**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

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

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].
