Acquisition and synthesis of folates by obligate intracellular bacteria of the genus Chlamydia

Acquisition and synthesis of folates by obligate

intracellular bacteria of the genus Chlamydia.

H Fan, … , R C Brunham, G McClarty

J Clin Invest.

1992;

90(5)

:1803-1811.

https://doi.org/10.1172/JCI116055

.

We undertook studies focused on folate acquisition by Chlamydia trachomatis L2,

Chlamydia psittaci 6BC, and C. psittaci francis. Results from in situ studies, using wild-type

host cells, confirmed that C. trachomatis L2 and C. psittaci 6BC are sensitive to

sulfonamides whereas C. psittaci francis is resistant. In addition C. trachomatis L2 and C.

psittaci francis were inhibited by methotrexate in situ whereas C. psittaci 6BC was not. In

contrast to C. trachomatis, neither C. psittaci strain was affected by trimethoprim.

Surprisingly our results indicate that all three strains are capable of efficient growth in

folate-depleted host cells. When growing in folate-folate-depleted cells C. psittaci francis becomes

sensitive to sulfonamide. The ability of all three strains to carry out de novo folate synthesis

was demonstrated by following the incorporation of exogenous [3H]pABA into intracellular

folates and by detecting dihydropteroate synthase activity in reticulate body crude extract.

Dihydrofolate reductase activity was also detected in reticulate body extract. In aggregate

the results indicate that C. trachomatis L2, C. psittaci francis, and C. psittaci 6BC can all

synthesize folates de novo, however, strains differ in their ability to transport preformed

folates directly from the host cell.

Research Article

Acquisition and Synthesis of Folates by Obligate

Intracellular Bacteria of the Genus Chlamydia

Huizhou Fan, Robert C. Brunham, and Grant McClarty

DepartmentofMedical Microbiology, UniversityofManitoba, Winnipeg,Manitoba, CanadaR3E 0W3

Abstract

We undertook studies focused on folate acquisition by

Chla-mydia

trachomatis

L2,

Chlamydia psittaci 6BC,

and C.

psit-taci

francis. Results from in situ studies, using wild-type host cells,

confirmed

that

C.

trachomatis L2 and C. psittaci 6BC are sensitive to sulfonamides whereas C.

psittaci

francis is resis-tant. In addition

C.

trachomatis

L2 and C.

psittaci

francis were inhibited by

methotrexate

insitu whereas C.

psittaci

6BC was not. Incontrast to C.

trachomatis,

neither C. psittaci strain was

affected

by

trimethoprim.

Surprisingly our results indicate that all three strains are capable of efficient growth in

folate-de-pleted

host cells. When growing in

folate-depleted cells

C.

psit-taci

francis becomes sensitive to sulfonamide. The ability of all three strains to carry out de novo folate synthesis was demon-stratedby

following

the

incorporation

of exogenous

13HJpABA

intointracellular folates and by detecting

dihydropteroate

syn-thase activity in reticulate body crude extract.

Dihydrofolate

reductase activity was also detected in reticulate body extract. Inaggregate the results indicate that C.

trachomatis

L2, C.

psittaci

francis,

andC.

psittaci

6BCcan all

synthesize

folates de novo, however, strains differ in their ability to transport

preformed folates directly from the host cell. (J.

Clin.

Invest.

1992.90:1803-1811.)

Key

words:

parasite

*

dihydropteroate

synthase *

dihydrofolate

reductase * sulfonamide * methotrexate

Introduction

Chlamydiae

are

obligate intracellular bacterial parasites

that

infect

a

wide

range of host cells and are the causative agents of a

variety of

human, nonhuman mammal, and avian diseases ( I

-5). Chlamydiae display

a

unique developmental

cycle

consist-ing of

an

infectious extracellular metabolically

inert elemen-tary

body

(EB)'

and a

noninfectious intracellular

metaboli-cally active reticulate body (RB). The function of the

EB

is

to

survive transit

in

the

extracellular environment until

ahost

is

Addresscorrespondenceto Dr.GrantMcClarty, Department of Medi-calMicrobiology, University ofManitoba, Room 504, 730 William Avenue, Winnipeg, Manitoba, Canada R3E OW3.

Receivedfor publication 6February1992andinrevisedform21 May 1992.

1. Abbreviations used in this paper: CHO, Chinese hamster ovary; DHFR,dihydrofolate reductase; DHPS,dihydropteroatesynthase; EB, elementary body; H2folate, dihydrofolate; H4folate, tetrahydrofolate;

ID_0,

antimetabolite concentration requiredtoreduceincorporationof radiolabel into DNA by 50%; pABA, para-aminobenzoic acid; RB, reticulate body.

encountered.

Once

inside

a host cell EBs

differentiate

to RBs, which divide by binary fission within the confines of a mem-brane

bound cytoplasmic

vacuole.

Chlamydiae

have an abso-lute

nutritional

dependency on the host cell to provide a wide variety of

intermediates

of metabolism. After multiple rounds

of

division

RBs

differentiate

back to EBs, which are

subse-quently released from

the host cell to

begin

a new infection cycle.

The genus

chlamydiae

is currently

divided into

three

spe-cies, Chlamydia

trachomatis, Chlamydia

psittaci, and

Chia-mydia

pneumoniae (5-7). Classically, the species have been

differentiated by inclusion morphology

(diffuse

vs.

compact),

presence or absence

of

glycogen

within

the

inclusion

(as

deter-mined

by

iodine staining),

and

differing sensitivity

to

sulfa

drugs.

C.

trachomatis is sensitive

to

sulfonamides

andthey

de-velop

diffuse

glycogen

containing inclusions.

In contrast

C.

psittaci, with the

exception of strain 6BC, is resistant

to

sulfa

drugs

and

they give rise

todense

inclusions

that lack

glycogen.

C.

pneumoniae is also resistant to sulfonamides

and

they

yield

dense

inclusions

that do not

contain glycogen.

Sulfonamides

are

structural

analogues

and

competitive

an-tagonists of

para-aminobenzoic

acid

(pABA),

and thus pre-ventnormal

bacterial

use

of

pABA

for

the de novo

synthesis of

folic

acid (8).

More

specifically, sulfonamides

are

competitive

inhibitors of the bacterial

enzyme

dihydropteroate synthase

(DHPS), which

catalyzes the

incorporation of

pABA

into

di-hydropteroic acid,

the

immediate

precursor

of folic acid.

As

such,

microorganisms

that are

sensitive

to

sulfonamides

must

synthesize

their

own

folates and

those

that

can use

preformed

folates

are

resistant

to

sulfa

drugs.

Mammalian

cells are not

affected

by

sulfonamides

becausethey must

obtain

preformed

folates from

dietary

sources.

The

biologically active form of folate is tetrahydrofolate

(H4folate),

which functions

as a

one-carbon unit carrier in

a

variety of biosynthetic reactions, including methionine

biosyn-thesis,

thymidylate synthesis,

and

purine biosynthesis (9,

10).

