Persistence in Chlamydia

Abstract

Chlamydia spp. are important causes of acute and persistent/chronic infections. All Chlamydia spp. display a unique biphasic developmental cycle alternating between an infectious elementary body (EB) and a replicative form, the reticulate body (RB), followed by the multiplication of RBs by binary fission and progressive differentiation back into EBs. During its intracellular life, Chlamydia employs multiple mechanisms to ensure its persistence inside the host. These include evasion of diverse innate immune responses, modulation of host cell structure and endocytosis, inhibition of apoptosis, activation of pro-signaling pathways, and conversion to enlarged, non-replicative but viable “aberrant bodies” (ABs). Early research described several systems for Chlamydial persistence with a significant number of variables that make a direct comparison of results difficult. Now, emerging tools for genetic manipulations in Chlamydia and advances in global microarray, transcriptomics, and proteomics have opened new and exciting opportunities to understand the persistent state of Chlamydia and link the immune and molecular events of persistence with the pathogenesis of recurrent and chronic Chlamydial infections. This chapter reviews our current understanding and advances in the molecular biology of Chlamydia persistence.

1. Introduction

1.1 Overview of Chlamydial persistence

Persistence is the ability of bacteria to remain viable in the host for a prolonged period of time. Bacteria have evolved several strategies by which subpopulations can survive conditions that are lethal for most members of bacterial populations. Well-known examples are the formation of endospores in Bacillales and Clostridiales orders, the formation of exospores in Actinomycetales, the presence of “persister” cells occurring in most bacteria, and the formation of viable but non-culturable cells [1]. All the survival stages are characterized by partial or complete inhibition of metabolism and cell division. Common to all of these survival states is the ability of the “persister” cells to resume their developmental stage under favorable conditions [1].

In the context of Chlamydia, persistence or Chlamydial stress response is the reversible inhibition of cell division that interrupts the pathogen’s developmental cycle in the presence of unfavorable growth conditions [2]. Chlamydial persistence in vitro is characterized by the presence of a “viable but non-cultivable growth stage resulting in a long-term relationship with the infected cell” [3]. Persistence is an important cause of recurrent Chlamydial disease characterized by chronic inflammation and tissue damage in epithelial cells. This chapter will discuss the recent developments in our understanding of Chlamydia persistence, focusing on past and current insights that have been obtained into the molecular and immunological basis of this stage of Chlamydia development.

2. The Chlamydial developmental life cycle

Chlamydiae are gram-negative obligate intracellular pathogens characterized by their biphasic life cycle [4, 5, 6]. Chlamydiae primarily infect mucosal epithelial cells and alternate between two morphologic forms. The first, known as the elementary body (EB), is the infectious form that attaches to the cell membrane of a host cell. Shortly after interacting with host cell membrane receptors, the bacterial ligands induce endocytosis of the pathogenic EB, leading to the creation of an EB-containing vacuole known as the inclusion [4]. Once inside the cell, the EB within the inclusion takes around 6–8 hours to transform into the second morphologic subtype, known as the reticulate body (RB). The RB modifies the inclusion’s membrane to prevent its degradation, while also prompting migration of the inclusion toward the microtubule-organization center (MTOC) to facilitate movement toward nutrient-rich areas within the host cell (e.g., periphery of the Golgi apparatus) [4].

Due to the parasitic nature of Chlamydiae, these pathogens not only rely on essential nutrients from the host cell but also require several metabolic enzymes, which are subsequently hijacked from the host. Thus, approximately 8–16 hours after infection, the mid-cycle begins, where the RB produces effectors that facilitate the looting of nutrients and enzymatic hijacking [2, 4]. Finally, after 24 hours of replication and growth, RBs can revert to EBs via an asynchronous process that allows them to exit the host cell (e.g., cell lysis or extrusion). Chlamydiae can also transform into a third morphologic subtype under certain conditions. When Chlamydiae experience physiologic stressors, RBs can transform into abnormally large bacteria known as an aberrant body (AB) [4]. ABs are characterized by their non-infectious “hibernating” state, allowing them to re-enter the normal biphasic life cycle once the underlying stressor subsides to continue producing infectious EBs [4].

3. Overview of Chlamydia pathogenesis

3.1 Acute infections

Chlamydia species cause widespread infections in humans. Chlamydia trachomatis serovars A–C are the leading cause of non-congenital trachoma and are the major cause of blindness and visual impairment in developing nations [7, 8]. C. trachomatis serovars D–K are considered the world’s most common sexually transmitted pathogen causing disease in the genital tract and in men, are the primary cause of non-gonococcal urethritis [7, 9]. Following vertical transmission through an infected birth canal, C. trachomatis serovars D–K cause neonatal conjunctivitis and pneumonia. Respiratory infection with C. pneumoniae causes an average of 10% of community-acquired pneumonia cases and 5% of bronchitis and sinusitis cases. In addition, avian strains of C. psittaci have long been known to cause zoonotic respiratory illness in humans [10]. The C. trachomatis lymphogranuloma venereum (LGV) biovar (serovars L1–L3) causes invasive urogenital or anorectal infection [11].

