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Journal articleBrown RL, Clarke TB, 2016,
The regulation of host defences to infection by the microbiota
, Immunology, Vol: 150, Pages: 1-6, ISSN: 0019-2805The skin and mucosal epithelia of humans and other mammals are permanently colonised by large microbial communities (the microbiota). Due to this life-long association with the microbiota, these microbes have an extensive influence over the physiology of their host organism. It is now becoming apparent that nearly all tissues and organ systems, whether in direct contact with the microbiota, or in deeper host sites, are under microbial influence. The immune system is perhaps the most profoundly affected, with the microbiota programming both its innate and adaptive arms. The regulation of immunity by the microbiota helps protect the host against intestinal and extra-intestinal infection by many classes of pathogen. In this review, we will discuss the experimental evidence supporting a role for the microbiota in regulating host defences to extra-intestinal infection, draw together common mechanistic themes, including the central role of pattern recognition receptors, and outline outstanding questions which need to be answered. This article is protected by copyright. All rights reserved.
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Journal articleBrown DR, Sheppard CM, Matthews S, et al., 2016,
The Xp10 bacteriophage protein P7 inhibits transcription by the major and major variant forms of the host RNA polymerase via a common mechanism
, Journal of Molecular Biology, Vol: 428, Pages: 3911-3919, ISSN: 1089-8638The σ factor is a functionally obligatory subunit of the bacterial transcription machinery, the RNA polymerase. Bacteriophage-encoded small proteins that either modulate or inhibit the bacterial RNAP to allow the temporal regulation of bacteriophage gene expression often target the activity of the major bacterial σ factor, σ70. Previously, we showed that during Xanthomonas oryzae phage Xp10 infection, the phage protein P7 inhibits the host RNAP by preventing the productive engagement with the promoter and simultaneously displaces the σ70 factor from the RNAP. In this study, we demonstrate that P7 also inhibits the productive engagement of the bacterial RNAP containing the major variant bacterial σ factor, σ54, with its cognate promoter. The results suggest for the first time that the major variant form of the host RNAP can also be targeted by bacteriophage-encoded transcription regulatory proteins. Since the major and major variant σ factor interacting surfaces in the RNAP substantially overlap, but different regions of σ70 and σ54 are used for binding to the RNAP, our results further underscore the importance of the σ–RNAP interface in bacterial RNAP function and regulation and potentially for intervention by antibacterials.
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Journal articleLiang X, Liu B, Zhu F, et al., 2016,
A distinct sortase SrtB anchors and processes a streptococcal adhesin AbpA with a novel structural property.
, Scientific Reports, Vol: 6, ISSN: 2045-2322Surface display of proteins by sortases in Gram-positive bacteria is crucial for bacterial fitness and virulence. We found a unique gene locus encoding an amylase-binding adhesin AbpA and a sortase B in oral streptococci. AbpA possesses a new distinct C-terminal cell wall sorting signal. We demonstrated that this C-terminal motif is required for anchoring AbpA to cell wall. In vitro and in vivo studies revealed that SrtB has dual functions, anchoring AbpA to the cell wall and processing AbpA into a ladder profile. Solution structure of AbpA determined by NMR reveals a novel structure comprising a small globular α/β domain and an extended coiled-coil heliacal domain. Structural and biochemical studies identified key residues that are crucial for amylase binding. Taken together, our studies document a unique sortase/adhesion substrate system in streptococci adapted to the oral environment rich in salivary amylase.
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Journal articleLee W-C, Matthews S, Garnett JA, 2016,
Crystal structure and analysis of HdaB: the Enteroaggregative Escherichia coli AAF/IV pilus tip protein
, Protein Science, Vol: 25, Pages: 1898-1905, ISSN: 1469-896XEnteroaggregative Escherichia coli is the primary cause of pediatric diarrhea indeveloping countries and utilize aggregative adherence fimbriae (AAFs) to promoteinitial adherence to the host intestinal mucosa, promote the formation of biofilms andmediate host invasion. Five AAFs have been identified to date and AAF/IV is amongstthe most prevalent found in clinical isolates. Here we present the X-ray crystal structureof the AAF/IV tip protein HdaB at 2.0 Å resolution. It shares high structural homologywith members of the Afa/Dr superfamily of fimbriae, which are involved in hostinvasion. We highlight surface exposed residues that share sequence homology andpropose that these may function in invasion and also non-conserved regions that couldmediate HdaB specific adhesive functions.
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Journal articleRasheed M, Garnett J, Perez-Dorado I, et al., 2016,
Crystal structure of the CupB6 adhesive tip from the chaperone-usher family of pili from Pseudomonas aeruginosa
, Biochimica et Biophysica Acta - Protein Structure, Vol: 1864, Pages: 1500-1505, ISSN: 0005-2795Pseudomonas aeruginosa is a Gram-negative opportunistic bacterial pathogen that can cause chronicinfection of the lungs of cystic fibrosis patients. Chaperone-usher systems in P. aeruginosa are knownto translocate and assemble adhesive pili on the bacterial surface and contribute to biofilm formationwithin the host. Here, we report the crystal structure of the tip adhesion subunit CupB6 from thecupB1-6 gene cluster. The tip domain is connected to the pilus via the N-terminal donor strand fromthe main pilus subunit CupB1. Although the CupB6 adhesion domain bears structural features similarto other CU adhesins it displays an unusual polyproline helix adjacent to a prominent surface pocket,which are likely the site for receptor recognition.
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Journal articleKierdorf K, Dionne MS, 2016,
The software and hardware of macrophages: a diversity of options
, Developmental Cell, Vol: 38, Pages: 122-125, ISSN: 1878-1551Macrophages play important immune and homeostatic roles that depend on the ability to receive and interpret specific signals from environmental stimuli. Here we describe the different activation states these cells can exhibit in response to signals and how these states affect and can be affected by bacterial pathogens.
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Journal articlePruneda JN, Durkin CH, Geurink PP, et al., 2016,
The molecular basis for ubiquitin and ubiquitin-like specificities in bacterial effector proteases
, Molecular Cell, Vol: 63, Pages: 261-276, ISSN: 1097-2765Pathogenic bacteria rely on secreted effector proteins to manipulate host signaling pathways, often in creative ways. CE clan proteases, specific hydrolases for ubiquitin-like modifications (SUMO and NEDD8) in eukaryotes, reportedly serve as bacterial effector proteins with deSUMOylase, deubiquitinase, or, even, acetyltransferase activities. Here, we characterize bacterial CE protease activities, revealing K63-linkage-specific deubiquitinases in human pathogens, such as Salmonella, Escherichia, and Shigella, as well as ubiquitin/ubiquitin-like cross-reactive enzymes in Chlamydia, Rickettsia, and Xanthomonas. Five crystal structures, including ubiquitin/ubiquitin-like complexes, explain substrate specificities and redefine relationships across the CE clan. Importantly, this work identifies novel family members and provides key discoveries among previously reported effectors, such as the unexpected deubiquitinase activity in Xanthomonas XopD, contributed by an unstructured ubiquitin binding region. Furthermore, accessory domains regulate properties such as subcellular localization, as exemplified by a ubiquitin-binding domain in Salmonella Typhimurium SseL. Our work both highlights and explains the functional adaptations observed among diverse CE clan proteins.
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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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