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The canonical PX domain structure is composed of a three-stranded β-sheet (β1, β2 and β3) followed by three close-packed α-helices. The first and second α-helices are connected by an extended proline-rich sequence. Typically PX domains have been found to bind to the endosome-enriched lipid phosphatidylinositol-3-phosphate (PtdIns3P) via a basic pocket formed at the junction between the β3 strand, α1 helix and Pro-rich loop. In contrast SNX5, SNX6 and SNX32 possess major alterations in the PtdIns3P-binding pocket that preclude canonical lipid head-group docking (see below). In addition they possess a unique extended helix-turn-helix insert between the Pro-rich loop and α2 helix of unknown function (Figure 2B) (Koharudin et al., 2009).
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To determine the structure of the SNX5-IncE complex we generated a fusion protein encoding the human SNX5 PX domain (residues 22–170) and C. trachomatis IncE C-terminal sequence (residues 108–132) attached at the PX domain C-terminus Figure 4—figure supplement 1A). This construct readily crystallised in several crystal forms, and we were able to determine the structure of the complex in three different spacegroups (Figure 4; Table 3; Figure 4—figure supplement 1B). Confirming that the fusion does not alter complex formation, the short linker region is disordered, and the mode of IncE-binding to SNX5 is identical in all three structures (Figure 4—figure supplement 1C and D). Because of the higher resolution, we focus our discussions on the structure of the SNX5 PX-IncE complex observed in the P212121 crystal form. The first three IncE N-terminal residues (Pro107, Ala108, Asn109) and the last three IncE C-terminal residues (Val130, Thr131, Gln132) were not modeled due to lack of electron density, suggesting disorder and matching precisely with our mapping experiments showing these residues are not necessary for SNX5 association.10.7554/eLife.22311.011Figure 4.Structure of the SNX5 PX domain in complex with the IncE C-terminal domain.(A) Crystal structure of the SNX5 PX domain (yellow) in complex with IncE residues 107–132 (magenta) shown in cartoon representation. (B) Backbone atoms of the SNX5 and IncE proteins are shown to highlight the prominent β-sheet augmentation mediating the association between the two molecules. (C) Close up view of the SNX5-IncE interface highlighting specific contact areas at the N-terminus of the IncE peptide. (D) Close up of the SNX5-IncE interface highlighting specific contact areas at the hairpin loop of the IncE peptide shown at 90° to Figure 4C. (E). Close up of the SNX5-IncE interface highlighting contact areas at the C-terminus of the IncE peptide in approximately the same orientation as Figure 4C. Residues in SNX5 (Phe136) and IncE (Phe116) that are critical for binding based on mutagenesis are boxed.DOI: http://dx.doi.org/10.7554/eLife.22311.01110.7554/eLife.22311.012Figure 4—figure supplement 1.Supplementary images for SNX5-IncE crystal structures.(A) Sequence of the SNX5 PX domain fusion protein with the IncE C-terminal peptide. (B) Refined 2fo-fc electron density contoured at 1.5σ for the SNX5-IncE structure in spacegroup P212121. (C) Overlay of each independent SNX5-IncE complex observed in the three crystal forms. (D) Ribbon structures indicating the locations of the linker regions in each crystal form. The C-terminal SNX5 residues and the N-terminal IncE residues are shown by spheres with distances indicated.DOI: http://dx.doi.org/10.7554/eLife.22311.01210.7554/eLife.22311.013Table 3.Summary of crystallographic structure determination statistics*.DOI: http://dx.doi.org/10.7554/eLife.22311.013CrystalSNX5 PX-IncE Form 1SNX5 PX-IncE Form 2SNX5 PX-IncE Form 3PDB ID5TGI5TGJ5TGHData collectionWavelength (Å)0.953700.953700.95370Space groupP212121I2P32Cell dimensions a, b, c (Å)60.7, 67.5, 88.258.4, 80.3, 94.6100.6, 100.6, 71.7 α, β, γ (°)90, 90, 9090, 97.2, 9090, 90, 120Resolution (Å)60.7–1.98 (2.03–1.98)31.9–2.6 (2.72–2.60)50.3–2.80 (2.95–2.80)Rmerge0.104 (0.525)0.153 (0.659)0.101 (0.713)Rmeas0.112 (0.572)0.18 (0.777)0.124 (0.873)Rpim0.042 (0.225)0.096 (0.408)0.051 (0.363)<I> / σI12.4 (3.4)39.6 (3.2)11.7 (2.3)Total number reflections178868 (11000)46691 (5757)115149 (16861)Total unique reflections26075 (1805)13432 (1632)20001 (2923)Completeness (%)100 (100)99.9 (100.0)100 (100)Multiplicity6.9 (6.1)3.5 (3.5)5.8 (5.8)Half-set correlation (CC(1/2))0.997 (0.868)0.986 (0.55)0.997 (0.683)RefinementResolution (Å)45.1–1.98 (2.02–1.98)31.9–2.6 (2.69–2.60)41.2–2.8 (2.87–2.80)No. reflections/No. Rfree26021/200013421/1342 (1208/134)19975/1972 (1301/144)Rwork/Rfree0.192/0.214 (0.221/0.246)0.199/0.242 (0.276/0.332)0.236/0.254 (0.329/0.372)No. atoms Protein257926195189 Solvent281690Average B-factor (Å2)31.842.556.0R.m.s deviations Bond lengths (Å)0.0120.0110.015 Bond angles (°)1.271.151.27*Highest resolution shell is shown in parentheses.
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(A) Crystal structure of the SNX5 PX domain (yellow) in complex with IncE residues 107–132 (magenta) shown in cartoon representation. (B) Backbone atoms of the SNX5 and IncE proteins are shown to highlight the prominent β-sheet augmentation mediating the association between the two molecules. (C) Close up view of the SNX5-IncE interface highlighting specific contact areas at the N-terminus of the IncE peptide. (D) Close up of the SNX5-IncE interface highlighting specific contact areas at the hairpin loop of the IncE peptide shown at 90° to Figure 4C. (E). Close up of the SNX5-IncE interface highlighting contact areas at the C-terminus of the IncE peptide in approximately the same orientation as Figure 4C. Residues in SNX5 (Phe136) and IncE (Phe116) that are critical for binding based on mutagenesis are boxed.
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10.7554/eLife.22311.012Figure 4—figure supplement 1.Supplementary images for SNX5-IncE crystal structures.(A) Sequence of the SNX5 PX domain fusion protein with the IncE C-terminal peptide. (B) Refined 2fo-fc electron density contoured at 1.5σ for the SNX5-IncE structure in spacegroup P212121. (C) Overlay of each independent SNX5-IncE complex observed in the three crystal forms. (D) Ribbon structures indicating the locations of the linker regions in each crystal form. The C-terminal SNX5 residues and the N-terminal IncE residues are shown by spheres with distances indicated.DOI: http://dx.doi.org/10.7554/eLife.22311.012
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(A) Sequence of the SNX5 PX domain fusion protein with the IncE C-terminal peptide. (B) Refined 2fo-fc electron density contoured at 1.5σ for the SNX5-IncE structure in spacegroup P212121. (C) Overlay of each independent SNX5-IncE complex observed in the three crystal forms. (D) Ribbon structures indicating the locations of the linker regions in each crystal form. The C-terminal SNX5 residues and the N-terminal IncE residues are shown by spheres with distances indicated.
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The IncE sequence forms a long β-hairpin structure that binds within a complementary groove at the base of the extended α-helical insertion of the SNX5 PX domain and adjacent to the β-sheet sub-domain (Figure 4A; Video 2). The β-hairpin structure of IncE (N-terminal βA and C-terminal βB strands) is directly incorporated as a β-sheet augmentation of the β1, β2 and β3 strands of SNX5 (Figure 4B). The N-terminal βA strand of the IncE sequence (Gly111-Lys118) forms the primary interface with SNX5, making main-chain hydrogen bonds with the β1 strand of the SNX5 PX domain for the stable positioning of the IncE structure. The two anti-parallel β-strands of IncE are connected by a short loop (Gly119-Ala124) that makes no direct contact with the SNX5 protein, and the C-terminal IncE βB strand (Asp125-Val130) forms an interface with the extended α-helical region of the SNX5 PX domain.Video 2.Animation highlighting the mechanism of interaction between SNX5 and IncE.The SNX5 PX domain is shown in yellow ribbons and the IncE peptide is shown in magenta.DOI: http://dx.doi.org/10.7554/eLife.22311.01410.7554/eLife.22311.014
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Detailed views of the SNX5-IncE interface are shown in Figure 4C, D and E. Aside from main-chain hydrogen bonding to form the extended β-sheet, IncE engages in several critical side-chain interactions with the relatively hydrophobic SNX5 binding groove. At the N-terminus of the βA strand Val114 of IncE inserts into a pocket formed primarily by Tyr132 and Phe136 on the SNX5 α’’ helix (Figure 4C). A major contribution comes from IncE Phe116, with π-stacking occurring with the Phe136 side-chain and hydrophobic docking with Val140 of SNX5 (Figure 4D). Adjacent to IncE Phe116 at the end of the βA strand Lys118 makes an electrostatic contact with SNX5 Glu144. Finally, at the C-terminal end of the IncE βB strand Val127 and Leu129 contact an extended SNX5 surface composed of Leu133, Tyr132 and Met106 (Figure 4E).