Thymidylate

synthesis is unique

among the

biosynthetic

reac-tions

that

employ

H4folate

as

cofactor

in that

it involves

not

only

the

transfer of

aone-carbon

moiety

but also the

oxidation

of

the

carrier (

11

).

The

dihydrofolate (H2folate)

formed is

con-verted back

to

H4folate

by the enzyme

dihydrofolate

reductase

(DHFR). H4Folate is again

converted toa

cofactor

by

the

ad-dition

of

a

one-carbon unit

as

catalyzed by serine

hydroxy-methyltransferase.

Together these

reactions form

the

thymidy-late

cycle

as

represented schematically

in

Fig.

1.

Both

C.

psittaci

and

C.

trachomatis

have been shown to

contain folates different from

those present in

their

host cells (

12, 13).

In

addition

several

studies

haveshown that

chlamyd-iae cannotuse

medium-supplied thymidine ( 14-17), however,

they

can

incorporate

exogenously supplied

uridine into

para-siteDNA

(

1

5-17).

Taken

together

these

results

imply

that

chla-mydiae

must

contain

a

thymidylate synthase. Recently

we

have

shown

that

C.

trachomatis

does encode a

thymidylate

J.Clin.Invest.

©The AmericanSocietyfor Clinical

Investigation,

Inc.

0021-9738/92/11/1803/09 $2.00

FULATIE FOLATE

de

_novoSAVG

SYNTHESIS SALVAGE

GTP

PABA glutamate

v

\,\

H2Pt

_CH20PP@

_T H2Pteroate . _{FAH2 FAH2}

at0) s (I® (methotrexate)

Sulfonamide

methotrexateo dTMP

trimethopnm 2 dUMP

FAH4 C

OH2-

FAH4

serine _glycne

THYMIDYLATE CYCLE

Figure

1.Schematic

_diagram

of the

thymidylate

cycle

andits relation

tofolate denovo

synthesis

and

salvage.

Not all

possible

routesof metabolismare

included, just

major

routesrelevanttothis

_study.

Squiggly

arrowsrepresent stepsinhibited

_{by sulfisoxazole,}

trimetho-prim,

andmethotrexate.

Important

enzymesarenumberedasfollows:

1,

dihydropteroate

synthase;

2,

dihydrofolate

reductase;

3, serine

hy-droxymethyltransferase;

4,

thymidylate

synthase;

and5,amembrane

transport systemfor folates.

FAH2,

dihydrofolate;

FAH4,

tetrahydro-folate;

and

_CH2-FAH4,

5,

10-methylene tetrahydrofolate.

synthase

for

_synthesis

of dTMP from dUMP

(17),

thus

con-firming

that a

folate-requiring

reaction exists in

_chlamydiae.

The sulfonamide inhibition studies mentioned above

_suggest

that

_C.

trachomatis is

_capable

of de novo folate

synthesis

whereas

C.

psittaci

is not

₍

12,

18-20).

Since

C.

psittaci

also

requires

folates for

_{thymidine synthesis}

it has been

suggested

that

_{they likely}

have the

_capacity

to

_transport

folates

_directly

from

the

host

cell

_{cytoplasm (5, 12,}

_19).

Since folates

are

essen-tial for

_{chlamydial growth}

and

_important

intaxonomic

classifi-cationwewantedto

_clarify

thenumerousinconsistencies inthe

existing

literature

_concerning

folate

metabolism

in

_chlamydiae

(for

reviewsee reference

_5).

Ourresults indicate that both

C.

trachomatis and

_C.

_psittaci

can

synthesize

folates de

_novo,

how-ever,thereappearstobea

considerable difference

in

their

abil-ity

toobtain

preformed

folates from thehost.

Methods

Materials.

_{[6-3H]Uridine}

₍₂₀

_Ci/mmol),

_[3,5-3H]pABA

₍₅₀

_Ci/

mmol),

[3',5'7,9-3H]dihydrofolic

acid

(H2folate,

38

_Ci/mmol)

and

[

3',5'7,9-3H]

folic acid

₍₄₀

_Ci/mmol)

were

purchased

from Moravek Biochemicals

(Brea,

CA).

Unlabeled

pABA,

para-aminobenzoyl-glu-tamic acid

_{(pABA-glutamate),}

folic

acid,

H2folate,

tetrahydrofolic

acid

(H4folate),

5-formyl-tetrahydrofolic

acid

_{(5-CHO-H4folate),}

sulfisox-azole, methotrexate,

and

_trimethoprim

were

_purchased

from

_Sigma

Chemical Co.

_{(St. Louis, MO).}

_5,

_{10-Methylenetetrahydrofolic}

acid

(5,

10-CH2-H4folate)

was

synthesized

from

_H4folate

in the_{presence of}

formaldehyde

as

_previously

described

(

17).

10-Formyl-tetrahydrofolic

acid

_{(I0-CHO-H4folate)}

was

synthesized

from 5-CHO-H4folate

_by

published

procedures

(21).

_{6-Hydroxymethyl-7,8-dihydropterin}

pyro-phosphate

(H2PtCH2OPP)

was

kindly

_{provided by}

C.

_Allegra,

Medi-cine

Branch,

National Cancer

_{Institute, Bethesda,}

MD.All other chemi-calswereof the

highest

obtainable

_purity.

Celllines and culture conditions. The

_wild-type

Chinese hamster ovary

(CHO)

Kl

celllinewas

purchased

from American

_Type

Culture Collection

_{(ATCC; Rockville, MD).}

The mutant CHO

Kl

subline

deficientin DHFR

_activity

was

kindly

provided

_by

R.Johnson

(22).

The

wild-type

mouseL cellswere

_{kindly provided by}

_K.

_Coombs,

De-partment of Medical

Microbiology, University

of Manitoba

(Winni-peg,Manitoba,Canada).

ThemouseL cellswereroutinelycultured insuspensionwith

mini-mumessential medium supplementedwith 10% fetal bovineserum

and 0.2 mMglutamine.The CHO KI cellsweremaintainedas

mono-layersat370Conthe surface ofplastictissue culture flasks

(Coming

GlassWorks,Coming Medical andScientific,Coming,NY).Since the CHOKI cell line is auxotrophic for proline, itwasmaintained in mini-mumessential mediumsupplemented with 10% fetal bovine serum, 0.2 mM glutamine, and 0.3 mMproline. CHO DHFR- cellswere

maintainedasmonolayer cultures in thesamemedium

supplemented

with 10% fetal bovine serum, 0.3 mMproline, 0.3 mMglycine,30zM hypoxanthine, and 30 MM thymidine. All cell lineswere

_routinely

checked formycoplasma contamination.