3.2 Persistent and chronic Chlamydial infections

C. trachomatis serovars D–K are responsible for about 15–40% of ascending upper genital tract infections leading to serious complications in women, such as salpingitis, pelvic inflammatory disease, ectopic pregnancy, epididymitis in men, and infertility in women and men [8]. C. trachomatis originating from the genital tract is also associated with reactive arthritis, which develops in 1-3% of patients after genital Chlamydial infection [9]. C. pneumoniae, which can also disseminate from the site of the initial infection, is linked to several chronic diseases, including asthma, atherosclerosis, arthritis, cardiovascular disease, and even late-onset Alzheimer’s disease [12]. In addition, unresolved respiratory C. pneumoniae infection may contribute to the pathogenesis of chronic inflammatory lung diseases, such as asthma and chronic obstructive pulmonary disease [12]. Further, C. trachomatis impedes human papillomavirus (HPV)-induced mechanisms that maintain cellular and genomic integrity, and it may be linked to cervical cancer [13].

Clinical conditions associated with inapparent Chlamydial infections include asymptomatic urethritis in male individuals and cervicitis in female individuals, and silent pelvic inflammatory disease in female individuals [3, 14]. The clinical significance of persistent infection is associated with the reactivation of infection after weeks or months in individuals treated with antibiotics, and negative culture results for individuals with strong serological titers and epidemiological associations [3].

4. Structural elements contributing to persistent infection in Chlamydia

4.1 The Chlamydial inclusion

In Chlamydia, a large part of the intracellular survival strategy involves the formation of a unique membrane-bound vacuole called an inclusion. The inclusion represents the ideal “protected niche” that ensures Chlamydia its survival by evading the endolysosomal pathway and the innate immune responses of the cell and favoring its growth by modulating host cell processes. Active transcription and translation within the lumen of the inclusion are required for the transition from the non-replicative ER to the replicative, morphologically larger RB. Concomitantly, the nascent Chlamydia-containing inclusions traffic along microtubules from the cell periphery to the microtubule organizing center (MTOC), where the inclusion resides for the duration of the life cycle [15].

The inclusion membrane (IM) serves as the means by which the bacterium communicates with the host cell. A notable component of the IM is the Chlamydia-specific Type III secretion (T3SS) effector transmembrane Inclusion membrane proteins (Incs) [16], Reviewed in [17]. Bioinformatic studies have estimated that C. trachomatis encodes 50–100 putative Incs proteins, which represent approximately 6% of the coding capacity of the organism [18]. At least, three classes of Incs have been identified during the Chlamydial developmental cycle: early-cycle Incs (highest mRNA levels between ~2 and 6 h post-infection); mid-cycle Incs (highest mRNA levels between 6 and 20 h post-infection); and late-cycle Incs (highest mRNA levels after ~20 h post-infection) [19]. The activity of some of these Incs proteins is important to ensure the Chlamydia long-term survival through the acquisition of nutrients, avoidance of fusion of the inclusion with lysosomes, stability of the inclusion membrane, and modulation of host cell death. For instance, C. trachomatis CpoS (Chlamydia promoter of Survival), Inclusion membrane C and CT383 have been reported to inhibit host cell death processes in Chlamydia-infected cells by controlling inclusion membrane stability [20, 21]. In addition, some Chlamydial Incs interfere with the innate host immune signaling [22]. Recent studies using conditional Incs mutants in C. trachomatis and Chlamydia muridarum has identified Incs as a key effector in the transition from infectious (EB) to replicative (RB) during the early stages of Chlamydia development in vivo [23]. Although only a few Incs have been characterized to date, the role of many Incs remains largely unknown.

4.1.1 Role of actin in the inclusion maturation

After the invasion, Chlamydia continues to manipulate the host cytoskeleton by assembling and maintaining an actin-rich cage around the Chlamydial inclusion [24]. One of the components of the actin cage (F-actin ring) provides structural rigidity and stability to the mature inclusion, as demonstrated by its resistance to nonionic detergents and the antimitotic agent nocodazole [25]. Intermediate filaments have also been shown to contribute to the stability and function of the inclusion cage by providing additional rigidity. Once the invasion is complete, actin is recruited via a RhoA/ROCK-mediated actin contraction signal pathway to the maturing inclusion alongside the intermediate filaments and septins, providing dynamic structural reinforcement to Chlamydia’s replicative niche [25]. Other studies suggest that the actin-ring cage formation may depend upon the de novo, unbranched polymerization of actin at inclusions [26]. The two proposed models of actin cage suggest that mechanism of cage assembly changes with the maturation of the inclusion. While the precise mechanism of F-actin synthesis and regulation within the actin cage is somewhat unclear, further study will give an insight into the dynamics of the inclusion vacuole during the Chlamydial persistent stage.

