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Conference paperRouse SL, Hawthorne W, Berry J, et al., 2017,
Structural and Mechanistic Insights into Transport of Functional Amyloid Subunits across the Pseudomonas Outer Membrane
, 61st Annual Meeting of the Biophysical-Society, Publisher: CELL PRESS, Pages: 188A-188A, ISSN: 0006-3495 -
Journal articleEldridge MJG, Sanchez Garrido J, Hoben GF, et al., 2017,
The atypical ubiquitin E2 conjugase UBE2L3 is an indirect caspase-1 target and controls IL-1beta secretion by inflammasomes
, Cell Reports, Vol: 18, Pages: 1285-1297, ISSN: 2211-1247Caspase-1 activation by inflammasome signalling scaffoldsinitiates inflammation and antimicrobial responses. Caspase-1 proteolytically converts newly induced pro-IL-1α into its mature form and directs its secretion, triggers pyroptosis and the release of non-substrate alarmins such as IL-1α and HMGB1. While somecaspase-1 substrates involved in these events are known, the identities and roles of non-proteolytic targets remain unknown. Here we report using unbiased proteomics that the UBE2L3 ubiquitin conjugase is an indirect target of caspase-1. Caspase-1, but not caspase-4, controlled pyroptosis-and ubiquitin-independent proteasomal degradation of UBE2L3 upon canonical and non-canonical inflammasome activation by sterile danger signals and bacterial infection. Mechanistically, UBE2L3 acted post-translationally to promote K48-ubiquitylation and turnover of pro-IL-1β and dampen mature-IL-1β production. UBE2L3 depletion increased pro-IL-1β levels and mature-IL-1βsecretion by inflammasomes. These findings on UBE2L3 as a molecular rheostat have implications for IL-1-driven pathology in hereditary fever syndromes, and autoinflammatory conditions associated with UBE2L3 polymorphisms.
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Journal articleBosi E, Fondi M, Orlandini V, et al., 2017,
The pangenome of (Antarctic) Pseudoalteromonas bacteria: evolutionary and functional insights
, BMC Genomics, Vol: 18, ISSN: 1471-2164Background:Pseudoalteromonas is a genus of ubiquitous marine bacteria used as model organisms to study the biological mechanisms involved in the adaptation to cold conditions. A remarkable feature shared by these bacteria is their ability to produce secondary metabolites with a strong antimicrobial and antitumor activity. Despite their biotechnological relevance, representatives of this genus are still lacking (with few exceptions) an extensive genomic characterization, including features involved in the evolution of secondary metabolites production. Indeed, biotechnological applications would greatly benefit from such analysis.Results:Here, we analyzed the genomes of 38 strains belonging to different Pseudoalteromonas species and isolated from diverse ecological niches, including extreme ones (i.e. Antarctica). These sequences were used to reconstruct the largest Pseudoalteromonas pangenome computed so far, including also the two main groups of Pseudoalteromonas strains (pigmented and not pigmented strains). The downstream analyses were conducted to describe the genomic diversity, both at genus and group levels. This allowed highlighting a remarkable genomic heterogeneity, even for closely related strains. We drafted all the main evolutionary steps that led to the current structure and gene content of Pseudoalteromonas representatives. These, most likely, included an extensive genome reduction and a strong contribution of Horizontal Gene Transfer (HGT), which affected biotechnologically relevant gene sets and occurred in a strain-specific fashion. Furthermore, this study also identified the genomic determinants related to some of the most interesting features of the Pseudoalteromonas representatives, such as the production of secondary metabolites, the adaptation to cold temperatures and the resistance to abiotic compounds.Conclusions:This study poses the bases for a comprehensive understanding of the evolutionary trajectories followed in time by this peculiar bact
