Dynamic diversity of the tryptophan pathway in chlamydiae: reductive evolution and a novel operon for tryptophan recapture

Abstract

Background: Complete genomic sequences of closely related organisms, such as the chlamydiae, afford the opportunity to assess significant strain differences against a background of many shared characteristics. The chlamydiae are ubiquitous intracellular parasites that are important pathogens of humans and other organisms. Tryptophan limitation caused by production of interferon-gamma by the host and subsequent induction of indoleamine dioxygenase is a key aspect of the host-parasite interaction. It appears that the chlamydiae have learned to recognize tryptophan depletion as a signal for developmental remodeling. The consequent non-cultivable state of persistence can be increasingly equated to chronic disease conditions.

Results: The genes encoding enzymes of tryptophan biosynthesis were the focal point of this study. Chlamydophila psittaci was found to possess a compact operon containing PRPP synthase, kynureninase, and genes encoding all but the first step of tryptophan biosynthesis. All but one of the genes exhibited translational coupling. Other chlamydiae (Chlamydia trachomatis, C. muridarum and Chlamydophila pneumoniae) lack genes encoding PRPP synthase, kynureninase, and either lack tryptophan-pathway genes altogether or exhibit various stages of reductive loss. The origin of the genes comprising the trp operon does not seem to have been from lateral gene transfer.

Conclusions: The factors that accommodate the transition of different chlamydial species to the persistent (chronic) state of pathogenesis include marked differences in strategies deployed to obtain tryptophan from host resources. C. psittaci appears to have a novel mechanism for intercepting an early intermediate of tryptophan catabolism and recycling it back to tryptophan. In effect, a host-parasite metabolic mosaic has evolved for tryptophan recycling.

Figure 1

The tryptophan-recapture operon of Chlamydophila psittaci (Cps). Genes are color-coded as shown in the key. Arrows indicate the direction of transcription. The nomenclature for tryptophan-pathway genes is that elaborated by Xie et al. [30], whereby genes encoding the five enzymes are named in order of the pathway steps. The two subunits of the first and fifth steps are given additional lower-case identifiers, that is, trpAa/trpAb and trpEa and trpEb (for simplicity, the trp genes are labeled without the trp identifier). Nucleotide spacing between genes is shown; negative values indicate open reading frames that overlap (translational coupling). The membrane-attack complex/perforin family protein from the chlamydiae, which belongs to PFAM protein family HMM PFO1823, has been discussed by Ponting [59]. The gene encoding this protein in C. psittaci appears to have multiple frameshifts, a possible carboxy-terminal truncation, and a possible insertion. Whether sequencing errors might account for this is unknown at the present time. Cpn, Chlamydophila pneumoniae.

Figure 2

Chlamydia/host tryptophan cycle. Solid arrows indicate conversion by the enzyme in green on the arrow; dotted arrows show movement across membranes. Induction of increased levels of tryptophan dioxygenase by IFN-γ causes flux of the host pool of tryptophan into the kynurenine pathway, causing starvation of Chlamydia parasites for tryptophan. Starvation for tryptophan presumably derepresses the trpR-regulated operon of tryptophan biosynthesis (trpBDCEbEa kynU kprS) in C. psittaci. The host-pathway enzymes of interest are located in the cytosol or are located in the mitochondrial compartment. Genes of tryptophan biosynthesis are named in the order of the pathway steps, following the nomenclature used by Xie et al. [30]. Thus, trpAa and trpAb (absent in C. psittaci) encode the large (aminase) and small (glutamine-binding) subunits of anthranilate synthase, trpB encodes anthranilate phosphoribosyl transferase, trpC encodes phosphoribosyl-anthranilate isomerase, trpD encodes indoleglycerol phosphate synthase, and trpEa and trpEb encode the α and β subunits of tryptophan synthase, respectively. KynU encodes kynureninase. kprS (synonymous with prsA) encodes PRPP synthase. Orange arrows indicate induction of indoleamine 2,3-dioxygenase and nitric oxide synthase by IFN-γ. Blue lines indicate inhibition of the latter two enzymes by nitric oxide and 3-hydroxyanthranilate, respectively.

Figure 3

Comparison of codon usage (arginine, leucine, proline and valine) for kynU genes from C. psittaci and H. sapiens with the corresponding whole-genome codon usages.

