The ClpX and ClpP2 Orthologs of Chlamydia trachomatis Perform Discrete and Essential Functions in Organism Growth and Development

Abstract

24Chlamydia trachomatis (Ctr) is an obligate intracellular bacterium that undergoes a complex 25 developmental cycle in which the bacterium differentiates between two functionally and 26 morphologically distinct forms, each of which expresses its own specialized repertoire of 27 proteins. The transitions between the infectious, non-dividing elementary body (EB) and the non-28 infectious, replicative reticulate body (RB) are not mediated by division events that re-distribute 29 intracellular proteins. Rather, both primary (EB to RB) and secondary (RB to EB) differentiation 30 require protein turnover. The Clp protease system is well conserved in bacteria and, minimally, 31 relies on a serine protease subunit, ClpP, and a AAA+ ATPase, such as ClpX, that recognizes 32 and unfolds substrates for ClpP degradation. In Chlamydia, clpX is encoded within an operon 33 adjacent to clpP2. We present evidence that the chlamydial ClpX ortholog, and the co-34 transcribed ClpP2, play a key role in organism viability and development. We demonstrate here 35 that chlamydial ClpX is a functional ATPase and forms the expected homohexamer in vitro.

Inclusion Membrane Growth and Composition Are Altered by Overexpression of Specific Inclusion Membrane Proteins in Chlamydia trachomatis L2

“…S6)). Others have overexpressed non-T3S substrates (e.g., wild-type ClpP1, ClpP2, and ClpX) with no impact on inclusion size or progeny production (44,59). These data support the findings that the overexpression of certain Incs results in the misorganization of Incs in the inclusion membrane that negatively impacts development.…”

“…To determine if the overexpression of certain Incs negatively impacts chlamydial development, C. trachomatis L2 was transformed with anhydrotetracycline (aTc)-inducible plasmids encoding ct813-FLAG, incF-FLAG, ct226-FLAG, ct483-FLAG, or mCherry (empty vector control (44)). Inclusion size was used as a metric for normal chlamydial development.…”

“…Stable clones were plaque purified (65,67) and subsequently density gradient purified. C. trachomatis L2 pBOMB4-Tet-CT483-FLAG (CTL0744) was a kind gift from M. Weber (University of Iowa, Iowa City, IA) (23), and the C. trachomatis L2 pBOMB4-Tet-CT226-FLAG (CTL0478) strain (46) and C. trachomatis L2 pBOMB4-mCherry-GFP strain was described previously (44).…”