Abstract
BackgroundFlagellar secretion systems are utilized by a wide variety of bacteria to construct the flagellum, a conserved apparatus that allows for migration towards non-hostile, nutrient rich environments. Chlamydia pneumoniae is an obligate, intracellular pathogen whose genome contains at least three orthologs of flagellar proteins, namely FliI, FlhA and FliF, but the role of these proteins remains unknown.ResultsFull length FliI, and fragments of FlhA, FliF, and FliI, were cloned and expressed as either GST or His tagged proteins in E. coli. The GST-tagged full length FliI protein was shown to possess ATPase activity, hydrolyzing ATP at a rate of 0.15 ± .02 μmol min-1 mg-1 in a time- and dose-dependant manner. Using bacterial-2-hybrid and GST pull-down assays, the N-terminal domain of FliI was shown to interact with the cytoplasmic domain of FlhA, but not with FliF, and the cytoplasmic domain of FlhA was shown to interact with the C-terminus of FliF. The absence of other flagellar orthologs led us to explore cross-reaction of flagellar proteins with type III secretion proteins, and we found that FliI interacted with CdsL and CopN, while FlhA interacted with CdsL and Cpn0322 (YscU ortholog CdsU).ConclusionsThe specific interaction of the four orthologous flagellar proteins in C. pneumoniae suggests that they interact in vivo and, taken together with their conservation across members of the chlamydiae sps., and their interaction with T3S components, suggests a role in bacterial replication and/or intracellular survival.
Highlights
Flagellar secretion systems are utilized by a wide variety of bacteria to construct the flagellum, a conserved apparatus that allows for migration towards non-hostile, nutrient rich environments
FliI and FlhA interact with T3S components Since Chlamydia have no apparent flagella, we investigated whether the flagellar proteins FliI, FlhA and FliF interact with T3S components
We show that FliI interacts with CdsL and CopN, two T3S components, while FlhA interacts with CdsL and a third T3S component, CdsU
Summary
Flagellar secretion systems are utilized by a wide variety of bacteria to construct the flagellum, a conserved apparatus that allows for migration towards non-hostile, nutrient rich environments. Chlamydia pneumoniae is an obligate, intracellular pathogen whose genome contains at least three orthologs of flagellar proteins, namely FliI, FlhA and FliF, but the role of these proteins remains unknown. CopN, orthologous to YopN, is believed to function as a regulator of the system which plugs the injectisome pore prior to activation of T3S and is a known effector protein [18]. Several reports have emerged characterizing protein interactions within the C. pneumoniae T3SS, describing novel protein complexes that form at the inner membrane. Johnson et al have shown that CdsD, a unique protein orthologous to YscD that contains two fork-head associated domains, interacts with the predicted C. pneumoniae ATPase tethering protein, CdsL, and CdsQ, a cytosolic component of the inner membrane that presumably forms the bulk of the T3S C-ring [20]. Stone et al extended these findings to show that CdsN, the ATPase, is involved in this complex as well as interacting with the proposed plug protein, CopN [16]
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