Chlamydia trachomatis attachment to cells induces the secretion of the elementary body–associated protein TARP (Translocated Actin Recruiting Protein). TARP crosses the plasma membrane where it is immediately phosphorylated at tyrosine residues by unknown host kinases. The Rac GTPase is also activated, resulting in WAVE2 and Arp2/3-dependent recruitment of actin to the sites of chlamydia attachment. We show that TARP participates directly in chlamydial invasion activating the Rac-dependent signaling cascade to recruit actin. TARP functions by binding two distinct Rac guanine nucleotide exchange factors (GEFs), Sos1 and Vav2, in a phosphotyrosine-dependent manner. The tyrosine phosphorylation profile of the sequence YEPISTENIYESI within TARP, as well as the transient activation of the phosphatidylinositol 3-kinase (PI3-K), appears to determine which GEF is utilized to activate Rac. The first and second tyrosine residues, when phosphorylated, are utilized by the Sos1/Abi1/Eps8 and Vav2, respectively, with the latter requiring the lipid phosphatidylinositol 3,4,5-triphosphate. Depletion of these critical signaling molecules by siRNA resulted in inhibition of chlamydial invasion to varying degrees, owing to a possible functional redundancy of the two pathways. Collectively, these data implicate TARP in signaling to the actin cytoskeleton remodeling machinery, demonstrating a mechanism by which C. trachomatis invades non-phagocytic cells. Chlamydia trachomatis is the causative agent of the most prevalent bacterial sexually transmitted disease in industrialized nations, and of the preventable blinding condition trachoma in developing countries. Survival and replication of chlamydial species occur exclusively inside a host cell, and thus, gaining access to the protective intracellular niche is an absolute requirement. This report describes how the chlamydia protein TARP, which is secreted at the base of the bacteria and across the host membrane, acts as a scaffold to which host signaling proteins bind. This assembly of the complex of signaling proteins, which include Sos1, Abi1, Eps8, and Vav2 results in the remodeling of the host cytoskeleton to facilitate engulfment of the infecting chlamydia. We conclude that these proteins have a role in chlamydia based on a number of observations including their interaction with the TARP protein, their ability to switch on known signaling participants in chlamydia invasion, their localization at the site of chlamydia entry, and the inhibition of chlamydia invasion in their absence. Altogether, the data functionally link TARP with signaling pathways that function in chlamydial invasion, demonstrating the direct involvement of TARP in the invasion of host cells by C. trachomatis.The human pathogen  Chlamydia trachomatis are primarily pathogens of humans. Serological variants (serovar) A, B, Ba, and C are the etiologic agents of trachoma, the leading cause of preventable blindness worldwide. Serovars D to K are associated with sexually transmitted diseases, and serovars L1, L2, and L3 cause lymphogranuloma venereum, a more invasive sexually transmitted disease. Chlamydophila psittaci is a zoonotic agent that causes a pneumonia-like respiratory disease in humans. Chlamydophila pneumoniae is a causative agent of community-acquired pneumonia, and has recently been associated with cardiovascular diseases. The genii Chlamydia and Chlamydophila share several biological properties, including a biphasic developmental cycle that includes cell types adapted for extracellular survival (elementary bodies or EBs) or intracellular multiplication (reticulate bodies or RBs) Chlamydiae are obligate intracellular bacterial pathogens that are responsible for a number of human diseases Because of the obligate intracellular nature of chlamydiae, access to the inside of the cell is paramount to survival. To this end, Chlamydiae have evolved to efficiently invade non-phagocytic cells through a process that has been described as “parasite-specified phagocytosis” Chlamydial invasion is initiated by the electrostatic and reversible interaction of EBs mediated through host heparan sulfate-like proteoglycans, followed by an irreversible host-dependent step that leads to internalization of EBs C. trachomatis urogenital isolate, serovar D, containing three repeat units and an LGV strain with almost six full repeat units Recently, a chlamydial protein associated with the uptake of EBs was found to be translocated by a type III secretion system into the host cell at the site of entry YEPISTTENIYESIDDSSTSDPENTSGGAAALNSLRGSSYSNYD, with the relevant tyrosines underlined -containing adaptor proteins . The prederlined . These tExpression of the fusion proteins was allowed to continue for 24 h post-transfection. The cells were incubated for 2 h with 4.5 uM beads coated with anti-CD4 antibody molecules to induce aggregation of plasma membrane-localized CD4-1xR fusion proteins. The highly localized tyrosine phosphorylation in vivo were determined to be Tyr179 or Tyr189, using a numbering system that starts with the Asp174 residue of the second repeat of the serovar L2 TARP homolog. The tyrosine residues in the CD4-1xR fusion protein that were actually phosphorylated YEPISTTENIYESIDDSSTSDPENTSGGAAALNSLRGSSYSNYD were custom synthesized either as non-phosphorylated tyrosines (WT), individually phosphorylated tyrosines (pY1 and pY2), or in which phenylalanines have been substituted for the tyrosines (F1F2). The biotinylated oligopeptides were incubated with lysates from HeLa cells and the presence of molecules known to participate with Rac1 in signal transduction pathways, specifically the Rac guanine nucleotide exchange factors Vav2 and Sos1 were monitored by Western blotting. The specificity of Sos1 towards Rac1 is conferred by the Abi1 and Eps8 proteins Because one copy of the repeated unit is apparently sufficient to recruit actin and Rac1 in our cell culture assay, and the dependence of these activities on the phosphorylation of Tyr179 and Tyr189, it is hypothesized that the domain may act as a signaling platform to which host signaling molecules are recruited. To directly test this possibility, biotinylated oligopeptides with the sequence DAAAD33 analog, the Rac GTPase protein could be detected from the pulldown fractions from both pY1 and pY2. Thus, it appears that interaction of Rac with the phosphodomain of TARP via Vav2 required PI 3,4,5-P3.Interestingly, Rac was only detected in the pY1 fraction, coinciding with the presence of Sos1, Abi1, and Eps8 in the same fraction. While Vav2 was also present in the pY1, it is unlikely that Rac is binding to this protein, because Rac was not pulled down by pY2 despite the presence of Vav2. However, it has been reported previously that Rac association with Vav2 could be enhanced by the addition of PI 3,4,5-Pvice versa.A series of lysates was prepared from HeLa cells depleted of Sos1, Abi1, and Eps8, and the co-precipitation of these signaling molecules was performed to determine which proteins are required for the interaction of the complex with the pY1 oligopeptide . The dep3 appears to be necessary for optimum GEF activity of Vav2 towards Rac, the localized synthesis of this phospholipid at the site of chlamydial entry was investigated, using the probe BTK-PH-GFP, where the pleckstrin homology (PH) domain of Bruton's tyrosine kinase (BTK) was fused to GFP. This domain has been demonstrated to be specific for PI 3,4,5-P3C. trachomatis serovar L2, and monitored using live microscopy  and 2821 cpm vs. 28660 cpm (Cdc42)). The corresponding increases could be attributed to the presence of Vav2, which is a GEF for Rac, Cdc42, and RhoA. Indeed, for the exchange reaction using His-Rac1 as the target, Vav2 depletion by siRNA returned the 32P level to that of the untreated cell lysate, even in the presence of PI 3,4,5-P3. While a similar reaction was not performed using Cdc42 as the target, the significantly lower level of GEF activity in the absence of PI 3,4,5-P3 indicate that Vav2 may be involved as well. From the data, it appears that withdrawal of PI 3,4,5-P3 was functionally similar to the depletion of Vav2, as this GEF appears to be inefficient in catalyzing the exchange reaction in Rac1 and Cdc42 in the absence of PI 3,4,5-P3. Note that the remaining GEF activity found in the Vav2-depleted lysates was likely due to the presence of Sos1, because its depletion resulted in the further 10-fold decrease in retained 32P label. The pY1 oligopeptide contained Sos1 and Vav2 exchange activities.The different precipitates were evaluated for GEF activity towards purified His-tagged Rac1 or His-tagged Cdc42 . His-Rac3 -treated (31700 cpm) or untreated (7634 cpm) lysates. The former indicated the presence of a PI 3,4,5-P3 -dependent Vav2 activity, which was confirmed by the loss of retained label (870 cpm) when Vav2 was depleted even in the presence of PI 3,4,5-P3. Similar to pY1, the exchange reaction using Cdc42 as the target was stimulated by the addition of PI 3,4,5-P3, indicating the involvement of Vav2. That Vav2 depletion resulted in values that are similar to the background levels suggests that Vav2 is the only GEF towards Rac1 and Cdc42 in the pY2 pulldown. Interestingly, there was still a relatively high level of GEF activity in the pY2 pulldown from untreated lysates (7634 cpm) compared to background (722 cpm). This result was reproducible, and the significance of this observation is unclear. As expected, the oligopeptides in which the tyrosine residues have been replaced by phenylalanine failed to yield values that are statistically different from the negative controls. Taken together, the lysates pulled down by the phosphorylated peptides contained GEF activity towards His-Rac (pY1) and Cdc42 (pY1 and pY2), and that this GEF activity was due to the presence of Sos1 and Vav2, with the latter requiring the addition of PI 3,4,5-P3. The involvement of Cdc42 in the in vitro GEF reaction poses an interesting question as this GTPase has been shown not to be required in C. trachomatis invasion The pulldowns with pY2 yielded relatively high numbers with the PI 3,4,5-PPhalloidin and 4G10 antibodies also colocalize at the sites of entry, where TARP molecules are predicted to be translocated across the host plasma membrane Abi1 and Eps8 adaptor proteins, when in a complex with Sos1 can function as a Rac-specific guanine nucleotide exchange factor 3-kinase-independent GEF (Sos1/Eps8/Abi1) is truly functionally redundant to Vav2 with respect to chlamydial invasion. However, wortmannin treatment in conjunction with the depletion of Sos1 protein led to a significant decrease in chlamydial invasion efficiency. For comparison, cells treated with the actin filament destabilizing drug cytochalasin D were found to be the least able to support chlamydial invasionTo explore the possible cooperation between the two GEF activities in chlamydial invasion, Sos1-depleted cells were also treated with wortmannin, which is implicated in the Vav2-dependent activation of Rac. C. trachomatis invasion. Both the Sos1/Eps8/Abi1 multiprotein complex and Vav2 were found to associate with the phosphodomain of TARP in a phosphorylation-dependent manner. Optimal Rac activation by Vav2 in vitro also required the presence of the phospholipids PI 3,4,5-P3, which is generated at the site of chlamydial attachment by virtue of the interaction of the p85 subunit of PI3-kinase with the TARP phosphodomain. Localization to the sites of chlamydial entry and their functional involvement in chlamydial invasion demonstrate their importance in chlamydial invasion of non-phagocytic cells.Intracellular pathogens have evolved a number of different mechanisms to subvert the actin cytoskeleton and facilitate their uptake by the host cell C. trachomatis TARP are likely recognized by a number of signaling molecules containing Src homology 2 (SH2) domains, and thus act as a scaffolding to which the signaling proteins relevant to actin remodeling are recruited. The binding of the Vav2 GEF and the p85 subunit of PI3-kinase is likely due to their respective SH2 domains, which recognize phosphorylated tyrosines. There is, however, a level of specificity to this interaction as not all of the SH2 domain-containing proteins examined bound to the phosphorylated oligopeptides. For example, the adaptor protein Grb-2 failed to bind to either pY1 or pY2 oligopeptides at levels above background. Furthermore, depletion of Grb-2 by siRNA did not affect chlamydial invasion (data not shown).The multiple phosphotyrosine residues in the repeat region of C. trachomatis serovar L2 is Rac-dependent, and that WAVE2 and Abi1 are downstream effectors that activate the Arp2/3 complex It has been previously shown that invasion of HeLa cells by et. al. have previously reported the specificity of the Sos1 protein towards Rac is conferred by the Eps8 and Abi1 proteins Scita We have previously shown that expression of the dominant negative mutant of the Rac GTPase inhibited chlamydial invasion, and this inhibition correlated with the loss of actin localization at the sites of entry. The depletion of guanine nucleotide exchange factors that activate Rac, in essence is functionally equivalent to the dominant negative Rac mutant. Thus, it is likely that inhibition of the guanine nucleotide exchange factor activity towards Rac would result in the similar loss of actin localization at the sites of chlamydial invasion.C. trachomatis serovar L2 invasion on Rac1 seems to contradict our