Molecular Basis Defining Human Chlamydia trachomatisTissue TropismA POSSIBLE ROLE FOR TRYPTOPHAN SYNTHASE*

Received for publication, April 23, 2002, and in revised form, May 7, 2002Published, JBC Papers in Press, May 13, 2002, DOI 10.1074/jbc.M203937200

Christine Fehlner-Gardiner‡, Christine Roshick‡, John H. Carlson§, Scott Hughes§,Robert J. Belland¶, Harlan D. Caldwell§, and Grant McClarty‡�

From the ‡Department of Medical Microbiology, University of Manitoba and National Microbiology Laboratory,Health Canada, Winnipeg, Manitoba R3E 0W3, Canada and the §Laboratory of Intracellular Parasites and¶Laboratory of Human Bacterial Pathogenesis, Rocky Mountain Laboratories, NIAID, National Institutes of Health,Hamilton, Montana 59840

Here we report the cloning and sequencing of a regionof the chlamydiae chromosome termed the “plasticityzone” from all the human serovars of C. trachomatiscontaining the tryptophan biosynthesis genes. Our re-sults show that this region contains orthologues of thetryptophan repressor as well as the � and � subunits oftryptophan synthase. Results from reverse transcrip-tion-PCR and Western blot analyses indicate that thetrpBA genes are transcribed, and protein products areexpressed. The TrpB sequences from all serovars arehighly conserved. In comparison with other tryptophansynthase � subunits, the chlamydial TrpB subunit re-tains all conserved amino acid residues required for �reaction activity. In contrast, the chlamydial TrpA se-quences display numerous mutations, which distinguishthem from TrpA sequences of all other prokaryotes. Allocular serovars contain a deletion mutation resulting ina truncated TrpA protein, which lacks � reaction activ-ity. The TrpA protein from the genital serovars retainsconserved amino acids required for catalysis but hasmutated several active site residues involved in sub-strate binding. Complementation analysis in Escherchiacoli strains, with defined mutations in tryptophan bio-synthesis, and in vitro enzyme activity data, with clonedTrpB and TrpA proteins, indicate these mutations resultin a TrpA protein that is unable to utilize indole glycerol3-phosphate as substrate. In contrast, the chlamydialTrpB protein can carry out the � reaction, which cata-lyzes the formation of tryptophan from indole and ser-ine. The activity of the chlamydial Trp B protein differsfrom that of the well characterized E. coli and Salmo-nella TrpBs in displaying an absolute requirement forfull-length TrpA. Taken together our data indicate thatgenital, but not ocular, serovars are capable of utilizingexogenous indole for the biosynthesis of tryptophan.

Members of the genus Chlamydia are obligate intracellularbacteria that possess a unique biphasic developmental cycleconsisting of an extracellular, infectious, but metabolically in-active elementary body (EB)1 and an intracellular, non-infec-tious, replicative form called the reticulate body (RB). Chlamy-dial infection involves the attachment of the EB to a host celland its subsequent internalization into a membrane-boundvesicle known as the chlamydial inclusion. Within this inclu-sion the EB differentiates into an RB, which then multiplies bybinary fission. The daughter RBs then redifferentiate into EBsthat are able to initiate new rounds of infection after release byhost cell lysis (1).

Chlamydia consists of three species that are importantpathogens of humans. Chlamydia psittaci strains are primarilypathogens of birds and lower animals, but humans are occa-sional hosts of avian-acquired psittacosis (2, 3). The two majorpathogens of humans are Chlamydia trachomatis and Chlamy-dia pneumoniae. C. pneumoniae is an important cause of com-munity-acquired pneumoniae (4) and has been linked to theetiology of chronic heart disease (5–7). C. trachomatis com-prises a family of antigenically related yet divergent organismsserologically classified into 15 distinct serovars based on anti-genic variation of the major outer membrane protein of theorganism (8). Curiously, the 15 different C. trachomatis sero-vars exhibit an extraordinary specificity in tissue tropism. Forexample, serovars A, B, Ba, and C are pathogens of the eye,where they infect columnar epithelial cells of the conjunctivaecausing trachoma, a chronic inflammatory disease that is theleading cause of preventable blindness in the world (2, 3, 9).The trachoma serovars are rarely isolated from the genitaltract. On the other hand, serovars D-K are sexually transmit-ted pathogens that infect columnar epithelial cells of the gen-ital tract (2, 3). These infections are the most common bacterialcause of sexually transmitted disease and in females causepelvic inflammatory disease. The sexually transmitted diseaseserovars can cause neonatal conjunctivitis but have not beenassociated with blinding trachoma. Furthermore, although in-fections with both ocular (A-C) and genital serovars (D-K) arenon-invasive and are restricted to the mucosal epithelium,those caused by the sexually transmitted lymphogranulomavenereum (LGV) serovars (L1, L2, and L3) are invasive (2, 3).The LGV strains penetrate the submucosal tissue, infect mono-

* This work was supported by research Grant GR-13301 from theCanadian Institutes of Health Research (to G. M.) and a postdoctoralfellowship from the Manitoba Health Research Council (to C. F. G.).The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby marked“advertisement” in accordance with 18 U.S.C. Section 1734 solely toindicate this fact.

The nucleotide sequence(s) reported in this paper has been submittedto the GenBankTM/EBI Data Bank with accession number(s)AY096805(serovar Atrp) AY096806 (Batrp), AY096807 (Ctrp), AY096808 (Dtrp),AY096809 (Etrp), AY096810 (Ftrp), Y096811 (Gtrp), AY096812 (Htrp),AY096813 (Itrp), AY096814 (Jtrp), AY096815 (Ktrp), AY096816 (L1trp),AY096817 (L2trp), and AY096818 (L3trp).

� To whom correspondence should be addressed: Dept. of MedicalMicrobiology, University of Manitoba, Winnipeg, Manitoba R3E 0W3,Canada. Tel.: 204-789-3307; Fax: 204-789-3926; E-mail: mcclart@cc.umanitoba.ca.

1 The abbreviations used are: EB, elementary body; RB, reticulatebody; LGV, lymphogranuloma venereum; IFN, interferon; IGP, indoleglycerol 3-phosphate; MEM, minimal essential medium; FCS, fetal calfserum; nt, nucleotide(s); RT, reverse transcription; IFU, inclusion-form-ing units.

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 277, No. 30, Issue of July 26, pp. 26893–26903, 2002Printed in U.S.A.

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cytes and macrophages, and disseminate to the local draininglymph nodes, where they produce a chronic granulomatousdisease. The factor(s) that controls the non-invasive/invasiveproperties of these genital serovars has been correlated withthe production of a chlamydial cytotoxin (10); however, viru-lence factors that decide the distinctive ocular and genital tracttissue tropisms have yet to be discovered.

T cells play an important role in the development of adaptiveimmunity against C. trachomatis mucosal infection, and inter-feron � (IFN-�) is key to this protective function (11–13). Themechanism by which IFN-� controls infection in vitro is byinterfering with the replicative capacity of the parasite (14, 15).Through binding of the IFN-� receptor, IFN-� transcriptionallyactivates the expression of indoleamine-2,3-dioxygenase, whichdegrades L-tryptophan to L-kynurenine (16, 17). This cytokine-mediated host cell response deprives intracellular chlamydialRBs of tryptophan, which ultimately prevents their growth andreplicative capabilities. Treatment of epithelial cells with highlevels of IFN-� completely inhibits growth, whereas subinhibi-tory concentrations induce the development of morphologicallyaberrant viable RB forms that have been implicated in thedevelopment of persistence (15).

The complete genomic sequence of several Chlamydiaceaehas been determined, including C. trachomatis serovars D (18)and MoPn (19), C. pneumoniae strains CWL029 (20), AR39(19), and J138 (21). and C. psittaci strain GPIC (www.tigr.org).In addition, partial sequence information is available for C.trachomatis serovar L2 (chlamydia-www.Berkeley.edu:4231).