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To verify the crystal structure we mutated residues from both SNX5 and IncE and measured their affinities using ITC (Figure 5A and B; Table 2). At the interface between SNX5 and IncE several side chains make key contributions to peptide recognition. Because Leu133 and Phe136 residues in SNX5 are located at the core of the IncE-binding interface, and also due to the structural rearrangements these residues make on binding (see below), we reasoned that L133D and F136A mutations would inhibit the interaction. Indeed these mutants abolished association with the IncE peptide (Figure 5A). The reciprocal mutations in IncE residues F116A and V127D also abolished binding to the SNX5 PX domain (Figure 5B), and the SNX6 and SNX32 PX domains (Figure 5—figure supplement 1), demonstrating the importance of these hydrophobic and π-stacking interactions for stable complex formation. In contrast mutations predicted to disrupt an observed electrostatic contact (IncE K118A or SNX5 E144A) had little effect on binding. Thus the core hydrophobic interactions are critical for IncE binding but the peripheral electrostatic contact is not essential.10.7554/eLife.22311.015Figure 5.Mutations in the SNX5 and IncE proteins prevent complex formation in vitro.(A) ITC experiments testing the effect of SNX5 mutations on IncE binding. Both L133D and F136A mutations prevented IncE binding, but the A144A mutation had little effect. (B) ITC experiments testing the effect of IncE mutations on SNX5 binding. Both F116A and V127D blocked SNX5 interaction, while Q115A had a partial effect and K118A had no effect on association.DOI: http://dx.doi.org/10.7554/eLife.22311.01510.7554/eLife.22311.016Figure 5—figure supplement 1.SNX6 and SNX32 PX domains bind IncE at the same site as SNX5.ITC experiments testing the effect of IncE peptide mutations on binding to SNX6 and SNX32 PX domains. The IncE F116A mutation blocks interaction with both PX domains similarly to SNX5 (Figure 5B).DOI: http://dx.doi.org/10.7554/eLife.22311.016
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(A) ITC experiments testing the effect of SNX5 mutations on IncE binding. Both L133D and F136A mutations prevented IncE binding, but the A144A mutation had little effect. (B) ITC experiments testing the effect of IncE mutations on SNX5 binding. Both F116A and V127D blocked SNX5 interaction, while Q115A had a partial effect and K118A had no effect on association.
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10.7554/eLife.22311.016Figure 5—figure supplement 1.SNX6 and SNX32 PX domains bind IncE at the same site as SNX5.ITC experiments testing the effect of IncE peptide mutations on binding to SNX6 and SNX32 PX domains. The IncE F116A mutation blocks interaction with both PX domains similarly to SNX5 (Figure 5B).DOI: http://dx.doi.org/10.7554/eLife.22311.016
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To confirm the role of IncE in direct SNX5 protein recruitment to the chlamydial inclusion we examined the localisation of GFP-tagged SNX5 in HeLa cells infected with C. trachomatis L2 (CTL2) for 24 hr (MOI ~0.5). Cells expressing the GFP-SNX5 protein showed clear and uniform recruitment to the limiting membrane of the inclusion as defined by mCherry-Rab25 (Figure 6A), which is consistent with the localisation observed by others (Aeberhard et al., 2015; Mirrashidi et al., 2015). In contrast, the GFP-SNX5 (F136A) mutant protein showed no recruitment to the chlamydial inclusion. The change in relative distribution of these GFP-SNX5 proteins on the inclusion was quantified for wildtype SNX5 (Mander’s coefficient 0.67 ± 0.14) and GFP-SNX5 (F136A) (0.041 ± 0.051) (Figure 6—figure supplement 1A). Like wild-type GFP-SNX5 the GFP-SNX5 (F136A) mutant was recruited to punctate endosomal structures throughout the cytoplasm of these cells, and in addition was able to co-immunoprecipate endogenous SNX1 in heterodimeric complexes identically to the wild-type GFP-SNX5 protein (Figure 6—figure supplement 1B). This implies that BAR-domain mediated heterodimer formation with SNX1 or SNX2 is required for endosomal recruitment, and is not perturbed by the IncE-binding mutation in the PX domain. Lastly, we tested the importance of IncE residues for SNX interaction in situ by expressing the GFP-tagged IncE C-terminal domain. The wild-type GFP-IncE(91-132) was recruited to endosomal structures via its interaction with SNX5-related proteins in both uninfected and infected HeLa cells (Figure 6B; Figure 6—figure supplement 1C). In contrast however, GFP-IncE(91-132)(F116D), a SNX5-binding mutant, was exclusively cytosolic. Note that neither IncE construct is localised to the inclusion, as expected due to lack of signal peptides and transmembrane domains (Figure 6—figure supplement 1C).10.7554/eLife.22311.017Figure 6.Mutations in the SNX5 and IncE proteins prevents their interaction in cells.(A) Single amino-acid mutation in the PX domain of the SNX5 (F136A) abolishes recruitment to the chlamydial inclusion. HeLa cells stably expressing mCherry-Rab25 (red) were transfected transiently with GFP-SNX5 or GFP-SNX5 (F136A) (green) and infected with Chlamydia trachomatis L2 for 18–24 hr. The cells were fixed and the nucleic materials were counter-stained with DAPI (blue). (B) HeLa cells were transfected transiently with GFP-IncE(91-132) or GFP-IncE(91-132)(F116D) (green) and co-labelled for the early endosomal marker EEA1 (red). Mutation in the SNX5 binding IncE peptide (F116D) abolishes recruitment to endosomal structures. *Represents the inclusion. Scale bar 20 μm.DOI: http://dx.doi.org/10.7554/eLife.22311.01710.7554/eLife.22311.018Figure 6—figure supplement 1.GFP-IncE C-terminal domain is localised to endosomes but not inclusions.(A) Quantitation of the degree of overlap between the GFP-SNX5 constructs and the mCherry-Rab25 inclusion membrane marker from Figure 6A. Mander’s correlation coefficient of mChRab25 signal over GFP-SNX5 signals (10 cells per group; error bars, S.D). (B) Co-immunoprecipitation of GFP-SNX5 from HeLa cells shows that both the wild-type and mutant protein (F136A) interact equally with endogenous SNX1. This indicates that both proteins are correctly folded and otherwise functional. (C) HeLa cells stably expressing mCherry-Rab25 (red) were transfected transiently with GFP-IncE(91-132) or GFP-IncE(91-132)(F116D) (green) and infected with C. trachomatis L2 for 18–24 hr. Mutation in the SNX5 binding IncE peptide (F116D) abolishes recruitment to endosomal structures. Neither construct is recruited to inclusions, which is consistent with the lack of transmembrane regions. *Represents the inclusion. Scale bar 20 μm.DOI: http://dx.doi.org/10.7554/eLife.22311.018
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(A) Single amino-acid mutation in the PX domain of the SNX5 (F136A) abolishes recruitment to the chlamydial inclusion. HeLa cells stably expressing mCherry-Rab25 (red) were transfected transiently with GFP-SNX5 or GFP-SNX5 (F136A) (green) and infected with Chlamydia trachomatis L2 for 18–24 hr. The cells were fixed and the nucleic materials were counter-stained with DAPI (blue). (B) HeLa cells were transfected transiently with GFP-IncE(91-132) or GFP-IncE(91-132)(F116D) (green) and co-labelled for the early endosomal marker EEA1 (red). Mutation in the SNX5 binding IncE peptide (F116D) abolishes recruitment to endosomal structures. *Represents the inclusion. Scale bar 20 μm.
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10.7554/eLife.22311.018Figure 6—figure supplement 1.GFP-IncE C-terminal domain is localised to endosomes but not inclusions.(A) Quantitation of the degree of overlap between the GFP-SNX5 constructs and the mCherry-Rab25 inclusion membrane marker from Figure 6A. Mander’s correlation coefficient of mChRab25 signal over GFP-SNX5 signals (10 cells per group; error bars, S.D). (B) Co-immunoprecipitation of GFP-SNX5 from HeLa cells shows that both the wild-type and mutant protein (F136A) interact equally with endogenous SNX1. This indicates that both proteins are correctly folded and otherwise functional. (C) HeLa cells stably expressing mCherry-Rab25 (red) were transfected transiently with GFP-IncE(91-132) or GFP-IncE(91-132)(F116D) (green) and infected with C. trachomatis L2 for 18–24 hr. Mutation in the SNX5 binding IncE peptide (F116D) abolishes recruitment to endosomal structures. Neither construct is recruited to inclusions, which is consistent with the lack of transmembrane regions. *Represents the inclusion. Scale bar 20 μm.DOI: http://dx.doi.org/10.7554/eLife.22311.018
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(A) Quantitation of the degree of overlap between the GFP-SNX5 constructs and the mCherry-Rab25 inclusion membrane marker from Figure 6A. Mander’s correlation coefficient of mChRab25 signal over GFP-SNX5 signals (10 cells per group; error bars, S.D). (B) Co-immunoprecipitation of GFP-SNX5 from HeLa cells shows that both the wild-type and mutant protein (F136A) interact equally with endogenous SNX1. This indicates that both proteins are correctly folded and otherwise functional. (C) HeLa cells stably expressing mCherry-Rab25 (red) were transfected transiently with GFP-IncE(91-132) or GFP-IncE(91-132)(F116D) (green) and infected with C. trachomatis L2 for 18–24 hr. Mutation in the SNX5 binding IncE peptide (F116D) abolishes recruitment to endosomal structures. Neither construct is recruited to inclusions, which is consistent with the lack of transmembrane regions. *Represents the inclusion. Scale bar 20 μm.