Chlamydiaestrains andpropagation. C. trachomatis strain

L2/

434/Buwasoriginally obtained from C. C.Kuo, University of Wash-ington (Seattle,WA) and has been maintained inourlaboratorysince that time. C. psittacipsittacosis strain 6BC(catalog No. ATCC VR-125) and meningopneumonitis strain francis(catalog No. ATCC VR-122; also called C. psittaciCal-1O)werepurchased from American Type Culture Collection. The authenticity of these strainswas

periodi-cally confirmed by serologic typing with monoclonal antibodies

kindly

performed by A. Andersen, United States Department ofAgriculture,

National Animal Disease Center (Ames, IA). All chlamydial EB stocks weregrown inmonolayers of mouse L cells and purified byRenografin

density gradient centrifugation ( 17). EB infectivitywastiteredas

previ-ously described( 17). Confluentmonolayers(3-4 x 106 cells per 5-cm plate) of wild-type CHO Kl and DHFR-deficient CHO cellswere in-fected at a multiplicity of infection of threetofiveinclusion-forming

units per cell, which resulted in 90-100% infection with little host cell toxicity.C. trachomatis L2 andC.psittaci6BC and francisweregrown in the presence of cycloheximide, 1 ug/ml ofculture medium,as

previ-ously described ( 16, 17). Mock-infected host cell culturesweretreated in the same fashionasinfected cultures except thatchlamydiaewere

notadded.

Suspension cultures ofmouseL cells were usedashost for prepar-ing large batches of RBs, whichwerehighly purified through

Renogra-findensity gradients as described previously ( 17). Purified RBswere

lysed andextractfor enzyme assays was preparedasdescribedby Fanet

al. ( 17).

Measurementof chlamydialDNAsynthesis activity in situ.

Chla-mydialDNA synthesis activitywasmeasured in situ by monitoring the

incorporationof[6-3H]uridineinto DNA in the presence of

cyclohexi-mideaspreviously described ( 16, 17). This DNA synthesis assay specif-icallymeasureschlamydial DNAsynthesis activity and providesan

accurateand reliable estimation of chlamydial growth ( 16). Unless otherwiseindicated,all resultsareexpressed in I03dpmincorporated

per 106 cells. For antimetabolite-orantagonist-treatedcultures,

incor-porationvaluesareexpressedaspercentages of the amount of radiola-belincorporated into DNA by untreated controls. The ID50 value is the antimetabolite concentration requiredtoreduceincorporation of

ra-diolabel into DNA by 50%.

Incorporation of[3H]pABAintochlamydialfolatesin situ. Results frompreliminary experiments indicated that[3H]pABAincorporation bychlamydiaewasgreater if the host CHO Kl cells were depletedof intracellular folates. Asaresult, all

[3H]pABA-labeling

experiments

weredone with CHO Kl cells that had been starved for folates before

radiolabeling.TodepleteCHO Kl cells of intracellular folates, cultures

weregrown for 10 passages in folate- andpABA-free Dulbecco modi-fied Eagle medium (DME H-2 I), obtained from the Tissue Culture

Facility, UniversityofCalifornia (San Francisco, CA), supplemented with 10%extensively dialyzedfetal bovine serum, 0.3 mM proline, 0.3 mMglycine,30 MMhypoxanthine, and 30 ,Mthymidine. Since

chla-mydiaeareauxotrophicforpurine ribonucleotides,glycine, and pro-line itwasnecessarytokeep these supplements in the culture medium after infection withchlamydiaeandduringthesubsequent

radiolabel-ing

period.

[3H]pABA-labeling experiments were performed with parallel flasks(150cm2)of mock-infected andchlamydiae-infected

folate-de-pleted

CHO Kl cells

(30-40

x 106 cells per 150-cm2flask).

atelyafter infection with chlamydiaethe cellmonolayer wasrinsed with Hanks'buffered saline, then15mlof DME H-21medium

supple-mentedwith 10% dialyzed fetal bovine serum, 0.3 mMproline,0.3 mM glycine,30MuMhypoxanthine,I

Ag/ml

cycloheximide,and30 sCi

[3H]pABAwas added to each flask. The cultures, both mock- and chlamydiae-infected,wereincubated at370Cfor 24handthen the cells were harvested andintracellular folates were extracted as previously described (23).Briefly,the monolayerswerewashedfive times with ice-cold PBS then the cells were harvested in 1 ml of PBS by scraping thesurface of theflask witharubberpoliceman. The cells were heated at1000Cfor 1 min in 3% sodium ascorbate, pH 6.0, and 3% 2-mercap-toethanol andthen the celldebris were removed by centrifugation. The cell supernatantwastreated with 0.5mlofpartially purified hog kidney polyglutamate hydrolase, prepared according to the method of McMartin et al. (24), at 370C for 30 min to convert all folates to monoglutamates. After an additional boiling with ascorbate and 2-mercaptoethanol, the folates were extracted into methanol using a C-18cartridge (Sep-Pak; Waters Chromatography Division, Milford, MA) and concentrated underasteady streamof nitrogen. The dried samplewasdissolved in 100 lof 5 mM PICA(Waters Chromatogra-phyDivision) and theindividual folates were resolved by HPLC using aC-8 ,uBondapak column (12.5 cm; Whatman International,Clifton, NJ) underisocraticconditions; the mobile phase consisted of 22.5% methanol and 77.5% 5 mM PIC A, pH 5.5. Isotopeincorporation into individual folates was determined byin-line radioactive flow detection ( 171 detector; BeckmanInstruments, Fullerton, CA). The identity of theradioactive peaks wasconfirmed by simultaneously monitoring the

A290

( 1066 UV detector;Beckman Instruments) of known unlabeled folate standards thatwerecoinjected with each sample. Datawere col-lected and processedwith anIBMPC 50using Beckman System Gold software.

AssayofDHPS activity in vitro. DHPSwasassayed bypreviously described procedures (25) with the following modifications. The DHPS assay mix contained, in afinal volume of 100

_Al,

100 mM Tris-HCl (pH 8.5), 5 mM NaF, 10 mMMgCl2, 10

_AM

H2PtCH2OPP, 1MM

[3H]pABA

(10

MCi/ml),

and 5 mMdithiothreitol. Thereaction wasinitiated by the addition of150

ig

RB extractprotein as a source of enzyme and thenwasallowed toproceedat 37°C for 60 min. The reactionwasterminated by the addition of 100A of 3% ascorbate/3%

2-mercaptoethanolfollowed byboiling for 1 min. The resulting precipi-tate wasremovedbycentrifugation(14,000g for 10min)and then 50

jAl

of the supernatantwasspottedonto3X30-cmstripsof 3MM chro-matography paper (Whatman International). Thestripswere devel-oped inadescending chromatographytankusingamobilephasebuffer of 0.1 MKH2PO4, pH 7.0. Once the buffer front had traveled 20 cm, the paperstripwasremoved from thechromatography tank,theorigin containingthelabeledproductwascutfrom thestrip, dried,andplaced

inascintillation vialcontaining 10 mlcocktail(Universol;ICN Bio-medicals, Inc., Costa Mesa, CA). The vialwasleftat roomtemperature for 16 h and thenitwascounted inaliquidscintillationcounter(LS 5000;BeckmanInstruments).