The gene order and content among these organisms are re-markably similar, with the exception of a region termed theplasticity zone, which has undergone genetic reorganization toa greater extent than the rest of the chromosome (19). Genesencoding enzymes required for the biosynthesis of tryptophanare found within the plasticity zone. However, the complementof trp genes within this region varies among the chlamydialspecies characterized to date. C. psittaci GPIC contains all ofthe genes of the tryptophan biosynthesis pathway with theexception of the first two enzymes encoded by trpE/G. In con-trast, C. trachomatis serovar MoPn and C. pneumoniae do notencode any trp genes in the plasticity zone. Interestingly, C.trachomatis serovars D and L2 contain only a subset of trpgenes in their plasticity zone, trpR, encoding a putative tryp-tophan repressor, and trpA and trpB, respectively, encodinghomologues of the � (TrpA) and � (TrpB) subunits of trypto-phan synthase (Fig. 1). This is an unusual circumstance in thatmost other organisms studied to date, both prokaryotic andeukaryotic, have either the full complement of trp genes or lackthem altogether. A further heterogeneity within the trpA geneof C. trachomatis has been identified by Shaw et al. (22) in thatserovar A and C appear to encode a truncated version of TrpAcompared with serovars D and L2 (22). The differences in thetrp gene complement among the chlamydiae characterized thusfar suggest that the ability to synthesize tryptophan de novo isnot required and raises the possibility that these genes are inthe process of being lost from the genome. Alternatively, thesedifferences may be important with respect to the unique tissue

FIG. 1. Reaction scheme for the bio-synthesis of tryptophan. Genes in boldare located in the plasticity zone and arecommon to both C. trachomatis serovar Dand C. psittaci strain GPIC. C. psittacistrain GPIC has all genes with the excep-tion of trpE and trpG.

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tropism of chlamydial strains and permit serovar-specific sur-vival or growth within different microenvironments of the host.

Tryptophan synthase is a tetramer consisting of two � sub-units and two � subunits (23–25). This bifunctional enzymecatalyzes the two final steps in the biosynthesis of tryptophan(Fig. 1), which are the cleavage of indole glycerol 3-phosphate(IGP) to indole and glyceraldehyde 3-phosphate (termed the �

reaction and catalyzed by TrpA) followed by the �-replacementreaction of indole with serine to form tryptophan (the � reac-tion, catalyzed by TrpB). Extensive characterization of theEscherichia coli and Salmonella enzymes has demonstrated alarge degree of allosteric regulation and cooperativity betweenthe � and � subunits (23–25). In fact, TrpA and TrpB exhibitlittle activity in their respective reactions in the absence of theother subunit (26–28). Given the association of IFN-� withchlamydial infections and its effect on tryptophan levels in thehost cell and, thus, on chlamydial growth, encoding functionaltryptophan synthase may be a survival factor for intracellularchlamydiae. However, IGP substrate for the � reaction in E.coli and Salmonella is supplied by the sequential activity of theother genes of the tryptophan biosynthesis pathway (TrpE, D,FC) (Fig. 1). C. trachomatis does not encode orthologues ofTrpE, G, D, or C, although paradoxically, it does have anorthologue of TrpF, the gene for which lies outside the plastic-ity zone (18, 19). Because mammalian cells lack the ability tobiosynthesize tryptophan and C. trachomatis appears to lack

the capability of IGP synthesis, it is unclear what the substratefor chlamydial TrpA would be.

The present work involved a study of the diversity within thetrp region among all 15 C. trachomatis serovars and character-ization of the functionality of the tryptophan synthase encodedtherein. Here we report that all the C. trachomatis type strainserovars, with the exception of B and MoPn, encode homo-logues of trpB and trpA, that the gene products are found inboth EBs and RBs, and that the ability to synthesize trypto-phan differs among ocular and genital serovars. Furthermore,we provide evidence that the � subunit of C. trachomatis tryp-tophan synthase differs from that of other Gram-negative bac-teria with respect to the utilization of IGP as a substrate.

MATERIALS AND METHODS

Bacterial Strains, Plasmids, and Antibodies—The bacterial strainsand plasmids utilized in this study are listed in Table I. E. coli strainswere grown in Luria-Bertani (LB) broth or on LB agar and in thepresence of 100 �g ml�1 ampicillin in the case of pQE-80L transfor-mants. C. trachomatis serovars were propagated in the HeLa 229 cer-vical carcinoma cell line (ATCC) maintained in minimal essential me-dium (MEM, Invitrogen) supplemented with 10% heat-inactivated fetalcalf serum (FCS) as described previously (29). For growth of C. tracho-matis under tryptophan-free conditions, dialyzed FCS was used. C.trachomatis EBs were purified by density gradient centrifugation ac-cording to established procedures and stored in sucrose phosphateglycerol medium at �80 °C (30). Polyclonal antiserum against C. tra-chomatis TrpA was raised in rabbits by immunization with purified

TABLE IBacterial strains and plasmids used in this study

In vitro passagesa Source

Bacterial strainC. trachomatis

A/Har-13 Y27H28 Harlan CaldwellB/TW-5 E9H52 Harlan CaldwellBa/AP2 Y5H10 Harlan CaldwellC/TW-3 E6H45 Harlan CaldwellD/UW-3 E10H17 Harlan CaldwellE/Bour Y15H6 Harlan CaldwellF/IC Cal-3 Y5H6 Harlan CaldwellG/UW-524 H16 Harlan CaldwellH/UW-4 E7H26 Harlan CaldwellIUW-12 E9H18 Harlan CaldwellJ/UW-36 E5H34 Harlan CaldwellK/UW-31 M2H42 Harlan CaldwellL1/LGV-440 Y5H5 Harlan CaldwellL2/LGV-434 E10H48 Harlan CaldwellL3/LGV-404 Y5H7 Harlan Caldwell

Genotype Source

Bacterial strainE. coli

KS463 F�, LAM�, trpA33, IN(rrnD-rrnE)1, rha-7 EGSCb

BW7622 Hfr, e14-, trpB114�Tn10, relA1, spoT1, thi-1, rph-1, IN(rrnD-rrnE)1 EGSCCY15077 F�, LAM�, DE(trpA-trpE)872, rph-1, tna-2 EGSC

Description Source

PlasmidpQE-80L E. coli expression vector, AmpR QiagenpCFG2 pQE-80L expressing E. coli trp This studypCR1 pQE-80L expressing C. trachomatis ser. L2 trpA This studypCR2 pQE-80L expressing C. trachomatis ser. L2 trpB This studypCR3 pQE-80L expressing C. trachomatis ser. L2 trpBA This studypCFG5 pQE-80L expressing C. trachomatis ser. A trpA This studypCFG9 pQE-80L expressing C. trachomatis ser. A trpB This studypCFG6 pQE-80L expressing C. trachomatis ser. A trpBA This studypCFG7 pQE-80L expressing C. trachomatis ser. A trpB and ser. L2 trpA This studypCFG8 pQE-80L expressing C. trachomatis ser. L2 trpB and ser. A trpA This studypCR4 pQE-80L expressing C. trachomatis ser. I trpA This studypCR5 pQE-80L expressing C. trachomatis ser. I trpB This studypCFG13 pQE-80L expressing C. trachomatis ser. I trpBA This study

a Number of in vitro passages of laboratory strains in either embryonating eggs (Y), McCoy (M), or HeLa 229 (H) cells.b EGSC, E. coli Genetic Stock Center.

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recombinant serovar L2 TrpA. Mouse ascites polyclonal anti-TrpB wasraised against recombinant serovar L2 TrpB by following the procedureof Lacy and Voss (31).

Sequence Analysis of C. trachomatis trp Genes—DNA between CT175and CT167 of serovar D (18), containing the C. trachomatis trp genes,was amplified from chromosomal DNA of all 15 C. trachomatis serovarsusing primers 00.11 and JHC258 (Table II) and Expand High Fidelitypolymerase according to manufacturer’s instructions (Roche MolecularBiochemicals). After gel purification, the PCR products were cloned intopCR-XL-TOPO using the kit from Invitrogen, and the constructs weretransformed into DH10B cells for propagation. Cloned insert DNA wassequenced by a commercial company (SeqWright, Houston, TX). Thenucleotide (nt) and deduced amino acid sequences were aligned usingClustalW version 1.8. The following sequences have been submitted toGenBankTM: accession numbers AY096805 (serovar Atrp) AY096806(Batrp), AY096807 (Ctrp), AY096808 (Dtrp), AY096809 (Etrp),AY096810 (Ftrp), Y096811 (Gtrp), AY096812 (Htrp), AY096813 (Itrp),AY096814 (Jtrp), AY096815 (Ktrp), AY096816 (L1trp), AY096817(L2trp), and AY096818 (L3trp).

