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Journal articleYu XJ, Liu M, Holden D, 2016,
Salmonella Effectors SseF and SseG Interact with Mammalian Protein ACBD3 (GCP60) To Anchor Salmonella-Containing Vacuoles at the Golgi Network
, mBio, Vol: 7, ISSN: 2161-2129Following infection of mammalian cells, Salmonella enterica serovar Typhimurium (S. Typhimurium) replicates within membrane-bound compartments known as Salmonella-containing vacuoles (SCVs). The Salmonella pathogenicity island 2 type III secretion system (SPI-2 T3SS) translocates approximately 30 different effectors across the vacuolar membrane. SseF and SseG are two such effectors that are required for SCVs to localize close to the Golgi network in infected epithelial cells. In a yeast two-hybrid assay, SseG and an N-terminal variant of SseF interacted directly with mammalian ACBD3, a multifunctional cytosolic Golgi network-associated protein. Knockdown of ACBD3 by small interfering RNA (siRNA) reduced epithelial cell Golgi network association of wild-type bacteria, phenocopying the effect of null mutations of sseG or sseF. Binding of SseF to ACBD3 in infected cells required the presence of SseG. A single-amino-acid mutant of SseG and a double-amino-acid mutant of SseF were obtained that did not interact with ACBD3 in Saccharomyces cerevisiae. When either of these was produced together with the corresponding wild-type effector by Salmonella in infected cells, they enabled SCV-Golgi network association and interacted with ACBD3. However, these properties were lost and bacteria displayed an intracellular replication defect when cells were infected with Salmonella carrying both mutant genes. Knockdown of ACBD3 resulted in a replication defect of wild-type bacteria but did not further attenuate the growth defect of a ΔsseFG mutant strain. We propose a model in which interaction between SseF and SseG enables both proteins to bind ACBD3, thereby anchoring SCVs at the Golgi network and facilitating bacterial replication.
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Journal articleSirianni A, Krokowski S, Lobato-Márquez D, et al., 2016,
Mitochondria mediate septin cage assembly to promote autophagy of Shigella
, EMBO Reports, Vol: 17, Pages: 1-15, ISSN: 1469-221XSeptins, cytoskeletal proteins with well-characterised roles in cytokinesis, form cage-like structures around cytosolic Shigella flexneri and promote their targeting to autophagosomes. However, the processes underlying septin cage assembly, and whether they influence S. flexneri proliferation, remain to be established. Using single cell analysis, we show that septin cages inhibit S. flexneri proliferation. To study mechanisms of septin cage assembly, we used proteomics and found mitochondrial proteins associate with septins in S. flexneriinfected cells. Strikingly, mitochondria associated with S. flexneri promote septin assembly into the cages that entrap bacteria for autophagy. We demonstrate that the cytosolic GTPase dynamin-related protein 1 (Drp1) interacts with septins to enhance mitochondrial fission. To avoid autophagy, actin-polymerising Shigella fragment mitochondria to escape from septin caging. Our results have demonstrated a role for mitochondria in anti-Shigella autophagy, and uncovered a fundamental link between septin assembly and mitochondria.
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Journal articleCheverton AM, Gollan B, Przydacz M, et al., 2016,
A Salmonella Toxin Promotes Persister Formation through Acetylation of tRNA.
, Molecular cell, Vol: 63, Pages: 86-96, ISSN: 1097-2765The recalcitrance of many bacterial infections to antibiotic treatment is thought to be due to the presence of persisters that are non-growing, antibiotic-insensitive cells. Eventually, persisters resume growth, accounting for relapses of infection. Salmonella is an important pathogen that causes disease through its ability to survive inside macrophages. After macrophage phagocytosis, a significant proportion of the Salmonella population forms non-growing persisters through the action of toxin-antitoxin modules. Here we reveal that one such toxin, TacT, is an acetyltransferase that blocks the primary amine group of amino acids on charged tRNA molecules, thereby inhibiting translation and promoting persister formation. Furthermore, we report the crystal structure of TacT and note unique structural features, including two positively charged surface patches that are essential for toxicity. Finally, we identify a detoxifying mechanism in Salmonella wherein peptidyl-tRNA hydrolase counteracts TacT-dependent growth arrest, explaining how bacterial persisters can resume growth.
