Faculty

General Research Interests

My laboratory explores the molecular mechanisms by which enteric bacterial pathogens cause disease. More specifically, we examine the proteins secreted by important diarrheal pathogens in terms of their structure, function, and potential interactions with host cells.

Current Research

IpaC-Mediated Invasion of Epithelial Cells by Shigella
Gastrointestinal illnesses are a serious worldwide public health problem and Shigella flexneri is an important agent of infectious diarrhea. S. flexneri is a gram negative enteric bacterium that breaches the colonic epithelium via M cells to come into contact with subepithelial macrophages that attack the bacterium; however, S. flexneri escapes being killed killing by inducing macrophage apoptosis. These events provide S. flexneri with access to the basal region of the epithelial layer where it invades the epithelial cells. The resulting cytokine responses promote PMN infiltration and evoke a severe localized inflammation.

S. flexneri invasion of epithelial cells is characterized by induced actin polymerization within the host cell at the point of pathogen contact. Cytoskeletal rearrangements lead to the formation of filopodia that coalesce to form membrane ruffles that surround the pathogen and trap it in a membrane-bound vacuole. Genes required for the S. flexneri invasive phenotype include the ipa genes, which encode four secreted proteins called invasion plasmid antigens (Ipa) ABCD, and the mix/spa genes which encode a type III secretion system (TTSS). A major point of divergence in known TTSSs is the nature of the secreted targets. For Shigella, the Ipa proteins are the major targets of the Mxi/Spa TTSS, which delivers these proteins to the host-pathogen interface following pathogen contact.

Research in my laboratory is directed at identifying important functional and structural properties of purified IpaC. Following secretion, IpaB and IpaC form a complex that is responsible for pathogen entry into host cells. This complex interacts with host cells via the hyaluronidate receptor (CD44), which results in IpaC penetration of the host cytoplasmic membrane. IpaC then triggers the activation of a host GTPase called Cdc42, which triggers filopodia formation. We have generated a large number of IpaC mutants, which have been useful for defining the functional significance of different regions of IpaC. From these mutants, we now know that a unique sequence at the immediate N-terminus of IpaC is required for secretion by the Shigella TTSS. Next to this secretion signal is an apparent translocation domain that which allows formation of IpaB/IpaC complexes. Also near the N-terminus is a hydrophobic region that is believed to allow membrane penetration and vacuolar escape. At the C-terminus, a predicted coiled-coil domain may allow IpaC oligomerization, which may result in the generatation of a structure interacts with Cdc42 to trigger bacterial entry.

