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 between mock infected HeLa cells versus HeLa cells infected with L2 (24 hpi) (A), mock infected HeLa cells versus HeLa cells infected with a cdu1 null strain (cdu1::GII) (24 hpi) (B), and HeLa cells infected with L2 (24 hpi) versus HeLa cells infected with a cdu1 null strain (24 hpi) (C). All infections were performed for 24 hours. 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 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 L2 inclusion membrane during infection of HeLa cells at 24 hpi. HeLa cells infected with an inaC null strain (M407), an ipaM null strain (ipaM::GII), and L2 pBOMB were used as antibody specificity controls. 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 InaC in HeLa cells infected for 24 hours with L2, inaC null, and cdu1 null 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 3 independent experiments. (B) Western blot analysis of endogenous IpaM in HeLa cells infected with L2, cdu1 null, and ipaM null strains for 24, 36, and 48 hours. Western blot images are representative of 2 independent experiments. (C) 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 L2 inclusion membrane. Arrowheads highlight cdu1 null inclusions lacking CTL0480 at 36 hpi. DNA stained with Hoechst is shown in blue. Scale bar: 10μm. (D) Quantification of CTL0480 localization at Ct inclusion membranes as shown in (C). 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 required to stabilize Cdu1, InaC, and IpaM.

(A) Western blot analysis of endogenous InaC and Cdu1-Flag catalytic variants expressed from a plasmid. HeLa cells were infected for 36 hours with L2, a cdu1 null strain, 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). Protein lysates from HeLa cells infected with M407 (inaC null) 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 wild type L2 background after infection of HeLa cells for 24 hours. Both Cdu1 variants are stabilized by endogenous Cdu1. (D) Western blot analysis of Cdu1-Flag and Cdu1C345A-Flag expressed in a cdu1 null strain and Cdu1C345A-Flag expressed in wild type L2 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 proteins acetylated at lysines from inclusion membrane subcellular fractions derived from HeLa cells infected with WT L2 for 24 hours. Western blot image is representative of two independent experiments. (F) Western blot analysis (Flag WB) of acetylated lysine immunoprecipitates generated from inclusion membrane subcellular fractions derived from HeLa cells infected with WT L2 strains expressing Cdu1-Flag, CTL0480-Flag, and IpaM-Flag at 40 hpi. Western blot image is representative of two independent experiments. (G) Flag WB of acetylated lysine immunoprecipitates generated from inclusion membrane subcellular fractions derived from HeLa cells infected with Rif-R L2 (M407 parental strain) and with M407 (inaC null) expressing InaC-Flag at 24 hpi. Western blot image is representative of two independent experiments. (H) The initiator methionine, Lys10, Lys126, and Lys263 of Cdu1 are acetylated during Ct infection of HeLa cells (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.

(A) Loss of Cdu1’s acetylase activity results in the accumulation of Ub (magenta signal) at the periphery of the inclusion. HeLa cells were infected for 24 hours. 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 in (A) and quantification of ubiquitinated inclusions in (B) 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) HeLa cells co-infected with cdu1::GII (cdu1⊗) and incA null (incA⊗, 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 incA⊗ strains did not block ubiquitination events at or near the inclusion membranes of neighboring cdu1⊗ strains (mCherry signal from pBOMB4-MCI plasmid). DNA stained with Hoechst is shown in blue. Scale bar: 10μm. (D) Quantification of cdu1⊗ inclusions as shown in (C) in which ubiquitination events are observed in regions of cdu1⊗ inclusion membranes that are in direct apposition to incA⊗ inclusion membranes (junctions). The total number of cdu1⊗ inclusions ubiquitinated at inclusion junctions was divided by the total number of inclusions analyzed (Cap1 staining). 66% and 61% of cdu1⊗ inclusions were decorated with Ub at junctions in HeLa cells co-infected with incA⊗ and cdu1⊗ strains or with incA⊗ and a cdu1⊗ strain ectopically expressing Cdu1K268E (Act-) respectively. Representative images in (C) and quantification of cdu1⊗ inclusions ubiquitinated at junctions in (D) 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.

The DUB activity of Cdu1 is not required for assembly of F-actin or Golgi ministack repositioning around the Ct inclusion while Cdu1 is required for MYPT1 recruitment to the inclusion.

Representative images of F-actin (green, Alexa FluorTM Phalloidin) assembled around the Ct inclusion (magenta, anti Cdu1 and Cap1 staining) in HeLa cells infected for 40 hours. (B) Representative images of Golgi (anti GM130 staining, magenta) around Ct inclusions (arrowheads) in HeLa cells infected for 24 hours. HeLa cells were infected with an inaC null strain transformed with empty p2TK2 vector (M407 p2TK2), the wild type strain in which M407 was derived from (Rif-R L2) and M407 ectopically expressing full length InaC (M407 p2TK2-InaC). HeLa cells were also infected with L2 pBOMB, cdu1::GII pBOMB, and cdu1 null strains ectopically expressing Cdu1-Flag (Cdu1), and the Cdu1 variants C345A-Flag (DUB-, Act-), I225A-Flag (DUB-), and K268E-Flag (Act-). (C) 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 A-C. Scale bar: 10μm in panels A-C. (D) Quantification of Golgi dispersal around the Ct inclusion as shown in (B). 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). Representative images in (B) and Golgi dispersal quantified in (D) were performed from inclusions imaged in 6 fields across 3 independent biological replicates. p values were determined by a student paired t-test. (E) Quantification of Ct inclusion surrounded by F-actin as shown in (A) normalized to total inclusions analyzed. Representative images in (A) and quantification of F-actin surrounding Ct inclusions in (E) 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. (F) Quantification of MYPT1 at Ct inclusions as shown in (C). Representative images in (C) 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 translocated Ct effectors InaC, IpaM, and CTL0480 from degradation.

The human cellular Ub machinery targets C. trachomatis effectors, including the inclusion membrane proteins InaC, IpaM, and CTL0480 for ubiquitination and subsequent protein degradation. C. trachomatis has evolved strategies to counter such defense mechanisms by translocating the effector Cdu1. Cdu1 utilizes its acetylase activity rather than its deubiquitinase activity to protect itself and all three inclusion membrane proteins from being targeted for ubiquitination and degradation. Cdu1 protects InaC in order to promote 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 also participate in promoting extrusion of C. trachomatis inclusions late in infection. Image created 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.

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.

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.