4.2 Aberrant bodies

Under non-bacteriocidal stress conditions, Chlamydia responds by markedly arresting RB division and differentiates into an atypical morphology referred to as aberrant body (AB) [3]. ABs are capable of remaining viable within the inclusion vacuole for extended period of time. Aberrant bodies were first described in 1993 on Chlamydia cultured on McCoy cells and incubated in Eagle’s minimal essential medium lacking all 13 amino acids [27]. Chlamydia AB formation is also induced in vitro by antibiotics (beta-lactam antibiotics, fosfomycin, novobiocin, fosmidomycin, and Azithromycin) [28, 29, 30, 31, 32, 33]), depletion of essential nutrients (i.e., iron, amino acids, and glucose), heat shock, coinfection with Herpes Simplex virus [27, 34, 35, 36, 37, 38, 39], infection of monocytes and macrophages [40, 41, 42], cytokines (Interferon-gamma and IFN-γ) [43], and a number of other pressures [44, 45]. AB in certain Chlamydial species is also induced by treatment with LPC-011 (LPC), a potent inhibitor of the zinc-dependent cytoplasmic deacetylase LpxC, which catalyzed the first step in the Chlamydial lipooligosaccharide (LOS) biosynthesis pathway [46]. When the stress stimulus is removed, cell division in the ABs resumes, allowing Chlamydia to complete the developmental cycle. ABs have been classically distinguished by their enlarged size (2–10 mm; for reference, the EB is ≤0.5 μm and the RB is ∼1 μm), the inhibition of cell division, and the inhibition of EB production [3]. However, a recent study using immunolabeling has shown that bacterial cell enlargement is not a prerequisite for persistence in C. trachomatis [2]. In addition, aberrant Chlamydia exhibits differences in the capacity to synthesize the cell wall polymer peptidoglycan in the presence of different aberrance-inducing conditions [2]. Moreover, some AB inducers halt the peptidoglycan biosynthesis pathway early enough to prevent the synthesis and release of the peptidoglycan component, muramyl tripeptide. These immunostimulatory components are ligands that activate the intracellular NOD1/NF-κB-mediated IL-8 inflammatory immune response to Chlamydial infections, and the prevention of this signaling pathway by a subset of persistent forms of Chlamydia inhibiting PG synthesis may confer an immunoevasive advantage during aberrancy [2]. In addition, ABs incorporate Incs effector proteins at various stages of the Chlamydial AB formation which suggests that persistent forms of Chlamydia exhibit differences in their abilities to undergo homotypic fusion and induce actin cage formation [2].

In addition to differences in the AB physiology, other studies have found that the transcriptional and translational responses of Chlamydiae differ according to the persistence-inducing stimuli [Reviewed in [47]]. For instance, different models of AB induction in vitro and in vivo data using Chlamydia-infected tissues revealed differences in the relative levels of expression in the major outer membrane protein (MOMP), Chlamydial heat shock protein 60 (cHSP60), and the three groEL genes (encoding cHSP60 homologs) [48, 49, 50]. Other studies analyzed the patterns of expression in genes related to cell division and chromosome replication in the Chlamydia ABs. These studies analyzed expression of genes encoding products predicted to function in DNA replication (polA, dnaA, and mutS), chromosome partitioning (parB and minD), and cell division (ftsK and ftsW) in various in vitro AB inducible systems and in vivo [51, 52]. These studies demonstrated mixed data in the expression patterns of the chromosome segregation gene, ftsK, and septum-peptidoglycan biosynthetic protein, ftsW. Similarly, DNA replication gene expression profiles were varied in the microarray study of IFN-exposed C. trachomatis, with some genes upregulated (dnaB, topA, and xerC) and others downregulated (dnaA-2, dnlJ, and ihfA) [51]. The varied data regarding cell division and DNA replication gene expression during persistence may indicate that RBs show different morphological alterations during the establishment of persistence.

Several studies on AB-inducible systems have reported variations in expression of genes involved in energy metabolism in vitro and in vivo [48]. Genes encoding enzymes belonging to glycolysis (pyk, gap, and pgk) and the pentose phosphate pathway (gnd and tal) were found to be selectively downregulated in vitro and in vivo relative to genes encoding enzymes in the tricarboxylic acid cycle (mdhC and fumC) [48]. The microarray expression data for genes encoding tricarboxylic acid cycle enzymes in IFN-induced persistence of C. trachomatis were mixed. Genes encoding 2-oxoglutarate dehydrogenase (sucA, sucB-1, and sucB-2) and succinate thiokinase (sucC and sucD) were downregulated. In contrast, genes encoding other enzymes in the cycle were either upregulated (fumC and sdhB) or unchanged (mdhC, sdhA, and sdhC) [48].

