**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*) [28–33]), depletion of essential nutrients (i.e., iron, amino acids, and glucose), heat shock, coinfection with Herpes Simplex virus [27, 34–39], infection of monocytes and macrophages [40–42], 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

#### *Persistence in* Chlamydia *DOI: http://dx.doi.org/10.5772/intechopen.109299*

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) [48–50]. 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 *Chlamydia*e, 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* [57–59].
