c-Myc plays a key role in IFN-γ-induced persistence of Chlamydia trachomatis
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Evaluation Summary:
This paper will be of interest to scientists working to understand Chlamydia trachomatis persistence, and host pathogen interaction in general. The authors report the surprising observation that the mechanism of restriction of bacterial growth is through the inhibition of c-Myc signaling by IFNg as opposed to IDO-dependent depletion of tryptophan levels, as had been previously suggested.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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Abstract
Chlamydia trachomatis (Ctr) can persist over extended times within their host cell and thereby establish chronic infections. One of the major inducers of chlamydial persistence is interferon-gamma (IFN-γ) released by immune cells as a mechanism of immune defence. IFN-γ activates the catabolic depletion of L-tryptophan (Trp) via indoleamine-2,3-dioxygenase (IDO), resulting in persistent Ctr . Here, we show that IFN-γ induces the downregulation of c-Myc, the key regulator of host cell metabolism, in a STAT1-dependent manner. Expression of c-Myc rescued Ctr from IFN-γ-induced persistence in cell lines and human fallopian tube organoids. Trp concentrations control c-Myc levels most likely via the PI3K-GSK3β axis. Unbiased metabolic analysis revealed that Ctr infection reprograms the host cell tricarboxylic acid (TCA) cycle to support pyrimidine biosynthesis. Addition of TCA cycle intermediates or pyrimidine/purine nucleosides to infected cells rescued Ctr from IFN-γ-induced persistence. Thus, our results challenge the longstanding hypothesis of Trp depletion through IDO as the major mechanism of IFN-γ-induced metabolic immune defence and significantly extends the understanding of the role of IFN-γ as a broad modulator of host cell metabolism.
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Evaluation Summary:
This paper will be of interest to scientists working to understand Chlamydia trachomatis persistence, and host pathogen interaction in general. The authors report the surprising observation that the mechanism of restriction of bacterial growth is through the inhibition of c-Myc signaling by IFNg as opposed to IDO-dependent depletion of tryptophan levels, as had been previously suggested.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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Reviewer #1 (Public Review):
In "c-Myc plays a key role in IFNγ-induced persistence of Chlamydia trachomatis", Vollmuth et al. investigate how IFNγ controls Chlamydia trachomatis development and persistence via regulation of the transcription c-Myc following infection of human epithelial cells and human fallopian tube organoids. The authors show that: IFNγ induces the downregulation of c-Myc, a key regulator of host cell metabolism, in a STAT1-dependent manner; and that constitutive expression of c-Myc rescues Chlamydia trachomatis from IFNγ-induced persistence. The authors then investigate the relationship between IFNγ-mediated c-Myc repression and IFNγ-mediated tryptophan depletion on Chlamydia trachomatis infection, showing that Chlamydia trachomatis development necessitates both exogenous tryptophan/indole and stabilisation of c-Myc …
Reviewer #1 (Public Review):
In "c-Myc plays a key role in IFNγ-induced persistence of Chlamydia trachomatis", Vollmuth et al. investigate how IFNγ controls Chlamydia trachomatis development and persistence via regulation of the transcription c-Myc following infection of human epithelial cells and human fallopian tube organoids. The authors show that: IFNγ induces the downregulation of c-Myc, a key regulator of host cell metabolism, in a STAT1-dependent manner; and that constitutive expression of c-Myc rescues Chlamydia trachomatis from IFNγ-induced persistence. The authors then investigate the relationship between IFNγ-mediated c-Myc repression and IFNγ-mediated tryptophan depletion on Chlamydia trachomatis infection, showing that Chlamydia trachomatis development necessitates both exogenous tryptophan/indole and stabilisation of c-Myc to overcome IFNγ-mediated persistence. The authors show that supplementation with tryptophan stabilises c-Myc, but conversely, that c-Myc exerts only a limited effect on tryptophan levels in IFNγ-treated Chlamydia trachomatis-infected host cells. Together, this suggested that c-Myc plays a role downstream of tryptophan-/indole mediated rescue of Chlamydia trachomatis development in IFNγ-treated cells. To further characterise how c-Myc achieves this, the authors perform metabolomic profiling in IFNγ-treated Chlamydia trachomatis-infected cells with or without c-Myc-induced rescue, identifying c-Myc dependent increases in TCA cycle intermediates and nucleoside metabolism as potential mediators. Finally, the authors show that IFNγ-mediated suppression of Chlamydia trachomatis development can be overcome in the absence of both constitutive c-Myc expression or tryptophan supplementation via supplementation of Chlamydia trachomatis-infected IFNγ-treated cells with TCA cycle intermediates or nucleosides.
Collectively, this work represents a promising study delineating the pathway by which IFNγ suppresses Chlamydia development during epithelial cell infection, and provides convincing evidence for involvement of the host transcription factor c-Myc. Although promising, the work does suffer from some limitations and concerns. These include an over-reliance at times on Western blots to assess Chlamydia proliferation (most notably in Figure 4 and Figure S4); these results would greatly benefit from additional microscopy-based analysis of Chlamydia proliferation, which the authors use to great effect in Figures 1-3. Additionally, the model linking IFNγ-induced tryptophan limitation to destabilisation of the c-Myc transcription factor via PI3K-GSK3beta is not fully supported by the current evidence. In particular, the authors suggest that c-Myc is destabilised by a lack of tryptophan in IFNγ-treated host epithelial cells during Chlamydia trachomatis infection via a pathway involving first dephosphorylation of GSK3beta that, in turn, results in phosphorylation of c-Myc on threonine 58, and thus ubiquitination and proteasomal degradation. However, the authors have not actually provided direct evidence of either increased phosphorylation of threonine 58 on c-Myc or increased proteasomal degradation of c-Myc as a result of decreased levels of tryptophan upon IFNγ treatment. Rather, the authors themselves provide evidence potentially contradicting such a model, showing that the phosphorylation of threonine 58 is unchanged upon IFNγ treatment. Despite the current limitations, the paper nonetheless shows promise and should be of broad interest.
