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  • Journal article
    Gunster R, Matthews SA, Holden DW, Thurston Tet 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: 1098-5522

    Within 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.

  • Journal article
    Bernal P, Allsopp LP, Filloux AAM, Llamas MAet al., 2017,

    The Pseudomonas putida T6SS is a plant warden against phytopathogens

    , The ISME Journal, Vol: 11, Pages: 972-987, ISSN: 1751-7362

    Bacterial 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.

  • Journal article
    Jønsson R, Liu B, Struve C, Yang Y, Jenssen H, Krogfelt K, Matthews SJet 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: 1570-9639

    Enteroaggregative 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

  • Journal article
    Johnson R, Byrne A, Berger CN, Klemm E, Crepin VF, Dougan G, Frankel Get al., 2016,

    The type III secretion system effector SptP of Salmonella enterica serovar Typhi

    , Journal of Bacteriology, Vol: 199, ISSN: 1098-5530

    Strains 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

  • Journal article
    Hawthorne W, Rouse S, Sewell L, Matthews SJet al., 2016,

    Structural insights into functional amyloid inhibition in Gram –ve bacteria

    , Biochemical Society Transactions, Vol: 44, Pages: 1643-1649, ISSN: 1470-8752

    Amyloids 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.

  • Journal article
    Matthews SJ, rouse S, hawthorne, Lambert, hare, morgan Met 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: 2053-230X

    Bacteria 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.

  • Journal article
    Valentini M, Laventie BJ, Moscoso JA, Jenal U, Filloux Aet al., 2016,

    Correction: The Diguanylate Cyclase HsbD Intersects with the HptB Regulatory Cascade to Control Pseudomonas aeruginosa Biofilm and Motility.

    , PLOS Genetics, Vol: 12, ISSN: 1553-7390
  • Journal article
    Valentini M, Laventie BJ, Moscoso J, Jenal U, Filloux Aet al., 2016,

    The Diguanylate Cyclase HsbD Intersects with the HptB Regulatory Cascade to Control Pseudomonas aeruginosa Biofilm and Motility.

    , PLOS Genetics, Vol: 12, ISSN: 1553-7390

    The molecular basis of second messenger signaling relies on an array of proteins that synthesize, degrade or bind the molecule to produce coherent functional outputs. Cyclic di-GMP (c-di-GMP) has emerged as a eubacterial nucleotide second messenger regulating a plethora of key behaviors, like the transition from planktonic cells to biofilm communities. The striking multiplicity of c-di-GMP control modules and regulated cellular functions raised the question of signaling specificity. Are c-di-GMP signaling routes exclusively dependent on a central hub or can they be locally administrated? In this study, we show an example of how c-di-GMP signaling gains output specificity in Pseudomonas aeruginosa. We observed the occurrence in P. aeruginosa of a c-di-GMP synthase gene, hsbD, in the proximity of the hptB and flagellar genes cluster. We show that the HptB pathway controls biofilm formation and motility by involving both HsbD and the anti-anti-sigma factor HsbA. The rewiring of c-di-GMP signaling into the HptB cascade relies on the original interaction between HsbD and HsbA and on the control of HsbD dynamic localization at the cell poles.

  • Journal article
    Zhang Y, Agrebi R, Bellows LE, Collet JF, Kaever V, Gründling Aet al., 2016,

    Evolutionary adaptation of the essential tRNA methyltransferase TrmD to the signaling molecule 3,5-cAMP in bacteria.

    , Journal of Biological Chemistry, Vol: 292, Pages: 313-327, ISSN: 1083-351X

    The nucleotide signaling molecule 3',5'-cyclic adenosine monophosphate (3',5'-cAMP) plays important physiological roles, ranging from carbon catabolite repression in bacteria to mediating the action of hormones in higher eukaryotes, including human. However, it remains unclear whether 3',5'-cAMP is universally present in the Firmicutes group of bacteria. We hypothesized that searching for proteins that bind 3',5'-cAMP might provide new insight into this question. Accordingly, we performed a genome-wide screen, and identified the essential Staphylococcus aureus tRNA m1G37 methyltransferase enzyme TrmD, which is conserved in all three domains of life, as a tight 3',5'-cAMP binding protein. TrmD enzymes are known to use S-adenosyl-L-methionine (AdoMet) as substrate; we shown that 3',5'-cAMP binds competitively with AdoMet to the S. aureus TrmD protein, indicating an overlapping binding site. However, the physiological relevance of this discovery remained unclear, as we were unable to identify a functional adenylate cyclase in S. aureus and only detected 2',3'-cAMP but not 3',5'-cAMP in cellular extracts. Interestingly, TrmD proteins from Escherichia coli and Mycobacterium tuberculosis, organisms known to synthesize 3',5'-cAMP, did not bind this signaling nucleotide. Comparative bioinformatics, mutagenesis and biochemical analyses revealed that the highly conserved Tyr86 residue in E. coli TrmD is essential to discriminate between 3',5'-cAMP and the native substrate AdoMet. Combined with a phylogenetic analysis, these results suggest that amino acids in the substrate binding pocket of TrmD underwent an adaptive evolution to accommodate the emergence of adenylate cyclases and thus the signaling molecule 3',5'-cAMP. Altogether this further indicates that S. aureus does not produce 3',5'-cAMP, which would otherwise competitively inhibit an essential enzyme.

  • Journal article
    Jia Y, Benjamin S, Liu Q, Xu Y, Dogga SK, Liu J, Matthews S, Soldati-Favre Det al., 2016,

    Toxoplasma gondii immune mapped protein 1 is anchored to the inner leaflet of the plasma membrane and adopts a novel protein fold

    , Biochimica et Biophysica Acta - Proteins and Proteomics, Vol: 1865, Pages: 208-219, ISSN: 1570-9639

    The immune mapped protein 1 (IMP1) was first identified as a protective antigen in Eimeria maxima and described as vaccine candidate and invasion factor in Toxoplasma gondii. We show here that TgIMP1 localizes to the inner leaflet of plasma membrane (PM) via dual acylation. Mutations either in the N-terminal myristoylation or palmitoylation sites (G2 and C5) cause relocalization of TgIMP1 to the cytosol. The first 11 amino acids are sufficient for PM targeting and the presence of lysine (K7) is critical. Disruption of TgIMP1 gene by double homologous recombination revealed no invasion defect or any measurable alteration in the lytic cycle of tachyzoites. Following immunization with TgIMP1 DNA vaccine, mice challenged with either wild type or IMP1-ko parasites showed no significant difference in protection. The sequence analysis identified a structured C-terminal domain that is present in a broader family of IMP1-like proteins conserved across the members of Apicomplexa. We present the solution structure of this domain determined from NMR data and describe a new protein fold not seen before.

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