data 3 at the site of entry is extremely short-lived, resulting in the transient interaction of Vav2 with its downstream GTPase effectors. Thirdly, there is a precedent for a difference in the duration of signals transduced by GTPase protein. An excellent example is the difference in Rac and Ras signaling, in which Ras signaling, mediated by the Sos1/Grb-2 complex during growth factor stimulation, was short-lived relative to signaling by Rac, which was mediated by Sos1/Abi1/Eps8, under similar conditions of growth factor stimulation 3 may explain the apparent lack of Cdc42 involvement in chlamydial invasion. On the other hand, Rac1 could be activated by two distinct pathways; one of which is independent of PI 3,4,5-P3. This Rac activation pathway would be expected to predominate during conditions of high PI 3,4,5-P3 turnover. The presence of a PI 3,4,5-P3 -independent pathway of Rac1 activation would be expected to confer a wortmannin-insensitive invasion mechanism. Indeed, entry of serovar L2 is insensitive to treatment with 100 nM wortmannin. During inhibition of PI 3,4,5-P3 synthesis, the Sos1/Abi1/Eps8 pathway of Rac activation remains functional, with the host cell still supporting chlamydial uptake.Vav2 demonstrated GEF activity towards both Rac1 and Cdc42. Our results are consistent with previous data that characterized Vav2 specificity 3 at the sites of chlamydial entry. This implies the recruitment of the regulatory subunit (p85) of PI3-kinase and its binding partner, the catalytic (p110) subunit. Indeed, the p85 subunit was found to bind preferentially the pY1 oligopeptide. In conjunction with the observed interaction of Vav2 with the pY2 oligopeptide, these two components may bind independently to two distinct binding sites, but cooperate to activate the Vav2-dependent pathway. That the PI3-kinase p85 subunit displayed the same preferential binding to pY1, as the Sos1/Abi1/Eps8 complex is suggestive of a competition or a hierarchical control that possibly determines which pathway is utilized for chlamydia uptake. The reproducible decrease in invasion efficiency during depletion of the Sos1 protein indicate the preferential utilization of this pathway compared to the Vav2 pathway. In addition, because TARP homologs within the C. trachomatis species have at least two phosphodomains, it is intriguing to speculate that the domains cooperate with each other to recruit signaling molecules, and bring into proximity components to stabilize the signaling complex and/or transduce the desired signal. However, the presence of multiple potential binding sites adds another level of complexity to this signaling process in the regulation of this signaling pathway.We observed highly localized and transient bursts of synthesis of PI 3,4,5-PChlamydophila species that possess TARP homologs, but do not contain the repeated phosphodomain clearly demonstrate that alternative mechanisms of actin remodeling and recruitment exist. An attractive scenario is the requirement for an additional bacterial factor that interacts with a conserved region of TARP and substitute for the tyrosine phosphorylation during recruitment of signaling molecules. The enterohemorrhagic E. coli (EHEC) EspFu/TccP protein directly binds to the GTPase binding domain of N-WASP to induce pedestal formation independent of Tir tyrosine phosphorylation and Nck recruitment C. trachomatis serovar L2 depends on Arp2/3. A model that we favor is a cooperative one, in which the activation of the Arp2/3 complex by the Rac-dependent signaling pathway initiates actin nucleation forming short actin filaments. These nascent filaments are then bound by the WH2 motifs within the C-terminal domain of TARP, nucleating spontaneous actin polymerization. This model fits a number of critical observations – a) C. trachomatis invasion is Arp2/3-dependent; b) actin polymerization can be mediated by a minimal TARP domain that contains the WH2 motifs in an Arp2/3-independent manner; and c) the C-terminal domain has a higher affinity for F-actin. How these two mechanisms cooperate is under investigation.Equally important is how the actin nucleating function of the conserved C-terminal domain of TARP fit with the model that requires signaling to the Arp2/3 complex to activate its actin-nucleating activity. It is unlikely that the Arp2/3-independent actin nucleating activity of the C-terminal domain of TARP is sufficient for chlamydia-induced actin recruitment and invasion. It has been reported that the invasion of Chlamydia trachomatis Type III effector TARP in invasion by identifying the host signaling molecules that link TARP to the actin remodeling machinery. The potential for TARP to be differentially phosphorylated at the two tyrosine residues described in this report and the presence of multiple phosphodomains together imply the presence of a control mechanism that fine tunes the function of TARP in chlamydia invasion. This modulation may be at the level of phosphorylation, binding of the signaling complexes, their respective stability, or the ability of the signaling pathway to cooperate with the nucleating function of the C-terminal domain of TARP. Clearly, many questions still need to be answered to gain a full understanding of the invasion process of this medically important obligate intracellular pathogen.In summary, this report is the first to directly implicate the C. trachomatis serovars L2 (LGV-434) were grown in and harvested from HeLa 229 cells as previously described Antiphosphotyrosine monoclonal antibody (MAb) clone 4G10 and FITC-conjugated 4G10 were purchased from Upstate USA ; anti-Sos1 monoclonal, anti-Abi1 monoclonal, anti-p85, and anti-Eps8 polyclonal antibodies were from and Abcam; anti-Vav2 rabbit polyclonal antibody was from Zymed ; and anti-Rac1 monoclonal antibody was purchased from Cytoskeleton. Secondary antibodies for immunoblotting were horseradish peroxidase-conjugated anti-mouse or anti-rabbit . GFP-actin , GFP-BTK-PH  and GFP-Rac  were described previously 5′-ATGGTACCATGACGAATTCTATATCAGGTG-3′ and the BamH1 linker-containing reverse primer 5′-ATGGATCCTGTTCTCCTTTGCTTTTTA-3′ with the PCR product digested with KpnI (New England Biolabs) and BamH1 (New England Biolabs) for an overnight ligation into the pEGFP-C1 vector (Clontech), which was pre-digested with the same restriction enzymes and dephosphorylated using calf intestinal alkaline phosphatase (New England Biolabs). Ligation was performed at 15°C for 16 h.Synthesis of pEGFP-C1 L2 TARP was performed as follows. The full length L2 TARP gene was amplified using KpnI linker-containing FWD primer 5′-CACCATGAACCGGGGAGTCCCTTTT-3′ and 5′-AAGCTTCTTCTACGGATCGGGTTACTT-3′ into the pcDNA3.1 TOPO vector . The resulting vector was digested with Kpn1 to accommodate the PCR fragment containing one repeated unit (amino acids 174–222) of the TARP phosphodomain region. This PCR fragment was generated by the amplification of the pEGFP-C1 L2 TARP using the primers 5′-ATGGTACCCTTCAGAAAGCTCAGAAACTA-3′ and 5′-ATGGTACCGTAGGAGGAGCCTCTTAGA-3′ containing Kpn1 linker sequences. The orientation of the insert relative to the the CD4 reading frame was determine by colony PCR using the primers CD4 forward primer 5′-CACCATGAACCGGGGAGTCCCTTTT-3′ and 5′-CTTAGTCATCAATACTCTCATAAATATTTTCAGTAGTGCTTATCG-3′, which annealed to an internal region of the 1xR sequence.The N-terminal domain of CD4 (amino acids 1–372) was subcloned from pCMV-Sport XL5-CD4 purchased from Origene by PCR using the primers HeLa 229 or Cos7 cells were seeded on 12-mm glass coverslips in 24-well plates to obtain a monolayer of approximately 50% confluence. Transfections of plasmid constructs were performed using FuGene  according to the manufacturer's instructions. The transfection mixture was prepared as follows. The FuGene reagent (3 µl was diluted into 97 µl Optimem (Invitrogen) serum-free media, and to which DNA (1.0 µg) and added. After a 20-minute incubation at room temperature, the complexes were added to 1 well of a 24-well plate containing 100 µl of Optimem. The transfection cocktail was incubated at 37°C. After 4 hours, the transfection medium was removed and antibiotic-free RPMI media with 10% fetal bovine serum was added. Expression from the transfection vectors was allowed to proceed for 24 hours at 37°C.siRNA transfection with the transfection reagent Ribojuice was performed per the manufacturer's instructions. Briefly, 5 nM siRNA was incubated with 4 ul (for a well in a 24-well plate) or 100 ul (for a 100 mm dish) of Ribojuice in OPTI-MEM and incubated at room temperature for 30 min. HeLa cells at 80% confluency were washed once with 1× HBSS and incubated in 100 ul or 1 ml OPTI-MEM. The siRNA transfection solution was added to the cells and incubated for 4 h. The transfection medium was removed and replaced with complete RPMI . At 48 h post-transfection, the levels of the proteins of interest were evaluated by Western blot.Briefly, post-nuclear supernatants were prepared as described previously Proteins were separated on a 10.5–14% continuous gradient sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels  and transferred to a 0.45-µm Trans-blot nitrocellulose membrane (Bio-Rad). Immunoblots were developed using Super Signal West Femto chemiluminescence reagent  per the manufacturer's instructions.in vitro GDP-GTP exchange assay 32P]GTP  and 83 pmol of cold GDP (10-fold excess over labeled), and 1 µg of Rac-His or Cdc42-His (Cytoskeleton) were added to 200 µl of exchange buffer . 10 µl of this solution was added to the oligopeptide pulldown fraction. The reaction was incubated at room temperature for 30 min, and terminated by the addition of 1 ml of the stop buffer  and immediate incubation on ice. The beads containing the oligopeptide and associated proteins were pelleted , and the supernatant were loaded onto nitrocellulose filters using a vacuum manifold (Bio-Rad). The filters were washed three times with ice cold PBS with 5 mM MgCl2. Radioactivity retained on the filters was counted by scintillation.The oligopeptide pulldown fractions described above were subjected to an Cells grown on coverslips were fixed in freshly prepared 4% paraformaldehyde in PBS for at least 1 h at 4°C. If required, the fixed cells were permeabilized with 0.1% Triton X-100 in PBS for 2 min at RT. The permeabilization buffer was removed and the cells rinsed three times with 1× PBS. Primary antibodies were diluted to their respective working concentrations (α-Sos1 1∶250 (Abcam), α-Abi1 1∶1000 (Abcam), α-Eps8 1∶250 (Abcam), α-Vav2 1∶500 (Zymed), α-L2 EB, 1∶1000 from Ted Hackstadt, RML, NIAID), added to the fixed cells, and incubated at 4°C overnight. Anti-rabbit or anti-mouse IgG secondary antibodies used were conjugated to either Alexa 488 or Alexa 594 (Invitrogen). Coverslips were mounted using Mowiol on glass slides, and samples visualized using the Olympus Fluoview 500 Laser Scanning Microscope. Images were processed using Adobe Photoshop Creative Suite.C. trachomatis LGV serovar L2 EBs, and observed at 5 s interval using an UltraView Live Cell Imaging system fitted with a Bioptechs Delta T4 objective heater. Images were assembled into a time lapse Quicktime movie using NIH ImageJ Cells grown on Delta T culture dish (0.17 mm thick) (Bioptechs) overnight and transfected with a GFP-BTK-PH expression construct. At 18 h post-transfection, the cells were infected with CMTPX-labeled Assay for invasion was performed as described previously Figure S1Scanning electron micrograph of cells mock-treated (left), treated with 4 µM cytochalasin D (middle), and 30 min after cytochalasin D has been removed. Removal of the F-actin-destabilizing drug resulted in the preferential reformation of microvilli at the sites of chlamydia attachment, indicating the induction of signaling directly underneath the EBs and the restriction of lateral mobility of the signaling complex. Scale bar = 1 µm.(2.04 MB TIF)Click here for additional data file.Figure S23 at the sites of chlamydial entry. Cos-7 cells expressing the pleckstrin homology domain of Bruton's tyrosine kinase fused to green fluorescent protein (BTK-PH-GFP) were monitored by live cell microscopy upon infection with CMTPX-labeled C. trachomatis serovar L2 elementary bodies. Images were acquired at 5 s intervals and compiled using the NIH Image J software. Note that the recruitment of the fluorescent probe is short-lived, indicating the transient nature of the signaling cascade. It is important to note that not all of the EB particles induced the synthesis of PI 3,4,5-P3 because there likely were non-infectious particles in the EB preparations. Loss of infectivity occurs during harvest, purification, and labeling of elementary bodies. A) Mock-treated cells; B) Wortmannin (100 nM)-treated cells.Localized synthesis of PI 3,4,5-P(3.72 MB MOV)Click here for additional data file.Figure S3C. trachomatis serovar L2 elementary bodies. Images were acquired at 5 s intervals and compiled using the NIH Image J software.Wortmannin (100 nM) treatment of Cos-7 cells expressing the BTK-PH-GFP probe abolished recruitment of the probe at the sites of attachment of CMTPX-labeled (3.57 MB MOV)Click here for additional data file.Figure S4Representative Western blots demonstrating the efficiency of depletion by siRNA of proteins of interest. HeLa cells were mock-transfected, transfected with a scrambled siRNA or cocktail of three siRNA for each target. Protein levels were analyzed by Western blot at 48 h post transfection.(1.01 MB TIF)Click here for additional data file.


