Human endocervical epithelial HPV-16 E6/E7 transformed End1s (End1 E6/E7, ATCC CRL-2615) were cultured at 37° C with 5% atmospheric CO₂ in Keratinocyte Serum-Free Medium (KSFM, Thermo Fisher Scientific) supplemented with human recombinant epidermal growth factor, bovine pituitary extract, 5 micrograms/mL gentamicin, and 0.4 mM CaCl₂ (unless otherwise indicated). For all experiments, End1s were cultured between passages 3 and 15. Primary human cervical epithelial cells (HCECs, ATCC PCS-0480-011, Lot 80306190) were cultured at 37° C with 5% atmospheric CO₂ in Cervical Epithelial Cell Basal Medium (CECBM, ATCC PCS-480-032) supplemented with all contents of a Cervical Epithelial Growth Kit (ATCC PCS-080-042). Raft cultures were prepared as previously described by our laboratory (Nogueira et al., 2017), exchanging HaCaT cells for End1s where indicated, and cultured over 19 days before being fixed for 30 minutes in 4% paraformaldehyde, OCT-embedded, cryo-sectioned, and stained with hematoxylin and eosin.

Both FLAG-YAP1-WT and FLAG-YAP1-Y357F were generated via the simplified FastCloning method of Gibson assembly (Li et al., 2011); briefly, PCR-amplified recombinant DNA was treated with DpnI (NEB Biolabs) for 3 h, then transformed into Stellar chemically competent E. coli (Clontech 636763) via heat shock, followed by Kanamycin selection, screening via colony PCR (where applicable), and sequence confirmation. FLAG-YAP1-WT was generated via amplification of pEGFP-C3-HYAP1, a gift from Marius Sudol (Addgene plasmid # 17843; http://n2t.net/addgene:17843; RRID : Addgene_17843), using the following primers: FWD: 5’-GACTACAAAGACGATGACGACAAGGATCCCGGGCAGCAGCCG-3’; REV: 5’-CTTGTCGTCATCGTCTTTGTAGTCCATGGTGGCGACCGGTAGCG-3’. FLAG-YAP1-Y357F was generated from FLAG-YAP1-WT via site-directed mutagenesis of the Y357 codon (TAC to TTC), using the following primers: FWD: 5’-GCAGCTTCAGTGTCCCTCGA-3’, REV: 5’- ACACTGAAGCTGCTCATGCTTAGTC-3’.

End1s were transfected with plasmid DNA via electroporation: after harvest via trypsinization, 2x10⁶ End1s per construct were resuspended in 400 μL Opti-MEM at room temperature (Thermo Fisher Scientific 31985062) containing 20 ug of plasmid DNA and 3 μL sheared salmon sperm DNA (Thermo Fisher Scientific AM9680). Suspensions were then transferred to individual GenePulser 0.4 cm cuvettes (Bio-Rad 1652088), then electroporated using a GenePulser XCell (Bio-Rad) using the following parameters: exponential decay program template, 225 V, 850 μF capacitance, infinite resistance. 100 μL aliquots of the resulting electroporants were transferred to individual wells of a 24-well plate containing cover slips and pre-warmed KSFM, then incubated until confluent monolayers had formed (24-48h) for subsequent infection as previously described.

For siRNA-mediated knockdown experiments, End1s were transfected with either an ON-TARGETplus non-targeting siRNA pool (Horizon Discovery D-001810-10-05) or an ON-TARGETplus YAP1-targeting siRNA SMARTpool (Horizon Discovery L-012200-00-0005) using Lipofectamine 3000 (Thermo Fisher Scientific L3000008), per manufacturer’s instructions, at an empirically determined optimal concentration of 10 nM. At 16 h post-seeding of End1s at 125% of confluence on 6-well plates as described above, siRNA was combined in Opti-MEM (Thermo Fisher Scientific 31985062) with the Lipofectamine 3000 reagent, incubated for 5 m at room temperature to allow for liposome formation, then added to wells dropwise with mixing. Transfected End1s were then incubated for 24 h prior to infection with Chlamydia (see below).

