The C. trachomatis inclusion membrane proteins InaC, IpaM, and CTL0480 are ubiquitinated in the absence of Cdu1.

(A-C) Volcano plots (pairwise comparisons) of the relative abundance of human and Ct ubiquitinated peptides.(A) Mock infected HeLa cells versus HeLa cells infected with WT Ct (L2 434 Bu pBOMB) (24 hpi). (B) Mock infected HeLa cells versus HeLa cells infected with a cdu1 null strain (cdu1::GII pBOMB) (24 hpi) (C) HeLa cells infected with WT Ct (24 hpi) versus HeLa cells infected with a cdu1 null strain (24 hpi). Significance values were interpolated from 3 independent biological replicates. Ubiquitinated proteins were enriched with magnetic TUBE 1 beads (binds to polyubiquitinated proteins) and peptides identified by quantitative LC MS/MS analysis. Three Ct inclusion membrane proteins, InaC, IpaM, and CTL0480 were differentially ubiquitinated in the absence of Cdu1. (D) InaC was ubiquitinated at K104, K107, and K149, IpaM at K290, and CTL0480 at K115 in the absence of Cdu1. TMD: Transmembrane domain. Numbering corresponds to amino acids in the protein sequence of each respective inclusion membrane protein.

Cdu1 associates with InaC, IpaM, and CTL0480.

(A) Co-localization of Cdu1(magenta) with endogenous InaC (green), IpaM (green), and ectopically expressed CTL0480-Flag (green) at the Ct (L2) inclusion membrane of HeLa cells infected for 24 h. HeLa cells infected with an inaC null strain (M407), an ipaM null strain (ipaM::GII), and WT Ct (L2 434 Bu pBOMB) were used as controls for antibody specificity. DNA stained with Hoechst is shown in blue. Scale bar: 10μm. Images are representative of multiple images captured across three independent replicates. (B) Line scan profiles of fluorescent signal intensities displayed in (A) showing co-localization of fluorescence intensities for endogenous Cdu1 with endogenous InaC and IpaM, and with CTL0480-Flag along the L2 inclusion membrane. (C) Schematic of Cdu1-GFP(C) (L2) fusion (Cdu1-GFP) and Cdu1-GFP variants used in co-transfections of HEK 293 cells. GFP: Green fluorescent protein. TMD: Transmembrane domain. PRD: Proline rich domain. CD: Catalytic domain. FL: Full length. TMD-: Cdu1-GFP variant lacking TMD domain. CD-: Cdu1-GFP variant lacking CD domain. (D-G) Western blot analysis of GFP immunoprecipitates from HEK 293 cells co-transfected with mammalian plasmids expressing: Cdu1-GFP variants and (D) truncated 3XFlag(N)-InaC (D/UW-3/CX CT813, amino acids 96-264), (E) V5(N)-IpaM (L2, full length), (F) V5(N)-CTL0480 (L2, full length), and (G) V5(N)-CpoS (L2, full length). Vec1: Empty pOPINN-GFP vector. Vec2: Empty pDEST53 vector. Vec3: Empty pcDNATM3.1/nV5-DEST vector. Western blot images are representative from two independent experiments.

Cdu1 stabilizes InaC, IpaM, and CTL0480.

(A) Western blot analysis of endogenous InaC in HeLa cells infected for 24, 36, and 48 hours with Wt Ct (L2 pBOMB), cdu1 null (cdu1::GII pBOMB), and inaC null (M407) strains. Ct Slc1 and human alpha tubulin were used to determine Ct burdens and equal loading of protein extracts respectively. Western blot images are representative of 2 independent experiments. (B) Quantification of InaC abundance (InaC western blot signal from (A)) normalized to Slc1 western blot signal (from panel A) in Hela cells infectedwith a cdu1 null strain, relative to normalized InaC abundance in Hela cells infected with Wt Ct. (C) Western blot analysis of endogenous IpaM in HeLa cells infected for 24, 36, and 48 hours with Wt Ct, cdu1 null, and ipam null (ipam::GII) strains. Western blot images are representative of 2 independent experiments. (D) Quantification of normalized IpaM abundance (from (C) in Hela cells infectedwith a cdu1 null strain, relative to normalized IpaM abundance in Hela cells infected with Wt Ct. (E) Localization of CTL0480 during Ct infection of HeLa cells at 24, 36, and 48 hpi. CTL0480 signal (green) co-localizes with the inclusion membrane protein IncA (magenta) at the Ct inclusion membrane. Arrowheads highlight cdu1 null inclusions lacking CTL0480 at 36 hpi. DNA stained with Hoechst is shown in blue. Scale bar: 10μm. Quantification of MYPT1 localization at inclusion membranes can be found in Supplemental Figure 5

The acetylase activity of Cdu1 is required to stabilize Cdu1, InaC, and IpaM.