Leptin is an adipocyte-derived hormone that acts via its hypothalamic receptor (LEPRb) to regulate energy balance. A downstream effect essential for the weight-regulatory action of leptin is the phosphorylation and activation of the latent transcription factor STAT3 by LEPRb-associated Janus kinases (JAKs). Obesity is typically associated with chronically elevated leptin levels and a decreased ability of LEPRb to activate intracellular signal transduction pathways (leptin resistance). Here we have studied the roles of the intracellular tyrosine residues in the negative feedback regulation of LEPRb-signaling under chronic leptin stimulation.Mutational analysis showed that the presence of either Tyr985 and Tyr1077 in the intracellular domain of LEPRb was sufficient for the attenuation of STAT3 phosphorylation, whereas mutation of both tyrosines rendered LEPRb resistant to feedback regulation. Overexpression and RNA interference-mediated downregulation of suppressor of cytokine signaling 3 (SOCS3) revealed that both Tyr985 and Tyr1077 were capable of supporting the negative modulatory effect of SOCS3 in reporter gene assays. In contrast, the inhibitory effect of SOCS1 was enhanced by the presence of Tyr985 but not Tyr1077. Finally, the reduction of the STAT-phosphorylating activity of the LEPRb complex after 2 h of leptin stimulation was not accompanied by the dephosphorylation or degradation of LEPRb or the receptor-associated JAK molecule, but depended on Tyr985 and/or Tyr1077.Both Tyr985 and Tyr1077 contribute to the negative regulation of LEPRb signaling. The inhibitory effects of SOCS1 and SOCS3 differ in the dependence on the tyrosine residues in the intracellular domain of LEPRb. Leptin ernators . Mechaniernators -19. Regaernators . Howevervia the Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway. Leptin stimulation leads to the activation of JAKs that are constitutively associated with LEPRb and in turn phosphorylate tyrosine residues in the intracellular domain of the receptor [The leptin receptor belongs to the cytokine receptor superfamily and exists in several splicing variants. Leptin binding to the long leptin receptor isoform (LEPRb) activates cytokine-like signal transduction receptor . The phoreceptor ,17,21). receptor ,23.in vivo [in vivo and in cultured cells [via binding to specific phosphotyrosine motifs  was sufficient for the attenuation of STAT3 activation, and that either tyrosine residue can support the suppression of LEPRb signaling by SOCS3. Finally, we show that the receptor-associated JAK kinase has reduced STAT-phosphorylating activity after two hours of continuous receptor stimulation. However, the kinase is not dephosphorylated or degraded, and the inhibition of activity depends on the presence of Tyr985 and Tyr1077 in LEPRb.To examine the role of Tyr985 and Tyr1077 in the attenuation of LEPRb signaling during prolonged ligand stimulation, we determined the kinetics of leptin-induced STAT3 tyrosine phosphorylation in RINm5F cells. We have previously established the insulinoma cell line RINm5F as a model system to define the differential effects of the intracellular tyrosines in LEPRb signal transduction and their effects on gene expression ,44. We uWe determined the surface expression of LEPRb in untreated and leptin-treated RINm5F cells to examine whether leptin signaling was downregulated by receptor internalization and/or degradation. After 2 h of leptin treatment, binding assays showed only a weakly reduced leptin binding to the cell surface, which could not account for the reduced STAT3 phosphorylation Figure . Next weNext we analyzed the roles of Tyr985 and Tyr1077 in the negative regulation of LEPRb signaling with the help of STAT3-dependent reporter gene assays. These assays were performed in the hepatoma cell line HepG2 to allow for ensuing knockdown experiments with shRNA vectors directed against human SOCS1 and SOCS3 (see below). As shown in Figure To determine the requirement of Tyr985 and Tyr1077 for the inhibitory effects of SOCS proteins, we studied the effects of SOCS3 and SOCS1 overexpression on the activation of the STAT3-dependent promoter by the LEPRb mutants. Both SOCS proteins were expressed from the same vector and at comparable levels  The presence of either Tyr985 or Tyr1077 in the cytoplasmic domain of LEPRb is sufficient for the attenuation of leptin-induced STAT3 phosphorylation Figure , suggestet al. , which s [et al. , we shows Figure . Whereass Figure , confirmet al. [Taken together, these results establish a role of pTyr1077 in the negative regulation of the LEPRb receptor complex, thus extending the prevailing view that Tyr985 is critical for the negative feedback regulation of leptin signaling. De Souza et al.  have detet al. [LeprLeu985 mice display a phenotype of increased leptin sensitivity, the effect of this mutation was only significant in females and under conditions of high fat diet [The capacity of Tyr1077 to substitute for the mutated Tyr985 provides an explanation for the Tyr985-independent attenuation of leptin signaling observed by Dunn et al. . Furtheret al. , as compet al. -54. In cfat diet . This mivs. SOCS3 SH2 domain [We have shown by direct comparisons that SOCS1 inhibits LEPRb→STAT3 signaling as potently as SOCS3. Furthermore, our results challenge the prevailing view that SOCS1 inhibits JAK kinases by directly binding to the activation loop, independent of receptor phosphotyrosine motifs ,38,55. E2 domain . Notably2 domain . Hence, 2 domain , criticae.g. IL-1β or IFNγ) rather than as a feedback mechanism. Interestingly, SOCS1 has been reported to be upregulated in adipose tissue in two different models of obesity associated with leptin resistance [The inhibitory effect of SOCS1 on LEPRb signaling has not previously been analyzed as a mechanism of leptin resistance, because leptin does not induce expression of SOCS1 . The dee.g. Hek293 [Taken together, we conclude from the available evidence and our present results that the first step in the attenuation of LEPRb→STAT3 signaling in cells chronically stimulated by leptin occurs through the fast upregulation of the negative feedback regulator SOCS3. SOCS3 is recruited to either pTyr985 or pTyr1077 in the receptor chain and inhibits the receptor-associated JAK molecules by direct interaction of its kinase inhibitory region (KIR) with the activation loop of the kinase. The next step is putatively the dephosphorylation of the JAK molecules and/or the receptor, which is a fast event in some cell systems (. Hek293 ) but wasRecombinant murine leptin was purchased from PeproTech  and recombinant human IL-1β from Roche Molecular Biochemicals . The following primary antibodies were used: polyclonal rabbit antibodies directed against p(Tyr701)-STAT1, p(Tyr705)-STAT3, STAT1, STAT3 , p(Tyr1007/Tyr1008)-JAK2  and SOCS3 ; goat anti mouse LEPR from R&D Systems , mouse monoclonal FLAG-M2 from Sigma-Aldrich , secondary horseradish peroxidase-labeled antibodies from Pierce Chemical Co. , and Dianova . Detection of immunoprecipitated SOCS3 was performed with biotinylated SOCS3-specific antibodies  and HRP-coupled anti-biotin antibody .2. HIT-T15 hamster insulinoma cells were cultivated under the same conditions except that 5% horse serum was added to the medium. Transient transfections were performed by the polyethyleneimine method . The RINm5F cell lines stably expressing WT or mutant LEPRb have been described previously [Rat RINm5F insulinoma cells and HepG2 hepatoma cells were grown in RPMI 1640 medium with L-glutamine, 10% (v/v) fetal bovine serum, 100 units/ml penicillin, and 100 mg/ml streptomycin at 37°C and 5% COeviously ,44. The RINm5F cells expressing WT LEPRb in 6-well plates  were serum-starved for 4 h before they were stimulated with leptin (20 ng/ml). Control cells were prepared identically but were not treated with leptin. After 2 h, cells were washed with ice-cold phosphate buffered saline (PBS) and then with ice-cold acetic acid  to remove receptor-bound leptin . After t2 macroglobulin gene  were transfected with 0.3 μg of pMET7-LEPRb expression plasmids  along wi-215Luc; ) and β-g-215Luc; ) were co32P-labeled probes were generated from the inserts of cDNA clones pEF-SOCS1 and pEF-SOCS3 by random oligonucleotide priming. Blots were rehybridized with additional cDNA probes following probe removal with 0.5% SDS at 90-100°C.RINm5F-LEPRb cells grown to 80% confluence were incubated in serum-free medium for 20 h before they were stimulated with leptin (100 ng/ml) or left unstimulated for variable times. Total RNA was isolated using the RNeasy Midi Kit , and samples (10 μg) of total RNA were separated by denaturing formaldehyde electrophoresis on 1% (w/v) agarose gels and transferred by capillary blot onto nylon membranes . 3VO4, 1 mM PMSF and 5 μg/ml each of aprotinin and leupeptin). Insoluble material was removed by centrifugation  before 12 μl of agarose-coupled c-myc antibody  was added to the supernatant and incubated for 4 h at 4°C. For detection of SOCS3, immunoprecipitations were performed with 1% Nonidet P-40 instead of Brij97 in the lysis buffer and 1 μg of SOCS3 antibody per 1 ml of lysate. The resin was washed twice with lysis buffer and twice with lysis buffer without detergent, and the bound proteins were eluted with denaturing gel loading buffer.For immunoprecipitation of LEPRb receptor complexes, RINm5F-LEPRb cells from a near-confluent 85-mm plate were lysed on ice for 30 min in 1 ml of native lysis buffer  Brij97, 1 mM EDTA, 10 mM NaF, 1 mM NaTotal cellular lysates for the detection of STAT3 tyrosyl phosphorylation were prepared using denaturing lysis buffer  and incubation in a boiling water bath for 5 min. For Western blot analysis, samples were separated by SDS-PAGE (8% gels), blotted onto nitrocellulose, and positive signals were revealed by chemiluminescence using the indicated primary antibodies and horseradish peroxidase- labeled secondary antibody. Blots were re-probed after removing the primary antibody by incubating the membrane in stripping buffer  for 20-30 min at room temperature.2, 5 mM MnCl2, 0.4 mM DTT, 1 mM Na3VO4, 5 μg/ml aprotinin, 5 μg/ml leupeptin). For the assay, 2.2 μg of recombinant STAT1 and 28 nmol ATP in 30 μl kinase buffer were added to the pellets . After incubation at 30°C for 45 min, supernatants were removed for Western blot analysis of STAT1 tyrosine phosphorylation. The resin with the bound receptor complexes was washed twice with wash buffer , eluted, and subjected to Western blot analysis.LEPRb complexes were immunoprecipitated using agarose coupled c-myc antibody (described above) and washed twice with kinase buffer  and the inhibitory effect in reporter gene assays and the expression level of SOCS3 in HIT-T15 cell .Click here for file


