Inhibition of tRNA Synthetases Induces Persistence inChlamydia

Abstract

18Chlamydia trachomatis is the leading cause of bacterial sexually transmitted infections, and C. 19 pneumoniae causes community-acquired respiratory infections. In vivo, the host immune system 20 will release interferon-gamma (IFNγ) to combat infection. IFNγ activates human cells to produce 21 the tryptophan (trp) catabolizing enzyme, IDO. Consequently, there is a reduction in cytosolic trp 22 in IFNγ-activated host cells. In evolving to obligate intracellular dependence, Chlamydia has 23 significantly reduced its genome size and content as it relies on the host cell for various nutrients. 24 Importantly, C. trachomatis and C. pneumoniae are trp auxotrophs and are starved for this essential 25 nutrient when the human host cell is exposed to IFNγ. To survive this, chlamydiae enter an 26 alternative growth state referred to as persistence. Chlamydial persistence is characterized by a 27 halt in the division cycle, aberrant morphology, and, in the case of IFNγ-induced persistence, trp 28 codon-dependent changes in transcription. We hypothesize that these changes in transcription are 29 dependent on the particular amino acid starvation state. To investigate the chlamydial response 30 mechanisms acting when other amino acids become limiting, we tested the efficacy of prokaryotic 31 specific tRNA synthetase inhibitors, indolmycin and AN3365, to mimic starvation of trp and 32 leucine, respectively. We show that these drugs block chlamydial growth and induce changes in 33 morphology and transcription consistent with persistence. Importantly, growth inhibition was 34 reversed when the compounds were removed from the medium. With these data, we find that 35 indolmycin and AN3365 are valid tools that can be used to mimic the persistent state independently 36 of IFNγ. 37 Word Count: 248/250 38 39 3 Importance 40The obligate intracellular pathogen Chlamydia trachomatis, although treatable, remains a 41 major public health concern due to rising infection rates. The asymptomatic nature of most 42 Chlamydia infections is hypothesized to be a product of its ability to transition into a slow-growing 43 state referred to as persistence. The most physiologically relevant inducer of persistence is the 44 immune cytokine IFNγ, which in humans activates an enzyme that degrades tryptophan, an 45 essential amino acid that Chlamydia scavenges from the host cell. Unfortunately, the exact timing 46 at which Chlamydia is starved after IFNγ treatment is inexact. To mechanistically study 47 48 at specific times is needed. Here, we demonstrate the capability of tRNA synthetase inhibitors, 49 indolmycin and AN3365, to model persistence independently from the use of IFNγ. These tools 50 will also allow comparisons between amino acid stress responses in this unique bacterium. 51 Word Count: 149/150 52 53Chlamydial diseases are significant causes of morbidity in humans. Chlamydia trachomatis 54 is the leading cause of bacterial sexually transmitted infections in the world. In 2017, the U.S. 55Centers for Disease Control and Prevention ...

Inclusion Membrane Growth and Composition Are Altered by Overexpression of Specific Inclusion Membrane Proteins in Chlamydia trachomatis L2

“…Prior to the collection of nucleic acids, coverslips in each well were fixed and stained for immunofluorescence to confirm the expression of the construct. RNA was extracted from infected HeLa cells using TRIzol (Invitrogen) as previously described (68). Briefly, RNA samples were treated with Turbo DNAfree (Ambion/Thermo Fisher) following the manufacturer's instructions to remove DNA contamination, and cDNA was synthesized from DNA-free RNA using random nonamers (New England BioLabs, Ipswich, MA) and SuperScript III reverse transcriptase (RT; Invitrogen/Thermo Fisher) per the manufacturer's instructions.…”