Vol. 175, No. 15JOURNAL OF BACTERIOLOGY, Aug. 1993, p. 4662-46690021-9193/93/154662-08$02.00/0Copyright X 1993, American Society for Microbiology

Purine Metabolism by Intracellular Chlamydia psittaciGRANT McCLARTY* AND HUIZHOU FAN

Department ofMedical Microbiology, University ofManitoba, 730 William Avenue,Winnipeg, Manitoba, Canada R3E OW3

Received 12 March 1993/Accepted 27 May 1993

Purine metabolism was studied in the obligate intracellular bacterium Chlamydia psittaci AA Mp in the wildtype and a variety of mutant host cell lines with well-defined deficiencies in purine metabolism. C. psittaci AAMp cannot synthesize purines de novo, as assessed by its inability to incorporate exogenous glycine into nucleicacid purines. C. psittaci AA Mp can take ATP and GTP, but not dATP or dGTP, directly from the host cell.Exogenous hypoxanthine and inosine were not utilized by the parasite. In contrast, exogenous adenine,adenosine, and guanine were directly salvaged by C. psittaci AA Mp. Crude extract prepared from highlypurified C. psittaci AA Mp reticulate bodies contained adenine and guanine but no hypoxanthine phosphori-bosyltransferase activity. Adenosine kinase activity was detected, but guanosine kinase activity was not. Therewas no competition for incorporation into nucleic acid between adenine and guanine, and high-performanceliquid chromatography profiles of radiolabelled nucleic acid nucleobases indicated that adenine, adenosine, anddeoxyadenosine were incorporated only into adenine and that guanine, guanosine, and deoxyguanosine wereincorporated only into guanine. Thus, there is no interconversion of nucleotides. Deoxyadenosine anddeoxyguanosine were cleaved to adenine and guanine before being utilized, and purine (deoxy)nucleosidephosphorylase activity was present in reticulate body extract.

Chlamydiae are obligate intracellular eubacterial parasitesthat infect a wide range of eukaryotic host cells (5, 21, 28,29). Chlamydiae reproduce, within the confines of a mem-brane bound cytoplasmic vacuole, by means of a uniquedevelopmental cycle. Two different cell types, the infec-tious, metabolically inert elementary body and the vegeta-tive noninfectious reticulate body (RB), alternate within thelife cycle (21, 28). In adapting to intracellular life, chlamyd-iae appear to have lost whatever energy-producing systemsthey might once have had. The ATP required to fuel meta-bolic processes is obtained directly from the host cell by anATP/ADP translocase (13), similar to that found in mito-chondria (33) and rickettsiae (36). Although it providesphosphate bond energy, the ATP/ADP translocase does notprovide a net gain of adenine nucleotides.

In contrast to free-living bacteria (24) and protozoanparasites (9), Chlamydia trachomatis L2 (4, 20, 26) andChlamydia psittaci 6BC (2, 10, 11) are incapable of salvagingpreformed purine or pyrimidine nucleobases or (deoxy)nu-cleosides. Similar findings have been reported for rickettsiae(35, 36). In spite of the fact that most prokaryotes andeukaryotes cannot transport highly charged molecules likeribonucleotides (24), C. trachomatis L2 (20) and C. psittaci6BC (10) have the capacity to acquire all four ribonucleosidetriphosphates directly from the host cell. However, to date,no parasite-specific transport proteins have been identified.These findings lead to the hypothesis that chlamydiae areauxotrophic for ribonucleotides.

Recently, we identified two avian C. psittaci isolates thatwere unlike other chlamydiae in that they could not takepyrimidine ribonucleotides from the host (18). Pyrimidinemetabolism in C. psittaci AA Mp, a representative isolate,was addressed in the accompanying paper (19). In this studywe report on the pathways of purine salvage metabolism inC. psittaci AA Mp.

* Corresponding author.

MATERIALS AND METHODS

Materials. [2,8-3H]adenine (46 Ci/mmol), [2,8-3H]adeno-sine (46 Ci/mmol), [2,8-3H]deoxyadenosine (50 Ci/mmol),[2'-3H]deoxyadenosine (12 Ci/mmol), [8-3Hjguanine (14 Ci/mmol), [8-3Hjguanosine (15 Ci/mmol), [8- Hjdeoxyguano-sine (9 Ci/mmol), [8-3H]hypoxanthine (20 Ci/mmol), [2,8-3H]inosine (23 Ci/mmol), and [8-3H]xanthine (9 Ci/mmol)were purchased from Moravek Biochemicals Inc. [U-14C]glycine (110 mCi/mmol) was purchased from DuPont NewEngland Nuclear. All tissue culture media and supplementswere from Flow Laboratories. Fetal bovine serum wasbought from Intergen. All unlabelled nucleobases, (deoxy)nucleosides, and 8-aminoguanosine (8-AG) were purchasedfrom Sigma. Deoxycoformycin (dCF) was a gift from J.Johnston, Manitoba Institute of Cell Biology, Winnipeg,Manitoba, Canada. All other chemicals were of the highestpurity obtainable.