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Superposition of the SNX5-IncE complex with the SNX5 PX domain in the apo state (Koharudin et al., 2009) reveals a significant conformational change in the α-helical extension, as well as localized alterations in the loop between the β1 and β2 strands to accommodate peptide binding (Figure 7A). In essence the IncE β-hairpin acts as a tether between the core PX fold and extended α-helical hairpin, pulling the two sub-structures closer together. Overall the α-helical extension undergoes a maximal movement of ~8–10 Å at the furthest tip, facilitated by the flexibility of the structure following the Pro-rich linker and an apparent hinge-point at Pro97 (Figure 7A upper panel). In the immediate vicinity of Pro97 the SNX5 loop that encompasses Asp43 is significantly shifted and stabilized by the repositioning of Arg103. At both the start of the first α’ helix of the extension and the end of the second α’’ helix more subtle changes occur in the positions of Met106, Leu128, Tyr132, Leu133 and Phe136. These changes result in formation of the hydrophobic pocket that engages the IncE side-chains Val114, Phe116, Val127 and Leu129 (Figure 7A, middle and lower panels).10.7554/eLife.22311.019Figure 7.Conformational changes in SNX5 and a model for SNX-BAR recruitment to inclusion membranes.(A) Comparison of the SNX5-IncE complex (yellow-magenta) with the previously reported apo- SNX5 PX domain crystal structure (blue) (PDB ID 3HPB)(Koharudin et al., 2009). The α-helical extension undergoes a significant displacement in the bound state. The enlarged panels to the right show several close-up views of the binding pocket highlighting conformational changes that are required to accommodate IncE. (B) A model for the SNX5-SNX1 PX-BAR heterodimer and its interaction with IncE at the inclusion membrane. The PX-BAR structure was modeled in silico (see methods). The left panel shows cartoon representations of the structure, viewed from the side and from the membrane surface. Middle panels show the same structures in electrostatic surface representation (red, negative; blue positive). The right panels show close ups of the putative PtdIns3P-binding pocket in SNX1 and SNX5, with a PtdIns3P head-group (shown in spheres) docked by aligning the previous SNX9 crystal structure (Pylypenko et al., 2007). SNX1 has a canonical PtdIns3P pocket, while SNX5 lacks a clear site for lipid head-group binding. (C) Sequence conservation of SNX5-related proteins was calculated and plotted using CONSURF. The surface representation indicates exposed side-chains that are evolutionarily conserved in green. The IncE peptide binds to a highly conserved surface groove, while the putative phosphoinositide binding region (Koharudin et al., 2009) on the opposite face is neither highly conserved nor poised to allow docking. (D) Cartoon model depicting the recruitment of SNX5 and related proteins to the inclusion membrane. Heterodimers with SNX1 or SNX2 will be recruited via IncE in infected cells, and this recruitment will be in competition with the binding of SNX1 and SNX2 to PtdIns3P for normal endosomal association, as well as interactions with other proteins including retromer and unidentified molecules that potentially bind to the conserved groove of the SNX5 PX domain.DOI: http://dx.doi.org/10.7554/eLife.22311.019
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(A) Comparison of the SNX5-IncE complex (yellow-magenta) with the previously reported apo- SNX5 PX domain crystal structure (blue) (PDB ID 3HPB)(Koharudin et al., 2009). The α-helical extension undergoes a significant displacement in the bound state. The enlarged panels to the right show several close-up views of the binding pocket highlighting conformational changes that are required to accommodate IncE. (B) A model for the SNX5-SNX1 PX-BAR heterodimer and its interaction with IncE at the inclusion membrane. The PX-BAR structure was modeled in silico (see methods). The left panel shows cartoon representations of the structure, viewed from the side and from the membrane surface. Middle panels show the same structures in electrostatic surface representation (red, negative; blue positive). The right panels show close ups of the putative PtdIns3P-binding pocket in SNX1 and SNX5, with a PtdIns3P head-group (shown in spheres) docked by aligning the previous SNX9 crystal structure (Pylypenko et al., 2007). SNX1 has a canonical PtdIns3P pocket, while SNX5 lacks a clear site for lipid head-group binding. (C) Sequence conservation of SNX5-related proteins was calculated and plotted using CONSURF. The surface representation indicates exposed side-chains that are evolutionarily conserved in green. The IncE peptide binds to a highly conserved surface groove, while the putative phosphoinositide binding region (Koharudin et al., 2009) on the opposite face is neither highly conserved nor poised to allow docking. (D) Cartoon model depicting the recruitment of SNX5 and related proteins to the inclusion membrane. Heterodimers with SNX1 or SNX2 will be recruited via IncE in infected cells, and this recruitment will be in competition with the binding of SNX1 and SNX2 to PtdIns3P for normal endosomal association, as well as interactions with other proteins including retromer and unidentified molecules that potentially bind to the conserved groove of the SNX5 PX domain.
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To better understand how IncE can recruit the SNX5-containing SNX-BAR complexes to inclusion membranes we constructed an in silico model of the SNX5-SNX1 heterodimeric PX-BAR proteins (Figure 7B). Consistent with the length of the IncE C-terminal cytoplasmic sequence the model predicts that the IncE sequence will bind to the surface of SNX5 close to, but oriented away from, the inclusion membrane. While PX domains are commonly able to recognise PtdIns3P lipid headgroups, SNX5-related proteins lack the typical binding pocket (Figure 7B right panel), and there is some controversy regarding their ability to mediate specific membrane interactions (Koharudin et al., 2009; Teasdale and Collins, 2012). We propose that in the context of C. trachomatis infection, SNX5-related proteins are directly associated with the inclusion via IncE protein-protein interactions in a phosphoinositide-independent manner, and are able to recruit their heterodimeric partners SNX1 and SNX2 (Sierecki et al., 2014; van Weering et al., 2012; Wassmer et al., 2009). The PX-BAR-domain containing complexes are then localised to the inclusion in a retromer-independent manner (Mirrashidi et al., 2015), and may contribute to the formation of the dynamic inclusion-associated membrane tubules.
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Interestingly, when a cross-species evolutionary analysis of side-chain conservation in the SNX5-related proteins is performed it is clear that the IncE peptide binds a hydrophobic surface groove that is strictly conserved in this protein family (Figure 7C). This very strongly implies that the site is normally engaged in a protein-protein interaction with an as yet unidentified binding partner(s) required for SNX5’s regular biological function, and that IncE is directly competing for this interface.
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Although more than fifty putative Incs have been identified in C. trachomatis, the exact roles of these inclusion membrane proteins are still poorly understood. Chlamydiae manipulate the host cellular and signaling networks via interactions between the cytoplasmic region of Incs and numerous host cell proteins. Recent studies reported retrograde trafficking proteins as significant components of the inclusion, with sorting nexin family members being particularly enriched (Aeberhard et al., 2015; Mirrashidi et al., 2015). In this study, we present the first reported crystal structure of a chlamydial inclusion protein (IncE) binding to its host effector protein (SNX5). While the detailed mechanism of IncE-mediated protein recruitment will be specific to this family member, the principle of extended cytoplasmic Inc sequences engaging with cellular host proteins on the inclusion is certain to be a general one. A simple analogy would be to consider the Inc proteins as being like a molecular ‘velcro’ that recognises and attaches host machinery needed for bacterial replication and survival.
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The manipulation of endocytic transport machinery is clearly critical for the obligate intracellular survival of C. trachomatis (Aeberhard et al., 2015; Mirrashidi et al., 2015; Moore and Ouellette, 2014). In addition to C. trachomatis, SNX1, SNX2, SNX5, SNX6 and the associated retromer complex have also been directly implicated in the cellular pathogenesis of Coxiella burnetii (McDonough et al., 2013), Salmonella enterica serovar Typhimurium (Bujny et al., 2008), hepatitis C virus (Yin et al., 2016), human papilloma virus (Ganti et al., 2016; Popa et al., 2015), and Legionella pneumophila (Finsel et al., 2013). Broadly then the manipulation of SNX proteins and endosomal trafficking machinery by viral and bacterial pathogens is a common occurrence during intracellular infection, and points to a wide-ranging role in host-pathogen interactions.
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Typically PX domains of sorting nexins, including SNX1 and SNX2 (Cozier et al., 2002; Zhong et al., 2005), play an important role in endosomal membrane recruitment by binding the endosome-enriched lipid PtdIns3P through four conserved residues (Mas et al., 2014; Teasdale and Collins, 2012). These residues are conserved in most PX domains including in SNX1 and SNX2, but are entirely absent in SNX5, SNX6 and SNX32. Although there is evidence for the weak association of the SNX5 PX domain with the lipid PtdIns(4,5)P2 from nuclear magnetic resonance (NMR) spectroscopy experiments (Koharudin et al., 2009), the crystal structure does not point to a clear binding mechanism. A second feature that sets SNX5-related proteins apart from the rest of the SNX family is the presence of an extended α-helical insertion. Our work confirms the central importance of this unique insert for the binding of the IncE inclusion protein, and provides the first clear description of how a PX domain can function as a protein-protein interaction scaffold as opposed to a lipid-binding domain.
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The high degree of conservation in the IncE binding surface of SNX5 implies that this site is critical for the normal function of SNX5 and its homologs. Previously, the expression of a GFP-tagged IncE C-terminal domain was shown to interfere with the SNX5/SNX6-dependent retrograde trafficking of the cation-independent mannose-6-phosphate receptor (CI-MPR) (Mirrashidi et al., 2015). Combined with our structural data, this infers that IncE is mimicking and interfering with SNX5/SNX6-mediated protein interactions, with a ligand(s) required for normal endosomal trafficking that remains to be discovered. Once recruited to the inclusion, SNX-BAR proteins are localized to the bulk membrane and dynamic tubules. While it is logical to imagine they could play a positive role in the sculpting of the inclusion, this is somewhat difficult to reconcile with the effect of SNX5 and SNX6 knockdown, which results in an increased production of C. trachomatis infectious progeny. Alternatively, although a pool of SNX5/SNX6 and associated SNX1/SNX2 proteins remain on endosomes in C. trachomatis infected cells, their sequestering by the chlamydial inclusion may interfere with normal endosomal trafficking (Figure 7D). It was thus proposed that the role of IncE could be to compete for SNX-retromer endosomal interactions, resulting in the breakdown of normal trafficking of the CI-MPR and lysosomal hydrolases and hence perturbation of the endolysosomal system’s capacity for bacterial destruction (Aeberhard et al., 2015; Mirrashidi et al., 2015). Defining the precise role of SNX proteins and other endocytic machinery in chlamydial infection will clearly require further study.
