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Tryptophan recycling is responsible for the interferon-γ resistance of Chlamydia psittaci GPIC in indoleamine dioxygenase-expressing host cells (2004)

Wood, Heidi ; Roshick, Christine ; McClarty, Grant

Oxford, UK : Blackwell Science Ltd

Molecular microbiology 52 (2004), S. 0

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ISSN: 1365-2958

Source: Blackwell Publishing Journal Backfiles 1879-2005

Topics: Biology , Medicine

Notes: Comparative genomics indicates that vast differences in Chlamydia sp. host range and disease characteristics can be traced back to subtle variations in gene content within a region of the chromosome termed the plasticity zone. Genes required for tryptophan biosynthesis are located in the plasticity zone; however, the complement of genes encoded varies depending on the chlamydial species examined. Of the sequenced chlamydia genomes, Chlamydia psittaci GPIC contains the most complete tryptophan biosynthesis operon, encoding trpRDCFBA. Immediately downstream of the trp operon are genes encoding kynureninase and ribose phosphate pyrophosphokinase. Here, we show that, in GPIC, these genes are transcribed as a single transcript, the expression of which is regulated by tryptophan. Complementation analyses, using various mutant Escherichia coli isolates, indicate that the tryptophan biosynthesis, kynureninase and ribose phosphate pyrophosphokinase gene products are functional. Furthermore, growth of C. psittaci GPIC in HeLa cells, cultured in tryptophan-free medium, could be rescued by the addition of anthranilate, kynurenine or indole. In total, our results indicate that this complement of genes enables GPIC to recycle tryptophan and thus accounts for the interferon-γ resistant phenotype displayed in indoleamine-2,3-dioxygenase-expressing host cells.

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1111/j.1365-2958.2004.04029.x

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Pyruvate kinase from Chlamydia trachomatis is activated by fructose-2,6-bisphosphate (2002)

Iliffe-Lee, Emma R. ; McClarty, Grant

Oxford, UK : Blackwell Science Ltd.

Molecular microbiology 44 (2002), S. 0

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ISSN: 1365-2958

Source: Blackwell Publishing Journal Backfiles 1879-2005

Topics: Biology , Medicine

Notes: Pyruvate kinase is the final regulatory point in the catabolic Embden–Meyerhoff–Parnas pathway, which controls the carbon flux of glycolytic intermediates and regulates the level of ATP in the cell. In a previous study, we identified, cloned and sequenced pyruvate kinase from the obligate intracellular bacterium Chlamydia trachomatis and demonstrated that the enzyme was active in crude extract. Here, we report the kinetic properties of highly purified C. tracho-matis pyruvate kinase. The results indicate that C. trachomatis pyruvate kinase is 53.5 kDa with a pH optima of 7.3. Kinetic studies show that C. trachomatis pyruvate kinase requires both K+ and Mg2+ ions for activity, exhibits sigmoidal kinetics with respect to phosphoenolpyruvate and Michaelis–Menten kinetics with respect to ADP. In addition, C. trachomatis pyruvate kinase is able to use alternative nucleoside diphosphates as phosphate acceptors, although it shows the greatest activity with ADP. In contrast to other bacterial pyruvate kinases that are activated by AMP, our data show that AMP, in addition to ATP and GTP, inhibits C. trachomatis pyruvate kinase. Surprisingly, unlike any other known bacterial pyruvate kinase, C. trachomatis pyruvate kinase was alloste-rically activated by fructose-2,6-bisphosphate, an important regulatory metabolite that has only been reported in eukaryotes.

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1046/j.1365-2958.2002.02924.x

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Regulation of tryptophan synthase gene expression in Chlamydia trachomatis (2003)

Wood, Heidi ; Fehlner-Gardner, Christine ; Berry, Jody ; [et al.]

Oxford, UK : Blackwell Science Ltd

Molecular microbiology 49 (2003), S. 0

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ISSN: 1365-2958

Source: Blackwell Publishing Journal Backfiles 1879-2005

Topics: Biology , Medicine

Notes: We previously reported that Chlamydia trachomatis expresses the genes encoding tryptophan synthase (trpA and trpB). The results presented here indicate that C. trachomatis also expresses the tryptophan repressor gene (trpR). The complement of genes regulated by tryptophan levels in C. trachomatis is limited to trpBA and trpR. trp gene expression was repressed if chlamydiae-infected HeLa cells were cultured the presence of tryptophan and induced if grown in tryptophan-depleted medium or in the presence of IFN-γ. Furthermore, expression of the trp genes in strains which encode a functional tryptophan synthase is repressed when infected cells are cultured in the presence of the tryptophan precursor indole. Results from experiments with cycloheximide, an inhibitor of eukaryotic protein synthesis, indicate that in addition to the absolute size of the intracellular tryptophan pool, host competition for available tryptophan plays a key role in regulating expression of the trp genes. The tryptophan analogue, 5-fluorotryptophan, repressed trp gene expression and induced the formation of aberrant organisms of C. trachomatis. The growth-inhibitory properties of 5-fluorotryptophan could be reversed with exogenous tryptophan but not indole. In total, our results indicate that the ability to regulate trp gene expression in response to tryptophan availability is advantageous for the intracellular survival of this organism. Furthermore, the fact that C. trachomatis has retained the capacity to respond to tryptophan limitation supports the view that the in vivo antichlamydial effect of IFN-γ is via the induction of the tryptophan-degrading enzyme, indoleamine 2,3-dioxygenase.

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1046/j.1365-2958.2003.03638.x

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Regulation of carbon metabolism in Chlamydia trachomatis (2000)

Iliffe-Lee, Emma R. ; McClarty, Grant

Oxford, UK : Blackwell Science Ltd

Molecular microbiology 38 (2000), S. 0

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ISSN: 1365-2958

Source: Blackwell Publishing Journal Backfiles 1879-2005

Topics: Biology , Medicine

Notes: The biological significance of glycogen accumulation and how the process is regulated in Chlamydia trachomatis remains poorly defined. C. trachomatis-infected HeLa cells were cultured in medium containing various glucose concentrations (0, 0.1, 1 or 10 mg ml−1) or in the presence of gluconeogenic carbon sources (20 mM glutamate, 20 mM malate, 20 mM α-ketoglutarate or 20 mM oxaloacetate), and the effects of these different culture conditions on the production of infectious chlamydial elementary bodies and glycogen accumulation were monitored. When chlamydiae were cultured in glucose concentrations greater than 1 mg ml−1, optimal growth and maximal glycogen accumulation occurred. In contrast to uninfected HeLa cells, which increased their glycogen stores when grown in the presence of high glucose concentrations, chlamydial glycogen accumulation remained essentially constant. When cultured in medium supplemented with either reduced glucose concentrations or any of the gluconeogenic carbon sources, chlamydiae still grew; however, the yield of elementary bodies was substantially decreased, and there was no significant amount of glycogen accumulated by host HeLa cells or C. trachomatis. This suggests that glycogen accumulation may not be essential for chlamydial survival. Reverse transcriptase–polymerase chain reaction (RT–PCR) results indicated that, despite the fact that the source and amount of carbon available in the medium affected chlamydial glycogen accumulation, the expression of genes required for glycogen metabolism was not significantly changed. Similarly, the expression of several genes encoding key enzymes of central metabolism was not affected by alterations in carbon source or availability. Taken together, the data suggest that, unlike most free-living bacteria, chlamydia are unable to alter the expression of genes involved in carbon metabolism in response to changes in environmental conditions.

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1046/j.1365-2958.2000.02102.x

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