(A) Western blot analysis of endogenous InaC and Cdu1-Flag catalytic variants expressed from a plasmid (pBOMB). HeLa cells were infected for 36 hours with WT Ct (L2 434 Bu pBOMB), a cdu1 null strain (cdu1::GII pBOMB), and cdu1 null strains expressing wild type Cdu1-Flag and the Cdu1 variants C345A-Flag (catalytic inactive), I225A-Flag (DUB deficient), and K268E-Flag (Act deficient). Cdu1-Flag variants were expressed from a pBOMB shuttle plasmid. Protein lysates from HeLa cells infected with an inaC null (M407) strain were used to control for the specificity of anti-InaC antibodies. Western blot images are representative of two independent experiments. (B) Western blot analysis of endogenous IpaM and Cdu1-FLAG variants in crude extracts of HeLa cells infected for 48 hours with the same strains as describe in (A). Infection of HeLa cells with ipaM::GII was used to test for the specificity of the anti-IpaM antibody. Western blot images are representative of two independent experiments. (C) Western blot analysis of Cdu1C345A-Flag (catalytic inactive) and Cdu1K268E-Flag (Act deficient) expressed in a cdu1 null strain or WT Ct (L2 434 Bu) background after infection of HeLa cells for 24 hours. Both Cdu1 variants are stabilized by Cdu1 expressed in WT Ct. (D) Western blot analysis of Cdu1-Flag and Cdu1C345A-Flag expressed in a cdu1 null strain and Cdu1C345A-Flag expressed in WT Ct (L2 434 Bu) following immunoprecipitation (anti-Flag) from HeLa cell extracts after infection for 24 hours and treatment with MG132 (25 μM, 5 hours). Western blot image is a representative blot from at least three independent experiments. (E) Western blot analysis of endogenous InaC and IpaM following immunoprecipitation of inclusion membrane enriched subcellular fractions (24hpi) with anti acetylated lysine antibodies. Western blot image is representative of two independent experiments. (F) Western blot analysis (Flag WB) of acetylated lysine immunoprecipitates generated from inclusion membrane enriched subcellular fractions (40 hpi) derived from HeLa cells infected with WT Ct strains expressing Cdu1-Flag, CTL0480-Flag, or IpaM-Flag. Western blot image is representative of two independent experiments. (G) WB of acetylated lysine immunoprecipitates of inclusion membrane enriched fractions (24 hpi) of HeLa cells infected with WT Ct and with an inaC null strain (M407) expressing InaC-Flag. Western blot image is representative of two independent experiments. (H) The initiator methionine, Lys10, Lys126, and Lys263 of Cdu1 are acetylated by 24 hpi. One tyrosine (Y) residue and multiple serine (S) and threonine (T) residues in Cdu1 are also phosphorylated during Ct infection of HeLa cells (24 hpi). Three PX(S/T)P MAPK phosphorylation consensus sequence motifs were identified in the proline rich domain of Cdu1. Modified residues were identified by quantitative LC MS/MS analysis of immunoprecipitated Cdu1-Flag across 3 independent biological replicates. TMD: Transmembrane domain. PR: Proline rich. PSTP: PX(S/T)P motifs. Ac: Acetylation. P: Phosphorylation. Numbering corresponds to amino acids in Cdu1 protein sequence.

The DUB activity of Cdu1 is not required for blocking the ubiquitination of inclusion membranes and Cdu1 is not required for protection against IFNγ mediated cellular immunity.