Flavonoids, a large group of polyphenolic metabolites derived from plants have received a great deal of attention over the last several decades for their properties in inflammation and allergy. Quercetin, the most abundant of plant flavonoids, exerts a modulatory action at nanomolar concentrations on human basophils. As this mechanism needs to be elucidated, in this study we focused the possible signal transduction pathways which may be affected by this compound. Methods: K2-EDTA derived leukocyte buffy coats enriched in basophil granulocytes were treated with different concentrations of quercetin and triggered with anti-IgE, fMLP, the calcium ionophore A23187 and the phorbol ester PMA in different experimental conditions. Basophils were captured in a flow cytometry analysis as CD123bright/HLADRnon expressing cells and fluorescence values of the activation markers CD63-FITC or CD203c-PE were used to produce dose response curves. The same population was assayed for histamine release.Quercetin inhibited the expression of CD63 and CD203c and the histamine release in basophils activated with anti-IgE or with the ionophore: the IC50 in the anti-IgE model was higher than in the ionophore model and the effects were more pronounced for CD63 than for CD203c. Nanomolar concentrations of quercetin were able to prime both markers expression and histamine release in the fMLP activation model while no effect of quercetin was observed when basophils were activated with PMA. The specific phosphoinositide-3 kinase (PI3K) inhibitor wortmannin exhibited the same behavior of quercetin in anti-IgE and fMLP activation, thus suggesting a role for PI3K involvement in the priming mechanism.These results rule out a possible role of protein kinase C in the complex response of basophil to quercetin, while indirectly suggest PI3K as the major intracellular target of this compound also in human basophils. Flavonoids include a large group of low molecular weight polyphenolic secondary plant metabolites which can be found in fruits and vegetables, and plant derived beverages such as tea, wine and coffee -3. More Aiming at understanding the bimodal mechanism by which quercetin acts on human basophils, in this study we investigated some basic signaling events in basophil activation, such as calcium, protein kinase C (PKC) and PI3K. From a molecular point of view, quercetin has a significant bulk of intracellular targets, mainly serine/threonine and tyrosine kinases, which is very difficult to disentangle . In baso3- ethylendiaminetetraacetic acid (EDTA), sodium heparin, trypan blue and distilled water  were all purchased from Sigma . Goat anti-human IgE was purchased from Invitrogen-Caltag Laboratories, (UK). Histamine enzyme-linked immunosorbent assay (ELISA) releasing test was purchased from Labor Diagnostika Nord GmbH & Co., Germany. Mouse anti-human monoclonal antibodies for flow cytometry evaluation CD123-PECy5 , CD45-APCCy7 (isotype IgG1 clone HI30), CD203c-PE (isotype IgG1 clone NP4D6), CD63-FITC (isotype IgG1 clone MEM-259) were purchased from Biolegend, San Diego CA, USA. HLA-DR-PECy7 (isotype IgG2a clone L243) was purchased from Becton Dickinson, Pharmigen CA, USA. Pure quercetin was dissolved in DMSO at a stock solution of 1 mg/ml and stored at +4°C for a maximum of 6 days; wortmannin was dissolved in DMSO at the stock concentration of 2 × 10-3 M, stored at -20°C and thawed before use. Working solutions were made into HEPES modified buffer  (HBE). fMLP was dissolved in dimethylsulfoxide (DMSO) as a 2 × 10-2 M stock solution, stored at -20°C and thawed before use. The calcium ionophore A23187 and PMA were both dissolved in DMSO as stock solutions of 1 mg/ml (1.91 mM and 1.62 mM respectively), stored at -20°C and thawed before use. Working solutions of fMLP, anti-IgE, A23187 and PMA were freshly prepared in HBE supplemented with 5 mM CaCl2 and 2 mM MgCl2 (HBC buffer). All reagents were pure and quality checked; whenever necessary disposable plastic ware and sterile apyrogenic solutions were used.N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), quercetin dihydrate (minimum 98% HPLC), phorbol-12-myristate-13-acetate (PMA), the ionophore A23187, the PI3K inhibitor wortmannin, NaA total of 70 blood donors volunteers  were enrolled in this study. Recruitment was randomized and encompassed an age range from 24 to 65 yrs (mean 44.61 ± 4.57 SD) in order to have a wide experimental population and to prevent age influence on cell releasability . All the2-EDTA anticoagulated peripheral blood from four screened healthy donors in each experiment performed, according to previously described methods [3-EDTA). Then the samples were put on ice and stained with monoclonal antibodies (20 minutes at +4°C), according to previously published methods [4Cl, 10 mM Na2HCO3, 0.10 mM Na3EDTA, pH = 7.2) for 4 minutes at +4°C; then samples were centrifuged at 700 g and pellets recovered and re-suspended in a PBS-buffered saline solution (pH 7.4) for flow cytometry reading.Basophils were collected as leukocyte-enriched buffy coats from venous K methods . Buffy c methods . The vol methods . AfterwaCells treated with different concentration of quercetin and activated with the indicated agonists, were pelleted at 6000 rpm for 5 minutes and surnatants collected for a competitive histamine ELISA test. 25 μl of each sample was treated with buffers and an acylation reagent, incubated for 1 hour at r.t. and diluted with 200 μl of distilled water. Aliquots of 20 μl of these acylated samples were incubated overnight with 100 μl of antiserum, washed 3 times, incubated for 1 hr at r.t. with a horseradish peroxidase-conjugate, washed 3 times with washing buffer, incubated for 30 min at r.t. with the colorimetric substrate tetramethylbenzidine and reaction stopped. The absorbance of the solution in each wells was read within 10 minutes at 450 nm with a reference wavelength of 620 nm. Histamine was calculated as ng/ml of the released amine against the corresponding standard concentrations in the calibration curve.positive cells) counted to the right of a threshold that was established including the main peak of fluorescence of a sample of resting cells. In order to reduce standard deviation due to positive fluorescent cells respect to negative or dimly ones, a logarithmic scale and a coefficient of variation to measure variability dispersion were used.Basophil membrane markers were evaluated by flow cytometry using a five-color fluorochrome panel including CD45-APCCy7, CD123-PECy5 and HLA-DR-PECy7 as phenotyping markers and CD63-FITC and CD203c-PE as activation ones . Flow anp < 0.05 was considered statistically significant. IC50 was calculated for each curve of percentage of effect/control by a linear regression calculation according to the four parameter logistic model (4PL), also called the Hill-Slope model.Data were analyzed using the software SPSS, version 11 for Windows, Chicago, IL. Dose response curves were obtained by plotting the triplicate data and their mean values and S.E.M. for each experiment using the Sigma plot 10 software. Kolmogorov-Smirnov and Shapiro-Wilk goodness-of-fit tests were performed to determine whether the sample population followed a Gaussian distribution. Differences between quercetin-treated and non-treated cells were analyzed by using a one-way analysis of variance (ANOVA) followed by Fisher LSD test. A value of 50 able to inhibit CD63 expression was about 0.132 μM  while IC50 was about 6-fold higher (0.775 μM) for CD203c. In basophils activated with formylated peptides the flavonoid inhibited the activation markers at concentrations higher than 1.0 μM while it enhanced the same response at the doses of 0.033 μM and 0.33 μM  and to diacylglycerol (DAG) from the precursor phosphatidylinosytol-4,5-biphosphate   which activates extracellular calcium influx by membrane Ca++ channels [Q1) Effects of quercetin in the FcεRI-anti-IgE activation model. Previously published reports have shown that quercetin is able to inhibit PI3K by binding to the catalytic pocket of the enzyme: as for instance, LY294002, a synthetic inhibitor of PI3K, has actually a chemical kinship with the flavonoid quercetin . The IC5.8-20 μM , which c.8-20 μM  which in) Figure . While D release . Warner channels .3-calcium signaling or by the activation of DAG-PKC pathway), so resulting in a priming effect.Q2) Effects of quercetin in the FPR-fMLP activation model. Figure Q3) Effect of quercetin on protein kinase C (PKC) activation pathway. In our assay system the flavonol proved insensitive to target protein kinase C (PKC), as resulted from the use of PMA as basophil stimulant, thus confirming previous reports : quercetQ4) Effect of quercetin on basophils triggered with calcium ionophore A23187. In response to calcium ionophore A23187 the expression of both the activation markers CD63 and CD203c was markedly up-regulated, but quercetin exerted a significant inhibitory action, even at nanomolar doses, only on CD63, so dissecting the response of the two activation markers to the ionophore. Previous evidence has reported that CD203c and CD63 upregulation in response to calcium signal by A23187 showed different kinetics , an evidThese hypotheses and models need for further investigation on a molecular level such as a direct demonstration of the kinases isolated from or detected in the purified basophils and/or by using isoform-selective inhibitors of PI3K and to assay calmodulin involvement in the A23187 activation pathway inhibited by quercetin. What is really interesting is that the observed modulatory biphasic (hormetic) mechanism can be related to the inhibition of PI3K by quercetin and that the efficacious doses are within the nanomolar plasma concentrations reported in several pharmacokinetic and bioavailability studies about this flavonoid -53. At tAllergy is a cause for concern, mainly due to its rising prevalence inside the population and to the increasing difficulty in treating chronic allergy. Quercetin might be a good candidate with the potential to counter this trend: an appropriate intake of this flavonol from food and beverage or from supplemental administration could be expected to improve allergy, to help anti-inflammatory and anti-oxidative responses by the organism and to prevent the onset of allergic chronic diseases. However our results introduce a caveat: although basophils play an important role in mediating allergic response and quercetin has proved to have an inhibitory action on basophils following stimulation with anti-IgE and calcium ionophore A23187, the existing bimodal effects of the flavonol and the complex nature of hypersensitivity reactions would require researchers be more cautious before considering quercetin in the practical use of the therapy and prevention of allergy. To achieve this goal, further research insights about cell signaling and about quercetin intracellular targets and studies in animal models are required.3: phosphatidylinositol-3,4,5-triphosphate; RBL-2H3: rat basophilic leukemia cell line.DMSO: dimethylsulfoxide; EDTA: ethylendiaminetetraacetic acid; ELISA = Enzyme-linked immunosorbent assay; ENPP-3: ectonucleotide pyrophosphatase phosphodiesterase-3. DAG: diacylglicerol; fMLP: N-formyl-L-methionyl-L-leucyl-L-phenylalanine; GRK: G-coupled receptor kinase; HEPES: 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; HMC-1: human mast cell line-1; LAMP-3: lysosome associated membrane protein-3; MFI: Mean of fluorescence intensity; PI3K: phosphoinositide-3 kinase; PKC: protein kinase C; PLC: phospholypase C; PMA: phorbol-12-myristate-13-acetate; p38MAPK: p38-mitogen activated protein kinase; PtdIns3,4,5-PThe authors declare that they have no competing interests.All the authors read and approved the final manuscript. SC designed the research, conducted in vitro analysis, discussed the results and wrote the manuscript. MM conducted some in vitro analysis and discussed the manuscript; AC discussed the pharmacological aspects of the manuscript; RO and AV managed the cytofluorimetric analysis; PB directed the research and revised the manuscript.