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In conclusion, our work provides novel molecular insights into the mechanism of SNX protein coercion by the IncE chlamydial effector, and presents a blueprint for future studies of other inclusion protein activities. In addition, our results provide a possible clue to understanding how SNX5-related molecules mediate protein interactions required for canonical cell trafficking pathways.
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All synthetic peptides used for isothermal titration were purchased from Genscript (USA). For ITC experiments, peptides were weighed and dissolved in 50 mM Tris (pH 8.0) and 100 mM NaCl (ITC buffer) to make a stock peptide concentration of 2 mM, which was diluted to 0.75 mM before use.
|
study
| 96.56 |
Polyclonal antibodies against C. trachomatis HtrA were generated previously (Huston et al., 2008). Monoclonal antibodies against EEA1 (610457, 1:100), SNX1 (611483,1:200) and SNX2 (611308, 1:200) were supplied by BD Bioscience. Monoclonal antibodies against the myc epiptope (9B11, 1:2000) were supplied by Abcam. Rabbit polyclonal antibodies against GFP (A-6455, 1:500) were purchased from Molecular Probes (Invitrogen). Rabbit polyclonal antibodies against Rab5 (C8B1, 1:100) were from Cell Signaling Technology. Goat polyclonal antibodies against Vps35 (IMG-3575, 1:400) were from Imgenex. Secondary antibodies were purchased from Molecular Probes (Life Technologies) and Li-Cor Bioscience. Wortmannin was supplied by Sigma-Aldrich (W1628). VPS34-In1 was from Merck Millipore (532628).
|
other
| 72.44 |
The IncE sequence used in this study is from the L3 serovar L3/404/LN (NCBI reference WP_015506602) (Harris et al., 2012). The pGEX-4T-2 bacterial expression plasmid encoding the human SNX5 PX domain (residues 22–170) was generated using a standard PCR-based cloning strategy, and its identity confirmed by sequencing. All other bacterial expression constructs for human SNX proteins were synthesized and cloned into pGEX-4T-2 by Genscript (USA). These included the SNX5 PX domain IncE fusion (SNX5 residues 22–170 with IncE residues 108–132 fused at the C-terminus (Figure 4—figure supplement 1A), SNX6 (residues 29–170), SNX32 (residues 17–166), and SNX5 PX domain mutants. The pcDNA3.1-N-eGFP mammalian expression constructs encoding full-length human SNX5, SNX5(F136A), IncE(91-132) and IncE(91-132)(F116D) with N-terminal GFP-tags were generated by Genscript (USA). The pCMU-myc-SNX5 was as described previously (Kerr et al., 2006), and the SNX6 and SNX32 genes cloned into the pcDNA3.1-nMyc vector at BamHI and XhoI restriction sites (Kerr et al., 2012). SNX5, SNX32 and SNX8 were also cloned by polymerase chain reaction, restriction digest and ligation into pEGFP-C1 for expression with N-terminal GFP tags as described previously (Wang et al., 2010).
|
study
| 100.0 |
All proteins except SNX5 PX domain mutants were expressed in Escherichia coli Rosetta cells, whereas mutant constructs were expressed in BL21 Codon Plus supplemented with appropriate antibiotics. Single colonies from cultures grown on LB agar plates were inoculated into 50 mL LB2+ with ampicillin (0.1 mg/mL) and chloramphenicol (0.1 mg/mL), and grown at 37°C with shaking overnight. The following day, 30 mL from the overnight culture was used to inoculate 1 L LB media containing ampicillin (0.1 mg/mL) and chloramphenicol (0.1 mg/mL) and incubated at 37°C. Cells were grown to an optical density (OD) of 0.5–0.6 at 600 nm and induced with 0.5 mM isopropyl-β-D-thiogalactopyranoside (IPTG) (except for the SNX5-IncE fusion, where expression was induced at OD600 of 0.8 with 1 mM IPTG). Cultures were incubated with shaking overnight at 18°C until the cells reach an O.D of 3.0 (~24 hr). Cells were harvested using a Beckman rotor JLA 8.1000 at 4000 RPM for 30 min at 4°C. Pellets were resuspended in 10 mL lysis buffer (50 mM Tris (pH 8.0), 100 mM NaCl, 5% glycerol, 1 mM DTT, 0.1 mg/ml benzamidine, 0.1 mg/ml DNase) per litre of culture. The cells were subjected to cell disruption and centrifugation at 18,000 RPM for 30 min at 4°C. The soluble fractions were first purified using affinity chromatography with glutathione-sepharose, and when required the GST tags were cleaved by thrombin while still bound to the column. The proteins were eluted in 50 mM Tris (pH 8.0), 100 mM NaCl, 5% glycerol, and 1 mM DTT, and then further polished using gel filtration chromatography (Superdex 200, GE healthcare) in a buffer containing 50 mM Tris (pH 8.0), 100 mM NaCl. The fractions corresponding to the respective proteins were then pooled and used directly for ITC or were further concentrated for crystallization.
|
study
| 100.0 |
ITC experiments were performed on a Microcal iTC200 instrument at 25°C. The proteins were buffer exchanged into ITC buffer (50 mM Tris (pH 8.0) and 100 mM NaCl) by gel filtration prior to ITC experiments. IncE peptides at 750 µM were titrated into 50 µM PX domain samples. The binding data was processed using ORIGIN 7.0 with a single site binding model to determine the stoichiometry (n), the equilibrium association constant Ka (1/Kd), and the enthalpy (△H). The Gibbs free energy (△G) was calculated using the equation △G = −RTIn(Ka); binding entropy (△S) was calculated by △G = △H – T△S. Three experiments were performed for each set of samples to determine the average ± standard error of the mean (SEM) for thermodynamic quantities, except for the peptide truncation experiments where only single experiments were performed. For these truncated peptide experiments, all experiments were performed using the same batch of protein to allow direct comparions to be made.
|
study
| 100.0 |
The SNX5 PX domain fusion with IncE was concentrated to 15 mg/ml for crystallization. Eight 96-well crystallization hanging-drop screens were set up using a Mosquito Liquid Handling robot (TTP LabTech) at 20°C. Optimized diffraction-quality crystals were obtained using streak seeding in sitting drop vapor diffusion plates. The crystallisation solution for crystal form 1 was 0.2 M KSCN, 25% PEG 2K MME, 100 mM sodium acetate (pH 5.5), for crystal form 2 was 0.1 M NaCl, 0.1 M MgCl2, 0.1 M Nacitrate (pH 3.5), 12 % PEG 4000, and for crystal form 3 was 1.26 M (NH4)2SO4, acetate (pH 4.5), 0.2 M NaCl. Data were collected at the Australian Synchrotron MX1 and MX2 Beamlines, integrated with iMOSFLM (Battye et al., 2011), and scaled with AIMLESS (Evans and Murshudov, 2013) in the CCP4 suite (Winn et al., 2011). The structures were initially solved by molecular replacement with PHASER (McCoy et al., 2007) using the apo-SNX5 PX domain crystal structure as the input model (PDB code 3HPB), minus the extended α-helical domain. The resulting model was rebuilt with COOT (Emsley et al., 2010), followed by repeated rounds of refinement with PHENIX (Adams et al., 2011). All structural figures were generated using PyMOL (DeLano scientific).
|
study
| 100.0 |
HeLa cells stably expressing mCherry-Rab25 were previously generated within the lab (Teo et al., 2016) and were maintained in DMEM (Gibco) supplemented with 10% (v/v) FCS (Bovogen) and 2 mM L-glutamine (Invitrogen) in a humidified air/atmosphere (5% CO2) at 37°C. Cells were transfected at 70% confluence with pcDNA3.1-N-eGFP plasmid constructs using Lipofectamine 2000 as per manufacturer’s protocol (Invitrogen) and examined 18–24 hr later. The HeLa cell line used in this study was from America Type Culture Collection (#ATCC CCL2). Parental and stable cells lines were negative for mycoplasma by DAPI staining, and authenticated by STR profiling (Cell Bank Australia). For inhibitor treatments, cells were treated with either 100 nM wortmannin or 1 µM Vps34-IN1 for 1 hr.
|
study
| 100.0 |
C.C. trachomatis serovar L2 (ATCC VR-902B) was used to infect cells at a multiplicity of infection (MOI) of ~0.5. Cells were infected 2 hr post-transfection in normal DMEM (Gibco) supplemented with 10% (v/v) FCS (Bovogen) and 2 mM L-glutamine (Invitrogen) in a humidified air/atmosphere (5% CO2) incubator at 37°C. After 2 hr media was replaced with fresh media.
|
study
| 99.94 |
Transfected and infected cells (18–24 hr post-infection) were fixed with 4% paraformaldehyde, permeabilised using TritonX-100 (Sigma) and immunolabeled as described previously (Teo et al., 2016) and counter-stained with DAPI. The coverslips were imaged using a confocal laser-scanning microscope (LSM 710 meta, Zeiss) with 63x oil immersion objective. Time-lapse videomicroscopy was carried out on individual live cells using a Nikon Ti-E inverted deconvolution microscope using a 40x, 0.9 Plan Apo DIC objective, a Hamamatsu Flash 4.0 4Mp sCMOS monochrome camera and 37°C incubated chamber with 5% CO2. GFP was excited with a 485/20 nm LED and captured using a 525/30 nm emission filter, and mCherry was excited using a 560/25 nm LED and captured using a 607/36 nm emission filter. Data was processed using ImageJ (https://imagej.nih.gov/ij/) and compiled using Adobe Illustrator CS6.
