Rickettsia prowazekiiUses ansn-Glycerol-3-Phosphate Dehydrogenase and a Novel Dihydroxyacetone Phosphate Transport System To Supply Triose Phosphate for Phospholipid Biosynthesis

Rickettsia prowazekii is an obligate intracellular pathogen that possesses a small genome and a highly refined repertoire of biochemical pathways compared to those of free-living bacteria. Here we describe a novel biochemical pathway that relies on rickettsial transport of host cytosolic dihydroxyacetone phosphate (DHAP) and its subsequent conversion tosn -glycerol-3-phosphate (G3P) for synthesis of phospholipids. This rickettsial pathway compensates for the evolutionary loss of rickettsial glycolysis/gluconeogenesis, the typical endogenous source of G3P. One of the components of this pathway isR. prowazekii open reading frame RP442, which is annotated GpsA, a G3P dehydrogenase (G3PDH). Purified recombinant rickettsial GpsA was shown to specifically catalyze the conversion of DHAP to G3Pin vitro . The products of the GpsA assay were monitored spectrophotometrically, and the identity of the reaction product was verified by paper chromatography. In addition, heterologous expression of theR. prowazekii gpsA gene functioned to complement anEscherichia coli gpsA mutant. Furthermore,gpsA mRNA was detected inR. prowazekii purified from hen egg yolk sacs, and G3PDH activity was assayable inR. prowazekii lysed-cell extracts. Together, these data strongly suggested thatR. prowazekii encodes and synthesizes a functional GpsA enzyme, yetR. prowazekii is unable to synthesize DHAP as a substrate for the GpsA enzymatic reaction. On the basis of the fact that intracellular organisms often avail themselves of resources in the host cell cytosol via the activity of novel carrier-mediated transport systems, we reasoned thatR. prowazekii transports DHAP to supply substrate for GpsA. In support of this hypothesis, we show that purifiedR. prowazekii transported and incorporated DHAP into phospholipids, thus implicating a role for GpsAin vivo as part of a novel rickettsial G3P acquisition pathway for phospholipid biosynthesis.