Was the Chlamydial Adaptative Strategy to Tryptophan Starvation an Early Determinant of Plastid Endosymbiosis?

Chlamydiales were recently proposed to have sheltered the future cyanobacterial ancestor of plastids in a common inclusion. The intracellular pathogens are thought to have donated those critical transporters that triggered the efflux of photosynthetic carbon and the consequent onset of symbiosis. Chlamydiales are also suspected to have encoded glycogen metabolism TTS (Type Three Secretion) effectors responsible for photosynthetic carbon assimilation in the eukaryotic cytosol. We now review the reasons underlying other chlamydial lateral gene transfers evidenced in the descendants of plastid endosymbiosis. In particular we show that half of the genes encoding enzymes of tryptophan synthesis in Archaeplastida are of chlamydial origin. Tryptophan concentration is an essential cue triggering two alternative modes of replication in Chlamydiales. In addition, sophisticated tryptophan starvation mechanisms are known to act as antibacterial defenses in animal hosts. We propose that Chlamydiales have donated their tryptophan operon to the emerging plastid to ensure increased synthesis of tryptophan by the plastid ancestor. This would have allowed massive expression of the tryptophan rich chlamydial transporters responsible for symbiosis. It would also have allowed possible export of this valuable amino-acid in the inclusion of the tryptophan hungry pathogens. Free-living single cell cyanobacteria are devoid of proteins able to transport this amino-acid. We therefore investigated the phylogeny of the Tyr/Trp transporters homologous to E. coli TyrP/Mre and found yet another LGT from Chlamydiales to Archaeplastida thereby considerably strengthening our proposal.

Horizontal gene transfer of a Chlamydial tRNA-guanine transglycosylase gene to eukaryotic microbes.

tRNA-guanine transglycosylases are found in all domains of life and mediate the base exchange of guanine with queuine in the anticodon loop of tRNAs. They can also regulate virulence in bacteria such as Shigella flexneri, which has prompted the development of drugs that inhibit the function of these enzymes. Here we report a group of tRNA-guanine transglycosylases in eukaryotic microbes (algae and protozoa) which are more similar to their bacterial counterparts than previously characterized eukaryotic tRNA-guanine transglycosylases. We provide evidence demonstrating that the genes encoding these enzymes were acquired by these eukaryotic lineages via horizontal gene transfer from the Chlamydiae group of bacteria. Given that the S. flexneri tRNA-guanine transglycosylase can be targeted by drugs, we propose that the bacterial-like tRNA-guanine transglycosylases could potentially be targeted in a similar fashion in pathogenic amoebae that possess these enzymes such as Acanthamoeba castellanii. This work also presents ancient prokaryote-to-eukaryote horizontal gene transfer events as an untapped resource of potential drug target identification in pathogenic eukaryotes.

Evidence of a conserved role for Chlamydia HtrA in the replication phase of the chlamydial developmental cycle.

Identification of the HtrA inhibitor JO146 previously enabled us to demonstrate an essential function for HtrA during the mid-replicative phase of the Chlamydia trachomatis developmental cycle. Here we extend our investigations to other members of the Chlamydia genus. C. trachomatis isolates with distinct replicative phase growth kinetics showed significant loss of viable infectious progeny after HtrA was inhibited during the replicative phase. Mid-replicative phase addition of JO146 was also significantly detrimental to Chlamydia pecorum, Chlamydia suis and Chlamydia cavie. These data combined indicate that HtrA has a conserved critical role during the replicative phase of the chlamydial developmental cycle.

Promiscuous and adaptable enzymes fill "holes" in the tetrahydrofolate pathway in Chlamydia species.

