R A Pfuetzner

University of British Columbia - Vancouver, Vancouver, British Columbia, Canada

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Publications (32)385.58 Total impact

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    ABSTRACT: The T3SS injectisome is a syringe-shaped macromolecular assembly found in pathogenic Gram-negative bacteria that allows for the direct delivery of virulence effectors into host cells. It is composed of a "basal body", a lock-nut structure spanning both bacterial membranes, and a "needle" that protrudes away from the bacterial surface. A hollow channel spans throughout the apparatus, permitting the translocation of effector proteins from the bacterial cytosol to the host plasma membrane. The basal body is composed largely of three membrane-embedded proteins that form oligomerized concentric rings. Here, we report the crystal structures of three domains of the prototypical Salmonella SPI-1 basal body, and use a new approach incorporating symmetric flexible backbone docking and EM data to produce a model for their oligomeric assembly. The obtained models, validated by biochemical and in vivo assays, reveal the molecular details of the interactions driving basal body assembly, and notably demonstrate a conserved oligomerization mechanism.
    PLoS Pathogens 04/2013; 9(4):e1003307. · 8.14 Impact Factor
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    ABSTRACT: Genome-wide association studies can identify common differences that contribute to human phenotypic diversity and disease. When genome-wide association studies are combined with approaches that test how variants alter physiology, biological insights can emerge. Here, we used such an approach to reveal regulation of cell death by the methionine salvage pathway. A common SNP associated with reduced expression of a putative methionine salvage pathway dehydratase, apoptotic protease activating factor 1 (APAF1)-interacting protein (APIP), was associated with increased caspase-1-mediated cell death in response to Salmonella. The role of APIP in methionine salvage was confirmed by growth assays with methionine-deficient media and quantitation of the methionine salvage substrate, 5'-methylthioadenosine. Reducing expression of APIP or exogenous addition of 5'-methylthioadenosine increased Salmonellae-induced cell death. Consistent with APIP originally being identified as an inhibitor of caspase-9-dependent apoptosis, the same allele was also associated with increased sensitivity to the chemotherapeutic agent carboplatin. Our results show that common human variation affecting expression of a single gene can alter susceptibility to two distinct cell death programs. Furthermore, the same allele that promotes cell death is associated with improved survival of individuals with systemic inflammatory response syndrome, suggesting a possible evolutionary pressure that may explain the geographic pattern observed for the frequency of this SNP. Our study shows that in vitro association screens of disease-related traits can not only reveal human genetic differences that contribute to disease but also provide unexpected insights into cell biology.
    Proceedings of the National Academy of Sciences 07/2012; 109(35):E2343-52. · 9.81 Impact Factor
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    ABSTRACT: Salmonella enterica species are exposed to envelope stresses due to their environmental and infectious lifestyles. Such stresses include amphipathic cationic antimicrobial peptides (CAMPs), and resistance to these peptides is an important property for microbial virulence for animals. Bacterial mechanisms used to sense and respond to CAMP-induced envelope stress include the RcsFCDB phosphorelay, which contributes to survival from polymyxin B exposure. The Rcs phosphorelay includes two inner membrane (IM) proteins, RcsC and RcsD; the response regulator RcsB; the accessory coregulator RcsA; and an outer membrane bound lipoprotein, RcsF. Transcriptional activation of the Rcs regulon occurred within minutes of exposure to CAMP and during the first detectable signs of CAMP-induced membrane disorder. Rcs transcriptional activation by CAMPs required RcsF and preservation of its two internal disulfide linkages. The rerouting of RcsF to the inner membrane or its synthesis as an unanchored periplasmic protein resulted in constitutive activation of the Rcs regulon and RcsCD-dependent phosphorylation. These findings suggest that RcsFCDB activation in response to CAMP-induced membrane disorder is a result of a change in structure or availability of RcsF to the IM signaling constituents of the Rcs phosphorelay.