AssayofDHFRactivity invitro. DHFR assayswere carriedout

essentiallyasdescribedbyBaccanarietal.(26).Thecompletereaction mixturecontained, inatotalvolume of 100

AI,

50 mMTris-HCl (pH 7.5), 1 mMdithiothreitol, 200,uMNADPH2, 100MM

_[3H]H2folate

(10

MCi/ml)

or

[3H]folate

(10

MCi/ml),

and 150

Mg

ofRBextract

proteinas a sourceof enzyme. The reactionwasallowedtoproceedat

30°C for 10 min and then the reactionwasterminatedbytheaddition of 100 AL of 3%ascorbate/3%2-mercaptoethanolfollowedby

boiling

for 1 min.Precipitatedproteinwasremovedby

centrifugation

andthe radiolabeled folicacid,H2folate,andH4folatepresent in the superna-tantwereresolved by HPLCusingaC-18

MBondapak

column(12.5 cm; Whatman International) under isocratic conditions; the mobile phase consisted of5 mMPIC A, 10mM(NH4) H2PO4 (pH 7.3),20% methanol,and5%acetonitrile. Theidentity of the radioactive folate peakswasconfirmed bysimultaneously monitoringtheA290of known

folate, H2folate,andH4folatestandardscoinjectedwith each sample. Datawerecollected and

analyzed

asdescribedabove.

Results

Effect

ofvarious

inhibitors offolate metabolism

on

chlamydiae

growth. Initiallywewantedtodeterminetheeffects ofvarious

inhibitors of folate

metabolismonthe growthof C.trachomatis andC. psittaci. Chlamydial growthwasmonitored by measur-ing theincorporation of[3H ]uridine intoDNAinthepresence

of the eucaryotic protein synthesis inhibitor cycloheximide ( 16). For historicalreasons weused the commonlystudied C.

trachomatis strain

L2 as well as C. psittaci psittacosis strain 6BC and C. psittaci meningopneumonitis strain francis (fre-quently referred to as C. psittaci Cal- 10). Three drugs that targetfolate metabolismweretested.Sulfisoxazole,a

competi-tive inhibitor of dihydropteroate synthase, inhibits denovo fo-late synthesis (8); trimethoprim, an inhibitor of bacterial DHFR thatenterscells by simple diffusion (27);and

metho-trexate,anaminopterin analogue that inhibits both mamma-lian and bacterial DHFR (28, 29).Invivomethotrexateisonly

effective against cells that haveatransport

system(s)

forfolates (28,30).

Results of experiments determining the effect of various concentrations of these three inhibitorsonchlamydialgrowth inwild-type CHO KIcellsareshownin Fig. 2.Inkeeping with earlierfindings ( 12, 18-20), sulfisoxazolewas aneffective

in-hibitor of

both C.

trachomatis

strain L2 and C.psittacistrain 6BC growth. The concentration of sulfisoxazole required to

inhibit DNA synthesis by 50%

(ID50)

was

0.4,uM

for C. tracho-matis L2 and 0.5 MM

for

C. psittaci 6BC. Also inagreement

with previousreports(5, 31 )wefoundthatsulfisoxazolehad

noeffectonC. psittaci francis DNA synthesis. Trimethoprim was

effective

against C.

trachomatis

L2, havinganID50of0.5

,uM.

Incontrastneither of the

C.

psittaci

strains

were

sensitive

to

trimethoprim.

Both

C.

trachomatis

L2 andC. psittaci fran-ciswereinhibited by methotrexate, having

IDm

valuesof 3.2

and0.3

,M,

respectively. Growth of C. psittaci 6BCwas unaf-fectedby methotrexateevenatconcentrationsashighas 100

MM (data

not

_shown).

Since

the

chlamydial ID50

values

for

methotrexatearemuch higher than the ID50 values for mam-malian cell lines (28, 29) it is difficultto determine whether methotrexate inhibits chlamydiae directlyorindirectly viaan effectonthehost cell line. This is particularly relevant when oneconsiders that methotrexate inhibits denovopurine biosyn-thesisin mammalian cells(28, 29)andchlamydiaeare

auxo-trophic for purine ribonucleotides (4, 5). To determine

whether methotrexate directly affects chlamydiae replication,

weusedaDHFR-deficient CHOcell lineas ahosttosupport parasite growth.Asaresult of the DHFRdeficiencythis cell

line isunabletoregenerateH4folatefromH2folateand is

unaf-fectedbymethotrexate( 17, 22).Methotrexatewas aneffective

inhibitorof

chlamydial

growth in this cell line(Fig. 3). The concentration of methotrexate requiredto inhibit C.

tracho-matis L2 and C.

psittaci

francis DNA

synthesis activity by

50% in this celllinewas4.8 and 2.0 MM, respectively. Aswasthe

case with wild-typecellsashost, C. psittaci6BCgrowth was

unaffected by methotrexate in the DHFR-deficient cell line

(datanotshown).

Growth

of chlamydiae

in host cells

depleted offolates

and pABA. To evaluate therequirementofchlamydiaefor

exoge-nous folates we tested the ability of the parasite togrow in

wild-type CHO KI cells with depleted intracellular folate

pools.To achievemaximal folatedepletionwegrewtheCHO

sup-Figure 2. Effect of

sulfi-A soxazole, trimethoprim,

and methotrexateon

[6-3H]uridine

incorpo-ration into DNA in (A)

C. trachomatis L2-, (B)C. psittaci6BC-,

and (C)

C. psittaci

francis-infected wild-type CHO K1 cells(4.0

I X 106cells per plate cultured in the presence of 1

Mig

cycloheximide/

ml). Theindicated concentrationsof sulfi-B soxazole

(o),

trimetho-prim

(-A),

or metho-trexate (o) were added immediately after infec-tion withchlamydiae,

i.e., 2 hpostinfection (p.i.). Radiolabeled uri-dine(final concentra-tion 0.3

MM)

wasadded at 20 hp.i.Cell culture =t conditions, chlamydiae

infection procedure, C and

3H-labeling

proce-dureareasdescribed

in Methods and refer-ence 17. Theamount of radiolabel

incorpo-rated into DNA is ex-pressedas apercentage of theuninhibited con-trol. Thefollowingare s 100%control values: C. 00 1 ;.0 5.0

10sI

trachomatis

L2-in-0.1 0 10.0

fected cultures,

Concentration (pM) 158,954±17,963dpm/ 103 cells; C. psittaci 6BC-infected cultures, 178,692±22,753 dpm/ 106cells; and C. psittaci francis-infected cultures, 143,650±13,098 dpm/ 106cells. The data represent theaverageoftwodeterminations. Bars, SD.

plemented

with

hypoxanthine,

proline, glycine, and thymi-dine. Results

presented

inTable I indicate that all three chla-mydial strainsgrew aswell in CHO Kl cells extensively starved

for folates and pABA asthey did in host cells that had been

previously cultured

incomplete medium. The observation that

C.

trachomatis

L2

and C.

psittaci

6BC

couldgrow

in

folate-de-pleted host cells is in keeping with their sulfa sensitivity and

furthersupportsthe suggestion that thesetwostrainscan

syn-thesizefolatesdenovo.However, given that C.psittacifrancis was resistant to sulfonamide (a result that suggested that it

could obtain preformed folates from the host)we were

sur-prised that it couldgrowsowell in host cellsdepletedof folates.