Chlamydia trachomatis serovar L2 (434/Bu) was originally obtained from Dr. Ted Hackstadt (Rocky Mountain National Laboratory, NIAID). Chlamydial EBs were isolated from infected, cycloheximide-treated McCoy cells at 36-40 hours post-infection (hpi) and purified by density gradient centrifugation as previously described (Caldwell et al., 1981). For infection on 6- and 24-well tissue culture plates (Greiner Bio-One 657160 and 662160), End1s were seeded at 125% of confluence to ensure uniform formation of monolayers occurred in the event of any cell death prior to infection. HCECs were grown to confluence after seeding at a density of 5000 cells/cm², per manufacturer’s recommendations. End1 (16 h post-seeding) and HCEC monolayers (6-7 days post-seeding) were washed with pre-warmed Hanks Buffered Saline Solution (HBSS) prior to inoculation with Chlamydia-containing KSFM or CECBM. To ensure uniformity of infection between individual cells for measurement of YAP nuclear translocation and/or phosphorylation, all infections were carried out at a multiplicity of infection (MOI) of 5, unless otherwise indicated. Tissue culture plates were centrifuged at 4° C and 500 rcf (Eppendorf 5810 R tabletop centrifuge, A-4-81 rotor) for 15 minutes to synchronize infection. Inoculum was then aspirated, and cells were washed with chilled HBSS prior to the addition of pre-warmed KSFM or CECBM.

Where indicated, media was replaced with fresh, pre-warmed KSFM containing DMSO (Thermo Fisher Scientific D12345, 1:1000), verteporfin (Cayman Chemical Company 17334, 5 μM), chloramphenicol (Sigma-Aldrich C0378, 50 μg/mL), sodium orthovanadate (Sigma-Aldrich 450243, 5 μM), RK-24466 (Cayman Chemical Company 15135, 10 nM), or PP2 (EMD Millipore 529576, 10 μM). Infected cultures were then returned to the tissue culture incubator until the indicated times post-infection.

Prior reports of host gene expression during C. trachomatis infection have employed an MOI of 1 (Humphrys et al., 2013; Porcella et al., 2015); given the potential for low MOI to produce a heterogeneous population of infected and uninfected cells, we opted to employ an MOI of 2 as a compromise between consistency with past work and ensuring uniformity of infection (and, thereby, identification of host transcription factors modulated by infection). Thus, End1s seeded on fibronectin-coated 6-well plates (Corning 354402) were infected at an MOI of 2 as described above (pre/post-washing, 15 m centrifugation at 500 rcf and 4° C) and harvested for RNA using TRIzol (Thermo Fisher Scientific 15596026) and the DNA-free DNA removal kit (Thermo Fisher Scientific AM1906), according to manufacturers’ protocols. Extracted RNA was subsequently enriched for polyadenylated mRNA transcripts using the NucleoTrap mRNA enrichment kit (Macherey-Nagel) according to the manufacturer’s protocol. For the comparison of mock- and Ct serovar L2-infected transcriptomes, RNA samples were subsequently assayed for fragmentation using an Agilent 5200 Fragment Analyzer; intact samples were enriched for polyadenylated transcripts via the NucleoTrap kit a second time due to the high incidence of ribosomal RNA in the fragment analysis results. cDNA library preparation was performed using the Ion Xpress Plus Fragment Library kit (Thermo Fisher Scientific 4471269), and sequencing of cDNA libraries was performed using the Ion Torrent system (Life Technologies). For the bulk RNA-sequencing of primary cells, HCECs infected at an MOI of 2 as described above (pre/post-infection washing, 15 m centrifugation at 500 rcf and 4° C) were harvested for RNA samples via TRIzol and the DNA-free DNA removal kit as described above, and were subsequently assayed for fragmentation using an Agilent Bioanalyzer 2100. cDNA library preparation of intact samples was performed using the NuGEN Universal mRNA-Seq Library Preparation kit, and sequencing of cDNA libraries was performed using the NextSeq 550 system (Illumina).