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Journal articleFilloux A, Freemont P, 2016,
Structural biology: baseplates in contractile machines
, Nature Microbiology, Vol: 1, ISSN: 2058-5276 -
Journal articleTaglialegna A, Navarro S, Ventura S, et al., 2016,
Staphylococcal Bap Proteins Build Amyloid Scaffold Biofilm Matrices in Response to Environmental Signals
, PLOS Pathogens, Vol: 12, ISSN: 1553-7366Biofilms are communities of bacteria that grow encased in an extracellular matrix that often contains proteins. The spatial organization and the molecular interactions between matrix scaffold proteins remain in most cases largely unknown. Here, we report that Bap protein of Staphylococcus aureus self-assembles into functional amyloid aggregates to build the biofilm matrix in response to environmental conditions. Specifically, Bap is processed and fragments containing at least the N-terminus of the protein become aggregation-prone and self-assemble into amyloid-like structures under acidic pHs and low concentrations of calcium. The molten globule-like state of Bap fragments is stabilized upon binding of the cation, hindering its self-assembly into amyloid fibers. These findings define a dual function for Bap, first as a sensor and then as a scaffold protein to promote biofilm development under specific environmental conditions. Since the pH-driven multicellular behavior mediated by Bap occurs in coagulase-negative staphylococci and many other bacteria exploit Bap-like proteins to build a biofilm matrix, the mechanism of amyloid-like aggregation described here may be widespread among pathogenic bacteria.
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Journal articlePlanamente S, Salih O, Manoli E, et al., 2016,
TssA forms a gp6-like ring attached to the type VI secretion sheath
, EMBO Journal, Vol: 35, Pages: 1613-1627, ISSN: 0261-4189The type VI secretion system (T6SS) is a supra-molecular bacterial complex that resembles phage tails. It is a killing machine which fires toxins into target cells upon contraction of its TssBC sheath. Here, we show that TssA1 is a T6SS component forming dodecameric ring structures whose dimensions match those of the TssBC sheath and which can accommodate the inner Hcp tube. The TssA1 ring complex binds the T6SS sheath and impacts its behaviour in vivo. In the phage, the first disc of the gp18 sheath sits on a baseplate wherein gp6 is a dodecameric ring. We found remarkable sequence and structural similarities between TssA1 and gp6 C-termini, and propose that TssA1 could be a baseplate component of the T6SS. Furthermore, we identified similarities between TssK1 and gp8, the former interacting with TssA1 while the latter is found in the outer radius of the gp6 ring. These observations, combined with similarities between TssF and gp6N-terminus or TssG and gp53, lead us to propose a comparative model between the phage baseplate and the T6SS.
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Journal articleHolmes AH, Gill SK, Hui K, et al., 2016,
Increased airway glucose increases airway bacterial load in hyperglycaemia
, Scientific Reports, Vol: 6, ISSN: 2045-2322Diabetes is associated with increased frequency of hospitalization due to bacterial lung infection.We hypothesize that increased airway glucose caused by hyperglycaemia leads to increasedbacterial loads. In critical care patients, we observed that respiratory tract bacterial colonisationis significantly more likely when blood glucose is high. We engineered mutants in genesaffecting glucose uptake and metabolism (oprB, gltK, gtrS and glk) in Pseudomonas aeruginosa,strain PAO1. These mutants displayed attenuated growth in minimal medium supplemented withglucose as the sole carbon source. The effect of glucose on growth in vivo was tested usingstreptozocin-induced, hyperglycaemic mice, which have significantly greater airway glucose.Bacterial burden in hyperglycaemic animals was greater than control animals when infected withwild type but not mutant PAO1. Metformin pre-treatment of hyperglycaemic animals reducedboth airway glucose and bacterial load. These data support airway glucose as a criticaldeterminant of increased bacterial load during diabetes.