RT-PCR Analysis of trp Gene Expression—Monolayers of HeLa 229cells in T-175 flasks were infected with C. trachomatis EBs at a multi-plicity of infection (m.o.i) of 3–5 inclusion-forming units (IFU) cell�1, aspreviously described (29). As a negative control, a mock-infected flaskwas prepared in the same manner but without the addition of EBs. Thecells were incubated for 24 h at 37 °C, and then total RNA was preparedusing Trizol reagent according to the manufacturer’s instructions (In-vitrogen). After treatment with amplification-grade DNase I (Invitro-gen), 1 �g of RNA was reverse-transcribed using random hexamerprimers and Thermoscript reverse transcriptase (Invitrogen) and thentreated with RNase H (Invitrogen). Primers specific for 16 S rRNA andthe trpB-trpA junction (Table II) were used to amplify products in PCRreaction mixtures containing 2 �l of cDNA, 0.2 �M primers, 0.2 mM

dNTPs, 1.5 mM MgCl2, 1� Taq reaction buffer, and 5 units of Taq DNApolymerase (Invitrogen). The cycling program was 3 min at 95 °C fol-lowed by 30 cycles of 30 s at 95 °C, 30 s at 60 °C, and 1.5 min at 72 °C.Products were separated on a 1.5% agarose-Tris-buffered EDTA gel andvisualized by ethidium bromide staining.

Western Blot Analyses—Purified EBs were lysed by suspension inLaemmli buffer followed by incubation at 95 °C for 10 min. Solubleproteins were fractionated by SDS-PAGE and electrophoretically trans-ferred to nitrocellulose membranes. The membranes were blocked with5% skim milk and then incubated with anti-TrpA antiserum followed byhorseradish peroxidase (HRP)-conjugated goat anti-rabbit immuno-globulin or with anti-TrpB ascites followed by goat anti-mouse HRP.Bound antibodies were detected by enhanced chemiluminescence ac-cording to manufacturer’s instructions (Amersham Biosciences).

Expression Cloning of trpB and trpA—C. trachomatis and E. coli trpgenes were amplified by PCR from purified chromosomal DNA usingthe reagent concentrations described for RT-PCR and the cycling pro-gram 3 min at 95 °C followed by 30 cycles of 1 min at 95 °C, 30 s at50 °C, and 2 min at 72 °C. The PCR primer sequences are listed in TableII and were designed to include unique restriction sites for cloning. Forconstruction of plasmids to co-express trpB and trpA, the 5�-CttrpB andthe 3�-CttrpA primers were used in the PCR reaction. The PCR productswere gel-purified, restricted with KpnI and SalI (for C. trachomatis) orBamHI and KpnI (for E. coli) and ligated to expression vector pQE-80L(Qiagen) cut with the corresponding restriction enzymes. Constructswere transformed into DH5� for screening, purified by miniprep, and

then used to transform E. coli mutant strains for complementationassays. Constructs co-expressing serovar A trpB with serovar L2 trpAand vice versa were prepared as follows. The C. trachomatis trpA genehas a unique SpeI site 73 bp downstream of the start codon in a regionof sequence identity among all of the serovars. Plasmids pCFG6 (sero-var A trpBA) and pCR3 (serovar L2 trpBA) were restricted with SpeIand KpnI, and the fragments were gel-purified. The trpA-containingfragment from pCR3 was then ligated to the trpB-containing fragmentfrom pCFG6 to generate pCFG7. Similarly, pCFG8 was constructed byligating the trpA-containing fragment from pCFG6 to the trpB-contain-ing fragment from pCR3.

Complementation Assays—The cells from stationary phase cultures ofE. coli trp transformants were harvested by centrifugation and washedthree times with sterile phosphate-buffered saline. The cell suspensionswere then streaked onto minimal agar (1� M9 salts, 0.2% glucose, 0.2%casamino acids, 2 mM MgSO4, 0.2 mM L-serine, 100 �g ml�1 ampicillin,and 50 �g ml�1 each thiamine, cysteine, and uracil) containing 100 �M

indole, 50 �g ml�1 L-tryptophan or without additional supplements. Theplates were incubated for 48 h at 37 °C and then photographed.

Preparation of Cell Lysates for Enzyme Assays—Five ml of stationaryphase cultures of CY15077 trp transformants were used to inoculate 50ml of LB broth containing 100 �g ml�1 ampicillin. After incubation withaeration for 2 h at 37 °C, the cultures were cooled to 18 °C, isopropyl-1-thio-�-D-galactopyranoside (Invitrogen) was added to a final concen-tration of 100 �M, and the cultures were incubated with aeration for afurther 18 h at 18 °C. The cells were then harvested by centrifugation,resuspended in 3 ml of 10 mM Tris-HCl, pH 7.8, and lysed by sonicationon ice. Cell debris was removed by centrifugation, and the clearedlysates were kept on ice. Protein concentration was determined byBradford assay using a commercial kit (Bio-Rad).

Enzyme Assays—One unit of activity is defined as the appearance of0.1 �mol of product (� reaction) or the disappearance of 0.1 �M sub-strate (� and �� reactions) in 20 min at 37 °C. The � reaction assays andthe �� reaction assays were performed using the methods of Smith andYanofsky (32). The � reaction mixture contained 0.3 �mol of IGP, 100�mol of phosphate buffer, pH 7.0, 2 �mol of NH2OH, and 70 �l of celllysate in a final volume of 0.5 ml. The �� reaction mixture contained 0.4�mol of IGP, 80 �mol of L-serine, 0.03 �mol of pyridoxal phosphate, 100�mol of Tris buffer, pH 7.8, 30 �l of saturated NaCl, and 70 �l of celllysate in a final volume of 1 ml. The � reaction assays were performedusing the method of Miles (33). The � reaction mixture contained 0.1�mol of indole, 20 �mol of L-serine, 0.0075 �mol of pyridoxal phosphate,25 �mol of Tris buffer, pH 7.8, 7.5 �l of saturated NaCl, and 50 �l of celllysate in a final volume of 250 �l. Specific activity (units mg�1) isreported as the average of triplicate determinations.

C. trachomatis Growth Assays—Monolayers of HeLa 229 cells in6-well plates were infected with C. trachomatis EBs at an m.o.i. of 3–5IFU cell�1 in MEM plus 10% dialyzed fetal bovine serum supplementedwith tryptophan (10 mg liter�1) lacking tryptophan or lacking trypto-phan but supplemented with indole (100 �M) or varying concentrationsof anthranilate. For tryptophan-free conditions, HeLa cells were incu-bated for 6 h in tryptophan-free MEM plus 10% dialyzed fetal bovineserum before infection with C. trachomatis to ensure depletion of en-dogenous tryptophan. Infected monolayers were incubated at 37 °C for48 h (LGV serovars) or 72 h (genital and ocular serovars), after whichtime the medium was collected, and the cells were lysed in cold, distilledwater. Aliquots of the combined HeLa cell lysates and culture mediumwere used to infect fresh HeLa cell monolayers. Recoverable IFU wereenumerated as previously described (30).

Indole Incorporation Assays—HeLa cell monolayers in 6-well plateswere infected with C. trachomatis EBs at an m.o.i. of 3–5 IFU cell�1 in theabsence or presence of tryptophan (10 mg liter�1) in MEM plus 10%dialyzed fetal bovine serum supplemented with 100 �M [14C]indole (0.1�Ci �M�1). Where indicated, the cells were incubated for 6 h in trypto-phan-free MEM plus 10% dialyzed fetal bovine serum before infection.After incubation for 36 h (serovar L2) or 48 h (serovars A, D, and I) at37 °C, the medium was removed, the cells were washed with Hanks’buffered saline solution, and then the cells were lysed in cold, distilledwater. Proteins in the cell lysate were precipitated with 10% trichloroace-tic acid, and incorporated 14C was quantified by scintillation counting(Beckman LS 5000). Data are expressed as dpm incorporated per 104 cells.