Figure 4

Alignment of E. coli (Eco) TrpC from the protein database (PDB code 1PII) with TrpC proteins from the indicated chlamydiae (Cmu, C. muridarum; Cps, C. psittaci; Ctr, C. trachomatis). The TrpC domain shown for Eco TrpC is the carboxy-terminal domain of a fusion protein (TrpD•TrpC) whose amino-terminal domain is TrpD. An extensive multiple alignment of TrpC proteins was carried out, and several potential pseudogenes were excluded. The gaps shown reflect the gaps required to align the Eco•TrpC with other sequences in the latter alignment (available on request). Invariant residues are highlighted yellow, and near-invariant (one exception tolerated) residues are shaded gray. Active-site residues are marked with a red triangle and and residues interacting with ligand with blue dots [60]. See Table 2 for gene identification numbers.

Figure 5

Alignment of TrpEa from S. typhimurium (Sty) with the TrpEa proteins of C. trachomatis (Ctr) and C. psittaci (Cps). The multiple alignment of numerous TrpEa proteins performed as described by Xie et al. [30] was used to assess the retention of critical residues by Ctr and Cps. Catalytic residues established in Sty (see [30]) and conserved throughout the multiple alignment are in blue. Residues interacting with the β subunit are in green. Additional residues in yellow are invariant. The gaps represent the gaps obtained in the overall multiple alignment (data not shown). Arrows point to probable inactivating changes in the Ctr sequence. See Table 2 for organism acronyms and gene identification numbers.

Figure 6

Alignment of TrpEb from S. typhimurium (Sty) with the TrpEb proteins from C. trachomatis (Ctr) and C. psittaci (Cps). Cps TrpEb1 is the paralog within the operon shown in Figure 1. Residues in the Sty sequence that are invariant in an extensive multiple alignment are marked in yellow and near-invariant residues are shaded in gray. Substrate, cofactor and metal-coordinating residues are color-coded as indicated by the key at bottom right. The movable COMM domain that interacts with the α subunit is marked, as well as residues K167 and S178 (not conserved) which contact the indicated residues of the α subunit. Residues critical for formation of internal salt bridges are also shown. See Xie et al. [30] for a detailed analysis. See Table 2 for organism acronyms and gene identification numbers.

Figure 7

Unrooted phylogenetic tree (radial view) of the TyrP family of transport proteins. A multiple alignment was obtained by input of the indicated sequences into the ClustalW program (version 1.4). Manual alignment adjustments were made with the assistance of the BioEdit multiple alignment tool of Hall [61]. The refined multiple alignment was used as input for generation of a phylogenetic tree using the program packages PHYLIP [62] and PHYLO_WIN [63]. The neigbor-joining and Fitch programs were used to obtain distance-based trees. The distance matrix was obtained using Protdist with a Dayhoff PAM matrix. The Seqboot and Consense programs were then used to assess the statistical strength of the tree using bootstrap resampling. Neighbor-joining (shown) and Fitch trees yielded similar clusters and arrangement of taxa within them. Bootstrap values of 1,000 per 1,000 iterations (indicated with blue circles) supported the major nodes, one of which contains all of the chlamydial proteins. The experimentally documented E. coli proteins TyrP (tyrosine transport), Mtr (high-affinity tryptophan transport) and TraB (low-affinity tryptophan transport) are highlighted in yellow. See Table 2 for organism acronyms and gene identification numbers. In contrast to the two paralogs of tyrP present in C. pneumoniae CWLO29, as shown, some intra-species variation occurs in that C. pneumoniae J138 has only a single tyrP gene, and a small hypothetical gene is inserted between the two paralog tyrP genes of C. pneumoniae AR39.

Figure 8

Unrooted phylogenetic tree (radial view) of the SstT family of transport genes. The data analysis was performed as described in the legend for Figure 7. The experimentally documented SstT proteins of serine transport in E. coli (Eco) and Porphyromonas gingivalis (Pgin) are highlighted in yellow. Bootstrap values at the major node positions are shown, and those nodes supported by 1,000 of 1,000 iterations are denoted with blue circles. See Table 2 for organism acronyms and gene identification numbers.