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Dr. E. Fidelma Boyd
Assistant Professor
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Boyd
Office: 328 Wolf Hall
Lab: 341 Wolf Hall
Mailing address:
Department of Biological Sciences
Wolf Hall
University of Delaware
Newark, DE 19716
Phone: (302) 831-1088
Fax: (302) 831-2281
E-mail: fboyd@udel.edu
Education
B.S., Ph.D.: National University of Ireland - Galway, Ireland
Postdoctoral: The Pennsylvania State University
Postdoctoral: Harvard University
Postdoctoral: Tufts University School of Medicine
Research Interests
Vibrio cholerae, the causative agent of the dreaded diarrheal disease cholera
Cholera is still a major scourge for developing countries. This severe diarrheal disease is caused by the gram-negative bacterium Vibrio cholerae, which is a natural inhabitant of brackish and estuarine waters. In its natural environment V. cholerae can be found either in a free-living stage, forming biofilms on biotic and abiotic surfaces, or associated with fish, crustaceans, algae, moults of copepods, and chironomids egg masses. Of the more than 200 O anitgens described, only two serogroups, O1 and O139, are known to cause epidemic cholera. Two virulence factors are indispensable to develop the disease, cholera Toxin (CT), which is the main cause of the profuse diarrhea, and the Toxin Corregulated Pilus (TCP), which is essential for attachment to the human intestine. Both CT and TCP are encoded on mobile DNA regions acquired by O1 and O139 isolates; the CTXphi phage and the TCP island or Vibrio Pathogenicity Island-1 (VPI-1).
Recently, my group identified a second pathogenicity island named VPI-2, which is present among O1 serogroup isolates (Jermyn and Boyd, 2002, 2005). VPI-2 is a 57.3 Kb genetic element that contains, among others, genes putatively involved in sialic acid transport (dctPQM) and degradation of sialic acid (nanA, nanEK, nagA). Adjacent this nan-nag region is the gene that encode for a neuraminidase (nanH) (Jermyn and Boyd, 2002, 2005). NanH removes two sialic acid residues from the trisialogangliosides found in the intestinal mucin, converting them into GM1 gangliosides, which are the receptors of CT. NanH is thought to enhance the severity of the diarrhea by increasing the number of GM1 receptors. We hypothesize that carriage of the VPI-2 region by V. cholerae O1 isolates gives them a fitness advantage and loss of the region from O139 isolates has resulted in their disappearance as a major cause of cholera.
Vibrio pathogenicity island-2 role in V. cholerae survival and fitness. One of our research goals is to determine the function role of VPI-2 in vitro and in vivo. The release of free sialic acid by neuraminidase should allow O1 serogroup strains to uptake and catobolize sialic acid as a carbon and nitrogen source giving them a competitive advantage (Almagro-Moreno and Boyd, unpublished data).
Pathogenicity island evolution,: VPI-2 excision dynamics. Surprisingly little is known of pathogenicity island evolution and the mechanisms of acquisition or mobility within Vibrio cholerae. By using an inverse nested PCR approach, we found that VPI-2 can form an extra chromosomal circular molecule after precise excision from its tRNA-serine attachment site (Murphy and Boyd, 2008). We determined the role of the VPI-2 encoded P4-like integrase in the excision process by constructing a knockout mutant in VC1758 (integrase) in V. cholerae strain N16961 and examined the ability of VPI-2 to excise in this strain (Murphy and Boyd, 2008). No excision product was found indicating that a functional cognate VPI-2 integrase is required (Murphy and Boyd, 2008). A second research goal is to ascertain pathogenicity islands stability and mobility, by examining VPI-2 excision dynamics and the role of island-encoded and host-encoded factors in island excision and integration.
Vibrio seventh pandemic island-II (VSP-II). Two additional regions, Vibrio seventh pandemic island-I (VSP-I) and VSP-II, are present exclusively in V. cholerae O1 El tor isolates. My group recently described VSP-II as a 27-kb region that encodes a P4-like integrase and is flanked by direct repeats (O'Shea et al., 2004). We examined the ability of VSP-II to excise from the genome and found similar to VPI-2, VSP-II excises and forms a CI. Molecular analysis of the cognate VSP-II integrase indicates that it too is required for excision (Murphy and Boyd, 2008).