RESULTS

Sequence Analysis of trp Genes from C. trachomatis Ocularand Genital Serovars—Chlamydiae sequence data are availablefor both tryptophan synthase subunits from the serovar D ge-nome sequencing project (18). Serovar D TrpB contains 392

TABLE IIPrimer sequences for nucleotide sequencing RT-PCR and cloning

Primer Sequence (5� 3 3�)

Sequencing00.11 CAT TTG CTT CCG TTC TTG GGT AGJHC258 TGA CAG ATC GCA ATC CGC

RT-PCR5�-RT16SRNA GGA GAA AAG GGA ATT TCA CG3�-RT16SRNA TCC ACA TCA AGT ATG CAT CG5�-RTtrpBA GCA TTG GAG TCT TCA CAT GC3�-RTtrpBA ACA CCT CCT TGA ATC AGA GC

Cloning5�-CttrpA CCC CGG TAC CAT GAT GAA ATT AAC C3�-CttrpA CCC CGT CGA CTT ATC CAG GAA TAA AC5�-CttrpB CCC CGG TAC CAT GTT CAA ACA TAA AC3�-CttrpB CCC CGT CTT ACT CAT AAA TTC C5�-EctrpA AGC GGA TCC GAA CGC TAC GAA TCT3�-EctrpA GGG GTA CCT AAG CGA AAC GGT AAA

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amino acids, giving a calculated molecular mass of 42.6 kDa,similar to the E. coli TrpB (34). A comparison of the completeamino acid sequence of the serovar D TrpB with that of repre-sentative TrpBs in the public databases (Fig. 2A) indicates thatthe proteins are �54% identical. Most importantly amino acidresidues identified as essential for enzyme activity, indole bind-ing, and pyridoxal phosphate-Lys87 Schiff base complex forma-tion in E. coli TrpB (His86, Lys87, Glu109, Arg148, Leu188, Cys230,

Asp305, Phe306, Glu350) (25, 33, 35–39) are conserved in serovar DTrpB.

Serovar D TrpA protein contains 253 amino acids, a sizesimilar to that of E. coli TrpA (40). A comparison of the com-plete amino acid sequence of serovar D TrpA with that ofrepresentative TrpAs in the public databases shows that theoverall level of homology is low (27% identity, Fig. 2B). The twoamino acids identified as essential for catalytic activity

FIG. 2. Comparison of the C. tracho-matis (CtD) serovar D tryptophansynthase with that from E. coli (Ec)(34, 40), Bacillus subtilis (Bs) (67),and Methanococcus jannaschii (Mj)(68). A, TrpB alignment. Critical con-served residues identified as necessaryfor TrpB activity including His86, Lys87,Glu109, Arg148, Leu188, Cys230, Asp305,Phe306, and Glu350 are in bold. B, TrpAalignment. Conserved catalytic amino ac-ids Glu49 and Asp60 are in bold and un-derlined. Critical residues in the activesite that have been shown to be necessaryfor TrpA activity, most of which are notconserved including Phe22, Thr183, Gly211,Gly213, Gly234, and Ser235 are in bold.Amino acids in loop 6 are overlined, andresidues that are typically invariant butnot conserved in chlamydial TrpA are inbold and italics. See “Results” for details.

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Glu49and Asp60 (41–43), are conserved in serovar D TrpA.Surprisingly several amino acids which form the active sitepocket and/or have been identified by mutagenesis as essentialfor TrpA activity in E. coli (Phe22, Thr183, Gly211, Gly213,Gly234, Ser235) (23–25, 39, 44–47) are not conserved in serovarD TrpA. Interestingly, of all the TrpA sequences deposited inthe public databases, only the chlamydial TrpAs show theseamino acid changes in the active site.

The unusual primary structure of the serovar D TrpA andthe previously reported truncation of TrpA in serovars A and C(22) prompted us to extend our investigations on tryptophansynthase to all human C. trachomatis serovars. We sequencedthe trpB and trpA genes from the laboratory-type strains of all15 C. trachomatis serovars to determine the diversity withinthis region. Consistent with the results of Shaw et al. (22), wewere unable to amplify products from serovar B chromosomalDNA, indicating a deletion of the trp region from this isolate.The sequences of the trpB genes from the 14 serovars areremarkably similar, with only 6 single nucleotide polymor-phisms present in 1179 nt (data not shown). One point muta-tion (nt 1017) does not alter the amino acid sequence (Asn339),a second point mutation (nt 206) is conservative, resulting in achange from Arg to Lys at position 69 in serovars H, J, L2, andL3, a third point mutation (nt 696) results in a change from Serto Phe at position 232 in serovar L1, and a fourth point muta-tion (nt 1143) results in a change from Pro to Ser at position381 in serovar Ba. The remaining two point mutations at nt107 and 179 result in the conversion of Ser36 to Asn in serovarsH, J, L2, and L3 and Asn60 to Ser in serovars A and C,respectively.

Examination of the nucleotide alignment of the 14 C. tracho-matis trpA sequences revealed both single nucleotide polymor-phisms and deletion mutations in the 762-nt gene (all number-ing is based on genital serovar sequences unless otherwisenoted). Of the 11 point mutations identified, 4 (nt 10, 120, 477,and 699) are silent, 2 (nt 110 and 344) result in conservativeamino acid changes, Gln 3 Arg and Ala 3 Val, in serovars H,J, L2, L3, and three (nt 39) result in changes, Leu 3 Pro inserovars D and K (data not shown). The remaining mutationsillustrated in Fig. 3 are more likely to have effects on enzymestructure and/or function. The point mutations at nt 499 and511 result in non-conservative amino acid substitutions andcluster the serovars into two groups. Thus, all of the ocularserovars encode His167 and Leu171, whereas all of the genitalserovars encode Tyr and Phe at the corresponding positions.The ocular and genital serovars also differ in sequence at nt408–410; these nt are deleted in the ocular serovars, resultingin the loss of Phe136 from the protein encoded by these genes(Fig. 3). The trpA genes of the ocular serovars also have a singlent deletion at position 528 (ocular serovar numbering) thatresults in a frameshift generating a putative stop codon at nt550–552 (ocular serovar numbering). These deletion mutationswere previously reported for serovars A and C (22); here wedemonstrate that they are also found in serovar Ba. The dele-tion mutation found at nt 528 in the ocular serovars lies withina mutation “hotspot” for trpA. Thus, in the genital serovarsthere are two point mutations found within this same region atnt 530 and 532 encompassing two codons at amino acid posi-tions 177 and 178. The net result of these mutations is that allLGV serovars encode Tyr177Glu178, serovars D, K, and E en-

FIG. 3. Alignment of partial sequences from trpA gene (A) and protein (B) of the 14 reference serovars for which trpA gene couldbe amplified by PCR. ClustalW alignment of nucleotide and the corresponding protein regions containing sequence polymorphisms areillustrated. The ocular serovar specific 3 base deletion (nt 408–410), missense mutation (nt 499 and 511), and single nucleotide deletion that resultsin a truncated protein (stop codon nt 550–552) are in bold and italics. Genital serovar specific missense mutations (nt 530 and 532) that result inamino acid changes (177 and 178) in loop6 of TrpA are shown in bold. See “Results” for details.

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code Cys177-Gln178, and serovars G, F, I, H, and J encodeTyr177-Gln178 (Fig. 3).

Expression of trp Genes during HeLa Cell Infection and inPurified EBs—To determine whether the trpB and trpA geneswere expressed in the various C. trachomatis serovars, RT-PCRand Western blot analyses were carried out on type strainsrepresentative of the LGV (L2), ocular (A), and genital serovars(D and I). For RT-PCR analysis, RNA was prepared from mid-phase (24 h) infected HeLa cell cultures. After reverse tran-scription, the cDNA was amplified using a forward primercomplementary to the 3� end of trpB and a reverse primercomplementary to the 5� end of trpA. PCR products of theexpected size were amplified from cDNA derived from HeLacells infected with serovars L2, A, I, and D as well as from aplasmid (pCR3) containing full-length L2 trpB and trpA (Fig.4A). These primers were specific for C. trachomatis-derivedmRNA, as no product was amplified from cDNA prepared frommock-infected HeLa cells. Similarly, primers specific for C.trachomatis 16 S rRNA only amplified products from C. tracho-matis-infected HeLa cell cDNA and a plasmid control but notthe mock-infected sample. These data indicate that typestrains representative of the serovars causing human diseaseall express trpB and trpA and that these genes are transcribedas an operon. Furthermore, the single base deletion mutationfound in serovar A trpA does not appear to affect transcriptionof trpBA mRNA.

