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FEMS Immunol Med Microbiol. Author manuscript; available in PMC Aug 16, 2010.
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PMCID: PMC2921798
NIHMSID: NIHMS222210

Chlamydia pneumoniae encodes a functional aromatic amino acid hydroxylase

Abstract

Chlamydia pneumoniae is a community-acquired respiratory pathogen that has been associated with the development of atherosclerosis. Analysis of the C. pneumoniae genome identified a gene (Cpn1046) homologous to eukaryotic aromatic amino acid hydroxylases. Aromatic amino acid hydroxylases (AroAA-H) hydroxylate phenylalanine, tyrosine, and tryptophan into tyrosine, dihydroxyphenylalanine (L-DOPA), and 5-hydroxytryptophan, respectively. Sequence analysis of Cpn1046 demonstrated that residues essential for AroAA-H enzymatic function are conserved and that a subset of Chlamydia species contain an AroAA-H homolog. The chlamydial AroAA-H are transcriptionally linked to a putative bacterial membrane transport protein. We determined that recombinant Cpn1046 is able to hydroxylate phenylalanine, tyrosine, and tryptophan with roughly equivalent activity for all three substrates. Cpn1046 is expressed within 24 h of infection, allowing C. pneumoniae to hydroxylae host stores of aromatic amino acids during the period of logarithmic bacterial growth. From these results we can conclude that C. pneumoniae, as well as a subset of other Chlamydia species, encode an AroAA-H that is able to use all three aromatic amino acids as substrates. The maintenance of this gene within a number of Chlamydia suggests that the enzyme may have an important role in shaping the metabolism or overall pathogenesis of these bacteria.

Keywords: bacteria, metabolism, hydroxylase

INTRODUCTION

Chlamydia is an obligate intracellular bacterium that infects a wide range of hosts. There are two species of Chlamydia that predominately cause disease in humans: C. trachomatis and C. pneumoniae. C.trachomatis is the leading cause of infectious blindness as well as the most common bacterial STD (WHO, 2001, Mabey, et al., 2003). C. pneumoniae is a community acquired respiratory pathogen, it is responsible for 10% of the pneumonia and 5% of the bronchitis cases that occur each year in the United States (Kuo, et al., 1995). Chlamydia has also been linked to a number of chronic diseases such as atherosclerosis and reactive arthritis (Schachter, 1999, Kalayoglu, et al., 2002).

The genomes of C. trachomatis and C. pneumoniae are highly similar. 92% of C. trachomatis genes have orthologs in C. pneumoniae and 80% of the C. pneumoniae genome is orthologous to that of C. trachomatis (Kalman, et al., 1999). In light of the fact that the genomes are so similar those genes unique to each species are likely key to the differences in tissue tropism and disease spectrum between C. pneumoniae and C. trachomatis. Cpn1046, a homolog to eukaryotic aromatic amino acid hydroxylases, is found in C. pneumoniae but not C. trachomatis (Kalman, et al., 1999).

There are three eukaryotic aromatic amino acid hydroxylases (AroAA-H); they function to hydroxylate phenylalanine, tyrosine, and tryptophan into tyrosine, dihydroxyphenylalanine (L-DOPA), and 5-hydroxytryptophan, respectively. These enzymes have been well characterized in eukaryotes due to their role in the synthesis of physiologically important products, including serotonin and melatonin. All three AroAA-Hs are tetrahydropterin dependent; the hydroxylation of the side chain of the aromatic amino acid utilizes a tetrahydropterin as the source of the two electrons required to reduce the other atom of oxygen to the level of water (Fitzpatrick, 1999). AroAA-Hs contain metal and are regulated by phosphorylation at serines in their N-termini (Fitzpatrick, 1999). The eukaryotic AroAA-H are homotetramers that contain a N-terminal regulatory domain and a C-terminal catalytic domain (Fitzpatrick, 1999). AroAA-Hs are typically promiscuous in their substrate specificity and can use various alternative amino acid substrates (Fitzpatrick, 1999).

Homologs of phenylalanine hydroxylase (PheH) are present in about 20% of the bacterial genomes that have been sequenced (Leiros, et al., 2007); however, little is known about the importance of these enzymes in bacteria. To date only the PheH of Chromabacterium violaceum (Chen & Frey, 1998, Erlandsen, et al., 2002, Volner, et al., 2003, Zoidakis, et al., 2005), Pseudomonas spp. (Zhao, et al., 1994, Herrera & Ramos, 2007), and Colwellia psychrerythraea (Leiros, et al., 2007) have been studied in detail. The bacterial PheH differ from mammalian PheH in that they lack the regulatory and tetramerization domains; however, residues key for ligand and metal binding are conserved between eukaryotes and bacteria (Erlandsen, et al., 2002).