Read alignment and downstream analysis of both experiments was performed using CLC Genomics Workbench (Qiagen); each treatment group was comprised of libraries from three biological replicates, each with a minimum of 30 million reads (unstranded single read, mean length 150 bp), genes with an FDRP ≤ 0.05 were considered differentially expressed. ChEA crossreferencing was performed in Python, identifying the number of genes induced (FC ≥ 1.5, FDRP ≤ 0.05) and repressed (FC ≤ 1.5, FDRP ≤ 0.05) by infection for each transcription factor’s gene targets (Lachmann et al., 2010). Pearson’s correlation coefficients between End1 and HCEC expression of YAP-responsive genes were subsequently calculated in R, using log₂-transformed fold changes of all genes differentially expressed in at least one data set. GO-BP term enrichment analysis was performed in R using the clusterProfiler gene set enrichment analysis package (Yu et al., 2012). The bulk RNA-sequencing datasets generated and analyzed for this study can be found in the Gene Expression Omnibus (Edgar et al., 2002), GEO Series accession number GSE180784 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE180784).

End1s seeded on fibronectin-coated 6-well plates (Corning 354402) and infected at an MOI of 5 as described above (pre/post-infection washing, 15 m centrifugation at 500 rcf and 4° C) were harvested for RNA using TRIzol (Thermo Fisher Scientific 15596026) and the DNA-free DNA removal kit (Thermo Fisher Scientific AM1906), according to manufacturers’ protocols. cDNA libraries were subsequently prepared using SuperScript IV Reverse Transcriptase (Thermo Fisher Scientific 11766050) according to the manufacturer’s protocol. Quantitative real-time PCR was performed on a QuantStudio 3 (Thermo Fisher Scientific) using TaqMan assay kits (Thermo Fisher Scientific) of the following genes: CTGF (Hs01026927_g1), CYR61 (Hs00998500_g1), INHBA (Hs01081598_m1), BMP2 (Hs00154192_m1), and the housekeeping gene HPRT (Hs02800695_m1). Statistical analysis was performed in R, using pairwise Student’s t-tests and Bonferroni’s correction for multiple comparisons; p-values less than 0.05 were considered statistically significant.

End1s or HCECs seeded on glass cover slips (VWR) coated with fibronectin (Corning 354008) or fibronectin-deposited micropatterned chips (CYTOO) and infected at an MOI of 5 as described above (pre/post-infection washing, 15 m centrifugation at 500 rcf and 4° C) were fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS) for 10 minutes at 37° C. After permeabilization via a 5 m incubation in PBS containing 0.25% Triton X-100, cover slips were washed in PBS, then blocked in 5% bovine serum albumin (BSA) in PBS for 1 hour at room temperature. Fixed and blocked cover slips were subsequently incubated overnight at 4° C with primary antibodies in 1% BSA-PBS: mouse anti-Ct Hsp60 (Invitrogen MA3-023, 1:250 dilution), goat anti-MOMP (Meridian Life Science B65266G, 1:1000 dilution), rabbit anti-YAP (CST 4912, 1:100 dilution), rabbit anti-E-Cadherin (CST 3195, 1:200 dilution). Cover slips were again washed in PBS, then incubated for 1 hour at room temperature with the following fluorophore-conjugated antibodies/dyes in 1% BSA-PBS: donkey anti-mouse Alexa-488 conjugate (Thermo Fisher Scientific A-21202, 1:1000 dilution), chicken anti-goat Alexa-594 conjugate (Thermo Fisher Scientific A-21468, 1:1000 dilution), goat anti-rabbit Alexa-488 conjugate (Thermo Fisher Scientific A-11034, 1:1000 dilution), phalloidin Alexa-594 conjugate (Thermo Fisher Scientific A-12381, 1:120 dilution), DAPI (Sigma-Aldrich 10236276001, 1:1000 dilution). Afterward, cover slips were washed in PBS and ultrapure water, then mounted on microscope slides using Shandon Immu-Mount (ThermoFisher Scientific 9990402).

