Ingrid Swanson Pultz.
I finished my graduate work at Miller lab and moved to David Baker lab at Dept of Biochemistry, University of Washington.
Research at Miller lab: c-di-GMP signaling in Salmonella Typhimurium. Cyclic diguanylic acid (c-di-GMP) is an important bacterial second messenger that controls a number of important bacterial processes such as motility, exopolysaccharide production, adherence to surfaces, the cell cycle, antibiotic resistance, and virulence. My thesis project involves studying the mechanisms by which c-di-GMP exerts its effects in Salmonella Typhimurium. In addition, I am working on developing new tools to measure the intracellular concentrations of c-di-GMP.
Pultz IS, Christen M, Kulasekara HD, Kennard A, Kulasekara B, Miller SI. Mol Microbiol. 2012 Dec;86(6):1424-40.
2011 Cell Microbiol – Mills E*, Pultz IS*,, Kulasekara HD, Miller SI. “The bacterial second messenger c-di-GMP: mechanisms of signaling.” Review discussing the diversity of c-di-GMP-metabolizing enzymes and mechanisms of action of this second messenger. *These two authors contributed equally to this work.
2008-2011 Founded the University of Washington International Genetically Engineered Machine (iGEM) team in 2008, and acted as their advisor for the 2008, 2009, 2010, and 2011 teams. The 2010 won Best Health or Medicine Project at the iGEM Jamboree. Visit their wiki here!
2010 PNAS – Costa KC, Wong PM, Wang T, Lie TJ, Dodsworth JA, Swanson I, Burn JA, Hackett M, Leigh JA ”Protein complexing in a methanogen suggests electron bifurcation and electron delivery from formate to heterodisulfide reductase.” Work from my second graduate student rotation (in the lab of Dr. John Leigh) published, studying how energy conservation occurs in a model archaea.
2007 Awarded a National Science Foundation Graduate Research Fellowship (NSF GRFP).
2005 As an ARCS Foundation scholar, joined the Department of Microbiology at the University of Washington in the Fall of 2005.
2006 J Virol – Swanson I, Jude BA, Zhang AR, Pucker A, Smith ZE, Golovkina TV ”Sequences within the gag gene of mouse mammary tumor virus needed for mammary gland cell transformation.” Although technically employed as a lab tech, I underwent intensive graduate-student-like training in the lab of Tanya Golovkina, studying how the Mouse Mammary Tumor Virus Gag protein effects tumor biogenesis in mice.
University: I always loved molecular biology, and this matured at Wellesley College where I took courses in molecular and cellular biology, biochemistry, organic chemistry, and microbiology courses – and graduated cum laude with a degree in Biology.
Dennis Ko, MD. PhD.
Beginning September 1, 2012, I will be continuing my studies of human variation in host-pathogen interactions as an Assistant Professor at Duke University. The appointment is in the Departments of Molecular Genetics & Microbiology (http://mgm.duke.edu/), Medicine (Infectious Disease), and the Center for Human Genome Variation (http://humangenome.duke.edu/). I can be contacted at denniscko [ a t ] gmail.com
The goals of my project, titled Hi-HOST for human in vitro susceptibility testing, are to 1) identify aspects of bacterial infection in human cells that exhibit heritable variation, 2) determine the genetic changes that are responsible, and 3) assess the relevance of this variation on the fitness of whole organisms.Using cells derived from hundreds of normal individuals, we have measured the naturally occurring variation for several intermediate phenotypes of susceptibility. Family-based association analyses are being used to correlate values from these assays with SNPs on a genome-wide scale. Causation of the identified SNPs is being established by a combination of expression analysis, RNA interference, overexpression experiments, pharmacologic inhibition, and mechanistic studies tailored to the individual SNPs. Finally, relevance to health and disease is being measured using association analysis of clinical phenotypes and phenotypic measurements of knockout and transgenic mice.
Articles employing the Hi-HOST screening approach:
- Ko DC, Gamazon ER, Shukla KP, Pfuetzner RA, Whittington D, Holden TD, Brittnacher MJ, Fong C, Radey M, Ogohara C, Stark AL, Akey JM, Dolan ME, Wurfel MM, Miller SI. (2012) A functional genetic screen of human diversity reveals a methionine salvage enzyme regulates inflammatory cell death. PNAS (in press).
- Ko DC, Shukla KP, Fong C, Wasnick M, Brittnacher MJ, Wurfel MM, Holden TD, O’Keefe GE, Van Yserloo B, Akey JM, Miller SI. (2009) A genome-wide in vitro bacterial-infection screen reveals human variation in the host response associated with inflammatory disease. Am J Hum Genet. 85(2):214-27.
Bioinformatics tool developed for analysis:
- Fong C, Ko DC, Wasnick M, Radey M, Miller SI, Brittnacher M. (2010) GWAS Analyzer: integrating genotype, phenotype and public annotation data for genome-wide association study analysis. Bioinformatics. 26(4):560-4.