The Phage, The Pilus and allelic variation: Bacteriophage-Host interaction in Vibrio cholerae. CTXphi interacts with the bacterial host receptor TCP (a type IV pilus) and co-receptor TolA. Although the pIIICTX locus, proposed to mimic the E. coli pIII mediator of Ff phage infection, is well conserved among V. cholerae strains, the tcpA locus, which encodes the major pilin protein of TCP, has significant sequence divergence (Boyd and Waldor, 2002). To investigate the interaction between CTXphi and TCP, we examined the specificity of the pIIICTX-TcpA and the pIIICTX-TcpA/TolA interactions. We utilized both genetic and proteomic approaches, first we took a range of diverse TcpA alleles and cloned them into a classical O395 isogenic background (Reen and Boyd). In addition, we cloned TcpA and TolA constructs into the O395 background. We used a Yeast two-hybrid system to study pIIICTX-TcpA and pIIICTX-TcpA/TolA interactions and we also studied their ability to transduce pIIICTX relative to wild-type (Reen and Boyd).
Our results suggest that the divergence at the tcpA locus is not reflected in the the pilin- pIIICTX interaction. The varied transduction efficiencies observed in wild-type strains were not observed in the isogenic mutants, which were found to be similar. In the Yeast two-hybrid system screen, the co-receptor TolA was shown to be unable to bind pIIICTX in the absence of TcpA, confirming the requirement for conformational change. Several truncated hybrid peptides of pIIICTX, incorporating the TcpA and TolA binding domains, are being characterized for the effects of allelic variation on the pilin-toxin interaction. This work, involving detailed investigation of the significance of genetic variation at the tcpA and tolA loci, is leading to a better understanding of the pathogenic potential that exists in the diverse species V. cholerae.
Vibrio parahaemolyticus, the causative agent of seafood borne diarrheal disease
V. parahaemolyticus is a halophilic and mesophilic organism that is presence in all coastal water around the United States. V. parahaemolyticus is most prevalent in the warmer summer months, especially in the US Gulf coast region where it occurs in high numbers. In the past decade, the geographic distribution of V. parahaemolyticus has been extended into more northerly climes, in particular the Pacific Northwest. This is most likely due to global warming and therefore the occurrence and prevalence of the organism is likely to continue to expand. V. parahaemolyticus is an enteric pathogen that causes gastroenteritis after the consumption of contaminated seafood, such as shellfish.
Comparative genomic analysis of V. parahaemolyticus. We identified seven regions that were acquired by the new O3:K6 pandemic clone, which may play a role in the emergence and pathogenesis of these strains (Hurley et al., 2006). The seven regions ranged in size from 10 kb to 81 kb and had the characteristics of regions that were acquired by horizontal gene transfer such as aberrant base composition compared to the core genome, presence of phage-like integrases, flanked by direct repeats and the absence of these regions from closely related species. We named these regions Vibrio parahaemolyticus island-1 (VPaI-1) to VPaI-7 (Hurley et al., 2006). Molecular analysis of a worldwide collection of isolates of V. parahaemolyticus demonstrated that the VPaI-1, VPaI-4, VPaI-5 and VPaI-6 regions are present exclusively in new O3:K6 and related strains recovered after 1995. Thus, these four VPaI regions examined, represents DNA acquired by the pandemic O3:K6 clone that may increase their fitness either in the aquatic environment or in their ability to infect humans. A number of the proteins encoded by these regions have attributes that could be involved in virulence (Hurley et al., 2006). A four-way BLAST analysis of V. parahaemolyticus RIMD2210633 genome sequence verses the four published Vibrio species genomes, and identified 24 regions of greater than 10 kb that are unique to RIMD2210633 (Boyd and Parent, unpublished data). Comparative whole genome analysis of RIMD2210633 with the genome of V. parahaemolyticus strain AQ3810, an O3:K6 isolate recovered in 1983, identified at least four regions unique to each strain (Boyd, E.F, unpublished data). We found that the majority of the 24 regions are present in our diverse collection of strains, whose phylogenetic relationships were reconstructed using multilocus sequence analysis (Boyd E.F., unpublished data). However, the seven VPaI regions are highly variable among our collection of isolates. Our data suggest that the new highly virulent O3:K6 clone arose from an O3:K6 isolate that acquired at least seven novel regions (Boyd E.F., unpublished data). Next, we will examine their gene expression profile in vitro and in vivo.