Cell lines and culture conditions. The wild-type mouse L929 cell line was provided by K. Coombs, Department ofMedical Microbiology, University of Manitoba, Winnipeg,Manitoba, Canada. The mutant mouse cell line deficient inhypoxanthine-guanine phosphoribosyltransferase activity(HGPRT-) and in adenine phosphoribosyltransferase(APRT) activity (APRT-) (line A-9; catalogue numberGM00346B) was obtained from the National Institute ofGeneral Medical Sciences Human Genetic Mutant Cell Re-pository, Camden, N.J. Both the wild-type and HGPRT-APRT- mouse cell lines were routinely maintained as mono-layer cultures on plastic tissue culture dishes (Corning GlassWorks) and as suspension cultures with minimal essentialmedium supplemented with 10% fetal bovine serum and 0.2mM glutamine. The hypoxanthine-requiring mutant Chinesehamster ovary (CHO) K1 (Ade-F) cell line (25) was kindlyprovided by D. Patterson, Eleanor Roosevelt Institute forCancer Research, Denver, Colo. The Ade-F cell line wasgrown in Dulbecco's modified Eagle medium-10% fetalbovine serum-0.2 mM glutamine, 300 ,uM proline supple-mented with 30 jxM hypoxanthine. The purine auxotroph

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Ade-F cell line cannot synthesize phosphoribosylformami-doimidazolecarboxamide from phosphoribosylaminoimida-zolecarboxamide because it lacks a functional phosphoribo-sylaminoimidazolecarboxamide formyltransferase (25). Theadenosine kinase-deficient mutant CHO K1 (AK-) cell line(7) was kindly supplied by R. Gupta, Department of Bio-chemistry, McMaster University, Hamilton, Ontario, Can-ada. The AK- cell line was grown in minimal essentialmedium supplemented with 0.2 mM glutamine, 0.3 mMproline, and 10% fetal bovine serum. All cell lines wereroutinely checked for mycoplasma contamination.

C. psittaci strain and propagation. C. psittaci meningo-pneumonitis strain francis (catalog number ATCC VR-122)isolate AA Mp was obtained from the American TypeCulture Collection, Rockville, Md. The origin and initialgenetic, biochemical, and immunological characterization ofC. psittaci AA Mp have been published elsewhere (18).Pyrimidine metabolism in C. psittaci AA Mp was describedin the accompanying paper (19). C. psittaci AA Mp wasgrown as described previously (19, 26), and unless otherwiseindicated, 1 ,ug of cycloheximide per ml was present in thepostinfection (p.i.) growth medium. Mock-infected (MI) hostcell cultures were treated in the same fashion as infectedcultures except that chlamydiae were not added. Cyclohex-imide (1 pug/ml) was always present in MI cell culturemedium.

Incorporation of radiolabelled precursors into C. psidtaci AAMp nucleic acids. Radiolabelling experiments were per-formed as described by McClarty and Tipples (20) and in theaccompanying paper (19). For C. psittaci-infected culturesincorporation of precursor into nucleic acid was measured at22 h p.i., a time when parasite RNA synthesis and DNAsynthesis are maximal (4). The values presented for incor-poration of radiolabel into C. psittaciAA Mp RNA and DNArepresent the actual value obtained for a chlamydia-infectedculture minus the value obtained for an identically treatedMI control culture.Acid hydrolysis of nucleic acid and subsequent nucleobase

analysis. MI and C. psittaci-infected cultures were radiola-belled with nucleic acid precursors, and then total nucleicacid was extracted and acid hydrolyzed to nucleobases asdescribed in the accompanying paper (19). Isotope incorpo-ration into nucleobases was monitored by on-line radioactiveflow detection (Beckman 171 flow detector) after separationof the nucleobases by high-performance liquid chromatogra-phy (HPLC) (4, 19). The identity of the radioactive peakswas confirmed by simultaneously monitoring the A254S ofknown nucleobase standards. Data were analyzed with anIBM PC50 using Beckman System Gold software.Enzymatic hydrolysis of DNA and subsequent deoxynucle-

oside analysis. MI and C. psittaci AA Mp-infected cultureswere radiolabeled with nucleic acid precursors, then totalnucleic acids were extracted, and DNA was isolated andenzymatically degraded to deoxynucleosides (19). Isotopeincorporation into deoxynucleosides was monitored by on-line radioactive flow detection (Beckman 171 flow detector)after separation of the deoxynucleosides by HPLC (19). Theidentity of the radioactive peaks was confirmed by simulta-neously monitoring the A254S of known deoxynucleosidestandards. Data were analyzed as described above.