Folates are tripartite molecules comprising pterin, para-aminobenzoate (PABA), and glutamate moieties, which are essential cofactors involved in DNA and amino acid synthesis. The obligately intracellular Chlamydia species have lost several biosynthetic pathways for essential nutrients which they can obtain from their host but have retained the capacity to synthesize folate. In most bacteria, synthesis of the pterin moiety of folate requires the FolEQBK enzymes, while synthesis of the PABA moiety is carried out by the PabABC enzymes. Bioinformatic analyses reveal that while members of Chlamydia are missing the genes for FolE (GTP cyclohydrolase) and FolQ, which catalyze the initial steps in de novo synthesis of the pterin moiety, they have genes for the rest of the pterin pathway. We screened a chlamydial genomic library in deletion mutants of Escherichia coli to identify the "missing genes" and identified a novel enzyme, TrpFCtL2, which has broad substrate specificity. TrpFCtL2, in combination with GTP cyclohydrolase II (RibA), the first enzyme of riboflavin synthesis, provides a bypass of the first two canonical steps in folate synthesis catalyzed by FolE and FolQ. Notably, TrpFCtL2 retains the phosphoribosyl anthranilate isomerase activity of the original annotation. Additionally, we independently confirmed the recent discovery of a novel enzyme, CT610, which uses an unknown precursor to synthesize PABA and complements E. coli mutants with deletions of pabA, pabB, or pabC. Thus, Chlamydia species have evolved a variant folate synthesis pathway that employs a patchwork of promiscuous and adaptable enzymes recruited from other biosynthetic pathways. Importance: Collectively, the involvement of TrpFCtL2 and CT610 in the tetrahydrofolate pathway completes our understanding of folate biosynthesis in Chlamydia. Moreover, the novel roles for TrpFCtL2 and CT610 in the tetrahydrofolate pathway are sophisticated examples of how enzyme evolution plays a vital role in the adaptation of obligately intracellular organisms to host-specific niches. Enzymes like TrpFCtL2 which possess an enzyme fold common to many other enzymes are highly versatile and possess the capacity to evolve to catalyze related reactions in two different metabolic pathways. The continued identification of unique enzymes such as these in bacterial pathogens is important for development of antimicrobial compounds, as drugs that inhibit such enzymes would likely not have any targets in the host or the host's normal microbial flora.

FtsZ-independent septal recruitment and function of cell wall remodelling enzymes in chlamydial pathogens.

The nature and assembly of the chlamydial division septum is poorly defined due to the paucity of a detectable peptidoglycan (PG)-based cell wall, the inhibition of constriction by penicillin and the presence of coding sequences for cell wall precursor and remodelling enzymes in the reduced chlamydial (pan-)genome. Here we show that the chlamydial amidase (AmiA) is active and remodels PG in Escherichia coli. Moreover, forward genetics using an E. coli amidase mutant as entry point reveals that the chlamydial LysM-domain protein NlpD is active in an E. coli reporter strain for PG endopeptidase activity (ΔnlpI). Immunolocalization unveils NlpD as the first septal (cell-wall-binding) protein in Chlamydiae and we show that its septal sequestration depends on prior cell wall synthesis. Since AmiA assembles into peripheral clusters, trimming of a PG-like polymer or precursors occurs throughout the chlamydial envelope, while NlpD targets PG-like peptide crosslinks at the chlamydial septum during constriction.

Transition from glycogen to starch metabolism in Archaeplastida.

In this opinion article we propose a scenario detailing how two crucial components have evolved simultaneously to ensure the transition of glycogen to starch in the cytosol of the Archaeplastida last common ancestor: (i) the recruitment of an enzyme from intracellular Chlamydiae pathogens to facilitate crystallization of α-glucan chains; and (ii) the evolution of novel types of polysaccharide (de)phosphorylating enzymes from preexisting glycogen (de)phosphorylation host pathways to allow the turnover of such crystals. We speculate that the transition to starch benefitted Archaeplastida in three ways: more carbon could be packed into osmotically inert material; the host could resume control of carbon assimilation from the chlamydial pathogen that triggered plastid endosymbiosis; and cyanobacterial photosynthate export could be integrated in the emerging Archaeplastida.