Western blot analyses were used to determine whether thetrp gene messages expressed by serovars L2, A, I, and D weretranslated to protein products. As shown in Fig. 4B, immuno-reactive material of the same electrophoretic mobility as re-combinant L2 TrpB was detected in purified EBs from serovarsL2, A, I, and D. Similarly, material with the same mobility asrecombinant L2 TrpA was detected in EB lysates of serovarsL2, I, and D. Serovar A EBs also had anti-TrpA immunoreac-tive material but of lower molecular weight than that of theother serovars. This result indicates that the frameshift muta-

tion in serovar A trpA results in the production of a truncatedversion of TrpA, consistent with the observations of Shaw et al.(22). No immunoreactive material of the appropriate size foreither TrpB or TrpA was detectable in EBs from serovar B orbiovar MoPn (data not shown), consistent with our inability toamplify products from these isolates using trpB- or trpA-spe-cific primers (data not shown) and the absence of trpBA genesin the MoPn genome (19).

Genetic Complementation and in Vitro Enzyme Assays—Todetermine whether the TrpB and TrpA proteins expressed byC. trachomatis were catalytically active, a heterologous comple-mentation system was utilized. The trp genes from serovars L2,A, and I were cloned into an E. coli expression vector andtransformed into E. coli mutants lacking various components ofthe tryptophan biosynthesis pathway. The ability of the E. colimutants expressing C. trachomatis trp genes to grow on mini-mal medium was then assessed (Fig. 5). The E. coli mutantKS463 expresses a non-functional TrpA but expresses activeTrpB. KS463 cells transformed with either expression vectoralone or constructs expressing C. trachomatis (serovars L2, A,or I) or E. coli trpA were able to grow on minimal mediumsupplemented with indole. These data are consistent with pub-lished observations indicating that E. coli TrpB can utilizeindole in the absence of functional TrpA (26–28). Similarly, allKS463 transformants were able to grow on minimal mediumsupplemented with tryptophan, as expected. However, KS463failed to grow on minimal medium when transformed witheither the expression vector alone or any of the constructsexpressing C. trachomatis trpA regardless of the serovar oforigin. In contrast, KS463 transformed with the E. coli trpAconstruct did grow on minimal medium. These data suggestedthat C. trachomatis TrpA could not efficiently utilize the IGPproduced by KS463, either due to a loss of catalytic activity forthis substrate or due to an inability to interact with and, thus,be activated by E. coli TrpB.

To distinguish between these two possibilities, the E. colitrpB transposon mutant BW7622, which does not express trpBor trpA, was transformed with constructs co-expressing C. tra-chomatis trpB and trpA. This eliminated the requirement for C.trachomatis TrpA to interact with a heterologous TrpB. Therewas no detectable growth of any of the transformants on min-imal medium, whereas expression of tryptophan synthase de-rived from serovars L2 and I complemented the growth ofBW7622 on indole-supplemented media (Fig. 5). These datasuggest that C. trachomatis tryptophan synthase is unable toutilize IGP or does so at levels insufficient to complementgrowth of BW7622. Furthermore, efficient utilization of indoleby C. trachomatis TrpB appeared to require the presence offull-length TrpA since serovar A tryptophan synthase expres-sion did not rescue the growth of BW7622 on indole, althoughthis transformant was able to grow on tryptophan-supple-mented media. This conclusion was confirmed by complemen-tation experiments carried out in an E. coli deletion mutant,CY15077, that lacks the entire tryptophan biosynthesis operon.Thus, transformation of CY15077 with constructs expressingonly C. trachomatis trpB did not complement growth on mini-mal medium supplemented with indole. However, co-expres-sion of trpB and trpA from serovars L2 and I did rescue thegrowth of CY15077 on indole-supplemented media. Similar tothe results observed with BW7622, co-expression of serovar AtrpB and trpA did not allow for growth of CY15077 on indole.This was likely due to the inability of serovar A TrpA to acti-vate serovar A TrpB, since the co-expression of serovar A TrpBwith serovar L2 TrpA permitted the growth of CY15077 onindole. In contrast, expression of serovar L2 TrpB with serovarA TrpA failed to rescue the growth of CY15077 on indole.

FIG. 4. Expression of trpB and trpA in C. trachomatis serovarsL2, A, I, and D. A, RT-PCR analysis of trp transcript expression. HeLacells were infected with C. trachomatis reference serovars, and RNAwas isolated 24 h post-infection. The RNA was reverse-transcribedusing random hexamer primers, and the cDNA was amplified withprimers specific for C. trachomatis 16 S rRNA or sequences flanking thetrpB-trpA junction. As a negative control, RNA from non-infected HeLacells was analyzed (mock), and as positive control, plasmid DNA con-taining L2 trpBA or 16 S rRNA genes were amplified in the PCRreaction. B, Western blot analysis of TrpB and TrpA expression inpurified EBs. EBs were lysed in Laemmli buffer, and proteins wereseparated by SDS-PAGE (12%) and then transferred to nitrocellulose.Proteins were detected using polyclonal antibodies raised against re-combinant (recomb) L2 TrpB or TrpA. The respective recombinantproteins were included as positive controls.

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In addition to the genetic complementation studies, in vitroactivity in the �, �, and �� reactions was determined for cel-lular extracts prepared from E. coli CY15077 co-expressing C.trachomatis trpB and trpA (Table III). As positive control,activity in all three reactions was detected for purified trypto-phan synthase from Salmonella enterica ser. Typhimurium. Noactivity was detected in any of the assays using cellular ex-tracts from CY15077 cells transformed with the expressionvector alone. As expected from the results of the complemen-tation studies, no activity for the � or �� reactions was detect-able in any of the lysates of cells expressing C. trachomatisproteins. In contrast, activity for the � reaction was readilydetectable in lysates containing TrpB and TrpA from serovars

L2 and I, whereas the � reaction activity of the lysate of serovarA-expressing cells was very low. Taken together, results fromthe complementation studies and in vitro enzyme assays sug-gest that unlike the Salmonella and E. coli enzymes, trypto-phan synthase from C. trachomatis is unable to efficientlycatalyze the conversion of IGP to indole (� reaction) and reac-tion of indole with serine to form tryptophan (� reaction) re-quires the presence of a full-length TrpA. Because the specificactivity in the � reaction for L2 and I extracts was low com-pared with the purified Salmonella enzyme, further studieswith purified C. trachomatis enzymes will be required to con-firm whether there is indeed no � activity or whether it is justtoo low to be detected in cellular extracts.

TABLE IIITryptophan synthase activity in lysates of CY15077 trpBA transformants

Enzyme sourceSpecific activitya

� reaction � reaction �� reaction

units mg�1

Vector alone NDAb NDA NDAL2 1.06 � 0.02c NDA NDAA 0.08 � 0.16 NDA NDAI 3.43 � 1.35 NDA NDAPurified Salmonella trp synthase 43.2 12.1 22.4

a 1 unit is the amount of enzyme that catalyses the disappearance (� reaction) or appearance (� reaction) of 0.1 �mol of indole in 20 min at 37 °C.b NDA, no detectable activity.c Specific activity �1 S.D.

FIG. 5. Analysis of C. trachomatis TrpB and TrpA enzymatic function by genetic complementation. trp genes from C. trachomatisserovars L2, A, and I were cloned into the E. coli expression vector pQE-80L either individually or together (trpBA). E. coli trpA was also clonedfor use as a positive control in the KS463 mutant. Constructs were transformed into E. coli mutants KS463 (trpA33), BW7622 (trpB::Tn10), orCY15077 (�trpE-A), and growth of the transformants was assessed on M9 minimal agar containing 100 �M indole, 50 �g ml�1 tryptophan or noadditional supplements. *, vector contains trpB from serovar A and trpA from serovar L2. **, vector contains trpB from serovar L2 and trpA fromserovar A.

