
==== Front
Pathog Dis
Pathog Dis
femspd
Pathogens and Disease
2049-632X
Oxford University Press

39210512
10.1093/femspd/ftae019
ftae019
Minireview
AcademicSubjects/SCI01150
Restriction and evasion: a review of IFNγ-mediated cell-autonomous defense pathways during genital Chlamydia infection
Reitano Jeffrey R Writing - original draft Writing - review & editing Department of Integrative Immunobiology, Duke University Medical School, 207 Research Dr. Box 3010, Durham, NC 27710, United States

https://orcid.org/0000-0001-8707-4608
Coers Jörn Writing - review & editing Department of Integrative Immunobiology, Duke University Medical School, 207 Research Dr. Box 3010, Durham, NC 27710, United States
Department of Molecular Genetics and Microbiology, Duke University Medical School, 213 Research Dr. Box 3054, Durham, NC 27710, United States

Corresponding author. Department of Integrative Immunobiology, Duke University Medical School, 207 Research Dr. Box 3010, Durham, NC 27710, United States. E-mails: jeffrey.reitano@duke.edu
Corresponding author. jorn.coers@duke.edu
2024
29 8 2024
29 8 2024
82 ftae01913 6 2024
09 8 2024
22 8 2024
17 9 2024
© The Author(s) 2024. Published by Oxford University Press on behalf of FEMS.
2024
https://creativecommons.org/licenses/by-nc/4.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (https://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com

Abstract

Chlamydia trachomatis is the most common cause of bacterial sexually transmitted infection (STI) in the USA. As an STI, C. trachomatis infections can cause inflammatory damage to the female reproductive tract and downstream sequelae including infertility. No vaccine currently exists to C. trachomatis, which evades sterilizing immune responses in its human host. A better understanding of this evasion will greatly benefit the production of anti-Chlamydia therapeutics and vaccination strategies. This minireview will discuss a single branch of the immune system, which activates in response to genital Chlamydia infection: so-called “cell-autonomous immunity” activated by the cytokine interferon-gamma. We will also discuss the mechanisms by which human and mouse-adapted Chlamydia species evade cell-autonomous immune responses in their native hosts. This minireview will examine five pathways of host defense and their evasion: (i) depletion of tryptophan and other nutrients, (ii) immunity-related GTPase-mediated defense, (iii) production of nitric oxide, (iv) IFNγ-induced cell death, and (v) RNF213-mediated destruction of inclusions.

This review will discuss a single branch of the immune system, so-called “cell-autonomous immunity” and how it is counteracted by Chlamydia species.

cell-autonomous immunity
Chlamydia trachomatis
interferon-γ
ubiquitylation
innate immunity
RNF213
National Institutes of Health 10.13039/100000002 AI103197
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pmcIntroduction

Chlamydia trachomatis (Ct) is the most common sexually transmitted bacterial infection (STI) in the USA (US Centers for Disease Control and Prevention 2024). Additionally, Ct causes ocular infections, which represent the leading cause of infectious blindness worldwide (Resnikoff et al. 2004, Mariotti et al. 2009). As an STI, Ct causes infections in the genital tract, which are often asymptomatic, allowing the bacterium to spread undetected (US Centers for Disease Control and Prevention 2024). A subset of Ct patients develops severe sequelae including pelvic inflammatory disease, ectopic pregnancy, and infertility (Haggerty et al. 2010). These sequelae in particular contribute to Chlamydia-associated medical costs and patient morbidity (Kumar et al. 2018). While effective antibiotics against Ct exist, they are unable to repair the inflammatory damage to the genital tract that Ct causes. Furthermore, antibiotic use has not significantly prevented the spread of Ct over the past several decades (US Centers for Disease Control and Prevention 2024). Instead, many researchers have turned to the development of a vaccine as the best way to prevent the spread and substantial health burden of this disease (Murray and Mckay 2021). This endeavor remains unsuccessful, in part because Ct is adept at evading our immune system’s defenses during infection (Murray and Mckay 2021). The exact mechanisms of Ct immune evasion are still under investigation, but several of these mechanisms will be discussed in this review.

Chlamydia species are obligate intracellular pathogens, which alternate between two developmental forms. Elementary bodies (EBs) are a nondividing infectious form of Chlamydia, which utilize a type III secretion system to invade host cells. Once inside the host cell, EBs inhabit a membrane-bound vacuole termed an “inclusion.” Inside the inclusion, EBs differentiate into a replicating form of Chlamydia called a reticulate body (RB) (Elwell et al. 2016). These RBs multiply within the inclusion for most of their developmental cycle before eventually differentiating back into infectious EBs. Finally, infectious EBs are released from the host cell into the extracellular space, where they infect additional cells or spread to a new host (Elwell et al. 2016). This review will focus on two closely related Chlamydia species, Ct, which has evolved to infect humans, and Chlamydia muridarum (Cm), which has evolved to infect mice. For the human-adapted pathogen Ct, different serovars exhibit differences in tissue tropism. Serovars A–C infect conjunctival epithelial cells causing blinding trachoma. Serovars D–K infect a range of cells in the male genital epithelium and female reproductive tract. Serovars L1–L3 invade the same tissues as D–K, but can also disseminate into lymph nodes to cause lymphogranuloma venereum (Elwell et al. 2016). In mice, the rodent-adapted pathogen Cm can infect the genital epithelium and macrophages and disseminates from the genital tract to the gastrointestinal tract during infection (Elwell et al. 2016, Dockterman and Coers 2021).

