Esther Bullitt, Ph.D.

Esther Bullitt, Ph.D.

Associate Professor

A.B. Grinnell College
Ph.D. Brandeis University

Phone: (617) 638-5037
Fax: (617) 638-4041
E-mail: bullitt@bu.edu
Address: see below
Link to BU Faculty Profile
Link to ORCID
Lab Website

 

 

 

 

 

 

Research

Protein Structure Facilitates Function: Using Electron Microscopy and Quantitative Image Analysis to see how Macromolecular Assemblies Work

Structural studies of biological macromolecular assemblies are providing an understanding of cellular function. In our laboratory, we utilize electron microscopy and image reconstruction to investigate questions about how adhesion pili aid pathogenic bacterial survival under harsh physiological conditions.

TYPE III SECRETION

MxiH_IpaDplusminusTEV_most
Type III Secretion System Needle Tips: A) Nascent needle tip with wild type IpaD B) Immature needle tip with only needle proteins C) Nascent needle tip (mesh) with distal domain of IpaD cleaved (green) D) Superposition of nascent (mesh) and immature (magenta) needle tips

Only 10 bacteria (or fewer!) are needed for Shigella flexneri to cause dysentery (bloody diarrhea). This disease is initiated via the type III secretion system, which is used to secrete both toxins and bacterial effectors that alter normal host cell functions to promote bacterial growth and spread.

IpaD_ribbon_confchange_and_segmented_EM_map
Distal Domain of IpaD is flipped up in nascent needle tip: A segmented map of the nascent needle tip shows that a conformational change is needed to fit the crystal structure (pdb 2j0o) into the EM density map


To understand (and disrupt) the process of infection, we are examining assembly intermediates of the T3SS syringe-like needle and its tip structure. Through our collaborations with the Picking lab at Oklahoma State University Stillwater and the Geisbrecht lab at the University of Missouri Kansas City we have shown that IpaD, the first protein that localizes to the needle tip, is present as a pentamer, and undergoes a dramatic conformational change as compared to the structure that has been solved by x-ray crystallography.

The structures of three-dimensional reconstructions of immature and nascent needle tips show clearly IpaD as an elongated pentameric structure.

This new result demonstrates that the distal domain of IpaD flips up, as compared to the structure solved by X-ray crystallography (pdb 2j0o).

Diagram to the right shows the conformational change of the distal domain that is needed to fit the crystal structure of IpaD into our density map of 3-dimensional reconstructions from electron microscopy data.

Epler, C.R., N.E. Dickenson, E. Bullitt*, W.L. Picking* (2012). Ultrastructural analysis of IpaD at the tip of the nascent MxiH type III secretion apparatus of Shigella flexneri. J. Mol. Biol. 420:29-39. PMID: 22480614 *corresponding authors


POLIOVIRUS REPLICATION

Schematic model of lattice formed by poliovirus 3Dpol (pdb 1rdr) superposed on an electron micrograph of negatively stained helical tubes, two-dimensional lattice, and twisted sheets of 3Dpol.

The poliovirus polymerase, 3Dpol, is an RNA-dependent RNA polymerase. Its structure is similar to other polymerases, and can be described as a right hand. We are investigating the role of two-dimensional 3Dpol lattices in replication, using electron microscopy and image processing of tubes and sheets formed in vitro. The crystal structure shown was solved in Steve Schultz’s lab, pdb 1rdr.

1ra6_fingerscoloredthenupdownIntIbkgwhite
Poliovirus Polymerase (1ra6) showing Interface I contacts: Arranged on a 21 screw axis, the first two pols are colored thumb blue, pinky pink, palm grey. Last four are palm down dark blue, palm up sky blue.

In collaboration with Dr. Karla Kirkegaard’s lab at Stanford University we have shown that

1) 3Dpol forms two-dimensional lattices that we expect facilitate replication by concentrating substrate and enzyme onto a two-dimensional surface, the vesicular membrane,

2) “Zombie” 3Dpol protein (dead active site) can support assembly of oligomers and restore replication when the wild type protein concentration is too low to do so.

Lyle, J.M.*, E. Bullitt*, K. Bienz, K. Kirkegaard (2002). Visualization and functional analysis of RNA-dependent RNA polymerase lattices. Science 296:2218-2222. *these authors contributed equally
PMID: 12077417

Spagnolo, J.F., E. Rossignol, E. Bullitt, K. Kirkegaard (2010). Enzymatic and non-enzymatic functions of viral RNA-dependent RNA polymerases within oligomeric arrays. RNA 16:382-393. PMCID: PMC2811667

Tellez, A.B*., J. Wang*, E.J. Tanner, J.F. Spagnolo, K. Kirkegaard+ and E. Bullitt+ (2011). “Interstitial Contacts in an RNA-Dependent RNA Polymerase Lattice.” J Mol Biol 412(4): 737-50. PMID: 21839092. http://www.ncbi.nlm.nih.gov/pubmed/21839092.
*these authors contributed equally, +corresponding authors

BACTERIAL ADHESION PILI

ecoli_18hr_001
Pathogenic bacteria express pili (also called ‘fimbriae’) on their surface for adhesion to their target cell: Shown here is an enterotoxigenic E. coli (ETEC) expressing CFA/I pili. Bacterium is ~ 1 µM by 3 µM, and pili are helical filaments ~8 nm in diameter and over 1 µM long

Fibers on the surfaces of many pathogenic bacteria can overextend like a toy Slinky, whereas homologous proteins on bacteria that cause pneumonia, ear infections, and meningitis assemble into a 3-stranded rope-like fibers (blue). In collaboration with Dr. Magnus Andersson’s lab at Umeå University in Umeå Sweden we have shown that the forces required for unwinding pili (also called ‘fimbriae’) are specific for the pilus-type, and thus vary with the microenvironment expected to be encountered. For example, CFA/I pili on diarrhea-causing intestinal bacteria unwind at less than one-third the force required to unwind P-pili expressed on urinary tract infection bacteria.