Bordetella pertussis adenylate cyclase toxin, cholera toxin and forskolin.Anthrax edema toxin (EdTx) is an adenylate cyclase which operates in the perinuclear region of host cells. However, the action of EdTx is poorly understood, especially at molecular level. The ability of EdTx to modulate cAMP-dependent signaling was studied in Jurkat T cells and was compared with that of other cAMP-rising agents: EdTx caused a prolonged increase of the intracellular cAMP concentration. This led to nuclear translocation of the cAMP-dependent protein kinase (PKA) catalytic subunit, phosphorylation of cAMP response element binding protein (CREB) and expression of a reporter gene under control of the cAMP response element. Neither p90 ribosomal S6 kinase nor mitogen- and stress-activated kinase, which mediate CREB phosphorylation during T cell activation, were involved. The duration of phospho-CREB binding to chromatin correlated with the spatio-temporal rise of cAMP levels. Strikingly, EdTx pre-treated T cells were unresponsive to other stimuli involving CREB phosphorylation such as addition of forskolin or T cell receptor cross-linking.We concluded that, in a first intoxication phase, EdTx induces PKA-dependent signaling, which culminates in CREB phosphorylation and activation of gene transcription. Subsequently CREB phosphorylation is impaired and therefore T cells are not able to respond to cues involving CREB. The present data functionally link the perinuclear localization of EdTx to its intoxication mechanism, indicating that this is a specific feature of its intoxication mechanism. Bacillus anthracis, a Gram-positive, sporulating bacterium. This organism is commonly found in soil and it mainly infects large herbivores, but occasionally also humans. The spores can enter the body from skin abrasions, the gastro-intestinal tract, or the lungs. Whilst cutaneous anthrax has only local effects, the gastro-intestinal and inhalation forms may lead to a systemic infection, causing septicemia and toxemia. Fatality of inhalation anthrax is very high B. anthracis is sensitive to different antibiotics, their therapeutic benefit is frequently diminished by the late onset of symptoms. Therefore, in recent years much research focused at finding new therapeutics that block the action of anthrax toxins, which are major virulence factors of B. anthracisAnthrax is caused by B. anthracis harbor three plasmid-encoded virulence factors: a polyglutamic capsule and two A–B toxins Pathogenic strains of B. anthracis to survive the host defense mechanisms. In some cell types, the two toxins act in synergism EF and LF act on many cell types, but their action on cells of both innate and adaptive immunity appears particularly relevant as it allows + T cells were identified as targets of anthrax toxins in vitro and ex vivoEdTx-induced alterations of cell signaling are generally thought to be inhibitory and to be mediated by cAMP-dependent protein kinase (PKA) Using Jurkat T cells, we studied the impact of EdTx-mediated intracellular cAMP elevation on PKA- and cAMP response element binding protein (CREB)-dependent signaling and demonstrated that EdTx activates gene expression. We report that EdTx generates an extremely enduring increase in cAMP levels, which stimulates nuclear translocation of the PKA catalytic subunit and phosphorylation of CREB. This in turn allows binding of phosphorylated CREB (pCREB) to chromatin and transcription of a reporter gene under control of the cAMP response element (CRE). Further, we found that pre-treatment with EdTx prevents phosphorylation of CREB induced by a cAMP stimulus or by TCR cross-linking. Comparison with other cAMP-elevating agents showed that length and site of cAMP synthesis are crucial to modulation of gene transcription, which indicates that prolonged perinuclear production of cAMP is a specific and functional feature of EdTx intoxication mechanism.All chemicals were p.a. grade or higher and purchased from Sigma, together with forskolin, cholera toxin (CT), and poly-D-lysine. Protease inhibitor tablets  were purchased from Roche. Adefovir dipivoxil was a kind gift of Gilead . Roswell Park Memorial Institute Medium (RPMI) 1640 and fetal bovine serum (FBS) were bought from Invitrogen. Antibiotics and L-glutamine were purchased from GIBCO. Antibodies against CREB and pCREB (phospho-S133) were purchased from Upstate, against tetra-acetyl-histone H4, pRSK1/2 p90 (phospho-S380), pMSK1 (phospho-T581) from Abcam, against CD3 (OKT3) from eBioscience, against actin from Sigma. Secondary antibodies and H-89 dihydrochloride were obtained from Calbiochem. Plasmids pcDNA3-RII-CFP and pcDNA3-C-YFP coding, respectively, for the regulatory subunit of PKA fused to cyan fluorescent protein and for the catalytic subunit of PKA fused to yellow fluorescent protein were as in 2 in a humid environment.E6.1 Jurkat T cells were kept in RPMI 1640, supplemented with 100 U/ml penicillin, 100 µg/ml streptomycin, 2 mM L-glutamine, 10 mM Hepes and 10% heat-inactivated FBS. Cells were maintained at 37°C and 5% COViability of Jurkat cells was assessed by measuring the oxidation of 3--5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) (MTS) using the CellTiter96 Aqueous One Solution Cell Proliferation Assay (Promega) 12 h, 24 h, 48 h, and 72 h after treatment with 10 nM EF+40 nM PA , 3 nM CT, 5 nM CyaA, 25 µM forskolin , 1∶400 (vol/vol) DMSO, 10 nM EF, or 40 nM PA.5 Jurkat cells in 200 µl were prepared the evening before the determination and stimulated for the indicated times with 10 nM EF+40 nM PA, 3 nM CT, 5 nM CyaA, 25 µM forskolin, or left untreated. 5 µM adefovir dipivoxil  was added 2 to 12 h before stimulation with EdTx and was reapplied 4 h after the first addition. Samples were prepared in triplicates. The culture medium was removed after 10 min of centrifugation at 1300 rpm and 4°C and the cells immediately processed for intracellular cAMP measurement by enzyme-linked immunoassay following the manufacturer's instruction . The number of cells present in each sample was determined separately using a Bürker chamber.5×102-competent Escherichia coli XL-1Blue cells that were transformed by the heat shock method 6 of Jurkat cells in 30 ml of culture medium were prepared the evening before transfection. 20 µg each of pcDNA3-RII-CFP and pcDNA3-C-YFP or 20 µg pCRE-Luc and 1 µg pRL-TK were introduced into cells kept in 400 µl of culture medium devoid of FBS giving an electric shock at 250 V and 950 F in electroporation cuvettes with 0.4 cm gap (Bio-Rad) using a GenePulser Xcell electroporator (Bio-Rad). The FBS content was brought back to 10% and cells allowed to grow a few hours at a concentration of 5×105 cells/ml.Plasmids pcDNA3-C-YFP, pcDNA3-RII-CFP, pCRE-Luc, and pRL-TK were propagated in CaCl48 h after transfection with pcDNA3-RII-CFP and pcDNA3-C-YFP, cells were stimulated with 10 nM EF+40 nM PA, 3 nM CT, 5 nM CyaA, 25 µM forskolin, or left untreated and at the indicated times allowed to adhere for 10 min to cover slips coated with poly-D-lysine (50 µg/ml). Cells were paraformaldehyde-fixed according to standard protocols. Z-stacks of samples with 0.27 µm width were acquired at 490 nm on a DMIRE2 fluorescence microscope (Leica), equipped with a Leica DC 500 CCD camera, using a 63× oil immersion objective with NA 1.4 and 1.5× magnification. Images were deconvoluted with the software LeicaDeblur and the maximal projection created in WCIF Image J 5 cells were lysed on ice for 10 min under rotation in the presence of protease inhibitors and heated for 8 min to 95°C in 60 µl Laemmli buffer Jurkat cells were treated for the indicated times with 10 nM EF+40 nM PA, 3 nM CT, 5 nM CyaA, 25 µM forskolin, or left untreated. To induce TCR cross-linking, cells were transferred for 30 min to plates coated over night with antibody against CD3 (10 µg/ml in PBS). Samples containing 105 cells were present. Samples were prepared in triplicates. For every sample, both the ratio between the luminescence of firefly and renilla luciferases and the firefly luciferase activity per µg of protein in cell lysates were obtained. Proteins were quantified by the Bradford method (Biorad Protein Assay).The day after transfection with pCRE-Luc and pRL-TK, cells were stimulated with 10 nM EF+40 nM PA, 3 nM CT, 5 nM CyaA, or 25 µM forskolin. As controls, 1∶400 (vol/vol) DMSO, 10 nM EF, or 40 nM PA were added or cells were left untreated. Cells were lysed on ice for 10 min in the presence of protease inhibitors and luciferase activity was assayed immediately using the Dual-Luciferase Reporter Assay System (Promega) as described by the manufacturer. For each time point, 105 Jurkat cells were inoculated into 2 ml culture medium. The following day, they were treated for the indicated times with 10 nM EF+40 nM PA, or 3 nM CT, or 5 nM CyaA, or 25 µM forskolin, or DMSO (1∶400 vol/vol), or left untreated. Proteins and DNA were cross-linked by addition of 1% (w/v) formaldehyde directly to the culture medium and incubation for 10 min at 37°C. The reaction was stopped with 125 mM glycine in PBS. Cells were washed with ice-cold PBS containing protease inhibitors and lysed on ice for 10 min under rotation in the presence of protease inhibitors. DNA was sheared to fragments of about 600 bp by three cycles of 10 s of ultrasonic dispersion using a Fisher Sonic Dismembrator Model 300 at 30% power output with a 4 mm tip. The lysates were subjected to chromatin immunoprecipitation according to the manufacturer instructions using the kit produced by Upstate, including standard controls such as absence of antibody and presence of unspecific rabbit IgG. 15 µl of anti-pCREB antibody were employed. Samples were processed for SDS-PAGE and subjected to western blotting with an antibody against tetra-acetyl-histone H4.The evening before the experiment, 5×10in vivo. In particular, the present concentration of 10 nM EdTx was chosen on the basis of our previous cellular studies The concentrations of the toxins and drugs used here were deduced from the literature. In fact, the concentration of EdTx, CT, and CyaA in circulating fluids during pathogenesis is not yet known. The selected toxin concentrations were either within or below the affinity range of these toxins for their respective cell surface receptor 5 Jurkat cells. Using FRET-based live imaging of EdTx activity, we previously found that cAMP levels start rising 30–40 min after toxin addition 5 cells) and then slowly decayed. Addition of 5 nM CyaA induced an immediate rise in cAMP concentration that reached its maximum 30–60 min after addition with about 450 fmol per 105 Jurkat cells, maintained these cAMP levels for another 1.5 h, and then progressively decayed. Forskolin (25 µM) caused a rapid (15 min) and sharp peak of cAMP concentration with 4400 fmol of cAMP per 105 Jurkat cells.The time course of total intracellular cAMP synthesis in Jurkat T cells following treatment with EdTx, CT, CyaA and forskolin was determined by competition ELISA. Although this approach is not as sensitive as the previously used FRET-based live imaging of intracellular cAMP production Next, we tested the possibility that the toxin- or forskolin-induced rise in cAMP concentration could activate PKA and drive the consequent nuclear translocation of its catalytic subunit. We imaged PKA in cells by transiently expressing a fluorescent probe consisting of the PKA catalytic subunit fused to YFP. As expected, in resting Jurkat cells the catalytic subunit of PKA resided in the ring-shaped cytosol . To the This result suggested to study the functional consequence of the nuclear migration of PKA on the phosphorylation of CREB, which is the best characterized cAMP-regulated transcription factor We further addressed the question whether phosphorylation of CREB depended only on PKA or also on other factors that could be activated by cAMP in Jurkat T cells. To this end, we monitored EdTx-, CT-, CyaA-, and forskolin-mediated CREB phosphorylation in the presence and absence of the inhibitor H-89 dihydrochloride, which is commonly