Molecular and Cellular Regulation of the Mucosal Immune Response
and
  Molecular Pathogenesis of Bordetella spp.:
Bacteria for Childhood Respiratory Disease

Research efforts in my laboratory are focused both in Immunologyand Bacterial Pathogenesis, two diverse fields of biomedical research for which I have two separate research groups.  Projects in both fields are available for graduate students and post-doctoral fellows.      

Regulation of Mucosal Immune Responses. A major focus of my laboratory is to investigate the cellular and molecular events which modulate mucosal immune responses.  We have demonstrated that LT-IIa and LT-IIb, two Type II heat-labile enterotoxins of Escherichia coli, are potent oral and nasal immunogens that have the capacity for augmenting humoral and cellular immune responses (See Figure below).  Using a variety of immunological and cellular techniques (including flow cytometry, FRET fluorescent detection, cytokine multiplex analysis, mutagenesis, and the use of transgenic mice), we are evaluating the manners in which these two immunomodulators productively interact with various lymphoid cells such as T cells, B cells, and dendritic cells to induce or suppress cytokine production, co-stimulatory ligand expression, and proliferation.  A practical outgrowth of these experiments is the potential to engineer novel recombinant vaccines by genetically fusing antigens from different pathogens to the immunomodulatory enterotoxins. We have shown that upon nasal immunization, these genetically-fused proteins elicit protective immune responses in animal challenge models. Other fusion proteins being produced are incorporating antigens from Haemophilus influenzae, hepatitis virus, and tuberculosis in an effort to produce new and effective vaccines.

Iron acquisition in the Bordetellae. A second focus of my laboratory is to investigate the mechanisms by which Bordetella pertussis, B. bronchiseptica, and B. avium, three gram-negative bacteria that infect the upper respiratory tract of children and domesticated animals, acquire essential nutrients required to colonize the respiratory tract and to sustain progressive infections. Iron (Fe) is one of the most important nutrients required by most, if not all infectious bacteria which must be obtained from the tissues and fluids of the infected host. Using proteomic, recombinant, immunological, and biochemical methods, we are unraveling the genetic and molecular components of these bacteria that mediate acquisition of Fe from host-derived heme, myoglobin, and hemoglobin.  We have identified a cluster of genes (bhuRSTUV) which encodes five proteins required for uptake of heme and a second gene cluster (rhuIR) which encodes for two heme-dependent regulatory proteins which control heme-dependent expression of the uptake systems (See Figure below).  Our current research is focused on analysis of these seven genes and expressed proteins.

PROFESSIONAL ACTIVITIES:

Editorial Board, Infection and Immunity, The American Society

Reviewer for:
European Journal of Immunology, Microbial Pathogenesis, Gene, Journal of Bacteriology, BioTechniques, Immunological Investigations, Medical Science Monitor, Microbiology, Trends in Microbiology, Molecular Microbiology, Applied and Environmental Microbiology, Archives of Microbiology

Panelist:
Grant reviewpanel HIBP, Host Interactions with Bacterial Pathogens, Center for Scientific Review, National Institutes of Health

PUBLICATIONS:

Connell, T.D. and R.K. Holmes. 1995. Mutational analysis of the ganglioside-binding activity of the type II  Escherichia coli heat-labile enterotoxin LT_IIb. Molecular Microbiology 16:21-31.

Connell, T.D., D. Metzger, M. Wang, M.G. Jobling, and R.K. Holmes. 1995. Initial studies of the structural signal for extracellular transport of cholera toxin and other proteins recognized by Vibrio cholerae. Infection & Immunity 63:4091-8.

van den Akker, F., S. Sarfaty, E.M. Twiddy, T.D. Connell, R.K. Holmes, W.G.J. Hol. 1996. Crystal structure of a new heat-labile enterotoxin, LT-IIb. Structure 4:665-78.

Connell, T.D., D. Metzger, C. Sfintescu, and R.T. Evans. 1998. Immunostimulatory activity of LT_IIa, a type II heat-labile enterotoxin of Escherichia coli. Immunology Letters 62: 119-122.

Connell, T.D., A. Dickenson, A.J. Martone, K. Militello, M.J. Filiatraut, M.L. Hayman, and J. Pitula. (1998). Iron-starvation of Bordetella avium stimulates expression of five outer membrane proteins and regulates a gene involved in acquiring iron from serum. Infection & Immunity 66:3597-3605.

Connell, T.D., D. Metzger, J. Lynch, and J. Folster. (1998). Endochitinase is transported to the extracellular milieu by the eps-encoded General Secretory Pathway of Vibrio cholerae. J. Bacteriology 180:5591-5600.

Murphy, E.R., A. Dickenson, K. Militello, and T.D. Connell. 1999. Genetic characterization of wild type and mutant fur genes of Bordetella avium. Infection & Immunity. 67:3160_3165.