(A) Representative images of Ct inclusions decorated with ubiquitin during infection of HeLa cells with a cdu1::GII strain for 24 hours. Representative images of infected Hela cells used for quantification of ubiquitin decorated inclusions in (B) are shown in Supplemental Figure 6A. Antisera against the membrane protein Cap1 (green) was used to mark inclusion membranes. DNA stained with Hoechst is shown in blue. Scale bar: 10μm. (B) Quantification of ubiquitinated inclusions as shown in (A). The Ub fluorescent signal was used to determine the number of infected cells with Ub decorated inclusions. The total number of ubiquitinated inclusions was divided by the total number of inclusions analyzed (defined by Cap1 staining). 87%, 86%, and 91% of inclusions were decorated with Ub in HeLa cells infected with a cdu1 null strain and cdu1 null strains expressing Cdu1C345A (DUB-, Act-), and Cdu1K268E (Act-) variants respectively. Representative images (panel (A) and Supplemental Figure 6A) and quantification of ubiquitinated inclusions were obtained from inclusions imaged in 10 fields across 3 independent biological replicates for each strain. p values were determined by a student paired t-test. (C) Representative images of HeLa cells co-infected with cdu1::GII (cdu1Δ) and incA null (incAΔ, M923) strains at 24 hpi. IncA-deficient inclusions do not fuse with other inclusions. In co-infected cells, Cdu1 present on the inclusion membranes of incAΔ strains did not block ubiquitination events at or near the inclusion membranes of neighboring cdu1Δ strains (mCherry signal from pBOMB4-MCI plasmid). Representative images of Hela cells infected with strains quantified in (D) are shown in Supplemental Figure 6B. DNA stained with Hoechst is shown in blue. Scale bar: 10μm. (D) Quantification of cdu1Δ inclusions as shown in (C) in which ubiquitination events are observed in regions of cdu1Δ inclusion membranes that are in direct apposition to incAΔ inclusion membranes (junctions). The total number of cdu1Δ inclusions ubiquitinated at inclusion junctions was divided by the total number of inclusions analyzed (Cap1 staining). 66% and 61% of cdu1Δ inclusions were decorated with Ub at junctions in HeLa cells co-infected with incAΔ and cdu1Δ strains or with incAΔ and a cdu1Δ strain ectopically expressing Cdu1K268E (Act-) respectively. Representative images (panel (C) and Supplemental Figure 6B) and quantification of cdu1Δ inclusions ubiquitinated at junctions are derived from inclusions imaged in 6 fields across 3 independent biological replicates for each condition. p values were determined by a student paired t-test. (E) Quantification of Ct inclusion production during infection of unprimed and IFNγ-primed (100 U/mL) A549 cells at 24 hours post infection. Inclusions were quantified by high-content imaging analysis. Plot reflects inclusion counts across 9 fields of view and 3 independent biological replicates. Inclusion counts by each strain in unprimed A549 cells were set to 100%. Inclusion counts resulting from cdu1::GII and garD::GII strains were normalized to corresponding parental Ct inclusion (100%) counts in unprimed cells. P-values were calculated by 2 way ANOVA analysis. (F) Representative images of RNF213 localizing to inclusions of WT Ct, garD::GII, and cdu1::GII strains during infection of A549 cells primed with IFNγ (100 U/mL). (G) Quantification of RNF213 localizing to Ct inclusions during infection of unprimed and IFNγ-primed (100 U/mL) A549 cells at 24 hours post infection. Plot reflects inclusion counts across 6 fields and 3 independent biological replicates.

Cdu1 is required for assembly of F-actin, Golgi ministack repositioning, and MYPT1 recruitment to the inclusion.

(A) Examples of representative images of F-actin (arrowheads) (green, Alexa FluorTM Phalloidin) assembled around the Ct inclusion (magenta, anti Cdu1 and Cap1 staining) in HeLa cells infected for 40 hours. Representative images for each strain analyzed can be found in Supplemental Figure 7. (B) Quantification of Ct inclusion surrounded by F-actin normalized to the total number of inclusions analyzed during infection of Hela cells at 40 hpi. Quantification of surrounding F-actin were obtained from inclusions imaged in 6 fields across 3 independent biological replicates. p values were determined by one-way ANOVAs with a Student-Newman-Keuls post hoc test. Strains used: WT L2 (Rif-R 434 Bu, parent of M407), M407 (inaC null strain) p2TK2, M407 p2TK2-InaC, WT L2 (434 Bu) pBOMB, cdu1::GII pBOMB, cdu1::GII pBOMB-Cdu1 Flag, cdu1::GII pBOMB-Cdu1C345A Flag, cdu1::GII pBOMB-Cdu1I225A Flag, and cdu1::GII pBOMB-Cdu1K268E Flag. (C) Sample representative images of Golgi (anti GM130 staining, magenta) around Ct inclusions in HeLa cells infected for 24 hours. Representative images for each strain analyzed can be found in Supplemental Figure 8. (D) Quantification of Golgi dispersal around the Ct inclusion during infection of Hela cells for 24 hpi. The length of Golgi dispersed around each Ct inclusion imaged was measured and normalized to the perimeter length of each inclusion (% Golgi dispersion around the inclusion). Golgi dispersal around Ct inclusions was quantified from inclusions imaged in 6 fields across 3 independent biological replicates. p values were determined by a student paired t-test. Strains analyzed were the same ones as mentioned in (B). (E) Representative images of MYPT1 (magenta) at Ct inclusions (green, anti-IncA staining). Arrowheads represent cdu1 null inclusions with low MYPT1 signal. DNA stained with Hoechst is shown in blue in panels C and E. Scale bar: 10μm. (F) Quantification of MYPT1 at Ct inclusions as shown in (E). Representative images in (E) and quantification of MYPT1 recruitment in (F) were obtained from inclusions imaged in 6 fields across 3 independent replicates. p values were determined by a student paired t-test.

