Fig. S1. Local and transient accumulation of poly-ubiquitinated proteins at Chlamydia entry sites. Cells were infected or not with unlabelled C. caviae GPIC, following the procedure described in Fig. 1. Ubiquitinated proteins were stained with FK1 antibodies (Enzo Life Sciences), which stains poly-ubiquitinated proteins (middle column), followed with Alexa647-conjugated anti-IgM antibodies. F-actin was stained with Alexa4880-conjugated phalloidin (left column). The inset shows a twofold magnification of an entry site. Bar = 5 μM.


Fig. S2. Sequence alignment of the N-terminal portion of CPn0483 and its orthologues. The amino acid sequences of C. pneumoniae CPn0483, C. abortus CAB255, C. caviae CCA00261 and C. felix CF0747 were aligned using the PROMAL software. Only the first third of the proteins are shown, as no particular feature was identified in the rest of the sequence. Hydrophobic residues forming a putative transmembrane domain are highlighted in green, prolines of the proline-rich domain in red, and the OTU-like domain in yellow, with predicted catalytic residues in bold letter. A graphical representation of the similarity along the set of aligned sequences obtained by Plotcon (EMBOSS-package) is shown below.


Fig. S3. Production and purification of recombinant proteins.

A. Schematic representation of the constructs used in this study.

B. Purification of recombinant proteins used in this study.

6xHIS-tagged CPn0483 truncated of its first 192 amino acids (Δ192CPn0483) and the OTU domain region only (CPn0483-OTU) were expressed in E. coli Rosetta-Gami 2 (DE3)pLys (Novagen) and purified by affinity chromatography in a soluble form. Samples were run on SDS-PAGE and proteins stained with Coomassie blue. SF, soluble fraction; FL, flow-through; E, eluted fractions.


Fig. S4.In vitro assays using C. caviae ChlaOTU.

A. GFP-tagged proteins were expressed in HeLa cells and immunoprecipitated with anti-GFP. Immunoprecipitates were incubated with 1 μg of HAUb-Br2 for 1 h at 37°C, subjected to 10% SDS-PAGE and immunoblotted with antibodies against GFP (left panel) and against HA (right panel). Arrowhead points to HA-Ub linked to GFPΔ188CCA00261. The mutation cysteine 286 to alanine abolished the ability for GFPΔ188CCA00261 to bind the probe, strongly supporting the identification of cysteine 286 as a catalytic residue, as suggested by the alignment shown in Fig. S1.

B. GFP-tagged proteins were expressed in HeLa cells for 24 h by transfection. Whole-cell lysates were subjected to SDS-PAGE and immunoblotted with antibodies against mono- and poly-ubiquitinated proteins (P4D1). No change in the overall profile of ubiquitinated proteins was observed, indicating that ChlaOTU reacts only on specific target(s).


Table S1. Primers used for the generation of plasmids.

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