The effector TepP mediates the recruitment and activation of Phosphoinositide 3 Kinase on early Chlamydia trachomatis vacuoles

Chlamydia trachomatis delivers multiple Type 3 secreted effector proteins to host epithelial cells to manipulate cytoskeletal functions, membrane dynamics and signaling pathways. TepP is the most abundant effector protein secreted early in infection but its molecular function is poorly understood. In this report, we provide evidence that TepP is important for bacterial replication in cervical epithelial cells, the activation of Type I IFN genes, and the recruitment of Class I phosphoinositide 3 kinases (PI3K) and the signaling adaptor protein CrkL to nascent pathogen-containing vacuoles (inclusions). We also show that TepP is a target of tyrosine phosphorylation by Src kinases but these modifications do not appear to influence the recruitment of PI3K or CrkL. The translocation of TepP correlated with an increase in the intracellular pools of phosphoinositide 3,4,5 triphosphate but not the activation of the pro-survival kinase Akt, suggesting that TepP-mediated activation of PI3K is spatially restricted to early inclusions. Furthermore, we linked PI3K activity to the dampening of transcription of Type I IFN induced genes early in infection. Overall, these findings indicate that TepP can modulate cell signaling and potentially membrane trafficking events by spatially restricted activation of PI3K.


INTRODUCTION
Chlamydia trachomatis is an obligate intracellular bacterial pathogen of significant socio-economic and medical importance. C. trachomatis is the leading causative agent of preventative blindness worldwide and the most prevalent sexually transmitted infection (STI) in the western world (1, 2). C. trachomatis undergoes two main developmental transitions with an infectious form, the elementary body (EB), and a replicative form, the reticulate body (RB). Both RB and EB forms of the pathogen manipulate host cellular functions by delivering Type 3 Secretion (T3S) effector proteins directly into the target cells' membranes and cytoplasm (3). The Chlamydia T3S system shares many functional features with T3S systems from other Gram negative bacteria, including the requirement for accessory chaperones that stabilize effectors and enhance their secretion (3-5). One of these T3S chaperones, Slc1, interacts with and enhances the secretion of multiple EB effectors (6). For instance, the effector Tarp (Translocated actin recruiting phosphoprotein) is delivered into epithelial cells within 5 minutes of EB attachment and phosphorylated at tyrosine residues (7-9). Multiple proteins with Src homology 2 (SH2) domains can bind in vitro to peptides representing the phosphorylated forms of Tarp (10,11). This includes the E3 ligase Cbl, the Rac1 exchange factor Vav2, the p85 regulatory subunit of phosphoinositide 3 kinase (PI3K), the signaling adaptors Shc1, Nck2 and CrkL, and the kinase Syk (12). Various tyrosine kinases can phosphorylate Tarp in vitro and in vivo, including Src, Abl and Syk (7,8). Tarp nucleates F-actin assembly, independently of its tyrosine phosphorylation status, through its F-actin-binding domain and interaction with the Arp2/3 complex, and by activating focal adhesion kinases (FAK) through molecular mimicry of the cofactor paxillin (9,11,(13)(14)(15)(16). Not surprisingly, this multifunctional effector is important for Chlamydia infection as microinjection of anti-Tarp antibodies into epithelial cells or expression of dominant negative Tarp constructs in Chlamydia inhibit bacterial invasion (11,17).
On a molar basis, TepP is one of the most abundant Chlamydia-specific proteins found within EBs (18). Once translocated into the host cell TepP is phosphorylated by host cell kinases at multiple tyrosine and serine residues (6). Two tyrosine phosphorylation sites generate consensus pYxxP motifs that provide docking site for proteins with SH2 domains including two spliceforms of the signaling adaptor protein Crk (Crk I-II) (reviewed in (19)). Indeed, CrkI and CrkII co-immunoprecipitate with TepP and are recruited to nascent C. trachomatis inclusions in a TepP-dependent manner (6). A comparison of the transcriptional responses of epithelial cells to infection with a tepP deficient mutant and TepP over-expressing strains indicated a role for TepP in the induction of a subset of genes associated with Type I IFN responses, including interferon-induced peptides with tetratricopeptide repeats (IFIT (20)) (6).
To address the mechanism by which TepP modulates host cellular functions, we identified host proteins that associate with TepP during the early stages of bacterial invasion and establishment of inclusions. We determined that CrkL and Class I phosphoinositide 3 kinases (PI3K) (21) are the major proteins that co-purify with TepP and that these proteins are recruited to nascent inclusion in a TepP-dependent manner.