Martin, M., D.J. Metzger, S.M. Michalek, T.D. Connell, and M.W. Russell. 2000. Comparative analysis of the mucosal adjuvanticity of the Type II heat-labile enterotoxins LT-IIa and LT-IIb. Infection & Immunity 68:281-287.

Ali, A., J.A. Johnson, A.A. Franco, D. Metzger, T.D. Connell, J.G., Morris, Jr., and S. Sozhamannen. 2000. Mutations in the extracellular protein secretion pathway genes (eps) interfere with rugose polysaccharide production in and motility of Vibrio cholerae. Infection &Immunity 68:1967-74.

Martin, M., G. Hajishengallis, D. Metzger, S. Michalek, T.D. Connell, and R.W. Russell. 2001. Recombinant antigen_enterotoxin A2/B chimeric mucosal immunogens differentially enhance antibody responses and B7-dependent co-stimulation of CD4+ T cells. Infection & Immunity 69:252-261.

Martin, M., G. Hajishengallis, D. Metzger, S. Michalek, T.D. Connell, and M.W. Russell.2001. Distinct cytokine regulation by cholera toxin and type II heat-labile enterotoxins involves differential regulation of CD40 ligand on CD4+ T cells. Infection & Immunity 69:4486-92.

Kirby, A.E., D. J. Metzger, E.M. Murphy, and T.D. Connell. 2001. RhuI, an extracytoplasmic function sigma factor, regulates heme utilization in Bordetella avium. Infection & Immunity 69:6951-6961.

Folster, J. and T.D. Connell. 2002. The extracellular transport signal of the Vibrio cholerae endochitinase (ChiA) is a structural motif located between amino acids 75‑555. Journal of Bacteriology 184:2225‑2234.

Drahushuk, K., T. D. Connell, and D. Higgins.  2002. Pituitary adenylate cyclase activating peptide and vasoactive intestinal peptide inhibit dendritic growth in cultured sympathetic neurons.  Journal of Neuroscience 22:6560‑6569.

Murphy, E.R., R.E. Sacco, A. Dickenson, D.J. Metzger, Y. Hu, P.E. Orndorff, and T.D. Connell. 2002.  BhuR, an virulence‑associated outer membrane protein of Bordetella avium, is required for acquisition of heme and hemoproteins.  Infection & Immunity 70: 5390 5403.

Kirby, A.E. and T.D. Connell.  2004.  RhuR, an extracytoplasmic function (ECF) sigma factor activator, is essential for heme dependent expression of the outer membrane heme and hemoprotein receptor of Bordetella avium. Infection & Immunity 72:896‑907.

Hajishengallis, G., H. Nawar, R. Tapping, M.W. Russell, and T.D. Connell.  2004.  Cytokine induction and regulation by the Type II heat‑labile enterotoxins in human monocytic cells.  Infection & Immunity 72:6351-58.

Hajishengallis, G., R.I. Tapping, M. H. Martin, H. Nawar, E. A. Lyle, M. W. Russell, and T. D. Connell.  2005.  Toll-like receptor 2 mediates cellular activation by the B subunits of type II heat- labile enterotoxins.  Infection & Immunity.  73: 1343-1349.

King, N.D., A.E. Kirby, and T.D. Connell.  2005.  Transcriptional control of the rhuIR bhuRSTUV heme acquisition locus in Bordetella avium.  Infection & Immunity.  73: 1613-1624.

Nawar, H.F., S. Arce, M.W. Russell, and T.D. Connell.  2005. Mucosal adjuvant properties of mutant LT-IIa and LT-IIb enterotoxins that exhibit altered ganglioside-binding activities.      Infection & Immunity.  73: 1330-1342.

Arce, S., H.F. Nawar, M.W. Russell, and T.D. Connell.   2005. Differential binding of Escherichia coli enterotoxins LT-IIa and LT-IIb and of cholera toxin elicits differences in apoptosis, proliferation, and activation of lymphoid cells.  Infection & Immunity.  73:2718-27.

Hajishengallis, G., S. Arce, C. M. Gockel, T. D. Connell, and M.W. Russell.  2005.  Immunomodulation with enterotoxins for the generation of secretory immunity or tolerance: applications for oral infections. Crit. Rev. Oral Biol. Med. 84:1104-1116.

van Ginkel, F.W., R. J. Jackson, N. Yoshino, Y. Hagiwara, D.J. Metzger, T.D. Connell, H.L. Vu, M. Martin, K. Fujihashi, and J.R. McGhee.  2005.  Enterotoxin-based mucosal adjuvants alter antigen trafficking and induce inflammatory responses in the nasal tract. Infection & Immunity.  73: 6892-6902.

Sebaihia, M.,, J. Preston, D.J. Maskell, H. Kuzmiak, T.D. Connell, N.D. King, P.E. Orndorff, D.M. Miyamoto, N.R. Thomson, D.  Harris, A. Goble, A. Lord, L. Murphy, M.A. Quail, S. Rutter, R. Squares, S. Squares, J. Woodward, J. Parkhill, and L. M. Temple.  2006.  Comparison of the genome sequence of the poultry pathogen Bordetella avium with those of B. bronchiseptica, B. pertussis, and B. parapertussis reveals extensive diversity in surface structures associated with host interaction.  J. Bacteriology.  188:6002-15.

