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.
more »
« less
Characterization of Chlamydial Rho and the Role of Rho-Mediated Transcriptional Polarity during Interferon Gamma-Mediated Tryptophan Limitation
ABSTRACT As an obligate intracellular, developmentally regulated bacterium, Chlamydia is sensitive to amino acid fluctuations within its host cell. When human epithelial cells are treated with the cytokine interferon gamma (IFN-γ), the tryptophan (Trp)-degrading enzyme, indoleamine-2,3-dioxygenase, is induced. Chlamydiae within such cells are starved for Trp and enter a state of so-called persistence. Chlamydia lacks the stringent response used by many eubacteria to respond to this stress. Unusually, chlamydial transcription is globally elevated during Trp starvation with transcripts for Trp codon-containing genes disproportionately increased. Yet, the presence of Trp codons destabilized 3′ ends of transcripts in operons or large genes. We initially hypothesized that ribosome stalling on Trp codons rendered the 3′ ends sensitive to RNase activity. The half-life of chlamydial transcripts containing different numbers of Trp codons was thus measured in untreated and IFN-γ-treated infected cells to determine whether Trp codons influenced the stability of transcripts. However, no effect of Trp codon content was detected. Therefore, we investigated whether Rho-dependent transcription termination could play a role in mediating transcript instability. Rho is expressed as a midcycle gene product, interacts with itself as predicted, and is present in all chlamydial species. Inhibition of Rho via the Rho-specific antibiotic, bicyclomycin, and overexpression of Rho are detrimental to chlamydiae. Finally, when we measured transcript abundance 3′ to Trp codons in the presence of bicyclomycin, we observed that transcript abundance increased. These data are the first to demonstrate the importance of Rho in Chlamydia and the role of Rho-dependent transcription polarity during persistence.
more »
« less
- Award ID(s):
- 1810599
- NSF-PAR ID:
- 10089571
- Date Published:
- Journal Name:
- Infection and Immunity
- Volume:
- 86
- Issue:
- 7
- ISSN:
- 0019-9567
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
More Like this
-
-
van Wezel, Gilles P. (Ed.)ABSTRACT Chlamydia trachomatis and Streptococcus pyogenes are among the most prevalent bacterial pathogens of humans. Interestingly, both pathogens are tryptophan (Trp) auxotrophs and must acquire this essential amino acid from their environment. For Chlamydia , an obligate intracellular bacterium, this means scavenging Trp from the host cell in which they reside. For Streptococcus , a primarily extracellular bacterium, this means scavenging Trp from the local environment. In the course of a natural immune response, both pathogens can be exposed to Trp-limiting conditions through the action of the interferon gamma-inducible IDO1 enzyme, which catabolizes Trp to N -formylkynurenine. How these pathogens respond to Trp starvation is incompletely understood. However, we have previously demonstrated that genes enriched in Trp codons were preferentially transcribed in C. pneumoniae during Trp limitation. Chlamydia , but not Streptococcus , lacks a stringent response, which is a global regulon activated by uncharged tRNAs binding in the A site of the ribosome. We hypothesized that the chlamydial response to Trp limitation is a consequence of lacking a stringent response. To test this, we compared global transcription profiles of C. trachomatis to both wild-type and stringent response mutant strains of Streptococcus during Trp starvation. We observed that both Trp auxotrophs respond with codon-dependent changes in their transcriptional profiles that correlate with Trp codon content but not transcript stability. Importantly, the stringent response had no impact on these transcriptional changes, suggesting an evolutionarily conserved adaptation to Trp starvation. Therefore, we have revealed a novel response of Trp auxotrophic pathogens in response to Trp starvation. IMPORTANCE Chlamydia trachomatis and Streptococcus pyogenes are important pathogens of humans. Interestingly, both are auxotrophic for tryptophan and acquire this essential amino acid from the host environment. However, part of the host defense against pathogens includes the degradation of tryptophan pools. Therefore, Chlamydia and Streptococcus are particularly susceptible to tryptophan starvation. Most model bacteria respond to amino acid starvation by using a global regulon called the stringent response. However, Chlamydia lacks a stringent response. Here, we investigated the chlamydial response to tryptophan starvation and compared it to both wild-type and stringent response mutant strains of S. pyogenes to determine what role a functional stringent response plays during tryptophan starvation in these pathogens. We determined that both of these pathogens respond to tryptophan starvation by increasing transcription of tryptophan codon-rich genes. This effect was not dependent on the stringent response and highlights a previously unrecognized and potentially evolutionarily conserved mechanism for surviving tryptophan starvation.more » « less
-
