Dual Mechanisms of Metabolite Acquisition by the Obligate Intracytosolic Pathogen Rickettsia prowazekii Reveal Novel Aspects of Triose Phosphate Transport

Abstract

Rickettsia prowazekii is an obligate intracytosolic pathogen and the causative agent of epidemic typhus fever in humans. As an evolutionary model of intracellular pathogenesis, rickettsiae are notorious for their use of transport systems that parasitize eukaryotic host cell biochemical pathways. Rickettsial transport systems for substrates found only in eukaryotic cell cytoplasm are uncommon among free-living microorganisms and often possess distinctive mechanisms. We previously reported that R. prowazekii acquires triose phosphates for phospholipid biosynthesis via the coordinated activities of a novel dihydroxyacetone phosphate transport system and an sn-glycerol-3-phosphate dehydrogenase (K. M. Frohlich et al., J. Bacteriol. 192:4281-4288, 2010). In the present study, we have determined that R. prowazekii utilizes a second, independent triose phosphate acquisition pathway whereby sn-glycerol-3-phosphate is directly transported and incorporated into phospholipids. Herein we describe the sn-glycerol-3-phosphate and dihydroxyacetone phosphate transport systems in isolated R. prowazekii with respect to kinetics, energy coupling, transport mechanisms, and substrate specificity. These data suggest the existence of multiple rickettsial triose phosphate transport systems. Furthermore, the R. prowazekii dihydroxyacetone phosphate transport systems displayed unexpected mechanistic properties compared to well-characterized triose phosphate transport systems from plant plastids. Questions regarding possible roles for dual-substrate acquisition pathways as metabolic virulence factors in the context of a pathogen undergoing reductive evolution are discussed. Rickettsia prowazekii is the causative agent of louse-borne typhus in humans and is designated a select agent. Members of the genus Rickettsia are Gram-negative alphaproteobacteria that grow only within the cytosol of a eukaryotic host cell (for reviews, see references 1 to 5). Rickettsiae are evolutionarily related to mitochondria (6) and present a fascinating biological paradigmorganisms that grow with cytoplasm on both sides of their cell membrane. Obligate intracytosolic growth is thought to be a primary driver of rickettsial genome reductive evolution, where many genes encoding de novo biosynthetic pathways have been lost (6-9). Instead, rickettsiae avail themselves of the metabolite pools of the host cell through the use of carrier-mediated transport systems (10-15). In essence, the eukaryotic host cell provides the metabolites that rickettsiae would otherwise have to synthesize themselves, thus creating a selective environment conducive to rickettsial genome reduction and metabolic streamlining (1). Conversely, environmental fluctuations, perturbations, and instabilities negate such a narrow strategy for the free-living microorganism, which has instead maintained coordinated, adaptive responses to furnish metabolic flexibility.Despite the paring effects of reductive evolution, some rickettsial metabolic pathways have retained complexity. The classical example is...

WhollyRickettsia! Reconstructed Metabolic Profile of the Quintessential Bacterial Parasite of Eukaryotic Cells

“…Acyl chain incorporation into lipid A follows the structure deduced for R. typhi ( 171 ). While both DHAP and G3P are known to be imported from the host ( 51 , 52 ), Rickettsia species lack enzymes to generate LPA via the first incorporation of hexadecanoyl-ACP (the gray circle represents this pathway hole). All enzymes subsequent to this step are highly conserved (see Fig.…”

“…Phylogenomics studies observed additional depleted metabolic pathways, particularly those for B vitamins, many cofactors, and pentose phosphates ( 48 – 50 ). Efforts in the postgenomics era identified rickettsial import of the glycerophospholipid precursors dihydroxyacetone phosphate (DHAP) and sn -glycerol 3-phosphate (G3P) ( 51 , 52 ) and also the cosubstrate S -adenosylmethionine (SAM), for which a novel transporter (EamA) was characterized ( 53 ). Additionally, substrate ranges of four Tlc1 paralogs (Tlc2 to Tlc5) were investigated, revealing that none function in energy exchange, yet two import host ribonucleotides (Tlc4 [CTP, UTP, and GDP] and Tlc5 [GTP and GDP]) ( 54 ).…”

Genomes of Candidatus Wolbachia bourtzisii wDacA and Candidatus Wolbachia pipientis wDacB from the Cochineal Insect Dactylopius coccus (Hemiptera: Dactylopiidae)

“…On the other hand, strain w DacB possessed three uptake systems of the drug/metabolite transporter (DMT) superfamily, and two genes for organophosphate:phosphate MFS antiporters that were not present in w DacA. The former DMT transporters were > 75% similar to the S-adenosylmethionine (SAM) uptake transporter of Rickettsia prowazekii ( Tucker et al 2003 ), and the highest similarities (∼49%) of the latter MFS antiporters were to proteins of R. prowazekii which have been implicated in triose phosphate uptake used for phospholipid biosynthesis ( Frohlich and Audia 2013 ). No hexose transporter genes were found, supporting the theory that there is no glycolysis in both strains.…”

Metabolic Adaptations of Intracellullar Bacterial Pathogens and their Mammalian Host Cells during Infection (“Pathometabolism”)

“…Mammalian Host Cells during Infection ("Pathometabolism") existing rickettsial triose phosphate transport systems (108). The existence of a large number of transporters for catabolic intermediates and anabolic monomers is indeed a hallmark of R. rickettsiae (105,(109)(110)(111)(112).…”

“…The hitherto poorly understood regulation of the rickettsial metabolite transporters in the course of the host cell infection is probably decisive for the balanced interaction of the host and the parasite since both partners compete, more or less, for the same nutrient pool. In this context, it has been proposed that the rickettsial transport systems might even be considered as metabolic virulence factors (108).…”