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  • 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 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
    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
    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
    Willis A, Moore C, Mazon Moya M, Krokowski S, Lambert C, Till R, Mostowy S, Sockett REet al., 2016,

    Injections of predatory bacteria work alongside host immune cells to treat Shigella Infection in zebrafish larvae

    , Current Biology, Vol: 26, Pages: 3343-3351, ISSN: 1879-0445

    Bdellovibrio bacteriovorus are predatory bacteria that invade and kill a range of Gram- negative bacterial pathogens in natural environments and in vitro [1, 2]. In this study we investigated Bdellovibrio as an injected, antibacterial-treatment, in vivo using zebrafish (Danio rerio) larvae infected with an antibiotic-resistant strain of the human pathogen Shigella flexneri. When injected alone Bdellovibrio can persist for more than 24 hours in vivo however exert no pathogenic effects on zebrafish larvae. Bdellovibrio injection of zebrafish containing a lethal dose of Shigella promotes pathogen killing, leading to increased zebrafish survival. Live-cell imaging of infected zebrafish reveals that Shigella undergo rounding induced by the invasive predation from Bdellovibrio in vivo. Furthermore, Shigella-dependent replication of Bdellovibrio was captured inside the zebrafish larvae indicating active predation in vivo. Bdellovibrio can be engulfed and ultimately eliminated by host neutrophils and macrophages, yet have a sufficient dwell time to prey upon pathogens. Experiments in immune-compromised zebrafish reveal that maximal therapeutic benefits of Bdellovibrio result from the synergy of both bacterial predation and host immunity, but that in vivo predation contributes significantly to the survival outcome. Our results demonstrate that successful antibacterial therapy can be achieved via the host immune system working together with bacterial predation by Bdellovibrio. Such cooperation may be important to consider in the fight against antibiotic resistant infections in vivo.

  • 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.

  • Journal article
    Saliba A-E, Li L, Westermann AJ, Appenzeller S, Stapels DAC, Schulte LN, Helaine S, Vogel Jet al., 2016,

    Single-cell RNA-seq ties macrophage polarization to growth rate of intracellular Salmonella

    , Nature Microbiology, Vol: 2, ISSN: 2058-5276

    Intracellular bacterial pathogens can exhibit large heterogeneity in growth rate inside host cells, with major consequences for the infection outcome. If and how the host responds to this heterogeneity remains poorly understood. Here, we combined a fluorescent reporter of bacterial cell division with single-cell RNA-sequencing analysis to study the macrophage response to different intracellular states of the model pathogen Salmonella enterica serovar Typhimurium. The transcriptomes of individual infected macrophages revealed a spectrum of functional host response states to growing and non-growing bacteria. Intriguingly, macrophages harbouring non-growing Salmonella display hallmarks of the proinflammatory M1 polarization state and differ little from bystander cells, suggesting that non-growing bacteria evade recognition by intracellular immune receptors. By contrast, macrophages containing growing bacteria have turned into an anti-inflammatory, M2-like state, as if fast-growing intracellular Salmonella overcome host defence by reprogramming macrophage polarization. Additionally, our clustering approach reveals intermediate host functional states between these extremes. Altogether, our data suggest that gene expression variability in infected host cells shapes different cellular environments, some of which may favour a growth arrest of Salmonella facilitating immune evasion and the establishment of a long-term niche, while others allow Salmonella to escape intracellular antimicrobial activity and proliferate.

  • Journal article
    Torraca V, Mostowy S, 2016,

    Septins and bacterial infection

    , Frontiers in Cell and Developmental Biology, Vol: 4, ISSN: 2296-634X

    Septins, a unique cytoskeletal component associated with cellular membranes, are increasingly recognized as having important roles in host defense against bacterial infection. A role for septins duringinvasion of Listeria monocytogenes into host cells was first proposed in 2002. Since then, work has shown that septins assemble in response to a wide variety of invasive bacterial pathogens, and septin assemblies can have different roles during the bacterial infection process. Here we review the interplay between septins and bacterial pathogens, highlighting septins as a structural determinant of host defense. We also discuss how investigation of septin assembly in response to bacterial infection can yield insight into basic cellular processes including phagocytosis, autophagy, and mitochondrial dynamics.

This data is extracted from the Web of Science and reproduced under a licence from Thomson Reuters. You may not copy or re-distribute this data in whole or in part without the written consent of the Science business of Thomson Reuters.

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