Purification, crystallization and preliminary crystallographic analysis of Chlamydophila pneumoniae AP endonuclease IV.

Base excision is a crucial DNA repair process mediated by endonuclease IV in nucleotide excision. In Chlamydia pneumoniae, CpendoIV is the exclusive AP endonuclease IV, exhibiting DNA replication error-proofreading capabilities, making it a promising target for anti-chlamydial drug development. Predicting the structure of CpendoIV, molecular docking with DNA was performed, analyzing complex binding sites and protein surface electrostatic potential. Comparative structural studies were conducted with E. coli EndoIV and DNA complex containing AP sites.CpendoIV was cloned, expressed in E. coli, and purified via Ni-NTA chelation and size-exclusion chromatography. Low NaCl concentrations induced aggregation during purification, while high concentrations enhanced purity.CpendoIV recognizes and cleaving AP sites on dsDNA, and Zn2+ influences the activity. Crystallization was achieved under 8% (v/v) Tacsimate pH 5.2, 25% (w/v) polyethylene glycol 3350, and 1.91 Å resolution X-ray diffraction data was obtained at 100 K. This research is significant for provides a deeper understanding of CpendoIV involvement in the base excision repair process, offering insights into Chlamydia pneumoniae.

Inhibition of tRNA Synthetases Induces Persistence in Chlamydia.

Chlamydia trachomatis is the leading cause of bacterial sexually transmitted infections, and Chlamydia pneumoniae causes community-acquired respiratory infections. In vivo, the host immune system will release gamma interferon (IFN-γ) to combat infection. IFN-γ activates human cells to produce the tryptophan (Trp)-catabolizing enzyme indoleamine 2,3-dioxygenase (IDO). Consequently, there is a reduction in cytosolic Trp in IFN-γ-activated host cells. In evolving to obligate intracellular dependence, Chlamydia has significantly reduced its genome size and content, as it relies on the host cell for various nutrients. Importantly, C. trachomatis and C. pneumoniae are Trp auxotrophs and are starved for this essential nutrient when the human host cell is exposed to IFN-γ. To survive this, chlamydiae enter an alternative developmental state referred to as persistence. Chlamydial persistence is characterized by a halt in the division cycle, aberrant morphology, and, in the case of IFN-γ-induced persistence, Trp codon-dependent changes in transcription. We hypothesize that these changes in transcription are dependent on the particular amino acid starvation state. To investigate the chlamydial response mechanisms acting when other amino acids become limiting, we tested the efficacy of prokaryote-specific tRNA synthetase inhibitors, indolmycin and AN3365, to mimic starvation of Trp and leucine, respectively. We show that these drugs block chlamydial growth and induce changes in morphology and transcription consistent with persistence. Importantly, growth inhibition was reversed when the compounds were removed from the medium. With these data, we find that indolmycin and AN3365 are valid tools that can be used to mimic the persistent state independently of IFN-γ.

Chlamydia Serine Protease Inhibitor, targeting HtrA, as a New Treatment for Koala Chlamydia infection.

The koala, an iconic marsupial native to Australia, is a threatened species in many parts of the country. One major factor in the decline is disease caused by infection with Chlamydia. Current therapeutic strategies to treat chlamydiosis in the koala are limited. This study examines the effectiveness of an inhibitor, JO146, which targets the HtrA serine protease for treatment of C. pecorum and C. pneumoniae in vitro and ex vivo with the aim of developing a novel therapeutic for koala Chlamydia infections. Clinical isolates from koalas were examined for their susceptibility to JO146. In vitro studies demonstrated that treatment with JO146 during the mid-replicative phase of C. pecorum or C. pneumoniae infections resulted in a significant loss of infectious progeny. Ex vivo primary koala tissue cultures were used to demonstrate the efficacy of JO146 and the non-toxic nature of this compound on peripheral blood mononuclear cells and primary cell lines established from koala tissues collected at necropsy. Our results suggest that inhibition of the serine protease HtrA could be a novel treatment strategy for chlamydiosis in koalas.

Lipid Requirements for the Enzymatic Activity of MraY Translocases and in Vitro Reconstitution of the Lipid II Synthesis Pathway.

