-vaccinated ones, showed reduced parasite burden by 59% and68%, respectively. The protective effect was related to the cellular and humoral immuneresponses induced by vaccination and included the release of Th1 cytokines IFN-γ and IL-12, antigen-stimulated spleen cell proliferation, and production of antigen-specific serumantibodies. Our results demonstrate that microneme proteins are potential vaccines againstT. gondii, since their inoculation prevents or decreases the deleterious effects of theinfection.
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Journal articleCagin U, Duncan OF, Gatt AP, et al., 2015,
Mitochondrial retrograde signaling regulates neuronal function
, Proceedings of the National Academy of Sciences of the United States of America, Vol: 112, Pages: E6000-E6009, ISSN: 0027-8424Mitochondria are key regulators of cellular homeostasis, and mitochondrial dysfunction is strongly linked to neurodegenerative diseases, including Alzheimer’s and Parkinson’s. Mitochondria communicate their bioenergetic status to the cell via mitochondrial retrograde signaling. To investigate the role of mitochondrial retrograde signaling in neurons, we induced mitochondrial dysfunction in the Drosophila nervous system. Neuronal mitochondrial dysfunction causes reduced viability, defects in neuronal function, decreased redox potential, and reduced numbers of presynaptic mitochondria and active zones. We find that neuronal mitochondrial dysfunction stimulates a retrograde signaling response that controls the expression of several hundred nuclear genes. We show that the Drosophila hypoxia inducible factor alpha (HIFα) ortholog Similar (Sima) regulates the expression of several of these retrograde genes, suggesting that Sima mediates mitochondrial retrograde signaling. Remarkably, knockdown of Sima restores neuronal function without affecting the primary mitochondrial defect, demonstrating that mitochondrial retrograde signaling is partly responsible for neuronal dysfunction. Sima knockdown also restores function in a Drosophila model of the mitochondrial disease Leigh syndrome and in a Drosophila model of familial Parkinson’s disease. Thus, mitochondrial retrograde signaling regulates neuronal activity and can be manipulated to enhance neuronal function, despite mitochondrial impairment.
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Journal articleSchroeder GN, Frankel G, Tate EW, et al., 2015,
The Legionella pneumophila effector LpdA is a palmitoylated phospholipase D virulence factor
, Infection and Immunity, Vol: 83, Pages: 3989-4002, ISSN: 1098-5522Legionella pneumophila is a bacterial pathogen that thrives in alveolar macrophages, causing a severe pneumonia. The virulence of L. pneumophila depends on its Dot/Icm type IV secretion system (T4SS), which delivers more than 300 effector proteins into the host, where they rewire cellular signaling to establish a replication-permissive niche, the Legionella-containing vacuole (LCV). Biogenesis of the LCV requires substantial redirection of vesicle trafficking and remodeling of intracellular membranes. In order to achieve this, several T4SS effectors target regulators of membrane trafficking, while others resemble lipases. Here, we characterized LpdA, a phospholipase D effector, which was previously proposed to modulate the lipid composition of the LCV. We found that ectopically expressed LpdA was targeted to the plasma membrane and Rab4- and Rab14-containing vesicles. Subcellular targeting of LpdA required a C-terminal motif, which is posttranslationally modified by S-palmitoylation. Substrate specificity assays showed that LpdA hydrolyzed phosphatidylinositol, -inositol-3- and -4-phosphate, and phosphatidylglycerol to phosphatidic acid (PA) in vitro. In HeLa cells, LpdA generated PA at vesicles and the plasma membrane. Imaging of different phosphatidylinositol phosphate (PIP) and organelle markers revealed that while LpdA did not impact on membrane association of various PIP probes, it triggered fragmentation of the Golgi apparatus. Importantly, although LpdA is translocated inefficiently into cultured cells, an L. pneumophila ΔlpdA mutant displayed reduced replication in murine lungs, suggesting that it is a virulence factor contributing to L. pneumophila infection in vivo.