Our structural studies have shown that the mechanisms by which pili assemble on the bacterial outer membane include a loop region that allows rotation into a helical filament (P-pili) and, in collaboration with Capt. Stephen Savarino’s lab at the Naval Medical Reseach Center in Silver Spring, a proline trans to cis isomeration (CFA/I pili).

As more bacteria become resistant to antibiotics, it is essential to develop novel approaches for prevention and treatment of infection. Structural studies are vital for discovering clues to how bacteria bind, and how they remain attached while the host is trying to remove them. We are examining the architecture of bacterial adhesion pili and investigating small molecules that disrupt their assembly and/or function.

CfaB_fit_in_CFA1_SupplFigure_B
Three-dimensional reconstruction of CFA/I pili (blue mesh and yellow density) with fit of CfaB pilin (pdb 3f85)
ComparePili_Feb2010_b
Structures of bacterial adhesion pili are optimized for their target microenvironment

Bullitt, E., L. Makowski (1995). Structural polymorphism of bacterial adhesion pili. Nature 373:164-167. PMID: 7816100

Mu, X.Q. and E. Bullitt (2006). Structure and assembly of P-pili: a protruding hinge region used for assembly of a bacterial adhesion filament. Proc. Natl. Acad. Sci. USA 103:9861-9866. PMCID: PMC1502544

Mu, X.Q., S.J. Savarino, E. Bullitt (2008). The three-dimensional structure of CFA/I adhesion pili: Traveler’s Diarrhea Bacteria Hang on by a Spring. J. Mol. Biol. 376:614-620. PMCID: PMC2265596

Li, Y-F, S. Poole, K. Nishio, K. Jang, F. Rasulova, A. McVeigh, S.J. Savarino, D. Xia, E. Bullitt (2009) Structure of CFA/I fimbriae from enterotoxigenic Escherichia coli. Proc. Natl. Acad. Sci. USA 106: 10793-10798. PMCID: PMC2705562

Andersson M, O. Björnham, M. Svantesson, A. Badahdah, B.E. Uhlin, E. Bullitt (2012). A Structural Basis for Sustained Bacterial Adhesion: Biomechanical Properties of CFA/I Pili. J. Mo.l Biol. 415: 918-928. PMCID: PMC3267891


Selected Publications

Andersson M, Björnham O, Svantesson M, Badahdah A, Uhlin BE, Bullitt E. (2012). A Structural Basis for Sustained Bacterial Adhesion: Biomechanical Properties of CFA/I Pili., J Mol Biol. 2012 Feb 3;415(5):918-28.

Tellez AB, Wang J, Tanner EJ, Spagnolo JF, Kirkegaard K, Bullitt E. (2011). Interstitial contacts in an RNA-dependent RNA polymerase lattice. J Mol Biol. 2011 Sep 30;412(4):737-50.

Li, Y-F, S. Poole, K. Nishio, K. Jang, F. Rasulova, A. McVeigh, S.J. Savarino, D. Xia, E. Bullitt (2009). Structure of CFA/I fimbriae from enterotoxigenic Escherichia coli. Proc. Natl. Acad. Sci. 106:10793-10798.

Mu X.Q., S.J. Savarino, E. Bullitt (2008). The three-dimensional structurel of CFA/I adhesion pili: Traveler’s diarrhea bacteria hang on by a spring. J. Mol. Biol. 376:614-620.

Verger, D., E. Bullitt, S.J. Hultgren, G. Waksman (2007). Crystal structure of the P pilus rod subunit PapA. PLoS Pathogens 3:e73(674-682).

Mu XQ and E. Bullitt (2006). Structure and assembly of P-pili: a protruding hinge region used for assembly of a bacterial adhesion filament. Proc. Natl. Acad. Sci.103:9861-9866.

Mu X.Q., E.H. Egelman, E. Bullitt (2002). Structure and Function of Hib Pili from Haemophilus influenzae Type b. J Bacteriol. 184:4868-74.

Lyle J.M., E. Bullitt, K. Bienz, K. Kirkegaard (2002). Visualization and functional analysis of RNA-dependent RNA polymerase lattices. Science. Jun 21;296(5576):2218-22.

Bullitt E., M.P. Rout, J.V. Kilmartin, C.W. Akey(1997). The yeast spindle pole body is assembled around a central crystal of Spc42p. Cell Jun 27;89(7):1077-86.

Bullitt E, L. Makowski (1995). Structural polymorphism of bacterial adhesion pili. Nature Jan 12;373(6510):164-7.

Please click here for a complete list of citations on PubMed

Contact Us

Department of Physiology and Biophysics
Boston University School of Medicine
700 Albany Street
Boston MA 02118-2526

Phone: (617) 638-5037
Fax: (617) 638-4041
e-mail: bullitt@bu.edu

Directory|BUMC
February 21, 2014
Primary teaching affiliate
of BU School of Medicine