Activation of dimanganese class Ib ribonucleotide reductase by hydrogen peroxide: mechanistic insights from density functional theory.

Activation of manganese-dependent class Ib ribonucleotide reductase by hydrogen peroxide was modeled using B3LYP* hybrid density functional theory. Class Ib ribonucleotide reductase R2 subunit (R2F) does not react with molecular oxygen. Instead R2F is proposed to react with H2O2 or HO2(-), provided by the unusual flavodoxin protein NrdI, to generate the observed manganese(III) manganese(III) tyrosyl-radical state. On the basis of the calculations, an energetically feasible reaction mechanism is suggested for activation by H2O2, which proceeds through two reductive half-reactions. In the first reductive half-reaction, H2O2 is cleaved with a barrier of 13.1 kcal mol(-1) [Mn(II)Mn(II) → Mn(III)Mn(III)], and in the second reductive half-reaction, H2O2 is cleaved with a barrier of 17.0 kcal mol(-1) [Mn(III)Mn(III) → Mn(IV)Mn(IV)]. Tyrosyl-radical formation from both the Mn(IV)Mn(IV) state and a Mn(III)Mn(IV) state, where an electron and proton have been taken up, is both kinetically and thermodynamically accessible. Hence, chemically, H2O2 is a possible oxidant for the manganese-dependent R2F. The selectivity between the second reductive half-reaction and a competing oxidative reaction, as in manganese catalase, may be the time scale for the availability of H2O2. The role of NrdI may be to provide H2O2 on the correct time scale.

Characterization of the activity and expression of arginine decarboxylase in human and animal Chlamydia pathogens.

Chlamydia pneumoniae encodes a functional arginine decarboxylase (ArgDC), AaxB, that activates upon self-cleavage and converts l-arginine to agmatine. In contrast, most Chlamydia trachomatis serovars carry a missense or nonsense mutation in aaxB abrogating activity. The G115R missense mutation was not predicted to impact AaxB functionality, making it unclear whether AaxB variations in other Chlamydia species also result in enzyme inactivation. To address the impact of gene polymorphism on functionality, we investigated the activity and production of the Chlamydia AaxB variants. Because ArgDC plays a critical role in the Escherichia coli acid stress response, we studied the ability of these Chlamydia variants to complement an E. coli ArgDC mutant in an acid shock assay. Active AaxB was detected in four additional species: Chlamydia caviae, Chlamydia pecorum, Chlamydia psittaci, and Chlamydia muridarum. Of the C. trachomatis serovars, only E appears to encode active enzyme. To determine when functional enzyme is present during the chlamydial developmental cycle, we utilized an anti-AaxB antibody to detect both uncleaved and cleaved enzyme throughout infection. Uncleaved enzyme production peaked around 20 h postinfection, with optimal cleavage around 44 h. While the role ArgDC plays in Chlamydia survival or virulence is unclear, our data suggest a niche-specific function.

Species-specific interactions of Src family tyrosine kinases regulate Chlamydia intracellular growth and trafficking.

Src family kinases (SFKs) regulate key cellular processes and are emerging as important targets for intracellular pathogens. In this commentary, we briefly review the role of SFKs in bacterial pathogenesis and highlight new work on the role of SFKs during the intracellular cycle of Chlamydia species.

Diverse requirements for SRC-family tyrosine kinases distinguish chlamydial species.

