Localization and characterization of a putative cysteine desulfurase in Chlamydia psittaci.

Chlamydia psittaci is an obligate intracellular pathogen with a biphasic developmental life cycle. It is auxotrophic for a variety of essential metabolites and obtains amino acids from eukaryotic host cells. Chlamydia can develop inside host cells within chlamydial inclusions. A pathway secreting proteins from inclusions into the host cellular cytoplasm is the type III secretion system (T3SS). The T3SS is universal among several Gram-negative bacteria. Here, we show that CPSIT_0959 of C. psittaci is expressed midcycle and secreted into the infected cellular cytoplasm via the T3SS. Recombinant CPSIT_0959 possesses cysteine desulfurase and PLP-binding activity, which removes sulfur from cysteine to produce alanine, and helps chlamydial replication. Our study shows that CPSIT_0959 improve the infectivity of offspring elementary bodies and seems to promote the replication by its product. This phenomenon has inhibited by the PLP-dependent enzymes inhibitor. Moreover, CPSIT_0959 increased expression of Bim and tBid, and decreased the mitochondrial membrane potential of host mitochondria to induce apoptosis in the latecycle for release of offspring. These results demonstrate that CPSIT_0959 has cysteine desulfurase and PLP-binding activity and is likely to contribute to apoptosis of the infected cells via a mitochondria-mediated pathway to improve the infectivity of progeny.

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

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.

Tumor necrosis factor-alpha and lipopolysaccharide enhance interferon-induced antichlamydial indoleamine dioxygenase activity independently.

In macrophages, interleukin-1 (IL-1) and lipopolysaccharide (LPS) enhance the antichlamydial effect of interferon-gamma (IFN-gamma) by increasing indoleamine 2,3-dioxygenase (IDO) activity in a dose-dependent manner. Our objectives were to characterize the antichlamydial effect of tumor necrosis factor-alpha (TNF-alpha) on IFN-induced IDO activity and to establish the relationship between LPS and TNF-alpha in IDO potentiation. TNF-alpha inhibited chlamydial growth in a dose-dependent manner only in IFN-treated macrophages. Furthermore, excess tryptophan reversed the effect of combined cytokine treatment, indicating that IDO alone was responsible for chlamydial inhibition. The promonocyte THP-1 cell line, previously used to model the effect of IL-1 on IDO mRNA expression, was treated with IFN-gamma and increasing concentrations of LPS or TNF-alpha. IDO mRNA was quantified by RT-PCR, and IDO activity was measured by HPLC at 24 and 48 h after treatment, respectively. Both LPS and TNF-alpha enhanced IDO activity and IDO mRNA expression, with maximal IDO induction at 100 ng/ml LPS or 5 ng/ml TNF-alpha. Anti-TNF-alpha failed to neutralize the effects of LPS treatment, and insufficient TNF-alpha or IL-1 was produced by LPS-treated THP-1 cells to account for the enhancing effect of LPS, indicating that the effect of LPS on IDO was independent of TNF-alpha and IL-1.

Molecular cloning, sequence analysis and functional characterization of the gene kdsA, encoding 3-deoxy-D-manno-2-octulosonate-8-phosphate synthase of Chlamydia psittaci 6BC.

The kdsA gene encoding 3-deoxy-D-manno-2-octulosonate-8-phosphate (Kdo-8-P) synthase of Chlamydia psittaci 6BC was cloned by complementing the temperature-sensitive kdsA mutant Salmonella enterica serovar Typhimurium AG701i50. The sequence analysis of a recombinant DNA fragment revealed an open reading frame of 807 nucleotides which codes for a polypeptide of 269 amino acids with a high degree of similarity to known KdsA proteins. In addition, alignments of Kdo-8-P synthases with bacterial and fungal 3-deoxy-D-arabino-2-heptulosonate-7-phosphate (Dha-7-P) synthases suggested that both classes of enzymes are structurally related and may belong to a family of 2-keto-3-deoxy-aldonic acid synthases. The chlamydial protein was overexpressed and functionally characterized in vitro to synthesize Kdo-8-P from D-arabinose 5-phosphate and phosphoenolpyruvate. A chlamydial DNA region upstream of the gene exhibiting similarities to the consensus sequence of sigma 70 promoters of Escherichia coli was responsible for the heterologous expression of kdsA.

