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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. We use molecular genetic, biochemical, and physiological studies as well as virulence testing to study selected virulence mechanisms of this organism. We have research projects ongoing in several areas - two are described below.
A. Iron 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 acquisition in pathogenesis. The ultimate goals of this work are to (1) biochemically characterize the components of iron uptake systems; (2) define their regulation at the molecular level; and (3) determine the relative importance of these components to the virulence of Y. pestis. The genome sequence of Y. pestis strains and our studies have identified 13 proven or putative inorganic iron transport systems and two heme transport systems. We are investigating the yersiniabactin system and three ferrous iron transporters.
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 ybt and possibly other genes (Fig. 1B) and may have a role in pathogenesis in lung infections that is independent of its role in iron acquisition.
A separate inorganic iron transport system (Yfe) is an ABC transporter that accumulates ferrous iron and Mn. 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 third system, FetMP, also plays a role in ferrous iron acquisition. The cloned locus restores iron-deficient, microaerophilic growth of the yfe feo double mutant. A yfe mutant has an ~10-fold reduction of virulence in mice infected subcutaneously while a yfe ybt fetMP 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 and FetMP systems may be more 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, and 3) organ systems in which these systems are required for growth.
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 but plays no role in early phase transmission by unblocked fleas. Biofilm development does play a role in the persistence of infection in fleas and likely long-term survival in nature. The biofilm system plays no 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), a second messenger signal molecule. 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. Finally, we have performed a comprehensive analysis of c-di-GMP metabolism enzymes in Y. pestis. While many bacteria have multiple diguanylate cyclases (DGCs) for synthesizing c-di-GMP and phosphodiesterase (PDEs) for degrading it, Y. pestis has only two functional DGCs (HmsT and Y3730) and one functional PDE (HmsP). We have also shown that c-di-GMP signaling does not play a significant role in the pathogenesis of Y. pestis. 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.
NIH; RO1 AI33481; Yersiniabactin and Plague Pathogenesis; $1,250,000 direct costs from 12/10/08 to 11/3013
NIH; R29 AI25098; Plague Biofilm Formation – Regulation and Function; $758,333 direct costs from 3/1/09 to 2/27/14
Fetherston, J.D., I. Mier, Jr., H. Truszczynska, and R.D. Perry. The Yfe and Feo transporters are involved in microaerobic growth and the virulence of Yersinia pestis in bubonic plague. Infect. Immun. 80 (11): in press, 2012.
Perry, R.D., S.K. Craig, J. Abney, A.G. Bobrov, O. Kirillina, I. Mier, Jr., H. Truszczynska, and J.D. Fetherston. Manganese transporters Yfe and MntH are Fur regulated and important for the virulence of Yersinia pestis. Microbiology 158:816-825, 2012
Perry, R.D., and K.A. McDonough. Iron regulation and virulence in Gram negative pathogens with Yersinia pestis as a paradigm. In: M. Vasil and A. Darwin (Ed.), Regulation of Bacterial Virulence, ASM Press, 2012 (in press).
Perry, R.D., A.G. Bobrov, O. Kirillina, E.R. Rhodes, L.A. Actis, and J.D. Fetherston. Yersinia pestis transition metal divalent cation transporters. Adv. Exp. Med. Biol. 954:267-279, 2012.
Perry, R.D., and J.D. Fetherston.Yersiniabactin iron uptake: mechanisms and role in Yersinia pestis pathogenesis. Microbes Infect., 13:808-817, 2011.
Bobrov, G.A., O. Kirillina, D.A. Ryjenkov, C.M. Waters, P.A. Price, J.D. Fetherston, D. Mack, W.E. Goldman, M. Gomelsky, and R.D. Perry. Systematic analysis of cyclic di-GMP signaling enzymes and their role in biofilm development and virulence in Yersinia pestis. Mol. Microbiol. 79:533-551, 2011.
Branger, C.G., W. Sun, A. Torres-Escobar, R. Perry, D.L. Roland, J. Fetherston, R. Curtiss, III. Evaluation of Psn, HmuR and a modified LcrV protein delivered to mice by live attenuated Salmonella as a vaccine against bubonic and pneumonic Yersinia pestis challenge. Vaccine 29:274-282, 2011.
Desrosiers, D.C., Bearden, S.W., I. Mier, Jr., J. Abney, J.T. Paulley, J.D. Fetherston, J.C. Salazar, J.D. Radolf, and R.D. Perry. Znu is the predominant zinc importer in Yersinia pestis during in vitro growth but is not essential for virulence.Infect. Immun. 78:5163-5177, 2010.
Vetter, S.M., R.J. Eisen, A.M. Schotthoefer, J.A. Montenieri, J.L. Holmes, A.G. Bobrov, S.W. Bearden, R.D. Perry, and K.L. Gage. Biofilm formation is not required for early-phase transmission of Yersinia pestis. Microbiology 156:2216-2225, 2010.