Osmotolerance in Vibrio parahaemolyticus. Salinity is an absolute requirement for growth and proliferation and V. parahaemolyticus can grow in up to 10% NaCl; however our knowledge of the mechanisms of osmotolerance in V. parahaemolyticus is limited. We have identified in the genome of V. parahaemolyticus a unique clustering of genes that show homology to osmotolerance systems from Escherichia coli and Bacillus. On chromosome I, a putative ectoine synthesis system is clustered with a ProVWX transporter and a BCCT transporter. On chromosome II, a putative betaine synthesis system is clustered with a second ProVWX transporter. V. parahaemolyticus also encodes two additional BCCT transporters and a PutP transporter, bringing to eight the number of systems identified, double the number present in V. vulnificus, V. cholerae or V. fischeri. Our hypothesis is that this unique clustering and number of operons enhances V. parahaemolyticus ability to grow in fluctuating saline environments. Comparative physiological analysis of V. parahaemolyticus with V. vulnificus, V. cholerae and V. fischeri grown under varying NaCl concentrations and temperatures found that in all cases V. parahaemolyticus had a growth advantage. The growth advantage was most evident at low temperatures. V. cholerae, which encoded the least number of osmotolerance systems (three), displayed the greatest growth disadvantage at 20°C. In addition, we show that knockout mutants of key osmotolerance systems on chromosome I display growth defects at high salt concentrations.
Teaching
- BISC 667 - Molecular Mechanisms of Pathogens
Research Group
- AnaLuisa Santos Ferreira de Vasoncelos Cohen, M.S. - Graduate Student (M.S., University of Reading, United Kingdom). Evolution and Emergence of the human pathogen Vibrio vulnificus.
- Salvador Almagro Moreno, M.S. - Graduate Student (M.S., National University of Ireland - Cork, Ireland). Sialic acid metabolism in Vibrio cholerae.
- Jennifer Moynihan, B.S. - Graduate Student (B.S., National University of Ireland - Cork, Ireland). Pseudmonas genomics.
- Lynn Naughton, B.S. - Graduate Student (B.S., National University of Ireland - Cork, Ireland). Vibrio parahaemolyticus osmoregulation.
Selected Publications
Sheikh, M.A., J.A. Potter, K.A. Johnson, R.B. Sim, E.F. Boyd, and G.L. Taylor. 2008. Crystal structure of VC1805, a conserved hypothetical protein from a Vibrio cholerae pathogenicity island, reveals homology to human p32. Proteins: Structure, Function, and Bioinformatics. In Press.
Boyd, E.F. 2008. Bacteriophages in Vibrio cholerae genetics and evolution. In Vibrio cholerae: Genomics and Molecular Biology. Ed. G.B. Nair and S. Faruque. Humana Press.
Murphy, R.A. and E.F. Boyd. 2008. Three pathogenicity islands of Vibrio cholerae excise from the chromosome and form circular intermediates. J. Bacteriology 190(2):636-647.
Cohen, A.L., J.D. Oliver, A. DePaola, E. Feil, and E.F. Boyd. 2007. Molecular phylogeny of Vibrio vulnificus based on multilocus sequence analysis and a 33 kb genomic island correlates with pathogenic potential. Applied Environmental Microbiology 73:5553-5565.
Murphy, B.P., R. O'Mahony, J.F. Buckley, P. Shine, E.F. Boyd, D. Gilroy, S. Fanning. 2007. Investigation of a global collection of nontyphoidal Salmonella of various serotypes cultured between 1953 and 2004 for the presence of class 1 integrons. FEMS Microbiol Lett. 266(2):170-176.
Reen, F.J., S.A. Moreno, D. Ussery, and E.F. Boyd. 2006. The Genomic code: Inferring Vibrionaceae niche specialization. Nat. Rev. Microbiol. 4(9):697-704.