Tohelp clarify thisparadoxwechecked the sulfonamide

sensi-tivity of C. psittaci francis growing in hostcells depleted of

folates.Theresults clearly showed that, incontrast tothe find-ings with folate-repletecells,C.psittacifranciswashighly

sus-ceptible to sulfisoxazole inhibition when grown in

folate-starvedcells(Fig. 4). With C. psittaci francis-infected folate-starvedcellstheID50 for

sulfonamide

was 1.0

gM.

Incorporation of

[3H]pABA

into

chlamydialfolates.

To

di-rectly test

if

chlamydiae

could

synthesize

folates de

novo we

determined

the

ability

of all three strains

to

incorporate

radio-labeled

pABA

into

their folate pools.

For

these studies

all

chla-mydial strains

weregrown in

folate-depleted

CHO

Kl

cells in

the presence of

_[3HIpABA.

Fig.

5

shows

typical

elution

profiles

obtained

after HPLC

separation

of radiolabeled folates

ex-tracted from

C.

trachomatis L2-, C.

psittaci 6BC-,

and C.

psittaci

francis-infected folate-starved

CHO Kl

cells. As

ex-pected, CHO

KI

cells

were

unable

touse

[3H]pABA

for the

synthesis

of folates (data

not

shown). All three

chlamydial

strains incorporated

[3H]pABA

into their folate

pools;

how-ever, there are

obvious differences in the elution

profiles

ob-tained for reduced folates when

C. trachomatis

and

C. psittaci

are

compared.

The

major

reduced folates

produced by

C.

tra-chomatis

L2

were

H4folate

and

l0-CHO-H4folate;

variable

amounts

of

5-CH3-H4folate

or

5,l0-CH2-H4folate

(the

two

peaks coeluted)

were also

routinely

detected.

In contrast, the

predominant reduced folate produced by C. psittaci strain 6BC

was

lO-CHO-H4folate;

with variable

amounts

of

H4folate,

5-CHO-H4folate,

and

5-CH3H4folate

also

being

detected.

C.

psittaci strain francis

produced

variable

amounts

of

10-CHO-H4folate, H4folate,

and

5-CH3folate and/or

5,lO-CH2-H4folate. Sulfisoxazole (

IOgM)

was

effective

in

preventing

the

incorporation of

[3HI

pABA into folates by

all

three

chlamyd-ial

strains.

° 100. 80 0-0

80-

\

Z

0 c

60-OD

0 0 C OD

20-.2

co

cc

0

I

0

1.0

2.5

5.0

10.0

Concentration

(pM)

Figure 3. Effect of methotrexate on

[6-3H]uridine

incorporation into DNA in

C.

trachomatis L2-

(.)

and

C.

psittaci francis (o) -infected

DHFR-deficient CHO

KI

cells (4 x

106

cells per plate cultured in complete medium supplemented with proline, glycine, and hypoxan-thine in thepresence ofI

Ag cycloheximide/

ml). The indicated con-centrations of methotrexate were added to the culture medium at 2 h

p.i.Radiolabeleduridine was added at 20 h p.i. Cell culture condi-tions, chlamydiae infection procedure, and

3H-labeling

conditions are asdescribed in Methods and reference 17. The amount of radiolabel incorporated into DNA is expressed as a percentage of the uninhibited control.Thefollowing are 100% control values:

C.

trachomatis L2-infected cultures, 124,763±14,980dpm/106cells and

C.

psittaci francis-infected cultures, 142,822±16,587dpm/106cells. The data represent the average of two determinations. Bars, SD.

iCDO'L 10 so-1-0 20.. ~~~~~I 100 -3 z 0 U z 0 cs co 0 C. 0 0 0D co cc 80- 60- 40- 2 0-100 80- 60- 40-

20-%p%F

TableI.

Effect

of

Exogenous Folate onthe Growth of Chlamydiae in Chinese Hamster Ovary

KJ

Cells

DNAsynthesist

Culture Mock C. trachomatis C.psittaci C. psittaci

Cell line medium* infected L2 6BC francis

CHO Ki Folatecontaining 0.9±0.1 162.6±16.8 197.9±10.7 189.4±9.2

CHO KI Folatefree 0.7±0.2 151.2±12.1 191.0±16.1 197.4±16.8

* Before chlamydial infection, CHO

KI

cells were cultured in complete medium containing 2.2

MM

folate or were depleted of intracellular folates by passage in folate- and pABA-free medium. For details, see methods and text. *_{The effect of exogenous folate onchlamydiaegrowth was}

assessedby measuring[6-3H]uridine incorporation into DNA at 20 h p.i. Folate-replete or folate-depleted CHO Ki cells were either mock- or chlamydiae-infected confluent monolayers (3.0 x 106cells per plate cultured in the presence of 1 ug cycloheximide/ml). For details see Methods andreference 17. Each value represents the mean±SD from two experiments. Results are expressed in103dpm per106cells.

Detection

of

in vitro DHPS activity in

chlamydial

extracts. To

conclusively

show that chlamydiae contain DHPS, we pre-paredextracts

from

highly purified C. trachomatis L2 and

C.

psittaci strains

6BC

and

francis

RBs and then assayed for DHPSactivity in vitro. DHPS activity was measured by follow-ing the synthesis ofdihydropteroate from [ 3H

_I

pABA and

6-hy-droxymethyl-7,8-dihydropterin

pyrophosphate. We consis-tentlydetected DHPS activity using RB extract prepared from any one

of

the threechlamydial strains as a source of enzyme. RBextracts prepared from C

trachomatis

L2,

C.

psittaci6BC,

"

100.

C

0

<

80

-Z

0

0

.

601

Cu 0 0

._2

~0

C.) co

a:

0-0.1

1.0

5.0

10.0

Concentration

(pM)

Figure 4. The effect of sulfisoxazoleon [6-3H]uridine incorporation

into DNA inC. psittaci francis-infectedfolate- andpABA-depleted

wild-typeCHO

Kl

cells(4X 106cellsperplate cultured in folate-andpABA-freemediumsupplementedwith

proline,

glycine,and hy-poxanthinein the presence of 1Ag

cycloheximide/ml).