Screening of new compounds directed against key protein targets must continually keep pace with emerging antibiotic resistances. Although periplasmic enzymes of bacterial cell wall biosynthesis have been among the first drug targets, compounds directed against the membrane-integrated catalysts are hardly available. A promising future target is the integral membrane protein MraY catalyzing the first membrane associated step within the cytoplasmic pathway of bacterial peptidoglycan biosynthesis. However, the expression of most MraY homologues in cellular expression systems is challenging and limits biochemical analysis. We report the efficient production of MraY homologues from various human pathogens by synthetic cell-free expression approaches and their subsequent characterization. MraY homologues originating from Bordetella pertussis, Helicobacter pylori, Chlamydia pneumoniae, Borrelia burgdorferi, and Escherichia coli as well as Bacillus subtilis were co-translationally solubilized using either detergent micelles or preformed nanodiscs assembled with defined membranes. All MraY enzymes originating from Gram-negative bacteria were sensitive to detergents and required nanodiscs containing negatively charged lipids for obtaining a stable and functionally folded conformation. In contrast, the Gram-positive B. subtilis MraY not only tolerates detergent but is also less specific for its lipid environment. The MraY·nanodisc complexes were able to reconstitute a complete in vitro lipid I and lipid II forming pipeline in combination with the cell-free expressed soluble enzymes MurA-F and with the membrane-associated protein MurG. As a proof of principle for future screening platforms, we demonstrate the inhibition of the in vitro lipid II biosynthesis with the specific inhibitors fosfomycin, feglymycin, and tunicamycin.

Enzymatic characterization of Chlamydophila pneumoniae phospholipase D.

Chlamydophila pneumoniae, an aetiological agent of respiratory infection, is also thought to play an immuno-pathogenetic role in atherosclerosis by contributing to inflammation and plaque instability. Phospholipase D (PLD) is an enzyme involved in lipid metabolism and may have a direct or indirect impact on virulence and the inflammatory response. Some aspects of the developmental cycle of C. pneumoniae suggest a direct implication of its PLD (CpPLD) in the pathogenesis, specifically by affecting the regulation of lipid metabolism and lipid exchange between C. pneumoniae and host cells. Our previous studies disclosed a specific anti-CpPLD antibody response in patients with acute coronary syndromes chronically infected with C. pneumoniae, and demonstrated that this antigen is a factor able to drive the inflammatory process in atherosclerosis. Due to the intriguing aspects of the CpPLD, the present study investigated CpPLD enzymatic activity of the protein and the two domains that include one HKD motif each polypeptide. Our results showed that CpPLD was able to synthesize the cardiolipin (CL) but unable to hydrolyze phospholipids. It was also observed that each single HKD motif has an independent CL synthetase activity. This enzymatic activity of CpPLD could be important in the inflammatory process within the atherothrombotic events.

In vitro activity of AZD0914, a novel DNA gyrase inhibitor, against Chlamydia trachomatis and Chlamydia pneumoniae.

The in vitro activities of AZD0914, levofloxacin, azithromycin, and doxycycline against 10 isolates each of Chlamydia trachomatis and Chlamydia pneumoniae were tested. For AZD0914, the MIC90s for C. trachomatis and C. pneumoniae were 0.25 µg/ml (range, 0.06 to 0.5 µg/ml) and 1 µg/ml (range, 0.25 to 1 µg/ml), respectively, and the minimal bactericidal concentrations at which 90% of the isolates were killed (MBC90s) were 0.5 µg/ml for C. trachomatis (range, 0.125 to 1 µg/ml) and 2 µg/ml for C. pneumoniae (range, 0.5 to 2 µg/ml).

AmiA is a penicillin target enzyme with dual activity in the intracellular pathogen Chlamydia pneumoniae.

Intracellular Chlamydiaceae do not need to resist osmotic challenges and a functional cell wall was not detected in these pathogens. Nevertheless, a recent study revealed evidence for circular peptidoglycan-like structures in Chlamydiaceae and penicillin inhibits cytokinesis, a phenomenon known as the chlamydial anomaly. Here, by characterizing a cell wall precursor-processing enzyme, we provide insights into the mechanisms underlying this mystery. We show that AmiA from Chlamydia pneumoniae separates daughter cells in an Escherichia coli amidase mutant. Contrary to homologues from free-living bacteria, chlamydial AmiA uses lipid II as a substrate and has dual activity, acting as an amidase and a carboxypeptidase. The latter function is penicillin sensitive and assigned to a penicillin-binding protein motif. Consistent with the lack of a regulatory domain in AmiA, chlamydial CPn0902, annotated as NlpD, is a carboxypeptidase, rather than an amidase activator, which is the case for E. coli NlpD. Functional conservation of AmiA implicates a role in cytokinesis and host response modulation.