Nawar, H.F., S. Arce, M.W. Russell, and T.D. Connell.  2007.  Mutants of Type II heat-labile enterotoxin LT-IIa exhibiting altered ganglioside-binding activities and diminished toxicities are potent mucosal adjuvants.  Infection & Immunity  72: 621-33.
 
Arce, S., H.E. Nawar, G. Muehlinghaus, M.W. Russell, T.D Connell.  2007.  "In vitro induction of  IgA- and IgM-secreting cells by cholera toxin depends on T-cell help and is mediated by CD154 up-regulation".  Infection & Immunity 75:1413-23.
 
Shuang, L.,  M. Wang, R. I. Tapping, V. Stepensky, H.F. Nawar, M. Triantafilou, K. Triantafilou, T. D. Connell, and G. Hajishengallis.  2007.  Ganglioside GD1a is an essential coreceptor for Toll-like Receptor  2 signaling in response to the B subunit of Type IIb enterotoxin.  J. of Biological Chemistry 282:7532-42.
 
Shuang, L. S., M. Wang, K. Triantafilou, M. Triantafilou, H.F. Nawar, M.W. Russell, T.D. Connell, and G. Hajishengallis.  2007.  The A subunit of Type IIb enterotoxin (LT-IIb) suppresses the proinflammatory potential of the B subunit and its ability to recruit and interact with TLR2.  J. of Immunology 178: 4811-19.
 
King, N.D., K.F. Smith, and T.D. Connell.  2007.  "Expression of hurP, a gene encoding a prospective site 2 protease, is essential for heme-dependent induction of bhuR in Bordetella bronchiseptica."   J. of Bacteriology.  In press.
 
Mocny, J., J. Olson, and T.D.  Connell.  2007.  The role of spontaneous loss of heme from hemoglobin and myoglobin in iron acquisition by Bordetella bronchiseptica.  Infection & Immunity.  In press.
 
Connell, T.D.  2007.  "Cholera toxin, LT-I, LT-IIa, and LT-IIb: the critical role of ganglioside-binding in  immunomodulation by Type I and Type II heat-labile enterotoxins".  Expert Review of Vaccines.  (Invited review, peer reviewed).  In press.

Nawar HF. Arce S. Russell MW. Connell TD. Mutants of type II heat-labile enterotoxin LT-IIa with altered ganglioside-binding activities and diminished toxicity are potent mucosal adjuvants. Infeciton & Immunity. 75(2):621-33, 2007 Feb.

Arce S. Nawar HF. Muehlinghaus G. Russell MW. Connell TD. In vitro induction of immunoglobulin A (IgA) - and IgM-secreting plasma blasts by cholera toxin depends on T-cell help and is mediated by CD154 up-regulation and inhibition of gamma interferon synthesis. Infection and Immunity. 75(3):1413, 2007 Mar.

Liang S. Wang M. triantafilou K. Triantafilou M. Nawar HF. Russell MW. Connell TD. Hajishengallis G. The A subunit of type IIb enterotoxin (LT-IIb) suppresses the proinflammatory potential of the B subunit and its ability to recruit and interact with TLR2. Journal of Immunology. 178(8):4811-9, 2007 April 15.

Liang S. Wang M. Tapping RI. Stepensky V. Nawar HF. Triantafilou M. Triantafilou K. Connell TD. Hajishengallis G. Ganglioside GD1a is an essential coreceptor for Toll-like receptor 2 signaling in response to the B subunit of type IIb enterotoxin. Journal of Biological Chemistry. 282(10):7532-42, 2007 Mar 9.

Sebaihia M. Preston A. Maskell DJ. Kuzmiak H. Connell TD. King ND. Orndorff PE. Miyamoto DM. Thomson NR. Harris d. Goble A. Lord A. Murphy L. Quail MA. Rutter S. Squares R. Squares S. Woodward J. Parkhill J. Temple LM. Comparison of the genome sequence of the poultry pathogen Bordetella avium with those of B. bronchiseptica, B. pertussis, and B. parapertussis reveals extensive diversity in surface structures associated with host interaction. Journal of Bacteriology. 188(16):6002-15, 2006 Aug.

Mocny JC. Olson JS. Connell TD. Passively released heme from hemoglobin and myoglobin is a potential source of nutrient iron for Bordetella bronchiseptica. Infection & Immunity. 75(10):4857-66, 2007 Oct.

King-Lyons ND. Smith KF. Connell TD. Expression of hurP, a gene encoding a prospective site 2 protease, is essential for heme-dependent induction of bhuR in Bordetella bronchiseptica. Journal of Bacteriology. 189(17):6266-75, 2007 Sep.