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C. trachomatis Tryptophan Synthase Activity in Vivo—It isclear from the complementation and in vitro activity studiesthat the trpB genes of C. trachomatis serovars L2, A, and Iencode functional enzymes that require the presence of full-length TrpA for detectable activity. It has been previouslyreported that growth of most human serovars of C. trachomatisare tryptophan-dependent (48–50). To determine whether C.trachomatis tryptophan synthase could function in vivo, HeLacell infections were carried out under tryptophan-free condi-tions, and chlamydial growth was assessed after supplementa-tion of the media with potential tryptophan precursors. It hasbeen proposed that anthranilate may serve as a precursor fortryptophan biosynthesis in C. trachomatis (51). However, thedecrease in recoverable IFU for serovar L2 grown in trypto-phan-free media did not change after supplementation withanthranilate (Fig. 6). Thus, anthranilate cannot by used by C.trachomatis for tryptophan synthesis. In addition, kynurenine,another tryptophan degradation product, was also unable torescue C. trachomatis growth in tryptophan-efficient medium(data not shown).

Results from the in vitro enzyme assays indicated that C.trachomatis TrpB could utilize indole for the synthesis of tryp-tophan. As shown in Fig. 7, the level of recoverable IFU undertryptophan-replete conditions varied depending upon the sero-var. However, growth of all serovars was inhibited by theremoval of tryptophan from the cell culture medium. In thepresence of 100 �M indole, growth of all the genital (D-K,L1-L3) serovars recovered to tryptophan-replete levels or bet-ter, whereas there was no effect of indole supplementation onthe growth of the ocular serovars (A-C, Ba). To confirm thatindole was being utilized by C. trachomatis and was not con-verted into a tryptophan precursor by the host cell, HeLa cellinfections were carried out in the presence of radiolabeledindole. All C. trachomatis serovars grown in the presence oftryptophan failed to incorporate 14C-labeled indole, suggestingthat there may be some regulation of tryptophan synthase bytryptophan levels in the cell. Under tryptophan-free conditions,serovars L2, D, and I were able to incorporate 14C-labeled

indole, whereas serovar A and mock-infected HeLa cellsshowed no indole incorporation (Fig. 8). Thus, it would appearthat in vivo, C. trachomatis genital serovars are able to syn-thesize tryptophan directly from indole and that lack of afull-length TrpA results in the inability of ocular serovars toutilize indole.

DISCUSSION

Host response to chlamydial infection involves production ofthe protective cytokine IFN-�, which induces the expression ofindoleamine-2,3-dioxygenase and, thus, promotes tryptophandegradation in the host cells (14, 15, 52). Thus, the ability tosynthesize tryptophan may be an important survival factor forC. trachomatis during the course of infection, allowing theintracellular bacteria to persist in the presence of IFN-�-in-duced tryptophan limitation. This study was undertaken todetermine the extent of heterogeneity in the C. trachomatis trpregion and to determine whether trpB and trpA, respectively,encoding the � and � subunits of tryptophan synthase, wereexpressed as functional enzymes.

Both trpB and trpA were expressed in C. trachomatis as shownby RT-PCR analysis of transcripts from infected HeLa cells.Results indicate that trpB and trpA can be expressed as a singletranscript, similar to what has been observed for the trp operonof other bacteria (53, 54). C. trachomatis encodes a putativetryptophan repressor (trpR), which presents the possibility thattranscription of the trp genes may be regulated by the tryptophanconcentration in the host cell. High levels transcriptional repres-sion of the trp operon by the TrpR-tryptophan repressor complexhas been observed in many Gram-negative bacteria (55). How-ever, results from the present study indicate that there must bea basal level of trp gene expression in C. trachomatis, as we coulddetect both TrpB and TrpA in purified EBs obtained from HeLacells infected in tryptophan-replete medium.

The C. trachomatis trpB gene sequences from the 15 refer-ence serovars were nearly identical, with only four single nu-cleotide polymorphisms observed. All of the amino acids essen-tial for activity, as identified in E. coli TrpB (His86, Lys87,Glu109, Arg148, Leu188, Cys230, Asp305, Phe306, Glu350) (33, 35–38) are conserved in the C. trachomatis proteins, suggestingthat they should have enzymatic activity. This was indeed thecase as shown by both genetic complementation studies and invitro assays of � reaction activity in crude cell lysates. TrpBfrom serovars L2, A, and I was capable of catalyzing the �-re-placement reaction of indole and serine to form tryptophan.However, our results suggested a unique property of the C.trachomatis TrpB compared with that characterized from otherGram-negative bacteria. Thus, C. trachomatis TrpB appearedto have an absolute requirement for TrpA for function; no �reaction activity was detectable in the absence of TrpA or in thepresence of truncated TrpA from serovar A. In contrast, E. coliTrpB has been shown to have activity in the absence of TrpA,albeit at a lower level than observed in its presence (26–28).Therefore, the requirement for TrpA activation of TrpB ap-pears to be more stringent in the C. trachomatis enzyme thanin that of E. coli or Salmonella.

A larger number of polymorphisms were found in the nucle-otide sequences of C. trachomatis trpA compared with trpB;however, there was still greater than 98% identity among thesequences from the various serovars. Interestingly, TrpA fromall serovars retained the invariant catalytic residues, Glu49

and Asp60, but had changed most of the other highly conservedamino acids (Phe22, Thr183, Gly211, Gly213, Gly234, Ser235) in theactive site pocket. These residues have been shown by mu-tagenesis to be critical for TrpA activity (41, 42, 44–46, 56) andare key residues involved in binding IGP in the SalmonellaTrpBA crystal structure (25, 39, 47). Given the changes in

FIG. 6. Effect of anthranilate on the growth of C. trachomatisserovar L2 under tryptophan-free conditions. HeLa cells wereinfected with serovar L2 EBs at an m.o.i. of 3–5 IFU cell�1 in MEM, 10%dialyzed FCS containing 10 mg liter�1 tryptophan (�TRP), lackingtryptophan (�TRP), or lacking tryptophan but supplemented with in-creasing concentrations of anthranilate (AN). After 48 h, infected cellsand culture supernatants were collected and used to infect a secondHeLa cell monolayer for enumeration of recoverable IFU. Data arepresented as the mean IFU (log10) of triplicate determinations fromthree separate experiments. Error bars have been omitted for clarity.The S.E. of any determination never exceeded 0.5 log10.

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these key amino acids, it is not surprising that we did notdetect � reaction activity in the genetic complementation stud-ies nor the in vitro activity assays of lysates from cells overex-pressing the C. trachomatis tryptophan synthase.

A polymorphic mutational “hot spot” was identified in trpAfrom the genital chlamydiae serovars at nt 530 and 532, result-ing in three possible amino acids combinations at positions 177and 178 in the translated protein. These amino acids lie inTrpA loop 6, a region identified in the Salmonella tryptophansynthase crystal structure as being highly flexible and impor-tant for subunit-subunit interactions between TrpB and TrpA,metabolite channeling, and substrate binding (23, 24, 39, 57–61). It is possible that the sequence polymorphisms observed inthe TrpA loop 6 region may affect interactions between the �and � subunits and thus influence TrpB activity. In total, theunusual primary structure of TrpA suggests that the mainfunction of the C. trachomatis �-subunit could be to positionTrpB in the appropriate or favorable conformation to efficientlycarry out the � reaction. Detailed kinetic characterization ofpurified TrpB and TrpA from the different serovars will berequired to determine this.

The deletion and frameshift mutations in trpA from serovarsA and C, originally identified by Shaw et al. (22), were alsofound in serovar Ba in the present study. Interestingly, these

mutations appear to be predictive for the tissue tropism of theisolates. All of the ocular serovars had a 3-base deletion (nt408–410) and a single nucleotide deletion (nt 528), resulting ina truncated TrpA, whereas none of the genital serovars did.Similarly, non-conservative point mutations also cluster theserovars into ocular and genital strains (i.e. at nt 499 and 511).We are currently investigating whether this correlation be-tween trpA sequence and serovar tissue tropism holds true forclinical isolates.