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Reviewer #2 (Public Review):
This work provides data supporting a new hypothesis for IFN-γ-induced persistence in C. trachomatis infection. Persistent infections are an important cause of tissue damage in the fallopian tubes, which can result in infertility and ectopic pregnancies. Previous work has shown that IFN-γ induces persistence by activating indoleamine 2,3-dioxygenase (IDO), which depletes L-tryptophan, a necessary amino acid that C. trachomatis gets from the host. C. trachomatis will enter a dormant, persistent state in response to nutrient starvation and antibiotic challenge. In previous work examining the alterations of host cell metabolism due to C. trachomatis infection, the authors discovered C. trachomatis infection increases levels of c-myc. Since IFN-γ is known to decrease c-myc levels, the authors made the logical …
Reviewer #2 (Public Review):
This work provides data supporting a new hypothesis for IFN-γ-induced persistence in C. trachomatis infection. Persistent infections are an important cause of tissue damage in the fallopian tubes, which can result in infertility and ectopic pregnancies. Previous work has shown that IFN-γ induces persistence by activating indoleamine 2,3-dioxygenase (IDO), which depletes L-tryptophan, a necessary amino acid that C. trachomatis gets from the host. C. trachomatis will enter a dormant, persistent state in response to nutrient starvation and antibiotic challenge. In previous work examining the alterations of host cell metabolism due to C. trachomatis infection, the authors discovered C. trachomatis infection increases levels of c-myc. Since IFN-γ is known to decrease c-myc levels, the authors made the logical connection that c-myc expression could be altered by IFN-γ during C. trachomatis infection, which could be an additional cause of persistence.
The authors' present work uses well-controlled and designed in vitro assays to study the effects of C. trachomatis infection on c-myc in the presence of IFN-γ. Using cells with inducible silencing of c-myc, as well as those with inducible c-myc expression, enabled the authors to show that c-myc expression can rescue C. trachomatis from IFN-γ-induced persistence. In addition to cell lines, the authors created human fallopian tube organoids and showed C. trachomatis infection in the presence of IFN-γ also resulted in the prevention of c-myc induction.
Since much of the literature of IFN-γ-induced persistence of C. trachomatis is based on tryptophan depletion, the authors set out to determine if there was any interplay between c-myc and tryptophan. Through various in vitro experiments, they found that both viable bacteria and the presence of tryptophan are needed to stabilize c-myc. In addition, they show that restoration of tryptophan resulted in phosphorylation of GSK3β, which consequently led to elevated c-myc levels. Since c-myc is involved in amino acid transport, they investigated and found that c-myc expression does not increase intracellular amounts of tryptophan, showing that restoration of tryptophan metabolism is not the main mechanism c-myc expression uses to result in rescue from persistence.
The authors then use metabolomics analysis to investigate the effect of c-myc expression on C. trachomatis infection with IFN-γ treatment. They found that IFN-γ treatment results in reduced c-myc, which causes a reduction in metabolites, nucleotides, and nucleosides. Supplementing these precursors resulted in partial restoration of C. trachomatis development.
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Reviewer #3 (Public Review):
The authors describe a new mechanism by which an antimicrobial cytokine, IFNg, restricts the growth of the intracellular pathogen Chlamydia trachomatis. IFNg antagonizes the expression of c-myc, a transcription factor associated with cellular growth and metabolism. The authors previously showed that Chlamydia activates the expression of c-myc to promote bacterial replication. By inhibiting c-myc activity, IFNg limits the ability of Chlamydia to replicate. This constitutes a potentially new mechanism of pathogen restriction by IFNg. Major strengths of the study include the use of engineered cell lines to dissect the role of c-myc in Chlamydia replication and how metabolite restriction in the context of IFNg limits Chlamydia replication. Another strength is the use of Chlamydia mutants to discover that …
Reviewer #3 (Public Review):
The authors describe a new mechanism by which an antimicrobial cytokine, IFNg, restricts the growth of the intracellular pathogen Chlamydia trachomatis. IFNg antagonizes the expression of c-myc, a transcription factor associated with cellular growth and metabolism. The authors previously showed that Chlamydia activates the expression of c-myc to promote bacterial replication. By inhibiting c-myc activity, IFNg limits the ability of Chlamydia to replicate. This constitutes a potentially new mechanism of pathogen restriction by IFNg. Major strengths of the study include the use of engineered cell lines to dissect the role of c-myc in Chlamydia replication and how metabolite restriction in the context of IFNg limits Chlamydia replication. Another strength is the use of Chlamydia mutants to discover that depletion of the amino acid tryptophan, which was believed to be critical to the anti-Chlamydia activity of IFNg, is not. Weaknesses of the study include reliance on quantification methods that may not be as accurate as stated and the tendency of some correlations to be stated as causal.
In general, the study is of high significance especially if some of the findings related to a c-myc-IFN axis and the regulation of metabolism can be extended to the controls of other pathogens.
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