Infections with intracellular pathogens like Chlamydia are sensed by innate and adaptive lymphocytes, which respond by producing the cytokine interferon-gamma (IFNγ) (Dockterman and Coers 2021). IFNγ binds and dimerizes the IFNγ receptor, leading to a cascade of events that activates and dimerizes the transcription factor STAT1 (Kak et al. 2018). Once active, STAT1 translocates into the nucleus, where it stimulates expression of hundreds of interferon-stimulated genes (ISGs) with diverse functions. Collectively, these ISGs work to defend IFNγ-primed cells against a variety of intracellular pathogens. This process, where infected cells defend against invading pathogens, is called “cell-autonomous immunity” (Randow et al. 2013). Chlamydia species, however, are highly adapted pathogens which have evolved to evade many mechanisms of cell-autonomous immunity downstream of IFNγ.

Fundamental principles of IFNγ-stimulated defense are conserved between different mammalian host species. However, the exact mechanisms of defense can differ greatly between distinct host species like human and mouse (Finethy and Coers 2016, Kak et al. 2018). Likewise, Chlamydia species have tailored their counter-immune strategies to the unique aspects of their preferred host’s immune system (Elwell et al. 2016, Finethy and Coers 2016). Thus, the human pathogen Ct has specifically evolved to evade many mechanisms of IFNγ-stimulated cell-autonomous immunity in humans but is vulnerable to analogous defenses in mice (Johansson et al. 1997). Likewise, the rodent pathogen Cm is effective at evading mechanisms of IFNγ-stimulated cell-autonomous immunity in mice but is vulnerable to IFNγ in human cells (Nelson et al. 2005, Roshick et al. 2006). On the one hand, these specific host adaptations represent a limitation of using mouse models to understand human infection with Chlamydia. On the other hand, research into these relationships has greatly informed our understanding of the coevolution of host and pathogen, in addition to deepening our understanding of immune evasion during Chlamydia infection.

This minireview will discuss mechanisms of IFNγ-stimulated cell-autonomous immunity against genital Chlamydia infection in humans and mice. We will cover five broad pathways of IFNγ-stimulated defense against Chlamydia (Fig. 1), beginning with the first identified, best-studied pathways of defense and ending with newer discoveries and open questions in the field. In humans, key pathways include the limitation of nutrients and RNF213-mediated ubiquitylation. In mice, defense is mediated by the immunity-related GTPase (IRG) defense pathway and inducible nitric oxide synthase 2 (NOS2). In both species, IFNγ induces anti-Chlamydia cell death. We will also discuss the mechanisms by which two Chlamydia species, the human-adapted Ct and the rodent-adapted Cm, evade these various defenses in their natural hosts.

Figure 1. IFNγ-stimulated cell autonomous defenses against Chlamydia. (i) IDO in human cells degrades l-tryptophan, and additional mechanisms restrict a variety of intracellular nutrients. (ii) IRGMs and ATG8 guide GKS proteins, ubiquitin, p62, and guanylate-binding proteins (GBPs) to the inclusion, leading to inclusion lysis in mouse cells. (iii) NOS2 produces Reactive Nitrogen Species (RNS) in mouse cells, which restrict Chlamydia via direct and/or indirect mechanisms. (iv) Anti-Chlamydia cell death is activated by GBP-driven pyroptosis or caspase-driven inclusion lysis. (v) The human E3 ligase RNF213 ubiquitylates Chlamydia inclusions, leading to bacterial restriction via autophagy.

Indoleamine 2,3-dioxygenase and nutrient limitation

As obligate intracellular bacteria, Chlamydia species have a vastly reduced genome (∼1 Mb) and heavily rely on their host cells for the acquisition of essential nutrients (Elwell et al. 2016). One such nutrient is l-tryptophan, which most Chlamydia species cannot synthesize de novo (Aiyar et al. 2014). In human cells, the most extensively studied IFNγ-stimulated response to Chlamydia involves the enzyme indoleamine 2,3-dioxygenase (IDO). IDO catalyses the breakdown of l-tryptophan to N-formylkynurenine, thus depleting the intracellular environment of an essential amino acid (Dai and Gupta 1990). In human macrophages and epithelial cells, IDO expression is strongly induced by the cytokine IFNγ. This provides an effective method for the human host to “starve” tryptophan-auxotrophic pathogens such as Chlamydia of an essential nutrient and restrict pathogen growth within the cell.

The role of IDO in anti-Chlamydia defense was first discovered using the zoonotic pulmonary pathogen Chlamydia psittaci. Byrne et al. (1986, 1989) were the first to identify that IFNγ-primed human macrophages and epithelial cells restrict growth of C. psittaci through tryptophan depletion, and found that tryptophan supplementation restored growth of the pathogen. IDO was later identified as the enzyme responsible for this IFNγ-dependent tryptophan depletion and restriction of C. psittaci (Thomas et al. 1993). Soon after, it was discovered that IDO also drives restriction of two genital tract Chlamydia species in human epithelial cells: the human-adapted pathogen Ct and murine-adapted pathogen Cm (Nelson et al. 2005).