It has been postulated that C. trachomatis may be able toscavenge tryptophan degradation products such as anthrani-late from the host cell for use as precursors in tryptophanbiosynthesis (51). Results from the present study clearly indi-cate that anthranilate could not rescue C. trachomatis growthin HeLa cells grown in tryptophan-free medium. This is notsurprising given the absence of several key enzymes in thetryptophan biosynthesis pathway. Although C. trachomatis en-codes a phosphoribosyl anthranilate isomerase (trpF), it lacksthe gene for anthranilate phosphoribosyltransferase requiredfor conversion of anthranilate to phosphoribosyl anthranilateas well as the gene for IGP synthase (trpC), required for theconversion of 1-(o-carboxylphenylamino)-1-deoxyribulose-5-phosphate to IGP (see Fig. 1).

Because of the lack of tools for genetic manipulation, it hasnot been possible to produce site-specific mutants in C. tracho-matis. Despite this limitation, our results indicate that thetryptophan synthase detected in C. trachomatis RBs appears tofunction similarly to the recombinant enzymes expressed in E.coli. Thus, C. trachomatis growing within HeLa cells was ableto utilize indole for growth in the absence of tryptophan. Onlygenital serovars could utilize indole, consistent with the obser-vations that in vitro the truncated TrpA (found in ocular sero-vars) could not enhance TrpB activity in the � reaction. Indolewas used directly by C. trachomatis and was not processed bythe HeLa cells into some other precursor molecule, as mock-infected cells exhibited no [14C]indole incorporation. In addi-tion, C. trachomatis serovar A could not incorporate ]14C]in-dole, further confirming that its TrpB is unable to function inthe absence of full-length TrpA.

Taken together, our results indicate that tryptophan syn-thase encoded by C. trachomatis trpBA is functional for con-version of indole to tryptophan, which permits the growth ofgenital serovars under conditions of tryptophan starvation. Instark contrast, ocular serovars A, Ba, and C, with a mutation intrpA, resulting in the production of a truncated protein, areunable to utilize indole for growth in the absence of tryptophan.

FIG. 7. Effect of indole on thegrowth of 15 C. trachomatis refer-ence serovars under tryptophan-freeconditions. HeLa cells were infectedwith EBs at an m.o.i. of 3–5 IFU cell�1 inMEM, 10% dialyzed FCS containing 10mg liter�1 tryptophan (�TRP), lackingtryptophan (�TRP), or lacking trypto-phan but supplemented with 100 �M in-dole (�TRP�IND). After 48–72 h, in-fected cells and culture supernatantswere collected and used to infect a secondHeLa cell monolayer for enumeration ofrecoverable IFU. Data are presented asthe mean IFU (log10) of triplicate deter-minations from three separate experi-ments. Error bars have been omitted forclarity. The S.E. of any determinationnever exceeded 0.5 log10.

FIG. 8. Utilization of indole by C. trachomatis during HeLa cellinfection under tryptophan-free conditions. HeLa cells were in-fected with C. trachomatis serovar L2, D, A, and I EBs at an m.o.i. of3–5 IFU cell�1 or left uninfected (mock) in MEM, 10% dialyzed FCSsupplemented with 10 �Ci of 14C-indole and containing 10 mg liter�1

tryptophan (TRP�) or lacking tryptophan (TRP�). After 36–48 h ofincubation at 37 C, the infected cells were lysed, and incorporatedradioactivity was determined by scintillation counting.

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What might the clinical significance of these findings be? It iswell known that ocular serovars rarely cause genital infections,and genital serovars are rarely associated with blinding tra-choma (2, 3, 9). The molecular basis for this distinct tissuetropism has never been defined. To our knowledge the differingabilities to synthesize tryptophan is the first demonstration ofa distinction in the biosynthetic capacity between the ocularand genital serovars.

The unusual properties of the chlamydial tryptophan syn-thase raise the question as to what might be the true substratein vivo. Our findings support the hypothesis that it is likelyindole. Under normal physiological conditions, indole is notreadily available as a metabolite in mammalian cells. Indole is,however, a major byproduct of tryptophan degradation in bac-teria encoding the enzyme tryptophanase (62). Common entericbacteria such as E. coli and Proteus sp. are known producers ofindole (62, 63). The same two organisms are also part of thenormal genital microflora and important urogenital pathogens(64, 65). Other indole-producing organisms (66) known to col-onize the female genital tract (65) include Peptostreptococcusasaccharolyticus, Fusobacterium species, Bacteroides species,Haemophilus influenza, and Weeksella virosa. This raises theintriguing possibility that C. trachomatis serovars that infectthe genital tract may be able to use indole produced by othermicroflora, either endogenous or the result of infection, as asubstrate to synthesize their own tryptophan for growth. Co-infection with indole-producing organisms may allow for therescue of chlamydial organisms persisting in a non-replicatingform in response to host IFN-� and its subsequent effect onintracellular tryptophan levels. The ability to synthesize tryp-tophan from indole may be important for the persistence of C.trachomatis within the genital tract epithelium, with impor-tant consequences for disease transmission as well as for theinflammatory sequelae associated with chronic infection. Be-cause the eye is normally a sterile niche, ocular serovars areless likely to encounter sources of indole during the course ofinfection, thereby eliminating the selective pressure to main-tain a functional tryptophan synthase.

Acknowledgments—We thank Dr. Edith Wilson Miles for providingus with purified Salmonella tryptophan synthase and IGP and Dr.Charles Yanofsky for several E. coli mutants. We are also indebted toboth for helpful advice.

REFERENCES

1. Moulder, J. W. (1991) Microbiol. Rev. 55, 143–1902. Schachter, J. (1989) Pathol. Immunopathol. Res. 8, 206–2203. Schachter, J. (1999) Chlamydia Intracellular Biology, Pathogenesis, and Im-

munity (Stephens, R. S., ed) pp. 139–170, American Society for Microbiol-ogy, Washington, D. C.

4. Kuo, C. C., Jackson, L. A., Campbell, L. A., and Grayston, J. T. (1995) Clin.Microbiol. Rev. 8, 451–461

5. Campbell, L. A., Rosenfeld, M., and Kuo, C. C. (2000) Trends Microbiol. 8,255–257

6. Kuo, C., and Campbell, L. A. (1998) Mol. Med. Today 4, 426–4307. Grayston, J. T., Kuo, C. C., Campbell, L. A., Wang, S. P., and Jackson, L. A.

(1997) Cardiologia 42, 1145–11518. Stephens, R. (1989) Antigenic Variation of Chlamydia trachomatis (Moulder,

J.W., ed) CRC Press, Inc., Boca Raton, FL9. Mabey, D., and Bailey, R. (1999) Br. J. Ophthalmol. 83, 1261–1263

10. Belland, R. J., Scidmore, M. A., Crane, D. D., Hogan, D. M., Whitmire, W.,McClarty, G., and Caldwell, H. D. (2001) Proc. Natl. Acad. Sci. U. S. A. 98,13984–13989

11. Brunham, R. C. (1999) Chlamydia Intracellular Biology, Pathogenesis, andImmunity (Stephens, R. S., ed) pp. 211–238, American Society for Microbi-ology, Washington, D. C.

12. Kim, S. K., and DeMars, R. (2001) Curr. Opin. Immunol. 13, 429–43613. Loomis, W. P., and Starnbach, M. N. (2002) Curr. Opin. Microbiol. 5, 87–9114. Beatty, W. L., Morrison, R. P., and Byrne, G. I. (1994) Microbiol. Rev. 58,

686–69915. Beatty, W. L., Belanger, T. A., Desai, A. A., Morrison, R. P., and Byrne, G. I.