    Journal of bacteriology 10/2010; 192(19):4894-903. · 3.94 Impact Factor
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    ABSTRACT: The type III secretion system (T3SS) is an interspecies protein transport machine that plays a major role in interactions of Gram-negative bacteria with animals and plants by delivering bacterial effector proteins into host cells. T3SSs span both membranes of Gram-negative bacteria by forming a structure of connected oligomeric rings termed the needle complex (NC). Here, the localization of subunits in the Salmonella enterica serovar Typhimurium T3SS NC were probed via mass spectrometry-assisted identification of chemical cross-links in intact NC preparations. Cross-links between amino acids near the amino terminus of the outer membrane ring component InvG and the carboxyl terminus of the inner membrane ring component PrgH and between the two inner membrane components PrgH and PrgK allowed for spatial localization of the three ring components within the electron density map structures of NCs. Mutational and biochemical analysis demonstrated that the amino terminus of InvG and the carboxyl terminus of PrgH play a critical role in the assembly and function of the T3SS apparatus. Analysis of an InvG mutant indicates that the structure of the InvG oligomer can affect the switching of the T3SS substrate to translocon and effector components. This study provides insights into how structural organization of needle complex base components promotes T3SS assembly and function.
    mBio 01/2010; 1(3). · 6.88 Impact Factor
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    ABSTRACT: The type III secretion system (T3SS) is a macromolecular 'injectisome' that allows bacterial pathogens to transport virulence proteins into the eukaryotic host cell. This macromolecular complex is composed of connected ring-like structures that span both bacterial membranes. The crystal structures of the periplasmic domain of the outer membrane secretin EscC and the inner membrane protein PrgH reveal the conservation of a modular fold among the three proteins that form the outer membrane and inner membrane rings of the T3SS. This leads to the hypothesis that this conserved fold provides a common ring-building motif that allows for the assembly of the variably sized outer membrane and inner membrane rings characteristic of the T3SS. Using an integrated structural and experimental approach, we generated ring models for the periplasmic domain of EscC and placed them in the context of the assembled T3SS, providing evidence for direct interaction between the outer membrane and inner membrane ring components and an unprecedented span of the outer membrane secretin.
    Nature Structural & Molecular Biology 05/2009; 16(5):468-76. · 11.90 Impact Factor
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    ABSTRACT: The Rho family of guanosine triphosphatases (GTPases) are essential eukaryotic signaling molecules that regulate cellular physiology. Virulence factors from various pathogens alter the signaling of GTPases by acting as GTPase activating factors, guanine nucleotide exchange factors, or direct covalent modifiers; however, bacterial virulence factors that sense rather than alter the signaling states of Rho GTPases have not been previously described. Here, we report that the translocated Salmonellae virulence factor SseJ binds to the guanosine triphosphate-bound form of RhoA. This interaction stimulates the lipase activity of SseJ, which results in the esterification of cholesterol in the host cell membrane. Our results suggest that the activation of molecules downstream of GTPases is not exclusive to eukaryotic proteins, and that a bacterial protein has evolved to recognize the activation state of RhoA, which regulates its enzymatic activity as part of the host-pathogen interaction.
    Science Signaling 01/2009; 2(95):ra71. · 7.65 Impact Factor
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    ABSTRACT: Bacterial virulence mechanisms are attractive targets for antibiotic development because they are required for the pathogenesis of numerous global infectious disease agents. The bacterial secretion systems used to assemble the surface structures that promote adherence and deliver protein virulence effectors to host cells could comprise one such therapeutic target. In this study, we developed and performed a high-throughput screen of small molecule libraries and identified one compound, a 2-imino-5-arylidene thiazolidinone that blocked secretion and virulence functions of a wide array of animal and plant Gram-negative bacterial pathogens. This compound inhibited type III secretion-dependent functions, with the exception of flagellar motility, and type II secretion-dependent functions, suggesting that its target could be an outer membrane component conserved between these two secretion systems. This work provides a proof of concept that compounds with a broad spectrum of activity against Gram-negative bacterial secretion systems could be developed to prevent and treat bacterial diseases.