A minimum of 5 fields per cover slip were imaged using a SP-8 Lightning Confocal Microscope (Leica) or CSU-W1 Spinning-Disk Confocal Microscope (Nikon). To account for variations in nuclear size and avoid biased selection of cells for measurement, blinded image quantification was performed by assigning image filenames randomized number codes, selecting 10 nuclei at random per field using only the DAPI channel, manually masking the nuclear area, and recording the mean YAP fluorescence intensity per nucleus. To account for variation in total YAP between cells, staining efficiency between cover slips, and compression of the nuclear/cytosolic compartments by the chlamydial inclusion, 5 cytosolic regions not occluded by an inclusion body and adjacent to measured nuclei were selected per field, with the mean YAP fluorescence intensity of these regions averaged to produce a per-field measurement of mean cytosolic YAP fluorescence intensity; nuclear translocation of YAP was thereby expressed as a ratio of mean nuclear fluorescence to mean cytosolic fluorescence. Statistical analysis was performed in R, using a Kruskal-Wallis test to first verify a statistically significant (p-value < 0.05) difference between treatment groups. Subsequent pairwise comparisons were performed using a Wilcoxon rank sum test and Bonferroni’s correction for multiple comparisons, with p-values less than 0.05 being considered statistically significant.

To minimize activity of the chlamydial protease CPAF, End1s seeded on 6-well plates and infected at an MOI of 5 as described above (pre/post-infection washing, 15 m centrifugation at 500 rcf and 4° C) were subsequently lysed in 1% SDS buffer heated to 95° C, as previously described (Johnson et al., 2015). After treatment with Pierce Universal Nuclease (Thermo Fisher Scientific, 1:1000 dilution) for 5 minutes at room temperature, lysates were combined with 4X Laemmli Sample Buffer (Bio-Rad 1610747) for loading on a 7.5% acrylamide/bis-acrylamide gel for SDS-PAGE (1.5 hours, 120V). Gels were then transferred to PVDF membranes (Bio-Rad 1620177) using a semi-dry transfer method. After blocking in 5% BSA in PBST (PBS containing 0.1% Tween-20) for 1 hour at room temperature, membranes were incubated overnight in 5% BSA-PBST at 4° C with primary antibodies: rabbit anti-YAP (CST 4912, 1:1000 dilution), rabbit anti-S127-pYAP (CST 13008, 1:1000 dilution), rabbit anti-Y357-pYAP (Abcam ab62571, 1:1000 dilution), mouse anti-Ct Hsp60 (Invitrogen MA3-023, 1:1000 dilution in). Membranes were subsequently washed in PBST, then incubated in 5%-BSA PBST with either a goat anti-rabbit HRP-conjugated secondary antibody (Dako P0448, 1:2000 dilution) or rabbit anti-mouse HRP-conjugated secondary antibody (Dak P0161, 1:4000 dilution) for 2 hours at room temperature. After additional washing in PBST, membranes were imaged using Immobilon HRP Substrate (Millipore Sigma WBKLS0500) or an Azure Biosystems c600. Images were analyzed using the ImageJ gel analysis tool to quantify the fluorescence density of phospho-analyte bands relative to the YAP total protein loading control. Statistical analysis was performed in R, using pairwise Student’s t-tests with Bonferroni’s correction for multiple comparisons; p-values less than 0.05 were considered statistically significant.

