**2. The bacteria: Intracellular life cycle,** *Chlamydia trachomatis* **serovars and virulence factors**

#### **2.1. Chlamydial developmental cycle and intracellular niche**

CT is a highly evolved pathogen that has a reduced genome, first sequenced by Stephens and collaborators in 1998. Its chromosome consists of approximately one million base pairs and encodes for up to 600 proteins [3]. Analysis of chlamydial genes reveals that this bacterium heavily depends on host cell for nutrition and replication, indicating a complex evolution for adaptation to an obligate intracellular lifestyle.

CT has tropism for genital mucosal epithelium, which promotes its own uptake into nonphagocytic cells. Chlamydial infection and propagation rely upon a unique biphasic life cycle that begins by contact of infectious, environmentally resistant, elementary bodies (EBs) with the apical surface of the epithelial cell. Several mechanisms are involved in the invasion of host cell, likely parasite-specified phagocytosis and receptor-mediated endocytosis [4]. Multiple receptors have been proposed to mediate the interaction between the EB and the host cell, among them, the mannose receptor, the mannose 6-phosphate receptor, and the estrogen receptor [5]. Other host molecules such as heparan sulfate proteoglycans [6,7], and protein disulfide isomerase also participate in EB binding to the eukaryotic cells [8]. Concomitantly, multiple bacterial adhesins and ligands such as glycosaminoglycan [9], the major outer membrane protein (MOMP) [10], OmcB [11], and PmpD [12] facilitate EB attachment to host cells. Translocated actin-recruiting phosphoprotein (TARP) is a bacterial protein that nucleates actin and promotes host cell cytoskeleton remodeling to force bacterium uptake [13–15].

The infectious EBs enter the host cell in membrane-bound vesicles that travel toward the perinucleus and fuse to form a single vacuole termed the inclusion. Once inside this modified phagosome, EBs differentiate into metabolically active but non-infectious reticulate bodies (RBs) that are the replicative bacterial forms. RBs asynchronously multiply by binary fission within the confines of the growing inclusion. After numerous rounds of replication, RBs redifferentiate back into infectious EBs to be ready for spreading to adjacent cells [16,17]. The ability of CT to cycle between resting and replicating organisms accounts for a drawback in the eradication of this intracellular pathogen. Finally, the infectious bacteria are released by two independent mechanisms, the host cell lysis, or the extrusion of the inclusion [18]. A scheme of chlamydial developmental life cycle is shown in Figure 1.

infections, and the pathogen factors that may be involved in the damage of female reproductive tract. Then, we analyze the host factors that may contribute to the development of infertility, mainly immune response and genetic predisposition, hormonal status, and sexual behavior. Undiagnosed and untreated infections, repeat and persistent infections, and coinfections are likely responsible for the detrimental sequelae on woman fertility of CT pathogenesis.

This chapter specially focuses on the consequences of chronic diseases after CT infections, mainly pelvic inflammatory disease, tubal infertility, and adverse pregnancy outcome, which

**2. The bacteria: Intracellular life cycle,** *Chlamydia trachomatis* **serovars and**

CT is a highly evolved pathogen that has a reduced genome, first sequenced by Stephens and collaborators in 1998. Its chromosome consists of approximately one million base pairs and encodes for up to 600 proteins [3]. Analysis of chlamydial genes reveals that this bacterium heavily depends on host cell for nutrition and replication, indicating a complex evolution for

CT has tropism for genital mucosal epithelium, which promotes its own uptake into nonphagocytic cells. Chlamydial infection and propagation rely upon a unique biphasic life cycle that begins by contact of infectious, environmentally resistant, elementary bodies (EBs) with the apical surface of the epithelial cell. Several mechanisms are involved in the invasion of host cell, likely parasite-specified phagocytosis and receptor-mediated endocytosis [4]. Multiple receptors have been proposed to mediate the interaction between the EB and the host cell, among them, the mannose receptor, the mannose 6-phosphate receptor, and the estrogen receptor [5]. Other host molecules such as heparan sulfate proteoglycans [6,7], and protein disulfide isomerase also participate in EB binding to the eukaryotic cells [8]. Concomitantly, multiple bacterial adhesins and ligands such as glycosaminoglycan [9], the major outer membrane protein (MOMP) [10], OmcB [11], and PmpD [12] facilitate EB attachment to host cells. Translocated actin-recruiting phosphoprotein (TARP) is a bacterial protein that nucleates actin and promotes host cell cytoskeleton remodeling to force bacterium uptake [13–15].