Preparation of RB extracts. Suspension cultures of mouseL 929 cells were used as the host for preparing large batchesof RBs which were highly purified through Renografin den-sity gradients as described previously (1). Purified RBs werelysed, and crude cell extract was prepared as described byFan et al. (4). Purified sham extracts were prepared from MI

mouse L 929 cells by the same procedure used to purify RBsfrom infected mouse cells.Enzyme assays. Purine phosphoribosyltransferase activi-

ties were assayed by a modified procedure of Green andMartin (6). The reaction mixture consisted of (in a totalvolume of 100 pl) 50 mM Tris-HCl (pH 7.6), 5 mM MgCl2, 1mM 5-phosphoribosyl-1-PPi containing bovine serum albu-min at 50 pug/ml, and 100 FM [2,8- H]adenine (50 mCi/mmol), 100 p.M [2,8-3Hlhypoxanthine (50 mCi/mmol), or 100p.M [8-3Hlguanine (50 mCi/mmol). The reaction was initiatedby the addition of RB cell extract (50 pug of protein) and thenallowed to proceed at 370C for 20 min. The reaction wasterminated by placing 50 ptl of the reaction solution onto a25-mm ion-exchange disc (DE-81; Whatman), and the ad-sorbed radioactivity was determined by liquid scintillationcounting (6).

Nucleoside kinase activity was assayed according to themethod of Nelson et al. (23). The reaction mixture consistedof (in a total volume of 100 RI) 0.10 M Tris-HCl (pH 7.5), 5mM ATP, 5 mM MgCl2, 4 IU of pyruvate kinase per ml, 250p.M phosphoenolpyruvate, and 500 p.M [2,8-3H]adenosine(100 mCi/mmol) or [8-3H]guanosine (50 mCi/mmol). Thereaction was initiated by the addition of RB cell extract (50jig of protein), the mixture was incubated at 370C for 20 min,then the reaction was terminated, and the DE-81-adsorbableradioactivity was determined (6). The first time phosphori-bosyltransferase and nucleoside kinase assays were run, theradiolabelled nucleotide products formed were separatedand identified by HPLC (19).Nucleoside phosphorylase activity was assayed according

to the method of Reyes et al. (27) with the followingmodifications. The reaction mixture consisted of (in a totalvolume of 100 RI) 0.1 M Tris-HCl (pH 7.6), 4 mM MgCl2, 50mM potassium phosphate, and 100 p.M [2,8-3H]adenosine(100 mCi/mmol), 100 p.M [2,8-3H]deoxyadenosine (100 mCi/mmol), [8-3H]guanosine (50 mCi/mmol), or [8-3H]deoxygua-nosine (50 Ci/mmol). The reaction was initiated by theaddition of RB cell extract (50 pug of protein) and thenallowed to proceed at 370C for 20 min. The reaction wasterminated by adding ice-cold trichloroacetic acid (finalconcentration, 10%), and then the tubes were placed on icefor 30 min. After centrifugation to remove precipitatedmaterial, the supernatant was neutralized by extraction withtri-n-octylamine-freon (16). The neutralized extract was an-alyzed by reverse-phase HPLC with A254 monitoring andon-line radioactive flow detection for identification andquantitation of the purine bases (4).