    Cell host & microbe 11/2008; 4(4):325-36. · 13.02 Impact Factor
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    ABSTRACT: Protein−protein interactions are key to function and regulation of many biological pathways. To facilitate characterization of protein−protein interactions using mass spectrometry, a new data acquisition/analysis pipeline was designed. The goal for this pipeline was to provide a generic strategy for identifying cross-linked peptides from single LC/MS/MS data sets, without using specialized cross-linkers or custom-written software. To achieve this, each peptide in the pair of cross-linked peptides was considered to be “post-translationally” modified with an unknown mass at an unknown amino acid. This allowed use of an open-modification search engine, Popitam, to interpret the tandem mass spectra of cross-linked peptides. False positives were reduced and database selectivity increased by acquiring precursors and fragments at high mass accuracy. Additionally, a high-charge-state-driven data acquisition scheme was utilized to enrich data sets for cross-linked peptides. This open-modification search based pipeline was shown to be useful for characterizing both chemical as well as native cross-links in proteins. The pipeline was validated by characterizing the known interactions in the chemically cross-linked CYP2E1−b5 complex. Utility of this method in identifying native cross-links was demonstrated by mapping disulfide bridges in RcsF, an outer membrane lipoprotein involved in Rcs phosphorelay.
    Analytical Chemistry 10/2008; 80(22). · 5.82 Impact Factor
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    ABSTRACT: Type III secretion systems (TTSSs) are multi-protein macromolecular 'machines' that have a central function in the virulence of many Gram-negative pathogens by directly mediating the secretion and translocation of bacterial proteins (termed effectors) into the cytoplasm of eukaryotic cells. Most of the 20 unique structural components constituting this secretion apparatus are highly conserved among animal and plant pathogens and are also evolutionarily related to proteins in the flagellar-specific export system. Recent electron microscopy experiments have revealed the gross 'needle-shaped' morphology of the TTSS, yet a detailed understanding of the structural characteristics and organization of these protein components within the bacterial membranes is lacking. Here we report the 1.8-A crystal structure of EscJ from enteropathogenic Escherichia coli (EPEC), a member of the YscJ/PrgK family whose oligomerization represents one of the earliest events in TTSS assembly. Crystal packing analysis and molecular modelling indicate that EscJ could form a large 24-subunit 'ring' superstructure with extensive grooves, ridges and electrostatic features. Electron microscopy, labelling and mass spectrometry studies on the orthologous Salmonella typhimurium PrgK within the context of the assembled TTSS support the stoichiometry, membrane association and surface accessibility of the modelled ring. We propose that the YscJ/PrgK protein family functions as an essential molecular platform for TTSS assembly.
    Nature 07/2005; 435(7042):702-7. · 38.60 Impact Factor
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    Journal of Biomolecular NMR 05/2005; 31(4):371-2. · 2.85 Impact Factor
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    ABSTRACT: Human enteropathogenic Escherichia coli (EPEC), enterohemorrhagic E. coli (EHEC), and the mouse pathogen Citrobacter rodentium (CR) belong to the family of attaching and effacing (A/E) bacterial pathogens. They possess the locus of enterocyte effacement (LEE) pathogenicity island, which encodes a type III secretion system. These pathogens secrete a number of proteins into culture media, including type III effector proteins and translocators that are required for the translocation of effectors into host cells. Preliminary evidence indicated that the LEE-encoded SepL and Rorf6/SepD may form a molecular switch that controls the secretion of translocators and effectors in CR. Here, we show that SepL and SepD indeed perform this function in A/E pathogens such as EHEC and EPEC. Their sepL and sepD mutants do not secrete translocators but exhibit enhanced secretion of effectors. We demonstrate that SepL and SepD interact with each other and that both SepL and SepD are localized to the bacterial membranes. Furthermore, we demonstrate that culture media influence the type III secretion profile of EHEC, EPEC, and CR and that low-calcium concentrations inhibit secretion of translocators but promote the secretion of effectors, similar to effects on type III secretion by mutations in sepL and sepD. However, the secretion profile of the sepD and sepL mutants is not affected by these culture conditions. Collectively, our results suggest that SepL and SepD not only are necessary for efficient translocator secretion in A/E pathogens but also control a switch from translocator to effector secretion by sensing certain environmental signals such as low calcium.
    Infection and Immunity 05/2005; 73(4):2135-46. · 4.07 Impact Factor
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    ABSTRACT: The ability to translocate virulence proteins into host cells through a type III secretion apparatus (TTSS) is a hallmark of several Gram-negative pathogens including Shigella, Salmonella, Yersinia, Pseudomonas, and enteropathogenic Escherichia coli. In common with other types of bacterial secretion apparatus, the assembly of the TTSS complex requires the preceding formation of its integral outer membrane secretin ring component. We have determined at 1.5 Å the structure of MxiM28–142, the Shigella pilot protein that is essential for the assembly and membrane association of the Shigella secretin, MxiD. This represents the first atomic structure of a secretin pilot protein from the several bacterial secretion systems containing an orthologous secretin component. A deep hydrophobic cavity is observed in the novel 'cracked barrel' structure of MxiM, providing a specific binding domain for the acyl chains of bacterial lipids, a proposal that is supported by our various lipid/MxiM complex structures. Isothermal titration analysis shows that the C-terminal domain of the secretin, MxiD525–570, hinders lipid binding to MxiM.