Representative Publications

• Birket, S., Harrington, A.T., Espina, M., Smith, N., Terry, C.M., Darboe, N., Markham, A.P., Middaugh, C.R., Picking, W.L., Middaugh, C.R., and Picking, W.D. (2007) Preparation and Characterization of Translocator/Chaperone Complexes and Their Component Proteins from Shigella flexneri. Biochemistry 46:8128-8137.
• Olive, A.J., Kenjale, R., Espina, M., Moore, D.S., Picking, W.L. and Picking, W.D. (2007) Bile salts stimulate recruitment of IpaB to the Shigella surface where it co-localizes with IpaD at the tip of the type III secretion needle. Infect. Immun. 75:2626-2629.
• Espina, M., Ausar, S.F., Middaugh, C.R., Baxter, M.A., Picking, W.D., and Picking, W.L. (2007) Conformational stability and differential structural analysis of LcrV, PcrV, BipD, and SipD from type III secretion systems. Prot. Sci. 16:704-714.
• Johnson, S., Roversi, P., Espina, M., Olive, A., Deane, J.E., Birket, S., Field, T., Picking, W.D., Blocker, A.J., Galyov, E.E., Picking, W.L. and Lea, S.M. (2007) Self-chaperoning of the type III secretion system needle tip proteins IpaD and BipD. J. Biol. Chem. 282:4035-4044.
• Deane, JE; Roversi, P; Cordes, FS; Johnson, S; Kenjale, R; Daniell, S; Booy, F; Picking, WD; Picking, WL; Blocker, AJ and Lea, SM. (2006) Molecular model of a Type Three Secretion System needle: implications for host-cell sensing. Proc. Natl. Acad. Sci. USA 103:12529-12533.
• Espina, M., Ausar, F.S., Picking, W.D., Middaugh, C.R., and Picking, W.L. (2006) Spectroscopic and calorimetric analyses of invasion plasmid antigen D (IpaD) from Shigella flexneri reveal the presence of two structural domains. Biochemistry 45:9219-9227.
• Espina, M., Olive, A.J., Kenjale, R., Moore, D.S., Ausar, S.F., Kaminski, R.W., Oaks, E.V., Middaugh, C.R., Picking, W.D., and Picking, W.L. (2006) IpaD localizes to the tip of the type III secretion system needle of Shigella flexneri. Infect. Immun., 74(8):4391-4400.
• Darboe, N., Kenjale, R., Picking, W.L., Picking, W.D., and Middaugh, C.R. (2006) Physical characterization of MxiH and PrgI, the needle component of the type III secretion apparatus from Shigella and Salmonella. Prot. Sci., 15:543-552.
• Zhang, L., Wang, Y., Picking, W.L., Picking, W. D., and De Guzman, R.N. (2006) Solution Structure of Monomeric BsaL, the Type III secretion Needle Protein of Burkholderia pseudomallei. J. Mol. Biol. 359:322-330.
• Harrington, A.T., Darboe, N., Picking, W.L., Middaugh, C.R., Birket, S., and Picking, W.D. (2006) Characterization of the Interaction of Single Tryptophan Containing Mutants of IpaC from Shigella flexneri with Phospholipid Membranes. Biochemistry, 45:626-636.
• Cordes, F.S., Daniell, S., Kenjale, R., Saurya, S., Picking, W.L., Picking, W.D., Booy, F., Lea, S.M., and Blocker, A. (2005) Helical packing of needles from functionally altered Shigella type III secretion systems. J. Mol. Biol., 354:206-11.
• Kenjale, R., Wilson, J., Zenk, S.F., Saurya, S., Picking, W.L., Picking, W.D., and Blocker, A. (2005) The needle component of the type III secreton of Shigella regulates the activity of the secretion apparatus, J. Biol. Chem., 280:42929-37.
• Picking, W.L., Nishioka, H., Hearn, P.D., Baxter, M.A., Harrington, A.T., Blocker, A., and Picking, W.D. (2005) IpaD of Shigella flexneri is independently required for regulation of Ipa protein secretion and efficient insertion of IpaB and IpaC into host membranes, Infect. Immun., 73:1432-1440. PMCID: 1064949
• Berring, E., Brancato, S., Grant, K., Schaper, E., Kadavil, S., Smagin, H., Hatic II, S.O., Picking, W., and Serfis. (2004) Destabilization of phospholipid model membranes by YplA, a phospholipase A2 secreted by Yersinia enterocolitica. Chem. Phys. Lipids 131:135-149.
• Harrington, A.T., Hearn, P.D., Picking, W.L., Barker, J.R., Wessel, A., Picking, W.D. (2003) Structural Characterization of the N-terminus of IpaC from Shigella flexneri. Infect. Immun., 71:1255-1264. PMCID: 148864