Theindicated concentrations of sulfisoxazolewereaddedtothe culture mediumat

2 hp.i.Radiolabeleduridinewasaddedat20hp.i.Cellculture

con-ditions,chlamydiae infectionprocedure,and3H-labeling procedure

were asdescribed in Methods and reference 17.The amountof

ra-diolabelincorporatedintoDNAisexpressedas apercentage of the uninhibited control. Thefollowingis the 100% control value: C.

psit-tacifrancis-infected cultures,169,094±15,386dpm/ 106 cells.The data represent the average oftwodeterminations.Bars, SD.

and C.

psittaci

francis catalyzed the synthesis of 3.1±0.5,

6.5±1.6, and 2.8±0.3 pmol dihydropteroate product/min

per mg

protein, respectively.

The

DHPS

activity

detected

from

all

strains

was

inhibited

. 90% by

10 MM

sulfisoxazole.

0.5- Figure 5. (A)

Ultravio-E3 6 A let(A290) absorption

a)124 5 I profileof folate

stan-cN

ll l dardsseparated by

o

0.25-

HPLC. Peaks identified

al l q \ 11 7 were:

1,

pABA;

2,

D X S l J wt A

pABA-glutamate;

3,

10-n" I

CHO-H4folate;

U J C 4,

H4fo-0

late;

5,

5-CHO-H4folate;

2.0 6,

H2folate;

7,

5-CH3-4 B

H4folate

and/or

5,10-CH2-H4folate. (B)

Ra-3 dioactiveprofileafter

21

2 incorporation of

[3H]-1.0-

Nu \

pABA into folates by

C.

trachomatisL2-, (C)

|1

\ 7 C.psittaci

6BC-,

and

(D)

C.

psittaci francis

rr

...*...*...

0

-infected

folate-and

0- pABA-depleted

wild-typeCHO

_Kl

cells

3 C _(30.0 x 106 cells per

C, ^flaskcultured in

folate-0 _{ll andpABA-freemedium}

5

5 supplementedwith

pro-line, glycine, and

hypo-4

|¢ xanthine in the presence

1 5 8 of 1

Mg

cycloheximide/

ml). Cell culture

condi-... ....; _ tions,

;.

chlamydiae

infec-0 _{tion procedure,}

[3H]-pABA-labeling

3 D _{conditions, folate}

ex-tractionprocedure,and HPLC conditionsare

0.5 2 4 asdescribed in Methods

1 5 and text. Solidline

rep-7 resentsradioactivity de-tected from folates

iso----...

_---

lated from

chlamydiae-0 5 _{10 15 20}

₂₅

_{infected control cultures} and the brokenline

rep-Retention time (minutes) _{resentsradioactivity}

Reversal of

suifisoxazole

inhibition by pABA andfolates. It has beenshown withnumerousexperimental systems that the inhibitory action of sulfa drugs can be antagonized by pABA (8). Results presented in Fig. 6 indicate that, with folate- and pABA-depleted CHO Ki cells as host, the growth inhibition

caused

by

1 MM

sulfisoxazole

(Fig.

6

A-C,

hatched

bars)

onall three strains ofchlamydiaecanbecompletelyreversedby 0.1

,uM

pABA

(Fig.

6

A-C,

cross-hatched

bars).

With

C.

psittaci

francis, 10 MM folic acid completely reversed the inhibition

caused by

1 MM

sulfisoxazole

(Fig.

6

C, square-checked bar).

Even 1 MM folic acid was sufficient to reverse 1 MM sulfisoxa-zole-induced inhibition (data not shown). In contrast, folic

acid was much less effectiveatreversingtheeffectsof sulfa on

C.

trachomatis L2

and

C. psittaci

6BC, showing essentially

no

antagonism

at 10 MM

(Fig.

6 Aand

B, square-checked bars)

and only partial reversionat 100 MM(datanotshown). We found that the inhibitory effects of 1 MMsulfisoxazole on C.

trachomatis

L2

and

C.

psittaci

francis could be

partially

and completely reversed, respectively, by 1 MuM 5-CHO-H4folate

(Fig. 6

A and

C, dotted

bar).

At a concentration of 10

MM,

75

120-CAB 0

*,0 100

z

80-0

Cd

0 CL

C ~ ~ A

Figure 6. (A) Effect of exogenous pABA and folates on the sulfisoxa-zole induced inhibition of C. trachomatis L2, (B) C. psittaci 6BC, and (C) C. psittaci francis DNA synthesis. Chlamydiae-infected folate and pABA-depleted wild-type CHOKlcells (4.0 x 106 cells per plate cultured in folate- and pABA-free medium supplemented with pro-line, glycine, and hypoxanthine in the presence of 1jig cyclohexi-mide/ml) were incubated in the absence or presence of sulfisoxazole, pABA, and/or folates. The indicated componentswere added at 2 h p.i., and then at 20 h p.i. the cultures were pulsed with radiolabeled uridine. Cell cultureconditions, chlamydiae infection procedure, and 3H-labeling conditions are as described in Methods and reference 17. The amount of radiolabel incorporated into DNA is expressed as a percentage of the uninhibited controls. The 100% control values are as follows: C. trachomatis L2-infected cultures, 138,034±15,984

dpm/ 106 cells; C. psittaci 6BC-infected cultures, 165,428±19,683

dpm/ 106 cells, and C. psittaci francis-infectedcultures,

167,592±20,849dpm/ 106 cells. The data represent the average of two determinations. Bars, SD. Chlamydiae-infected control cultures, (solid bars); chlamydiae-infected cultures plus 1.0jiM sulfisoxazole, (hatched bars); chlamydiae-infected cultures plus 1.0jiM

sulfisoxa-zole and 0.1

jiM

pABA, (cross-hatched bars); chlamydiae-infected

cultures plus 1.0

jiM

sulfisoxazole and 1.0

jiM

5-CHO-H4folate, (dotted bars); and chlamydiae-infected cultures plus 1.0jiM sulfi-soxazole and 10.0

jiM

5-CHO-H4folate, (open bars).

5-CHO-H4folate completely reversedtheinhibitoryeffectsof1

,qM

sulfisoxazole on C. trachomatis L2 (Fig. 6 A, openbar).

Surprisingly,

even though methotrexate did not inhibit the growth of C. psittaci strain 6BC (Fig. 2),wefound that 10jM 5-CHO-H4folate couldreversetheeffects of1

gM

sulfisoxazole (Fig. 6

B,

openbar).

Ourcommercial

preparation

of folic acidwas 98%pure,

therefore itwaspossible thatasmallamountofcontaminating pABA mayhave been present in our folate preparations. Since pABA was 2 100 times more effective atantagonizing sulfa activity compared with 5-CHO-H4folate itwaspossible that the reversionbrought about by folateswasreallycausedby

contam-inating pABA. To eliminate this possibilitywetested theability of folinic acid to reverse the inhibitoryaction of

trimethoprim/

sulfisoxazole against C. trachomatis L2. The results clearly show that5-CHO-H4folate can antagonizethe combined

activ-ity of the DHFR inhibitor trimethoprim and the DHPS inhibi-torsulfisoxazole (Table II). As expected folic acid could not

reversetrimethoprim inhibition of C. trachomatisL2growth (data not shown).