UNLABELLED: Chlamydiae are well known for their species specificity and tissue tropism, and yet the individual species and strains show remarkable genomic synteny and share an intracellular developmental cycle unique in the microbial world. Only a relatively few chlamydial genes have been linked to specific disease or tissue tropism. Here we show that chlamydial species associated with human infections, Chlamydia trachomatis and C. pneumoniae, exhibit unique requirements for Src-family kinases throughout their developmental cycle. Utilization of Src-family kinases by C. trachomatis includes tyrosine phosphorylation of the secreted effector Tarp during the entry process, a functional role in microtubule-dependent trafficking to the microtubule organizing center, and a requirement for Src-family kinases for successful initiation of development. Nonhuman chlamydial species C. caviae and C. muridarum show none of these requirements and, instead, appear to be growth restricted by the activities of Src-family kinases. Depletion of Src-family kinases triggers a more rapid development of C. caviae with up to an 800% increase in infectious progeny production. Collectively, the results suggest that human chlamydial species have evolved requirements for tyrosine phosphorylation by Src-family kinases that are not seen in other chlamydial species. The requirement for Src-family kinases thus represents a fundamental distinction between chlamydial species that would not be readily apparent in genomic comparisons and may provide insights into chlamydial disease association and species specificity. IMPORTANCE: Chlamydiae are well known for their species specificity and tissue tropism as well as their association with unique diseases. A paradox in the field relates to the remarkable genomic synteny shown among chlamydiae and the very few chlamydial genes linked to specific diseases. We have found that different chlamydial species exhibit unique requirements for Src-family kinases. These differing requirements for Src-family kinases would not be apparent in genomic comparisons and appear to be a previously unrecognized distinction that may provide insights to guide research in chlamydial pathogenesis.

Chlamydia abortus YhbZ, a truncated Obg family GTPase, associates with the Escherichia coli large ribosomal subunit.

The stringent stress response is vital for bacterial survival under adverse environmental conditions. Obligate intracellular Chlamydia lack key stringent response proteins, but nevertheless can interrupt the cell cycle and enter stasis or persistence upon amino acid starvation. A possible key protein retained is YhbZ, a homologue of the ObgE guanosine triphosphatase (GTPase) superfamily connecting the stringent stress response to ribosome maturation. Curiously, chlamydial YhbZ lacks the ObgE C-terminal domain thought to be essential for binding the large ribosomal subunit. We expressed recombinant Chlamydia abortus YhbZ and showed it to be a functional GTPase, with similar activity to other Obg GTPase family members. As Chlamydia are resistant to genetic manipulation, we performed heterologous expression and gradient centrifugation experiments in Escherichia coli and found that, despite the missing C-terminal domain, C. abortus YhbZ co-fractionates with the E. coli 50S large ribosomal subunit. In addition, overexpression of chlamydial YhbZ in E. coli leads to growth defects and elongation, as reported for other Obg members. YhbZ did not complement an E. coli obgE temperature-sensitive mutant, indicating the C-terminal acidic domain may have an additional role. This data supports a role for YhbZ linking the chlamydial stress response to ribosome function and cellular growth.

An alternative menaquinone biosynthetic pathway operating in microorganisms: an attractive target for drug discovery to pathogenic Helicobacter and Chlamydia strains.

Menaquinone is an essential vitamin as an obligatory component of the electron transfer pathway in microorganisms. Menaquinone has been shown to be derived from chorismate by eight enzymes, designated MenA to -H in Escherichia coli. However, bioinformatic analyses of whole-genome sequences have suggested that some microorganisms, such as Helicobacter pylori and Campylobacter jejuni, which are known to cause gastric carcinoma and diarrhea, respectively, do not have orthologs of most of the men genes, although they synthesize menaquinone. The (13)C-labeling pattern of menaquinone purified from Streptomyces coelicolor A3(2) grown on [U-(13)C]glucose was quite different from that of E. coli, suggesting that an alternative pathway was operating in the strain. We searched for candidate genes participating in the alternative pathway by in silico screening, and the involvement of these genes in the pathway was confirmed by gene-disruption experiments. We also used mutagenesis to isolate mutants that required menaquinone for their growth and used these mutants as hosts for shotgun cloning experiments. Metabolites that accumulated in the culture broth of mutants were isolated and their structures were determined. Taking these results together, we deduced the outline of the alternative pathway, which branched at chorismate in a similar manner to the known pathway but then followed a completely different pathway. As humans and some useful intestinal bacteria, such as lactobacilli, lack the alternative pathway, it would be an attractive target for the development of chemotherapeutics.