Cdu1, InaC, and IpaM are required for optimal extrusion of Ct inclusions from HeLa cells.

(A) Representative images of extrusions isolated from HeLa cell monolayers infected with Ct strains for 52 hours. Scale bar: 200 μm (B) Quantification of the number of extruded inclusions produced by infected HeLa cell monolayers. p values were determined by a student paired t-test. (C) Quantification of the size of extruded inclusions quantified in (B). Extruded inclusions varied in size among cells infected with wild type L2 (average :43 μm), ipaM mutants (average: 56 μm) and cdu1 mutants complemented wild type Cdu1 (average: 52 μm) and Cdu1I225A (DUB-) (average: 60 μm). p values were determined by one-way ANOVAs with a Student-Newman-Keuls post hoc test. Representative images in (A) and quantification of extrusion number in (B) and extrusion size in (C) are based on images obtained from 6 fields across 3 independent biological replicates.

A model for acetylation mediated protection of the Inc proteins InaC, IpaM, and Ctl0480 from degradation.

The cellular Ub machinery targets C. trachomatis effectors, including the Inc proteins InaC, IpaM, and CTL0480 for ubiquitination and subsequent protein degradation. C. trachomatis counters such defense mechanisms by translocating Cdu1 which protect itself and all three Inc proteins from being targeted for ubiquitination and degradation through its acetylase activity. Cdu1 protects InaC and enables the recruitment of F-actin scaffolds and Golgi ministacks to the inclusion perimeter and CTL0480 to recruit the Myosin II regulator MYPT1. All three inclusion proteins and Cdu1 promote extrusion and dissemination of C. trachomatis inclusions late in infection. Image generated with BioRender.com.

Key Resource Table

TargeTron mediated disruption of the L2 cdu1 ORF.

(A) Depiction of the cdu1 ORFs in WT L2 and cdu1::GII strains with corresponding diagnostic PCR amplicons. (B) PCR analysis of the disrupted cdu1 ORF in the cdu1::GII strain. *Amplification of cdu1 ORF in cdu1::GII strain was unsuccessful due to size of amplicon. + control: WT cdu2.

Generation of a cdu1 null strain in C. trachomatis (L2).

(A) Western blot analysis of protein lysates derived from HeLa cells infected with a Ct L2 434 Bu parental strain (L2) and an L2 cdu1::GII aadA (cdu1::GII) strain for 24 hours. Blots were probed with antibodies raised against recombinant Cdu1 (amino acids 71-401), Ct RpoB (bacterial RNA polymerase), and human α tubulin. (B) HeLa cells infected with L2 transformed with pBOMB4-MCI empty vector (L2 pBOMB) and cdu1::GII transformed with empty pBOMB4-MCI (cdu1::GII pBOMB) were fixed at 24 hpi and stained with Cdu1 antisera. Cdu1 signal is depicted in green, Ct cells expressing mCherry (encoded in pBOMB4-MCI vector) are shown in red, and DNA stained with Hoechst in blue. Scale bar: 10μm. (C) Total RNA isolated from HeLa cells infected with L2 and cdu1::GII strains for 24 hours was used to synthesize cDNAs which served as templates for PCR analysis of cdu1, cdu2, and cdu1-cdu2 bicistronic expression. RT+: Total RNA + reverse transcriptase. RT-: Total RNA without reverse transcriptase. gDNA: Genomic DNA.

Proteins co-precipitating (non ubiquitinated) with human and Ct proteins enriched by TUBE 1 pulldowns.