The infectious EBs enter the host cell in membrane-bound vesicles that travel toward the perinucleus and fuse to form a single vacuole termed the inclusion. Once inside this modified phagosome, EBs differentiate into metabolically active but non-infectious reticulate bodies (RBs) that are the replicative bacterial forms. RBs asynchronously multiply by binary fission within the confines of the growing inclusion. After numerous rounds of replication, RBs redifferentiate back into infectious EBs to be ready for spreading to adjacent cells [16,17]. The ability of CT to cycle between resting and replicating organisms accounts for a drawback in the eradication of this intracellular pathogen. Finally, the infectious bacteria are released by

are of therapeutic interest in the reproduction field.

adaptation to an obligate intracellular lifestyle.

**2.1. Chlamydial developmental cycle and intracellular niche**

**virulence factors**

136 Genital Infections and Infertility

**Figure 1.** *Chlamydia trachomatis* developmental cycle. Chlamydial infection begins with attachment of the infectious bacterial form, the elementary body (EB) to uncharacterized host cell receptors. Signal transduction events are trig‐ gered, and EBs entry into the host cell in small vesicles. These CT-containing vesicles are actively modified by the bac‐ teria; they travel toward the perinucleus and fuse to form a single vacuole named "the inclusion". Once internalized, EB differentiates into the replicative bacterial form, the reticulate body (RB), which multiplies by binary fission. At the end, RBs re-differentiate to EBs that are released by host cell lysis or extrusion of the inclusion. The whole cycle is com‐ pleted in 40–72 hours. In a stressful environment, RBs enter a latent stage where it persists until more favorable grow‐ ing conditions. These aberrant bacteria (AB) are present in persistent and chronic infections.

In response to stress, CT enters into a low replicative viable state that is termed "persistent or aberrant bacterial form", which is able to resume normal replication as soon as conditions are again favorable. Among the inducers of the persistent bacterial state stand the sphingolipid deprivation and tryptophan lack, the presence of interferon gamma (IFN-γ), or certain antibiotics such as penicillin. The evasion strategy has been linked to the capacity of these bacteria to cause latent and chronic infections. Thus, the onset of the infection is generally not detected and re-infection may occur, especially when infected couples are involved. Further‐ more, the fact that CT clearance is rarely followed up, combined with the ability of this pathogen to persist, contributes to the occurrence of long-term infections [19].

Undoubtedly, an essential issue to chlamydial growth and development is the establishment of its intracellular niche. Early chlamydial gene expression is required to inclusion generation, to avoid immune system surveillance, and to hijack host cell functions [20]. These bacteria actively modify the inclusion membrane by exclusion or recruitment of selected host proteins, mainly Rab proteins, the master controllers of intracellular traffic [16,21]. Increasing evidence points out that the invading bacteria subvert trafficking not only to circumvent the lysosomal degradative route but also to facilitate the delivery of host nutrients to the growing inclusion [17,22]. As soon as the chlamydial inclusion is formed, it dissociates from the classical phag‐ ocytic pathway and barely interacts with endocytic vesicles [23]. Instead, chlamydial inclusion intersects Golgi-derived vesicles [24–27], multivesicular bodies [28], and lipid droplets [29,30]. By this strategy, these bacteria take over the infected cell for sphingomyelin, cholesterol, and neutral lipid acquisition like pirates [31–34]. In addition, CTs possess other mechanisms, such as transporter molecules, finely adapted to acquire amino acids, nucleotides, and energy from the host cell [22,35–38]. At present, the strategies developed by CTs to re-route intracellular trafficking and to co-opt host cell functions for their benefit are being actively studied.