Recently, there has been a growing appreciation for additional nutrients that are limited in response to IFNγ. In Ct-infected human epithelial cells, IFNγ drives depletion of amino acids glutamate, aspartate, glycine, and alanine; Krebs cycle intermediates citrate, aconitate, and α-ketoglutarate; and the nucleotide triphosphates adenosine triphosphate, cytidine triphosphate, and uridine triphosphate (Vollmuth et al. 2022, Banerjee et al. 2023). This broad metabolic reprogramming appears dependent on c-Myc, a transcriptional regulator of cell growth, death, and metabolism, which is downregulated by IFNγ (Dang 1999, Ramana et al. 2000, Vollmuth et al. 2022). Depletion of c-Myc is sufficient to restrict intracellular Ct growth, even in the absence of IFNγ, while overexpression of c-Myc protects Ct from IFNγ-mediated restriction within human epithelial cells and organoids (Vollmuth et al. 2022). Additionally, IFNγ has been shown to repress expression of the metabolic transcription factor subunit HIF-1α, thereby limiting intracellular glucose levels in human epithelial cells (Shima et al. 2018). This depletion of glucose provides additional nutritional immunity against intracellular Ct, which requires glucose for growth (Shima et al. 2018). Finally, a cocktail of IFNγ, TNFα, IL1, and LPS can drive iron-dependent restriction of Ct in human epithelial cells (Igietseme et al. 1998a). However, the effect of individual cytokines on this process, like IFNγ alone, remains unexplored.

Collectively, these reports demonstrate that IFNγ priming of human cells drives a metabolic shift that starves Chlamydia species of essential nutrients. This shift is classically characterized by the enzyme IDO and depletion of intracellular tryptophan. However, more recent research has shown restriction of chlamydial growth though the depletion of a variety of amino acids and nutrients dependent on metabolic transcription factors such as c-Myc and HIF-1α. Further research is required to better understand the respective importance of each of these nontryptophan metabolites.

The human pathogen Ct has evolved at least two strategies to evade IFNγ-stimulated nutrient deprivation. The first strategy is a specific adaption to IDO-mediated tryptophan depletion and is dependent on a partial Trp operon. Many bacterial species have a Trp operon: a set of enzymes under the same promoter that work together to synthesize tryptophan de novo (Merino et al. 2008). Ct has lost the majority of the genes in this operon. In place of a full operon, genital serovars of Ct including D and L2 utilize a partial Trp operon containing only three genes: the regulatory gene trpR and enzymes trpB/A. TrpR functions as a sensor of tryptophan concentration and repressor of tryptophan synthesis. In high concentrations of tryptophan, TrpR binds upstream of the Trp operon and reduces expression of the operon. During tryptophan limiting conditions, such as those induced by IDO, TrpR releases this upstream region and increases expression of genes trpR/B/A (Wood et al. 2003, Akers and Tan 2006). TrpB/A are enzymes that form a bifunctional, tetrameric enzyme complex (Merino et al. 2008). In most bacteria, TrpA converts indole-3-glycerol phosphate into indole, and TrpB uses this indole to produce tryptophan. However, chlamydial TrpA has lost its enzymatic activity and instead functions as a cofactor to TrpB, increasing the kinetics of tryptophan synthesis from indole (Fehlner-Gardiner et al. 2002, Michalska et al. 2021). Put together, the Ct Trp operon functions in tryptophan-limiting conditions to synthesize tryptophan from indole. This provides Ct with the ability to survive and proliferate in IFNγ-treated cells, as long as indole is present (Fehlner-Gardiner et al. 2002, Caldwell et al. 2003, Kari et al. 2011, O’neill et al. 2018).

What is the source of this indole during a typical human Chlamydia infection? The answer is unknown, but it has been hypothesized that indole originates from the vaginal microbiota (Caldwell et al. 2003, Aiyar et al. 2014). Unlike mammalian cells, which do not produce indole, many bacterial species that colonize the female genital tract are known indole producers (Caldwell et al. 2003). Supporting this hypothesis, bacterial vaginosis (i.e. overgrowth of anaerobic vaginal bacterial species like the indole-producing Prevotella) is markedly associated with increased risk of genital Ct infection (Wiesenfeld et al. 2003, Shipitsyna et al. 2020). Interestingly, ocular serovars of Ct lack the ability to produce tryptophan from indole, either due to a truncation of trpA (observed in serovars A and C) or a lack of the operon entirely (serovar B) (Shaw et al. 2000). The loss of an indole scavenging pathway in ocular Ct serovars is consistent with the absence of indole-producing microbiota in the eye. Like ocular Ct serovars, the rodent-adapted Cm also lacks a trp operon. The lack of a trp operon in Cm is likely not a tissue- but a host-adaptation, since IDO-mediated nutritional immunity is largely absent from mouse cells. While mice have a functional homolog of IDO, it is not induced by IFNγ in most murine cell lines (Roshick et al. 2006). Furthermore, clearance of Ct and Cm infection in the mouse female reproductive tract is independent of IDO and intracellular tryptophan (De La Maza et al. 1985, Nelson et al. 2005, Roshick et al. 2006). Together, these data support a model where genital Ct serovars greatly benefit from a partial Trp operon, since they inhabit a tissue that both limits tryptophan and contains indole-producing bacteria. In contrast, ocular Ct serovars or rodent-adapted Cm receive no selective pressure to maintain the indole scavenging pathway executed by TrpR/B/A.