Characterization of serine hydroxymethyltransferase GlyA as a potential source of D-alanine in Chlamydia pneumoniae.

For intracellular Chlamydiaceae, there is no need to withstand osmotic challenges, and a functional cell wall has not been detected in these pathogens so far. Nevertheless, penicillin inhibits cell division in Chlamydiaceae resulting in enlarged aberrant bodies, a phenomenon known as chlamydial anomaly. D-alanine is a unique and essential component in the biosynthesis of bacterial cell walls. In free-living bacteria like Escherichia coli, penicillin-binding proteins such as monofunctional transpeptidases PBP2 and PBP3, the putative targets of penicillin in Chlamydiaceae, cross-link adjacent peptidoglycan strands via meso-diaminopimelic acid and D-Ala-D-Ala moieties of pentapeptide side chains. In the absence of genes coding for alanine racemase Alr and DadX homologs, the source of D-Ala and thus the presence of substrates for PBP2 and PBP3 activity in Chlamydiaceae has puzzled researchers for years. Interestingly, Chlamydiaceae genomes encode GlyA, a serine hydroxymethyltransferase that has been shown to exhibit slow racemization of D- and L-alanine as a side reaction in E. coli. We show that GlyA from Chlamydia pneumoniae can serve as a source of D-Ala. GlyA partially reversed the D-Ala auxotrophic phenotype of an E. coli racemase double mutant. Moreover, purified chlamydial GlyA had racemase activity on L-Ala in vitro and was inhibited by D-cycloserine, identifying GlyA, besides D-Ala ligase MurC/Ddl, as an additional target of this competitive inhibitor in Chlamydiaceae. Proof of D-Ala biosynthesis in Chlamydiaceae helps to clarify the structure of cell wall precursor lipid II and the role of chlamydial penicillin-binding proteins in the development of non-dividing aberrant chlamydial bodies and persistence in the presence of penicillin.

MAPK kinase 3 potentiates Chlamydia HSP60-induced inflammatory response through distinct activation of NF-κB.

Chlamydia pneumonia (C. pneumonia) remains one of the leading causes of bacterial pneumonia and has been implicated in the pathogenesis of some inflammation-related diseases, such as asthma, chronic obstructive pulmonary disease, and vascular diseases. Heat shock protein 60 is one of the pathogenic components of C. pneumonia that is closely associated with the inflammatory disorders. However, the molecular basis for the immunopathologic property of chlamydial heat shock protein (cHSP60) has not been elucidated. In this article, we report that MAPK kinase 3 (MKK3) is essential for cHSP60-induced lung inflammation, because MKK3-knockout mice displayed significantly reduced lung neutrophil accumulation and decreased production of proinflammatory mediators, correlating with the alleviated inflammatory response in lung tissues. Mechanistically, p38 kinase was selectively activated by MKK3 in response to cHSP60 and activated NF-κB by stimulating the nuclear kinase, mitogen- and stress-activated protein kinase 1. The specific knockdown of mitogen- and stress-activated protein kinase 1 in macrophages resulted in a defective phosphorylation of NF-κB/RelA at Ser(276) but had no apparent effect on RelA translocation. Furthermore, TGF-ß-activated kinase 1 was found to relay the signal to MKK3 from TLR4, the major receptor that sensed cHSP60 in the initiation of the inflammatory response. Thus, we establish a critical role for MKK3 signaling in cHSP60 pathology and suggest a novel mechanism underlying C. pneumonia-associated inflammatory disorders.

Chlamydophila pneumoniae endonuclease IV prefers to remove mismatched 3' ribonucleotides: implication in proofreading mismatched 3'-terminal nucleotides in short-patch repair synthesis.