CrkL and PI3K co-purify with TepP translocated during infection.
TepP is phosphorylated at multiple tyrosine residues upon delivery into host cells (6) and may directly recruit Src-homology 2 (SH2) and phosphotyrosine binding (PTB) domain-containing proteins to assemble novel host cell signaling complexes (22,23).
To identify Chlamydia and host proteins associated with TepP-containing signaling complexes, we infected A2EN endocervical epithelial cells with the tepP null mutant strain CTL2-M062G1, and variants transformed with either an empty plasmid (pVec) or a plasmid expressing TepP-FLAG (pTepP). After 4 hours, infected cells were lysed under non-denaturing conditions and subjected to immunoprecipitation (IP) with anti-FLAG antibodies. All proteins in the IP were digested with trypsin and the resulting peptides identified by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). The major human proteins co-purifying exclusively with TepP-FLAG included the catalytic (p110α, p110β) and regulatory (p85α and p85β) subunits of PI3K, CrkL, and glycogen synthase kinase (GSK) (Fig. 1A and Table S1). The specificity of these interactions was verified by immunoblot analysis of subsequent IPs. CrkL, GSK and both PI3K subunits (p110 and p85) co-precipitated with TepP during infection (Fig. 1B).
Reciprocal IP of CrkL and p110α co-precipitated phospho-TepP from infected cells (Fig. 1C and D), validating the specificity of these interactions.

TepP-deficient mutants fail to recruit PI3K and CrkL to early inclusions.
To perform a clean phenotypic analysis of TepP mutants, we disrupted the tepP gene in CTL2 with a gene encoding β lactam resistance (bla) by Group II intron mediated gene insertion (TargeTron TM ) (24). TepP could not be detected in insertional mutants by immunoblot analysis (Fig. 2A). The resulting tepP::bla (ΔtepP) strain behaved as the CTL2-M062G1 tepP nonsense mutant in that it failed to induce the expression of Interferon induced peptides with tetratricopeptide repeats (IFIT) genes ( Fig. 2B) that are typically activated during the early stages of infection (6) and blocked the tyrosine phosphorylation of a subset of host proteins (Fig. 2C) (6). We next determined if TepP is important for efficient bacterial replication by comparing the generation of infectious particles in cell lines infected with either wild type or Δ tepP mutants. A2EN cells, a newly derived cervical epithelial cell line (25), were particularly restrictive for the replication of Δ tepP stains as compared to HeLa cells (Fig. 2D). This defect appears to be unrelated to invasion as Δ tepP mutants were not impaired for entry into cells (data not shown).
We next tested if PI3K and CrkL are recruited to early inclusions where TepP translocation is most apparent (6). We infected A2EN cells with wild type or Δ tepP C.
trachomatis and determined the extent of co-localization of TepP binding partners with intracellular bacteria by indirect immunofluorescence. Both PI3K (83%) and CrkL (90%) were strongly associated with clusters of intracellular bacteria that had migrated to a perinuclear region of host cells by 4 hpi (Fig. 2E). The accumulation of GSK at early inclusions was less prominent (48%). In contrast, we observed very little association of PI3K, CrkL or GSK with early inclusions formed by TepP-deficient Chlamydia (Fig. 2F).
Because PI3K, Crk adaptors and GSK are involved in multiple signaling pathways and can impact the organization of the cytoskeleton, activation of innate immune factors, and expression of pro-survival signals (19,21,26,27), our findings suggest that TepP recruits signaling proteins to influence signaling events.