Electron microscopic visualization in chronically diseased tissues shows similar morphologically aberrant forms resembling those observed in vitro, though the viability of these particles is uncertain. The presence of viable but atypical Chlamydiae in vivo is suggested by the detection of enlarged, pleomorphic RB within infected human-derived samples such as fibroblasts and macrophages in synovial membrane samples from patients with C. trachomatis-associated reactive arthritis or Reiter’s syndrome [53], macrophages in aortic valve samples from patients with degenerative aortic valve stenosis [54], and prostatic secretion samples from patients with chronic Chlamydial prostatitis [55]. Moreover, Chlamydial inclusions were found in the luminal epithelium of the oviducts of mice experimentally inoculated with the mouse pneumonitis (MoPn) biovar of C. trachomatis [56]. Aberrant bodies are not exclusive of human Chlamydiae, as members of the zoonotic Chlamydiales and “Chlamydia-related bacteria” also exhibit the persistent AB phenotype under several experimental conditions in vitro and in vivo [57, 58, 59].

5. Immunological basis of Chlamydia persistence

Chlamydia employs several mechanisms to interfere with the host innate immune response to persist within the host cell.

5.1 Modulation of proinflammatory signaling pathways

The epithelial cells of the urethra or vagina/endocervix represent the first contact and innate immune barrier against Chlamydia. The cells can recognize the pathogen through pattern recognition receptors (PRRs) such as Toll-like receptors (TLRs), nucleotide-binding oligomerization domain (NOD)-like receptors or cyclic guanosine monophosphate (GMP)-adenosine monophosphate (AMP) (cGAMP) synthase (cGAS), which induces the production of proinflammatory cytokines via nuclear factor-κB (NF-κB) or activator protein 1 (AP-1) signaling [Reviewed in 61]. The stimulation of cGAS by Chlamydia spp. DNA leads to the dimerization and activation of the IFN regulatory factor 3 (IRF3), which then translocates into the nucleus and promotes the transcription of type I IFN and IFN-inducible genes [60].

The NFκB pathway may be modulated by several different Chlamydial proteins and mechanisms, all of which can interfere with NFκB-mediated gene transcription and regulation. Some of these mechanisms include: (1) blocking the degradation of the NF-κB retention factor, IκBα via C. trachomatis deubiquitination (DUB) proteins ChlaDub1 and ChlaDub2 [61], (2) preventing the nuclear translocation of NF-κB, thus stopping or dampening NF-κB transcription, (3) sequestration of the NF-κB activator 1 (Act1) upon binding of the C. pneumonia-specific inclusion membrane protein (Inc) CP0236 [22], and (4) suppression of NF-κB signaling by Chlamydia secreted proteases (the tail-specific protease of C. trachomatis, CT441, and Chlamydial protease-like activity factor, CPAF) [62].

5.2 Interference with proinflammatory cytokines

Inflammation participates significantly not only in host defenses against Chlamydia spp., but it also contributes to the pathophysiology of infection. Chlamydia-infected host cells produce a number of cytokines and chemokines, including CXC-chemokine ligand 1 (CXCL1), CXCL8 (also known as interleukin-8, IL-8), TNF-α, and IL-1β and cause activation of various inflammasome pathways, including the NLRP3/ASC inflammasome [60]. These proinflammatory mediators recruit immune cells to the site of infection and cause local inflammation and tissue damage. Chlamydia employs several mechanisms to interfere with inflammation, promoting Chlamydial persistence. For instance, the C. trachomatis inclusion membrane protein CpoS can inhibit host inflammasome responses [20]. Another mechanism is the overexpression of the anti-inflammatory cytokine IL-10 [63]. This in vitro study was confirmed by findings of an increased in vivo expression of IL-10 in the semen and serum of patients infected with C. trachomatis [64]. Chlamydia CPAF contributes to the anti-inflammatory state required for persistence by inhibiting the IL-1ß-dependent secretion of IL-8 through cleavage of the transcription factor p65/RelA [65]. CPAF is also involved in the inhibition of the complement activation by cleavage of the complement factors B and C3 and attenuating the production of proinflammatory cytokines [66].

5.3 IFN-γ-induced persistence

IFN-γ is the major component of the innate immune response against Chlamydia and is the factor that has received the most research attention as a Chlamydial inducer of persistence [41, 43, 44, 67]. Various mechanisms of IFN-γ-induced persistence have been proposed. IFN-γ activates the catabolic depletion of L-tryptophan (Trp) via indoleamine-2,3-dioxygenase (IDO), the enzyme that degrades tryptophan. Since tryptophan is an essential amino acid for C. trachomatis, the presence of this enzyme induces a tryptophan starvation that inhibits the growth of Chlamydial RBs [41, 43]. IFN-γ is also involved in the inhibition of the transcription factor and proto-oncogene c-Myc, the key regulator of host cell metabolism and a central regulator of Chlamydia persistence [67].

5.4 Autophagy: mediated resistance

Autophagy is a physiological degradation process that occurs within the lysosomes of most cell types. Its main functions are to maintain cellular homeostasis and selectively remove intracellular bacteria or viruses. In C. trachomatis, Guanylate-binding proteins (GBPs) and the immunity-related GTPases (IRGs) such as GBP1, GBP2, Irga6, and Irgd, which can induce lysis and infection clearance by autophagy, were found to accumulate in the Chlamydial inclusions [68, 69], suggesting a role for these proteins in the autophagy-mediated resistance to C. trachomatis infection.