As a consequence of Cpn1046’s possible role in C. pneumoniae pathogenesis, Cpn1046 function and expression was analyzed. We determined that Cpn1046 functions as an AroAA-H and shows roughly equivalent activity with all three aromatic amino acids. Transcriptional linkage analysis demonstrated the chlamydial AroAA-H is co-transcribed with a putative membrane transport protein. Given its determined activity, we named the chlamydial protein encoded by Cpn1046 Aah for aromatic amino acid hydroxylase.

MATERIALS AND METHODS

Chemicals and reagents

Carbenicillin, IPTG, and DTT were purchased from Invitrogen (Carlsbad, CA). HEPES, Fe(NH4)2(SO4)2, EDTA and catalase were purchased from Sigma (St. Louis, MO). Tetrahydro-L-biopterin was purchased from Calbiochem (San Diego, CA). Activated charcole and PMSF were purchased from Fisher (Santa Clara, CA). All radiolabeled substrates; [4-3H]Phenylalanine, [3,5-3H]tyrosine, and [5-3H]tryptophan, were purchased from GE Healthcare Bio-Sciences Corp. (Piscataway, NJ).

Cell lines, chlamydial stocks, and infection

HEp-2 and L929 cells were grown in RPMI 1640 (Invitrogen) supplemented with 5% heat-inactivated fetal bovine serum (FBS) (Hyclone, Logan, UT). Cells were grown at 37°C in an atmosphere containing 5% CO2. C. pneumoniae CWL029 EB were purified from HEp-2 cells and C. trachomatis L2 EB were purified from L929 cells. EB purification was done using 30% and 30–44% discontinuous Renografin gradients (E. R. Squibb and Sons, Cranbury, NJ) as previously described (Koehler, et al., 1990). Bacterial preparations were stored at −80°C until use. HEp-2 cells were infected with C. pneumoniae by centrifuging bacteria onto cells at 900 g for 1 h; cells were then incubated with the inoculum for 1 h at 37°C in an atmosphere containing 5% CO2; the inoculum was then removed and fresh RPMI 1640 + 5% FBS was added. L929 cells were infected with C. trachomatis by incubating cells with bacteria for 1 h at 24°C; cells were then washed with PBS and fresh RPMI 1640 + 5% FBS was added.

Transcriptional linkage analysis

Reverse transcription coupled with PCR (RT-PCR) was used to determine if Cpn1045 and Cpn1046 were co-transcribed. Chlamydia RNA was extracted from Trizol (Invitrogen) according to the manufacturer’s instructions. Total RNA was RQ1 DNase (Promega, Madison, WI) treated for 2 h and then further purified using RNeasy MinElute Cleanup columns (Qiagen, Valencia, CA). cDNA was generated from 5 μg of total RNA using random hexamer primers (Invitrogen) and SuperScript III (Invitrogen) following manufacturer’s instructions. Reverse-transcription reactions without Superscript III were used as negative controls and to evaluate DNase treatment efficiency. Genomic DNA was isolated from C. pneumoniae infected HEp-2 cells 72 h post-infection and from C. trachomatis infected L929 cells 24 h post-infection. DNA isolation was done using the High Pure PCR Template kit (Roche, Indianapolis, IN) according to the manufacturer’s instructions. Following purification DNA was treated with RNase A (Invitrogen) to eliminate any remaining RNA. Primers annealing 400 bp upsteam (5′ -CAT AGA AAA AGT ACA AGC TCT TC- 3′) and 400 bp downstream (5′ -AAA GGA CGA TGT CTA ATT GCT- 3′) of the Cpn1046 transcriptional start site were used for PCR, with an 800 bp product being indicative of co-transcription. Amplicons were separated and visualized in ethidium bromide-stained 1% agarose gels.

Protein expression and purification

Cpn1046 was PCR amplified from C. pneumoniae genomic DNA using the forward primer Cpn1046F 5′-CAC CGT GCA CTA CTG CGA GAG AAC CC-3′ and reverse primer Cpn1046R 5′-CTA TTG GCA AAG TAC CTC AAA ACC-3′. The PCR product was purified using PCR-prep columns (Qiagen). The purified PCR product was ligated into the pET100/D-TOPO expression vector (Invitrogen) to generate Cpn1046 with a N-terminal His tag. The cloned Cpn1046 (rCpn1046) was then sequenced (University of California, Berkeley Sequencing Center) using pET TOPO forward and reverse sequencing primers (Invitrogen) to confirm the sequence and proper orientation.