Enhancement of in vitro transcription by addition of cloned, overexpressed major sigma factor of Chlamydia psittaci 6BC.

Obligate parasitic bacteria of the genus Chlamydia possess a developmental cycle that takes place entirely within eucaryotic host cells. Because standard methods of genetic analysis are not available for chlamydiae, an in vitro transcription system has been developed to elucidate the mechanisms by which chlamydiae regulate gene expression. The in vitro system is specific for chlamydial promoters but is inefficient, presumably because the RNA polymerase is not saturated with sigma factor. Therefore, we prepared recombinant Chlamydia psittaci 6BC major sigma factor to enhance transcription in the in vitro system. The gene encoding the major sigma factor (sigA) was identified by using an rpoD box oligonucleotide and was subsequently cloned and sequenced. It was found to encode a potential 571-amino-acid protein (sigma 66) that is greater than 90% identical to the previously identified major sigma factors from the L2 and MoPn strains of Chlamydia trachomatis. sigA was recloned into a T7 RNA polymerase expression system to produce large quantities of sigma 66 in Escherichia coli. Overexpressed sigma 66 was identified by immunoblot by using monoclonal antibodies 2G10 (reactive) and 2F8 (nonreactive) generated against E. coli sigma 70. After purification by polyacrylamide gel electrophoresis, the recombinant protein was found to stimulate, by 10-fold or more, promoter-specific in vitro transcription by C. psittaci 6BC and C. trachomatis L2 RNA polymerases. Transcription was dependent on added chlamydial sigma 66, rather than on potentially contaminating E. coli sigma 70 or other fortuitous activators, since the monoclonal antibody 2G10, and not 2F8, inhibited transcription initiation. Recombinant omega(66) had no effect on transcription by E. coli core polymerase. The addition of recombinant omega(66) to the in vitro system should be useful for distinguishing omega(66)-dependent transcription of developmentally regulated chlamydial genes from omega(66)-independent transcription.

Enzyme electrophoretic polymorphism differentiates invasive from non-invasive Chlamydia psittaci ruminant isolates.

A group of 24 Chlamydia psittaci strains isolated from ruminants, belonging to serotype 1 and previously classified as invasive in a mouse model of virulence, was compared to a group of 10 non-invasive strains belonging to serotype 2 by using determination of glucose-6-phosphate and L-malate dehydrogenase zymotypes resulting of the infection of cells by these strains. The serotype 1 or invasive isolates represent a homogeneous group by sharing a unique zymotype which was not observed in the non-invasive strains. On the contrary, the serotype 2 or non-invasive isolates constitute a heterogeneous group in generating 2 different zymotypes. Zymotyping clearly distinguishes the ruminant strains from an avian C. psittaci and two C. trachomatis isolates studied for comparison. Our results suggest the usefulness of the studied molecular approach for chlamydiae typing. Furthermore, it can be used as marker of virulence within the C. psittaci strains isolated from ruminants.

Use of HeLa cell guanine nucleotides by Chlamydia psittaci.

Exogenous guanine was found to be incorporated into the nucleic acids of Chlamydia psittaci when the parasite was grown in HeLa cells containing hypoxanthine guanine phosphoribosyltransferase (EC 2.4.2.8) activity but not when the parasite was grown in transferase-deficient HeLa cells. No evidence for a chlamydia-specific transferase activity was found in either transferase-containing or transferase-deficient infected HeLa cells. It is concluded that C. psittaci is incapable of metabolizing guanine, but that the parasite can use host-generated guanine nucleotides as precursors for nucleic acid synthesis.

Detection of DNA polymerase activity in Chlamydia psittaci.

DNA polymerase activities of intact and disrupted suspensions of the mature infectious, extracellular elementary bodies of the meningopneumonitis strain of Chlamydia psittaci were studied. Intact elementary bodies failed to incorporate labeled thymidine triphosphate (TTP), but homogenates of the organisms did incorporate TTP into the acid insoluble fraction; the reaction continued at a linear rate for 60 min and the newly synthesized DNA hybridized exclusively with DNA derived from C. psittaci elementary bodies. Synthesized DNA sedimented in 2 fractions in alkaline sucrose gradients, one in the 50S region and the other in the 10S region, corresponding to molecular weights of 2 X 10(7) and 5 X 10(5) daltons, respectively.