5.5 Interaction with innate immune cells

5.5.1 Macrophages (Mϕ)

Macrophages (Mϕ), unlike epithelial cells, are not a hospitable niche for Chlamydial intracellular replication. Mϕs migrate to Chlamydial infection sites, phagocytose bacteria, produce proinflammatory cytokines, and destroy C. trachomatis with host cell autophagy [69, 70]. Also, studies have demonstrated that Mϕ autophagy can enhance antigen presentation to T-cells [69]. Furthermore, IFN-γ has been shown to enhance both autophagy and upregulation of MHC class II molecules in Mϕ [71]. Several mechanisms of Chlamydia spp. persistence in macrophages have been described: (1) living as aberrant RBs; (2) interaction with organelles to acquire sufficient nutrients [72, 73]; (3) modulation of inflammatory cytokines such as TNF-α, IFN-γ, and ILs, to escape eradication via apoptosis or autophagy [74]; and (4) the production of adhesion molecules such as the intercellular cell adhesion molecule-1 (ICAM-1), to increase macrophage adherence, thus facilitating the migration of EBs to their preferred sites of replication [75].

Mϕs are involved in the engulfment and transient persistence of the Chlamydial extrusions [76]. Upon release from infected epithelial cells, Chlamydia-containing extrusions are engulfed by macrophages. Migration of these macrophages, followed by eventual escape of Chlamydia from them, can result in the dissemination of infectious C. trachomatis to more distant sites, for example, away from inflammatory foci surrounding the primary site of infection, to draining lymph nodes or to new hosts [76].

5.5.2 Monocytes and dendritic cells (DC)

Monocytes are responsible for spreading C. trachomatis throughout the body, while dendritic cells (DCs) play an important role in mediating immune response against bacterial infection. The C. trachomatis serovars Ba, D, and L2 can productively infect human peripheral blood monocytes and monocyte-derived DCs in a comparable manner [77]. Chlamydia Serovars Ba and D are able to persist on monocytes, while they degrade within DC’s [77]. The mechanism of persistence within monocytes is not known.

6. Molecular basis of Chlamydial persistence

During the persistent state, Chlamydiae can activate pro-survival pathways and inhibit apoptosis to ensure long-term survival inside the cells. Extensive research has established a strong correlation between inhibition of host cell apoptosis and persistent C. trachomatis infection.

6.1 Inhibition of apoptosis

Apoptosis is an active process of cellular death induced by both extrinsic (death receptor signaling) and intrinsic (mitochondrial) pathways in response to variety of physiological and stress stimuli. Host cell death has long been recognized as the final stage of the Chlamydia infection cycle, enabling the release of EBs and spreads of infection. However, Chlamydia must protect the host cell from succumbing to stress-induced death before the Chlamydial developmental cycle is complete. The ability of Chlamydiae to induce host-cell apoptosis under some circumstances and actively inhibit apoptosis to complete their obligate intracellular growth has been extensively studied for decades ([78]; reviewed in Ref. [79]). C. trachomatis inhibits specifically the mitochondrial pathway, while signals that originate at death receptors and bypass mitochondria are not blocked [80]. Apoptosis inhibition in Chlamydia is observed in a variety of cell lines and primary cells from diverse origins, including epithelial cells, fibroblasts, endothelial cells, monocytes, and lymphoid cells, and not only during active but also during persistent infection [78].

6.1.1 Interaction with mitochondria

Mitochondria play a central role in energy (ATP) metabolism via oxidative phosphorylation, biosynthesis of macromolecules, and cell death regulation. Within the host cell, the mitochondria constitute the primary target for C. trachomatis. Its high demand for metabolites during its inclusion phase induces massive stress in the host cell, eventually leading to the induction of apoptotic cell death as a cell autonomous defense mechanism. Accumulating evidence suggests that Chlamydia can manipulate the mitochondrial morphology to promote their own replication or to escape from host immune responses (Reviewed in Ref. [81]). Altered mitochondrial dynamics of fusion and fission allow Chlamydia to maintain the cycle of reproduction and growth. Chlamydia suppresses mitochondrial fission and promotes mitochondrial fusion in the host cell via lowering ROS generation, inhibiting the tumor suppressor protein P53 transcription, increasing P53 protein ubiquitination levels, and inhibiting the dynamin-related protein 1(DRP1) oligomerization [81, 82]. Another study provided evidence that Chlamydia promotes intracellular survival by inducing mitochondrial elongation during the early phase of infection via phosphorylated fission mediator protein Drp1 followed by a fragmentation phase at the late stages of infection [83].

