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The Perry/Fetherston laboratory studies the bacterium Yersinia pestis, the causative agent of bubonic and pneumonic plague. This organism has an obligate life cycle that alternates between rodents and their fleas. Our in vitro molecular genetic, biochemical, and physiological studies use avirulent strains of Y. pestis. We have research projects ongoing in several areas - two are described below.
A. Iron and Heme Acquisition Systems. Vertebrates present pathogens with a highly iron-deficient environment by chelating iron and heme to host proteins; bacteria unable to remove iron or heme from these host proteins are avirulent. My research program uses Y. pestis to study the role of bacterial iron and heme acquisition in pathogenesis. The ultimate goals of this work are to (1) biochemically characterize the components of the iron accumulation and storage systems; (2) define their regulation at the molecular level; and (3) determine the relative importance of these components to the virulence of Y. pestis. Our studies and the genome sequence of Y. pestis has identified 13 proven or putative inorganic iron transport systems and two heme transport systems. We are investigating each of these systems.
The Yersiniabactin (Ybt) siderophore-dependent system is encoded within a pathogenicity island. We have identified ten ybt genes that are required either for synthesis of the siderophore, uptake of the Ybt-iron complex, or regulation of Ybt system gene expression (Fig. 1). Mutations in these genes cause a defect in iron-deficient growth at 37°C, loss of either synthesis or utilization of yersiniabactin, and complete loss of virulence in mice infected subcutaneously while virulence in mice infected intravenously is unaffected. Ybt also plays an important role in pneumonic plague. The Ybt siderophore regulates transcription of the ybt and possibly other genes (Fig. 1B).
A separate inorganic iron transport system (Yfe) is an ABC transporter that accumulates Fe2+ and Mn2+. Mutations in the five yfe genes cause a defect in iron-deficient growth. Expression of yfe genes is repressed by Fur (Ferric Uptake Regulator) complexed with iron or manganese. A second ferrous iron transporter (Feo) plays role similar to that of Yfe – a Yfe- Feo- double mutant shows a more severe in vitro growth defect under iron-deficient, microaerophilic growth conditions than a single mutation in either system. A Yfe- mutant has an ~10-fold reduction of virulence in mice infected subcutaneously while a Yfe- Ybt- mutant is completely avirulent in mice infected intravenously. This suggests that the Ybt system is essential during the early stages of bubonic plague while the Yfe system is important during the later stages of infection. A Yfe- Feo- double mutant has an ~90-fold loss of virulence in the mouse model of bubonic plague. In contrast, intranasal infections with this double mutant show no loss of virulence for the pneumonic disease. For these iron transport systems, we are continuing to analyze 1) in vitro and in vivo regulatory mechanisms, 2) transport characteristics, 3) organ systems in which these systems are required for growth, and 4) surface-exposed components as potential subunit vaccine candidates. The Y. pestis KIM genome contains 12 other proven or potential iron/heme transporter systems. Of these, 3 have proven to be functional, 4 are untested, and the remaining 5 systems appear to be partially or completely nonfunctional due to mutations or insertion of an IS element. The three functional systems (Yfu, Yiu, and Hmu) appear to play no role in bubonic disease.
For these iron transport systems, we are continuing to analyze 1) in vitro and in vivo regulatory mechanisms, 2) transport characteristics, 3) organ systems in which these systems are required for growth, and 4) surface-exposed components as potential subunit vaccine candidates.
The Y. pestis KIM genome contains 12 other proven or potential iron/heme transporter systems. Of these, 3 have proven to be functional, 4 are untested, and the remaining 5 systems appear to be partially or completely nonfunctional due to mutations or insertion of an IS element. The three functional systems (Yfu, Yiu, and Hmu) appear to play no role in bubonic disease.