DNA polymerase I (DNApolI) catalyzes DNA synthesis during Okazaki fragment maturation, base excision repair, and nucleotide excision repair. Some bacterial DNApolIs are deficient in 3'-5' exonuclease, which is required for removing an incorrectly incorporated 3'-terminal nucleotide during DNA elongation by DNA polymerase activity. The key amino acid residues in the exonuclease center of Chlamydophila pneumoniae DNApolI (CpDNApolI) are naturally mutated, resulting in the loss of 3'-5' exonuclease. Hence, the manner by which CpDNApolI proofreads the incorrectly incorporated nucleotide during DNA synthesis warrants clarification. C. pneumoniae encodes three 3'-5' exonuclease activities: one endonuclease IV and two homologs of the epsilon subunit of replicative DNA polymerase III. The three proteins were biochemically characterized using single- and double-stranded DNA substrate. Among them, C. pneumoniae endonuclease IV (CpendoIV) possesses 3'-5' exonuclease activity that prefers to remove mismatched 3'-terminal nucleotides in the nick, gap, and 3' recess of a double-stranded DNA (dsDNA). Finally, we reconstituted the proofreading reaction of the mismatched 3'-terminal nucleotide using the dsDNA with a nick or 3' recess as substrate. Upon proofreading of the mismatched 3'-terminal nucleotide by CpendoIV, CpDNApolI can correctly reincorporate the matched nucleotide and the nick is further sealed by DNA ligase. Based on our biochemical results, we proposed that CpendoIV was responsible for proofreading the replication errors of CpDNApolI.

Effect of the termini of RNase Hs from Chlamydophila pneumoniae on enzymatic biochemical characterization.

A difference between prokaryotic RNase HII and HIII, which both belong to type 2 RNase H, is a long N-terminal extension of HIII; however, the main-fold structures of HII and HIII known as RNase H-fold are similar. To further understand the structure-function relationship of RNase HII and RNase HIII, biochemical analyses were carried out using N-terminal truncations of RNase HIII (IIIN56(Δ), IIIN81(Δ), and IIIN88(Δ)) and C-terminal truncation (IIC19(Δ)) of RNase HII from Chlamydophila pneumoniae. Compared with wild-type CpRNase HII/III, IIIN56(Δ) had no obvious variation on the cleavage site and efficiency of DNA-rN(1)-DNA/DNA (DR(1)D) and DNA-rN(4)-DNA/DNA (DR(4)D) substrates. IIC19(Δ) and IIIN81(Δ) both showed decreased activities, and IIIN88(Δ) exhibited little cleavage on these substrates. However, IIIN81(Δ) showed very different activities toward different substrates (20% for DR(1)D and 85% for DR(4)D). Moreover, IIC19(Δ)IIIN(82-88) mutant, prepared through adding N-terminal 82nd to 88th residues locating at the bound region of N- and C-terminal domains of CpRNase HIII to N-terminus of IIC19(Δ), cleaved DR(4)D substrate more efficiently and preferentially at the cleavage sites of CpRNase HIII but not those of CpRNase HII. These results indicated that C-termini of CpRNase HII, N-termini of CpRNase HIII, and bound region of N- and C-terminal domain are all important for enzymatic activities. Moreover, the 82nd to 88th residues of N-terminus of CpRNase HII are related with enzyme cleavage site specificity. These results will help to understand the importance of C-termini of CpRNase HII and N-termini of CpRNase HIII to the enzyme activities for DR(1)D and DR(4)D substrate.

Involvement of Ser94 in RNase HIII from Chlamydophila pneumoniae in the recognition of a single ribonucleotide misincorporated into double-stranded DNA.