Src family kinases mediate the tyrosine phosphorylation of TepP but Src activity is not required for the recruitment of PI3K and CrkL to early inclusions
Because Tarp is phosphorylated by Src and Abl kinases (7, 8), we next tested if these kinases also play a role in the phosphorylation of TepP (7,8,28) by performing in vitro phosphorylation assays. Hexahistidine-tagged TepP purified from E. coli was incubated with ATP and cytosolic extracts derived from the following: Vero cells, Abl -/Argmouse embryonic fibroblasts (MEFs), Src/Yes/ Fyn-deficient (SYF) MEFs, and their rescued counterparts. TepP was re-isolated and the extent of tyrosine phosphorylation assessed by immunoblot analysis with anti-phosphotyrosine antibodies. Recombinant TepP was phosphorylated after incubation with all cell lysates except for those derived from SYF MEFs, suggesting that Src-family kinases are required for TepP tyrosine phosphorylation in vitro (Fig. 3A). To determine if Src performs a similar function in vivo the same cells lines were infected with CTL2-M062G1 expressing TepP-FLAG. After IP with anti-FLAG antibodies, we assessed the extent of TepP tyrosine phosphorylation by immunoblot analysis (Fig. 3B). We observed diminished phosphorylation of TepP during infection of SYF MEFs, and increased phosphorylation upon overexpression of c-Src and to a lesser extent of Abl and Arg. Taken together, these findings suggests that the Src family kinases phosphorylate TepP during infection, although other kinases may also play an auxiliary role.
We hypothesized that Src-mediated phosphorylation of TepP would regulate the recruitment of SH2 and PTB domain proteins like PI3K (p85 subunit) and CrkL. We infected SYF and SYF+Src MEFs with wild type C. trachomatis and assessed the recruitment of PI3K and CrkL to nascent inclusions by immunofluorescence microscopy.
Surprisingly, Src kinases were not required for the recruitment of PI3K or CrkL to inclusions (Fig. 3C). As a complementary approach, we generated phenylalanine substitutions at the three tyrosine (Y43 Y496 and Y504) identified as TepP phosphorylation sites (6). The TepP Y43F/Y496F/Y504F variant was still capable of pulling down PI3K and CrkL and the mutant strain recruited these factors to early inclusions.
However, this TepP variant was still tyrosine phosphorylated during infection (Fig. S1), suggesting that additional tyrosine phosphorylated residues in TepP may mediate interactions with host proteins.
Taken all together, this data suggests that while Src kinases are important for the full phosphorylation of TepP, these modifications do not appear to be essential determinant for TepP to interact with PI3K or CrkL in infected cells.

PI3K association with TepP at early inclusions is independent of CrkL.
PI3K and Crk adaptors are important signal transducers in growth factor mediated activation of receptor tyrosine kinases (29, 30). Both p85 and CrkL have SH3 and SH2 domains that mediate protein-protein interactions. Proteins that bind to Crk proteins at both the SH2 and SH3 domains include p85 itself, paxillin, C3G, Abl, Arg, Cas, and Sos (19,22,(30)(31)(32). Similarly the p110 and p85 PI3K subunits interact with a variety of kinases including Fyn, Lyn,Src,Fak,and Bcr (33,34). Because CrkL and PI3K can interact with each other (19,22,31) we tested if the association of these proteins with TepP is co-operative. We generated HeLa cell lines lacking the p110α subunit of PI3K, CrkL, or CrkI/II by CRISPR/Cas9-mediated gene disruption (Fig. 4A). These cells were infected with wild type C. trachomatis and the degree of co-localization of PI3K and Crk proteins with nascent inclusions was determined by indirect immunofluorescence. We did not detect any major differences in the efficiency of co-localization of the bacteria with PI3K in CrkL-deficient HeLa cells and vice versa (Fig. 4B) (Fig. 4C). However, we did not observe differences in the generation of EBs between wild type and TepP-deficient Chlamydia among the various knockout HeLa cell lines, suggesting that the role of PI3K and Crk proteins in promoting Chlamydia replication, at least in HeLa cells, occurs independently of their interactions with TepP.