Identifying catalytic residues in CPAF, a Chlamydia-secreted protease.

A secreted chlamydial protease designated CPAF (Chlamydial Protease/proteasome-like Activity Factor) degrades host proteins, enabling Chlamydia to evade host defenses and replicate. The mechanistic details of CPAF action, however, remain obscure. We used a computational approach to search the protein databank for structures that are compatible with the CPAF amino acid sequence. The results reveal that CPAF possesses a fold similar to that of the catalytic domains of the tricorn protease from Thermoplasma acidophilum,and that CPAF residues H105, S499, and E558 are structurally analogous to the tricorn protease catalytic triad residues H746, S965, and D1023. Substitution of these putative CPAF catalytic residues blocked CPAF from degrading substrates in vitro, while the wild type and a noncatalytic control mutant of CPAF remained cleavage-competent. Substrate cleavage is also correlated with processing of CPAF into N-terminal (CPAFn) and C-terminal (CPAFc) fragments, suggesting that these putative catalytic residues may also be required for CPAF maturation.

Kdo-(2 --> 8)-Kdo-(2 --> 4)-Kdo but not Kdo-(2 --> 4)-Kdo-(2 --> 4)-Kdo is an acceptor for transfer of L-glycero-alpha-D-manno-heptose by Escherichia coli heptosyltransferase I (WaaC).

Early steps in the biosynthesis of lipopolysaccharide (LPS) involve the transfer of 3-deoxy-alpha-D-manno-oct-2-ulopyranosonic acid (Kdo) to lipid A. Whereas Kdo transferases (WaaA) of Escherichia coli generate a (2 --> 4)-linked Kdo disaccharide, Chlamydiae contain tri- or tetra-functional WaaA generating oligosaccharides with (2 --> 8)- and (2 --> 4)-linkages between Kdo. It has been suggested that the transfer of L-glycero-alpha-D-manno-heptose (Hep) to Kdo by an E. coli WaaC may not be possible in the presence of (2 --> 8)-linked Kdo. E. coli double-mutants deficient in heptosyltransferases I (waaC) and II (waaF) and expressing waaA of Chlamydiae instead of their own, make Chlamydia-type Kdo oligosaccharides which are attached to an E. coli lipid A. Using such strains expressing waaA of Chlamydophila pneumoniae, Chlamydophila psittaci, or Chlamydia trachomatis, we have studied the effect of E. coli waaC gene expression on LPS structure. Structural analyses revealed the formation of two novel oligosaccharides Hep-(1 --> 5)[Kdo-(2 --> 4)]-Kdo and Hep-(1 --> 5)[Kdo-(2 --> 8)-Kdo-(2 --> 4)]-Kdo showing that Hep is transferred in the presence of (2 --> 8)-linked Kdo. Surprisingly, the transfer of Hep onto Kdo-(2 --> 4)-Kdo-(2 --> 4)-Kdo did not occur, despite the fact that Hep-(1 --> 5)[Kdo-(2 --> 4)-Kdo-(2 --> 4)]-Kdo is found in nature as a partial structure of E. coli LPS. The premature end of the biosynthesis and incorporation of Hep into the LPS indicated that WaaC had access to the substrate before Kdo transfer was completed. We have observed differences between WaaA of C. trachomatis, C. pneumoniae and C. psittaci which indicate mechanistic differences between these Kdo transferases.

Cytopathicity of Chlamydia is largely reproduced by expression of a single chlamydial protease.

