Interactions between CdsD, CdsQ, and CdsL, Three PutativeChlamydophila pneumoniaeType III Secretion Proteins

Abstract

Chlamydophila pneumoniae is a gram-negative obligate intracellular bacterial pathogen that causes pneumonia and bronchitis and may contribute to atherosclerosis. The developmental cycle of C. pneumoniae includes a morphological transition from an infectious extracellular elementary body (EB) to a noninfectious intracellular reticulate body (RB) that divides by binary fission. The C. pneumoniae genome encodes a type III secretion (T3S) apparatus that may be used to infect eukaryotic cells and to evade the host immune response. In the present study, Cpn0712 (CdsD), Cpn0704 (CdsQ), and Cpn0826 (CdsL), three C. pneumoniae genes encoding yersiniae T3S YscD, YscQ, and YscL homologs, respectively, were cloned and expressed as histidineand glutathione S-transferase (GST)-tagged proteins in Escherichia coli. Purified recombinant proteins were used to raise hyper-immune polyclonal antiserum and were used in GST pull-down and copurification assays to identify protein-protein interactions. CdsD was detected in both EB and RB lysates by Western blot analyses, and immunofluorescent staining demonstrated the presence of CdsD within inclusions. Triton X-114 solubilization and phase separation of chlamydial EB proteins indicated that CdsD partitions with cytoplasmic proteins, suggesting it is not an integral membrane protein. GST pull-down assays indicated that recombinant CdsD interacts with CdsQ and CdsL, and copurification assays with chlamydial lysates confirmed that native CdsD interacts with CdsQ and CdsL. To the best of our knowledge, this is the first report demonstrating interactions between YscD, YscQ, and YscL homologs of bacterial T3S systems. These novel protein interactions may play important roles in the assembly or function of the chlamydial T3S apparatus.Gram-negative bacterial pathogens such as Salmonella, Shigella, Yersinia, and Escherichia coli use type III secretion (T3S) to deliver toxins and effector proteins directly into the cytoplasm of host cells, resulting in bacterial uptake, survival, and virulence. The T3S apparatus is composed of approximately 20 to 25 functionally conserved proteins that assemble to form a unified structure containing oligomeric protein complexes in the bacterial cytoplasm (C ring), inner membrane (inner ring), periplasm (inner rod), outer membrane (outer ring; secretin), extracellular space (needle, needle extension, and ruler), and host cell membrane (translocons). This elongated needle complex is also known as the injectisome, and upon cellular contact, the translocons insert into the host cell plasma membrane, forming a conduit for the delivery of effector proteins into the host cytoplasm (10).Chlamydophila pneumoniae is an obligate intracellular gramnegative pathogen with a unique biphasic developmental cycle. C. pneumoniae elementary bodies (EB) attach to host cells and are internalized via an unknown mechanism involving the activation of Rho family GTPases, actin polymerization, and the formation of microvilli (7,9,43). Once within cells, metabolically inert EB transform i...

Making connections: snapshots of chlamydial type III secretion systems in contact with host membranes

“…The structure of this translocon and the nature of its interaction with the host membrane remain unknown, as it is never co-isolated with the detergent-solubilised core T3SS complex. Chlamydiae encode homologues of core complex components [15,16,17], yet in comparison to other pathogens in which the genes encoding T3SSs are grouped together on pathogenicity islands, T3SS-related genes are distributed across the genome in four distinct clusters composed of at least ten separate operons [18]. Unusually, Chlamydiae also possess two copies of putative translocon components (CT578/CT579 and CT860/CT861) identified by primary sequence similarity to the Yersinia YopB and YopD translocon proteins [19], although the significance of this remains unresolved.…”

“…The structure of this translocon and the nature of its interaction 19 with the host membrane remain unknown, as it is never co-isolated with the 20 detergent-solubilised core T3SS complex. Chlamydiae encode homologues of core 21 complex components [15,16,17] membrane that initially loosely encloses the EB and co-envelopes host material 5 transitions to form a tight structure proximal to the EB surface. During this time, the 6 EBs lose their polarity, with an associated reduction of the pronounced periplasmic 7 widening and a decrease in assembled T3SSs (Figure 2) [26].…”

Forkhead-associated Domain-containing Protein Rv0019c and Polyketide-associated Protein PapA5, from Substrates of Serine/Threonine Protein Kinase PknB to Interacting Proteins of Mycobacterium tuberculosis

“…The information acquired in the past few years point toward their roles in secretion pathways, transcription, cellular metabolism, and signal transduction (17)(18)(19)(20)(21). Among prokaryotes, Mycobacterium tuberculosis has emerged as an attractive model system for understanding FHA domain-mediated signaling for two reasons: 1) M. tuberculosis encodes for 11 eukaryotic like STPKs (PknA to PknL, except PknC) and seven FHA domains in six proteins (22), thus offering an experimentally malleable system to understand molecular themes common to prokaryotes and eukaryotes.…”

Identification and biochemical characterization of the CHLAMYDIA TRACHOMATIS type III secretion chaperone, SLC1, and its role in the translocation of the invasion-associated effector TARP.

“…Most of our current knowledge of the structure of the chlamydial type III secretion apparatus comes from the discovery of in vitro protein-protein interactions between chlamydial type III components. [27,[32][33][34][35][36][37][38][39][40][41], while the detection of new chlamydial effectors has involved expression and secretion of recombinant chlamydial proteins in heterologous type III secretion systems of other bacteria [3 8, 40-43]. The predicted structure of the Chlamydia type III secretion apparatus and its components is visualized in Figure 1.…”

“…CdsD homo logs in other bacteria function as scaffolding proteins to link CdsC and other integral membrane components. CdsD has been shown to coimmunoprecipitate with other integral membrane proteins as expected [36], but unique features such as forkhead association domains (FHA) and in vitro phosphorylation are 7 puzzling as to the actual role of CdsD in type III secretion [ 46,47]. Homology with innermembrane components from Yersinia species indicate that CdsJ provides a scaffold for the inner membrane components CdsRlS/T/U and V [46].…”