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Journal articleLarrouy-Maumus GJ, Abigail Clements, Alain Filloux, et al., 2015,
Direct detection of lipid A on intact Gram-negative bacteria byMALDI-TOF mass spectrometry
, Journal of Microbiological Methods, Vol: 120, Pages: 68-71, ISSN: 1872-8359The purification and characterization of Gram-negative bacterial lipid A is tedious and time-consuming. Herein we report a rapid and sensitive method to identify lipid A directly on intact bacteria without any chemical treatment or purification, using an atypical solvent system to solubilize the matrix combined with MALDI-TOF mass spectrometry.
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Journal articleWitcomb LA, Collins JW, McCarthy AJ, et al., 2015,
Bioluminescent Imaging Reveals Novel Patterns of Colonization and Invasion in Systemic Escherichia coli K1 Experimental Infection in the Neonatal Rat
, Infection and Immunity, Vol: 83, Pages: 4528-4540, ISSN: 0019-9567Key features of Escherichia coli K1-mediated neonatal sepsis and meningitis, such as a strong age dependency and development along the gut-mesentery-blood-brain course of infection, can be replicated in the newborn rat. We examined temporal and spatial aspects of E. coli K1 infection following initiation of gastrointestinal colonization in 2-day-old (P2) rats after oral administration of E. coli K1 strain A192PP and a virulent bioluminescent derivative, E. coli A192PP-lux2. A combination of bacterial enumeration in the major organs, two-dimensional bioluminescence imaging, and three-dimensional diffuse light imaging tomography with integrated micro-computed tomography indicated multiple sites of colonization within the alimentary canal; these included the tongue, esophagus, and stomach in addition to the small intestine and colon. After invasion of the blood compartment, the bacteria entered the central nervous system, with restricted colonization of the brain, and also invaded the major organs, in line with increases in the severity of symptoms of infection. Both keratinized and nonkeratinized surfaces of esophagi were colonized to a considerably greater extent in susceptible P2 neonates than in corresponding tissues from infection-resistant 9-day-old rat pups; the bacteria appeared to damage and penetrate the nonkeratinized esophageal epithelium of infection-susceptible P2 animals, suggesting the esophagus represents a portal of entry for E. coli K1 into the systemic circulation. Thus, multimodality imaging of experimental systemic infections in real time indicates complex dynamic patterns of colonization and dissemination that provide new insights into the E. coli K1 infection of the neonatal rat.
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Conference paperShah A, Kannambath S, Herbst S, et al., 2015,
'THE KISS OF DEATH' - CALCINEURIN INHIBITORS PREVENT ACTIN-DEPENDENT LATERAL TRANSFER OF ASPERGILLUS FUMIGATUS IN NECROPTOTIC HUMAN MACROPHAGES
, Winter Meeting of the British-Thoracic-Society, Publisher: BMJ PUBLISHING GROUP, Pages: A48-A48, ISSN: 0040-6376 -
Journal articleFigueira R, Brown DR, Ferreira D, et al., 2015,
Adaptation to sustained nitrogen starvation by Escherichia coli requires the eukaryote-like serine/ threonine kinase YeaG
, Scientific Reports, Vol: 5, ISSN: 2045-2322 -
Journal articlePakharukova N, Garnett JA, Tuittila M, et al., 2015,
Structural Insight into Archaic and Alternative Chaperone-Usher Pathways Reveals a Novel Mechanism of Pilus Biogenesis.
, PLOS Pathogens, Vol: 11, ISSN: 1553-7366Gram-negative pathogens express fibrous adhesive organelles that mediate targeting to sites of infection. The major class of these organelles is assembled via the classical, alternative and archaic chaperone-usher pathways. Although non-classical systems share a wider phylogenetic distribution and are associated with a range of diseases, little is known about their assembly mechanisms. Here we report atomic-resolution insight into the structure and biogenesis of Acinetobacter baumannii Csu and Escherichia coli ECP biofilm-mediating pili. We show that the two non-classical systems are structurally related, but their assembly mechanism is strikingly different from the classical assembly pathway. Non-classical chaperones, unlike their classical counterparts, maintain subunits in a substantially disordered conformational state, akin to a molten globule. This is achieved by a unique binding mechanism involving the register-shifted donor strand complementation and a different subunit carboxylate anchor. The subunit lacks the classical pre-folded initiation site for donor strand exchange, suggesting that recognition of its exposed hydrophobic core starts the assembly process and provides fresh inspiration for the design of inhibitors targeting chaperone-usher systems.
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Journal articleCharlton T, Kovacs-Simon A, Michell S, et al., 2015,
Quantitative lipoproteomics in Clostridium difficile reveals a role for lipoproteins in sporulation
, Chemistry & Biology, Vol: 22, ISSN: 1074-5521Bacterial lipoproteins are surface exposed, anchored to the membrane by Sdiacylglyceryl modification of the N-terminal cysteine thiol. They play important roles inmany essential cellular processes and in bacterial pathogenesis. For example,Clostridium difficile is a Gram-positive anaerobe that causes severe gastrointestinaldisease, however, its lipoproteome remains poorly characterized. Here we describe theapplication of metabolic tagging with alkyne-tagged lipid analogues, in combinationwith quantitative proteomics, to profile protein lipidation across diverse C. difficilestrains and on inactivation of specific components of the lipoprotein biogenesispathway. These studies provide the first comprehensive map of the C. difficilelipoproteome, demonstrate the existence of two active lipoprotein signal peptidasesand provide insights into lipoprotein function, implicating the lipoproteome intransmission of this pathogen.
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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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