Ct employs an additional response to IFNγ-mediated nutrient starvation, called persistence (Beatty et al. 1993, 1994, Panzetta et al. 2018). Persistence is characterized by a halt in bacterial division, aberrant morphology, and vast transcriptional changes (Beatty et al. 1994, Belland et al. 2003, Panzetta et al. 2018). In addition to IFNγ, many stressors can induce chlamydial persistence, including antibiotic treatment, iron starvation, and heat shock (Panzetta et al. 2018). Importantly, chlamydial persistence is a temporary state. When a stressor is removed (or when nutrients such as tryptophan or indole are restored), Ct will exit persistence and resume its developmental cycle (Beatty et al. 1993, 1994, Kari et al. 2011).

Recent work in the field has focused on two facets of IFNγ-induced chlamydial persistence: (1) how Ct initiates persistence following IFNγ treatment, and (2) how it recovers from persistence after IFNγ is removed. A possible mechanism of persistence initiation was recently discovered, involving changes in expression of chlamydial cell division proteins. Several cell division proteins, including FtsI/Pbp3, Pbp2, RodA, RodZ, and the peptidoglycan synthesis protein MraY were found to be especially rich in Trp codons. Upon tryptophan starvation, transcription of these proteins was maintained, but translation was reduced. This defect in translation is dependent on the high tryptophan content of these proteins, because substitution of Trp codons in rodZ with Phe codons was sufficient to restore protein expression (Riffaud et al. 2023). This defect in expression is likely due to ribosomal “stalling” on Trp codons under tryptophan-depleted conditions, thus preventing translation of these proteins. However, there may also be a role for the transcription termination factor Rho, which was recently shown in Ct to destabilize the 3′ ends of Trp codon-rich transcripts during tryptophan starvation (Ouellette et al. 2018). It is still unknown whether the diminished expression of these specific cell division proteins is necessary or sufficient to induce persistence, or whether additional mechanisms are required.

A chemical mutagenesis screen of Ct L2 recently identified two chlamydial proteins involved in recovery from persistence: CTL0694 and CTL0225. Mutants in CTL0694 or CTL0225 retain the ability to enter a persistent state following IFNγ treatment but are unable to “recover” from persistence after restoration of tryptophan (Muramatsu et al. 2016). These defects in recovery are different from one another in nature. CTL0225 is a putative amino acid transporter, sharing homology with the neutral amino acid transporter SnatA from Thermococcus spp. (Banerjee et al. 2023). Following IFNγ-mediated persistence, the CTL0225 missense mutant is unable to resume replication within the inclusion, even when excess tryptophan or indole are provided. However, addition of alanine to tryptophan-replete media restores growth of the CTL0225 mutant to wild-type levels (Banerjee et al. 2023). These data support the hypothesis that CTL0225 transports amino acids such as alanine, and missense mutations in CTL0225 partially inhibit this function. Additionally, these data highlight the importance of alanine depletion in human IFNγ-mediated defense. Unlike the CTL0225 mutant, a CTL0694 missense mutant retains the ability to replicate after tryptophan restoration. However, the CTL0694 mutant is unable to transition from replicating RBs into infectious EBs following tryptophan restoration. Thus, similar to the CTL0225 mutant, the CTL0694 mutant produces less infectious progeny after recovery from persistence (Muramatsu et al. 2016). CTL0694 is predicted to be an oxidoreductase, but the connection between this predicted function and recovery from persistence remains unknown (Muramatsu et al. 2016).

IRGs in murine host defense

In mice, the human-adapted pathogen Ct is killed by a pathway involving the p47 IRGs (Nelson et al. 2005, Bernstein-Hanley et al. 2006). The murine-adapted Cm, meanwhile, has evolved to fully evade restriction by this pathway (Coers et al. 2008). IRGs are a family of IFNγ-inducible GTPases related to dynamins. Mice have about 20 IRGs, split into two subfamilies: the antimicrobial effector “GKS” proteins (defined by a canonical GxxxxGKS amino acid sequence in the P-loop of their GTP binding domain), and the regulatory “IRGM” proteins (which instead contain a GxxxxGMS amino acid sequence) (Bekpen et al. 2005). Together, IRG proteins regulate a cascade of events that leads to recognition and clearance of a wide variety of bacterial, protozoan, and viral pathogens, including Ct (Dockterman and Coers 2022).

At the top of this cascade are the regulatory IRGM (Immunity-Related GTPase clade M) proteins, specifically IRGM1 and IRGM3. These two proteins direct downstream immune effectors to localize to the Ct inclusion in infected cells such as fibroblasts. They are thus required for (1) targeting of immune effectors to intracellular Ct inclusions, (2) IFNγ-mediated restriction of Ct in cell culture, and (3) early control of Ct infection in an in vivo mouse model (Coers et al. 2008, Haldar et al. 2013). Interestingly, IRGM proteins do not firmly attach to Ct inclusions themselves. Instead, IRGMs reside on a variety of host membranes including the endoplasmic reticulum, Golgi, mitochondria, and lipid droplets (Dockterman and Coers 2022). By marking “self” membranes, IRGM1/3 divert the localization of downstream host effectors to “non-self” membranes like the Ct inclusion (Hunn et al. 2008, Haldar et al. 2013). This function of IRGM1/3 is nonredundant, and individual knockouts of either Irgm1 or Irgm3 exhibit notably reduced defense against Ct infection (Bernstein-Hanley et al. 2006, Coers et al. 2008). IRGM1/3 drive several interdependent downstream responses that converge at the Ct inclusion, including the delivery of GKS IRG proteins, ubiquitin, and guanylate-binding proteins (GBPs). These responses together drive lytic destruction of the Ct inclusion (Haldar et al. 2015).