rCpn1046 was transformed into Escherichia coli BL21 Star (DE3) cells (Invitrogen) and cells were grown in LB-medium containing 100 μg ml−1 carbenicillin. Overnight culture were diluted 1:30 and grown shaking at 23°C for 2 h. Protein expression was induced by addition of IPTG at a final concentration of 0.1 mM, and cells were harvested 3 h after induction and stored at −20°C. The cell pellet from 1 L of bacteria was suspended in 25 ml 4°C Novagen (Madison, WI) His-Bind® buffer supplemented with 100 μg ml−1 lysozyme and protease inhibitor cocktail (Sigma). The resuspended pellet was incubated 15 min in ice and 0.1% sarkosyl was added. Cells were lysed by sonication and then centrifuged 10 min at 9,000 g at 4°C. The supernatant was than loaded on a Novagen (Madison, WI) His-Bind® column and His tagged protein was purified according to manufacturers’ instructions. Following protein elution fractions enriched for tagged protein were dialyzed in AroAA-H buffer (50 mM NaHEPES pH 6.8, 200 mM NaCl, 0.4 mM FeSO4, 1 mM EDTA, 0.1% Tween 20, 10% glycerol, 1 mM PMSF) (Yang & Kaufman, 1994) and concentrated using 35 kDa polyethylene glycol. Fractions eluted from the column were assessed for presence of recombinant protein (rCpn1046) by SDS-PAGE followed by Commassie staining or immunoblot using anti-HisG antibody (Invitrogen) and goat anti-mouse IgG HRP (Invitrogen).

Rat aromatic amino acid hydroxylase preparation

Virgin Lewis rat brains were generously provided the Satyabrata Nandi laboratory Univ. of California, Berkeley. The animals were purchased from Harlan Sprague-Dawley (Indianapolis and San Diego) and housed in a temperature-controlled room with 12 h light/dark schedule. They were fed food (Teklad 8640; Teklad, Madison, WI) and water ad libitum. All of the procedures followed Univ. of California Animal Care and Use Committee guidelines. Crude extracts of rat AroAA-H were prepared following the method described by Beevers et al. (1983) (Beevers, et al., 1983). The brainstem was dissected out in 4°C PBS and was then homogenized in 3 volumes of 50 mM TrisCl pH 7.6, 2 mM DTT, at 4°C. Homogenates were centrifuged for 30 min at 40,000 g at 4°C; the supernatant was then stored at −80°C for later use in enzyme activity assays. The brain stem is highly enriched in TyrH and TrpH; both of these enzymes can also use phenylalanine as a substrate (Fitzpatrick, 1999), allowing for the use of brainstem homogenate as a positive control for the activity assay with all three aromatic amino acids.

Aromatic amino acid activity assay

Activity of recombinant Cpn1046 (rCpn1046) and rat AroAA-H was determined by using a method modeled after one reported by Vrana et al. (1993) for trypotphan hydroxylase activity and one by Reinhard et al. (1986) for tyrosine hydroxylase activity. For each reaction rat brainstem homogenate or dialyzed rCpn1046 was added to 100 μl of 2x reaction mixture (HEPES pH 7.0 100 mM, DTT 10 mM, Fe(NH4)2(SO4)2 0.02 mM, catalase 0.2 mg ml−1) (Vrana, et al., 1993, Zoidakis, et al., 2005). Substrate ([4-3H]phenylalanine, [3,5-3H]tyrosine, or [5-3H]tryptophan) was added to the reaction at a final concentration of 1 mM and the reaction was initiated by addition of 0.5 mM tetrahydro-L-biopterin (BH4) the total reaction volume was 200 μl. Each reaction was incubated shaking at 37°C for 0.5 h to 4 h for rCpn1046. 50 μl of 60% HClO4 was then added and incubation was continued for an additional 20 min at 37°C. The reactions were cooled to room temperature and 500 μl of activated charcoal (50 mg ml−1 in H2O) was added. The mixture was briefly vortex and then centrifuged 5 min at 12,000 g at 4°C. The supernatant was loaded onto a 0.22 μm centrifuge tube filter (Fisher) and centrifuged an additional 5 min at 12,000 g at 4°C to remove any remaining charcoal. 250 μl of the filtered supernatant was added to 2 ml of ICN (Irvine, CA) EcoLite scintillation cocktail. A Beckman LS6000 series liquid scintillation counter was used for counting. Background blank determinates were made by performing the assay with H2O instead of enzyme (data not shown) or by leaving out cofactor (BH4) from the reaction.

Quantitative gene expression analysis

RNA extraction and cDNA generation was performed as described for transcriptional linkage analysis. The relative abundance of specific mRNA sequences was ascertained by real-time PCR using the ABI PRISM 7500 (Applied BioSystems, Foster City, CA). The reaction mixture was as follows: 13 μl SYBR Green PCR master mix (Applied BioSystems), 4 μl water, 8 μl template, and 1 μl primer. The reaction conditions were: 95°C for 10 min; 40 cycles 95°C for 15 s and 60°C for 60 s; with dissociation protocol. Primers used for real-time PCR were designed using ABI Primer Express Software. Threshold fluorescence was determined during the geometric phase of logarithmic gene amplification, from this the cycle threshold (CT) was set. Standard curves for 16S rRNA and Cpn1046 were generated by plotting log genomic DNA versus (CT) and these plots were used to ensure that equivalent amounts of cDNA were added to each reaction. The relative level of Cpn1046 in samples was determined by converting transcript level to a log2 ratio of 16S rRNA expression.