Detection

of

invitro DHFR

activity

in

chlamydial

extracts.

To directly demonstrate that chlamydiae encode DHFR we

conducted in vitro assays for DHFR using extract prepared from highly purified RBs as a sourceofenzyme(Table III).As

acontrol

experiment

we conductedDHFRassayswith crude

extract

prepared

from

logarithmically

growing wild-type mouse L

cells.

We

consistently

detected DHFR

activity

in ex-tracts prepared from C. trachomatis L2 as well as C. psittaci 6BC and francis RBs. The formation oftetrahydrofolate was

dependent on the presence of RB extract, NADPH2, and H2fo-late (data not shown). No

activity

wasdetected iffolic acid was used as substrate. Mock infected mouse cell extract had essen-tially no DHFR activity.

Similar

to insitu

results

we

found that

trimethoprim

was a

highly

effective inhibitor of C.

trachomatis

L2 DHFRactivity in

vitro,

however,

it

was less

effective

against

C.

psittaci 6BC

Table II.Effect of

5-CHO-Hfolate

onTrimethoprim/

Sulfisoxazole-induced

Inhibition

of

C. trachomatis Growth

Addition to growthmedium* DNAsynthesist

5-CHO-H4 C.irachomatis Percent Sulfisoxazole Trimethoprim folate infected cells activity

MM

- -

145.2±20.4

100

1.0 1.0 - 19.0±8.5 13

1.0 1.0 1.0 67.4±8.6 46

1.0 1.0 10.0 147.4±12.3 102

*_{Theindicated concentration of sulfisoxazole, trimethoprim, and/or} 5-CHO-H4folatewas added tothe culture medium immediately after infection (2hp.i.) withC.trachomatis L2. *_{The effect of the various}

agentsonchlamydiae growth was assessed by measuring

[6-3H]-uridineincorporation intoDNA at 20 h p.i. Confluent monolayers of CHOKI cells(3.0 x 106cellsper plate cultured in the presence of 1 jg cycloheximide/ml)wereinfected withC.trachomatis L2. For detailsseeMethods andreference 17. Each value represents the mean±SD from two experiments. Results are expressed in 103dpm

per

106

cells. § Theeffect of the various agents on the incorporation ofradiolabel intoDNAis expressed as the percentage of the

Table III. Dihydrofolate Reductase Activity in Crude Extracts Preparedfrom Logarithmically Growing Host Cells

and

Purified

ChlamydiaeReticulate Bodies

Sourceofenzyme'

Mock-infected

Log growing mousecells mousecells C.trachomatisL2 C. psittaci 6BC C.psitaaci francis

DHFR DHFR DHFR DHFR DHFR

Substrate* Inhibitort activityl % activity % activity % activity % activity %

H2folate 3.65±0.56 100 <0.01 100 2.34±0.56 100 0.37±0.05 100 1.87±0.39 100

TMP 4.18±0.71 114 ND' ND 0.05±0.02 2 0.25±0.04 65 1.53±0.11 82

MTX 0.45±0.06 12 ND ND 1.26±0.15 54 0.03±0.02 8 1.03±0.12 55

Folic acid 0.26±0.05 7.1 ND ND <0.01 <1 <0.01 <1 <0.01 <1

*_{The complete DHFR reaction mix contained either}

100I

MM

H2folate

_or

100

MM

_{folic acid as the substrate. For details of assay conditions, see}

Method. t To assess the effect of trimethoprim (TMP) and methotrexate (MTX) on DHFR activity, a complete reaction mix minus substrate was incubated in the presence of 10 nM TMP or 1 nM MTX for 10 min at

4VC.

Thereaction was initiated by the addition ofH2folate

substrateandincubation was at 30'C. I_{Crude extracts were prepared from the various sources as described in reference 17.} 11Reactions were carried out at30Cfor 10 min. DHFR activity is expressed as nmolH4folateformed/mg protein permin. Each value represents the mean±SD from two experiments. 'ND,not determined.

and francis DHFR

activity

in vitro

(Table III).

In

agreement

with previous observations that methotrexate is an effective in vitro inhibitor of DHFR activity from most bacterial and

mammalian

sources(32), we found that it was active against in vitro DHFR activity of all three chlamydial strains.

Discussion

Although there was no

conclusive evidence

until recently ( 17),

it

has been

assumed

that

chlamydiae require folates for the

generation

of

thymidine

nucleotides(5, 15). Furthermore,

it

has

generally been accepted that all C.

trachomatis

strains

are

sensitive

to

sulfonamides

whereas all

C. psittaci strains, with

the

exception of 6BC,

are

resistant

tosulfaaction

(

1, 4,

5, 12,

18-20). Areasonable

explanation for

these

findings

wasthat C.

trachomatis

strains

andC.

psittaci 6BC

were

capable

of

syn-thesizing folates

denovowhereas the remainder of the C.

psit-taci

strains

were not.Unlike the

simple interpretation required

to

explain

the

action of sulfonamides against chlamydiae

ithas proven

difficult

to

interpret

results

obtained

using antifols

that target DHFR

(5, 19, 33). One

mustbecautious when

compar-ing various

results

obtained with

DHFR

inhibitors

because many

different

host cell systems,

i.e.,

chicken

embryo

and

tis-sueculture cell

lines

from different

mammalian

species,

have been

employed

and it

has

recently

been shown that variations in

methodology markedly influence chlamydial antimicrobial

susceptibility

results

(34).

In agreement

with earlier

in

situ

observations

(

12, 18),

we

found

that

C.

trachomatis

L2 and C.

psittaci

6BC were

sensi-tive

to

sulfonamides.

Furthermore,

C.

psittaci

franciswas

resis-tant to

sulfonamide,

so

long

asfolateswerepresentinthe

cul-ture

medium.

In

addition

ourresults indicate that

trimetho-prim

was

active

against C. trachomatis

L2insitu but hadno

effect

against

either

C.

psittaci

strain. Methotrexate inhibited the

growth of C. trachomatis

L2 andC.

psittaci

francis but did

noteffect

C.

psittaci 6BC

growth.

Previously

Morgan

(

19)

re-ported

thatC.

psittaci

6BCwassensitiveto

aminopterin.

We have

consistently

found

that the

growth

of

C.

psittaci

6BC is

resistant

to a

wide

variety

of

antifols, including

aminopterin

(data

not

shown), methotrexate,

and

trimethoprim.

It is

sur-prising

that

C.

psittaci 6BC was completely resistant to metho-trexate

especially since

we

found that

5-CHO- [

H4] folate

could reverse

sulfa

inhibition of

C.

psittaci 6BC growth. One possible

explanation for this finding is

that

C.

psittaci 6BC may only be capable

of transporting

reduced folates.

This

would also

ex-plain

why

folinic acid

was much more effective at reversing

sulfa action

against C.

psittaci

6BC

than was

folic acid.