In uninfected cells, GKS IRGs typically reside in the cytosol as GDP-bound monomers. IRGMs maintain this equilibrium by residing on host membranes and transiently interacting with GKS proteins, preventing them from binding guanosine triphosphate (GTP) (Hunn et al. 2008). This maintains GKS proteins as inactive, cytosolic monomers, and protects host organelles from targeting and activation of GKS proteins. During Ct infection, GKS proteins come into contact with an additional membrane, the inclusion membrane, which lacks IRGMs. Without IRGMs to inhibit GKS activation, GKS proteins bind GTP, target the inclusion membrane, and oligomerize on this membrane in a GTP-dependent manner (Hunn et al. 2008, Haldar et al. 2013). GKS proteins also require an additional “second signal” to target the Ct inclusion which is mediated by the autophagy machinery (Zhao et al. 2008, Al-Zeer et al. 2009). In an uninfected cell, the autophagy machinery conjugates the Atg8 family of proteins to phosphatidylethanolamine to form autophagosomes that degrade intracellular waste (Li et al. 2022). During Ct infection, this machinery targets Ct inclusions and is required for the recruitment of GKS proteins (Al-Zeer et al. 2009, Haldar et al. 2014). Components of the autophagy machinery, too, are regulated by the IRGM family, and overexpression or knockout of IRGM1 is sufficient to alter the intracellular localization of specific autophagy proteins (Gutierrez et al. 2004, Traver et al. 2011). IRGMs thus drive trafficking of GKS proteins to the Ct inclusion through both direct and indirect mechanisms. Of the many GKS proteins, IRGA6, IRGB6, and IRGB10 are all known to target the Ct inclusion in mouse cells. Reports differ as to the relative importance of each protein, but the deletion or overexpression of several individual GKS proteins has been shown to alter cell-autonomous defense against Ct infection (Nelson et al. 2005, Coers et al. 2008, Al-Zeer et al. 2009).

The IRGMs and GKS proteins collectively recruit ubiquitin to the Ct inclusion (Haldar et al. 2015, Lee et al. 2020). Ubiquitin is a small protein that can be covalently bound to other proteins by an E3 ubiquitin ligase. In the case of Ct infection, ubiquitin ligases TRAF6 and TRIM21 are both recruited to Ct inclusions where they deposit ubiquitin on the inclusion membrane (Haldar et al. 2015). The substrate of this ubiquitylation is unknown, although it has been hypothesized that ubiquitin is ligated to the GKS proteins themselves (Traver et al. 2011, Finethy and Coers 2016).

Ubiquitin on the Ct inclusion then recruits the adaptor protein p62, which in turn recruits GBPs. Like IRGs, GBPs are a family of IFNγ-inducible GTPases in the dynamin superfamily (Prakash et al. 2000, Kutsch and Coers 2021). GBPs are also similar to GKS proteins in that they inhabit the cytosol as GDP-bound monomers but can form dimers with membrane affinity in a GTP-dependent manner (Vöpel et al. 2010). When GBPs come into contact with intracellular pathogens, they oligomerize and can activate several defense mechanisms, including recruitment of NADPH oxidase, recruitment of autolysosomes, and direct lysis of host and pathogen membranes via surfactant-like action (Kim et al. 2011, Meunier et al. 2014, 2015, Kutsch et al. 2020). In the case of Ct infection, GBPs are recruited to the inclusion by p62, ubiquitin, and IRG proteins (Haldar et al. 2013, 2015). Once there, they assist in the recruitment of GKS proteins, continuing this anti-Ct feedback loop (Haldar et al. 2014). Together, this suite of downstream effectors (GKS, ubiquitin, p62, and GBPs) drives lysis of the inclusion membrane, releasing Ct into the cytosol where it is vulnerable to destruction (Fig. 1; section II) (Haldar et al. 2015).

Unlike mice, humans only have a single IRGM protein and lack IFNγ-inducible GKS effectors. Human IRGM has significantly diverged from its murine orthologs and plays no appreciable role in restriction or ubiquitylation of intracellular Chlamydia inclusions (Bekpen et al. 2005, Haldar et al. 2016). Human cells instead employ IRGM-independent ubiquitylation in response to Chlamydia infection (see: the section “RNF213-mediated ubiquitylation”). Human IRGM does, however, play a role in limiting inflammation, and polymorphisms in this gene are associated with increased risk to autoimmune disease and sepsis (Mccarroll et al. 2008, Kimura et al. 2014, Yao et al. 2018). This anti-inflammatory function of IRGM appears conserved in mice, which require IRGMs to limit inflammasome activation and to limit pathology in the Chlamydia-infected female reproductive tract, independent of cell-autonomous immunity (Mehto et al. 2019, Eren et al. 2020, Finethy et al. 2020, Dockterman et al. 2024). Thus, while human IRGM has lost its cell-autonomous immune function against Chlamydia, it appears to exhibit similar anti-inflammatory effects as the murine IRGM proteins.