To identify host transcription factors modulated by Chlamydia infection, we opted to model infection of the upper female genital tract using End1/E6E7 immortalized, non-transformed endocervical epithelial cells (Fichorova et al., 1997). We first confirmed the non-tumorigenic growth properties of End1 cells via organotypic raft culture: first seeding End1 cells on collagen gels in a transwell insert, then inducing differentiation via exposure to the air-liquid interface (Nogueira et al., 2017). Transformed cervical epithelial cells form irregular layers when induced to differentiate via air-liquid interface (Aasen et al., 2003); by contrast, differentiating End1s grown via raft culture formed epithelial tissues of uniform thickness ( Supplementary Figure S1 ), consistent with previous reports of organotypic culture with this cell line (Gali et al., 2010). Having thus validated the non-tumorigenic phenotype of our experimental model, we then infected End1s with the lymphogranuloma venereum Chlamydia trachomatis serovar L2, then harvested polyadenylated RNA for bulk RNA-sequencing at 24 hours post-infection (hpi). This timepoint occurs well after chlamydial differentiation into proliferative and metabolically active reticulate bodies (Lee et al., 2018), thus allowing for production of any chlamydial factors modulating host gene expression. That being said, factors produced by C. trachomatis at later stages of infection may also modulate host gene expression; thus, we cannot at this time dismiss the possibility of additional infection-mediated alterations to the host transcriptome occurring later than 24 hpi. Nevertheless, infection at this time point induced the differential expression (FDRP ≤ 0.05) of 3611 genes relative to a mock-infected control ( Figure 1A ; Supplementary Data S1 , GEO Accession: GSE180784).

To then determine potential transcription factors driving the host response to infection, we cross-referenced this differentially expressed gene set with the ChEA database of transcription factor gene targets, derived from gene set enrichment analysis of published ChIP-chip, ChIP-seq, ChIP-PET, and DamID data (Lachmann et al., 2010). Of the 199 transcription factors included in ChEA, 149 exhibited twice as many induced target genes (FC ≥ 1.5) as repressed target genes (FC ≤ 1.5), suggesting a general chlamydial induction of transcription ( Supplementary Data S2 ). Critically, targets of transcription factors known to regulate inflammation (IRF1, STAT1, STAT3) and fibrosis (SMAD2/3, EGR1, TEAD4, YAP1) appeared to be highly induced in the host ( Figure 1B ), consistent with prior reports of infection-dependent induction of pro-inflammatory and pro-fibrotic gene expression (Carlson et al., 2005; Lad et al., 2005; Humphrys et al., 2013; Sun et al., 2017).

Intriguingly, the fibrosis-associated transcription factors identified by this analysis (SMAD2/3, EGR1, and TEAD4) are known binding partners of the transcriptional coactivator YAP1 (Zagurovskaya et al., 2009; Zanconato et al., 2015; Szeto et al., 2016). Importantly, 81 YAP target genes (as identified by ChEA) were induced in Chlamydia-infected End1s, suggesting infection may modulate YAP activity. To assess the physiological relevance of apparent modulation of YAP target genes, we sequenced polyadenylated RNA from primary human endocervical epithelial cells (HCECs) infected with Ct serovar L2 ( Supplementary Data S3 , GEO Accession: GSE180784). Infected HCECs exhibited differential expression (FDRP ≤ 0.05) of 8241 genes ( Figure 1C ), of which 830 (10.1%) mapped to the ChEA database of YAP-responsive genes ( Figure 1D ). Expression of YAP-responsive genes by infected End1s and HCECs exhibited significant correlation ( Figure 1E ), suggesting that infection of these cell types activates a conserved, putatively YAP-mediated transcriptional program. Chlamydia-infected End1 cells and HCECs exhibited both induction and repression of YAP target genes ( Figure 1F ; Supplementary Data S4 ), in accordance with recent work indicating YAP can situationally act as a transcriptional corepressor (Kim et al., 2015; Hoxha et al., 2020; Ma et al., 2022). Functional characterization of YAP-associated gene expression in both infections via GO-BP (gene ontology, biological process) term enrichment analysis identified terms associated with regulation of epithelial cell proliferation and organ morphogenesis ( Figure 1G ), consistent with prior reports implicating YAP in these processes (Zhao et al., 2008; Zhao et al., 2010). Taken together, these data imply that Chlamydia infection targets the YAP regulon to impact a subset of the transcriptome of host epithelial cells.