6.2 Modulation of Bcl-2 family pro-apoptotic proteins

The Bcl-2 family proteins and caspase-3 are critical regulatory proteins in cell apoptosis. Members of the Bcl-2 family can regulate the mitochondrial outer membrane permeability and control cell apoptosis by activating the caspase-3-mediated pathway [84]. Bcl-2 family can be divided into anti-apoptotic proteins (such as Bcl-2 and BclxL) and proapoptotic proteins (such as Bax and Bak). The ratio of anti-apoptotic to proapoptotic proteins is involved in the determination of cellular fate. Activated Bax/Bak induces the formation of oligomers that form pores in the mitochondrial outer membrane. These pores are channels for proapoptotic factors such as cytochrome c to translocate to the cytoplasm. The result is twofold: the loss of cytochrome c from mitochondria disables energy production, and cytosolic cytochrome c instigates a proteolytic cascade that dismantles the cell [85].

Various mechanisms of interference with pro-apoptotic BCL-2 family proteins have been described in Chlamydia: (1) sequestration of the BCL-2-associated agonist of cell death (BAD) to the inclusion membrane via the host-cell adapter 14-3-3β-binding, (2) prevention of cytochrome c release from the mitochondria by Chlamydia-dependent anti-apoptotic factors, (3) upregulation of the expression of genes that encode the myeloid leukemia cell differentiation protein (Mcl-1), an anti-apoptotic member of the BCL-2 family, and (4) upregulation of BCL-2-associated athanogene 1 (BAG1), aBCL-2 binding protein, via RAF/MEK/ERK signaling pathway [60, 79]. Recent data provided strong evidence that Chlamydial apoptosis inhibition in infected human cells occurs during the activation of Bax and Bak, and the Chlamydial porin OmpA can interfere with Bak activation [86].

Chlamydial plasmid-encoded secreted protein PGP3 also contributes to apoptosis inhibition by regulating expression levels of Bax and Bcl-2 and activation of caspase-3. Anti-apoptotic activity of PGP3 involves ERK activation via upregulation of caspase DJ-1 protein [87] and phosphorylation and nuclear entry of MDM2, and p53 degradation via activation of the PI3K/AKY signaling pathway [88].

6.3 Inactivation of pro-apoptosis factors by kinases

Kinases regulate host cell processes by phosphorylation of their target proteins and are fundamental for suppressing host cell apoptosis. A key subset of host proteins sequestered by Chlamydia during its survival and development within the inclusion include an assortment of host kinase signaling networks vital for many Chlamydial processes, including entry, nutrient acquisition, and suppression of host cell apoptosis (Reviewed in Ref. [89]).

The mitogen-activated protein-MAP kinase/extracellular signal-regulated kinase (MEK/ERK) and Phosphatidylinositol-3-kinase (PI3K) signaling pathways are among the most prominent kinase signaling networks utilized by Chlamydia in activating pro-survival mechanisms [89]. MEK/ERK signaling and P13K pathways are activated immediately after entry upon binding to host receptor tyrosine kinases. Phosphorylation of the Chlamydial TarP activates the MEK/ERK signaling through interaction with SRC homology 2 domain-containing transforming protein C1 (SHC1). ERK activation and upregulation of the BCL-2 family member MCL-1 are involved in the anti-apoptotic state by activating the PI3K pathway [89]. Activation of the PI3K pathway results in the phosphorylation and activation of the serine/threonine kinase (Akt) cell survival cascade. The PI13K/Akt complex maintains the BCL-2-associated agonist of cell death (BAD) in a phosphorylated state as it is sequestered by the host-cell adapter 14-3-3ß protein at the inclusion vacuole [90]. Depletion of AKT through short-interfering RNA reverses the resistance to apoptosis of C. trachomatis-infected cells. Other kinases (PKCδ, GSK3ß) interact with the inclusion by binding to diacylglycerol-enriched membranes and activating pro-apoptotic signals via different mechanisms [89].

Other pro-survival signaling pathway activated by Chlamydia is the Wnt/β-catenin signaling through the interaction of Chlamydia with fibroblast growth factor receptor (FGFR) or the receptor tyrosine kinases (RTKs) and the ephrin receptor A2 (EPHA2) [79].

6.4 Inhibition of apoptosis by non-coding RNA’s

Non-coding RNAs (ncRNAs) are a novel type of short RNAs that regulate gene expression at multiple levels via various mechanisms, thus influencing development, differentiation, and metabolism [91]. One type of ncRNAs, long non-coding RNA (lncRNAs) regulates gene expression and function, either positively or negatively, by interacting with DNA, RNA, and proteins and also modulate transcriptional, post-transcriptional, and post-translational processes [91].

Chlamydia trachomatis expresses distinct patterns of ncRNAs during normal development [92]. Expression of many ncRNAs is altered during growth stress stimuli that induce persistent growth, particularly IFN-γ and carbenicillin [92]. Recent findings provided evidence that lncRNAs are involved in regulating apoptosis pathways in Chlamydia [93, 94]. The anti-apoptotic activity of the lncRNAs includes modulation of the DNA replication and apoptosis of host cells via Wnt/β-catenin pathway [93] or downregulation of the Bcl-2/Bax ratio with a marked release of cytochrome c, resulting in a significantly elevated level of caspase-3 activation [94]. One of the Chlamydial targeted lncRNA (MIAT) was involved in regulating Chlamydial development during the persistent infection [94]. The discovery of non-coding circular RNAs (circRNAs) has opened the possibility of the role of these rare RNAs in Chlamydia persistence.