While the IRG protein family is highly divergent between mouse and human, the family of IFNγ-inducible GBPs is much more conserved between the two host species. Nonetheless, it has been debated whether endogenous human GBPs are able to directly target and restrict Chlamydia inclusions (Tietzel et al. 2009, Al-Zeer et al. 2013, Johnston et al. 2016). Regardless, both human and murine GBPs are definitively involved in execution of a cell death pathway which will be discussed later in this review (see: the section “IFNγ-stimulated cell death”).

Many questions remain about the murine IRG defense pathway. First, how do IRGMs differentiate between “self” membranes like the ER and “non-self” membranes like the Ct inclusion? We hypothesize that IRGM proteins may respond to differences in lipid composition between these membranes and may have higher affinity for lipids on “self” membranes. Second, which of the downstream effectors is responsible for lysis of the inclusion? This question has been particularly difficult to answer because these proteins interact with one another, and deletion of individual effectors can impair the localization of others. Third, what substrate on the inclusion membrane is ubiquitylated? Finally, how does Cm evade targeting and killing by the IRG defense pathway? We speculate that Cm may utilize an “anti-IRG” virulence factor, but more research is required to determine if such a factor exists, and if so, how it drives evasion of the IRGs.

Inducible nitric oxide synthase

The enzyme NOS2 (inducible nitric oxide synthase) represents an additional mechanism of IFNγ-stimulated host defense in mouse macrophages, epithelial cells, and fibroblasts. NOS2 catalyses the production of nitric oxide (NO), a reactive compound which is a precursor to a variety of reactive nitrogen species (RNS) (Macmicking et al. 1997). These RNS, including NO itself, can directly kill pathogens through chemical modification of microbial enzymes, virulence factors, nucleic acids, and other essential macromolecules. RNS also act against pathogens indirectly through effects on the host cell. These indirect effects include host cell death, autophagy, and immunoregulatory signaling (Bogdan 2015). Together, NOS2-dependent antimicrobial effects are able to defend against a broad range of pathogens (Macmicking et al. 1997, Bogdan 2015).

The importance of NOS2 during Chlamydia infection is still under investigation. In vitro data show that NOS2 drives clearance of Ct and Cm in mouse macrophages (Roshick et al. 2006, Zhang et al. 2012, Rajaram and Nelson 2015). NOS2 also drives clearance of Ct in some IFNγ-primed mouse fibroblast and epithelial cell lines (Mayer et al. 1993, Roshick et al. 2006), an effect that is further amplified by coculture with anti-Chlamydia T helper cells (Igietseme et al. 1996, 1997). In vivo, however, NOS2 appears less important. Several reports have shown that mice lacking NOS2 (either through chemical or genetic inhibition) exhibit wild-type clearance of genital infection with Cm or Ct (Igietseme et al. 1998b, Perry et al. 1998, Ramsey et al. 1998, 2001, Nelson et al. 2005). What is the explanation for this discrepancy between cell culture and in vivo mouse data? One possibility is that NOS2 functions in a redundant pathway in vivo, and that multiple pathways can individually eliminate genital Chlamydia infection in a mouse model. Indeed, Johnson et al. (2012) identified an independent anti-Chlamydia pathway in vivo mediated by the gene Placenta-specific 8 (Plac8). The exact role of Plac8 in Chlamydia infection is unclear, but it is thought to assist T cell degranulation (Johnson et al. 2012). Individual inhibition of Plac8 or NOS2 has no effect on clearance of Cm from the genital tract. However, inhibiting both pathways (combined knockout of Plac8 and chemical inhibition of NOS2) impairs clearance of Cm from the genital tract up to 60 days postinfection (Johnson et al. 2012). These data support the model that NOS2 guides a functional anti-Chlamydia pathway in the mouse, albeit a redundant one.

While NOS2 is not required for Chlamydia restriction in vivo, it does play an important role in Chlamydia-induced inflammation. Indeed, several reports indicate that deletion of NOS2 results in greater inflammation and damage in the genital tract, marked by increased hydrosalpinx, uterine distension, and severe endometrial cysts (Perry et al. 1998, Ramsey et al. 2001). The mechanism of this inflammation is poorly understood, but NOS2 may act by inhibiting expression of the T cell-stimulating cytokine IL-12. Supporting this hypothesis, NO has previously been shown to inhibit transcription of IL-12 in activated macrophages (Huang et al. 1998).

The relevance of NOS2 to human Chlamydia infection is also debated. In many human cell lines, IFNγ alone is insufficient to induce a detectable NOS2 response (Macmicking et al. 1997, Roshick et al. 2006, Bogdan 2015). Perhaps then it is unsurprising that inhibition of NOS2 does not impact IFNγ-mediated defense against Ct or Cm in several human cell lines (Roshick et al. 2006). One study in human mesenchymal cells challenges this paradigm. These researchers found that uninfected human mesenchymal cells express NOS2, but Ct infection triggered upregulation of the host enzyme ornithine decarboxylase and subsequent downregulation of NOS2 (Abu-Lubad et al. 2014). This research suggests a possible mechanism of NOS2 evasion by Ct, but also raises many questions. What is the identity of the Ct factor(s) that target NOS2? Are these bacterial factor(s) essential for evasion of IFNγ-driven host defense? Is this phenotype limited to mesenchymal stem cells or is it more broadly conserved in other human cell types? Finally, in both mouse and human, are the putative anti-Chlamydia effects of NOS2 through direct or indirect functions of RNS? Answering these questions will be important to better understand the relevance of NOS2 in Chlamydia infection.