more » « less
Abstract As a model organism for studies of cell and environmental biology, the free‐living and cosmopolitan ciliate
Euplotes vannus shows intriguing features like dual genome architecture (i.e., separate germline and somatic nuclei in each cell/organism), “gene‐sized” chromosomes, stop codon reassignment, programmed ribosomal frameshifting (PRF) and strong resistance to environmental stressors. However, the molecular mechanisms that account for these remarkable traits remain largely unknown. Here we report a combined analysis of de novo assembled high‐quality macronuclear (MAC; i.e., somatic) and partial micronuclear (MIC; i.e., germline) genome sequences forE. vannus , and transcriptome profiling data under varying conditions. The results demonstrate that: (a) the MAC genome contains more than 25,000 complete “gene‐sized” nanochromosomes (~85 Mb haploid genome size) with the N50 ~2.7 kb; (b) although there is a high frequency of frameshifting at stop codons UAA and UAG, we did not observe impaired transcript abundance as a result of PRF in this species as has been reported for other euplotids; (c) the sequence motif 5′‐TA‐3′ is conserved at nearly all internally‐eliminated sequence (IES) boundaries in the MIC genome, and chromosome breakage sites (CBSs) are duplicated and retained in the MAC genome; (d) by profiling the weighted correlation network of genes in the MAC under different environmental stressors, including nutrient scarcity, extreme temperature, salinity and the presence of ammonia, we identified gene clusters that respond to these external physical or chemical stimulations, and (e) we observed a dramatic increase in HSP70 gene transcription under salinity and chemical stresses but surprisingly, not under temperature changes; we link this temperature‐resistance to the evolved loss of temperature stress‐sensitive elements in regulatory regions. Together with the genome resources generated in this study, which are available online atEuplotes vannus Genome Database (http://evan.ciliate.org ), these data provide molecular evidence for understanding the unique biology of highly adaptable microorganisms. -
Margolin, William (Ed.)ABSTRACT Chlamydia is an obligate intracellular bacterial pathogen that has significantly reduced its genome size in adapting to its intracellular niche. Among the genes that Chlamydia has eliminated is ftsZ , encoding the central organizer of cell division that directs cell wall synthesis in the division septum. These Gram-negative pathogens have cell envelopes that lack peptidoglycan (PG), yet they use PG for cell division purposes. Recent research into chlamydial PG synthesis, components of the chlamydial divisome, and the mechanism of chlamydial division have significantly advanced our understanding of these processes in a unique and important pathogen. For example, it has been definitively confirmed that chlamydiae synthesize a canonical PG structure during cell division. Various studies have suggested and provided evidence that Chlamydia uses MreB to substitute for FtsZ in organizing and coordinating the divisome during division, components of which have been identified and characterized. Finally, as opposed to using an FtsZ-dependent binary fission process, Chlamydia employs an MreB-dependent polarized budding process to divide. A brief historical context for these key advances is presented along with a discussion of the current state of knowledge of chlamydial cell division.more » « less
-
The tnaC regulatory gene from the tna operon of Escherichia coli controls the transcription of its own operon through an attenuation mechanism relying on the accumulation of arrested ribosomes during inhibition of its own translation termination. This free L-Trp dependent mechanism of inhibition of translation termination remains unclear. Here, we analyzed the inhibitory effects of L-Trp on the function of two known E. coli translation termination factors, RF1 and RF2. Using a series of reporter genes, we found that the in vivo L-Trp sensitivity of tnaC gene expression is influenced by the identity of its stop codon, with the UGA stop codon producing higher expression efficiency of the tnaA-lacZ gene construct than the UAG stop codon. in vitro TnaC-peptidyl-tRNA accumulation and toeprinting assays confirmed that in the presence of L-Trp, the UGA stop codon generates higher accumulation of both TnaC-peptidyl-tRNA and arrested ribosomes than does the UAG stop codon. RF-mediated hydrolysis assays corroborated that L-Trp blocks RF2 function more than that of RF1. Mutational analyses disclosed that amino acids substitutions at the 246 and 256 residue positions surrounding the RF2-GGQ functional motif reduce L-Trp dependent expression of the tnaC(UGA) tnaA-lacZ construct and the ability of L-Trp to inhibit RF2-mediated cleavage of the TnaC-peptidyl-tRNA. Altogether, our results indicate that L-Trp preferentially blocks RF2 activity during translation termination of the tnaC gene. This inhibition depends on the identities of amino acid residues surrounding the RF2-GGQ functional motif.more » « less
- Free Publicly Accessible Full Text
-
Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1128/IAI.00240-18
