Background

Chlamydia trachomatis (C.t.) is the etiological agent of blinding trachoma and the most prevalent sexually transmitted disease in the world. Chlamydiae are gram-negative obligate intracellular bacteria that have evolved to occupy a niche within the host cytoplasm derived from a modified endocytic vesical called an inclusion. Chlamydia initiates entry into the host cell by binding to the sialic acid moieties of proteins on the host cell surface. The precise mechanisms regulating entry of C.t. into the host cell are poorly understood but several studies have implicated clathrin mediated endocytosis (Hodinka et al., 1988; Wyrick et al., 1989; Majeed and Kihlstrom, 1991), while others have suggested caveola mediated entry is involved (Boleti et al., 1999). C.t. is taken up into the cell within an endocytic vesicle which it heavily modified to form the chlamydial inclusion. Fusion of this compartment with lysosomes is blocked and the bacterium secretes numerous effector proteins that subvert host resources and redirect them to the inclusion to promote chlamydial proliferation. The Chlamydial lifecycle is biphasic, consisting of different infectious and proliferative stages. The infectious but metabolically inactive form is referred to as an elementary body (EB) while the metabolically active but non-infectious form is called the reticulate body (RB) (Figure 1). Chlamydial EB’s bind to sialic acid residues on cell surface receptors and through an unknown mechanism induce their uptake by the cell into an endocytic vesicle. The inclusion avoids fusion with lysosomes and degradative compartments while trafficking along microtubules to the peri Golgi region. By about 4 h post-infection, most of the EBs have converted to RBs. Following chlamydial cell division, the RB’s undergo asynchronous conversion to EB’s. Depending on stress, cellular environment, and nutrient availability the chlamydial replicative cycle takes 48-72 h to complete and chlamydia exits via host cell lysis or through extrusion. Of note, the EB is the form usually purified (Caldwell et al., 1981) although techniques have been described to purify RBs (Skipp et al., 2016). For C.t. purifications, the EB is most desirable, since it is the stage associated with infectivity.


Figure 1. Transmission electron micrograph of wild-type C.t. L2 at 36h post-infection. Scale bar is 1 µm.

Although one study reported that a medium had been developed that could support the axenic metabolic and biosynthetic activity from both EBs and RBs, to the best of our knowledge no lab has had success in using this media to support propagation of chlamydia (Omsland et al., 2012). Thus, in the research laboratory, C.t. is typically cultivated in HeLa 229, Hep2, McCoy, or Vero cells although other cell-types can be used they are less common. Until recently, C.t. was largely intractable to genetic manipulation but advances in tools to generate site-specific mutations and to overexpress proteins during infection have revolutionized the field and labs are now making recombinant C.t. clones regularly. In the past, large preparations of C.t. EBs could carry the lab for quite some time. Now, with the generation of new strains occurring on a regular basis, C.t. preparations are a much more common occurrence in the chlamydia laboratory. This is certainly a good thing but cultivating, purifying, and titering new C.t. strains on a regular basis can be a costly and time-consuming endeavor. Not to mention that a key reagent that has been used for years, Renografin, has been made extremely difficult to obtain due to Mallinckrodt ceasing production. Fortunately, a similar product has been identified and new sources discovered (this information will be provided in the materials section).