|
study
| 99.94 |
The immunofluorescence colocalisation of GFP-SNX5 with chlamydial inclusion membranes (Figure 6A; Figure 6—figure supplement 1A) imaged on a confocal microscope was measured by Mander’s correlation coefficient of red pixel (EEA1 or mCherry-Rab25) over green pixel (GFP-SNX5) signals, which were determined using ImageJ (https://imagej.nih.gov/ij/) with the JACoP plugin (Bolte and Cordelières, 2006). Punctate structures were automatically counted using ImageJ analyse particle tool across total of 10 cells from two biological replicates. To quantify the effect of PI3K inhibitors on SNX recruitment (Figure 1—figure supplement 2), Z-stacks were captured with a Zeiss 710 confocal laser scanning microscope using a 40x objective. Maximum projections were generated with FIJI (https://fiji.sc/) and Pearson’s correlation coefficients for individual cells determined using the FIJI ‘Coloc 2’ function with Costes threshold regression and 100 Costes randomisations. Co-localization analyses were conducted on two independent experiments from five images per condition each containing at least 20 cells (>100 cells analysed per condition).
|
study
| 100.0 |
HeLa cells were transfected with pcDNA3.1-N-eGFP plasmid constructs overnight at 70% confluence and the cells were lysed using lysis buffer (H2O, 50 mM HEPES, 150 mM NaCl, 1% Triton-X100, 10 mM Na4P2O7, 30 mM NaF, 2 mM Na3VO4, 10 mM EDTA, 0.5 mM AEBSF and protease inhibitor cocktail). Cell lysates were incubated with GFP nano-trap agarose beads (Protein Expression Facility, UQ) after preclear using protein G-agarose beads (Invitrogen). Protein complexes attached to the beads were detached by boiling for 5 min with 5x denaturing and reducing buffer (0.625 M Tris pH 6.8, 50% glycerol, 10% SDS, 0.25% Bromophenol blue and 500 mM DTT). Denatured and reduced proteins were separated by molecular mass using SDS-PAGE. Proteins were transferred onto PVDF-FL membrane (Immobilon) and were detected by immunoblotting with polyclonal anti-GFP and monoclonal SNX1 antibodies, and near-infrared fluorescent dyes (LI-COR). Immunolabelled proteins were visualised using LI-COR Odyssey imaging system.
|
study
| 99.94 |
Human SNX5 and SNX1 sequences were submitted to the PHYRE2 server for automated homology-based model building (Kelley et al., 2015). For both proteins the top scoring modelling template was the crystal structure of the SNX9 PX-BAR domains (PDB ID 2RAJ) (Pylypenko et al., 2007) with Confidence Scores of 100% (and sequence identities of 19% and 16% respectively). The PX domain of the SNX5 model generated using this structural template was missing the extended α-helical insert, so to complete the model the SNX5 PX domain-IncE complex was substituted and a dimer of SNX5 and SNX1 PX-BAR domains generated by overlaying with the SNX9 dimer in the PtdIns3P-bound state (PDB ID 2RAK) (Pylypenko et al., 2007). The resulting model was subjected to simple energy minimisation in PHENIX (Adams et al., 2011). Conservation of surface residues was computed using the CONSURF server (Ashkenazy et al., 2016).
|
study
| 100.0 |
In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.
|
other
| 99.94 |
Thank you for submitting your article "Structural basis for the hijacking of endosomal sorting nexin proteins by Chlamydia trachomatis" for consideration by eLife. You will be pleased to learn that your article has been favorably reviewed by three peer reviewers, one of whom, Suzanna Pfeffer, is a member of our Board of Reviewing Editors, and the evaluation has been overseen by Vivek Malhotra as the Senior Editor. The other following individuals involved in review of your submission have agreed to reveal their identity: Oliver Daumke and Volker Haucke.
|
other
| 99.94 |
Using quantitative proteomics, Heuer and colleagues previously found that SNX 5 and certain other SNX proteins are enriched on Chlamydia inclusions; in particular, SNX5 controls the C. trachomatis infection and retrograde trafficking is essential for infectious progeny formation. Engel and colleagues reported similar findings and more specifically, that the C-terminal portion of IncE binds to sorting nexins (SNXs) 5/6 of the retromer. Both reports showed that depletion of retromer components enhances progeny production, indicating that Chlamydia may hijack retromer to avoid degradative trafficking.
|
study
| 99.94 |
This manuscript reports the crystal structure of IncE C-terminal residues in complex with the PX domain of SNX5; in addition, it presents a protein binding interface for a PX domain in a subfamily of PX domain proteins that does not contain a functional phosphoinositide binding domain. Comparable binding affinity is seen for SNX 5, 6 and 32 PX domains but not that of SNX1. The story is compact and of high quality; it is an interesting variation on a theme of Chlamydial hijacking and the reviewers support presentation in eLife because it highlights an interesting biological interaction of medical importance: the elucidation of the molecular basis for hijacking of endosomal SNX5/6 and possibly 32 by bacterial IncE is important and of interest to the readers of eLife. Overall, the data support the main conclusions of the paper, however a few aspects should be strengthened by a few straightforward, additional experiments.
|
review
| 99.7 |
1) The authors provide qualitative data (e.g. Figure 4) from overexpression experiments in Chlamydia infected HeLa cells as an assay for IncE-mediated GFP-SNX5 recruitment. The staining in Figure 4 is hard to see and quantitative image analysis from multiple experiments will greatly strengthen the present story. The presence of endogenous endosomal SNX proteins that may interfere with recruitment and result in additional effects (e.g. as they may heterodimerize) that make the results difficult to interpret. Thus it would be worth testing IncE-mediated recruitment of SNX5 to bacterial inclusions using quantitative image analysis in HeLa cells depleted of endogenous SNX5/6/32 that express wt or mutant versions of SNX5 to verify the conclusions from transient overexpression and qualitative imaging. Moreover, do the mutations in the IncE binding interface equally hamper recruitment of SNX6 and SNX32, similar to what is seen for SNX5? Are other endogenous SNX proteins co-recruited to the inclusion in this case?
|
study
| 99.94 |
2) Figure 3 would benefit from rearrangement and clearer labels. The authors may consider splitting panel C into three separate panels that are the easier to refer to in the main text. Moreover, critical amino acids delineating the SNX5 interface for IncE binding should be highlighted: Yellow labels of amino acids are difficult to see. Moreover, a different view of the interface from another angle may help to bring the message across. In 3C, maybe remove the non-relevant background and shades. The orientation of the magnifications relative to Figure 3A is not clear; for better orientation, indicate the deduced membrane binding site of the PX domain.
|
other
| 99.56 |
3) Regarding the role of PI3P in SNX5 recruitment: The text in the Results section refers to Figure 1—figure supplement 2, which is missing from the PDF. As for the data shown in Figure 4 please re-investigate the role of endosomal PI3P by quantitative image analysis and application of available selective Vps34 inhibitors (e.g. Vps34-IN1). Finally, split and merged channels as well as blow-ups should be shown in the actual figure to bring across this important point.
|
other
| 99.06 |
The fusion protein should be more precisely described in the Results and Materials and Methods. How long is the linker, is the connection between C-terminus of PX domain and N-terminus of IncE long enough to account for the connection? Is the linker still intact upon crystallization? Are residues of the linker resolved in the crystal structure? Include the linker as dotted line in Figure 3A.
|
other
| 99.6 |
Thank you for resubmitting your work entitled "Structural basis for the hijacking of endosomal sorting nexin proteins by Chlamydia trachomatis" for further consideration at eLife. Your revised article has been favorably evaluated by Vivek Malhotra (Senior editor) and three reviewers, one of whom is a member of our Board of Reviewing Editors. Although eLife tries very hard not to ask for extra, unnecessary experiments, the reviewers did not feel that you had adequately address their concerns about the presentation and quantification of the images as described below. We would like to be able to publish this story when you are able to address these issues satisfactorily and will welcome one last round of revision before we reach a binding decision.
|
other
| 99.94 |
Unfortunately, the authors have really not adequately improved the quality or the quantification of the micrographs. I strongly suggest that the micrographs be shown as individual figures with larger images, and we need to know how many cells were counted and how the analyses were carried out before we can publish the story. I hate to ask for more work but the images are an important part of the overall proposal.
|
other
| 99.94 |
I have to say that I remain unsatisfied with the way the authors have addressed the questions regarding the binding properties and cellular localization of mutant SNX5. All they have done is adding some Mander's colocalization coefficient, but it is unclear from how many cells and how many independent experiments these are derived. More importantly, I would still like to see formal proof that the F136A mutation does not impair SNX5-SNX1 (or 2) heterodimer formation in vitro and its localization at endosomes in mammalian cells assayed quantitatively. This in my view is required to support the corresponding statement in the text. Also, some better high mag colocalization images illustrating SNX5 WT and mutant targeting to endosomes would help to convince readers of the paper.
|
other
| 99.3 |
Thank you for resubmitting your work entitled "Structural basis for the hijacking of endosomal sorting nexin proteins by Chlamydia trachomatis" for further consideration at eLife. Your revised article has been favorably evaluated by Vivek Malhotra (Senior editor), a Reviewing editor, and an additional reviewer. You will be pleased to learn that the reviewers found the revised manuscript to be much improved, and if you are able to make the following minor corrections, they have recommended in favor of publication in eLife.