RESULTS

Incorporation of purines into C. psittaci AA Mp nucleicacids. In the accompanying paper we reported on pyrimidinemetabolism in C. psittaci AA Mp (19). During the course ofthose studies we noted that C. psittaci AA Mp also pos-sessed purine-metabolizing capabilities previously not de-tected in C. trachomatis L2 (20, 26), C. psittaci 6BC (2, 10,12), or C. psittaci Cal-10 (31). The results of experimentsmonitoring the incorporation of exogenous purines intoRNA and DNA of C. psittaci AA Mp-infected wild-typemouse L cells and HGPRT- APRT- cells are shown inTable 1. The relative incorporation of radiolabel from thevarious purine precursors into RNA and DNA should beconsidered semiquantitative, since the specific activities ofthe parasite (deoxy)ribonucleoside triphosphate substrateswere not determined. Also shown in Table 1 is the pre-dominant (deoxy)ribonucleoside triphosphate(s) synthesized

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TABLE 1. Incorporation of purine nucleotide precursors into C psittaci AA Mp nucleic acids in C. psittaci-infected wild-type andHGPRT- APRT- cellsa

Results in:

PrecursoraddedbWild-type cells HGPRT- APRT- cells

PrecursoraddedbWidtpcelNucleotide Incorporation intod: Nucleotide Incorporation intod:availablec RNA DNA availablec RNA DNA

[2,8-3H]adenine ATP, GTP 470 317 235 158[2,8-3H]adenosine ATP, GTP 489 286 ATP, GTP 192 132[2,8-3H]deoxyadenosine (d)ATP, (d)GTP 362 236 dATP 195 121[2'-3H]deoxyadenosine *dAIP ND 0.3 *dATP ND 0.1(2,8-3H]hypoxanthine ATP, GTP 210 168 0.5 0.1[2,8-3H]inosine ATP, GTP 244 159 0.6 0.4[8-3H]guanine GTP 188 93 86 51[8-3H]guanosine GTP 173 97 78 48[8-3H]deoxyguanosine (d)GTP 175 110 dGTP 66 42[8-3H]xanthine 0.2 0.1 ND ND

a Parallel cultures of wild-type mouse cells and HGPRT- APRT- mouse cells were seeded, cultured, and infected with C. psittaci AA Mp as described inMaterials and Methods and references 20 and 26.

b The various 3H-labelled purine precursors were added at 22 h p.i. to achieve a final concentration of 0.3 FM, and incubation was continued for 2 h.c The primary nucleoside triphosphate(s) synthesized, from the added precursor, by the wild-type or HGPRT- APRT- host mouse cells. See the text for more

details. Asterisks indicates that the 3H label is at the 2' position on the deoxyribose of deoxyadenosine. -, precursor is not metabolized by the host cell.d Incorporation of radiolabel into C. psittaci RNA and DNA was determined as described in Materials and Methods and reference 19. All analyses were made

on duplicate dishes, with results varying by less than 10%. Results are expressed in 103 dpm/106 cells. ND, not determined.

from each precursor by the host cell and thereby madeavailable to chlamydiae. With the exception of [2'-3H]deoxy-adenosine and [8-3H]xanthine, all purine precursors testedwere readily incorporated into RNA and DNA of C psittaciAA Mp when wild-type mouse L cells were used as the host.When HGPRT- APRT- cells were used as the host, a

different precursor utilization pattern was observed. Both[2,8-3Hladenine and [8-3H]guanine were readily incorpo-rated into C. psittaci AA Mp RNA and DNA when HGPRT-APRT- cells were used as the host. Furthermore, [8-3H]gua-nosine, [8-3H~deoxyguanosine, and [2,8-3H]adenosine werealso incorporated into C. psittaci AA Mp RNA and DNA;however, [2,8-3H]hypoxanthine and [2,8-3H]inosine werenot utilized. [2,8-3H~deoxyadenosine labelled C. psittaci AAMp RNA and DNA, but [2'-3HHdeoxyadenosine did not getincorporated into parasite DNA.De novo purine synthesis. To test whether C. psittaci AA

Mp could synthesize purines de novo, we monitored theincorporation of [14C]glycine into nucleic acid bases. Ade-Fcells were used as the host for these experiments becausethey cannot de novo synthesize purines (25). MI and Cpsittaci AA Mp-infected Ade-F cells were incubated in thepresence of 1.2 ,uM [14C]glycine for 22 h, then the nucleicacids were extracted and acid hydrolyzed, and the resultingnucleobases were separated by HPLC. The HPLC eluentwas simultaneously monitored for UV absorbance and ra-dioactivity. As a control experiment we monitored theincorporation of [1"C]glycine into purines by wild-type CHOKi cells. As expected, wild-type CHO Ki cells incorporated[14C]glycine into adenine and guanine (Fig. la). In contrast,neither MI nor C psittaci AA Mp-infected Ade-F cellsshowed any significant radiolabelling of nucleic acid purines.