#### **2.2. Bacterial genotypes and virulence factors**

Different strains of CT have been described based on genome sequencing and the antigenic properties of the major outer membrane protein (MOMP) [39]. There are more than 20 distinct serovars (serologically variant strains) of CT currently identified, on the basis of monoclonal antibody-based typing assays [40–42]. In general, CTs have been grouped into three main pathobiotypes: ocular infections (serovars A to C), sexually transmitted diseases (D to K), and lymphogranuloma venereum (L1 to L3). Serovars A, B, and C have tropism for the ocular epithelium, causing from acute conjunctivitis to trachoma, a serious eye disease endemic in Africa and Asia that is characterized by chronic conjunctivitis and can lead to infectious blindness. Serovars D through K have emerged as the major causing agents of sexually transmitted diseases. They preferentially infect squamocolumnar epithelial cells of female reproductive system and the male genitourinary tract. E and D serovars are isolated from genital tract infections with the most frequency worldwide. Occasionally, they cause conjunc‐ tivitis or pneumonia in newborns infected during labor. Serovars L1 to L3 are responsible for a systemic illness, the lymphogranuloma venereum that is associated with genital ulcer disease in tropical countries [43,44] (Table 1).

*Chlamydia* genotyping is useful to determine tissue tropism [45–47]. Several studies attempted to directly link disease severity with CT serovars; however, they often failed because of small number of samples and high variability in results [48]. Intensive research is conducted to confidently associate CT serotypes to higher pathogenic potential, clinical course, or disease outcome. Nevertheless, at present, bacterial ability to ascend and colonize female upper reproductive tract is not clearly associated to a particular CT serovar.

On the other hand, CT gene polymorphisms determine distinct antigenic challenge to the immune system [49]. Certain bacterial polymorphisms may induce an altered immune response [50]. In consequence, they are able to cause immunological disorders, especially in susceptible individuals. Chlamydial infections often precede the initiation of autoimmune diseases, and frequently, these bacteria are found within autoimmune lesions. Bacterial proteins similar to host self-proteins might be the underlying cause of diverse autoimmune diseases [51,52]. This molecular mimicry may elicit an immune response to both self and microbial proteins. Chlamydial heat shock protein 60, DNA primase, and OmcB proteins represent the strongest cases for molecular mimicry [53]. The most frequent autoimmune diseases connected to chlamydial infections are intestinal inflammatory pathologies and rheumatic or connective-tissue diseases [54,55]. Further research is required to unravel the molecular machinery involved in the complex pathogen-host cell interaction.

actively modify the inclusion membrane by exclusion or recruitment of selected host proteins, mainly Rab proteins, the master controllers of intracellular traffic [16,21]. Increasing evidence points out that the invading bacteria subvert trafficking not only to circumvent the lysosomal degradative route but also to facilitate the delivery of host nutrients to the growing inclusion [17,22]. As soon as the chlamydial inclusion is formed, it dissociates from the classical phag‐ ocytic pathway and barely interacts with endocytic vesicles [23]. Instead, chlamydial inclusion intersects Golgi-derived vesicles [24–27], multivesicular bodies [28], and lipid droplets [29,30]. By this strategy, these bacteria take over the infected cell for sphingomyelin, cholesterol, and neutral lipid acquisition like pirates [31–34]. In addition, CTs possess other mechanisms, such as transporter molecules, finely adapted to acquire amino acids, nucleotides, and energy from the host cell [22,35–38]. At present, the strategies developed by CTs to re-route intracellular trafficking and to co-opt host cell functions for their benefit are being actively studied.