We recently provided the first report that RNase HIII can cleave a DNA-rN(1)-DNA/DNA substrate (rN(1), one ribonucleotide) in vitro. In the present study, mutagenesis analyses and molecular dynamics (MD) simulations were performed on RNase HIII from Chlamydophila pneumoniae AR39 (CpRNase HIII). Our results elucidate the mechanism of ribonucleotide recognition employed by CpRNase HIII, indicating that the G95/K96/G97 motif of CpRNase HIII represents the main surface interacting with single ribonucleotides, in a manner similar to that of the GR(K)G motif of RNase HIIs. However, CpRNase HIII lacks the specific tyrosine required for RNase HII to recognize single ribonucleotides in double-stranded DNA (dsDNA). Interestingly, MD shows that Ser94 of CpRNase HIII forms a stable hydrogen bond with the deoxyribonucleotide at the (5')RNA-DNA(3') junction, moving this nucleotide away from the chimeric ribonucleotide. This movement appears to deform the nucleic acid backbone at the RNA-DNA junction and allows the ribonucleotide to interact with the GKG motif. Based on the inferences drawn from MD simulations, biochemical results indicated that Ser94 was necessary for catalytic activity on the DNA-rN(1)-DNA/DNA substrate; mutant S94V could bind this substrate but exhibited no cleavage. Mismatches opposite the single ribonucleotide misincorporated in dsDNA inhibited cleavage by CpRNase HIII to varying degrees but did not interfere with CpRNase/substrate binding. Further MD results implied that mismatches impair the interaction between Ser94 and the deoxyribonucleotide at the RNA-DNA junction. Consequently, recognition of the misincorporated ribonucleotide was disturbed. Our results may help elucidate the distinct substrate-recognition properties of different RNase Hs.

RNase HIII from Chlamydophila pneumoniae can efficiently cleave double-stranded DNA carrying a chimeric ribonucleotide in the presence of manganese.

Two ribonuclease Hs (RNase Hs) have been found in Chlamydophila pneumoniae, CpRNase HII and CpRNase HIII. This work is the first report that CpRNase HIII can efficiently cleave DNA-rN(1) -DNA/DNA (rN(1) , monoribonucleotide) in vitro in the presence of Mn(2+) , whereas the enzymatic activity of CpRNase HII on the same substrate was inhibited by Mn(2+) and dependent on Mg(2+) . However, the ability of both CpRNase Hs to cleave other alternative substrates (RNA/DNA hybrids and Okazaki-like substrates), was insensitive to the divalent ions changes, suggesting that high concentrations of Mn(2+) specifically repressed the ability of CpRNase HII to cleave DNA-rN(1) -DNA/DNA but activated this function in CpRNase HIII. Further in vivo experiments showed that the CpRNase HII complementation of Escherichia coli rnh(-) mutations in an Mg(2+) environment was suppressed by Mn(2+) . In contrast, Mn(2+) was indispensable for CpRNase HIII to complement the same mutations. Further, the cell growth inhibition and the genomic DNA sensitivity to alkali in the bacterial strain lacking RNase HII activity could be relieved by functional CpRNase HII or HIII with its compatible ion. Therefore, CpRNase HIII can execute cleavage activity on DNA-rN(1) -DNA/DNA under a Mn(2+) -rich environment and may function as a substitute for CpRNase HII under special physiological states.

Chlamydophila pneumoniae phospholipase D (CpPLD) drives Th17 inflammation in human atherosclerosis.

Phospholipases are produced from bacterial pathogens causing very different diseases. One of the most intriguing aspects of phospholipases is their potential to interfere with cellular signaling cascades and to modulate the host-immune response. Here, we investigated the role of the innate and acquired immune responses elicited by Chlamydophila pneumoniae phospholipase D (CpPLD) in the pathogenesis of atherosclerosis. We evaluated the cytokine and chemokine production induced by CpPLD in healthy donors' monocytes and in vivo activated T cells specific for CpPLD that infiltrate atherosclerotic lesions of patients with C. pneumoniae antibodies. We also examined the helper function of CpPLD-specific T cells for monocyte matrix metalloproteinase (MMP)-9 and tissue factor (TF) production as well as the CpPLD-induced chemokine expression by human venular endothelial cells (HUVECs). We report here that CpPLD is a TLR4 agonist able to induce the expression of IL-23, IL-6, IL-1ß, TGF-ß, and CCL-20 in monocytes, as well as CXCL-9, CCL-20, CCL-4, CCL-2, ICAM-1, and VCAM-1 in HUVECs. Plaque-derived T cells produce IL-17 in response to CpPLD. Moreover, CpPLD-specific CD4(+) T lymphocytes display helper function for monocyte MMP-9 and TF production. CpPLD promotes Th17 cell migration through the induction of chemokine secretion and adhesion molecule expression on endothelial cells. These findings indicate that CpPLD is able to drive the expression of IL-23, IL-6, IL-1ß, TGF-ß, and CCL-20 by monocytes and to elicit a Th17 immune response that plays a key role in the genesis of atherosclerosis.