To confirm our RNA-sequencing analysis suggesting infection may promote YAP-dependent gene expression, we next assayed infection-dependent induction of known YAP target genes via RT-qPCR. Total RNA was harvested from confluent C. trachomatis-infected End1 cells at 24 hpi. Consistent with our bulk RNA sequencing results, infection increased expression of the direct YAP target CTGF (connective tissue growth factor), but not CYR61 (cysteine-rich angiogenic inducer 61) ( Figure 2A ), suggesting that infection potentially modulates the YAP regulon in a fashion distinct from previous reports of YAP activity. Critically, infection-dependent CTGF expression was sensitive to siRNA-mediated YAP knockdown ( Figure 2B ), confirming that chlamydial induction of this gene was YAP-dependent. A similar trend was exhibited by INHBA and BMP2 ( Supplementary Figure S2 ) - genes induced by infection in our bulk RNA-seq results previously shown to be YAP-responsive (Mo et al., 2012; Huang et al., 2016). As incomplete YAP knockdown may be insufficient to inhibit expression of genes indirectly regulated by YAP, we then opted to treat infected cells with the YAP-TEAD complex inhibitor verteporfin (10 μM for 16 h, starting at 8 hpi). Consistent with our results with siRNA, verteporfin treatment significantly inhibited CTGF expression in Chlamydia-infected cells, as well as inhibiting expression of INHBA and BMP2 ( Figure 2C ). Taken together, these results suggest that a component of the infected host cell transcriptome may be driven by infection-associated alteration of the YAP regulon.

Transcriptional regulation by YAP of target genes requires nuclear translocation (Pocaterra et al., 2020). Thus, we opted to confirm that chlamydial infection promotes YAP activity by assaying the nuclear incidence of YAP in serovar L2-infected confluent End1 monolayers via immunofluorescence. Infection was performed at a multiplicity of 5 to ensure that the majority of cells were equivalently infected, and thus any infection-associated effects upon nuclear incidence of YAP would be uniform. To account for variations in nuclear size and staining efficiency, we performed blind quantification of randomly selected nuclei from infected cells in each sample, measuring ratios of nuclear to cytosolic YAP, analogous to previous study of YAP activation (Zhao et al., 2007; Dupont et al., 2011; Das et al., 2016). C. trachomatis infection of End1s was readily visualized by the presence of a vacuole containing DAPI-positive puncta adjacent to the host cell nucleus and colocalizing with staining of chlamydial Hsp60 and LPS ( Supplementary Figure S3 ). Consistent with our initial observations of YAP target gene induction, nuclear YAP was increased in Chlamydia-infected End1/E6E7 cells at 24 hpi, relative to mock-infected control cells ( Figure 2D ). Indeed, infection was concomitant with a 1.5-fold increase in nuclear-to-cytosolic YAP ratio relative to mock-infected cells ( Figure 2E ). Given that expression of HPV protein E6 has been shown to stabilize YAP and thereby modulate its activity (Strickland et al., 2018), we assayed nuclear YAP in primary HPV-negative HCECs as well, observing an equivalent increase to that in E6-expressing End1s under similar infection conditions ( Figures 2F, G ). Collectively, these data indicate infection enhances YAP activation irrespective of HPV E6/E7 expression.