B. Biofilm formation and the transmission of bubonic plague. The hemin-storage phenotype (Hms) was named for the adsorption of enormous quantities of heme by Y. pestis cells grown at ambient temperatures but not at mammalian temperature. The Hms system synthesizes a biofilm necessary for colonization and blockage of the flea proventriculus. Blockage of this valvular organ between the esophagus and stomach causes an efflux of blood, contaminated with Y. pestis, back into the host of the feeding flea. Biofilm formation is crucial for one form of transmission of plague from fleas to mammals; it does not appear to play a significant role in the mammalian virulence of plague. Six genes in hmsT, hmsP, and hmsHFRS loci are required for biofilm formation and its regulation (Fig. 2). We have demonstrated interaction of inner membrane proteins HmsR, HmsS, HmsT, and HmsP. A second, separate outer membrane complex includes HmsH and HmsF, a porin and polysaccharide deacetylase, respectively. Temperature regulation, on at 21-34°C and off at 37°C, does not occur at the level of transcription or translation. Instead, HmsH, HmsR, and HmsT are selectively degraded at 37°C. HmsR possesses a glycosyl transferase domain that is likely involved in synthesis of an exopolysaccharide for the biofilm. HmsT and HmsP respectively synthesize and degrade cyclic-di-GMP (c-di-GMP). C-di-GMP has been implicated in regulating the synthesis of biofilms in other bacteria by affecting the activity of glycosyl transferase enzymes. In Y. pestis we have found that high c-di-GMP levels promote biofilm formation and increase the level of HmsR while low c-di-GMP levels have the opposite effect. In addition, the polyamine putrescine affects biofilm development by affecting the levels of HmsT and HmsR proteins. Future studies are 1) continuing analysis of the temperature regulation of Hms genes and proteins, 2) identifying additional gene products required for biofilm formation and regulation, 3) defining enzymatic activities of Hms proteins; and 4) continuing to analyze the mechanisms by which c-di-GMP affects biofilm development.
Perry, R.D., I. Mier, Jr., and J.D. Fetherston. Roles of the Yfe and Feo transporter of Yersinia pestis in iron uptake and intracellular growth. Biometals 20:699-703, 2007. PDF
Forman, S., M.J. Nagiec, J. Abney, R.D. Perry, and Jacqueline D. Fetherston. Analysis of the aerobactin and ferric hydroxamate uptake systems of Yersinia pestis. Microbiology 153: 2332-2341, 2007. PDF
Branger, C.G., J.D. Fetherston, R.D. Perry, and R. Curtiss III. Oral vaccination with different antigens from Yersinia pestis KIM delivered by live attenuated Salmonella Typhimurium elicits a protective immune response against plague. Adv. Expt. Med. Biol. 603:386-398, 2007.
Bobrov, A.G., S.W. Bearden, J.D. Fetherston, A. Abu Khweek, K.D. Parrish, and R.D. Perry. Functional quorum sensing systems affect biofilm formation and protein expression in Yersinia pestis. Adv. Expt. Med. Biol. 603:178-191, 2007.
Perry, R.D. and J.D. Fetherston (eds). 2007. The Genus Yersinia – From Genomics to Function. 429 pp. Springer Science+Business Media, LLC, New York, NY, USA
Forman, S. A.G. Bobrov, O. Kirillina, S.K. Craig, J. Abney, J.D. Fetherston, and R.D. Perry. Identification of critical amino acid residues in the plague biofilm Hms proteins. Microbiology 152:3399-3410, 2006. PDF
Miller, M.C., S. Parkin, J.D. Fetherston, R.D. Perry, and E. DeMoll. Crystal structure of ferric-yersiniabactin: a virulence factor of Yersinia pestis. J. Inorg. Biochem. 100:1495-1500, 2006. PDF
Patel, C. N., B. W. Wortham, J. L. Lines, J. D. Fetherston, R. D. Perry, and M. A. Oliveira. Polyamines are essential for the formation of plague biofilm. J. Bacteriol. 188:2355-2363, 2006. PDF
Simm, R., J. D. Fetherston, A. Kader, U. Römling, and R. D. Perry. Phenotypic convergence mediated by GGDEF-domain-containing proteins. J. Bacteriol. 187:6816-6823, 2005. PDF
O. Kirillina, A.G. Bobrov, J.D. Fetherston, and R.D. Perry. HmsP, a putative phosphodiesterase involved in controlling expression of the Hms extracellular matrix of Yersinia pestis. Mol. Microbiol. 54:75-88, 2004. PDF
Perry, R.D., A.G. Bobrov, O. Kirillina, H.A. Jones, L. Pedersen, J. Abney, J.D. Fetherston. Temperature regulation of the Haemin storage (Hms+) phenotype of Yersinia pestis is posttranscriptional. J. Bacteriol. 186:1638-1647, 2004. PDF
Perry, R.D. and J.D. Fetherston. Iron and heme uptake systems, p. 257-283. In: Carniel, E. and B.J. Hinnebusch (eds). Molecular and Cellular Biology of Pathogenic Yersinia , Horizon Sci. Press/Caister Acad. Press, 2004.