RESULTS

Cpn1046 is similar to known aromatic amino acid hydroxylases

The sequence of Cpn1046 was compared to known eukaryotic and prokaryotic aromatic amino acid hydroxylases and aligned based on protein sequence (Fig. 1). Cpn1046 exhibited 23% overall identity to P. fluorescens phenylalanine hydroxylase (PheH), 26% to human PheH, 28% to human tyrosine hydroxylase (TyrH), and 29% to human tryptophan hydroxylase (TrpH). Eukaryotic AroAA-Hs consist of an N-terminal regulatory domain and a C-terminal catalytic domain. In eukaryotes the regulatory domain is dispensable for catalytic activity and it is not conserved in characterized bacterial PheH (Zhao, et al., 1994, Erlandsen, et al., 2002, Leiros, et al., 2007). Alignment analysis demonstrated that Cpn1046 lacked significant similarity to the N-terminal region of eukaryotic AroAA-Hs, but showed striking similarity within the catalytic domain. AroAA-Hs require metal to function; the metal is bound within the catalytic domain of the enzyme by two histidines and one glutamic acid residue (His331, His336, and Glu376 in rat TyrH) (Fitzpatrick, 1999). These residues have been shown to be essential for iron binding by eukaryotic TyrH (Ramsey, et al., 1995). The metal binding histidines as well as the glutamic acid residue are conserved in Cpn1046 (Fig. 1).

FIG. 1
Alignment of Cpn1046 and known eukaryotic and bacterial aromatic amino acid hydroxylases. The catalytic domain is highlighted in yellow and the essential iron binding histidines and glutamic acid residues are marked with arrows. Alignment was performed ...

Genes similar to Cpn1046 are found in C. caviae, C. abortus, and C. felis; but not in C. trachomatis, C. muridarum or Parachlamydia

Several Chlamydia genomes have been published, these include a number of different Chlamydia species and serovars; the thirteen published Chlamydia genomes were searched for potential AroAA-H and genes similar to Cpn1046. Parachlamydia (Protochlamydia amoebophila UWE25), an endosymbiont of Acanthamoeba sp. (Horn, et al., 2004), contained no regions similar to known AroAA-H. C. muridarum strain Nigg and C. trachomatis serovars A, B, D, and L2 also contained no significant similarity. Genes similar to eukaryotic AroAA-H were identified in C. caviae, C. abortus, C. felis, and each C. pneumoniae strain. The chlamydial AroAA-H had a region of significant similarity to the catalytic domain of eukaryotic AroAA-H and contained the histidine and glutamic acid residues essential for iron binding by eukaryotic TyrH (Ramsey, et al., 1995) (Fig. 2).

FIG. 2
Homology to Cpn1046 in other Chlamydiae. The catalytic domain is highlighted in yellow and the essential iron binding histidines and glutamic acid residues are marked with arrows. The end of the extended C. pneumoniae N-terminal is indicated by an asterisk. ...

Although C. pneumoniae, C. caviae, C. abortus, and C. felis all contain genes similar to eukaryotic AroAA-H there appears to have been divergence in the gene following the split between C. pneumoniae and the other three Chlamydia species. C. caviae, C. abortus, and C. felis AroAA-H are very similar and share 73% to 75% protein identity with one another (Table 1). Whereas, Cpn1046 only has 43% identity with the AroAA-H of C. caviae and 44% identity with AroAA-H from C. abortus or C. felis (Table 1).

Another striking difference is that Cpn1046 contains an extended N-terminal region not found in the other chlamydial AroAA-H and this region is dissimilar to sequence from any other species (Fig. 2). This extended N-terminal region is not responsible for the decrease in similarity seen between Cpn1046 and the other chlamydial AroAA-H (Table 1). A genome for a strain of C. pneumoniae isolated from koala was recently sequenced (Timms et al., unpublished); prior to this all C. pneumoniae isolates had been of human origin. The Cpn1046 homolog from this C. pneumoniae biovar lacks the extended N-terminal region present in human C. pneumoniae isolates; however, unlike the genes from C. caviae, C. abortus, and C. felis, the koala C. pneumoniae encodes the last eleven residues of extended N-terminal region. This suggests the initial 93 residues of the extended N-terminal may have been lost from the koala isolate. The fact that the complete extended N-terminal region is only present in human C. pneumoniae isolates and was rapidly lost from the koala strain strongly supports the hypothesis that this region is specifically retained and tailored for C. pneumoniae life within the human host.