PI3K modulates TepP-dependent Type I IFN responses.
PI3K and CrkL participate in the activation of immune signaling (32,37,38). CrkL can interact with STAT5 to regulate the translocation of this transcription factor to the nucleus and the expression of Type I interferon-induced genes (32, 39). We tested if PI3K and Crk adaptors were required for the TepP-dependent induction of IFIT genes.
PI3K, CrkL and Crk I/II deficient HeLa cells were infected with either wild type or Δ tepP C. trachomatis and IFIT1 and IFIT2 expression was assessed at 16 hours post infection by quantitative PCR. CrkI/II and CrkL-deficient HeLa cells did not display any significant differences in their response to either wild type or mutant Chlamydia. In contrast, PI3K deficient cell lines displayed an exaggerated IFIT response to wild type bacteria (Fig. 5).
These cell lines also displayed a stronger response to cytoplasmic DNA, suggesting a broader role for PI3K activity in modulating response to cytosolic nucleic acids. Similar observations were made in HeLa cells treated with the PI3K inhibitor LY294002 in response to both DNA and Chlamydia infection (Fig. S2). Overall, these observations imply that one of the functions of p110α at nascent inclusions is to dampen Type I IFN response in response to TepP-expressing Chlamydia.
Activated Akt is an important regulator of multiple pro-survival signals (21,27,41). Upon C. trachomatis infection of HeLa cells, phosphorylation of Akt at Ser473 occurs with a bimodal distribution with early (<1h) and late (>12h) peaks of activation (42). We next tested if TepP contributed to the activation of PI3K and Akt. HeLa cells were infected with either wild type or Δ tepP C. trachomatis and the levels of Akt activation assessed by immunoblot analysis with anti phospho-Akt antibodies. We observed low levels of p-Akt levels at 4h post-infection and this activity was dependent on PI3K activity as the signal was blocked by pre-incubation with the inhibitor LY294002 (Fig. 6A). Infection with Δ tepP bacteria did not significantly alter in pAkt levels as compared to wild type C.
trachomatis suggesting that PI3K activation at the plasma membrane is not overtly affected by TepP. The activation of Akt during Chlamydia infection is mostly mediated by the p110α isoform as no phospho-Akt signal was detectable in p110α KO cells (data not shown).
Although Akt phosphorylation is a common readout of increased activation of Class I PI3Ks, additional co-factors like PDK1 are required to phosphorylate Akt (21, 43). The most direct measurement of PI3K activity is the generation of PIP3. To assess the levels and spatial distribution of PIP3 pools in infected cells, we transfected cells with constructs expressing EGFP fusions to the PH domains of Btk and Akt that specifically bind to PIP3 (44). We observed an accumulation of PIP3 positive intracellular puncta in cells infected with wild type but not Δ tepP C. trachomatis ( Fig. 6C-E). These puncta were observed in the vicinity of nascent inclusions but not always completely overlapping (Fig. 6D) and were not observed in PI3K deficient cells, suggesting that TepP mediates the localized formation of PIP3 though the activation of PI3K activity (Fig.   6E).

Discussion
The process of Chlamydia entry into cells and establishment of a nascent inclusion is mediated by the controlled reorganization of the cytoskeleton and membranes by effector proteins. We determined that the early secreted effector protein TepP recruits the adaptor protein CrkL and the lipid kinase PI3K, leading to the localized synthesis of PIP3 on early inclusions ( Fig. 1 and 2). Based on TepP's ability to stably recruit PI3K, CrkL and CrkI/II proteins, and the role that Crk proteins play as adaptors linking receptors to signaling outputs, we hypothesize that TepP acts as a scaffold for the localized reprogramming of signaling proteins early during infection.
We generated a strain with a bla gene insertion in tepP in a clean genetic background to assess the phenotypic difference between wild type C. trachomatis and strains deficient only for the expression of TepP. All TepP dependent phenotypes described previously in a chemically derived tepP mutant (6) were validated using this new deletion strain and further allowed us to reveal a contribution of TepP to bacterial replication in A2EN cells. A2EN cells were recently derived from cervical tissue explants and maintain phenotypic and functional characteristics of endocervix epithelial cells, including cell polarization, mucus secretion, and expression of anti-microbial peptides in response to microbial TLR agonists (45).