    The EMBO Journal 03/2005; 24(6):1111-1121. · 9.82 Impact Factor
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    Journal of Biomolecular NMR 01/2005; 31:371-372. · 2.85 Impact Factor
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    ABSTRACT: Several Gram-negative bacterial pathogens have evolved a type III secretion system to deliver virulence effector proteins directly into eukaryotic cells, a process essential for disease. This specialized secretion process requires customized chaperones specific for particular effector proteins. The crystal structures of the enterohemorrhagic Escherichia coli O157:H7 Tir-specific chaperone CesT and the Salmonella enterica SigD-specific chaperone SigE reveal a common overall fold and formation of homodimers. Site-directed mutagenesis suggests that variable, delocalized hydrophobic surfaces observed on the chaperone homodimers are responsible for specific binding to a particular effector protein. Isothermal titration calorimetry studies of Tir-CesT and enzymatic activity profiles of SigD-SigE indicate that the effector proteins are not globally unfolded in the presence of their cognate chaperones.
    Nature Structural Biology 01/2002; 8(12):1031-6.
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    ABSTRACT: LexA repressor undergoes a self-cleavage reaction. In vivo, this reaction requires an activated form of RecA, but it occurs spontaneously in vitro at high pH. Accordingly, LexA must both allow self-cleavage and yet prevent this reaction in the absence of a stimulus. We have solved the crystal structures of several mutant forms of LexA. Strikingly, two distinct conformations are observed, one compatible with cleavage, and the other in which the cleavage site is approximately 20 A from the catalytic center. Our analysis provides insight into the structural and energetic features that modulate the interconversion between these two forms and hence the rate of the self-cleavage reaction. We suggest RecA activates the self-cleavage of LexA and related proteins through selective stabilization of the cleavable conformation.
    Cell 10/2001; 106(5):585-94. · 31.96 Impact Factor
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    ABSTRACT: LexA repressor undergoes a self-cleavage reaction. In vivo, this reaction requires an activated form of RecA, but it occurs spontaneously in vitro at high pH. Accordingly, LexA must both allow self-cleavage and yet prevent this reaction in the absence of a stimulus. We have solved the crystal structures of several mutant forms of LexA. Strikingly, two distinct conformations are observed, one compatible with cleavage, and the other in which the cleavage site is ∼20 Å from the catalytic center. Our analysis provides insight into the structural and energetic features that modulate the interconversion between these two forms and hence the rate of the self-cleavage reaction. We suggest RecA activates the self-cleavage of LexA and related proteins through selective stabilization of the cleavable conformation.
    Cell. 01/2001; 106(5):585-594.
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    ABSTRACT: Intimin and its translocated intimin receptor (Tir) are bacterial proteins that mediate adhesion between mammalian cells and attaching and effacing (A/E) pathogens. Enteropathogenic Escherichia coli (EPEC) causes significant paediatric morbidity and mortality world-wide. A related A/E pathogen, enterohaemorrhagic E. coli (EHEC; O157:H7) is one of the most important food-borne pathogens in North America, Europe and Japan. A unique and essential feature of A/E bacterial pathogens is the formation of actin-rich pedestals beneath the intimately adherent bacteria and localized destruction of the intestinal brush border. The bacterial outer membrane adhesin, intimin, is necessary for the production of the A/E lesion and diarrhoea. The A/E bacteria translocate their own receptor for intimin, Tir, into the membrane of mammalian cells using the type III secretion system. The translocated Tir triggers additional host signalling events and actin nucleation, which are essential for lesion formation. Here we describe the the crystal structures of an EPEC intimin carboxy-terminal fragment alone and in complex with the EPEC Tir intimin-binding domain, giving insight into the molecular mechanisms of adhesion of A/E pathogens.