used to discriminate between the effects of PKA from those of other kinases, as well as of the guanine nucleotide exchange factor Epac. Epac can induce phosphorylation of CREB in a Rap1- and MAPK-dependent way To test whether the phosphorylation of CREB described above could lead to gene transcription through its cognate activator sequence cAMP response element (CRE), Jurkat cells were double-transfected with two plasmids, one coding for firefly luciferase under the control of CRE and the other coding for renilla luciferase under the control of a viral promoter with constitutive transcriptional activity. Stimulation of pCREB transcriptional activity in the transfected cells will result in an increase of the ratio of firefly luciferase activity to that of renilla luciferase activity. Indeed, as shown in The use of a reporter system has shown that EdTx, CT, CyaA, and forskolin are capable of inducing expression of a gene regulated by CRE. However, a CRE-driven reporter assay is a highly simplified system with respect to transcription of cellular genes, where several distinct promoter sequences control this process. Therefore, to test for the association of pCREB with transcriptionally active genomic DNA, we immunoprecipitated pCREB and determined the amount of associated chromatin by western blotting with an antibody specific for the acetylated histone H4, which is present in CREB-activated genes The fact that phosphorylation of CREB caused by EdTx was terminated after 14 h  in spiteEdTx-mediated suppression of T cell activity is generally thought to be achieved by inhibition of MAPK-dependent signaling. The present data indicate that EdTx also targets CREB-dependent T cell signaling. In fact, cAMP synthesis and phosphorylation of CREB are tightly regulated in space and time during T cell activation We report here that EdTx mediates an extremely prolonged rise of cAMP levels, whereas CT-, CyaA- and forskolin-induced elevations of cAMP concentration are transient. This sustained action might be explained either by enduring catalytic activity of EdTx in the cytosol due to high resistance to proteolytic degradation or be linked to its mode of cell entry. In fact, EdTx exploits the endocytic route to reach the cytosol and was proposed to associate with ILVs Pseudomonas aeruginosa ExoY has distinct effects on endothelial cell permeability with respect to the cAMP produced at the level of the plasma membrane The prolonged rise in cAMP levels elicited by EdTx also resulted in long-lasting phosphorylation of CREB. EdTx, CT, CyaA and forskolin were all able to induce transcription of a reporter gene. However, EdTx and, to a lesser extent, CT appeared to be more efficient in promoting enduring pCREB association with transcriptionally active chromatin. This is consistent with the generally accepted finding that stimuli can provoke phosphorylation of CREB with comparable kinetics and stoichiometry, yet not all stimuli are able to induce pCREB-mediated transcription to the same extent kip1, which block cell growth CREB mediates expression of a high number of genes that are implicated in a wide array of cell functions, including transcription, cell cycle, and immune regulation in vivo observation that the effects on T lymphocyte cytokine secretion change with the duration of EdTx intoxication We also report here that, although EdTx initially induces CREB phosphorylation and gene expression, after a prolonged incubation it clearly inhibits the subsequent phosphorylation of CREB induced by forskolin or by TCR cross-linking. This property likely depends on the massive and long-lasting cAMP synthesis caused by EdTx, which probably fuels negative feedback mechanisms of CREB phosphorylation In conclusion, we identify PKA and CREB as targets of inhibition of T cell function caused by EdTx. Our data suggest that EdTx displays its action in two distinct phases: first, EdTx induces PKA-dependent CREB phosphorylation and expression of genes under the control of CRE; then, EdTx prevents CREB phosphorylation in response to incoming stimuli. This is a novel mode of intoxication by bacterial toxins acting on cellular signaling. Comparison of EdTx activity with that of other cAMP-elevating agents such as CT, CyaA and forskolin showed that EdTx is particularly efficient in affecting T cell function, probably owing to the duration and site of cAMP production.


Chlamydophila pneumoniae contains a dual-specific Ser/Thr protein kinase that phosphorylates CdsD, a structural component of the type III secretion apparatus. To further study the role of PknD in growth and development we sought to identify a PknD inhibitor to determine whether PknD activity is required for replication.We have shown previously that in vitro kinase assay we screened 80 known eukaryotic protein kinase inhibitors for activity against PknD and identified a 3'-pyridyl oxindole compound that inhibited PknD autophosphorylation and phosphorylation of CdsD. The PknD inhibitor significantly retarded the growth rate of C. pneumoniae as evidenced by the presence of very small inclusions with a reduced number of bacteria as seen by electron microscopy. These inclusions contained the normal replicative forms including elementary bodies (EB), intermediate bodies (IB) and reticulate bodies (RB), but lacked persistent bodies (PB), indicating that induction of persistence was not the cause of reduced chlamydial growth. Blind passage of C. pneumoniae grown in the presence of this PknD inhibitor for 72 or 84 hr failed to produce inclusions, suggesting this compound blocks an essential step in the production of infectious chlamydial EB. The compound was not toxic to HeLa cells, did not block activation of the MEK/ERK pathway required for chlamydial invasion and did not block intracellular replication of either Chlamydia trachomatis serovar D or Salmonella enterica sv. Typhimurium suggesting that the inhibitory effect of the compound is specific for C. pneumoniae.Using an in vitro kinase activity of C. pneumoniae PknD and inhibits the growth and production of infectious C. pneumoniae progeny in HeLa cells. Together, these results suggest that PknD may play a key role in the developmental cycle of C. pneumoniae.We have identified a 3'-pyridyl oxindole compound that inhibits the  Chlamydophila pneumoniae is an important human respiratory pathogen that causes laryngitis, pharyngitis, bronchitis and community acquired pneumonia [C. pneumoniae to remain viable within lung macrophages [Chlamydia to other anatomical sites that may include the arterial wall [C. pneumoniae respiratory infections is therefore important to ensure complete clearance of the bacteria in order to avoid infections elsewhere in the body. Antibiotics such as azithromycin, clarithromycin, erythromycin, and doxycycline have been used to treat C. pneumoniae respiratory infections [Chlamydia resistant to azithromycin and erythromycin have been reported [C. pneumoniae develop resistance to antibiotics in vitro [in vivo may result in chlamydial persistence [Chlamydia persisting in the body for months or years [neumonia  and has neumonia ,3, atherneumonia -6, arthrneumonia ,7, Alzheneumonia ,9 and Muneumonia -13. The rophages -16 proviial wall  and the fections . Howeverreported , and somin vitro -25. Furtsistence , renderisistence ,28, and or years ,30. Givespp. exhibit a developmental cycle that begins when an infectious elementary body attaches to and invades a eukaryotic host cell. During invasion the EB becomes enveloped by the host cell plasma membrane, ultimately creating an intracellular vacuole known as an inclusion, within which the bacterium undergoes replication. The EB next transforms into a reticulate body, a developmental process that is characterized by reduction of EB outer membrane proteins [C. trachomatis and 6-7 hours for C. pneumoniae [Chlamydia may be induced to form persistent bodies, a morphological state not part of normal growth and development. The PB is an abnormally large form of chlamydia that occurs in response to interferon-γ [Chlamydia in cell culture. These include selective cycloxygenase inhibitors, rottlerin and inhibitors of type III secretion [C. pneumoniae in HeLa cells [Yersinia type III secretion, may target a bacterial-specific factor related to the type III secretion system and directly abrogate chlamydial growth in eukaryotic cells [All chlamydiae proteins -33 and Deumoniae -36. A haeumoniae . In addirferon-γ , antibiorferon-γ , or ironrferon-γ , and is rferon-γ . In addiecretion ,38-42. RLa cells . RottlerLa cells  and theric cells ,41,44. TC. pneumoniae PknD is a dual-specific protein kinase that autophosphorylates on threonine and tyrosine residues and phosphorylates serine and tyrosine residues of the FHA-2 domain of Cpn0712, a putative Yersinia YscD ortholog called CdsD [C. pneumoniae PknD activity. This compound prevented PknD autophosphorylation and phosphorylation of CdsD, a type III secretion apparatus protein. When added to infected HeLa cells, the compound retarded C. pneumoniae growth and significantly reduced the amount of infectious C. pneumoniae produced suggesting that PknD plays an important role in chlamydial replication.Chlamydial genomic studies have identified three Ser/Thr protein kinases, Pkn1, Pkn5, and PknD. Our laboratory has shown previously that led CdsD . In thisC. pneumoniae contains three Ser/Thr protein kinases [Chlamydia development, we screened an existing library of 80 small molecule kinase inhibitors, including inhibitors of eukaryotic receptor tyrosine kinases and atypical kinases, for their ability to inhibit PknD autophosphorylation in vitro. Recombinant GST-tagged PknD kinase domain (GST-PknD KD) was pre-incubated with 10 μM of each compound and reactions initiated with the addition of kinase assay buffer containing Mn2+ and ATP. SDS-PAGE and Western blotting followed by autoradiography was used to visualize the extent of PknD autophosphorylation in the presence of each compound. Nine compounds  of the 80 tested exhibited some level of inhibition of PknD autophosphorylation when tested at 10 μM (data not shown). Of these nine compounds only one, compound D7, a 3'-pyridyl oxindole, completely inhibited PknD autophosphorylation. Fig. We have recently shown that  kinases  and that kinases . In ordeC. pneumoniae [Compound D7 is ATP competitive and therefore it has the potential to inhibit other chlamydial enzymes that utilize ATP as a substrate. To determine if compound D7 could inhibit a chlamydial ATPase, we examined its effect on the activity of CdsN, the T3SS ATPase of eumoniae . The actTo assess whether compound D7 could be used in cell culture we first exposed the compound to reducing conditions similar to that found in eukaryotic cells, then tested its ability to inhibit PknD. Equivalent volumes of compound D7 (100 μM) and DTT (2 mM) were mixed on ice for 15 minutes prior to testing in the kinase assay. Compound D7 retained the ability to inhibit PknD autophosphorylation fig.  after exYersinia YscD, as a substrate of PknD and showed that PknD phosphorylated 2 FHA domains of CdsD [We recently identified CdsD, an ortholog of  of CdsD . We therC. pneumoniae. Since PknD may play a role at various times throughout the 72 hour developmental cycle we tested the effect of several compounds including compound D7 on the growth of C. pneumoniae in cell culture. Compounds were added to the cell culture media 1 hr prior to infection with C. pneumoniae and inclusions were visualized by immunofluorescent (IF) staining at 72 hr. Compound D7 retarded the growth of C. pneumoniae in HeLa cells  phosphorylation in the presence of compound D7. HeLa cells exposed to either 10 or 100 μM of compound D7 contained high levels of phosphorylated p44 and p42 MAP kinase following EGF stimulation. HeLa cells exposed to 10 or 25 μM U0126, a specific inhibitor of MEK1/2, were used as control and did not contain phosphorylated p44 or p42 MAP kinase following EGF stimulation , it is possible that the inhibitory effect occurs at a specific