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ABSTRACTGamma interferon (IFN-γ) induces expression of the tryptophan-catabolizing enzyme indoleamine 2,3-dioxygenase (IDO1) in human epithelial cells, the permissive cells for the obligate intracellular bacterium Chlamydia trachomatis. IDO1 depletes tryptophan by catabolizing it to kynurenine with consequences for C. trachomatis, which is a tryptophan auxotroph. In vitrostudies reveal that tryptophan depletion can result in the formation of persistent (viable but noncultivable) chlamydial forms. Here, we tested the effects of the IDO1 inhibitor, levo-1-methyl-tryptophan (L-1MT), on IFN-γ-induced C. trachomatispersistence. We found that addition of 0.2 mM L-1MT to IFN-γ-exposed infected HeLa cell cultures restricted IDO1 activity at the mid-stage (20 h postinfection [hpi]) of the chlamydial developmental cycle. This delayed tryptophan depletion until the late stage (38 hpi) of the cycle. Parallel morphological and gene expression studies indicated a consequence of the delay was a block in the induction of C. trachomatispersistence by IFN-γ. Furthermore, L-1MT addition allowed C. trachomatisto undergo secondary differentiation, albeit with limited productive multiplication of the bacterium. IFN-γ-induced persistent infections in epithelial cells have been previously reported to be more resistant to doxycycline than normal productive infections in vitro. Pertinent to this observation, we found that L-1MT significantly improved the efficacy of doxycycline in clearing persistent C. trachomatisforms. It has been postulated that persistent forms of C. trachomatismay contribute to chronic chlamydial disease. Our findings suggest that IDO1 inhibitors such as L-1MT might provide a novel means to investigate, and potentially target, persistent chlamydial forms, particularly in conjunction with conventional therapeutics.

ABSTRACTWe previously demonstrated that plasmid-deficient Chlamydia muridarumretains the ability to infect the murine genital tract but does not elicit oviduct pathology because it fails to activate Toll-like receptor 2 (TLR2). We derived a plasmid-cured derivative of the human genital isolate Chlamydia trachomatisD/UW-3/Cx, strain CTD153, which also fails to activate TLR2, indicating this virulence phenotype is associated with plasmid loss in both C. trachomatisand C. muridarum.As observed with plasmid-deficient C. muridarum, CTD153 displayed impaired accumulation of glycogen within inclusions. Transcriptional profiling of the plasmid-deficient strains by using custom microarrays identified a conserved group of chromosomal loci, the expression of which was similarly controlled in plasmid-deficient C. muridarumstrains CM972 and CM3.1 and plasmid-deficient C. trachomatisCTD153. However, although expression of glycogen synthase, encoded by glgA, was greatly reduced in CTD153, it was unaltered in plasmid-deficient C. muridarumstrains. Thus, additional plasmid-associated factors are required for glycogen accumulation by this chlamydial species. Furthermore, in C. trachomatis, glgAand other plasmid-responsive chromosomal loci (PRCLs) were transcriptionally responsive to glucose limitation, indicating that additional regulatory elements may be involved in the coordinated expression of these candidate virulence effectors. Glucose-limited C. trachomatisdisplayed reduced TLR2 stimulation in an in vitroassay. During human chlamydial infection, glucose limitation may decrease chlamydial virulence through its effects on plasmid-responsive chromosomal genes.

We previously demonstrated that plasmid-deficient Chlamydia muridarum retains the ability to infect the murine genital tract but does not elicit oviduct pathology because it fails to activate Toll-like receptor 2 (TLR2). We derived a plasmid-cured derivative of the human genital isolate Chlamydia trachomatis D/UW-3/Cx, strain CTD153, which also fails to activate TLR2, indicating this virulence phenotype is associated with plasmid loss in both C. trachomatis and C. muridarum. As observed with plasmid-deficient C. muridarum, CTD153 displayed impaired accumulation of glycogen within inclusions. Transcriptional profiling of the plasmid-deficient strains by using custom microarrays identified a conserved group of chromosomal loci, the expression of which was similarly controlled in plasmid-deficient C. muridarum strains CM972 and CM3.1 and plasmid-deficient C. trachomatis CTD153. However, although expression of glycogen synthase, encoded by glgA, was greatly reduced in CTD153, it was unaltered in plasmid-deficient C. muridarum strains. Thus, additional plasmid-associated factors are required for glycogen accumulation by this chlamydial species. Furthermore, in C. trachomatis, glgA and other plasmid-responsive chromosomal loci (PRCLs) were transcriptionally responsive to glucose limitation, indicating that additional regulatory elements may be involved in the coordinated expression of these candidate virulence effectors. Glucose-limited C. trachomatis displayed reduced TLR2 stimulation in an in vitro assay. During human chlamydial infection, glucose limitation may decrease chlamydial virulence through its effects on plasmid-responsive chromosomal genes.