Office phone: (859)323-6341
Fax: (859)257-8994
Lab phone: (859)323-1628
Selected publications
Research statement: My 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 ten 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. 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. However, virulence in mice infected intravenously is unaffected. The Ybt siderophore also plays a role in regulating transcription of the ybt genes.
A separate inorganic iron transport system (Yfe) is also an ABC transporter system. Transport studies indicate that Fe3+, Mn2+, and possibly Zn2+ compete for uptake via this system. Expression of yfe genes is repressed by Fur (Ferric Uptake Regulator) complexed with iron or manganese. Mutations in the five yfe genes also cause a defect in iron-deficient growth and an ~100-fold reduction of virulence in mice infected subcutaneously. 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. For both 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, 4) surface-exposed components as potential subunit vaccine candidates, and 6) enzymatic mechanisms for the synthesis of the Ybt siderophore.
A third inorganic iron transport system (Yfu) is highly related to a subfamily of ABC iron transporters that include Yfu of Yersinia enterocolitica, Hit of Hemophilus influenzae, and Sfu of Serratia marcescens. The yfuABC promoter is repressed by iron and Fur. The cloned system in E. coli transports iron and enhances growth in iron-deficient media and is functional in Y. pestis. We have begun studies several other putative iron transport systems identified by genomic analysis – FeoB, a ferrous iron transporter; three more ABC transporters (Fit, Fiu,Yiu), and an aerobactin siderophore synthesis (Iuc) and transport system (Fhu). We have shown that the Yiu and Feo systems are functional. Either the Feo or the Yfe system is required for intracellular growth in cells of the macrophage cell line J774; in contrast, the Ybt system is not required. The aerobactin biosynthetic system does not produce the siderophore but the Fhu transport system is able to transport aerobactin and another siderophore – ferrichrome. The heme transport system (hmuPRSTUV) is required for utilization of hemin, hemoglobin, myoglobin, and other hemoprotein complexes. This system operates at mammalian and flea temperatures and likely transports the intact porphyrin ring into the cell. Mutations that delete the entire locus or the gene encoding the outer membrane receptor, HmuR, result in an inability to use any heme source for growth. We have not demonstrated a role for the Hmu system in mice. However, this system is important for growth in J774.
A second putative heme and hemoprotein uptake system in Y. pestis has strong homologies to the Has hemophore system first described in Serratia marcescens. A small secreted peptide (HasA) acts in a manner similar to a siderophore - it is secreted from the cell and binds heme from the environment. HasDEF form a secretion complex for HasA. HasR is an outer membrane receptor for uptake of the HasA-heme complex and HasB is a TonB-like protein that may be required for translocation across the outer membrane. Although the has promoter is transcriptionally active and isolated HasA binds heme, we were not able to demonstrate heme uptake and utilization via this system in Y. pestis.
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. Consequently, biofilm formation is thought to be crucial for the transmission of plague from fleas to mammals; it does not appear to play a significant role in the mammalian virulence of plague. All five genes in the hmsT and hmsHFRS loci are required for biofilm formation. 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 a newly identified gene product, HmsP respectively synthesize and degrade cyclic-di-GMP. Cyclic-di-GMP has been implicated in regulating the synthesis of biofilms in other bacteria by affecting the activity of glycosyl transferase enzymes. Future studies are 1) continuing analysis of the temperature regulation of these proteins, 2) identifying additional gene products required for biofilm formation, 3) characterizing amino acids essential for the function of the Hms proteins; 4) determining whether Hms components will be useful as diagnostic reagents or as protective antigens, and 5) characterizing the synthesis and final structure of the Hms-dependent extracellular polysaccharide matrix.