(A) Volcano plots (pairwise comparisons) of human proteins (non-ubiquitinated) that co-precipitate with TUBE1 bound proteins in mock infected HeLa cells and HeLa cells infected with L2 or cdu1 null strains (24 hpi). (B) Volcano plots (pairwise comparisons) of Ct TUBE1 co-precipitating proteins (non-ubiquitinated) identified in mock infected HeLa cells and HeLa cells infected with L2 or cdu1 null strains (24 hpi).

Pathway enrichment analysis of human and Ct proteins that co-precipitate (non ubiquitinated) with TUBE1 bound proteins.

(A) Metascape functional enrichment analysis of human co-precipitating proteins in mock infected HeLa cells. (B) Metascape functional enrichment analysis of human co-precipitating proteins in cdu1 null infected HeLa cells. (C) DAVID pathway enrichment analysis of Ct co-precipitating proteins enriched in infected (L2 and cdu1 null) HeLa cells.

Quantification of CTL0480 at Ct inclusion membranes.

Quantification of CTL0480 localization at Ct inclusion membranes as shown in Figure 3E. CTL0480 fluorescent signal was used to determine the percent of inclusions displaying CTL0480 at the inclusion membranes. Representative images and quantification of CTL0480 positive inclusions are derived from Ct inclusions imaged in 10 fields across 3 independent biological replicates. p values were determined by a student paired t-test.

The Acetylase activity of Cdu1 is the predominant activity of Cdu1 responsible for protecting Ct inclusions from ubiquitination.

(A) Representative images of Ct inclusions (green, Cap1) decorated with Ubiquitin (magenta, FK2 antibody) in HeLa cells infected for 24 hours. Strains used: WT Ct (L2 434 Bu), cdu1::GII pBOMB MCI, cdu1::GII pBOMB MCI-Cdu1 Flag, cdu1::GII pBOMB MCI-Cdu1C345A Flag, cdu1::GII pBOMB MCI-Cdu1I225A Flag, and cdu1::GII pBOMB MCI-Cdu1K268E Flag. (B) Representative images of Hela cells co-infected with an incA null strain and cdu1::GII strains expressing Cdu1 variants for 24 hours. Strains used: cdu1::GII pBOMB MCI, inaC null (M923), cdu1::GII pBOMB MCI-Cdu1 Flag, cdu1::GII pBOMB MCI-Cdu1C345A Flag, cdu1::GII pBOMB MCI-Cdu1I225A Flag, and cdu1::GII pBOMB MCI-Cdu1K268E Flag. DNA stained with Hoechst is shown in blue. Scale bar: 10μm. MCI= mCherry.

The DUB activity of Cdu1 is not required for assembly of F-actin around the Ct inclusion.

Representative images of F-actin (arrowheads) (green, Alexa FluorTM Phalloidin) assembled around the Ct inclusion (magenta, anti Cdu1 and Cap1 staining) in HeLa cells infected for 40 hours. Strains used: WT L2 (Rif-R 434 Bu, parent of M407), M407 (inaC null strain) p2TK2, M407 p2TK2-InaC, WT L2 (434 Bu) pBOMB, cdu1::GII pBOMB, cdu1::GII pBOMB-Cdu1 Flag, cdu1::GII pBOMB-Cdu1C345A Flag, cdu1::GII pBOMB-Cdu1I225A Flag, and cdu1::GII pBOMB-Cdu1K268E Flag. DNA stained with Hoechst is shown in blue. Scale bar: 10μm.

The DUB activity of Cdu1 is not required for Golgi ministack repositioning around the Ct inclusion.

Representative images of Golgi (anti GM130 staining, magenta) around Ct inclusions in HeLa cells infected for 24 hours. Strains used: WT L2 (Rif-R 434 Bu, parent of M407), M407 (inaC null strain) p2TK2, M407 p2TK2-InaC, WT L2 (434 Bu) pBOMB, cdu1::GII pBOMB, cdu1::GII pBOMB-Cdu1 Flag, cdu1::GII pBOMB-Cdu1C345A Flag, cdu1::GII pBOMB-Cdu1I225A Flag, and cdu1::GII pBOMB-Cdu1K268E Flag. DNA stained with Hoechst is shown in blue. Scale bar: 10μm.

The number of inclusions and the size of inclusions in cdu1 null, inaC null, ipaM null, and ctl0480 null strains are similar across each strain

(A) Representative images of inclusions in infected HeLa cell monolayers at 48 hpi. Scale bar: 200 μm (B) Quantification of the number of inclusions in infected HeLa cell monolayers. (C) Quantification of inclusion size.