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Journal articleRolhion N, Furniss R, Grabe G, et al., 2016,
Inhibition of nuclear transport of NF-kB p65 by the Salmonella type III secretion system effector Sp
, Plos Pathogens, Vol: 12, Pages: 1-26, ISSN: 1553-7374Salmonella enterica replicates in macrophages through the action of effector proteins translocated across the vacuolar membrane by a type III secretion system (T3SS). Here we show that the SPI-2 T3SS effector SpvD suppresses proinflammatory immune responses. SpvD prevented activation of an NF-ĸB-dependent promoter and caused nuclear accumulation of importin-α, which is required for nuclear import of p65. SpvD interacted specifically with the exportin Xpo2, which mediates nuclear-cytoplasmic recycling of importins. We propose that interaction between SpvD and Xpo2 disrupts the normal recycling of importin-α from the nucleus, leading to a defect in nuclear translocation of p65 and inhibition of activation of NF-ĸB regulated promoters. SpvD down-regulated pro-inflammatory responses and contributed to systemic growth of bacteria in mice. This work shows that a bacterial pathogen can manipulate host cell immune responses by interfering with the nuclear transport machinery.
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Journal articleHergott CB, Roche AM, Tamashiro E, et al., 2016,
Detection of peptidoglycan from the gut microbiota governs the lifespan of circulating phagocytes at homeostasis
, Blood, Vol: 127, Pages: 2460-2471, ISSN: 1528-0020Maintenance of myeloid cell homeostasis requires continuous turnover of phagocytes from the bloodstream, yet whether environmental signals influence phagocyte longevity in the absence of inflammation remains unknown. Here, we show that the gut microbiota regulates the steady-state cellular lifespan of neutrophils and inflammatory monocytes, the two most abundant circulating myeloid cells and key contributors to inflammatory responses. Treatment of mice with broad-spectrum antibiotics, or with the gut-restricted aminoglycoside neomycin alone, accelerated phagocyte turnover and increased the rates of their spontaneous apoptosis. Metagenomic analyses revealed that neomycin altered the abundance of intestinal bacteria bearing γ-D-glutamyl-meso-diaminopimelic acid (iE-DAP), a ligand for the intracellular peptidoglycan sensor Nod1. Accordingly, signaling through Nod1 was both necessary and sufficient to mediate the stimulatory influence of the flora on myeloid cell longevity. Stimulation of Nod1 signaling increased the frequency of lymphocytes in the murine intestine producing the pro-inflammatory cytokine interleukin 17A (IL-17A), and liberation of IL-17A was required for transmission of Nod1-dependent signals to circulating phagocytes. Together, these results define a mechanism through which intestinal microbes govern a central component of myeloid homeostasis and suggest perturbations of commensal communities can influence steady-state regulation of cell fate.
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Journal articleSantos AJM, Durkin C, Helaine S, et al., 2016,
Clustered intracellular Salmonella Typhimurium Blocks Host Cell Cytokinesis
, Infection and Immunity, Vol: 84, Pages: 2149-2158, ISSN: 1098-5522Several bacterial pathogens and viruses interfere with the cell cycle of their host cells to enhance virulence. This is especially apparent in bacteria that colonise the gut epithelium, where inhibition of the cell cycle of infected cells enhances the intestinal colonisation. We found that intracellular Salmonella enterica Typhimurium induced the binucleation of a large proportion of epithelial cells by 14 hours post invasion, which was dependent on an intact Salmonella pathogenicity island-2 (SPI-2) type 3 secretion system. The SPI-2 effectors SseF and SseG were required to induce binucleation. SseF and SseG are known to maintain microcolonies of Salmonella-containing vacuoles close to the microtubule organising centre of infected epithelial cells. During host cell division these clustered microcolonies prevented the correct localisation of members of the chromosomal passenger complex and mitotic kinesin-like protein 1, and consequently prevented cytokinesis. Tetraploidy, arising from a cytokinesis defect, is known to have a deleterious effect on subsequent cell divisions, either resulting in chromosomal instabilities or cell cycle arrest. In infected mice, proliferation of small intestinal epithelial cells was compromised in an SseF/SseG-dependent manner, suggesting that cytokinesis failure caused by S. Typhimurium delays epithelial cell turnover in the intestine.
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