Chlamydiae replicate in a vacuole within epithelial cells and commonly induce cell damage and a deleterious inflammatory response of unknown molecular pathogenesis. The chlamydial protease-like activity factor (CPAF) translocates from the vacuole to the cytosol, where it cleaves several cellular proteins. CPAF is synthesized as an inactive precursor that is processed and activated during infection. Here, we show that CPAF can be activated in uninfected cells by experimentally induced oligomerization, reminiscent of the activation mode of initiator caspases. CPAF activity induces proteolysis of cellular substrates including two novel targets, cyclin B1 and PARP, and indirectly results in the processing of pro-apoptotic BH3-only proteins. CPAF activation induces striking morphological changes in the cell and, later, cell death. Biochemical and ultrastructural analysis of the cell death pathway identify the mechanism of cell death as nonapoptotic. Active CPAF in uninfected human cells thus mimics many features of chlamydial infection, implicating CPAF as a major factor of chlamydial pathogenicity, Chlamydia-associated cell damage, and inflammation.

Insight into disulfide bond catalysis in Chlamydia from the structure and function of DsbH, a novel oxidoreductase.

The Chlamydia family of human pathogens uses outer envelope proteins that are highly cross-linked by disulfide bonds but nevertheless keeps an unusually high number of unpaired cysteines in its secreted proteins. To gain insight into chlamydial disulfide bond catalysis, the structure, function, and substrate interaction of a novel periplasmic oxidoreductase, termed DsbH, were determined. The structure of DsbH, its redox potential of -269 mV, and its functional properties are similar to thioredoxin and the C-terminal domain of DsbD, i.e. characteristic of a disulfide reductase. As compared with these proteins, the two central residues of the DsbH catalytic motif (CMWC) shield the catalytic disulfide bond and are selectively perturbed by a peptide ligand. This shows that these oxidoreductase family characteristic residues are not only important in determining the redox potential of the catalytic disulfide bond but also in influencing substrate interactions. For DsbH, three functional roles are conceivable; that is, reducing intermolecular disulfides between proteins and small molecules, keeping a specific subset of exported proteins reduced, or maintaining the periplasm of Chlamydia in a generally reducing state.

Crystal structure of LL-diaminopimelate aminotransferase from Arabidopsis thaliana: a recently discovered enzyme in the biosynthesis of L-lysine by plants and Chlamydia.

The essential biosynthetic pathway to l-Lysine in bacteria and plants is an attractive target for the development of new antibiotics or herbicides because it is absent in humans, who must acquire this amino acid in their diet. Plants use a shortcut of a bacterial pathway to l-Lysine in which the pyridoxal-5'-phosphate (PLP)-dependent enzyme ll-diaminopimelate aminotransferase (LL-DAP-AT) transforms l-tetrahydrodipicolinic acid (L-THDP) directly to LL-DAP. In addition, LL-DAP-AT was recently found in Chlamydia sp., suggesting that inhibitors of this enzyme may also be effective against such organisms. In order to understand the mechanism of this enzyme and to assist in the design of inhibitors, the three-dimensional crystal structure of LL-DAP-AT was determined at 1.95 A resolution. The cDNA sequence of LL-DAP-AT from Arabidopsis thaliana (AtDAP-AT) was optimized for expression in bacteria and cloned in Escherichia coli without its leader sequence but with a C-terminal hexahistidine affinity tag to aid protein purification. The structure of AtDAP-AT was determined using the multiple-wavelength anomalous dispersion (MAD) method with a seleno-methionine derivative. AtDAP-AT is active as a homodimer with each subunit having PLP in the active site. It belongs to the family of type I fold PLP-dependent enzymes. Comparison of the active site residues of AtDAP-AT and aspartate aminotransferases revealed that the PLP binding residues in AtDAP-AT are well conserved in both enzymes. However, Glu97* and Asn309* in the active site of AtDAP-AT are not found at similar positions in aspartate aminotransferases, suggesting that specific substrate recognition may require these residues from the other monomer. A malate-bound structure of AtDAP-AT allowed LL-DAP and L-glutamate to be modelled into the active site. These initial three-dimensional structures of LL-DAP-AT provide insight into its substrate specificity and catalytic mechanism.