(1994) Infect. Immun. 62, 3705–371116. Taylor, M. W., and Feng, G. S. (1991) FASEB J. 5, 2516–252217. Boehm, U., Klamp, T., Groot, M., and Howard, J. C. (1997) Annu. Rev. Immu-

nol. 15, 749–79518. Stephens, R. S., Kalman, S., Lammel, C., Fan, J., Marathe, R., Aravind, L.,

Mitchell, W., Olinger, L., Tatusov, R. L., Zhao, Q., Koonin, E. V., and Davis,R. W. (1998) Science 282, 754–759

19. Read, T. D., Brunham, R. C., Shen, C., Gill, S. R., Heidelberg, J. F., White, O.,Hickey, E. K., Peterson, J., Utterback, T., Berry, K., Bass, S., Linher, K.,Weidman, J., Khouri, H., Craven, B., Bowman, C., Dodson, R., Gwinn, M.,Nelson, W., DeBoy, R., Kolonay, J., McClarty, G., Salzberg, S. L., Eisen, J.,and Fraser, C. M. (2000) Nucleic Acids Res. 28, 1397–1406

20. Kalman, S., Mitchell, W., Marathe, R., Lammel, C., Fan, J., Hyman, R. W.,Olinger, L., Grimwood, J., Davis, R. W., and Stephens, R. S. (1999) Nat.Genet. 21, 385–389

21. Shirai, M., Hirakawa, H., Kimoto, M., Tabuchi, M., Kishi, F., Ouchi, K., Shiba,T., Ishii, K., Hattori, M., Kuhara, S., and Nakazawa, T. (2000) Nucleic AcidsRes. 28, 2311–2314

22. Shaw, A. C., Christiansen, G., Roepstorff, P., and Birkelund, S. (2000) Mi-crobes Infect 2, 581–592

23. Miles, E. W. (1991) Adv. Enzymol. Relat. Areas Mol. Biol. 64, 93–17224. Miles, E. W. (1995) Subcell. Biochem. 24, 207–25425. Miles, E. W. (2001) Chem. Rec. 1, 140–15126. Lane, A. N., and Kirschner, K. (1983) Eur. J. Biochem. 129, 571–58227. Miles, E. W., and McPhie, P. (1974) J. Biol. Chem. 249, 2852–285728. Crawford, I. P., and Yanofsky, C. (1958) Proc. Natl. Acad. Sci. U. S. A. 44,

1161–117029. Tipples, G., and McClarty, G. (1991) J. Bacteriol. 173, 4932–494030. Caldwell, H. D., Kromhout, J., and Schachter, J. (1981) Infect. Immun. 31,

1161–117631. Lacy, M. J., and Voss, E. W., Jr. (1986) J. Immunol. Methods 87, 169–17732. Smith, O. H., and Yanofsky, C. (1962) Methods Enzymol. 5, 794–80633. Miles, E. W. (1970) J. Biol. Chem. 245, 6016–602534. Crawford, I. P., Nichols, B. P., and Yanofsky, C. (1980) J. Mol. Biol. 142,

489–50235. Fluri, R., Jackson, L. E., Lee, W. E., and Crawford, I. P. (1971) J. Biol. Chem.

246, 6620–662436. Tanizawa, K., and Miles, E. W. (1983) Biochemistry 22, 3594–360337. Miles, E. W., Kawasaki, H., Ahmed, S. A., Morita, H., and Nagata, S. (1989)

J. Biol. Chem. 264, 6280–628738. Higgins, W., Miles, E. W., and Fairwell, T. (1980) J. Biol. Chem. 255, 512–51739. Hyde, C. C., Ahmed, S. A., Padlan, E. A., Miles, E. W., and Davies, D. R. (1988)

J. Biol. Chem. 263, 17857–1787140. Nichols, B. P., and Yanofsky, C. (1979) Proc. Natl. Acad. Sci. U. S. A. 76,

5244–524841. Nagata, S., Hyde, C. C., and Miles, E. W. (1989) J. Biol. Chem. 264, 6288–629642. Milton, D. L., Napier, M. L., Myers, R. M., and Hardman, J. K. (1986) J. Biol.

Chem. 261, 16604–1661543. Shirvanee, L., Horn, V., and Yanofsky, C. (1990) J. Biol. Chem. 265,

6624–662544. Allen, M. K., and Yanofsky, C. (1963) Genetics 48, 1065–108345. Yanofsky, C., Helinski, D. R., and Maling, B. D. (1961) Cold Spring Harbor

Symp. Quant. Biol. 26, 11–2446. Yanofsky, C., and Horn, V. (1972) J. Biol. Chem. 247, 4494–449847. Weyand, M., and Schlichting, I. (1999) Biochemistry 38, 16469–1648048. Jones, M. L., Gaston, J. S., and Pearce, J. H. (2001) Microb. Pathog. 30,

299–30949. Coles, A. M., Reynolds, D. J., Harper, A., Devitt, A., and Pearce, J. H. (1993)

FEMS Microbiol. Lett. 106, 193–20050. Morrison, R. P. (2000) Infect. Immun. 68, 6038–604051. Stephens, R. S. (1999) Chlamydia Intracellular Biology, Pathogenesis, and

Immunity. (Stephens, R. S., ed) pp. 9–28, American Society for MicrobiologyPress, Washington, D. C.

52. Byrne, G. I., Lehmann, L. K., and Landry, G. J. (1986) Infect. Immun. 53,347–351

53. Crawford, I. P. (1975) Bacteriol. Rev. 39, 87–12054. Yanofsky, C. (1960) Bacteriol. Rev. 24, 221–24555. Rose, J. K., Squires, C. L., Yanofsky, C., Yang, H. L., and Zubay, G. (1973) Nat.

New Biol. 245, 133–13756. Lim, W. K., Sarkar, S. K., and Hardman, J. K. (1991) J. Biol. Chem. 266,

20205–2021257. Ruvinov, S. B., and Miles, E. W. (1992) FEBS Lett. 299, 197–20058. Brzovic, P. S., Sawa, Y., Hyde, C. C., Miles, E. W., and Dunn, M. F. (1992)

J. Biol. Chem. 267, 13028–1303859. Brzovic, P. S., Hyde, C. C., Miles, E. W., and Dunn, M. F. (1993) Biochemistry

32, 10404–1041360. Miles, E. W. (1991) J. Biol. Chem. 266, 10715–1071861. Schneider, T. R., Gerhardt, E., Lee, M., Liang, P. H., Anderson, K. S., and

Schlichting, I. (1998) Biochemistry 37, 5394–540662. Yanofsky, C. (2000) J. Bacteriol. 182, 1–863. Kamath, A. V., and Yanofsky, C. (1992) J. Biol. Chem. 267, 19978–1998564. Gibbs, R. S. (1987) Am. J. Obstet. Gynecol. 156, 491–49565. Larsen, B., and Monif, G. R. (2001) Clin. Infect. Dis. 32, 69–7766. Balows, A. (1991) The Prokaryotes (Balows, A., Truper, H. G., Dworkin, M.,

Harder, W., and Schleifer, K.-H., eds) pp. 1–4, Springer-Verlag New YorkInc., New York

67. Kunst, F., Ogasawara, N., Moszer, I., Albertini, A. M., Alloni, G., Azevedo, V.,Bertero, M. G., Bessieres, P., Bolotin, A., Borchert, S., Borriss, R., Boursier,L., Brans, A., Braun, M., Brignell, S. C., Bron, S., Brouillet, S., Bruschi,C. V., Caldwell, B., Capuano, V., Carter, N. M., Choi, S. K., Codani, J. J.,Connerton, I. F., Danchin, A., et al. (1997) Nature 390, 249–256

68. Bult, C. J., White, O., Olsen, G. J., Zhou, L., Fleischmann, R. D., Sutton, G. G.,Blake, J. A., FitzGerald, L. M., Clayton, R. A., Gocayne, J. D., Kerlavage,A. R., Dougherty, B. A., Tomb, J. F., Adams, M. D., Reich, C. I., Overbeek,R., Kirkness, E. F., Weinstock, K. G., Merrick, J. M., Glodek, A., Scott, J. L.,Geoghagen, N. S., and Venter, J. C. (1996) Science 273, 1058–1073

C. trachomatis Tryptophan Synthase 26903

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Belland, Harlan D. Caldwell and Grant McClartyChristine Fehlner-Gardiner, Christine Roshick, John H. Carlson, Scott Hughes, Robert J.

POSSIBLE ROLE FOR TRYPTOPHAN SYNTHASE Tissue Tropism: AChlamydia trachomatisMolecular Basis Defining Human

doi: 10.1074/jbc.M203937200 originally published online May 13, 20022002, 277:26893-26903.J. Biol. Chem. 

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