IFNγ-stimulated cell death

In response to Chlamydia infection, IFNγ galvanizes at least two pathways of cell death in both human and mouse cells. The first is a proinflammatory type of cell death termed pyroptosis, reviewed extensively by Li et al. (2023) and Newton et al. (2024). Pyroptosis can be activated by one of several “inflammasomes”: intracellular receptors which respond to a wide variety of signals. These inflammasomes drive two main functions: (1) cleavage and activation of cytokines IL-1β and IL-18 and (2) rupture of the plasma membrane leading to cell death. In primed mouse macrophages, Ct or Cm infection activates pyroptosis via three such inflammasomes: NLRP3, AIM2, and the noncanonical inflammasome (Abdul-Sater et al. 2010, He et al. 2010, Finethy et al. 2015). Human macrophages exhibit a similar response, albeit limited to the NLRP3 inflammasome alone (Xavier et al. 2020). The activation of these inflammasomes in response to Chlamydia species is dependent on the previously mentioned GBPs, and deletion of multiple GBP proteins in Chlamydia-infected macrophages results in a significant reduction in pyroptosis (Finethy et al. 2015, Xavier et al. 2020). Interestingly, this function of GBPs is independent from targeting or lysis of inclusions. This can be observed in Cm-infected mouse macrophages, which exhibit no targeting or lysis of inclusions by GBPs, but proper function of GBP-dependent inflammasome machinery (Finethy et al. 2015).

How do GBPs activate these various inflammasomes? This function of GBPs has been poorly characterized for AIM2 but is better understood for NLRP3 and the noncanonical inflammasome during Chlamydia infection. NLRP3 activates in response to a plethora of intracellular stimuli, including the metabolite uric acid. One of the major pathways of uric acid synthesis in mammals requires guanosine monophosphate (GMP) as a precursor. It was recently shown that GBP1 hydrolyzes GTP to GMP during Chlamydia infection, which drives production of uric acid and cell death via NLRP3 (Schwemmle and Staeheli 1994, Xavier et al. 2020). This process can be inhibited by eliminating downstream enzymes that produce uric acid from GMP and can be induced in a GBP1 knockout by exogenously restoring guanine or guanosine (Xavier et al. 2020). Unlike NLRP3, the noncanonical inflammasome activates in the presence of lipopolysaccharide (LPS), a large molecule found in the bacterial outer membrane of Gram-negative bacteria like Chlamydia (Agnew et al. 2021). During infection, Chlamydia species shed LPS into the host cytosol (Richmond and Stirling 1981, Nguyen et al. 2011). In a Shigella infection model, it was recently shown that GBPs can bind to cytosolic LPS, oligomerize, and form large LPS aggregates. These GBP-bound aggregates have a much greater ability to activate the noncanonical inflammasome than free LPS alone (Dickinson et al. 2023). Thus, current evidence suggests that GBPs induce pyroptosis during Chlamydia infection by controlling the production of uric acid (NLRP3) and by aggregating chlamydial LPS in the cytosol (noncanonical inflammasome). However, more research is required to directly show that GBPs can (1) aggregate chlamydial LPS specifically and (2) drive activation of the noncanonical inflammasome via those chlamydial LPS aggregates.

Another form of cell death, involving apoptotic caspases and inclusion lysis, was recently identified in IFNγ-primed murine cells infected with Chlamydia. Cm was shown to evade this form of cell death using the putative inclusion membrane protein TC0574. In short, Giebel et al. (2019) demonstrated that a TC0574 mutant Cm is vulnerable to restriction in IFNγ-primed mouse fibroblasts, while wild-type Cm is not. Similar to the IRG defense pathway, this restriction involves lysis of the Chlamydia inclusion. Unlike the IRG defense pathway, this process does not require IRGMs or autophagy and this inclusion lysis precedes host cell death (Giebel et al. 2019). Interestingly, inhibition of pro-apoptotic caspases 3, 8, and 9 partially blocked inclusion lysis and host cell death in this model. The Cm protein TC0574 seemingly blocks this defense pathway, which is not active against wild-type Cm (Giebel et al. 2019). This research raises several key questions. Do caspases directly cleave proteins on the inclusion membrane to lyse mutant inclusions, or is this interaction indirect? Additionally, how does TC0574 block inclusion lysis? Similar to Cm, the human-adapted pathogen Ct is not vulnerable to this pathway in mouse cells (Giebel et al. 2019). This begs the question whether an orthologous defense pathway exists in human cells, and if so, whether Ct evades it via a similar mechanism.

RNF213-mediated ubiquitylation

Humans, like mice, use ubiquitylation as an IFNγ-mediated defense. Unlike mice, human cells ubiquitylate inclusions of the murine-adapted pathogen Cm, but not the human-adapted pathogen Ct (Haldar et al. 2016). The mechanism of this ubiquitylation, and its evasion by Ct, has been recently characterized.