7. Molecular tools to study Chlamydia persistence

Historically, genetic manipulation of Chlamydia has been a challenge to scientists because of its obligate intracellular lifestyle, biphasic developmental cycle, and limited metabolic activity of EBs during persistence. On the other hand, scientists have presumed Chlamydia spp. to deliver over 100 proteins through its T3SS that interfere with normal host cell processes to promote invasion, intracellular replication, inclusion formation, and dissemination [17]. Bioinformatics has identified several C. trachomatis effector proteins (Reviewed in Ref. [95]), yet the biological role in persistence remains to be elucidated.

In the context of Chlamydial persistence, the two intracellular morphological forms (RB and AB) have features that render them more suitable than the infectious EB for genetic manipulation. Unlike the rigid cell-walled EB, the RBs slow levels of peptidoglycan in its cell wall, which could facilitate the uptake of DNA [96]. RBs also undergo cell division and express DNA repair enzymes that mediate the chromosomal integration of DNA by homologous recombination during division. Thus, RBs are likely to be naturally competent for transformation. However, one challenge in the genetic manipulation of the Chlamydial RBs in persistence studies is the fact that transformation within infected cells requires exogenous DNA to traverse through several other lipid bilayers (the host plasma membrane and the inclusion membrane) before encountering the RB outer and inner membranes and eventually the chromosome [96].

7.1 Molecular manipulation of Chlamydia

With the recent advances in the molecular genetic manipulation of Chlamydia, it is now possible to perform targeted gene inactivation, whole-genome sequencing to identify mutations, and plasmid transformation to generate fluorescent reporter strains to identify proteins involved in the pathogenesis of Chlamydia [96]. On the other hand, the ability to express exogenous proteins, epitope tags, and fluorescent and other reporter proteins in Chlamydia has expanded the repertoire of possible technologies to study the Chlamydia–host interface [97]. High-resolution microscopy has complemented the advances in genetic and biochemical approaches [96].

Molecular manipulation in Chlamydia takes advantage of the sequencing of the first Chlamydiae genome [98]. C. trachomatis can insert exogenous DNA into its genome because it encodes an intact DNA recombination machinery that facilitates the development of a stable transformation system of Chlamydia with recombinant DNA [99]. This transformation system has enabled the construction of a series of shuttle vectors for gene inactivation by targeted gene knockouts with versatile multiple-cloning sites (MCS), fluorescent protein reporters, inducible promoters, and new selectable markers. Strategies to mediate targeted genetic modifications, such as gene disruptions and gene replacements, include the Targeting Induced Local Lesions in Genomes (TILLING) technology and TargeTron, based on the transient transformation of Chlamydia with a plasmid that encodes an altered group-II intron (Reviewed in Ref. [100]). The recent development of a Fluorescence-reported allelic exchange mutagenesis (FRAEM) using the suicide vector pSUmC has allowed the generation of null mutation strains via the complete deletion of chromosomal genes in C. trachomatis [101].

7.2 Genome-scale analyses

7.2.1 Transcriptomics

High-throughput analysis of protein-encoding mRNA (transcriptomic approaches) has explored the differential expression of genes at different stages of the Chlamydial infectious cycle, allowing the identification of previously unrecognized early Chlamydial gene expression and complex host cell responses [102, 103, 104]. However, such bulk-cell approaches can potentially miss cell-cell variability or cells that contribute to overlapping phenotypic characteristics, potentially masking critical biological heterogeneity as irrelevant signals from non-participating cells that can skew the average [105].

Single-cell RNA sequencing (scRNA-seq) is an alternative to bulk cell populations as it can analyze RNA molecules in individual cells with high resolution and on a genomic scale [105]. The construction of a pilot dataset, applying scRNA-Seq to C. trachomatis infected and mock-infected epithelial cells (HEp-2) has allowed the differential expression of genes involved with cell cycle regulation, innate immune responses, cytoskeletal components, lipid biosynthesis, and cellular stress at early times of infection [105].

7.2.2 Whole-proteome microarrays

Proteome microarray is a novel alternative to gene expression profiling by microarrays for studying Chlamydia–host interaction. Proteins expressed on microarrays display antigenic epitopes, thereby providing an efficient method for immunoprofiling patients and allowing de novo identification of disease-related serum antibodies. The technology takes advantage of the recent construction of a whole-proteome microarray using on-chip protein expression of the C. trachomatis 895 proteins [106]. Comparison of antibody reactivity patterns allowed the identification of new antigens recognized by known C. trachomatis seropositive samples and antigens reacting only with samples from cervical cancer patients [106]. More recently, the whole C. trachomatis screening identified antibody patterns associated with pelvic inflammatory disease (PID), tubal factor infertility, chronic pelvic pain (CPP), and ectopic pregnancy that results from a Chlamydial persistent infection [106, 107]. Although protein microarrays have been used in the field of clinical diagnosis for de novo identification of antibodies associated with general infection and disease-related serum antibodies, the technique can easily be adapted to the identification of antigen biomarkers of Chlamydia persistence.