Interactions between flagellar and type III secretion proteins in Chlamydia pneumoniae.

BACKGROUND: Flagellar secretion systems are utilized by a wide variety of bacteria to construct the flagellum, a conserved apparatus that allows for migration towards non-hostile, nutrient rich environments. Chlamydia pneumoniae is an obligate, intracellular pathogen whose genome contains at least three orthologs of flagellar proteins, namely FliI, FlhA and FliF, but the role of these proteins remains unknown. RESULTS: Full length FliI, and fragments of FlhA, FliF, and FliI, were cloned and expressed as either GST or His tagged proteins in E. coli. The GST-tagged full length FliI protein was shown to possess ATPase activity, hydrolyzing ATP at a rate of 0.15 +/- .02 micromol min-1 mg-1 in a time- and dose-dependant manner. Using bacterial-2-hybrid and GST pull-down assays, the N-terminal domain of FliI was shown to interact with the cytoplasmic domain of FlhA, but not with FliF, and the cytoplasmic domain of FlhA was shown to interact with the C-terminus of FliF. The absence of other flagellar orthologs led us to explore cross-reaction of flagellar proteins with type III secretion proteins, and we found that FliI interacted with CdsL and CopN, while FlhA interacted with CdsL and Cpn0322 (YscU ortholog CdsU). CONCLUSIONS: The specific interaction of the four orthologous flagellar proteins in C. pneumoniae suggests that they interact in vivo and, taken together with their conservation across members of the chlamydiae sps., and their interaction with T3S components, suggests a role in bacterial replication and/or intracellular survival.

A novel inhibitor of Chlamydophila pneumoniae protein kinase D (PknD) inhibits phosphorylation of CdsD and suppresses bacterial replication.

BACKGROUND: We have shown previously that Chlamydophila pneumoniae contains a dual-specific Ser/Thr protein kinase that phosphorylates CdsD, a structural component of the type III secretion apparatus. To further study the role of PknD in growth and development we sought to identify a PknD inhibitor to determine whether PknD activity is required for replication. RESULTS: Using an in vitro kinase assay we screened 80 known eukaryotic protein kinase inhibitors for activity against PknD and identified a 3'-pyridyl oxindole compound that inhibited PknD autophosphorylation and phosphorylation of CdsD. The PknD inhibitor significantly retarded the growth rate of C. pneumoniae as evidenced by the presence of very small inclusions with a reduced number of bacteria as seen by electron microscopy. These inclusions contained the normal replicative forms including elementary bodies (EB), intermediate bodies (IB) and reticulate bodies (RB), but lacked persistent bodies (PB), indicating that induction of persistence was not the cause of reduced chlamydial growth. Blind passage of C. pneumoniae grown in the presence of this PknD inhibitor for 72 or 84 hr failed to produce inclusions, suggesting this compound blocks an essential step in the production of infectious chlamydial EB. The compound was not toxic to HeLa cells, did not block activation of the MEK/ERK pathway required for chlamydial invasion and did not block intracellular replication of either Chlamydia trachomatis serovar D or Salmonella enterica sv. Typhimurium suggesting that the inhibitory effect of the compound is specific for C. pneumoniae. CONCLUSION: We have identified a 3'-pyridyl oxindole compound that inhibits the in vitro kinase activity of C. pneumoniae PknD and inhibits the growth and production of infectious C. pneumoniae progeny in HeLa cells. Together, these results suggest that PknD may play a key role in the developmental cycle of C. pneumoniae.

Chlamydia pneumoniae encodes a functional aromatic amino acid hydroxylase.

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 (AroAA-Hs). AroAA-Hs hydroxylate phenylalanine, tyrosine, and tryptophan into tyrosine, dihydroxyphenylalanine, 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-Hs 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 hydroxylate 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.

Proteomic analysis of Chlamydia pneumoniae-infected HL cells reveals extensive degradation of cytoskeletal proteins.