Previous genome wide RNAi screens aimed at identifying host factors important for
Chlamydia proliferation identified PI3K and Crk proteins as important for Chlamydia entry and replication (35,46). These growth defects were confirmed in HeLa cells where p110α and Crk genes had been inactivated through CRISPR/Cas9-mediated gene editing, with p110α-deficient lines showing the greatest impairment (Fig 4). These host proteins may contribute to pathogen replication by facilitating entry, activation of pathways important for nutrient acquisition, and/or inactivation of cell defense mechanisms. However, although TepP is required for the recruitment of CrkL and PI3K to nascent inclusions early in infection, their contribution to enhancing bacterial replication in HeLa cells does not appear to occur through its interaction with TepP as Δ tepP mutants replicated to similar levels as wild type C. trachomatis in HeLa cells where Crk and PI3K had been inactivated (Fig. 4).
We expected that most host factors would specifically associate with phosphorylated versions of TepP. This appears not to be the case as the interaction between TepP and PI3K and CrkL, at least within the limits of our assays, was not affected by Src-mediated phosphorylation of TepP or by mutation of the three major tyrosines that are phosphorylated ( Fig. 3 and S1). It is possible that other tyrosine kinases or additional tyrosine phosphorylation sites may independently contribute to the recruitment of PI3K, Crk adaptors, and/or other SH2 and PTB domain-containing proteins. Because Crk proteins bridge phosphorylated RTKs to p85 to activate PI3K (33), we expected that the binding of the p85/p110 complex to TepP would be mediated by CrkL. However, we find that recruitment of these proteins to early inclusions occurred independently of each other. The influenza viral protein NS1 independently binds to p85 and CrkL to activate PI3K activity (47, 48). TepP could be performing a similar function by assembling a tripartite Crk-TepP-PI3K complex that potentiates kinase activity as exemplified by the marked accumulation of PIP3 positive vesicles at the periphery of early inclusions.
Alternatively, because the presence of TepP increased the efficiency of PI3K co-IP with CrkL ( Fig 1C) there's the possibility that Crk proteins further escalate the recruitment of additional p85/p110 to early inclusions and to enhance PI3K activity. We note that the activation of PI3K activity by TepP at intracellular sites did not lead to increased phosphorylation of Akt, suggesting that the activity of this kinase is uncoupled from events that may occur at the plasma membrane.
The prolonged recruitment of PI3K and Crk proteins on early inclusions that have migrated to perinuclear regions of infected cells correlates with the kinetics of TepP translocation after EB entry into cells (6). We predict that this recruitment and activation is independent of events that occur during the initial stages of infection (<1h) where engagement of RTKs like the EGFR (49), EphrRA2 (50) and PDGFR (35), and/or tyrosine phosphorylated Tarp may transiently recruit Crk proteins and PI3K to entry sites. The consequence of PI3K activation by TepP is the localized production of PIP3 at and in the vicinity of internalized bacteria (Fig. 6), which we predict lead to the recruitment of specialized PH domain-containing proteins that will further influence downstream signaling and/or cytoskeletal and membrane remodeling events. It remains to be determined how TepP orchestrates PI3K activity, Crk and GSK3 binding with the potential recruitment of additional factors. But the ultimate consequences of a failure to recruit all (or a subset of these factors) is a decrease in Chlamydia fitness in cervical epithelial cells and a decrease in the transcription of genes associated with Type I IFN responses.
Although PI3K and Crk proteins are important for full bacterial replication, as assessed by reduced growth in cells where these factors have been deleted, their role in promoting Chlamydia replication does not appear to be mediated by TepP, at least in HeLa cells. PI3K, but not Crk proteins, however, does appear to influence the magnitude of IFN responses to TepP-deficient bacteria (Fig. 5). PI3K knockout HeLa cells had a >5 fold in IFIT expression upon infection with TepP-deficient bacteria. PI3K regulates innate immunity in a variety of ways (48, 51) and inhibition of PI3K in dendritic cells enhances IFN-β transcription (52). It is possible that the absence of TepP increases the availability of microbial products from early inclusions to innate immune sensors or that PI3K activity modulates the function of sensors, which is consistent with the finding that basal expression of IFIT genes increases 2-3 fold in response to cytosolic DNA (Fig 5).
Overall, our findings begin to delineate important molecular components that are specifically assembled on C. trachomatis early inclusions that are ultimately important for fitness of the pathogen and for dampening of innate immune signaling in response to infection. The molecular players downstream of increased PI3K at intracellular sites and how they modulate the expression of immunity genes and bacterial survival in cervical epithelial cell remains to be determined.
The total number of infected progeny released (output) was divided by the total number of input infectious particles. All statistical analysis was performed using Graphpad Prism.

In vitro kinase Assays
His-TepP was purified from E.coli BL21 strain, grown in LB broth, with plasmid induction with IPTG (Sigma, 367-93-1) at a concentration of 0.5 mM for 4 hours at 37°C. Cells were lysed with lysis buffer (5M NaCl, 10% Triton-X100, 1M Imidazole, 250 mM PMSF, and 20 mM Phosphate) supplemented with Lysozyme (ThermoFisher Scientific, 89833), with sonication to reduce viscosity and centrifugation to pellet cell debris. Purification of His-TepP was preformed using nickel resin (ThermoFisher Scientific, 88221) and endover-end incubation at 4°C for 1 hour. Resin was washed 3X with lysis buffer and subsequently washed 3X with in vitro kinase buffer (1M Hepes, 2M MgCl₂, 250mM KCl, 1ATP, PMSF, Halt protease inhibitor cocktail (ThermoFisher Scientific, 78430), and cOmplete phosphatase inhibitor cocktail (Sigma, 4693159001)). Bound His-TepP was then incubated with whole cell lystates, lysed with in vitro kinase buffer and light sonication, from Vero, MEF, SYF, c+Src, Abl/Arg, Abl/Arg +Abl/Arg cells for 1 hour with end-over-end mixing at 4°C. Bound TepP was then washed 5X with in vitro kinase buffer without ATP. His-TepP was eluted from the nickel resin by incubation with Laemmli sample buffer and boiling. Levels of phosphorylation were assessed by immunoblot staining with anti-phosphotyrosine (Cell Signaling, 9411) and TepP levels assessed by immunoblot staining with anti-TepP (6).

