Loss-of-function haploid genetic screen with C. trachomatis L2. (A) Outline of the loss-of-function haploid genetic screen with C. trachomatis L2; see the text for details. (1) KBM7 cells are reprogrammed to adherent cells by OCT4, Sox-2, c-MYC, and KLF4. (2) HAP1 cells are transduced with the gene trap virus. (3) The mutated cells are inoculated with C. trachomatis L2. (4) Virtually all (99.99%) of the mutant cells die from Chlamydia infection. (5) Survivors are expanded, the DNA is extracted, and the DNA is sequenced. (B) Bubble plot of loss-of-function haploid genetic screen results. The x axis shows genes with at least one inactivating mutation ranked in order of chromosomal position. The y axis shows the −log of the P value of the enrichment of gene-trap insertions in the indicated gene compared with an unselected control dataset. The size of the bubble is correlated with the number of independent insertions. Genes with a P value < 0.0001 are labeled, and the number of independent insertions is given in brackets.

Cells without B3GAT3, B4GALT7, or SLC35B2 are resistant to death by Chlamydia infection. (A) RT-PCR analysis showing that the isolated null clones SLC35B2^GT, B4GALT7^GT, and B3GAT3^GT do not express any mRNA of their particular gene. (B) Bar graph of the survival of null clones inoculated with C. trachomatis L2. Retroviral expression of C-terminal HA-tagged B3GAT3, B4GALT7, and SLC35B2 fully restored susceptibility to killing by Chlamydia. HA-tagged enzymatically inactive forms of the encoded gene products did not restore susceptibility to Chlamydia, and these clones readily survived for 4 d after inoculation with Chlamydia.

Although the null clones resist Chlamydia infection, the Chlamydia that do enter the clones behave similarly to the WT clone. (A–C) Time course of C. trachomatis L2 replication as measured by Chlamydia DNA compared with a housekeeping gene (β-actin). (D and E) Transmission electron microscopy of WT and null clones (B3GAT3^GT, B4GALT7^GT, and SLC35B2^GT) at 36 h after infection with C. trachomatis L2. There were fewer inclusions in the null clones, and the inclusions that were seen were of similar size. The elementary bodies and reticulate bodies had equivalent morphology.

The null clones resist Chlamydia attachment. (A) Bar graph comparing the attachment of C. trachomatis L2 of the null clones and enzymatically inactive reconstituted clones with that of the clones reconstituted with the WT protein. (B) Immunofluorescence of the null clones, reconstituted clones with the WT protein, and reconstituted clones with enzymatically inactive mutants after infection with C. trachomatis L2 for 1 h at room temperature. The Chlamydia elementary bodies are stained with a monoclonal antibody and appear green; the clones are counterstained with Evans blue and appear purple.

The importance of sulfation beyond heparan sulfate in Chlamydia infection. (A) Bar graph comparing the attachment of C. trachomatis L2 of the null clones. A one-sided unpaired t test shows less attachment of chlamydia to SLC35B2^GT versus B3GAT3^GT, to B3GAT3^GT versus B4GALT7^GT, and to SLC35B2^GT versus B4GALT7^GT (P = 0.0418, 0.0232, and 0.0139, respectively). (B) Infectivity of C. trachomatis L2 recovered from WT and null clones (B3GAT3^GT, B4GALT7^GT, and SLC35B2^GT). At 36 h after infection of the clones, Chlamydia was harvested and used to inoculate McCoy cells for tittering. Chlamydia inclusions were detected by immunofluorescence and enumerated by counting inclusions (IFU). n = 3. The differences between B3GAT3^GT and SLC35B2^GT, between B3GAT3^GT and B4GALT7^GT, and between B4GALT7^GT and SLC35B2^GT are significant by the one-sided unpaired t test (P < 0.0001, < 0.0012, and < 0.0002, respectively). (C) FACS histograms of the null clones and WT clone stained with anti-heparan sulfate antibody before and after heparitinase digestion. The B3GAT3 and B4GALT7 clones are completely deficient in heparan sulfate proteins, whereas the SLC35B2 clone has ∼5% of cells positive for heparan sulfate. (D) (Left) Autoradiogram control showing equivalent incorporation of [³⁵S]met/[³⁵S]cys in the null clones and WT clone. (Right) Autoradiogram showing a 90–95% reduction in the incorporation of ³⁵SO₄ in the SLC35B2 null clone (SLC35B2^GT) compared with the WT clone. The SLC35B2 null clone has less sulfated proteins than the B3GAT3 null clone, which has less sulfated proteins than the B4GALT7 null clone.