**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 antiapoptotic 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 antiapoptotic 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/βcatenin 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, posttranscriptional, 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.
