Inhibition of tRNA Synthetases Induces Persistence inChlamydia

Abstract

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.

The iron-dependent repressor YtgR is a tryptophan-dependent attenuator of the trpRBA operon in Chlamydia trachomatis

“…4a). Treatment of Chlamydia-infected cells with indolmycin also polarizes transcription of the ytg operon, mimicking IFNγ-mediated depletion of host cell tryptophan pools 56 . This regulation is mediated by the chlamydial ortholog of the transcription termination factor Rho, whereby ribosome stalling at tryptophan codon-rich regions of a transcript enables binding of Rho to rho utilization (rut) sites to terminate downstream transcription 57 .…”

“…The W codon content and presence of sequential W codon motifs is indicated above the plots for each gene. Note that these samples were prepared for a prior publication 56 and thus the previously reported methodology applies. c Assessment of the involvement of the termination factor Rho in mediating transcriptional polarity for the genes described above in b.…”

Regulation of an iron-dependent repressor by tryptophan availability attenuates transcription of the tryptophan salvage genes inChlamydia trachomatis

“…Treated cells were then returned to the tissue culture incubator for the remainder of the experimental time course. For cell culture experiments, indolmycin treatment was performed essentially as described previously 45 . In brief, 120 mM indolmycin (Cayman Chemical, Ann Arbor, MI, USA; CAS: 21200-24-8) prepared in DMSO was added to tryptophandepleted DMEM-F12 at a working concentration of 120 µM at 8 hpi and proceeded for 16 or 20 hrs.…”

“…The W codon content and presence of sequential W codon motifs is indicated above the plots for each gene. Note that these samples were prepared for a prior publication 45 and thus the previously reported methodology applies. c, Assessment of the involvement of the termination factor Rho in mediating transcriptional polarity for the genes described above in b.…”

“…4a). Treatment of Chlamydia-infected cells with indolmycin also polarizes transcription of the ytg operon, mimicking IFNg-mediated depletion of host cell tryptophan pools 45 . This regulation is mediated by the chlamydial ortholog of the transcription termination factor Rho, whereby ribosome stalling at tryptophancodon rich regions of a transcript enables binding of Rho to rho utilization (rut) sites to terminate downstream transcription 46 .…”