Chlamydial AroAA-H are transcriptionally linked to a putative bacterial membrane transport protein

All of the chlamydial AroAA-H appear to be transcriptionally linked to a uncharacterized putative bacterial membrane transport protein (Fig. 3A); the transport protein is located at the 3′ end of the AroAA-H and apparently lacks an independent promoter. To test for transcriptional linkage, RT-PCR was done using primers that annealed 400 bp upstream or downstream of the translational start site of Cpn1046, C. pneumoniae genomic DNA was used as a positive control and C. trachomatis genomic DNA was used as a negative control. From the analysis we can conclude that Cpn1046 and Cpn1045, the membrane transport protein, are transcriptionally linked (Fig. 3B).

FIG. 3
Co-transcribed transport protein. (A) All of the Chlamydia containing an AroAA-H (aah) are transcriptionally linked to a putative membrane transport protein. (B) PCR based transcriptional linkage analysis confirmed that in C. pneumoniae the two proteins ...

This transport protein is not found in Chlamydia that lack AroAA-H. The physically-linked transport protein from all the aromatic amino acid containing Chlamydia were aligned based on protein sequence (Fig. 3C); they showed a high level of homology and consistent with the structure of know membrane transporters had multiple α-helices.

Cpn1046 hydroxylates phenylalanine, tyrosine, and tryptophan

To determine if Cpn1046 functions as an aromatic amino acid hydroxylase in Chlamydia, enzymatic activity assays were performed using recombinant Cpn1046 (rCpn1046) purified from E. coli. Enzymatic activity of rCpn1046 was measured as the specific incorporation of 3[H] into 3[H]-H2O from substrates (phenylalanine, tyrosine, or tryptophan) containing tritium at the site of hydroxylation (Fig. 4) (Reinhard, et al., 1986, Vrana, et al., 1993, Khan, 2004). This is a well established enzymatic assay wherein the formation of tritated water is stoichiometrically proportional to the activity of the enzyme (Reinhard, et al., 1986, Vrana, et al., 1993, Khan, 2004).

FIG. 4
Reactions catalyzed by aromatic amino acid hydroxylases. AroAA-H add a hydroxyl group to phenylalanine, tyrosine, and tryptophan; the reaction and the basis for tritium release assay is shown. For tritium release assays, radiolabled amino acids were used ...

rCpn1046 was column purified to eliminate E. coli proteins (Fig. 5A). There were a large number of proteins present in the whole cell lysate that cross-reacted with anti-His antibody; however, these were not retained in the purification process (Fig. 5A). Prior to analysis of rCpn1046 enzymatic activity, the radioassay was validated using rat brain stem homogenate (RBS). The brainstem is enriched in TyrH and TrpH, which are able to hydroxylate all three aromatic amino acids (Fitzpatrick, 1999). Increased enzymatic activity, denoted as fold increase CPM over reactions lacking cofactor, was seen with increasing amounts of RBS (Fig. 5B). rCpn1046 displayed significant enzymatic activity with all three aromatic amino acids, and the level of activity was roughly equivalent with each substrate (Fig. 5C). This analysis demonstrates that Cpn1046 is enzymatically active and has similar cofactor and metal requirements as mammalian AroAA-H. The presence of conserved active site residues and the functional ability to hydroxylate each of the three aromatic amino acids implicates the function of Cpn1046 is as an AroAA-H expressed by chlamydiae; thus, the protein encoded by Cpn1046 was named Aah (aromatic amino acid hydroxylase).

FIG. 5
Recombinant Cpn1046 (rCpn1046) is enzymaticaly activity. (A) The stages of protein chromatography were analyzed by Commassie stain (left panel) and immunoblot with His antibody (right panel). The soluble (lane 2), insoluble (lane 3), unbound (lane 4), ...

The aah gene is expressed during peak C. pneumoniae growth

Having established that Aah is enzymatically active, the kinetics of gene expression during the course of C. pneumoniae infection were evaluated. Chlamydia infects cells in a metabolically inactive form, within 2 to 6 h after infection the chlamydial developmental cycle begins. For the next 24 to 48 h the bacteria grows and replicates, showing optimal metabolic activity. After this period of exponential growth, bacterial replication diminishes as individual bacteria differentiate into the infectious metabolically inactive form of Chlamydia. Different species and serovars of Chlamydia progress through this development cycle at different rates. The growth dynamic of multiple C. pneumoniae strains have been analyzed and in all cases an initial lag phase is seen from 6 to 18 h post infection, this is then followed by a period of exponential growth that can last from 26 to 46 h (Bonanomi, et al., 2003). RNA was harvested 12, 24, 48, 72, and 96 h post-infection and aah expression was analyzed by quantitative RT-PCR. Upregulation of aah was first observed 24 h post-infection and was maximal at 48 h (Fig. 6). A similae upregulation of aah (Cpn1046) between 24 and 48 h post-infection was also reported by Mathews et al. (Mathews, et al., 2001), Aah is upregulated during the period of logarithmic bacterial growth (18 h to 70 h post-infection) and is downregulated as the organisms begin to redifferentiate into the metabolically inactive form in preparation for cellular exit.

