Identification of Metabolic Virulence Factors in the Epidemic Typhus Rickettsia

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DESCRIPTION (provided by applicant): The goal of this R21 proposal is to initiate a study that could ultimately lead to the identification of metabolic virulence factors in the obligate intracytoplasmic pathogen Rickettsia prowazekii, the louse-vectored agent of the human disease epidemic typhus. Obligate intracellular growth has led to the evolution of R. prowazekii as a quintessential metabolic scavenger; many essential metabolites are transported from the host cytosol in lieu of de novo synthesis. Although rickettsiae typify organisms undergoing reductive evolution they appear to have maintained mechanisms of metabolic redundancy with respect to scavenging of essential host cell metabolites. Annotation of the R. prowazekii genome revealed the presence of 'orphaned enzymes' - the sole remnants of biochemical pathways that have been otherwise lost in favour of a transporter for the end product of the pathway. Further, these orphaned enzymes are often predicted to catalyze the last step in the enzymatic pathway. I hypothesize that these so-called orphaned enzymes are functional and that rickettsiae have evolved novel transport systems for the substrates of these enzymes, thus providing alternate means to acquire essential metabolites from the host. These parallel scavenging mechanisms could be required for rickettsial competition with host cell enzymes for critical metabolites and are, thus, essential for growth. This study will focus on two pathways: 1) rickettsial acquisition of Coenzyme A (CoA) via transport CoA and 3'dephosphoCoA (DPC) and 2) rickettsial acquisition of sn-glycerol-3-phosphate (G3P) via transport of G3P and dihydroxyacetone phosphate (DHAP). I posit that these redundant pathways could represent targets for novel immuno/therapeutic targets to treat this Select Agent and be exploited to produce a R. prowazekii vaccine strain. Following the R21 granting mechanism philosophy, this exploratory project will identify and characterize the rickettsial transport systems involved in CoA/DPC and G3P/DHAP uptake to identify suitable candidates for a long-term study in which these systems will be inactivated and assayed for virulence. These studies represent important steps in developing countermeasures against bioterrorism threats. Aim I: Rickettsiae possess parallel metabolic pathways to procure G3P. Rickettsiae are able to transport G3P using a homologue of the bacterial GlpT transporter. However, rickettsiae are also able to convert dihydroxyacetone phosphate (DHAP) to G3P in a reaction catalyzed by the GpsA protein (a G3P dehydrogenase). I will use high-throughput screening of a rickettsial DNA library expressed in Escherichia coli to identify and characterize the rickettsial DHAP transporter. Aim II: Rickettsiae possess parallel pathways to obtain CoA. Purified rickettsiae transport CoA and its metabolic precursor, 3'dephosphocoenzyme A (DPC) using separate carrier-mediated transport systems. Bioinformatics has determined that rickettsiae possess an annotated DPC kinase to convert DPC to CoA. I will use the above mentioned screening method to identify and characterize these transporters. Rickettsia prowazekii is the louse-vectored agent of the human disease epidemic typhus and is categorized as a Category B select agent indicative of its potential subversion as an agent of bioterrorism. The focus of this study is to identify and characterize essential transport systems that are unique to R. prowazekii and are, thus, potential targets for the development of novel antimicrobials to treat this obligate intracytoplasmic pathogen.