stage viz. EB to RB differentiation or RB replication. Alternatively, a block in replication could be due to the induction of persistence which occurs under conditions of limiting tryptophan or iron. To determine whether compound D7 blocks chlamydial growth at a specific stage, we used electron microscopy to look for various developmental forms. Figs. C. pneumoniae-infected HeLa cells incubated in the presence of 10 μM compound D7. These inclusions are smaller and contain fewer bacteria compared with chlamydial inclusions in the absence of compound D7  were seen in the presence of compound D7, and no aberrant forms or PB were detected, indicating that the inhibition of chlamydial growth was not due to the induction of persistent bodies. These results show that compound D7 attenuates Chlamydia growth by decreasing the number of bacteria present in infected cells.Since the evidence indicates the inhibitory effect of compound D7 on D7 figs.  and 7D, Chlamydia progeny are infectious after exposure to compound D7, a blind passage experiment was performed. C. pneumoniae-infected HeLa cells were incubated in the presence of compound D7 or DMSO and the cells were lysed at 72 or 84 hr. Lysates containing chlamydiae were either undiluted, or diluted in media lacking compound D7 and blind passaged onto fresh HeLa cell monolayers. Compound D7 reduced the number of infectious chlamydiae compared with DMSO alone at both times by greater than 90% based on inclusion counts  for the in vitro kinase assays. HeLa 229 cells were obtained from ATCC (Manassas). Chlamydophila pneumoniae CWL029 and Chlamydia trachomatis serovar D were obtained from ATCC . E. coli Rosetta pLysS and BL21(DE3) pLysS were from Novagen (EMD). Epidermal growth factor (EGF) and the MEK inhibitor U0126 were from Sigma (Oakville). U0126 was resuspended in DMSO immediately prior to addition to cell culture in the MEK/ERK activation experiment.Minimal essential medium (MEM)  containing Earle's salts and L-glutamine was supplemented with 10% fetal bovine serum. The Calbiochem InhibitorSelect Protein Kinase Inhibitor Library I containing 80 receptor tyrosine kinase inhibitors and atypical kinase inhibitors was from EMD (San Diego). MP Biomedicals (Santa Ana) supplied radiolabelled ATP  supplemented with 1× complete EDTA-free protease inhibitors, unless otherwise noted. Reactions were incubated for 90 min. at 33°C, terminated with SDS-PAGE loading buffer, separated by 10% SDS-PAGE and transferred to polyvinyldinedifluoride (PVDF) membrane. Membranes were exposed to Kodak X-OMAT film for 1-12 hours at -80°C and subsequently developed using an X-ray processor.Eighty cell permeable and ATP competitive protein kinase inhibitors were purchased from EMD (San Diego). Each compound in the InhibitorSelect protein kinase library was screened at 10 μM (unless otherwise noted) in an 2, and 10 mM KCl. Compound D7 was added to final concentrations of 1 μM, 5 μM, 10 μM and 100 μM. The reaction mixture (50 μL) was incubated at 37°C for 30 min. The reaction was stopped by the addition of 10 μL of Malachite Green Reagent A followed by 10 μL of Malachite Green Reagent B and incubated at room temperature for one minute before an OD610 reading was taken, according to the manufacturer's instructions.ATP hydrolysis by GST-CdsN purified from glutathione-agarose beads was measured using a malachite green assay (R & D Systems). Reaction mixtures contained 100 ng of GST-CdsN, 4 mM ATP, 50 mM Tris-HCL pH 7.0, 5 mM MgCl5) on coverslips in shell vials were infected with C. pneumoniae CWL029 (MOI of 1) using centrifugation, and replacement media containing 2 μg/mL cycloheximide was added at 1 hpi. Protein kinase inhibitors  were added to the replacement media to a final concentration of 10 μM (unless otherwise noted), for the duration of the Chlamydia developmental cycle (72 hours). For time course immunofluorescence (IF) experiments, compound D7 was added at 1, 15 and 24 hpi. For IF staining cell monolayers were fixed in methanol for 10 minutes at 72 hpi for C. pneumoniae and at 48 hpi for C. trachomatis. Inclusions were stained with the Pathfinder reagent, a FITC-conjugated anti-LPS monoclonal antibody  containing Evan's Blue counterstain. Images were captured at 400× magnification using an Olympus BX51 fluorescent microscope equipped with a color camera . To determine the infectivity of Chlamydia grown in the presence of inhibitors, HeLa cells were infected with C. pneumoniae CWL029 and grown for 72 or 84 hrs in the presence of various compounds (used at 10 μM) or vehicle (DMSO 0.1%) then cells were lysed with glass beads into fresh MEM. Serial dilutions of lysates were used to infect fresh HeLa cells and inclusions were stained at 72 hr as described above.HeLa cells . The cytotoxicity assay was performed as per the manufacturer's protocol. Briefly, supernatants from HeLa cell cultures incubated in the presence of compound D7 or DMSO (in MEM containing cycloheximide) were tested for evidence of eukaryotic cell cytotoxicity. Aliquots (5 uL) of each supernatant were mixed with 25 uL of Adenylate Kinase Detection Reagent and samples were incubated at room temperature for 5 minutes. Relative light units (RLUs) were measured using a 20/20 n Single Tube Luminometer from Turner BioSystems . Assays were conducted in triplicate for each condition. Cell monolayers were washed with warm PBS. 0.75 mL of trypsin was added to each well, and 0.75 mL of MEM was added after complete trypsinization (trypsinization was monitored by light microscopy). Each sample was thoroughly resuspended and aliquoted into a plastic cuvette and the cell number immediately quantitated by determining the optical density at 800 nM [The effect of compound D7 on HeLa cell viability was determined. Briefly, 10 or 100 μM compound D7, or 0.1% DMSO, with or without cycloheximide in MEM, was added to subconfluent HeLa cells in 6-well plates. At 0, 22, 44 and 66 hours supernatants were harvested and tested for the presence of adenylyl kinase using a cytotoxicity assay  in 9 cm2 wells of six-well plates were infected with C. pneumoniae CWL029 at a multiplicity of infection of 1. Compounds were added at 1 hpi and cells harvested at 48 hpi. Cells were fixed overnight at 4°C in 0.1 M sodium cacodylate buffer containing 2% gluteraldehyde, embedded in araldite resin and thin sections were viewed using a Jeol JEM 1200EX electron microscope at 12,000× magnification.HeLa cells (1 × 10EB: elementary body; ECL: enhanced chemiluminescence; hpi: hours post infection; IB: intermediate body; IF: immunofluorescence; LB: Luria-Burtani broth; PB: persistent body; PknD: protein kinase D; RB: reticulate body; T3SS: type III secretion system.Salmonella growth experiment and prepared the associated bar graph. JBM contributed to study conception and design and drafting the manuscript. DLJ contributed to study conception and design, carried out all other experiments and drafted the manuscript. All authors read and approved the final manuscript.CBS conducted the ATPase assay and DCB conducted the HeLa cell cytotoxicity analysis and prepared the associated bar graph. BKC conducted the


The results indicated epimuqubilin A (1) possessed potent NO inhibitory activity against lipopolysaccharide (LPS)-induced nitric oxide release with an IC50 value of 7.4 μM, a level three times greater than the positive control, L-NG-monomethyl arginine citrate, followed by 6 , whereas other compounds exhibited only modest activity  a monocyclic carbon skeleton framework was essential for activity, (2) free acids gave higher activity, (3) the orientation of H3-22 with an equatorial position increased activity, and (4) a bicyclic structure reduced activity. This is the first report of a norsesterterpene peroxide with NO–inhibitory activity. In addition, compounds 1–7 were also evaluated for their inhibitory activities in the yeast glycogen synthase kinase-3β assay. In summary, several norsesterterpene peroxides showed novel biological activities of inhibition in NO production, suggesting that these might provide leads for anti-inflammatory or cancer chemopreventive agents.Seven norsesterterpene peroxides: epimuqubilin A (activity . These c Sesterterpenes isolated from marine organisms are often modified by the loss or addition of one or more carbon units. Norsesterterpene peroxides are characterized with a 2-substituted propionic acid or methyl propionate group attached to a 1,2-dioxane ring at the C-3 position.Sigmosceptrella D21+55.9 ; IR (film) νmax 3450, 2925, 1710, 1578, 1320 cm−1; ESIMS m/z 393.30 [M + H]+.Muqubilone B (2): Colorless viscous oil; [α]D21+52.5 ; IR (film) νmax 3422, 2953, 1700, 1498, 1321 cm−1; ESIMS m/z 425.30 [M + H]+.Unnamed cyclic peroxide ester (3): Colorless viscous oil; [α]D21−57.8 ; IR (film) νmax 2967, 1670, 1530, 1134 cm−1; ESIMS m/z 407.32 [M + H]+.Epimuqubilin B (4): Colorless viscous oil; [α]D21+ 15.8 ; IR (film) νmax 3445, 2929, 1685, 1540, 1130 cm−1; ESIMS m/z 423.31 [M + H]+.Sigmosceptrellin A methyl ester (5): Colorless viscous oil; [α]D21+57.9 ; IR (film) νmax 2935, 1665, 1434, 1150 cm−1; ESIMS m/z 407.32 [M + H]+.Sigmosceptrellin A (6): Colorless viscous oil; [α]D21+51.3 ; IR (film) νmax 3380, 2960, 1723, 1560, 1140 cm−1; ESIMS m/z 393.30 [M + H]+.Sigmosceptrellin B methyl ester (7): Colorless viscous oil; [α]D21−55.4 ; IR (film) νmax 2960, 1660, 1560, 1150 cm−1; ESIMS m/z 407.32 [M + H]+.5 cells/well and incubated for 24 h at 37°C in a humidified atmosphere containing 5% CO2. The cells were treated with samples dissolved in phenol red-free DMEM for 30 min followed by 1 μg/mL of LPS treatment for 20 h. The amount of NO in the cultured medium was measured by the Griess reagent. The standard curve was created by using known concentrations of sodium nitrite, and the absorbance was measured at 540 nm. To evaluate the cytotoxic effect of samples in RAW 264.7 cells in the assay condition, SRB assay was performed. Briefly, after the fixation with 10% trichloroacetic acid (TCA), cells were stained with 0.4% SRB solution in 1% acetic acid followed by dissolving bound SRB in 10 mM Tris-buffer. The optical density was determined at 515 nm.The inhibitory effects of samples on NO production were evaluated in LPS-activated murine macrophage RAW 264.7 cells, using a method modified from that previously reported . Brieflygsk-3 null mutant confers a temperature-sensitive phenotype (growth defect at 37ºC), that can be suppressed by the expression of cloned mammalian GSK-3β , was grown in 5 mL of liquid media of SC minus uracil, shaking, at 37ºC for 2 days. Four hundred-μL of the yeast culture was added to 100 mL of SC minus uracil agar . To perform the assay, compounds and extracts at a concentration of 80 μg per disk were dispensed on filter disks, and placed on plates containing yeast seeded into SC minus uracil media. The Petri dishes were incubated at both 25ºC and 37ºC for 72 hours. Three phenotypes are observed on yeast plates. A clear zone of inhibition (ZOI) at 37ºC and no ZOI at 25ºC indicate that the agent tested inhibits the GSK-3β pathway. The clear zones of inhibition at both 37 and 25ºC of equal width indicate that the agents are cytotoxic. Plates that have a negative phenotype are plates that have no observable ZOI. Active compounds were then tested at lower concentrations . The assays were performed in duplicate. GSK-3β inhibitor , and staurosporine were used as positive controls.Disruption of four yeast GSK-3 homologs  in n GSK-3β . Inhibit