It

is

of

interest to note that Pediococcus cerevisiae is resistant to

ami-nopterin

and methotrexate but it requires 5-CHO-

[H4]folate

for growth

and hasa

specific

transport system

for

reduced

fo-lates (35).

Since

C.

trachomatis

L2

and

C. psittaci 6BC

are

sensitive

to

sulfa action it

has

long

been

assumed that

they must

be capable

of

denovo

folate

synthesis.

We have

confirmed this by showing

that:

(a) both

these

strains

readily

grow

in

folate-depleted CHO

Kl

cells,

(b)

both

incorporate

exogenous

[3H]pABA

into

fo-lates, and (c) extracts

prepared from highly purified

RBs

of

both

strains contain

DHPS

activity.

Most

interestingly

our

re-sults

clearly indicate that,

when

growing

in

folate-depleted

CHO

Kl

cells,

the

normally sulfonamide-resistant

C.

psittaci

francis

becomes

sensitive

to the drug. The

ability

to

incorpo-rate

[3H

]

pABA into folates

and the

detection of in vitro

DHPS

activity

provides

conclusive evidence for

the

existence of

a

de

novo

synthesis pathway

in

C.

psittaci francis.

As

expected,

sul-fisoxazole

prevented

the

in situ

incorporation

of

[3H] pABA

into

folates in all three

chlamydial

strains.

With folate-starved CHO

Kl

cells

as hostwe

found

that

sulfonamide inhibition of

all

chlamydial

strains could

be

re-versed by the

addition of

exogenous

pABA. 5-CHO-H4Folate

was able to

antagonize sulfonamide activity

in all three

strains,

a result that supports the

suggestion

that all

strains

have the

capacity

to transport reduced

folates.

Interestingly,

even

though

itwasless

effective

on amolar

basis

than

5-CHO-H4fo-late, folic

acid could

also

effectively

reverse

sulfa inhibition of

C. psittaci francis

growth

butwasmuch less

effective

at

antago-nizing sulfa activity

against

C. trachomatis

L2 or C.

psittaci

6BC.

Although

many

interpretations

are

possible,

we

believe

that this result

likely

reflects

the

fact

that the host cell

folate

transporter hasalower

affinity

for

folic acid

than 5-CHO-

[

ob-taining

both reduced and nonreduced forms of folates from the hostcell than are

C. trachomatis L2

or

C. psittaci 6BC. This

hypothesis is also

supported by

the

observation

that when fo-latesare present in the culture medium C.

psittaci

francis does not

depend

on de novo folate

synthesis (as

indicated

by

sulfa

resistance) whereas C. trachomatis

L2 and

C.

psittaci

6BCdo (as

indicated by

sulfa

sensitivity).

We

consistently found

that

there

was a difference in the

composition

ofthe intracellular folate

pools

between C.

tracho-matis and C.

psittaci species. Although

reduced folates were

predominant

in

both

chlamydial species,

theC. trachomatis L2

folate pool

was

dominated by

H4folate

whereas the C.

psittaci

6BC folate

pool

wasdominated

by

reduced folates

carrying

a one-carbon unit

(i.e.,

10-CHO-H4folate).

At the present time the

significance

of this difference isnotknown.

However,

it is

interesting

that,

using the classical

microbiological

assay with

Lactobacillus casei and

Pediococcus cerevisiae,

Colon and Moulder(

31

)

also

detected

a

difference

in the

composition

of

chlamydial species folate pools.

Our

ability

to

detect

DHFR

activity

in RB extracts

from

all three

chlamydial

strains confirm that the

parasite

does encode a DHFR. Results

of

in

vitro

DHFR assays indicate that the enzyme from all three strains is sensitive to methotrexate. In agreement

with in situ results,

we

found

that

trimethoprim

was

a

good inhibitor

of C. trachomatis L2 DHFR

activity

in vitro. Both

strains of C.

psittaci were

resistant

to

trimethoprim in

situ and the

in vitro

DHFR

activities of

thesetwo

strains

wereless'

sensitive

to

trimethoprim

than was

C.

trachomatis

L2 DHFR

activity

in

vitro. However, the difference

in in

vitro sensitivity

between the

species

was not as great as

might

have been

ex-pected

given

the

large difference

in

trimethoprim sensitivity

in

situ. This

raises

the

possibility

that there could

be

differences in

the way C. trachomatis

and

C. psittaci metabolize

trimetho-prim

or

in

their intrinsic

permeability

to

the drug.

It

is

evident from

the

results

presented that no

simple

con-cluding

statement canbe made

with regard

to

folate

metabo-lism in chlamydiae.

The vast

majority of

free-living bacteria,

both

pathogenic

and

nonpathogenic,

lack transport

system(s)

for

preformed folates

and thus

depend

on de novo

synthesis.

Recent

studies

on a

variety of parasitic

protozoa have

shown

that

both de novo

synthesis

and

salvage pathways for

folates

exist in eucaryotic intracellular parasites (36-40). Intracellular

parasites

spend most

of their lives within host

cells

rich in

nu-trients.

To

obtain

nondiffusible nutrients from their host,

in-tracellular

parasites

must

evolve (or

obtain) suitable

transport systems.

Once

a

parasite has

acquired the ability

to

obtain

com-plex

nutrients from its

host

it

can

afford

to loose thecapability to

synthesize

the

given nutrient

denovo. There would

likely

be a

period of time

when both

capacities

overlap and in some

instances it

may be necessary for the parasite to retain both

pathways.

We believe that folate metabolism in chlamydiae is

currently

at

this

stage in evolution. All strains have an absolute

dependence

on

folates

for de novothymidine synthesis. Origi-nally

this

need waslikely fulfilled via de novo folate synthesis as suggested by the ability ofall strains tested to incorporate exoge-nous

pABA

into folates.

More recently chlamydiae has

ob-tained the

necessarygenetic

information

to allowthem to

ac-quire

preformed

folates

from their host. The current status of

the

folate

transport system(s) appears to vary from strain to strain. At one extreme

C. psittaci

francis fulfills its needs for

folate

_{strictly by transporting preformed}

host

folates but does

retain the

capacity

to

synthesize

denovo.At the other extreme C.

psittaci

6BC appears to

depend

almost

exclusively

onits ability to synthesize folates de novo, however, it also has the

capacity

totransport reduced folate toalimited extent. Much of the

discrepancy

in the literature

regarding

the effectiveness of antifols

against

chlamydiae,

both in situ and

clinically,

prob-ably

results from the

parasites

variable

dependence

onthe two folate

acquisition pathways.

Wethank C.AllegraforprovidinguswithH2PtCH20PPand A.

Ander-senforperformingthe monoclonal

antibody typing.

This researchwassupportedbygrantsprovided from the Manitoba

Health ResearchCouncil(G.

McClarty)

andMedical Research

Coun-cilof Canada(G. McClartyand R.

Brunham).

Grant

McClarty

isa

recipient ofa Manitoba Health Research Council

Scholarship

and

HuizhouFan isarecipient ofaManitoba Health Research Council Studentship.

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