• Kueltzo, L.A., Osiecki, J., Barker, J., Picking, W.L., Wessel, A., Picking, W.D., and Middaugh, C.R. (2003) Structure-function analysis of invasion plasmid antigen C (IpaC) from Shigella flexneri. J. Biol. Chem., 278:2792-2798.
• Hatic II, S.O., Picking, W.L., Young, B.M., Young, G.M., and Picking, W.D. (2002) Comparison of the Activity of Recombinant Variants of YplA, the Secreted Phospholipase of Yersinia enterocolitica. Biochem. Biophys. Res. Commun. 292:463-467.
• Picking, W.D. (2001) Cholera Toxin. The Encyclopedia of Molecular Medicine. pp. 754-756. (Creighton, T.E., Ed.) Wiley Press, New York, NY.
• Osiecki, J.C., Barker, J., Picking, W.L., Barnoski Serfis, A., Berring, E., Shah, S., and Picking, W.D. (2001) IpaC from Shigella and SipC from Salmonella Possess Similar Biochemical Properties But Are Functionally Distinct. Mol. Microbiol., 42:469-481.
• Hatic II, S.O., McCann, J.A., and Picking, W.D. (2001) Reconstitution of Novel Cholera Toxin Homologues In Vitro. Analyt. Biochem., 292:171-177.
• Picking, W.L., Coye, L., Osiecki, J.C., Barnoski Serfis, A., Schaper, E., and Picking, W.D. (2001) Identification of Functional Regions within Invasion Plasmid Antigen C (IpaC) of Shigella flexneri. Mol. Microbiol., 39:100-111.
• Picking, W.D. (2000) The Use of Fluorescence Resonance Energy Transfer to Identify Conformational Changes in Protein Toxins, Methods in Molecular Biology: Protein Toxins, 145:133-146.
• Tran, N., Serfis, A.B., Davis, R., Osiecki, J.C., Coye, L., Picking, W.L., and Picking, W.D. (2000) Interaction of Shigella flexneri IpaC with Model Membranes Correlates with Extracellular Effects on Cultured Cells. Infect. Immun., 68:3710-3715. PMCID: 97663
• Picking, W.D. and McCann, J.A. (1999) The Influence of Environmentally-Induced Changes in the Conformation of the Cholera Toxin B Subunit on Membrane Structure. pp. 167-182. Membrane Structure in Disease and Drug Therapy, Marcel Dekker, Inc. New York, NY.
• Picking, W.D., McCann, J.A., Nutikka, A., and Lingwood, C.A. (1999) Localization of the Gb3 Binding Site of Verotoxin 1 by Fluorescence Analysis. Biochemistry, 38:7177-7184.
• Davis, R., Marquart, M.E., Lucius, D., and Picking, W.D. (1998) Protein-Protein Inter-actions in the Assembly of Shigella flexneri Invasion Plasmid Antigens IpaB and IpaC into Protein Complexes. Biochim. Biophys. Acta, 1429:45-56.
• McCann, J.A., Mertz, J.A., and Picking, W.D. (1997) Structural Changes in the Cholera Toxin B Subunit and GM1-Containing Membranes Are Elicited by Environmental pH. Biochemistry, 36:9169-9178.
• McCann, J.A. and Picking, W.D. (1997) Purification of Recombinant Cholera Toxin Polypeptide A2 and Reconstitution with the Cholera Toxin B Subunit. Prot. Pept. Lett. 4:39-46.
• Picking, W.L., Mertz, J.A., Marquart, M.E., and Picking, W.D. (1996) Cloning, Expression, and Affinity Purification of Recombinant Shigella flexneri Invasion Plasmid Antigens IpaB and IpaC. Prot. Expr. Purif. 8:401-408.
• Marquart, M.E., Picking, W.L., and Picking, W.D. (1996) Soluble Invasion Plasmid Antigen C (IpaC) from Shigella flexneri Elicits Epithelial Cell Responses Related to Pathogen Invasion. Infect. Immun. 64:4182-4187. PMCID: 174354
• Oaks, E.V., Picking, W.D., and Picking, W.L. (1996) Antibody Response of Monkeys to Invasion Plasmid Antigen D after Infection with Shigella spp. Clin. Diagn. Lab. Immunol. 3:242-245. PMCID: 170291
• Marquart, M., Picking, W.L. and Picking, W.D. (1995) Structural Analysis of Invasion Plasmid Antigen D (IpaD) from Shigella flexneri. Biochem. Biophys. Res. Commun. 214:963-970.
• Picking, W.L., Moon, H.-Y., Wu, H., and Picking, W.D. (1995) Fluorescence Analysis of the Interaction Between GM1-Containing Phospholipid Vesicles and the B Subunit of Cholera Toxin. Biochim. Biophys. Acta 1247:65-73.