|
other
| 99.94 |
It appears that the statistical analysis of protein co-localization in Figure 1—figure supplement 2 and Figure 6—figure supplement 1 is based on the number of cells analyzed: 10 cells per condition – a fairly low number – which the authors say to derive from 2 independent experiments. According to convention in experimental biology individual cells from a cell line cannot be considered n for statistical analysis. Hence, n in fact equals "2" in this case, which does not allow for statistical testing by either parametric or non-parametric tests. However, as the phenotypes are clear-cut and in the interest of timely publication we recommend simply eliminating the statistical test from the figure, legend, and the text and simply leave the bar diagrams including error bars as a means of quantitation.
|
other
| 96.7 |
A last minor point: The heading of Figure 5 should read "Mutations […] formation in vitro" (e.g. delete "and in cells") as no cell data are contained in the respective figure. Provided these small changes we recommend acceptance and congratulate the authors on this nice piece of work.
|
other
| 99.94 |
This manuscript reports the crystal structure of IncE C-terminal residues in complex with the PX domain of SNX5; in addition, it presents a protein binding interface for a PX domain in a subfamily of PX domain proteins that does not contain a functional phosphoinositide binding domain. Comparable binding affinity is seen for SNX 5, 6 and 32 PX domains but not that of SNX1. The story is compact and of high quality; it is an interesting variation on a theme of Chlamydial hijacking and the reviewers support presentation in eLife because it highlights an interesting biological interaction of medical importance: the elucidation of the molecular basis for hijacking of endosomal SNX5/6 and possibly 32 by bacterial IncE is important and of interest to the readers of eLife. Overall, the data support the main conclusions of the paper, however a few aspects should be strengthened by a few straightforward, additional experiments.
|
review
| 99.7 |
1) The authors provide qualitative data (e.g. Figure 4) from overexpression experiments in Chlamydia infected HeLa cells as an assay for IncE-mediated GFP-SNX5 recruitment. The staining in Figure 4 is hard to see and quantitative image analysis from multiple experiments will greatly strengthen the present story.
|
other
| 96.56 |
We have provided Mander’s coefficients to measure the relative distribution of the GFP-SNX5 and mutant to the inclusion membrane as defined by mCherry-Rab25. This data has been added to the manuscript. With respect to “quantification” of the changes that occurs in the localization of IncE in Figure 4D and Figure 4—figure supplement 1B we do not consider any of the correlative methods appropriate for the following reasons. Firstly, the correlation of the IF signal for EEA1 and IncE is relatively low. They tend to be proximal to each other presumably as they are located on distinct subdomains of same endosomes (EEA1 – body; IncE/SNX5 – tubular domains). Secondly, the dramatic change of the mutant proteins to the cytoplasm results in a low level of correlation between endosome-associated proteins and the surrounding cytoplasm. We did calculate the number of punctate structures/per cell positive for GFP-IncE in an attempt to quantify any change in endosome association. For example, 29.556 +/- 4.558 punctate structures per cell were observed for IncE 91-132 while 0 +/- 0 for IncE 91-132 (F116D). However, as the analysis of the mutant proteins showed no punctate structures we felt documentation of this obvious difference provided little additional insight.
|
study
| 100.0 |
The presence of endogenous endosomal SNX proteins that may interfere with recruitment and result in additional effects (e.g. as they may heterodimerize) that make the results difficult to interpret. Thus it would be worth testing IncE-mediated recruitment of SNX5 to bacterial inclusions using quantitative image analysis in HeLa cells depleted of endogenous SNX5/6/32 that express wt or mutant versions of SNX5 to verify the conclusions from transient overexpression and qualitative imaging.
|
study
| 100.0 |
We requested some further clarification on this referee query from the editor, who stated “The reason the reviewer suggested this is that the mutation may affect dimerization and that's why the mutant SNX5 is not recruited to IncE inclusions. If the authors have other ways of addressing this point and are able to provide more quantitative data regarding the recruitment behavior of mutant SNX5 in cells this is fine.”
|
other
| 99.9 |
Our interpretation then is that the referees are concerned the mutant SNX5 (F136A) may also impact the folding and normal heterodimerisation of the SNX5 protein with either SNX1 or SNX2 Note that SNX5 does not homodimerise with itself, nor heterodimerise with SNX6 and SNX32 is not expressed endogenously in HeLa cells. We believe this concern is addressed by the fact that the SNX5 (F136A) mutant is recruited normally to endosomes (Figure 4C). As this will only occur in a heterodimeric complex with either SNX1 or SNX2 we therefore conclude that the mutant is properly folded and otherwise functional.
|
study
| 91.6 |
Because we are not performing a rescue experiment to determine if mutation results in recovery of a ‘normal’ SNX5 function, we believe that performing the C. trachomatis inclusion recruitment experiment in a knockout background would not be informative. Here we are simply assessing if the binding deficient mutants are capable of being recruited to the chlamydial inclusion (SNX5) or endosome (GFP-IncE) through reciprocal interactions. As they are clearly not recruited (and see additional quantitation in Figure 4D), it is not clear what additional information would be gained by also knocking out the endogenous proteins
|
study
| 100.0 |
To determine if mutation to the IncE binding interface also prevents interaction with SNX6 or SNX32 we performed additional in vitro experiments. Two new pieces of data clearly show that the IncE binding mechanism is identical in the three related SNX proteins. In new ITC data we find that mutation of the key Phe116 side chain in the IncE peptide blocks binding to both the SNX6 and SNX32 PX domains (new Figure 4 —figure supplement 1A), similarly to the interaction with SNX5 (Figure 4A). In addition, in unpublished results we recently determined a crystal structure of the SNX32 PX domain in complex with the peptide from IncE (see image showing an overlay of SNX32 and SNX5 complexes). In this case the structure was solved with a co-complex including a synthetic peptide rather than a fusion protein as done for SNX5 (resolution, 2.3 Å; R/Rfree 0.19/0.23). We have not included this data in the manuscript, but it clearly confirms for the referees that the binding site is essentially identical in both proteins. In summary the IncE binding interface is highly conserved and we are certain that mutation of the strictly conserved Phe136 side- chain in all three SNX proteins would perturb IncE binding and inclusion recruitment.
|
study
| 100.0 |
Within cells we did not provide any experimental data that expression of the fragment of IncE “hampered recruitment” of SNX proteins to the bacterial inclusion. In fact, expression of these fragments does not have any impact on recruitment of endogenous SNX5 to the inclusion as the full-length membrane anchored bacterially synthesized IncE is still present on the outer membrane of the inclusion.
|
study
| 100.0 |
2) Figure 3 would benefit from rearrangement and clearer labels. The authors may consider splitting panel C into three separate panels that are the easier to refer to in the main text. Moreover, critical amino acids delineating the SNX5 interface for IncE binding should be highlighted: Yellow labels of amino acids are difficult to see. Moreover, a different view of the interface from another angle may help to bring the message across. In 3C, maybe remove the non-relevant background and shades. The orientation of the magnifications relative to Figure 3A is not clear; for better orientation, indicate the deduced membrane binding site of the PX domain.
|
other
| 99.56 |
This figure has now been adjusted to improve clarity as requested. The three panels have been separated into Figure 3C, 3D and 3E and referred to separately in the text and figure legend. The background shading in these close up images has been reduced and the yellow text has been changed to orange to make it easier to see. The two side chains critical for binding in SNX5 (Phe136) and IncE (Phe116) have been highlighted for the reader. We have decided not to indicate where the putative membrane-binding site is located in these images, as we feel this is better addressed in Figure 5 and would be confusing to include in this figure.
|
other
| 99.9 |
3) Regarding the role of PI3P in SNX5 recruitment: The text in the Results section refers to Figure 1—figure supplement 2, which is missing from the PDF. As for the data shown in Figure 4 please re-investigate the role of endosomal PI3P by quantitative image analysis and application of available selective Vps34 inhibitors (e.g. Vps34-IN1). Finally, split and merged channels as well as blow-ups should be shown in the actual figure to bring across this important point.
|
other
| 99.06 |
Figure 1—figure supplement 2 has been renamed as Video 1 to avoid confusion. It is not a PDF but an attached video showing the disruption of endosomal recruitment of GFP-SNX5 following wortmannin treatment in HeLa cells, while recruitment to the inclusion membrane is not affected. We have added an additional sentence to the legend of Figure 1—figure supplement 1 in order to clarify this. The figure panels have been split into separate channels with merged images shown alongside.
|
other
| 99.9 |
In the current study we have blocked formation of all 3-phosphoinositide species using the pan-specific PI3-kinase inhibitor wortmannin. As C. trachomatis inclusion recruitment of SNX- BAR proteins is not perturbed this indicates that the inclusion recruitment of SNX5 is likely driven by a protein-protein interaction (later shown to be IncE by SNX5 mutagenesis), rather than via 3- phosphoinositides including both the early and late-endosome enriched PtdIns3P and PtdIns(3,5)P2. We altered the text to emphasise wortmannin’s broad inhibitory effect to avoid confusion: “Interestingly, when infected cells are treated with wortmannin, a pan-specific inhibitor of phosphoinositide-3-kinase (PI3K) activity, we see a loss of the SNX proteins from punctate endosomes, but not from the inclusion membrane”. The reviewers suggest that we perform additional experiments using a specific inhibitor such as the Class III PI3K inhibitor Vps34-IN1, which will block specific formation of PtdIns3P. As wortmannin has no effect on inclusion recruitment, Vps34-IN1 is unlikely to provide additional insights.