7.3 In vitro cell systems

The 2D in vitro cell-culture models have been the most widely used models for studying the dynamics of Chlamydial persistence, including its virulence factors and molecular and cellular pathways. The findings of altered morphological forms of C. psittaci in infected mouse fibroblasts (L cells) constituted the first in vitro model of Chlamydial persistence [108]. Since then, the induction of persistent C. trachomatis has been studied extensively using different in vitro cell lines (Reviewed in Ref. [3]). The different in vitro persistence systems have revealed altered Chlamydial growth characteristics, for example, enlarged pleomorphic inclusions with a loss of infectivity and cell division. These changes are generally reversible upon removal of the growth inhibitory factor. One advantage of these systems is that they can be used under highly controlled experimental conditions; however, they fail to mimic the complex and dynamically changing structure of in vivo human host tissues.

Three-dimensional (3D) cell-culture models based on primary cells are acquiring great importance as a new and robust platform for studying complex biological processes and might be a promising alternative in C. trachomatis pathogenetic studies (Reviewed in [109]). The 3D “organoid” models mimic the microenvironment that C. trachomatis encounters in the host tissue, allowing a deeper understanding of host–pathogen interactions by promoting direct cell-to-cell contact, interacting with cells of the extracellular matrix and allowing in vivo exchange of soluble factors. In addition, 3D cell culture models retain the cellular structural integrity resembling the in vivo parental tissue than the 2D cell culture models.

The recent development of Female Reproductive Tract (FRT) Organoid technology is opening up new possibilities to investigate the mechanisms of Chlamydia disease in the FRT [Reviewed in 113]. Human and mouse-derived primary cervical epithelial three-dimensional (3D) organoids resembling the in vivo FTR native tissue architecture offer a unique possibility to elucidate the dynamics and impact of different infections and co-infections in pathogenesis and carcinogenesis [110]. One advantage of using FTR organoids is that they can be propagated and expanded long term under their optimal culture conditions (≥ 6 months), thus providing the ideal model to study persistence in Chlamydia. For instance, in a human ectocervical organoid model, co-infection with Human papillomavirus (HPV)16 E6E7 slowed down the C. trachomatis developmental life cycle by inhibiting the redifferentiation of RBs into EBs, thus inducing persistence [111].

8. Animal models

Extending the in vitro observations of Chlamydial persistence to an animal infection model has remained challenging. The first and only animal model to study Chlamydia persistence was reported a decade ago [112]. The study showed that amoxicillin could induce persistence in BALB/c mice infected intravaginally with the murine pathogen C. muridarum, a close relative of C. trachomatis. Another relevant observation is that amoxicillin-induced persistence resulted in increased failure of subsequent treatment with the first-choice antiChlamydial antibiotic azithromycin [112]. Interestingly, a murine model of naturally chronic nonhuman Chlamydial infection has been recently developed [113].

Other animal models to study Chlamydia genital tract pathogenesis, including guinea pig, nonhuman primate, pig, rat, and the rabbit, have been developed (Reviewed in Ref. [114]). However, none of the animal models perfectly mimics the anatomy, histology, and endocrinology of the human reproductive system or the pathogenesis and immune responses occurring during a chronic human genital C. trachomatis infection [114]. In addition, the use of animal models possesses important ethical issues [114].

9. Concluding remarks

Chlamydia’s ability to manipulate the host cell biology, evade immunity, and undergo morphological aberrant conformations allows these successful intracellular pathogens to enter a persistent state. Persistence has been studied for decades by observing the ability of Chlamydia to survive for long periods of time in cell culture in response to stress stimuli. The stress responses that lead to persistence in Chlamydia comprise complex regulatory networks that control the expression of multiple genes to inhibit apoptosis and activate pro-signaling pathways and immunomodulation. The transcriptional response of Chlamydia differs according to the persistence-inducing stimuli, suggesting differences in the host cell response. On the other hand, isolated in vitro studies indicate common pathways that are down- or upregulated in a similar way by different stress conditions, which may interact and crosstalk between these regulons. Thus, an understanding of the morphological features, as well as the regulatory mechanisms and functional redundancies in pathways involved in persistence, is very critical for the design of novel anti-Chlamydial strategies.

Methodological advances in Chlamydial gene mutagenesis and DNA transformation, deep sequencing technologies, and the implementation of high-throughput genome-scale analysis and improvement in in vitro cell systems have opened new opportunities in our understanding of persistence. The genes encoding critical functional proteins are potential drug targets for treating persistent C. trachomatis infections. Understanding the gene-level changes that take place for Chlamydia to enter persistence could help researchers develop strategies to block these changes from occurring, making the organism more vulnerable to antibiotics and circumventing chronic Chlamydial infections.

Acknowledgments

We thank Dr. Estela S. Estape for her critical reading of the manuscript and the San Juan Bautista School of Medicine for its institutional support.

Conflict of interest

The authors declare no conflict of interest.