Incorporation of various purines into C. psittaci AA Mpnucleic acids. In wild-type mammalian cells exogenous hy-poxanthine is readily salvaged and gives rise to ATP andGTP. C. psittaci AA Mp-infected wild-type cells, but notHGPRT- APRT- cells (Table 1), readily incorporated exog-enous hypoxanthine into parasite RNA and DNA. Thisresult suggested that C. psittaci AA Mp could transportpurine ribonucleotide(s) from the host cell but could not

salvage hypoxanthine. To determine whether C psittaci AAMp was taking ATP and/or GTP from the host cell, weincubated MI and C psittaci AA Mp-infected wild-typemouse L cells in the presence of [2,8-3H]hypoxanthine, thenextracted and acid hydrolyzed the nucleic acids, and deter-mined the distribution of radioactivity into purine bases byHPLC analysis. The results shown in Fig. lb indicate thatradiolabel was detected in adenine and guanine, with C.psittaci AA Mp-infected cells incorporating approximatelyfour to five times more radiolabel than the MI control.The ability of the parasite to interconvert adenine and

guanine nucleotides was assessed by incubating MI and Cpsittaci-infected HGPRT- APRT- cells with [2,8-3H]ade-nine or [8-3H]guanine and then analyzing the total nucleicacids for distribution of radiolabel into purine bases. Asexpected, no radioactive peaks were detected from MIHGPRT- APRT- cultures incubated with [2,8-3H]adenine(Fig. 2a) or [8-3H]guanine (Fig. 2b). When C. psittaci AAMp-infected HGPRT- APRT- cells were incubated with[2,8-3H]adenine, radioactivity was detected only in nucleicacid adenine (Fig. 2a). Similarly, when infected HGPRT-APRT- cells were incubated with [8-3H]guanine, radioactiv-ity was detected only in nucleic acid guanine (Fig. 2b). Thedistribution of [2,8-3H]adenine and [8- H]guanine into purinedeoxynucleosides of DNA was also determined. MI and Cpsittaci AA Mp-infected HGPRT- APRT- cells were incu-bated in the presence of radiolabelled purine, then the DNAwas extracted and enzymatically hydrolyzed, and the distri-bution of radioactivity into purine deoxynucleosides wasanalyzed by HPLC. Results of these experiments indicatedthat radioactivity was detected only in deoxyadenosine from[2,8-3Hladenine-labelled C. psittaci AA Mp-infected cul-tures and in deoxyguanosine from [8-3H]guanine-labelledinfected cultures (data not shown).We also incubated C. psittaci AA Mp-infected HGPRT-

APRT- cells with [2,8-3H]deoxyadenosine and then deter-mined the distribution of radiolabel into purine deoxynucle-osides of DNA as well as purine bases of RNA. The resultsof these experiments indicated that only deoxyadenosine of

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0.60 b

45-

30-

15 4

2 4 6

Retention Time (min)FIG. 1. (a) Incorporation of ["4C]glycine acid into nucleic acid

bases of logarithmically growing wild-type CHO K1 cells (- - -), MIpurine auxotrophic Ade-F cells (.), and C. psittaci AA Mp-infected Ade-F cells (-). (b) Incorporation of [2,8-3H]hypoxan-thine into nucleic acid bases of MI (.) and C psittaci AAMp-infected (-) wild-type mouse L cells. All cell cultures wereincubated in the presence of radiolabel for 20 h. Radiolabel wasadded to C. psittaci AA Mp-infected cultures at 2 h p.i. Nucleicacids were extracted and acid hydrolyzed to nucleobases whichwere separated and analyzed by HPLC. For details, see Materialsand Methods and reference 19. The identity of the radioactive peakswas confirmed by monitoring the A2us of known adenine (A) andguanine (G) standards for nucleobases. The positions of standardsare indicated by arrows.

DNA and adenine of RNA were radiolabelled (data notshown).

Effect of dCF and 8-AG on the incorporation of adenosineby C. psittaci AA Mp. dCF and 8-AG are inhibitors of the twomain mammalian cell purine (deoxy)nucleoside catabolicpathway enzymes, (deoxy)adenosine deaminase and purinenucleoside phosphorylase, respectively (3, 15, 17). In thepresence of dCF and 8-AG, less adenosine is degraded tohypoxanthine and, as a result, more substrate is left foradenosine kinase. Results of experiments determining theeffect of dCF and 8-AG on [2,8-3Hjadenosine incorporationinto RNA and DNA of C. psittaci AA Mp-infected HGPRT-

E0.U~

Retention Time (min)FIG. 2. Incorporation of [2,8-3H]adenine (a) or [8-3H]guanine (b)

into nucleic acid bases of MI (...) and C. psittaci AA Mp-infected( ) HGPRT- APRT- cells. For details, see Materials and Meth-ods and reference 19. The positions of nucleobase standards areindicated by arrows.

