Bioinformatic analysis revealed a putative

Bioinformatic analysis revealed a putative coiled-coil SLD within CT229 which we show to be essential for intracellular replication and recruitment of Rab GTPases to the inclusion. Mutation of a single amino Zafirlukast residue in the SLD domain (CT229L120D) significantly reduced Rab binding and recruitment to the inclusion, suggesting that the SLD of CT229 is essential for interactions with host vesicle trafficking pathways. Eukaryotic SNARE proteins mediate fusion of vesicles with membranes and are not known for their ability to bind and capture Rab GTPases. It is possible that CT229 acts in concert with either a host SNARE or another Inc protein to mediate fusion of vesicles with the inclusion membrane. Multifunctionality of bacterial effector proteins is not uncommon and we speculate that CT229 may both capture and initiate fusion of certain host vesicles with the inclusion membrane. The CI-M6PR in the TGN recognizes M6P residues on lysosomal proteins and promotes their delivery to prelysosomal compartments, where the drop in pH promotes ligand dissociation and the CI-M6PR is subsequently recycled back to the TGN (Braulke and Bonifacino, 2009). During trafficking, the CI-M6PR associates with the retromer complex through interactions with sorting nexin (SNX)5 and SNX6. Using an affinity purification mass spectrometry (AP-MS) screen, an interaction between IncE and SNX5/6 was detected (Mirrashidi et al., 2015). Structural analysis of the IncE-SNX5 interaction revealed that IncE binds to a conserved hydrophobic groove in the PX domain, the same residues in which the CI-M6PR receptor binds (Elwell et al., 2017, Paul et al., 2017, Sun et al., 2017) The interaction with IncE disrupts CI-M6PR trafficking, which may act to decrease lysosome potency or to access critical nutrients (Elwell and Engel, 2018). Here, we demonstrate that CT229 is required to recruit CI-M6PR-containing vesicles to the vicinity of the inclusion. Whether CT229 interacts in a cooperative manner with IncE to tether vesicles to the inclusion membrane or if this is a compensatory mechanism occurring independently of IncE warrants further study. Acquisition of iron is typically an infection limiting step during pathogenic bacterial infections (Pokorzynski et al., 2017). As such, chlamydia requires iron to complete its replicative cycle; however, the mechanism(s) by which chlamydia acquires iron from the host cell is unknown. Iron is essential for productive chlamydial infection, and although it has been known for 20 years that transferrin localizes to the periphery of the inclusion membrane, the mechanisms utilized by the bacterium to recruit Tfn have remained elusive (Al-Younes et al., 2001, van Ooij et al., 1997, Scidmore et al., 1996). Although Tfn localizes to the periphery of the inclusion, it is not observed within the inclusion, suggesting that if Tfn represents a source of iron for the bacteria, it is freed either prior to or during fusion with the inclusion membrane. Once inside the inclusion, iron may be transported via unknown siderophores, carriers, or specific ion channels into the bacterial cell. Here, we show that interactions between CT229 and Rab4 or 35 positive vesicles are required for Tfn localization to the periphery of the inclusion. These data potentially provide an important clue about how C. trachomatis may subvert iron from the host without disrupting the cellular labile iron pool and homeostasis.
STAR★Methods
Acknowledgments This work was supported by startup funds from the University of Iowa to M.M.W. We thank Fabienne Paumet for assistance with bioinformatics. We also thank Janet Sager and Tina Clark for technical assistance.
Introduction Innate immune system is a set of a variety of cells and molecules, including toll-like receptors (TLRs) for pathogen recognition that are mainly expressed in macrophages, forming a part of a complex whole for the organism's defense. Upon recognition, TLRs prompt intracellular activation of critical downstream signaling pathways that eventually culminates the production of proinflammatory cytokines. Properly controlled activation of NF-κB and mitogen-activated protein kinases (MAPKs) is a result of coordinated recruitment of intracellular signaling molecules and cascade events involving mainly adaptor molecule MyD88, kinases IRAK1/4, ubiquitin ligase TRAF6, and TAK1 and IKK’s complex (Kawasaki and Kawai, 2014). This is imperative for governing the transcriptional activation of an array of genes encoding proinflammatory cytokines, including TNF and IL-6 (Beutler, 2009). Given its complexity, it is likely that cellular components other than from innate immune system pathway participate in regulating the production of inflammatory mediators.