As mentioned previously, ubiquitylation is the formation of a covalent bond between ubiquitin and a substrate, typically a protein. Ubiquitin E3 ligases are the final step in this process and are crucial for determining the ubiquitin substrate and linkage (Komander and Rape 2012). In human cells, Walsh et al. (2022) recently demonstrated that the IFNγ-inducible E3 ligase RNF213 is responsible for the ubiquitylation of Cm inclusions in human cells. RNF213 directly translocates to the Cm inclusion membrane in human epithelial cells, where it deposits ubiquitin on an unknown substrate. This inclusion-bound ubiquitin then recruits several autophagy adaptor proteins, including OPTN, NDP52, and TAX1BP1. These adaptors subsequently recruit autolysosomal markers LC3 and LAMP1 to the inclusion and trigger degradation of Chlamydia by autophagy (Fig. 1; section V) (Haldar et al. 2016, Walsh et al. 2022). This pathway is a conserved antimicrobial response, and recent reports have shown a role for RNF213-mediated ubiquitylation and restriction of many bacterial, viral, and protozoan pathogens (Otten et al. 2021, Thery et al. 2021, Hernandez et al. 2022).

Wild-type Ct expresses a secreted bacterial effector to evade this process: GarD. The effector GarD resides on the Ct inclusion membrane and blocks translocation of RNF213 to Ct. In blocking RNF213 translocation, GarD also blocks the downstream effects of this process including ubiquitylation, recruitment of the autophagy machinery, and restriction of Chlamydia burden (Walsh et al. 2022). Ct lacking the virulence factor GarD is vulnerable to this defense pathway. GarD-mediated evasion of RNF213 occurs in cis, meaning that a WT Ct inclusion can protect itself against RNF213, but cannot protect a GarD knockout Ct inclusion in the same cell. Together, these data support the hypothesis that GarD functions at the inclusion membrane to block RNF213 (Walsh et al. 2022). The importance of this evasion mechanism is demonstrated by the rapid clearance of GarD-defective Ct mutants in a primate infection model (Fernandez et al. 2024).

There are many unanswered questions about the relationship between RNF213 and GarD. What is the ubiquitylation substrate for RNF213 at the inclusion membrane? Are there adaptor proteins upstream from RNF213 that facilitate inclusion recognition? Mice have an RNF213 ortholog, and Cm has a GarD ortholog—do these proteins share the same relationship as human RNF213 and Ct GarD? Finally, how does Ct GarD block binding of human RNF213? We hypothesize that GarD outcompetes RNF213 for a shared binding substrate, but answering this question requires significantly more research.

Conclusions

In conclusion, there are at least five well-studied pathways of IFNγ-stimulated cell-autonomous defense against Chlamydia species (Fig. 1). In humans, restriction of essential nutrients like tryptophan is a potent host defense, but this response is counteracted by the human-adapted pathogen Ct, which synthesizes tryptophan from vaginal indole and can enter the nonreplicating state of persistence to overcome tryptophan starvation. Many questions remain surrounding the relative importance of nontryptophan metabolites in nutrient deprivation, and the mechanisms by which Ct enters and recovers from persistence. Additionally, humans defend against infection using the E3 ubiquitin ligase RNF213, which deposits ubiquitin on the inclusions of nonadapted Chlamydia and traffics these inclusions into the autophagy pathway. Ct uses the virulence factor GarD to prevent targeting and destruction by RNF213. The molecular function of GarD is still unresolved, and the exact downstream mechanisms of RNF213 require further investigation.

In the mouse, IRGM proteins control a cascade of downstream effectors including GKS proteins, ubiquitin, p62, and GBPs, which work together to lyse Ct inclusions. The murine-adapted pathogen Cm evades targeting and lysis of its inclusion. Many questions remain surrounding the importance and role of each downstream effector in this pathway, and the mechanism by which Cm evades this pathway. Mice also produce RNS to defend against Chlamydia infection, although this effect is not essential for anti-Chlamydia defense in vivo. It is still unclear if NOS2 works directly or indirectly to kill Chlamydia species, how its function is redundant with Plac8 in vivo, and whether NOS2 mediates anti-Chlamydia defense in the human host. Finally, both humans and mice utilize IFNγ-stimulated cell death to clear invading Chlamydia, either through GBP-stimulated pyroptosis or caspase-induced inclusion lysis. Cm evades the inclusion lysis pathway with the virulence factor TC0574. Further research is required to understand the function of this virulence factor and whether an orthologous mechanism exists in human Ct infections.

Humans have a tremendous number of bona fide IFNγ-stimulated genes, estimated to be around 2% of the total open reading frames in our genome (Abrams et al. 2020, Nurk et al. 2022). As such, it is likely that there are additional, undiscovered pathways of IFNγ-mediated cell-autonomous defense against Chlamydia species, and additional, undiscovered mechanisms of evasion by this highly evolved pathogen. Research into these potential pathways is essential to produce a more comprehensive picture of IFNγ-stimulated host–pathogen interactions during Chlamydia infection. Importantly, all five of the pathways discussed in this review are known to drive defense against additional, non-Chlamydia pathogens. Thus, further research into these pathways (and new, undiscovered pathways) will provide novel perspectives into the “molecular arms race” between mammalian hosts and a wide variety of pathogens. Finally, future work should also focus on the mechanisms by which Chlamydia species evade cell-autonomous immunity. We believe that this research will help us understand how Chlamydia evades the immune system at large and will provide key insights for the design of novel therapies and vaccination strategies against this widespread pathogen.

Acknowledgments

We would like to thank members of the Coers lab for providing valuable feedback. Figure created with Biorender.com.

Author contributions

Jeffrey R. Reitano (Writing – original draft, Writing – review & editing), and Jörn Coers (Writing – review & editing)

Conflict of interest

None declared.

Funding

This work was supported by the National Institutes of Health grant AI103197 (to J.C.).
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