APRT- cells and AK- cells are shown in Table 2. In thepresence of dCF and 8-AG incorporation of [2,8-3H]adeno-sine into C. psittaci AA Mp RNA and DNA increasedapproximately twofold. Similar results were obtained re-gardless of which cell line was used as the host.

Effect of excess purines on adenine and guanine incorpora-tion. Results of competition experiments between [2,8-3H]adenine or [8-3Hjguanine and a 50-fold-higher concentrationof unlabelled purines are shown in Table 3. These experi-ments were done with C. psittaci AA Mp-infected HGPRT-APRT- cells to minimize host purine base metabolism.Radiolabelled-adenine incorporation was inhibited by excessadenine but essentially unaffected by hypoxanthine or gua-nine. Similarly, radiolabelled-guanine incorporation was in-hibited by excess guanine but was unaffected by adenine orhypoxanthine. We also found that excess (deoxy)adenosineinhibited adenine incorporation and excess (deoxy)guano-sine inhibited guanine incorporation into C psittaci AA Mpnucleic acid (data not shown).

Purine metabolism by C. psittaci AA Mp RB extract. APRT

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TABLE 2. Effect of dCF and 8-AG on incorporation ofadenosine into nucleic acids of C. psiutaci AA Mp-infected

HGPRT- APRT- and AK- cells

Incorporation ofHost cell Addition to mediuma [2,8-3H]adenosine

line intob:dCF 8-AG RNA DNA

HGPRT- - - 184 142APRT-

+ + 381 228AK- - - 153 126

+ + 329 201

a dCF and 8-AG were not added (-) or were added (+) to achieve finalconcentrations of 200 and 7.4 FM, respectively, 20 h prior to the addition ofradiolabel.

b Parallel cultures of HGPRT- APRT- mouse cells or AK- CHO cells wereseeded, cultured, and infected with C. psittaci AA Mp as described inMaterials and Methods and references 20 and 26. [2,8-3H]adenosine wasadded at 22 h p.i., to achieve a final concentration of 0.3 PM, and incubationwas continued for 2 h. Incorporation of radiolabel into C. psittaci RNA andDNA was determined as described in Materials and Methods and reference19. All analyses were made on duplicate dishes, with results varying by lessthan 10%. Results are expressed in 103 dpm/106 cells.

and guanine phosphoribosyltransferase (GPRT) activitieswere detected in extract prepared from highly purified C.psittaci AA Mp RBs (Table 4). In contrast, no phosphoribo-syltransferase activity was detected with hypoxanthine asthe substrate. Purine (deoxy)ribonucleoside-cleaving activ-ity was also detected. These activities, cleaving (deoxy)ad-enosine and (deoxy)guanosine, were dependent on the pres-ence of phosphate and so are likely phosphorylases;however, purine (deoxy)nucleoside hydrolase activity mayalso be present. While guanosine kinase activity was notfound, a low level of adenosine kinase activity was detected.There was no measurable adenosine phosphotransferase oradenosine deaminase activity. Purified sham suspensions,prepared from MI mouse L cells, had less than 1% of theactivity detected for any of the above enzymes (data notshown).

TABLE 3. Effect of excess purine base on the incorporation ofadenine or guanine into nucleic acid of C. psittaci AA Mp-

infected HGPRT- APRT- cells

Addition to growth mediumb Incorporation of

Precursor addeda precursor into

Precursoruanine Hyaddedae nucleic acid'Adenine Guanine Hypoxanthine (% of control)

[2,8-3H]adenine - - - 301 (100)+ 57 (19)- + - 280 (93)- - + 319(106)

[8-3H]guanine - - - 118 (100)+ - - 127 (108)- + - 31 (26)

+ 114 (97)a Tritium-labelled adenine or guanine was added at 22 h p.i. to achieve a

final concentration of 0.3 pM, and incubation was continued for 2 h.b Competing adenine, guanine, or hypoxanthine was added (+), to achieve

a final concentration of 15 pM, at the same time as radiolabelled precursor (-,

no addition).I Radiolabelled precursor incorporation into total nucleic acid ofC psittaci

AA Mp-infected HGPRT- APRT- cells was determined as described inMaterials and Methods and reference 19. All analyses were made on duplicatedishes, with results varying by less than 10%. Results are expressed in 103dpm/106 cells.