Cytoskeletal proteins of HL cells, following Chlamydia pneumoniae infection, were studied by two-dimensional gel electrophoresis and two-dimensional difference gel electrophoresis. Proteome analyses of HL cells at 48 and 72 h postinfection revealed significant changes in important constituents of the intermediate filament and microtubulin networks. These cytoskeletal proteins, identified as keratin K8, keratin K18, vimentin and beta-tubulin, were represented by several distinct spots with different pI and molecular weight values, implying that they have undergone posttranslational modifications stimulated by the infection. According to MS analyses, these proteins appeared to be N- and/or C-terminally truncated. Additional immunoblot analyses suggested that inhibiting the activity of the chlamydial protease-like activity factor (CPAF) by lactacystin results in increased stability of keratin K18, vimentin and beta-tubulin in infected HL cells. Interestingly, primary amino acid sequence analyses revealed potential recognition/cleavage sites for CPAF in each of these cytoskeletal proteins. These results provide an insight into the pathogenic mechanisms exploited by chlamydia, and suggest that proteolytic modification of the indicated proteins may be involved in establishing a productive infection.

Chlamydophila pneumoniae HflX belongs to an uncharacterized family of conserved GTPases and associates with the Escherichia coli 50S large ribosomal subunit.

Predicted members of the HflX subfamily of phosphate-binding-loop guanosine triphosphatases (GTPases) are widely distributed in the bacterial kingdom but remain virtually uncharacterized. In an attempt to understand mechanisms used for regulation of growth and development in the chlamydiae, obligate intracellular and developmentally complex bacteria, we have begun investigations into chlamydial GTPases; we report here what appears to be the first analysis of a HflX family GTPase using a predicted homologue from Chlamydophila pneumoniae. In agreement with phylogenetic predictions for members of this GTPase family, purified recombinant Cp. pneumoniae HflX was specific for guanine nucleotides and exhibited a slow intrinsic GTPase activity when incubated with [gamma-(32)P]GTP. Using HflX-specific monoclonal antibodies, HflX could be detected by Western blotting and high-resolution confocal microscopy throughout the vegetative growth cycle of Cp. pneumoniae and, at early time points, appeared to partly localize to the membrane. Ectopic expression of Cp. pneumoniae HflX in Escherichia coli revealed co-sedimentation of HflX with the E. coli 50S large ribosomal subunit. The results of this work open up some intriguing possibilities for the role of GTPases belonging to this previously uncharacterized family of bacterial GTPases. Ribosome association is a feature shared by other important conserved GTPase families and more detailed investigations will be required to delineate the role of HflX in bacterial ribosome function.

Characterization of the putative type III secretion ATPase CdsN (Cpn0707) of Chlamydophila pneumoniae.

Type III secretion (T3S) is utilized by a wide range of gram-negative bacterial pathogens to allow the efficient delivery of effector proteins into the host cell cytoplasm through the use of a syringe-like injectisome. Chlamydophila pneumoniae is a gram-negative, obligate intracellular pathogen that has the structural genes coding for a T3S system, but the functionality of the system has not yet been demonstrated. T3S is dependent on ATPase activity, which catalyzes the unfolding of proteins and the secretion of effector proteins through the injectisome. CdsN (Cpn0707) is predicted to be the T3S ATPase of C. pneumoniae based on sequence similarity to other T3S ATPases. Full-length CdsN and a C-terminal truncation of CdsN were cloned as glutathione S-transferase (GST)-tagged constructs and expressed in Escherichia coli. The GST-tagged C-terminal truncation of CdsN possessed ATPase activity, catalyzing the release of ADP and P(i) from ATP at a rate of 0.55 +/- 0.07 micromol min(-1) mg(-1) in a time- and dose-dependent manner. CdsN formed oligomers and high-molecular-weight multimers, as assessed by formaldehyde fixation and nondenaturing polyacrylamide gel electrophoresis. Using bacterial two-hybrid and GST pull-down assays, CdsN was shown to interact with CdsD, CdsL, CdsQ, and CopN, four putative structural components of the C. pneumoniae T3S system. CdsN also interacted with an unannotated protein, Cpn0706, a putative CdsN chaperone. Interactions between CdsN, CdsD, and CopN represent novel interactions not previously reported for other bacterial T3S systems and may be important in the localization and/or function of the ATPase at the inner membrane of C. pneumoniae.