FIG. 6
Quantitative analysis of Cpn1046 expression. cDNA was generated from RNA isolated 12, 24, 48, 72, and 96 h post-infection and quantitative PCR was performed to determine the amount of Cpn1046 cDNA present at each time. Cpn1046 transcript level was transformed ...

DISCUSSION

Within the Chlamydia genus there are a number of different species of eukaryotic pathogens. Although there is a great deal of variety in host tropism and disease spectrum between these species they are all obligate intracellular pathogens with small genomes ranging from 1.04 Mb to 1.23 Mb. The similarity between the different species’ genomes suggests that those genes unique to a particular subset of species may be key to differences in pathology. Minor changes in Chlamydia metabolism can strongly influence species-specific biology; one such example is variations in tryptophan biosynthesis machinery. It is believed that changes in the trp operon are key to determining the host niche of some Chlamydia (Fehlner-Gardiner, et al., 2002), with modulation of tryptophan availability serving as a pathoadaptation to the environment. Similarly it has been suggested that the tyrP gene, a tyrosine/tryptophan permease, influences C. pneumoniae tissue tropism and pathogenicity (Gieffers, et al., 2003). Respiratory C. pneumoniae strains possess multiple copies of the tyrP gene; whereas, vascular strains only contain a single copy (Gieffers, et al., 2003). The possible influence of tyrP on C. pneumoniae tissue tropism underlies how minor changes in amino acid availability can greatly impact Chlamydia biology.

While Cpn1046 did show enzymatic activity with all three aromatic amino acids it’s rate of activity was much lower than that of rat AroAA-H. In tritium release assays with rat AroAA-H only a 30 min incubation was necessary to achieve significant c.p.m. readout; whereas, a 4 h incubation was required for rCpn1046. Substrate did not appear to be limiting during this time and increased substrate concentrations led to high levels of background in assays performed without cofactor. Variations in reaction conditions and cofactor did not improve rCpn1046 activity. This lower level of enzymatic activity could be due to partial enzyme inactivation during the purification process or to suboptimal conformation of the recombinant protein. Statistically significant enzymatic activity of rCpn1046 was observed with all three aromatic amino acids; however, it is likely that the native Chlamydia enzyme would have an increased rate of activity in vivo.

It does not appear that Chlamydia is able to use any aromatic amino acid as a carbon source as key enzymes in the degradation pathway are missing. The chlamydial AroAA-H can convert phenylalanine to tyrosine and the bacteria also encode a putative aromatic amino acid aminotransferase, allowing for the conversion of tyrosine into 4-hydroxyphenylpyruvate. 4-hydroxyphenylpyruvate dioxygenase is necessary for 4-hydroxyphenylpyruvate catabolic conversion into homogentisate but this enzyme is not present in Chlamydia. The chlamydial AroAA-H also converts tryptophan into 5-hydroxytryptophan. Chlamydia are not known to metabolize 5-hydroxytryptophan and it has been demonstrated that addition of 5-hydroxytryptophan to C. pneumoniae infected cells has an inhibitory effect on chlamydial infection (Rahman, et al., 2005). Thus it appears that chlamydiae no longer retain metabolic pathways for catabolism of aromatic amino acids. Nevertheless, it was established that C. pneumoniae Aah functions as an AroAA hydroxylase in vitro, and the physical linkage to a predicted AroAA transport protein suggests a similar function in vivo. 67% of the putative transport protein linked to chlamydial AroAA-H consists of a YhhQ family motif, this YhhQ motif is present in the aromatic amino acid transporter of Bacillus halodurans. It is reasonable to expect that the protein coupled to Chlamydia AroAA-H functions in amino acid transport, supplying aromatic amino acid substrates for the bacterial AroAA-H.

C. pneumoniae lacks the machinery to synthesize phenylalanine, tyrosine, and tryptophan. Presence of an AroAA-H in Chlamydia is consequently surprising as it is expected to deprive the bacteria of phenylalanine, tyrosine, and tryptophan without synthesizing any metabolites that are of apparent value to the organism. It is possible that Chlamydia Aah has reverse activity and is able to generate essential amino acids for bacterial metabolism from hydroxylated intermediates. However; this activity would require the availability and transport of host hyroxylated amino acids into the Chlamydia containing vacuole, which seems unlikely as they are substrates for cellular metabolic pathways. In addition, incidences of AroAA-H displaying such activity have not been reported.