TABLE 4. Purine salvage enzyme activities in crude extractprepared from highly purified C. psittaci AA Mp RBs

Sp actEnzyme type (nmol/min/mg

of protein)a

PhosphoribosyltransferasesAdenine................... 0.78 ± 0.18Hypoxanthine ................... NDGuanine................... 1.6 ± 0.24

Nucleoside cleavingAdenosine................... 3.4 +0.66Deoxyadenosine ................... 2.9 ± 0.42Guanosine................... 3.9 ± 0.77Deoxyguanosine ................... 2.2 ± 0.61

Nucleoside kinasesAdenosine................... 0.25 ± 0.08Guanosine................... ND

Enzyme reaction conditions were as described in Materials and Methods.Each value represents the mean ± standard deviation from three experiments.ND, not detected.

DISCUSSION

Using the purine auxotroph Ade-F line as a host, we haveconfirmed the absence of de novo purine synthesis in Cpsittaci AA Mp by showing that the parasite cannot incor-porate exogenous glycine into nucleic acid purines. Rickett-sia (35, 36), another obligate intracellular bacterium, and allparasitic protozoa studied to date (9) also lack the ability tosynthesize purines de novo.

In the accompanying paper (19) we reported that C.psittaci AA Mp did not take pyrimidine ribonucleotides fromthe host cell and, as a result, the parasite depends onpyrimidine salvage for survival. The situation appears quitedifferent with purines. Because of the extensive network ofdegradation and salvage enzymes present in wild-type mam-malian cells (17), the majority of the purine precursors testedgive rise, either directly or indirectly, to ATP and/or GTP.The only exceptions are xanthine, which is not anabolized inmammalian cells (32), and [2'-3H]deoxyadenosine, whichcan give rise only to labelled dATP. When growing inwild-type host cells, C psittaci AA Mp could utilize allpurine precursors with the exception of [8-3H]xanthine and[2'-3H]deoxyadenosine. Furthermore, even though C. psitt-aci AA Mp could not salvage hypoxanthine (see below),HPLC profiles of acid-hydrolyzed nucleic acids extractedfrom infected wild-type cells incubated with [2,8-3H]hypo-xanthine showed radioactivity in adenine and guanine.Taken together these results suggest that, similar to otherchlamydia isolates (4, 11, 20, 31), C. psittaci AA Mp canobtain purine ribonucleotides but not deoxyribonucleotidesdirectly from the host cell. The lack of direct utilization ofdeoxynucleosides, coupled with the incorporation of exoge-nous adenine and guanine into C. psittaci AA Mp DNA,suggests the presence of ribonucleotide reductase, an en-zyme known to exist in C. trachomatis L2 (30).

Results obtained from precursor incorporation studieswith HGPRT- APRT- cells suggested that C psittaci AAMp possessed a variety of purine salvage pathways. [2,8-3H]adenine and [8-3H~guanine, but not [2,8-3H]hypoxan-thine or [8-3H]xanthine, were readily incorporated into Cpsittaci AA Mp RNA and DNA despite the fact that no ATPand/or GTP was made by the HGPRT- APRT- host cells. Incontrast, C. trachomatis L2 (20, 26) and C. psittaci 6BC (2)cannot utilize any of the purine bases in HGPRT- and/orAPRT- cells. These results suggested the presence of phos-

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C. PSI7TACI PURINE METABOLISM 4667

phoribosyltransferases in C. psittaci AA Mp, and theirpresence was confirmed by enzyme assay. Results fromcompetition experiments indicate that adenine and guaninedo not compete with each other for incorporation intoparasite nucleic acids. In addition, hypoxanthine does notcompete with adenine or guanine, and inosine was notincorporated by C. psittaci AA Mp. Together these findingssuggest that C. psittaci AA Mp may contain two phospho-ribosyltransferases, one for adenine and one for guanine,and that neither recognizes hypoxanthine. This is furthersupported by the fact that we could not detect hypoxanthinephosphoribosyltransferase (HPRT) activity in C. psittaci AAMp RB extracts.Mammals have two distinct phosphoribosyltransferases,

one for adenine and one for hypoxanthine and guanine (22).In many procaryotes there are separate phosphoribosyl-transferases for each of the four bases adenine, guanine,hypoxanthine, and xanthine (24). The salvage of preformedpurines is essential for most parasitic protozoa because theylack the ability to synthesize purines de novo (9). The vastmajority of protozoan parasites can utilize hypoxanthine (9).An interesting exception is Giardia lamblia, which, like C.psittaci AA Mp, contains APRT and GPRT but no HPRT(34).The results of competition experiments and precursor