Different strains of CT have been described based on genome sequencing and the antigenic properties of the major outer membrane protein (MOMP) [39]. There are more than 20 distinct serovars (serologically variant strains) of CT currently identified, on the basis of monoclonal antibody-based typing assays [40–42]. In general, CTs have been grouped into three main pathobiotypes: ocular infections (serovars A to C), sexually transmitted diseases (D to K), and lymphogranuloma venereum (L1 to L3). Serovars A, B, and C have tropism for the ocular epithelium, causing from acute conjunctivitis to trachoma, a serious eye disease endemic in Africa and Asia that is characterized by chronic conjunctivitis and can lead to infectious blindness. Serovars D through K have emerged as the major causing agents of sexually transmitted diseases. They preferentially infect squamocolumnar epithelial cells of female reproductive system and the male genitourinary tract. E and D serovars are isolated from genital tract infections with the most frequency worldwide. Occasionally, they cause conjunc‐ tivitis or pneumonia in newborns infected during labor. Serovars L1 to L3 are responsible for a systemic illness, the lymphogranuloma venereum that is associated with genital ulcer disease

*Chlamydia* genotyping is useful to determine tissue tropism [45–47]. Several studies attempted to directly link disease severity with CT serovars; however, they often failed because of small number of samples and high variability in results [48]. Intensive research is conducted to confidently associate CT serotypes to higher pathogenic potential, clinical course, or disease outcome. Nevertheless, at present, bacterial ability to ascend and colonize female upper

On the other hand, CT gene polymorphisms determine distinct antigenic challenge to the immune system [49]. Certain bacterial polymorphisms may induce an altered immune response [50]. In consequence, they are able to cause immunological disorders, especially in susceptible individuals. Chlamydial infections often precede the initiation of autoimmune diseases, and frequently, these bacteria are found within autoimmune lesions. Bacterial proteins similar to host self-proteins might be the underlying cause of diverse autoimmune diseases [51,52]. This molecular mimicry may elicit an immune response to both self and

reproductive tract is not clearly associated to a particular CT serovar.

**2.2. Bacterial genotypes and virulence factors**

138 Genital Infections and Infertility

in tropical countries [43,44] (Table 1).


**Table 1.** Chlamydial serovars, tissue tropism and clinical diseases. Acute and chronic pathologies occur in men, women and newborns following CT infections. Serovars A to C have tropism for ocular epithelium, causing from acute conjunctivitis to infectious blindness or trachoma. Serovars D to K infect epithelial cells of the genitourinary system, generating a broad range of acute and chronic pathologies that damage reproductive tissue and may infect newborns during labor. Serovars L1–3 cause lymphogranuloma venereum. Several autoimmune diseases are associated to diverse CT strains.

Several putative virulence factors have been postulated, including the polymorphic outer membrane autotransporter family of proteins (pmp), type III secretion system (TTSS) effectors, a large cytotoxin, and stress response proteins may contribute to increase the CT-associated pathogenicity. Pmp proteins are strongly immunogenic and trigger pro-inflammatory cytokine responses [56]. Chlamydial TTSS effectors mediate the interaction with the host as they are injected to the cytoplasm and alter host cell functioning [57–59]. Important TTSS effectors are the inclusion (Inc) proteins that are bacterial proteins present at the inclusion membrane. For instance, IncA promotes the fusion of individual CT-containing vesicles to form a single inclusion [60,61]. Natural IncA bacterial mutants are associated with reduced virulence [62]. Another TTSS effector is TARP, mentioned in the previous section, as a bacterial protein that favors CT internalization via an actin recruiting mechanism [34,63]. Additionally, a chlamydial cytotoxin glycosylates the eukaryotic protein Rac1, and thereby induces actin reorganization and promotes the invasion of host cell [64,65]. Chlamydial glycolipid exoanti‐ gens [66] and the lipopolysaccharide [67] may constitute additional virulence factors. Other proteins encoded by the cryptic plasmid or related to the ability of the bacteria to survive under stressful metabolic conditions such as iron or tryptophan deprivation are thought to increase virulence and pathogenicity [68,69]. Chlamydial stress proteins, GroEL and GroES, may activate toll-like receptors and trigger a potent inflammatory response, injuring host repro‐ ductive tissues [70–73].

In addition to CT serovars and virulence molecules, other bacterial factors may be involved in the pathogenicity and chlamydial infection outcome, such as the pathogen load, route of infection, bacterial ability to enter persistent state, ascension capacity and strength to colonize genital upper tract, resistance to antibiotic treatment, and so on. Further studies are needed to determine the contribution of each bacterial factor to the development of severe damage on the female reproductive system.