Indirect immunofluorescence microscopy
A2EN cells were seeded at a density of approximately 8 x 10 4 cells/well on glass coverslips pre-coated with 30 μ g/mL collagen in 20 mM acetic acid (Spectrum) for 5 min and rinsed twice with fresh media. HeLa and MEF cells were seeded at a density of 5 x 10 4 cells/well on glass coverslips. The following day, CTL2 EBs were added at the indicated MOIs (20 or 100).
Infections in A2EN cells were synchronized by centrifugation (500 x g for 5 min) at 10°C whereas infections in HeLa cells and MEFs were synchronized by centrifugation (1,000 x g for 20 min) at 10°C.The media was replaced and the infected cells transferred to a 37°C, 5 % CO 2 humidified incubator.
Coverslips were fixed with ice-cold 100% methanol for 15 min, rehydrated with PBS (3 x 5 min washes), and blocked with 2% BSA in PBS for 20 min To assess the levels of PIP3 on early inclusions, HeLa cells were transfected with a plasmid encoding PH-Akt-GFP. After 24h, cells were infected with CTL2 or CTL2

Image Processing
All images were processed using ImageJ open source software (ImageJ). Linear adjustments were made for all images and max projections from Z-stacks are portrayed.
Quantification of recruitment of proteins to the nascent inclusion was performed in

RNA isolation and RT-qPCR
RNA was collected from 3 wells of a 6-well plate of infected cells at 16 hpi using the RNeasy plus mini kit (Qiagen, 74134). A2EN, HeLa-Cas9, and HeLa-Cas9 KO cell lines were seeded 24 hours prior to infection at a density of approximately 0.8 x 10 6 cells per well. The cells were then infected with gradient purified EBs of either CTL2 or CTL2 Δ tepP::bla strains at an MOI of 10. As controls, cells were also mock infected or transfected with 5ug of a linearized bacterial plasmid (pET-24d), using jetPRIME transfection system (Polyplus-transfection, VWR 89129-922). RT-qPCR was conducted using the Power SYBR Green RNA-to-CT™ 1-Step Kit (Thermo-Fisher Scientific). To assess differential gene expression of the IFIT1-2 (primers described in (6)

Generation of CRISPR/Cas9 KO cells
HeLa

Generation of TepP point mutants
TepP tyrosine phosphorylation mutants were generated using site directed mutagenesis with the Q5 site-directed mutagenesis kit (New England Biolabs, E0554S). Each tyrosine site (Y43, Y496, and Y504) was mutated individually to phenylalanines in the +pTepP vector (6). Each version of the plasmid was transformed into C. trachomatis L2-M062G1 (TepP deficient strain) using a modified version of the CaCl₂ transformation protocol previously described (54). Expression and translocation of TepP variants was verified with FLAG anti-bodies in immunoblot and immunofluorescence staining.

LY249002 Inhibitor treatment, RNA isolation, and RT-qPCR
HeLa-Cas cells were seeded 24 hours prior to infection at a density of approximately 0.8 x 10 6 cells per well. Cells were incubated with DMSO (solvent only control) or the PI3K kinase inhibitor LY294002 (Cell Signaling, 9901S) at a concentration of 20μM/mL for 1 hour prior to infection. Cells were then infected with gradient purified CTL2 or CTL2ΔtepP::bla EBs at an MOI of 10. As a control, cells were also mock infected or transfected with 5ug of a linearized bacterial plasmid (pET-24d) using jetPRIME transfection system (Polyplus-transfection, VWR 89129-922). RNA was collected from three wells of a 6-well plate at 16 hpi, DMSO or inhibitor treatment was maintained throughout infection. RT-qPCR was conducted using the Power SYBR Green RNA-to-CT™ 1-Step Kit (Thermo-Fisher Scientific). To assess differential gene expression of the IFITs (primers described in (6)  ( 2  0  0  6  )  R  e  c  r  u  i  t  m  e  n  t  o  f  B  A  D  b  y  t  h  e  C  h  l  a  m  y  d  i  a  t  r  a  c  h  o  m  a  t  i  s  v  a  c  u  o  l  e  c  o  r  r  e  l  a  t  e  s  w  i  t  h  h  o  s  t  -c  e  l  l  s  u  r  v  i  v  a