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Journal articleJennings LK, Storek KM, Ledvina HE, et al., 2015,
Pel is a cationic exopolysaccharide that cross-links extracellular DNA in the Pseudomonas aeruginosa biofilm matrix
, Proceedings of the National Academy of Sciences of the United States of America, Vol: 112, Pages: 11353-11358, ISSN: 1091-6490Biofilm formation is a complex, ordered process. In the opportunistic pathogen Pseudomonas aeruginosa, Psl and Pel exopolysaccharides and extracellular DNA (eDNA) serve as structural components of the biofilm matrix. Despite intensive study, Pel’s chemical structure and spatial localization within mature biofilms remain unknown. Using specialized carbohydrate chemical analyses, we unexpectedly found that Pel is a positively charged exopolysaccharide composed of partially acetylated 1→4 glycosidic linkages of N-acetylgalactosamine and N-acetylglucosamine. Guided by the knowledge of Pel’s sugar composition, we developed a tool for the direct visualization of Pel in biofilms by combining Pel-specific Wisteria floribunda lectin staining with confocal microscopy. The results indicate that Pel cross-links eDNA in the biofilm stalk via ionic interactions. Our data demonstrate that the cationic charge of Pel is distinct from that of other known P. aeruginosa exopolysaccharides and is instrumental in its ability to interact with other key biofilm matrix components.
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Journal articleSurana S, Shenoy AR, Krishnan Y, 2015,
Designing DNA nanodevices for compatibility with the immune system of higher organisms
, Nature Nanotechnology, Vol: 10, Pages: 741-747, ISSN: 1748-3395DNA is proving to be a powerful scaffold to construct molecularly precise designer DNA devices. Recent trends reveal their ever-increasing deployment within living systems as delivery devices that not only probe but also program and re-program a cell, or even whole organisms. Given that DNA is highly immunogenic, we outline the molecular, cellular and organismal response pathways that designer nucleic acid nanodevices are likely to elicit in living systems. We address safety issues applicable when such designer DNA nanodevices interact with the immune system. In light of this, we discuss possible molecular programming strategies that could be integrated with such designer nucleic acid scaffolds to either evade or stimulate the host response with a view to optimizing and widening their applications in higher organisms.
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Journal articleMostowy S, Shenoy AR, 2015,
The cytoskeleton in cell-autonomous immunity: structural determinants of host defence
, Nature Reviews Immunology, Vol: 15, Pages: 559-573, ISSN: 1474-1741Host cells use antimicrobial proteins, pathogen-restrictive compartmentalization and cell death in their defence against intracellular pathogens. Recent work has revealed that four components of the cytoskeleton — actin, microtubules, intermediate filaments and septins, which are well known for their roles in cell division, shape and movement — have important functions in innate immunity and cellular self-defence. Investigations using cellular and animal models have shown that these cytoskeletal proteins are crucial for sensing bacteria and for mobilizing effector mechanisms to eliminate them. In this Review, we highlight the emerging roles of the cytoskeleton as a structural determinant of cell-autonomous host defence.
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Journal articleSo EC, Mattheis C, Tate EW, et al., 2015,
Creating a customized intracellular niche: subversion of host cell signaling by Legionella type IV secretion system effectors
, Canadian Journal of Microbiology, Vol: 61, Pages: 617-635, ISSN: 1480-3275The Gram-negative facultative intracellular pathogen Legionella pneumophila infects a wide range of different protozoa in the environment and also human alveolar macrophages upon inhalation of contaminated aerosols. Inside its hosts, it creates a defined and unique compartment, termed the Legionella-containing vacuole (LCV), for survival and replication. To establish the LCV, L. pneumophila uses its Dot/Icm type IV secretion system (T4SS) to translocate more than 300 effector proteins into the host cell. Although it has become apparent in the past years that these effectors subvert a multitude of cellular processes and allow Legionella to take control of host cell vesicle trafficking, transcription, and translation, the exact function of the vast majority of effectors still remains unknown. This is partly due to high functional redundancy among the effectors, which renders conventional genetic approaches to elucidate their role ineffective. Here, we review the current knowledge about Legionella T4SS effectors, highlight open questions, and discuss new methods that promise to facilitate the characterization of T4SS effector functions in the future.