|
study
| 100.0 |
The fusion protein should be more precisely described in the Results and Materials and methods. How long is the linker, is the connection between C-terminus of PX domain and N-terminus of IncE long enough to account for the connection? Is the linker still intact upon crystallization? Are residues of the linker resolved in the crystal structure? Include the linker as dotted line in Figure 3A.
|
other
| 99.6 |
We have included additional panels in Figure 3—figure supplement 1 that show the precise fusion protein sequence used for crystallization, as well as the highlighting the distances between the SNX5 C-terminus and the IncE N-terminus visible in our crystal structures. In each structure there are approximately 3-4 residues that are disordered and are not visible in the electron density, but the distances between the SNX5 C-termini and IncE N-termini are consistent with the expected linker lengths.
|
study
| 100.0 |
Yes it includes the 2 complexes from the P212121 crystal form, 2 complexes from the I2 crystal form and the 4 complexes from the P32 crystal form. The close alignment of the eight separate complexes makes is a consequence of the close structural similarity. The different conformations for some side-chains of the IncE peptides in Figure 3—figure supplement 1 provide the clearest display of the eight overlaid structures.
|
study
| 99.94 |
Unfortunately, the authors have really not adequately improved the quality or the quantification of the micrographs. I strongly suggest that the micrographs be shown as individual figures with larger images, and we need to know how many cells were counted and how the analyses were carried out before we can publish the story. I hate to ask for more work but the images are an important part of the overall proposal.
|
other
| 99.94 |
As suggested by the reviewer we have now adjusted all immunofluorescence images so that they are single large figures for clarity. Immunofluorescence images in Figure 6 showing the effect of SNX5 and IncE mutagenesis have been recaptured, and we have provided details of the number of cells used in these analyses and more details related to the methods of quantification.
|
other
| 99.8 |
I have to say that I remain unsatisfied with the way the authors have addressed the questions regarding the binding properties and cellular localization of mutant SNX5. All they have done is adding some Mander's colocalization coefficient, but it is unclear from how many cells and how many independent experiments these are derived.
|
other
| 99.1 |
More importantly, I would still like to see formal proof that the F136A mutation does not impair SNX5-SNX1 (or 2) heterodimer formation in vitro and its localization at endosomes in mammalian cells assayed quantitatively. This in my view is required to support the corresponding statement in the text. Also, some better high mag colocalization images illustrating SNX5 WT and mutant targeting to endosomes would help to convince readers of the paper.
|
other
| 99.9 |
As discussed above, GFP-SNX5 wild-type and mutant are both localized to endosomal puncta as normal. In additional data we have performed co-immunoprecipitation of these GFP-SNX5 constructs and find that the F136A mutation does not affect the heterodimeric interaction with endogenous SNX1 as we predicted (Figure 6—figure supplement 1B). Overall we believe the data is conclusive that the F136A mutation has a specific affect on IncE interaction and inclusion membrane recruitment without perturbing other functions or folding of the SNX5 protein.
|
study
| 100.0 |
We have now further addressed the original comment by Reviewer 2 by both using the specific Vps34 PI3K inhibitor Vps34-IN1, as well as quantification of the loss of endosomal localization following depletion of 3-phosphoinositides. Pearson’s correlation coefficients have been calculated for the endosomal colocalization of SNX1, SNX2 and SNX5 with endogenous endosomal markers. This data has been added to Figure 2—figure supplement 2. The text has been modified in the results as follows: “Interestingly, when infected cells are treated with wortmannin, a pan-specific inhibitor of phosphoinositide-3-kinase (PI3K) activity, we see a loss of the SNX proteins from punctate endosomes, but not from the inclusion membrane (Figure 1—figure supplement 1; Video 1). A similar result is seen for specific inhibition of PtdIns3P production by Vps34 using Vps34-IN1.”
|
study
| 46.38 |
It appears that the statistical analysis of protein co-localization in Figure 1—figure supplement 2 and Figure 6—figure supplement 1 is based on the number of cells analyzed:10 cells per condition – a fairly low number – which the authors say to derive from 2 independent experiments. According to convention in experimental biology individual cells from a cell line cannot be considered n for statistical analysis. Hence, n in fact equals "2" in this case, which does not allow for statistical testing by either parametric or non-parametric tests. However, as the phenotypes are clear-cut and in the interest of timely publication we recommend simply eliminating the statistical test from the figure, legend, and the text and simply leave the bar diagrams including error bars as a means of quantitation.
|
other
| 96.7 |
A last minor point: The heading of Figure 5 should read "Mutations […] formation in vitro" (e.g. delete "and in cells") as no cell data are contained in the respective figure. Provided these small changes we recommend acceptance and congratulate the authors on this nice piece of work.
|
other
| 99.94 |
Community participation in health research serves as a catalyst for the development of best practices, systemic changes in healthcare delivery, and improved health outcomes [2, 28, 32]. Despite major advances in medicine and public health due to research , diverse representation in research participation remains a challenge. Researchers struggle to access, engage, recruit, and retain underrepresented populations such as racial and ethnic minorities, those from socio-economically disadvantaged backgrounds, and/or individuals with disabilities [3, 6, 15, 29]. From the research perspective, this limits understanding of the effectiveness of a given treatment across populations, discovery of new drugs and devices, improved healthcare delivery, and knowledge of behavioral and physical science [1, 3]. From the patient perspective, there is limited understanding of underrepresented population needs, and could be a major component of the observed health and healthcare disparities. Studies have suggested that their absence in research exacerbates their vulnerability to poor health outcomes and reduced access to healthcare services [1, 3]. Hence, increasing diversity in research via engaging underrepresented populations is important for reducing the unequal burden of disparities in health and healthcare .
|
review
| 99.75 |
There is a growing awareness by researchers of the critical need to engage underrepresented populations in research , resulting in studies to explore this phenomenon. Past findings suggest that underrepresented individuals are willing to participate in research [14, 23, 24], and have expressed positive views about research participation being an opportunity or even a right . However, barriers reduce researchers’ ability to engage with these groups. These individuals are often not in an environment where research recruitment occurs , and/or they lack awareness of research opportunities. Underrepresented groups sometimes feel that research is not relevant to them or their families . Limited knowledge about the nature of research is another barrier. For example, these community members often misunderstand the purpose of research and research procedures; perceive that clinical trials will not benefit them personally; feel frustrated due to poor dissemination of research findings to the community; and feel “left out” of the research process . As a result, a new approach is warranted to enhance research participation rates.
|
other
| 71.1 |
Community engagement has been identified as an effective, evidence-based mechanism to improve interest and trust in research . Community engaged research (CEnR) principles such as community member involvement in the research process, seeking input from community advisory boards, and peer-concordant, community health worker led study recruitment have shown promise to enhance clinical trial recruitment and retention [10, 13, 19, 22]. However, opportunities for underrepresented populations to engage in the entirety of the research process (i.e., involvement in early phases such as setting research agendas, or in advisory capacities) are limited. The goal of this study was to use community engaged research principles to identify research priorities and concerns of underrepresented populations. We believe our findings can be used to inform clinical research areas and study design to increase participation of underrepresented populations in health research which may, in turn, advance the understanding of health disparity origins.
|
study
| 99.94 |
One of the goals of the Meharry-Vanderbilt Community Engaged Research Core (CERC) is to build academic and community capacity to conduct patient and community engaged research. In order to promote this goal, a collaborative partnership between CERC academic partners, including the Vanderbilt Institute for Clinical and Translational Research (VICTR) and the Meharry Clinical and Translational Research Center (MeTRC), and a community partner, the Neighborhoods Resource Center (NRC) was formed. VICTR and MeTRC were developed with Clinical and Translational Science awards to Vanderbilt University Medical Center from NCATS and to Meharry from NIMHD, respectively. NRC is a community-based non-profit organization that works with residents and neighborhood associations to build stronger neighborhoods, particularly in low-income and marginalized communities. For this study, the NRC was engaged in all phases of this research, from conceptualization to dissemination. Members of the academic-community partnership engaged in several meetings prior to study implementation to plan study logistics. All study procedures were reviewed and approved by the Vanderbilt University Medical Center Institutional Review Board.
|
study
| 84.0 |
Underrepresented research populations residing in Nashville/Davidson County were sought as participants in this study, where individuals were considered underrepresented if they belonged to a group that does not typically participate in research due to cultural or socioeconomic barriers, or issues related to a physical/cognitive impairment . These populations were chosen based on a review of the literature that demonstrated their lack of participation in research [3, 6, 15, 29].
|
study
| 99.94 |
Participants were sought in specific Nashville urban core neighborhoods densely populated with underrepresented populations where NRC has community relationships and through NRC partner community organizations whose members are those likely to be underrepresented in research. For the data collection, 10 groups of hard-to-reach community members were convened, including low socioeconomic status African American and Spanish/English-speaking individuals, Spanish speaking individuals from a faith-based organization, individuals from economically disadvantaged (i.e., uninsured, HUD housing) backgrounds, and individuals with hearing impairments. Individuals with hearing impairments were not included in the original target population. We added this group of community members when we became aware of this population at CERC’s community research day, an interactive networking session between potential academic and research partners.
|
study
| 97.7 |
A key component of CEnR is engaging community members in prioritizing research topics . The academic-community partnership identified Community Listening Sessions (CLS) as the most appropriate data collection method for this study. Eleven CLSs with a total of 117 individuals were conducted in urban core neighborhoods highly populated with underrepresented populations. A CLS is a qualitative research method where a group of people of diverse backgrounds and perspectives express their views on an important issue. These sessions have shown success because they are led by a well-known, trained community leader and are less structured than focus groups, allowing individuals to express their views on an issue in a more wide-ranging manner ; focus groups are led by trained facilitators and involve a focused discussion topic [11, 30].