To determine if the observed increase in YAP activity was a Chlamydia-driven process requiring the action of one or more chlamydial effectors, we utilized chloramphenicol to inhibit chlamydial protein synthesis. Over the course of a 24-hour infection (at 2, 4, 8, 12, 18, and 24 hpi), we observed a significant increase in YAP nuclear localization by immunofluorescence between mock- and serovar L2-infected End1/E6E7 cells, starting as early as 8 hpi. A statistically significant increase between infected cells was first detected between 12 and 18 hpi, indicating that synthesis of YAP-modulating chlamydial effectors may occur at this stage of infection ( Figure 2H ). We next attempted to attenuate chlamydial YAP induction at 18 hpi via treatment with chloramphenicol (50 ug/mL). In agreement with our initial hypothesis, chloramphenicol treatment for one hour prior to fixation at 18 hpi was sufficient to significantly attenuate nuclear YAP relative to a DMSO-treated control ( Figures 2I, J ). Given these data, we conclude that YAP activation in infected cells requires de novo synthesis of chlamydial effectors.

The Hippo kinase cascade is the major regulator of YAP activity in confluent epithelial cells (Gumbiner and Kim, 2014). When stabilized by cell-cell contact, Hippo initiates a Ser/Thr kinase cascade that leads to the phosphorylation of S127 residue of YAP, promoting cytosolic sequestration via binding to 14-3-3 (Piccolo et al., 2014). Increased nuclear YAP in infected cells could thus be due to chlamydial inhibition of the Hippo kinase cascade. To determine if chlamydial YAP activation was responsive to adherens junction disruption, we assayed nuclear incidence of YAP in two complementary conditions of attenuated cell-cell contact: small cell clusters seeded 70-85 μm apart on 30 μm (1-3 cells/cluster) and 45 μm (2-4 cells/cluster) fibronectin micropatterns (Degot et al., 2010), as well as confluent monolayers in which cell-cell contacts were disassembled via calcium withdrawal (O’Keefe et al., 1987). If YAP activation in infected cells is mediated by chlamydial antagonism of Hippo, we would expect that an infection-associated increase in nuclear:cytosolic YAP would be lost in cell culture conditions of minimal cell-cell contact (and, by extension, minimal YAP inhibition by Hippo). Thus, we monitored YAP responsiveness to cell seeding configurations with differing degrees of cell-cell contact. YAP nuclear incidence was not increased in mock-infected cells relative to mock-infected confluent monolayers, suggesting that residual cell-cell contact in small cell clusters maintained Hippo activity ( Supplementary Figure S4 ). By contrast, nuclear:cytosolic YAP was increased in mock-infected monolayers cultured in calcium-deplete media relative those cultured in calcium-replete media ( Figures 3A, B ), confirming previous reports of adherens junction disruption being a known trigger of YAP activation, presumably through destabilization of the Hippo kinase cascade complex. Importantly, infected cells exhibited increased YAP nuclear translocation relative to mock-infected control cells in calcium-deplete conditions ( Figures 3A, B ), indicating that infection yielded an additional level of activation over calcium depletion alone.

This result suggests an activating process independent of the known YAP regulatory mechanism associated with adherens junctions, i.e. Hippo kinase cascade-dependent inhibition by phosphorylation at residue S127. Therefore, we sought to confirm independence of infection-associated YAP activation from the adherens junction-related regulatory mechanism. Downstream of Hippo complex formation at cell-cell contacts, complex members LATS1/2 inhibit YAP via phosphorylation at S127. YAP phosphorylated at this residue is bound by the adaptor protein 14-3-3, retaining YAP in the cytosol (Zhao et al., 2007; Piccolo et al., 2014). We collected whole cell lysates from mock- and Chlamydia-infected confluent End1 monolayers, assaying YAP S127 phosphorylation via Western blotting. Chlamydia-infected End1 monolayers exhibited equivalent levels of S127 phosphorylation relative to mock-infected controls ( Figures 3C, D ).

