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

Abstract

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 to sn-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 is R. 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 G3P in 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 the R. prowazekii gpsA gene functioned to complement an Escherichia coli gpsA mutant. Furthermore, gpsA mRNA was detected in R. prowazekii purified from hen egg yolk sacs, and G3PDH activity was assayable in R. prowazekii lysed-cell extracts. Together, these data strongly suggested that R. prowazekii encodes and synthesizes a functional GpsA enzyme, yet R. 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 that R. prowazekii transports DHAP to supply substrate for GpsA. In support of this hypothesis, we show that purified R. prowazekii transported and incorporated DHAP into phospholipids, thus implicating a role for GpsA in vivo as part of a novel rickettsial G3P acquisition pathway for phospholipid biosynthesis.

The Rickettsia prowazekii ExoU Homologue Possesses Phospholipase A 1 (PLA 1 ), PLA 2 , and Lyso-PLA 2 Activities and Can Function in the Absence of Any Eukaryotic Cofactors In Vitro

“…All experiments described in this article were performed using the Madrid E strain of R. prowazekii (hen egg yolk sac passage 282), purified as previously described (18). For immunoprecipitation (IP) of the RP534 protein from R. prowazekii, purified rickettsiae were washed and suspended in 4 ml of buffer A (135 mM NaCl, 2.7 mM KCl, 5.4 mM Na 2 HPO 4 , 1.8 mM KH 2 PO 4 , 5 mM ␤ME, and Roche's EDTA-free complete protease inhibitor cocktail [PIC] [at the manufacturer's suggested concentration], pH 7.4), lysed by ballistic shearing, and cleared of debris (18).…”

“…All experiments described in this article were performed using the Madrid E strain of R. prowazekii (hen egg yolk sac passage 282), purified as previously described (18). For immunoprecipitation (IP) of the RP534 protein from R. prowazekii, purified rickettsiae were washed and suspended in 4 ml of buffer A (135 mM NaCl, 2.7 mM KCl, 5.4 mM Na 2 HPO 4 , 1.8 mM KH 2 PO 4 , 5 mM ␤ME, and Roche's EDTA-free complete protease inhibitor cocktail [PIC] [at the manufacturer's suggested concentration], pH 7.4), lysed by ballistic shearing, and cleared of debris (18). R. prowazekii lysed cell extracts (ϳ5 mg total protein, measured using the Bio-Rad DC protein assay) were incubated with 10 l protein A Sepharose beads (Pierce) for 10 min at 4°C with agitation followed by incubation with 24 g of affinity-purified anti-RP534 antibody and 10 l protein A Sepharose beads for 18 h at 4°C with agitation.…”

“…Molecular cloning techniques were previously described (18). The R. prowazekii RP534 gene was cloned into the pET16b expression vector (Novagen) to incorporate an amino-terminal decahistidine (N-His 10 ) tag (plasmid designated pET16b-N-His 10 -RP534).…”

“…Transformants were not used for more than 1 week, but liquid cultures that had been induced for protein expression were routinely stored for weeks as cell pellets at Ϫ80°C prior to protein purification. Standard bacterial growth and protein purification conditions were as described previously (18), with the following modifications. Cell lysates were loaded onto a nickel-nitrilotriacetic acid (Ni-NTA) resin using a Bio-Rad Profinia protein purification system and sequentially washed with 0.5 M NaCl, 20 mM Tris-HCl, 10 mM imidazole, and 10 mM ␤-mercaptoethanol (␤ME), pH 8.0, with 0.5 M NaCl, 20 mM Tris-HCl, 30 mM imidazole, and 10 mM ␤ME, pH 8.0, and with 0.5 M NaCl, 20 mM Tris-HCl, 300 mM imidazole, and 10 mM ␤ME, pH 8.0.…”

Coxiella burnetii Expresses a Functional Δ24 Sterol Reductase

“…In summary, our data demonstrate that the intracellular bacterial pathogen C. burnetii expresses a sterol reductase capable of reducing the C24 double bond of ergosterol precursors. Both C. burnetii sterol reductases are considered "orphaned enzymes" because the bacterium lacks the remaining terminal enzymes in the cholesterol biosynthetic pathway (9). However, the fact that C. burnetii has retained sterol reductase genes suggests they serve a critical function.…”