Michael Jacobs, PhD.
Dr. Jacobs recently moved to Illumina Inc.
My research program is dedicated to describing the variation among bacterial isolates through whole genome sequencing. We use the latest technology as it becomes available, and currently use the Illumina GAIIx and HiSeq2000 platforms, the Life Sciences Ion Torrent, and the ABI 3730xl sequencing platforms. Our deep experience in whole genome sequencing and finishing is leveraged by the bioinformatics expertise of my collaborators. We have gone in a few short years from sequencing one genome at a time to dozens, and soon hundreds.
We are currently studying the evolution of a clonal epidemic of S. dysenteriae from the period of 1968 to 1992 in central America, in-patient diversification and selection of B. pseudomallei and the diversity found in non-typhoidal Salmonellae.
Karol, K.G., Jacobs MA, Zhou Y, Sims EH, Gillett WD, Cattolico RA. 2010. Comparative analysis of complete mitochondrial genome sequences from two geographically distinct Heterosigma akashiwo
(Raphidophyceae) strains. Proceedings of the Seventh International Chrysophyte Symposium. 261-282.
Kulasekara BR, Jacobs MA, Zhou Y, Wu Z, Sims E, Saenfigphimmachak C, Rohmer L, Ritchie JM, Radey M, McKevitt M, Freeman TL, Hayden HS, Haugen E, Gillett W, Fong C, Chang J, Beskhlebnaya V, Waldor MK, Samadpour M, Whittam TS, Kaul R, Olson MV, Brittnacher M, Miller SI. 2009. The complete genome sequence of the outbreak enterohemorrhagic Escherichia coli isolate TW14359 identifies genetic factors that may enhance virulence. Infec. And Immun. 77(9):3713-3721.
Hayden HS, Gillett W, Saenphimmachak C, Lim R., Zhou Y, Jacobs MA, Chang J, Rohmer L, D’Argenio DA, Palmieri A, Levy R, Haugen E, Wong GK, Brittnacher MJ, Burns JL, Miller SI, Olson MV, Kaul R. 2008. Large-insert genome analysis technology detects structural variation in Pseudomonas aeruginosa clinical strains from cystic fibrosis patients. Genomics. 291(6):530-7. Epub 2008 Apr 29.
Cattolico RA, Jacobs MA, Zhou Y, Chang J, Duplessis M, Lybrand T, McKay J, Ong HC, 2, Sims EH, Rocap G. 2008. Chloroplast genome sequence analysis using a fosmid cloning approach: Analysis of Heterosigma akashiwo CCMP452 (West Atlantic) and NIES 293 (West Pacific) strains. BMC Genomics. 9:211 (1-20).
Jacobs, MA. 2007. How to Make a Defined Near-Saturation Mutant Library. Case 1: Pseudomonas aeruginosa PAO1. Methods Mol Biol. 416: Microbial Gene Essentiality Eds. Osterman AL, Gerdes, SY Humana Press, Totowa NJ. Pgs. 133-52.
Jacobs M. and Manoil C. 2006. P. aeruginosa comprehensive mutant library, in: Pseudomonas IV, eds. Ramos and Levesque, Plenum Press, New York.
Miller DG, Trobridge GD, Petek LM, Jacobs MA, Kaul R, Russell DW. 2005. Large-scale analysis of adeno-associated virus vector integration sites in normal human cells. J Virol. 79(17):11434-42.
Wu, L., Estrada, O., Zaborina, O., Bains, M., Shen, L., Kohler, J. E., Patel, N., Musch, M. W., Chang, E. B., Fu, Y.-X., Jacobs, M. A., Nishimura, M. I., Hancock, R. E. W., Turner, J. R., Alverdy, J. C. 2005. Recognition of Host Immune Activation by Pseudomonas aeruginosa. Science 2005 309: p. 774-777.
Jacobs MA, Alwood A, Thaipisuttikul I, Spencer D, Haugen E, Ernst S, Will O, Kaul R, Raymond C, Levy R, Chun-Rong L, Guenthner D, Bovee D, Olson MV, Manoil C. 2003. Comprehensive transposon mutant library of Pseudomonas aeruginosa. Proc Natl Acad Sci U S A. 100(24):14339-44.
Richard moved to Axel Brunger lab at Standford.
Education: B.Sc McMaster University, Hamilton, Ontario, Canada
1992-1996- Aled M Edwards lab, McMaster University, Hamilton, Ontario, Canada-Purification, characterization and crystallization of DNA binding proteins
1996-1998- Roderick Mackinnon lab, Rockefeller University, New York, NY-Purification, characterization and crystallization of a bacterial potassium channel.
1998-2004- Natalie Strynadka lab, University of British Columbia, Vancouver, BC Canada-Purification, characterization and crystallization of various proteins involved in bacterial pathogenesis.