An alternative means of YAP activation was recently described, involving phosphorylation of YAP at Y357 (Smoot et al., 2018). While the specific mechanism of this effect remains unclear, it is suggested that YAP phosphorylation at Y357 and other tyrosine residues may promote YAP translocation into the nucleus (Elbediwy et al., 2018), or YAP retention in the nuclear compartment (Sugihara et al., 2018). To determine whether Chlamydia promotes YAP activation via this alternative mechanism, we assayed YAP phosphorylation at Y357 in infected End1 monolayers. In contrast to levels of S127-pYAP, Y357 phosphorylation was significantly increased in infected whole cell lysates relative to mock-infected controls ( Figures 3C, E ). This result was consistent with mock-infected cells treated with sodium orthovanadate (Na₃VO₄), a tyrosine phosphatase inhibitor that serves as a positive control for increased YAP tyrosine phosphorylation ( Figures 3C, E ) (Sugihara et al., 2018). Intriguingly, unlike nuclear incidence of YAP during infection, Y357 phosphorylation was not reduced by 1 hour of chloramphenicol treatment, suggesting that chlamydial enhancement of Y357 phosphorylation is less sensitive to inhibition of bacterial protein synthesis. Nevertheless, an 8-hour chloramphenicol treatment attenuated infection-associated Y357 phosphorylation, with attendant reduction in chlamydial Hsp60 expression indicating that this phenotype is sensitive to a delay in chlamydial development ( Figures 3F, G ). Collectively, these data demonstrate that infection-dependent YAP nuclear translocation bypasses S127 phospho-inhibition, instead correlating with increased YAP phosphorylation at Y357.

To determine the significance of Y357 phosphorylation, we ectopically expressed FLAG-tagged Y357F-mutant YAP in infected End1s via transient transfection, and compared nuclear incidence to an equivalent mock-infected control. Infected cells expressing the wild-type construct exhibited an increased ratio of nuclear:cytosolic YAP relative to mock-infected cells. However, infection did not increase nuclear incidence of the phospho-dead YAP-Y357F mutant, with infected cells exhibiting equivalent nuclear:cytosolic YAP to mock-infected cells expressing the same construct ( Figures 4A, B ). This result suggests that loss of Y357 ablates YAP nuclear translocation mediated by infection, highlighting involvement of an alternative strategy.

YAP phosphorylation at Y357 phosphorylation has been shown to be sensitive to inhibition of the Src family of tyrosine kinases; knockdown of Lck in particular is reported to decrease this phosphorylated species of YAP (Smoot et al., 2018; Sugihara et al., 2018). We therefore opted to assay YAP nuclear incidence in infected End1 monolayers treated with the Lck-specific small molecule inhibitor RK-24466. Surprisingly, the ratio of nuclear:cytoplasmic YAP in infected cells was unaffected by Lck inhibition ( Figures 4C, D ), suggesting a different mechanism of Y357 phosphorylation modulated by Chlamydia. We then assayed YAP nuclear incidence of infected cells treated with the pan-Src-family kinase (SFK) inhibitors PP2 and A-419259, as well as the dual-acting SFK/Abl inhibitor dasatinib (Wilson et al., 2002; Pene-Dumitrescu et al., 2008; Sugihara et al., 2018). In contrast with PP2 and A-419259 treatment, serovar L2-infected cells treated with dasatinib exhibited significant attenuation of YAP nuclear incidence relative to DMSO-treated cells, suggesting that infection-enhanced YAP nuclear incidence is mediated by Abl ( Figures 4E, F ). However, YAP phosphorylation at Y357 in infected cells was severely attenuated by treatment with all three inhibitors ( Figure 4G ), in accordance with studies directly implicating SFKs in YAP tyrosine phosphorylation (Li et al., 2016; Sugihara et al., 2018). Taken together, these data indicate that Chlamydia-mediated induction of YAP nuclear translocation requires host Abl activity, apparently altering translocation independent of Y357 phosphorylation.