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Journal articlePearson JS, Giogha C, Muhlen S, et al., 2017,
EspL is a bacterial cysteine protease effector that cleaves RHIM proteins to block necroptosis and inflammation
, NATURE MICROBIOLOGY, Vol: 2, ISSN: 2058-5276 -
Journal articleGunster R, Matthews SA, Holden DW, et al., 2017,
SseK1 and SseK3 T3SS effectors inhibit NF-kB signalling and necroptotic cell death in Salmonella-infected macrophages
, Infection and Immunity, Vol: 85, ISSN: 1098-5522Within host cells such as macrophages, Salmonella enterica translocates virulence (effector) proteins across its vacuolar membrane using the SPI-2 type III secretion system. Previously it has been shown that when expressed ectopically the effectors SseK1 and SseK3 inhibit TNFα-induced NF-κB activation. In this study we show that ectopically expressed SseK1, SseK2 and SseK3 suppressed TNFα-, but not TLR4-, or interleukin-induced NF-κB activation. Inhibition required a DXD motif, which in SseK1 and SseK3 is essential for protein Arginine-N-acetylglucosamine (GlcNAc)-ylation. During macrophage infection, SseK1 and SseK3 inhibited NF-κB activity in an additive manner. SseK3-mediated inhibition of NF-κB activation did not require the only known host-binding partner of this effector, the E3-ubiquitin ligase TRIM32. SseK proteins also inhibited TNFα-induced cell death during macrophage infection. Despite SseK1 and SseK3 inhibiting TNFα-induced apoptosis upon ectopic expression in HeLa cells, the percentage of infected macrophages undergoing apoptosis was SseK-independent. Instead, SseK proteins inhibited necroptotic cell death during macrophage infection. SseK1 and SseK3 caused GlcNAcylation of different proteins in infected macrophages suggesting that these effectors have distinct substrate specificities. Indeed, SseK1 caused the GlcNAcylation of the death domain containing proteins FADD and TRADD, whereas SseK3 expression resulted in weak GlcNAcylation of TRADD but not FADD. Additional, as yet unidentified substrates are likely to explain the additive phenotype of a Salmonella strain lacking both SseK1 and SseK3.
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Journal articleBernal P, Allsopp LP, Filloux AAM, et al., 2017,
The Pseudomonas putida T6SS is a plant warden against phytopathogens
, The ISME Journal, Vol: 11, Pages: 972-987, ISSN: 1751-7362Bacterial type VI secretion systems (T6SSs) are molecular weapons designed to deliver toxic effectors into prey cells. These nanomachines play an important role in inter-bacterial competition and provide advantages to T6SS active strains in polymicrobial environments. Here we analyse the genome of the biocontrol agent Pseudomonas putida KT2440 and identify three T6SS gene clusters (K1-, K2- and K3-T6SS). Besides, ten T6SS effector/immunity pairs were found, including putative nucleases and pore-forming colicins. We show that the K1-T6SS is a potent antibacterial device which secretes a toxic Rhs-type effector Tke2. Remarkably, P. putida eradicates a broad range of bacteria in a K1-T6SS-dependent manner, including resilient phytopathogens which demonstrates that the T6SS is instrumental to empower P. putida to fight against competitors. Furthermore, we observed a drastically reduced necrosis on the leaves of Nicotiana benthamiana during co-infection with P. putida and Xanthomonas campestris. Such protection is dependent on the activity of the P. putida T6SS. Many routes have been explored to develop biocontrol agents capable of manipulating the microbial composition of the rhizosphere and phyllosphere. Here we unveil a novel mechanism for plant biocontrol which needs to be considered for the selection of plant wardens whose mission is to prevent phytopathogen infections.