Research Summary at Miller Lab:
The type III secretion system is central to the pathogenesis of many infectious gram negative bacteria. This complex is composed of oligomeric rings of protein subunits embedded in the inner membrane, outer membrane and periplasm of the bacterium. The “needle complex” has an extension beyond the surface of the bacterium resulting in a syringe like assembly that can transfer protein effectors from the cytoplasm of the bacterium directly to the cytoplasm of the host cell.
In collaboration with the Goodlett lab and Baker lab at the UW and the Strynadka lab at the University of British Columbia, we have mapped detailed, high resolution protein-protein interactions within the base of the type III secretion “needle complex” using a combination of chemical cross linking, mass spectrometry, protein crystallography, and molecular modeling. . We have made significant advances in working out the details of how this important organelle is assembled. In addition to the work on the needle complex structure, various aspects of effector protein structure and function are being studied as well.
Dr. Didier Hocquet recently moved back to Besancon.
Areas of interest:
- Resistance of Pseudomonas aeruginosa to antibiotics by production of β-lactamases and efflux pumps. Characterization of new enzymes, detection of resistance mechanisms in the clinical laboratory, epidemiology.
- Adaptation of Pseudomonas aeruginosa during the infection (current project, granted by a Marie Curie fellowship from the European Community). We are characterizing melanogenic clinical isolates of P. aeruginosa, that regularly emerge in chronically-infected patients, especially those suffering from cystic fibrosis. The genome sequencing of wild-type clinical isolates and their melanogenic derivatives will identify the missing genes and help to address the genetic mechanism by which the deletion occurs. The new properties of the mutants were assessed using phenotypic microarrays (Biolog, Inc.).
Publications: link to Pubmed
Educational website (in French): Bacterioweb
Lisette Coye, Ph.D.
Research at Miller Lab: Salmonella Translocated Effectors
My research focuses on the SPI-2 effectors SseJ and SifA. SifA is a SPI-2 effector that is critical for intracellular survival in macrophages and for mouse virulence. Crystal structure analysis reveals that SifA has two domains. The amino terminal domain is known to bind the eukaryotic protein SKIP (SifA and kinesin interacting protein). SifA recruits SKIP to the SCV and SKIP in turn interacts with the motor protein kinesin linking SifA to the microtubular network. The carboxyl terminal domain of SifA has a guanine nucleotide exchange factor fold (GEF) similar to the SPI-1 effector SopE and binds RhoA-GDP. We have shown that the GEF domain is critical for intracellular replication. The focus of my research is to discover the GTPase targeted by SifA and further investigate the role of the GEF-like domain during intracellular infection.
SseJ is a SPI-2 effector that has glycerophospholipid :cholesterol acyltransferase (GCAT), phospholipase A1 and deacylase activity. A deletion of SseJ results in attenuation of virulence in mice and peritoneal macrophages. The eukaryotic signaling protein RhoA stimulates SseJ’s enzymatic activity resulting in an increase of intracellular cholesterol ester. My current research is to further investigate the role of SseJ during Salmonella infection.
Christen M.*, Coye LH.*, Hontz JS., LaRock D., Pfuetzner R., Megha, Miller SI. Activation of a bacterial virulence protein by the GTPase RhoA. Science Signaling. 2009 Nov: 2(95):ra71.* Co-first author
Dr. Itay Levin:
Dr. Levin recently moved back to Israel.
Research at Miller lab:
My main research focus is the function of the Salmonella typhimurium effector protein SspH2. SspH2 is a secreted toxin of the Salmonellapathogenicity island (SPI-2) type III secretion system of Salmonella enterica serovar Typhimurium, it is conserved in all pathogenic Salmonella species. Recently it has been reported that IpaH9.8, a Shigella SsPH2 homolog, have an E3 like ubiquitin ligase activity in vitro (Rohde et al Cell Host Microbe. 2007). To date the full machinery necessary for ubiquitination was found exclusively in Eukaryotes, which consistsof an enzyme cascade consisting of E1, E2 and E3 enzymes. Ubiquitination (Ub) can have several distinct effects on substrate proteins, the most common being degradation by the 26S proteasome. However, ubiquitination also regulates several other cellular processes, such as DNA repair, signaling, endocytosis, vesicular trafficking, and cell-cycle progression. Thus, since this class of E3s has a unique structure bacterial ubiquitin ligases might have a novel mechanism of ubiquitination but will have similar physiological effects. My research in collaboration with Dr. Peter Brzovic from Prof. Rachel Klevit’s group at the University of Washington has shown that although SspH2 utilize the mammalian ubiquitination machinery, its activity involves a novel mechanism; as opposed to mammalian E3s that bind E2s which are not charged with ubiquitin, SspH2 selectively binds the human E2 ∼ Ub conjugate recognizing regions of both the E2 (UbcH5c) and Ub surface. Surprisingly, intermediates in SspH2-directed reactions are activated poly-Ub chains directly tethered to the UbcH5 active site (UbcH5 ∼ Ubn) (Levin et al PNAS 2010). This mechanistic model was speculated but never demonstrated before for ubiquitin ligases.