Perry, R.D., J. Shah, S.W. Bearden, J.M. Thompson, and J.D. Fetherston. Yersinia pestis TonB: role in iron, heme and hemoprotein utilization. Infection Immun. 71: 4159-4162, 2003. PDF
Deng, W., V. Burland, G. Plunkett III, A. Boutin, G.F. Mayhew, P. Liss, N.T. Perna, D.J. Rose, B. Mau, S. Zhou, D.C. Schwartz, J.D. Fetherston, L.E. Lindler, R.R. Brubaker, G.V. Plano, S.C. Straley, K.A. McDonough, M.L. Nilles, J. S. Matson, F.R. Blattner, R.D. Perry. Genome sequence of Yersinia pestis KIM. J. Bacteriol. 184:4601-4611, 2002. PDF
Bobrov, A.G., V.A. Geoffroy, and R.D. Perry. Yersinia pestis YbtD – a putative phosphopantetheinylate transferase required for synthesis of the siderophore yersiniabactin. Infect. Immun. 70:4204-4214, 2002. PDF
Rossi, M.S., J.D. Fetherston, S. Létoffé, E. Carniel, R.D. Perry, and J.M. Ghigo. Isolation and characterization of the hemophore-dependent heme acquisition system of Yersinia pestis. Infect. Immun.69:6707-6717, 2001. PDF
Gong, S., S.W. Bearden, V.A. Geoffroy, J.D. Fetherston, and R.D. Perry. Characterization of the Yersinia pestis Yfu ABC inorganic iron transport system. Infect. Immun. 69:2829-2837, 2001. PDF
Geoffroy V.A., J.D.Fetherston and R.D. Perry. Yersinia pestis YbtU and YbtT are involved in synthesis of the siderophore yersiniabactin but have different effects on regulation. Infect. Immun. 68:4452-4461, 2000. PDF
Fetherston, J.D., V.J. Bertolino, and R.D. Perry. YbtP and YbtQ: two ABC transporter proteins required for iron uptake in Yersinia pestis. Mol. Microbiol.32:289-299, 1999. PDF
Gehring, A.M., E. DeMoll, J.D. Fetherston, I. Mori, G. Mayhew, F.R. Blattner, C.T. Walsh, and R.D. Perry. Iron acquisition in plague: modular logic in enzymatic biogenesis of yersiniabactin by Yersinia pestis. Chem. Biol. 5:573-586, 1998.
Lillard, J.W., Jr., J.D. Fetherston, L. Pedersen, M.L. Pendrak, and R.D. Perry. Sequence and genetic analysis of the hemin storage (hms) locus of Yersinia pestis. Gene 193:13-21, 1997. PDF
Bearden, S.W., R.D. Perry, and J.D. Fetherston. Genetic organization of the yersiniabactin biosynthetic region and construction of avirulent mutants in Yersinia pestis. Infect. Immun. 65:1659-1668, 1997. PDF
Fetherston, J.D., S.W. Bearden, and R.D. Perry.YbtA, an AraC-type regulator of the Yersinia pestis receptor for yersiniabactin and pesticin. Mol. Microbiol. 22:315-325, 1996. PDF