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Journal articleJønsson R, Liu B, Struve C, et al., 2016,
Structural and functional studies of Escherichia coli Aggregative Adherence Fimbriae (AAF/V) reveal a deficiency in extracellular matrix binding
, BBA Protein and Proteomics, Vol: 1865, Pages: 304-311, ISSN: 1570-9639Enteroaggregative Escherichia coli (EAEC) is an emerging cause of acute and persistent diarrhea worldwide. The pathogenesis of different EAEC stains is complicated, however, the early essential step begins with attachment of EAEC to intestinal mucosa via aggregative adherence fimbriae (AAFs). Currently, five different variants have been identified, which all share a degree of similarity in the gene organization of their operons and sequences. Here, we report the solution structure of Agg5A from the AAF/V variant. While preserving the major structural features shared by all AAF members, only Agg5A possesses an inserted helix at the beginning of the donor strand, which together with altered surface electrostatics, renders the protein unable to interact with fibronectin. Hence, here we characterize the first AAF variant with a binding mode that varies from previously described AAFs
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Journal articleJohnson R, Byrne A, Berger CN, et al., 2016,
The type III secretion system effector SptP of Salmonella enterica serovar Typhi
, Journal of Bacteriology, Vol: 199, ISSN: 1098-5530Strains of the various Salmonella enterica serovars cause gastroenteritis or typhoid fever in humans, with virulence depending on the action of two type III secretion systems (Salmonella pathogenicity island 1 [SPI-1] and SPI-2). SptP is a Salmonella SPI-1 effector, involved in mediating recovery of the host cytoskeleton postinfection. SptP requires a chaperone, SicP, for stability and secretion. SptP has 94% identity between S. enterica serovar Typhimurium and S Typhi; direct comparison of the protein sequences revealed that S Typhi SptP has numerous amino acid changes within its chaperone-binding domain. Subsequent comparison of ΔsptP S Typhi and S. Typhimurium strains demonstrated that, unlike SptP in S. Typhimurium, SptP in S Typhi was not involved in invasion or cytoskeletal recovery postinfection. Investigation of whether the observed amino acid changes within SptP of S Typhi affected its function revealed that S Typhi SptP was unable to complement S. Typhimurium ΔsptP due to an absence of secretion. We further demonstrated that while S. Typhimurium SptP is stable intracellularly within S Typhi, S Typhi SptP is unstable, although stability could be recovered following replacement of the chaperone-binding domain with that of S. Typhimurium. Direct assessment of the strength of the interaction between SptP and SicP of both serovars via bacterial two-hybrid analysis demonstrated that S Typhi SptP has a significantly weaker interaction with SicP than the equivalent proteins in S. Typhimurium. Taken together, our results suggest that changes within the chaperone-binding domain of SptP in S Typhi hinder binding to its chaperone, resulting in instability, preventing translocation, and therefore restricting the intracellular activity of this effector. IMPORTANCE: Studies investigating Salmonella pathogenesis typically rely on Salmonella Typhimurium, even though Salmonella Typhi causes the more severe disease in humans. As such, an understanding of S. Typhi
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Journal articleHawthorne W, Rouse S, Sewell L, et al., 2016,
Structural insights into functional amyloid inhibition in Gram –ve bacteria
, Biochemical Society Transactions, Vol: 44, Pages: 1643-1649, ISSN: 1470-8752Amyloids are proteinaceous aggregates known for their role in debilitating degenerative diseases involving protein dysfunction. Many forms of functional amyloid are also produced in nature and often these systems require careful control of their assembly to avoid the potentially toxic effects. The best-characterised functional amyloid system is the bacterial curli system. Three natural inhibitors of bacterial curli amyloid have been identified and recently characterised structurally. Here, we compare common structural features of CsgC, CsgE and CsgH and discuss the potential implications for general inhibition of amyloid.
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Journal articleMatthews SJ, rouse S, hawthorne, et al., 2016,
Purification, crystallization and characterization of the Pseudomonas outer membrane protein FapF, a functional amyloid transporter
, Acta Crystallographica Section F: Structural Biology Communications, Vol: F72, Pages: 892-896, ISSN: 2053-230XBacteria often produce extracellular amyloid fibresviaa multi-componentsecretion system. Aggregation-prone, unstructured subunits cross the periplasmand are secreted through the outer membrane, after which they self-assemble.Here, significant progress is presented towards solving the high-resolutioncrystal structure of the novel amyloid transporter FapF fromPseudomonas,which facilitates the secretion of the amyloid-forming polypeptide FapC acrossthe bacterial outer membrane. This represents the first step towards obtainingstructural insight into the products of thePseudomonas fapoperon. Initialattempts at crystallizing full-length and N-terminally truncated constructs byrefolding techniques were not successful; however, after preparing FapF106–430from the membrane fraction, reproducible crystals were obtained using thesitting-drop method of vapour diffusion. Diffraction data have been processedto 2.5 A ̊resolution. These crystals belonged to the monoclinic space groupC121,with unit-cell parametersa= 143.4,b= 124.6,c= 80.4 A ̊, = = 90, = 96.32 and three monomers in the asymmetric unit. It was found that the switch tocomplete detergent exchange into C8E4 was crucial for forming well diffractingcrystals, and it is suggested that this combined with limited proteolysis is apotentially useful protocol for membrane -barrel protein crystallography. Thethree-dimensional structure of FapF will provide invaluable information on themechanistic differences of biogenesis between the curli and Fap functionalamyloid systems.
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