    Nature 07/2000; 405(6790):1073-7. · 38.60 Impact Factor
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    ABSTRACT: Enteropathogenic Escherichia coli (EPEC) triggers a dramatic rearrangement of the host epithelial cell actin cytoskeleton to form an attaching and effacing lesion, or pedestal. The pathogen remains attached extracellularly to the host cell through the pedestal for the duration of the infection. At the tip of the pedestal is a bacterial protein, Tir, which is secreted from the bacterium into the host cell plasma membrane, where it functions as the receptor for an EPEC outer membrane protein, intimin [1]. Delivery of Tir to the host cell results in its tyrosine phosphorylation, followed by Tir-intimin binding. Tir is believed to anchor EPEC firmly to the host cell, although its direct linkage to the cytoskeleton is unknown. Here, we show that Tir directly binds the cytoskeletal protein alpha-actinin. alpha-Actinin is recruited to the pedestal in a Tir-dependent manner and colocalizes with Tir in infected host cells. Binding is mediated through the amino terminus of Tir. Recruitment of alpha-actinin occurs independently of Tir tyrosine phosphorylation. Recruitment of actin, VASP, and N-WASP, however, is abolished in the absence of this tyrosine phosphorylation. These results suggest that Tir plays at least three roles in the host cell during infection: binding intimin on EPEC; mediating a stable anchor with alpha-actinin through its amino terminus in a phosphotyrosine-independent manner; and recruiting additional cytoskeletal proteins at the carboxyl terminus in a phosphotyrosine-dependent manner. These findings demonstrate the first known direct linkage between extracellular EPEC, through the transmembrane protein Tir, to the host cell actin cytoskeleton via alpha-actinin.
    Current Biology 07/2000; 10(12):735-8. · 9.49 Impact Factor
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    ABSTRACT: Enteropathogenic Escherichia coli (EPEC) triggers a dramatic rearrangement of the host epithelial cell actin cytoskeleton to form an attaching and effacing lesion, or pedestal. The pathogen remains attached extracellularly to the host cell through the pedestal for the duration of the infection. At the tip of the pedestal is a bacterial protein, Tir, which is secreted from the bacterium into the host cell plasma membrane, where it functions as the receptor for an EPEC outer membrane protein, intimin [1]. Delivery of Tir to the host cell results in its tyrosine phosphorylation, followed by Tir–intimin binding. Tir is believed to anchor EPEC firmly to the host cell, although its direct linkage to the cytoskeleton is unknown. Here, we show that Tir directly binds the cytoskeletal protein α-actinin. α-Actinin is recruited to the pedestal in a Tir-dependent manner and colocalizes with Tir in infected host cells. Binding is mediated through the amino terminus of Tir. Recruitment of α-actinin occurs independently of Tir tyrosine phosphorylation. Recruitment of actin, VASP, and N-WASP, however, is abolished in the absence of this tyrosine phosphorylation. These results suggest that Tir plays at least three roles in the host cell during infection: binding intimin on EPEC; mediating a stable anchor with α-actinin through its amino terminus in a phosphotyrosine-independent manner; and recruiting additional cytoskeletal proteins at the carboxyl terminus in a phosphotyrosine-dependent manner. These findings demonstrate the first known direct linkage between extracellular EPEC, through the transmembrane protein Tir, to the host cell actin cytoskeleton via α-actinin.
    Current Biology 01/2000; 10(12):735-738. · 9.49 Impact Factor

Publication Stats

2k Citations
385.58 Total Impact Points

Institutions

  • 2000–2013
    • University of British Columbia - Vancouver
      • • Centre for Blood Research (CBR)
      • • Department of Biochemistry and Molecular Biology
      Vancouver, British Columbia, Canada
  • 2008–2012
    • University of Washington Seattle
      • • Department of Medicinal Chemistry
      • • Department of Immunology
      • • Department of Genome Sciences
      Seattle, WA, United States
  • 2005
    • The American Society for Biochemistry and Molecular Biology
      Fort Smith, Arkansas, United States
    • Universidad Nacional Autónoma de México
      • Department of Molecular Microbiology
      Ciudad de México, The Federal District, Mexico
  • 1988–1997
    • McMaster University
      • Department of Chemistry and Chemical Biology
      Hamilton, Ontario, Canada