The stacking of thylakoids from the stn8 and stn7stn8 mutants, deficient in STN8 kinase and consequently in light-dependent phosphorylation of PSII, was increased even in the absence of Mg++. Additional PSII protein phosphorylation in wild type plants exposed to high light enhanced Mg++-dependence of thylakoid stacking. Protein phosphorylation in the plant leaves was analyzed during day, night and prolonged darkness using three independent techniques: immunoblotting with anti-phosphothreonine antibodies; Diamond ProQ phosphoprotein staining; and quantitative mass spectrometry of peptides released from the thylakoid membranes by trypsin. All assays revealed dark/night-induced increase in phosphorylation of the 43 kDa chlorophyll-binding protein CP43, which compensated for decrease in phosphorylation of the other PSII proteins in wild type and stn7, but not in the stn8 and stn7stn8 mutants. Quantitative mass spectrometry determined that every PSII in wild type and stn7 contained on average 2.5±0.1 or 1.4±0.1 phosphoryl groups during day or night, correspondingly, while less than every second PSII had a phosphoryl group in stn8 and stn7stn8. It is postulated that functional cation-dependent stacking of plant thylakoid membranes requires at least one phosphoryl group per PSII, and increased phosphorylation of PSII in plants exposed to high light enhances stacking dynamics of the photosynthetic membranes.Phosphorylation of photosystem II (PSII) proteins affects macroscopic structure of thylakoid photosynthetic membranes in chloroplasts of the model plant Arabidopsis. In this study, light-scattering spectroscopy revealed that stacking of thylakoids isolated from wild type Arabidopsis and the mutant lacking STN7 protein kinase was highly influenced by cation (Mg Plant chloroplasts contain extremely large and well organized photosynthetic thylakoid membranes with highly stacked membrane layers Arabidospis thaliana affects macroscopic structure of thylakoids. The Arabidopsis stn8 and stn7stn8 mutants defective in phosphorylation of PSII have grana stacks that are markedly bigger than in thylakoids of wild type plants Arabidopsis mutants deficient in light-induced phosphorylation of PSII in vitro studies clearly demonstrated that electrostatic forces control the stacking and unstacking of isolated thylakoid membranes We recently demonstrated that phosphorylation of PSII proteins in the model plant Arabidopsis. The thylakoid associated Ser-Thr kinase STN7 is essential for phosphorylation of light harvesting complex polypeptides, LHCII, of the minor light harvesting protein CP29 and of TSP9, a soluble protein involved in regulation of light harvesting Thylakoid protein phosphorylation is mediated by a redox-sensitive regulatory system reflecting different light and other environmental conditions stn8 and stn7stn8 protein kinase mutants with the abnormal grana size of the tylakoids and the wild type and stn7 Arabidopsis plants with the normal membrane organization Phosphorylation of LHCII and PSII proteins is regulated according to dark and light conditions. However, LHCII is quickly phosphorylated under low light and dephosphorylated in darkness, while the PSII core has a more stable phosphorylation during the photoperiod Arabidopsis thaliana wild type and stn7, stn8, and stn7stn8 mutants at different photoperiodic time points and during prolonged darkness. For comprehensive analysis of the in vivo protein phosphorylation under various light/dark conditions we use three different complementary techniques: western blotting with two different anti-phosphothreonine antibodies; Diamond ProQ staining of phosphorylated proteins; and quantitative mass spectrometry of non-phosphorylated and phosphorylated peptides released from the thylakoid membranes. Furthermore, we also use light-scattering spectroscopy to monitor changes in thylakoid membrane structure depending on the protein phosphorylation state and varying concentrations of MgCl2, a salt important in maintaining grana structures in isolated thylakoid membranes ++-dependent stacking of Arabidopsis thylakoid membranes.In this study we analyze the phosphorylation of PSII proteins in leaves of 2 retained stacked thylakoids, while chloroplasts isolated in low salt media lost their grana structure and had a reduced absorbance at 580 nm stn7stn8 and stn8 mutants of Arabidopsis are deficient in light-induced phosphorylation of PSII and have increased size of stacked thylakoid membranes compared to wild-type and stn7 plants, as has been revealed by transmission electron microscopy stn7, stn8 and stn7stn8 membranes resuspended in a buffer containing 5mM MgCl2, to retain the native grana and stroma lamellae structures. It is clearly seen that the scattering properties of the thylakoids are different between wild type and stn7 on one hand and stn8 and stn7stn8 on the other hand. The difference is clear due to the higher absorbency of the stn8 and stn7stn8 membranes in the yellow/orange range of 570–620 nm. Notably, Thylakoid membranes carry net negative charges on their surfaces and electrostatic interactions control the stacking and unstacking of these membranes stn8 and stn7stn8 mutants . To preserve the in vivo protein phosphorylation we isolated thylakoid membranes in the presence of NaF, an inhibitor of thylakoid protein phosphatases stn7 and stn8 mutants at these time points representative for night and day , which differ from each other in sensitivity against major thylakoid phosphoproteins Arabidopsis plants to 3 hours of light , transferred plants to darkness for 4 hours, and then exposed them to light for 1 or 2 hours. The thylakoid membranes were isolated from the leaves harvested at these time points and analyzed by western blotting using anti-phosphothreonine antibody  and de-phospho (638.42+) forms of the CP43 peptide (Ac-TLFNGTLALAGR) at different time points and decrease in amount of phosphorylation upon the phosphatase treatment. The similar measurements were done for peptide/phosphopeptide pairs from the D1, D2 and PbsH proteins and their flyability ratios were calculated using 0, 10, 20, 40 min time points (HPLC in conjugation with Electro Spray Ionization Mass Spectrometry (ESI-MS) allows resolution and detection of the major phosphorylated peptides and their non-phosphorylated counterparts for determination of the phosphorylation stoichiometry for PSII proteins e points . These re points .stn7, stn8 and stn7stn8 plants. The results are presented in We performed LC-MS analyses of the peptide mixtures from thylakoids isolated from leaves harvested during the day or night. The typical chromatogram is shown in stn7 was very similar to that in wild type  plants, stn7 (SALK 073254) stn8 (SALK 060869) stn7stn8−2 s−1 and photoperiod of 8 h light and 16 h dark. In the case of high light experiments photosynthetic flux of 900 µmol photons m−2 s−1 has been used.Arabidopsis leaves harvested at time points indicated. The leaves were homogenized in 20 ml of ice-cold 25 mM Tricine (pH 7.8), 330 mM sorbitol, 1mM EDTA, 10mM KCl, 0.15% bovine serum albumin, 4 mM sodium ascorbate, 7mM L-cysteine in a metal blender for four periods of 1 sec at high speed. The homogenate was immediately filtered through four layers of nylon mesh (20 µm pore size), and the filtrate was centrifuged for 3 min at 1000 g. The pellet was resuspended in the same buffer to wash the chloroplasts and centrifuged for 5 min at 1000 g. The chloroplast pellet was resuspended in 10 mM Tricine, 5 mM MgCl2, and 10 mM NaF and allowed to stand for 5 min in the dark on ice in order to lyse the chloroplasts. Following lysis, the thylakoids were pelleted by centrifugation for 5 min at 6000 g. To wash the thylakoids the pellet was resuspended in 100 mM sorbitol, 25 mM Tricine (pH 7.8), 5 mM MgCl2, 10 mM KCl, and 10 mM NaF and centrifuged for 5 min at 6000 g. The pellet was resuspended in a small volume of the same buffer. NaF was used as a phosphatase inhibitor in the buffers when phosphorylation was quantified. All procedures were made under weak green light at 4°C, and the sample was kept on ice throughout the whole process.Four-week-old plants were used for the preparation of chloroplasts and thylakoids. The thylakoid membranes were isolated from 4g of Thylakoid membrane proteins were separated by SDS-PAGE (6% acrylamid stacking gel+14% separation gel+6M urea) and the proteins were transferred to a PVDF membrane . For the anti-phosphothreonine antibodies, purchased from Zymed Laboratories Inc. or New England Biolabs , the membranes were blocked with 5% bovine serum albumin. For specific antibodies against the DE-loop in D1 protein Proteins from thylakoids containing equal amounts of chlorophyll were separated by SDS-PAGE and stained with ProQ® Diamond phosphoprotein gel stain and SYPRO® Ruby protein gel stain according to manufacturer's instructions . The gels were fixated in 50% methanol with 10% acetic acid for 30 min, the solution was changed and the fixation continued overnight, followed by washing 3×10 min in MilliQ water. Immediately prior to use the ProQ Diamond stain was equilibrated to room temperature and vigorously mixed. Gels were stained for 90 min in darkness, followed by destaining three times for 30 min with 4% acetonitrile in 50 mM sodium acetate pH 4.0, and finally washed two times in MilliQ water prior to scanning on a FLA-5100 imaging system at 532 nm. The gels were then immediately put in Sypro Ruby total protein stain and incubated over night followed by two washes in 10% methanol, 7% acetic acid and finally rinsed twice with MilliQ water. Total protein was detected on FLA-5100 imaging system at 473 nm.stn8, stn7, stn7stn8 mutant plants were resuspended in 25 mM NH4HCO3, 10 mM NaF to a final concentration of 2.5 mg of chlorophyll/ml and incubated for 3 h at 22°C with a sequencing grade-modified trypsin from Promega  at 5 µg of enzyme/mg of chlorophyll The thylakoids isolated from the wild type or 4HCO3 (pH 8.0). The aliquots were taken at 0, 10, 20 and 40 min time points and dephosphorylation reactions were stopped by 1% formic acid (pH 4.0).Controlled dephosphorylation of the peptides derived from thylakoids by trypsin treatment, as described above, was performed using alkaline phosphatase at a concentration of 100 milliunits/µl  in a buffer containing 25 mM NH2, 10 mM KCl, and 10 mM NaF, which was used to preserve the native conformation of the membranes. For titration studies with different concentrations of MgCl2 the membranes were resuspended in the same buffer without MgCl2 or with MgCl2 at the corresponding concentration and incubated on ice for 5 min with occasional careful mixing of the solution. For pigment analysis the same membranes were dissolved in 80% acetone at 0.0125µg chlorophyll/µl.The spectroscopic measurements were performed using a PerkinElmer Lambda 25 spectrophotometer. The data were sampled at 2-nm intervals in the 400–750 nm range. Thylakoid membranes were resuspended at 0.0125µg chlorophyll/µl in a buffer containing 100 mM sorbitol, 25 mM Tricine (pH 7.8), 5 mM MgClFigure S1Thylakoid protein phosphorylation during the photoperiod. Immunoblot of SDS-PAGE separated thylakoid proteins with anti-phosphothreonine antibody from Zymed Laboratories. Positions of the phosphorylated thylakoid proteins and of the molecular mass markers are indicated. Thylakoids were prepared from leaves of the wild type plants harvested at the indicated time points during night or day .(0.88 MB EPS)Click here for additional data file.Figure S2Phosphorylation of thylakoid proteins during prolonged darkness or during dark/light transitions not related to photoperiod. Thylakoid membrane proteins from wild type plants were separated on SDS-PAGE and immunoblotted with anti-phosphothreonine antibody. A and B, thylakoids were isolated from leaves exposed for 4 hours to normal light of 120 µmol photons m-2 s-1 (Light) or for 4, 16, 20 and 24 hours of darkness, as indicated. A, immunoblot with anti-phosphothreonine antibody from Zymed Laboratories. B, immunoblot with anti-phosphothreonine antibody from Cell Signaling. Lower panels in A and B demonstrate immunoblots of the same membranes with antibodies against the CP43 and D1 proteins, correspondingly. C, thylakoids were isolated from the wild type plants exposed to normal light for 3 hours (Light), then incubated for 4 hours in darkness and re-exposed to normal light for 1 or 2 hours, as indicated. Thylakoid membrane proteins were separated on SDS-PAGE and immunoblotted with anti-phosphothreonine antibody from Zymed Laboratories.(1.18 MB EPS)Click here for additional data file.