utilization studies in HGPRT- APRT- cells also suggest thatC psittaci AA Mp lacks the enzymes necessary for inter-

conversion of purine ribonucleotides (AMPtIMPtGMP).Additional support for this hypothesis comes from HPLCprofiles of acid-hydrolyzed nucleic acids, extracted from[2,8-3H]adenine- or [8-3Hlguanine-labelled C. psittaci AAMp-infected HGPRT- APRT- cells, which show a completelack of interconversion between adenine and guanine nucle-otides. Of the protozoan parasites studied, only Tricho-monas vaginalis (14), G. lamblia (34), and Entamoebahistolytica (8) are reported to lack the enzymes required tointerconvert purine nucleotides. Given that C. psittaci AAMp lacks the enzymes for purine interconversion, it is notsurprising that it lacks HPRT.When the parasite was growing in AK- or HGPRT-

APRT- cells, dCF and 8-AG increased incorporation of[2,8-3H]adenosine into C. psittaci AA Mp nucleic acids. Thissuggested the presence of adenosine kinase, which has beenverified by enzyme assays. Adenosine kinase has beenreported in a wide variety of parasitic protozoa (9). Resultsfrom precursor incorporation experiments and HPLC pro-files (data not shown) indicate that deoxyadenosine can beutilized by C. psittaci AA Mp only after it is cleaved toadenine. Consistent with this was the detection of (deoxy)adenosine-cleaving activity in RB extracts. This activity wasphosphate dependent and therefore was likely a phosphory-lase. Adenosine was also readily cleaved to adenine, pre-sumably by the same enzyme. Apparently, C. psittaci AAMp can obtain adenine nucleotides by (i) taking ATP directly

FIG. 3. Proposed model for the acquisition of purines in C. psittaci AA Mp. C. psittaci AA Mp lacks the ability to transport, from the hostcell cytoplasm, the purine deoxyribonucleoside phosphates within the dashed box and lacks the intracellular enzymes necessary for themetabolism of the purine bases and nucleosides within the solid box. C. psittaci AA Mp has an ATP-GTP transport system(s) and theintracellular enzymes required for the metabolism of adenine, (deoxy)adenosine, (deoxy)guanosine, and guanine. Single arrows do notnecessarily imply one-step reactions. Abbreviations: AXP, GXP, dAXP, and dGXP, purine (deoxy)ribonucleoside phosphates; dA,deoxyadenosine; dG, deoxyguanosine; AK, adenosine kinase; RR, ribonucleotide reductase. For simplicity the chlamydial vacuolarmembrane has been omitted from the model. The functional significance of the vacuole and its membrane is currently unknown.

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from the host, (ii) salvaging adenine via APRT, or (iii)salvaging adenosine via adenosine kinase. It must be empha-sized, however, that since ATP also enters by way of theATP translocase (on a one-for-one exchange with ADP) (13)and there is likely free mixing of the total adenine nucleotidepool in an RB, it is hard to conclusively say that nettransport of ATP is occurring.

Since no guanosine kinase activity was detected andguanosine-cleaving activity and GPRT activity were present,we conclude that guanosine must be hydrolyzed to guaninebefore incorporation. Similarly, deoxyguanosine must behydrolyzed to guanine prior to incorporation. As mentionedabove, C. psittaci AA Mp also appears to be capable oftaking GTP directly from the host cell.A summary and model for purine salvage in C psittaci AA

Mp are shown in Fig. 3. There is no de novo synthesis ofpurines. Hypoxanthine, inosine, and xanthine are not uti-lized. Adenine is salvaged by APRT, and guanine is salvagedby GPRT. There is an adenosine kinase but no guanosinekinase. Deoxyadenosine, deoxyguanosine, and guanosineare apparently readily hydrolyzed to adenine and guanine byphosphorylase(s). In addition, C. psittaci AA Mp appears totransport GTP and ATP directly from the host cell. There-fore, in contrast to enzymes of pyrimidine salvage (19), thepurine salvage enzymes described may not be essential forparasite survival. We are presently trying to isolate Cpsittaci AA Mp mutants that are deficient in some of theseenzymes.

ACKNOWLEDGMENTS

We thank R. Brunham, G. Tipples, and B. Qin for helpfuldiscussions.

This research was supported by a grant provided from the MedicalResearch Council of Canada (G.M.). Grant McClarty was a recipi-ent of a Manitoba Health Research Council Scholarship, andHuizhou Fan is a recipient of a Manitoba Health Research CouncilStudentship.

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