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Journal articleThompson CC, Griffiths C, Nicod SS, et al., 2015,
The Rsb phosphoregulatory network controls availability of the primary sigma factor in Chlamydia trachomatis and influences the kinetics of growth and development
, PLOS Pathogens, Vol: 11, Pages: 1-22, ISSN: 1553-7366Chlamydia trachomatis is the leading cause of both bacterial sexually transmitted infection and infection-derived blindness world-wide. No vaccine has proven protective to date in humans. C. trachomatis only replicates from inside a host cell, and has evolved to acquire a variety of nutrients directly from its host. However, a typical human immune response will normally limit the availability of a variety of essential nutrients. Thus, it is thought that the success of C. trachomatis as a human pathogen may lie in its ability to survive these immunological stress situations by slowing growth and development until conditions in the cell have improved. This mode of growth is known as persistence and how C. trachomatis senses stress and responds in this manner is an important area of research. Our report characterizes a complete signaling module, the Rsb network, that is capable of controlling the growth rate or infectivity of Chlamydia. By manipulating the levels of different pathway components, we were able to accelerate and restrict the growth and development of this pathogen. Our results suggest a mechanism by which Chlamydia can tailor its growth rate to the conditions within the host cell. The disruption of this pathway could generate a strain incapable of surviving a typical human immune response and would represent an attractive candidate as an attenuated growth vaccine.
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Journal articleFairweather NF, Peltier J, Shaw HA, et al., 2015,
Cyclic diGMP regulates production of sortase substrates of Clostridium difficile and their surface exposure through ZmpI protease-mediated cleavage
, Journal of Biological Chemistry, Vol: 290, Pages: 24453-24469, ISSN: 1083-351XBackground: Bacteria use variousmechanisms to anchor their surface proteins,including a sortase enzyme.Results: Covalent anchoring of proteins to thepeptidoglycan in Clostridium difficile and itsregulation by cyclic-di-GMP and proteaseactivity are demonstrated.Conclusion: A novel regulatory mechanism ofcell wall protein anchoring is found.Significance: Elucidating how proteins areanchored may shed light on control of bacterialcolonization in vivo.
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ReportCurry S, Leen EN, Sorgeloos F, et al., 2015,
A conserved interaction between a C-terminal motif in Norovirus VPg and the HEAT-1 domain of eIF4G is essential for translation initiation
, Publisher: bioRxivTranslation initiation is a critical early step in the replication cycle of the positive-sense, single-stranded RNA genome of noroviruses, a major cause of gastroenteritis in humans. Norovirus RNA, which has neither a 5 ́ m7G cap nor an internal ribosome entry site (IRES), adopts an unusual mechanism to initiate protein synthesis that relies on interactions between the VPg protein covalently attached to the 5 ́-end of the viral RNA and eukaryotic initiation factors (eIFs) in the host cell. For murine norovirus (MNV) we previously showed that VPg binds to the middle fragment of eIF4G (4GM; residues 652-1132). Here we have used pull-down assays, fluorescence anisotropy, and isothermal titration calorimetry (ITC) to demonstrate that a stretch of ~20 amino acids at the C terminus of MNV VPg mediates direct and specific binding to the HEAT-1 domain within the 4GM fragment of eIF4G. Our analysis further reveals that the MNV C-terminus binds to eIF4G HEAT-1 via a motif that is conserved in all known noroviruses. Fine mutagenic mapping suggests that the MNV VPg C terminus may interact with eIF4G in a helical conformation. NMR spectroscopy was used to define the VPg binding site on eIF4G HEAT-1, which was confirmed by mutagenesis and binding assays. We have found that this site is non-overlapping with the binding site for eIF4A on eIF4G HEAT-1 by demonstrating that norovirus VPg can form ternary VPg-eIF4G-eIF4A complexes. The functional significance of the VPg-eIF4G interaction was shown by the ability of fusion proteins containing the C- terminal peptide of MNV VPg to inhibit translation of norovirus RNA but not cap- or IRES-dependent translation. These observations define important structural details of a functional interaction between norovirus VPg and eIF4G and reveal a binding interface that might be exploited as a target for antiviral therapy.
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