Genomic analyses done in this study demonstrated that C. caviae, C. abortus, and C. felis contain homologs to Cpn1046; whereas C. trachomatis, C. muridarum, and Parachlamydia do not. C. pneumoniae is a respiratory pathogen of humans and is also associated with the development of atherosclerosis. C. caviae is a pathogen of guinea pigs that causes inclusion conjunctivitis (Gordon, et al., 1966). C. abortus causes abortions and foetal loss in ruminants and has been linked to spontaneous abortions and respiratory disease in humans (Mare, 1994, Jorgensen, 1997, Walder, et al., 2003). C. felis is associated with pneumonia and conjunctivitis in cats (Sykes, 2005). The wide range in pathologies associated with Chlamydiae containing an AroAA-H and the fact that similar disease manifestations are seen in Chlamydia lacking an AroAA-H suggest that AroAA-H expression may not influence clinical presentation of infection. C. pneumoniae is the only Chlamydia that has been linked to atherosclerosis, the bacteria can infect vascular endothelial cells and has been isolated from atherosclerotic plaques (Kalayoglu, et al., 2002, Sessa, et al., 2003). The role of Aah in C. pneumoniae pathogenesis is enigmatic; however, the gene’s maintenance within multiple Chlamydia species and unique enzymatic role suggests it is important to the organism’s ecological success.

ACKNOWLEDGEMENTS

We thank Peter Timms for providing the alignment of human C. pneumoniae aah and koala C. pneumoniae aah. This work was supported by National Institutes of Health grants HL071730, AI042156, and AI032943 and training grant T32-AI007620.