Miller, M.C., J.D. Fetherston, C.L Pickett, A.G. Bobrov, R.H. Weaver, E. DeMoll, and R.D. Perry. Yersinia pestis irp1-TE and ybtT mutants produce low levels of the Ybt siderophore sufficient to activate transcription of yersiniabactin genes. Microbiology 156:2226-2238, 2010.
Wortham, B.W., M.A. Oliceira, J.D. Fetherston, and R.D. Perry. Polyamines are required for the expression of key Hms proteins important for Y. pestis biofilm formation. Environ. Microbiol. 12:3024-3047, 2010.
Fetherston,J.D., O. Kirillina, A.G. Bobrov, J.T. Paulley, and R.D. Perry. The yersiniabactin transport system is critical for the pathogenesis of bubonic and pneumonic plague. Infect. Immun, 78:2045-2052, 2010. PDF
Abu Khweek, A., J.D. Fetherston, and R.D. Perry. Analysis of HmsH and its role in plague biofilm formation. Microbiology 156:1424-1438, 2010. PDF
Forman, S., J.T. Paulley, J.D. Fetherston, Y-Q. Cheng, and R.D. Perry. Yersinia Ironomics – comparison of iron transporters among Yersinia pestis biotypes and its nearest neighbor, Yersinia pseudotuberculosis. BioMetals 23:275-294, 2010. PDF
Pieper, R., S.-T. Huang, P.P. Parmar, D.J. Clark, H. Alami, R.D. Fleischmann, R.D. Perry, and S.N. Peterson. Proteomic analysis of iron acquisition, metabolic and regulatory responses of Yersinia pestis to iron starvation. BMC Microbiology 10:30, 2010. PDF
Pieper, R., S.-T Huang., D.J. Clark, J.M. Robinson, P.P. Parmar, H. Alami, C.L. Bunai, R.D. Perry, R.D. Fleischmann, and S.N. Peterson. Characterizing the dynamic nature of the Yersinia pestis periplasmic proteome in response to nutrient exhaustion and temperature change. Proteomics. 8:1442-1458, 2008. PDF
Bobrov, A.G., O. Kirillina, S. Forman, D. Mack, and R.D. Perry. Insights into Yersinia pestis biofilm development: topology and co-interaction of Hms inner membrane proteins involved in biofilm exopolysaccharide production. Environ. Microbiol. 10:1419-1432, 2008. PDF
Bearden, S.W., and R.D. Perry. Laboratory maintenance and characterization of Yersinia pestis. Curr. Protocols Microbiol. Suppl. 11: Unit 5B1, 2008.
Perry, R.D., and S.W. Bearden. Isolation and confirmation of Yersina pestis mutants exempt from Select Agent regulations. Curr. Protocols Microbiol. Suppl. 11: Unit 5B2, 2008.
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
Bobrov, A.G., O. Kirillina, and R.D. Perry. Regulation of biofilm formation in Yersinia pestis. Adv. Expt. Med. Biol. 603: 201-210, 2007.
Forman, S., M.J. Nagiec, J. Abney, R.D. Perry, and J.D. Fetherston. Analysis of the aerobactin and ferric hydroxamate uptake systems of Yersinia pestis. Microbiology 153: 2332-2341, 2007. [Abstract]
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
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
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
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. [Abstract]
Kirillina, O., A.G. Bobrov, and R.D. Perry. A hierarchy of iron uptake systems: Yfu and Yiu are functional in Yersinia pestis. Infect. Immun. 74:6171-6178, 2006. PDF
Bobrov, A. G., O. Kirillina, and R. D. Perry. The phosphodiesterase activity of the HmsP EAL domain is required for negative regulation of biofilm formation in Yersinia pestis. FEMS Microbiol. Lett. 247:123-130, 2005. 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., 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
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
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
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
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
Lillard, J.W., Jr., and R.D. Perry. The haemin storage (Hms+) phenotype of Yersinia pestis is not essential for the pathogenesis of bubonic plague in mammals. Microbiology 145:197-209, 1999. 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
Bearden, S.W., and R.D. Perry. The Yfe system of Yersinia pestis transports iron and manganese and is required for full virulence of plague. Mol. Microbiol. 32:403-414, 1999. PDF
Jones, H.A., J.W. Lillard, Jr., and R.D. Perry. HmsT, a protein essential for expression of the haemin storage (Hms+) phenotype of Yersinia pestis. Microbiology 145:2117-2128, 1999. PDF
Bearden, S.W., T.M. Staggs, and R.D. Perry. An ABC transporter system of Yersinia pestis allows utilization of chelated iron by Escherichia coli. J. Bacteriol. 180:1135-1147, 1998. 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
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
Hinnebusch, B.J., R.D. Perry, and T.G. Schwan. Role of the Yersinia pestis hemin storage (hms) locus in the transmission of plague by fleas. Science 273:367-370, 1996.
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