Hurley, C.C., A.M. Quirke, F.J. Reen, and E.F. Boyd. 2006. Four genomic islands that mark pandemic Vibrio parahaemolyticus isolates. BMC Genomics. 7:104
Quirke, A.M., F.J. Reen, M.J. Claessen, and E.F. Boyd. 2006. Genomic island identification in Vibrio vulnificus reveals significant genome plasticity in the important human pathogen. Bioinformatics. 22:905-910.
Jermyn, W.S., Y.A. O'Shea, A.M. Quirke, and E.F. Boyd. 2006. Genomics and the emergence of pathogenic Vibrio cholerae. In "Bacterial Genomes and Infectious Diseases". Ed. Chan VL, Sherman PM, Bourke B. Humana Press.
Reen, F.J. and E.F. Boyd. 2005. Adaptation of Vibrio species to the environment and host. In "Understanding Pathogen Behaviour". Ed. M. Griffiths and F. Dodds, Woodhead Publishing.
Reen, F.J. and E.F. Boyd. 2005. Molecular typing of epidemic and nonepidemic Vibrio cholerae isolates and differentiation of V. cholerae and V. mimicus isolates by PCR-single-strand conformation polymorphism analysis. J. Appl. Microbiol. 98:544-555.
Reen, F.J., E.F. Boyd, S. Porwollik, B. Murphy, D. Gilroy, S. Fanning, and M. McClelland. 2005. Genomic comparisons of recent Salmonella enterica isolates in milk filters from Ireland using a Salmonella microarray. Appl. Environ. Microbiol. 71:3:00.
Jermyn, W.S. and E.F. Boyd. 2005. Molecular evolution of the Vibrio Pathogenicity Island-2 (VPI-2): Mosaic structure among Vibrio cholerae and Vibrio mimicus natural isolates. Microbiol. 151:311-322.
Boyd, E.F. 2004. Bacteriophages and bacterial virulence. In "Bacteriophages: Molecular Biology and Applications". Eds. E. Kutter and A. Sulakvelidze. CRC Press.
Finnan, S., J. Morrissey, F. O'Gara, and E.F. Boyd. 2004. Genomic diversity of Pseudomonas aeruginosa isolates from cystic fibrosis patients in Ireland. J. Clin. Microbiol. 42:5783-5792.
O'Shea, Y., S. Finnan, F.J. Reen, J. Morrissey, F. O'Gara, and E.F. Boyd. 2004. The Vibrio seventh pandemic island-II is a 26.9 kb genomic island present in V. cholerae El Tor and O139 serogroup isolates that shows homology to a 43.5 Kb island in V. vulnificus. Microbiol. 150:4053-4063.
Porwollik, S., E.F. Boyd, C. Choy, P. Cheng, L. Florea, E. Proctor, and M. McClelland. 2004. Characterization of Salmonella enterica subspecies I genovars using microarrays. J. Bacteriol. 186:5884-5894.
O'Shea, Y.A., F.J. Reen, AM. Quirke, and E.F. Boyd. 2004. Evolutionary genetic relationships among Vibrio cholerae natural isolates based on multilocus comparative sequence analysis and multilocus virulence gene profiles. J. Clin. Microbiol. 42:4657-4671.
Boyd, E.F., S. Porwollik, F. Blackmer, and M. McClelland. 2003. Differences in gene content among Salmonella enterica serovar Typhi isolates. J. Clin. Microbiol. 41:3823-3828.
Boyd E.F. and H. Brüssow. 2002. Common themes among bacteriophage-encoded virulence genes and diversity among the bacteriophages involved. Trends Microbiol. 10:521-529.
Jermyn, W.S. and E.F. Boyd. 2002. Characterization of a novel Vibrio pathogenicity island (VPI-2) encoding neuraminidase (nanH) among toxigenic Vibrio cholerae isolates. Microbiol. 148:3681-3693.
O'Shea Y.A. and E.F. Boyd. 2002. Mobilization of the Vibrio Pathogenicity Island (VPI) between Vibrio cholerae O1 isolates mediated by CP-T1 generalized transduction. FEMS Microbiol. Lett. 214:153.
Boyd, E.F. and M.K. Waldor. 2002. Evolutionary and functional analyses of variants of the toxin-coregulated pilus protein TcpA from toxigenic Vibrio cholerae non-O1/non-O139 serogroup isolates. Microbiol. 148:1655-1666.
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