REFERENCES

[1] Beevers SJ, Knowles RG, Pogson CI. A sensitive radiometric assay for tryptophan hydroxylase applicable to crude extracts. J Neurochem. 1983;40:894–897. [PubMed]
[2] Bonanomi A, Dohm C, Rickenbach Z, Altwegg M, Fischer J, Gygi D, Nadal D. Monitoring intracellular replication of Chlamydophila (Chlamydia) pneumoniae in cell cultures and comparing clinical samples by real-time PCR. Diagn Microbiol Infect Dis. 2003;46:39–47. [PubMed]
[3] Chen D, Frey PA. Phenylalanine hydroxylase from Chromobacterium violaceum. Uncoupled oxidation of tetrahydropterin and the role of iron in hyroxylation. J Biol Chem. 1998;273:25594–25601. [PubMed]
[4] Erlandsen H, Kim JY, Patch MG, Han A, Volner A, Abu-Omar MM, Stevens RC. Structural comparison of bacterial and human iron-dependent phenylalanine hydroxylases: similar fold, different stability and reaction rates. J Mol Biol. 2002;320:645–661. [PubMed]
[5] Fehlner-Gardiner C, Roshick C, Carlson JH, Hughes S, Belland RJ, Caldwell HD, McClarty G. Molecular basis defining human Chlamydia trachomatis tissue tropism. A possible role for tryptophan synthase. J Biol Chem. 2002;277:26893–26903. [PubMed]
[6] Fitzpatrick PF. Tetrahydropterin-dependent amino acid hydroxylases. Annu Rev Biochem. 1999;68:355–381. [PubMed]
[7] Gieffers J, Durling L, Ouellette SP, et al. Genotypic differences in the Chlamydia pneumoniae tyrP locus related to vascular tropism and pathogenicity. J Infect Dis. 2003;188:1085–1093. [PubMed]
[8] Gordon FB, Weiss E, Quan AL, Dressler HR. Observations on guinea pig inclusion conjunctivitis agent. J Infect Dis. 1966;116:203–207. [PubMed]
[9] Herrera MC, Ramos JL. Catabolism of phenylalanine by Pseudomonas putida: the NtrC-family PhhR regulator binds to two sites upstream from the phhA gene and stimulates transcription with sigma70. J Mol Biol. 2007;366:1374–1386. [PubMed]
[10] Horn M, Collingro A, Schmitz-Esser S, et al. Illuminating the evolutionary history of chlamydiae. Science. 2004;304:728–730. [PubMed]
[11] Jorgensen DM. Gestational psittacosis in a Montana sheep rancher. Emerg Infect Dis. 1997;3:191–194. [PMC free article] [PubMed]
[12] Kalayoglu MV, Libby P, Byrne GI. Chlamydia pneumoniae as an emerging risk factor in cardiovascular disease. Jama. 2002;288:2724–2731. [PubMed]
[13] Kalman S, Mitchell W, Marathe R, et al. Comparative genomes of Chlamydia pneumoniae and C. trachomatis. Nat Genet. 1999;21:385–389. [PubMed]
[14] Khan HA. Analytical characterization of a sensitive radioassay for tyrosine hydroxylase activity in rodent striatum. Neurochem Res. 2004;29:1467–1472. [PubMed]
[15] Koehler JE, Burgess RR, Thompson NE, Stephens RS. Chlamydia trachomatis RNA polymerase major sigma subunit. Sequence and structural comparison of conserved and unique regions with Escherichia coli sigma 70 and Bacillus subtilis sigma 43. J Biol Chem. 1990;265:13206–13214. [PubMed]
[16] Kuo CC, Jackson LA, Campbell LA, Grayston JT. Chlamydia pneumoniae (TWAR) Clin Microbiol Rev. 1995;8:451–461. [PMC free article] [PubMed]
[17] Leiros HK, Pey AL, Innselset M, Moe E, Leiros I, Steen IH, Martinez A. Structure of phenylalanine hydroxylase from Colwellia psychrerythraea 34H, a monomeric cold active enzyme with local flexibility around the active site and high overall stability. J Biol Chem. 2007;282:21973–21986. [PubMed]
[18] Mabey DC, Solomon AW, Foster A. Trachoma. Lancet. 2003;362:223–229. [PubMed]
[19] Mare CJ. Mammalian chlamydiosis. In: Beran GW, editor. Handbook of Zoonoses. CRC Press; Boca Raton: 1994. pp. 403–415.
[20] Mathews S, George C, Flegg C, Stenzel D, Timms P. Differential expression of ompA, ompB, pyk, nlpD and Cpn0585 genes between normal and interferon-gamma treated cultures of Chlamydia pneumoniae. Microb Pathog. 2001;30:337–345. [PubMed]
[21] Rahman MA, Azuma Y, Fukunaga H, et al. Serotonin and melatonin, neurohormones for homeostasis, as novel inhibitors of infections by the intracellular parasite Chlamydia. J Antimicrob Chemother. 2005;56:861–868. [PubMed]
[22] Ramsey AJ, Daubner SC, Ehrlich JI, Fitzpatrick PF. Identification of iron ligands in tyrosine hydroxylase by mutagenesis of conserved histidinyl residues. Protein Sci. 1995;4:2082–2086. [PMC free article] [PubMed]
[23] Reinhard JF, Jr., Smith GK, Nichol CA. A rapid and sensitive assay for tyrosine-3-monooxygenase based upon the release of 3H2O and adsorption of[3H]-tyrosine by charcoal. Life Sci. 1986;39:2185–2189. [PubMed]
[24] Schachter J. Infection and disease epidemiology. In: Stephens RS, editor. Chlamydia: Intracellular biology, pathogenesis, and immunity. American Society for Microbiology; Washington, D.C.: 1999. pp. 139–269.
[25] Sessa R, Di Pietro M, Schiavoni G, et al. Chlamydia pneumoniae DNA in patients with symptomatic carotid atherosclerotic disease. J Vasc Surg. 2003;37:1027–1031. [PubMed]
[26] Sykes JE. Feline chlamydiosis. Clin Tech Small Anim Pract. 2005;20:129–134. [PubMed]
[27] Volner A, Zoidakis J, Abu-Omar MM. Order of substrate binding in bacterial phenylalanine hydroxylase and its mechanistic implication for pterin-dependent oxygenases. J Biol Inorg Chem. 2003;8:121–128. [PubMed]
[28] Vrana SL, Dworkin SI, Vrana KE. Radioenzymatic assay for tryptophan hydroxylase:[3H]H2O release assessed by charcoal adsorption. J Neurosci Methods. 1993;48:123–129. [PubMed]
[29] Walder G, Meusburger H, Hotzel H, Oehme A, Neunteufel W, Dierich MP, Wurzner R. Chlamydophila abortus pelvic inflammatory disease. Emerg Infect Dis. 2003;9:1642–1644. [PMC free article] [PubMed]
[30] WHO . Global Prevalence and Incidence of Selected Curable Sexually Transmitted Infections: Overview and Estimates. World Health Organization; Geneva: 2001.
[31] Yang XJ, Kaufman S. High-level expression and deletion mutagenesis of human tryptophan hydroxylase. Proc Natl Acad Sci U S A. 1994;91:6659–6663. [PMC free article] [PubMed]
[32] Zhao G, Xia T, Song J, Jensen RA. Pseudomonas aeruginosa possesses homologues of mammalian phenylalanine hydroxylase and 4 alpha-carbinolamine dehydratase/DCoH as part of a three-component gene cluster. Proc Natl Acad Sci U S A. 1994;91:1366–1370. [PMC free article] [PubMed]
[33] Zoidakis J, Loaiza A, Vu K, Abu-Omar MM. Effect of temperature, pH, and metals on the stability and activity of phenylalanine hydroxylase from Chromobacterium violaceum. J Inorg Biochem. 2005;99:771–775. [PubMed]
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