STRUCTURAL AND BIOCHEMICAL CHARACTERIZATION OF THE CHLAMYDIA PNEUMONIAE TYPE III SECRETION SYSTEM

Abstract

<p><em>Chlamydia pneumoniae</em> is a Gram-negative intracellular pathogen that uses type III secretion to invade and survive within eukaryotic cells. The T3SS secretes specific effector proteins during the infection process to facilitate immune evasion and nutrient acquisition. Unfortunately, the genetic intractability and difficult culturing conditions of Chlamydiae has inhibited progress in the chlamydial T3S field. This thesis characterizes fundamental aspects of the <em>C. pneumoniae </em>injectisome such as the ATPase, the inner-membrane export apparatus, and a specific effector protein Cpn0803. Initially, we explored whether <em>C. pneumoniae</em> encodes a functional T3S ATPase and if it associates with other T3S components. We found that CdsN has enzymatic activity consistent with other Gram-negative T3S ATPases, and that CdsN associates with inner-membrane and soluble components such as CdsD, CdsQ, CopN and CdsL. We also found that CdsN has binding surfaces for either structural or putative effector / chaperone T3S proteins. Next, we explored the putative flagellar genes, which were of interest since <em>Chlamydia</em> is a non-motile bacteria that lacks flagellum. We found that the flagellar proteins associate with the T3S apparatus, suggesting that they play a role in T3S during the life-cycle. We extended this observation to show that CdsL, a T3S component, down-regulates both CdsN and FliI enzymatic activity, suggesting that the flagellar proteins are involved in T3S. Furthermore, we characterized Cpn0803 as an exemplary effector, which associates with both CdsN and FliI. We found that Cpn0803 is secreted into host cells upon<em> Chlamydia</em> infection. Cpn0803 was thought to be the T3S needle-tip protein; however, the crystal structure does not support this hypothesis. Presently, the actual role of Cpn0803 in the T3S apparatus remains unknown. Overall, our data suggests that CdsN and FliI both function during the chlamydial life-cycle in the T3S process, possibly coordinating effector proteins (such as Cpn0803) for secretion into host cells.</p>