|
study
| 98.56 |
A purposive sampling method was used to recruit participants. Each participant was screened for inclusion criteria (i.e., adults, targeted neighborhood resident or priority health issues, ethnicity, and ages 18 and over). All eligible participants resided in the targeted neighborhood except for deaf and hard of hearing participants, the Latino participants, and the African-American male participants. Recruitment for the deaf and hard of hearing group was conducted through Bridges, an organization that serves deaf and hard of hearing adults and children to ensure they reach their potential . Recruitment strategies included flyers as well as scripted phone and e-mail correspondence to NRC networks (e.g., church and neighborhood associations) to identify potential participants. The Latino participants were recruited using a Spanish-translated flyer, which was distributed in high density areas.
|
study
| 100.0 |
A facilitator’s guide, developed jointly by CERC and NRC, focused on the following topics: priority health concerns for research, perceptions towards research, methods or sources for receiving health information and barriers to research. Questions included: (1) What are the most important health concerns for you and your family? (2) What health concerns would you like to see being researched or addressed by the health systems? (3) How do you generally receive health information? (4) What is the most effective way for you, your family and community to receive health information? (5) Have you ever participated in research? If yes, was it a positive or negative experience? If you are comfortable, please elaborate. If no, have you ever been asked? Would you participate if asked? (6) In general, do you think research is important to improving health? (7) What concerns do you have about participating in research? (8) What are some of the barriers to participating in research?
|
other
| 99.9 |
Three NRC organizers were trained by CERC staff in reflective techniques that encouraged discussion and enhanced each participant’s sense of having been heard . All facilitators attended a 1.5 h training offered by Bridges to learn how to work with an interpreter and basic cultural competencies when working with the deaf and hard of hearing community. For the Latino CLS, a bicultural, bilingual interpreter/translator was recruited to assist in facilitating the session.
|
other
| 99.9 |
Nine community listening sessions were held in a community setting such as a familiar neighborhood community center or family resource center. The deaf and hard of hearing listening session was held at Bridges and the Latino listening session was held at a Hispanic church. Written informed consent was obtained from all participants. Community NRC organizers and CERC staff facilitated all sessions. Specifically, a bilingual trained facilitator and a CERC staff member conducted the Latino CLS in Spanish. The CLS for the hearing impaired group was conducted by a Bridges staff member and a CERC staff member using American Sign Language (ASL). The session was conducted by two interpreters, a trained facilitator, and CERC staff. Community Listening Sessions lasted approximately 1.5 h each. The sessions were audio-recorded and notes were scribed on a flip chart allowing participants to ensure an accurate representation of their comments. All sessions were commercially transcribed and used verbatim for analysis.
|
other
| 99.5 |
An inductive, qualitative content analysis approach was used to identify emerging themes . This is a bottom-up approach that inductively builds upon the data as opposed to establishing a deductive coding scheme in advance of the analysis. Two researchers performed coding consensus using a line-by-line open coding technique . Key concepts, ideas, beliefs, and/or events were coded with one or more codes. Codes were added or modified as necessary as new meanings emerged . To assess coding consistency, codes and their assignment to text were checked and rechecked. If any discrepancies arose, researchers discussed the codes until consensus was reached. Using a constant comparison method , codes were compared and queried to identify themes that emerged from the groups.
|
study
| 99.94 |
Eleven listening sessions were conducted with 117 participants from urban core neighborhoods highly populated with underrepresented populations. While the majority of the participants were African Americans (66 %), the sample was racially and ethnically diverse with Caucasians (15 %) and Hispanics (14 %). Seventy-one percent of participants were female. Many (41 %) of the participants were over the age of 50, followed by the age ranges of <30 (21 %), 30–39 (19 %), and 40–49 (16 %).
|
study
| 99.94 |
Four themes emerged from community member responses: (1) Uncertainties of underrepresented populations regarding research participation; (2) Ineffective communication about research opportunities and research findings; (3) High priorities for underrepresented populations in research and healthcare; and (4) Research teams need training in cultural competence and humility. These themes highlight barriers to research, illustrate research priorities and concerns, and provide methods to enhance engagement in research from the perspective of underrepresented populations (See Table 1 of how these themes compared across groups.)
|
other
| 82.25 |
Table 1Themes indicated across community listening sessionsGroup N = 117ThemesNumber of themes within groupUncertainties of underrepresented populations regarding research participationIneffective communication about research opportunities and research findingsResearch on primary care and prevention are priorities for underrepresented populations in researchResearch teams need training in communication and cultural humilityAfrican American Menn = 7XXX3Deaf and hard of hearingn = 10XXX3Low socio-economic status African Americansn = 11XXX3Low-income African American parentsn = 12XXX3Low socio-economic status Caucasiansn = 7XXXX4Uninsured Caucasians/African Americansn = 9XXX3Low socio-economic status college studentsn = 9XXX3Older Spanish speaking Hispanic Americansn = 16XXX3Low socio-economic status Caucasians/African Americansn = 13XXX3Low socio-economic status Caucasians/African Americansn = 11XXXX4Residents of HUD-housing (low-income housing)n = 12XXXX4Number of themes across groups911115
|
other
| 99.9 |
Community members expressed their insecurities as it relates to their research participation. Lack of trust was commonly cited among community members. In addition to lack of trust, community members expressed their concerns for safety and how it influences their participation or lack thereof in research. Each of these will be discussed in further detail below.
|
other
| 99.94 |
Lack of trust stemmed from past personal and familial experiences with medical research. These experiences resulted in community members perceiving both negative physical and emotional implications towards research. For members who had previously participated in research, there was a sense that researchers were not forthcoming about their research purposes, and in many cases, refrained from providing evidence of progress throughout the research study. Community members also reported a lack of knowledge regarding medications taken or administered as a part of research procedures. Failure to be provided with sufficient explanations of the medications’ purpose and side-effects was commonly reported by community members.
|
other
| 85.0 |
In addition to expressing lack of trust, community members conveyed concerns for safety. Concerns about safety were often generated from exposure to media outlets such as television and internet stories. The stories, both fictional and non-fictional, contributed to feelings of skepticism toward the true intentions of researchers and research objectives. Examples of these concerns included fear of being treated as “guinea pigs,” being provided with placebo medications, and loss of life resulting from research participation.
|
other
| 99.9 |
Consequences of ineffective communication result in limited reach of research opportunities to underrepresented populations. It also contributes to lack of research dissemination to these groups. The continuum of communication issues include the sources of communication to the methods (i.e., channels) of communication. The details of these issues are explained below.
|
other
| 99.7 |
A key aspect to engaging community members in research is connecting the community to research opportunities. Ineffective communication was cited as a substantial barrier to connecting underrepresented community members to research. A major contributing factor was lack of awareness of research studies and of the researchers who conduct the studies. Community members expressed that research information, such as project outcomes, is not adequately being disseminated throughout the community. In instances where research information reaches the community, it is not easily understood and is not user friendly for some groups. Additionally, those community members who felt their efforts were futile in identifying research opportunities cited that the information on research websites was inadequate, out of date, and/or too complex. Furthermore, they wanted the research outcomes to be circulated throughout the community to assist its members in making informed healthcare decisions.
|
other
| 99.9 |
Community members identified current and preferred methods for communicating research information and opportunities. Current methods of communication included websites, physicians, family, and friends; however, these methods were characterized as ineffective. Preferred methods of communication identified by community members consisted of community meetings, newsletters, and social media (e.g. YouTube, Facebook).
|
other
| 99.9 |
Research priorities among community members involved research on prevention, need for patient-centered care, and patient-centered outcomes in research, and improved patient-provider communication. Participants reflected on personal experiences and perceptions towards research participation to express their priorities. Each of these areas is discussed in further detail below.
|
other
| 99.9 |
Primary prevention was a theme that deeply resonated within the listening sessions. Particularly, community members provided their perspective on the need for research aimed at preventing chronic diseases. Diseases identified as priorities include heart disease, diabetes, cancer, and Alzheimer’s disease. Concern for these specific illnesses is a result of the experiences and even loss of many family and community members who suffered with these illnesses. There were also suggestions that exercise programs, organic foods, and natural supplements should be incorporated into the research agenda in an effort to prevent chronic disease. The Community further emphasized the need to identify and/or improve current treatments for these diseases to improve health-related quality of life.
|
other
| 99.7 |
In addition to establishing research priorities, participants indicated that research should focus on patient-centered care. Community members highlighted the need for patient-centeredness to improve the quality of healthcare. Community member discussions further emphasized their desire to be involved in the research process so that their needs and preferences would be part of improving healthcare delivery and outcomes. Lastly, many members emphasized that researchers and providers are not responsive to their medical needs regarding information delivery preferences and treatment options.
|
other
| 99.8 |
Community members expressed a profound need to improve patient-provider communication. Poor communication in medical settings was a common experience among community members in each CLS. Several participants suggested that their lack of participation in research was linked to ineffective communication with medical providers and/or researchers. Examples included inadequate information, insufficient explanations of blood test results and treatment options, insufficient time with their providers, and methods of follow-up.
|
other
| 99.9 |
The need to improve cultural competency among healthcare professionals and clinical research staff was a primary concern of community members. This perspective was evident in community member personal experiences with medical and research staff. Experiences indicating the need for cultural competence include poor treatment by research staff, cognitive bias among researchers, and lack of willingness to use devices supporting communicate with patients with disabilities. For some community members, these experiences were at their initial research encounter, subsequently tarnishing perceptions of research participation (see Table 2 for examples of participants’ quotes for each theme).
|
other
| 99.5 |
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