PHARMACEUTICAL COMPOSITION FOR THE TREATMENT OF CHLAMYDIAL INFECTION

Subject of the present invention is a pharmaceutical composition comprising at least one inhibitor of a microorganism selected from the family Chlamydiaceae, optionally together with pharmaceutically acceptable carriers, adjuvants, diluents or/and additives, wherein the inhibitor is selected from compounds capable of modulating the activity of a polypeptide selected from Table 1. Another subject of the present invention is screening method for identification of a compound suitable as inhibitor in a pharmaceutical composition defined herein, comprising the steps: (a) providing a eukaryotic host cell or/and a transgenic non-human animal capable of being infected with a microorganism selected from the family Chlamydiaceae, such as Chlamydia, in particular Chlamydia trachomatis, (b) contacting the cell or/and the transgenic animal of (a) with a microorganism selected from the family Chlamydiaceae, such as Chlamydia, in particular Chlamydia trachomatis, and contacting a compound with the cell or/and the transgenic non-human animal of (a), and (c) selecting a compound which inhibits the microorganism of (a).

Chlamydiae are Gram-negative, obligate, intracellular bacterial pathogens and the causative agents of a wide range of human and animal diseases. Chlamydia trachomatis (Ctr) is a human pathogen associated with several diseases, including sexually transmitted diseases (Brunham and Rey-Ladino, 2005) and preventable blindness (trachoma) (Wright et al., 2008). The developmental cycle of Ctr alternates between two functionally and morphologically distinct forms: the extracellular, infectious, metabolically inactive elementary body (EB) and the intracellular, metabolically active, replicating reticulate body (RB). EBs infect host cells and differentiate into RBs within a membrane-bound, protective vacuole called the inclusion. RBs multiply, and at the end of the cycle they redifferentiate into EBs, which are released from cells to initiate a new developmental cycle by infecting neighboring cells (Moulder, 1991).

Acivicin (L-[αS,5S]-α-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid) irreversibly inhibits the γ-glutamine amidotransferase activity of GMPS (Chittur et al., 2001). Acivicin is an α-amino acid produced by Streptomyces sviceus that contains the dihydroisoxazole ring as a mimic of the glutamine γ-carboxiamide group. Acivicin has been classified along with DON (6-diazo-5-oxo-L-norleucine) and azaserine as affinity analogues of glutamine amidotransferases (GATs) (O'Dwyer et al., 1984).

Acivicin inhibits each of the four amidotransferases of the novo pathway of purine and pyrimidine synthesis: phosphoribosyl pyrophosphate amidotransferase (PPAT), guanosine monophosphate synthase (GMPS), carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase (CAD), and UTP-ammonia ligase 1 (CTPS). The inhibition of these enzymes result in decrease of cellular UTP, CTP, and GMP concentrations, with no alteration in ATP or ITP pools (Neil et al., 1979).

The effect of acivicin on eukaryotic parasite growth has been investigated:

• • kills both the vector and the host form of Leishmania donovani (Mukherjee et al., 1990). Mukherjee at al. investigated acivicin in the context of inhibiting the carbamyl phosphate synthetase II, the first enzyme of the pyrimidine biosynthetic pathway.
  • has been shown to inhibit the growth of P. falciparum in vitro (Vilmont et al., 1990).
  • its CTPS inhibitory activity has been correlated to the observed antitrypanosomal activity against bloodstream T. brucei in culture and in a mouse model (Hofer et al., 2001, Fijolek et al. 2007).

There is one report of acivicin use in bacteria. Orth, R. et al. (2010) report the synthesis of acivicin inspired 3-chloro- and 3-bromo-dihydroisoxazole probes and their application in target profiling in non-pathogenic and as well as in pathogenic bacteria such as S. aureus and multiresistant S. aureus (MRSA).

Weber and others (1991) have demonstrated that in hepatoma and several other tumors, derived from experimental and human sources, the rate-limiting enzymes of nucleic acid biosynthesis show markedly increased activity.

The silencing of gene expression by RNA interference (RNAi) technology is proving to be a powerful tool to investigate the function of host proteins. Here, we present a systematic siRNA-based loss-of-function screen aimed at discovering host cell factors that interfere with the entry, survival, and replication of Ctr within human epithelial cells. We identified 59 host cell factors whose knockdown altered Ctr infectivity (see Table 1a). These factors included K-Ras and Raf-1, which when knocked down led to the increased growth of Ctr. Despite the depletion of K-Ras and Raf-1, ERK was still activated after the infection of cells with Ctr, which was accompanied by the strong stimulation of cPLA₂. This suggested that activation of ERK in Ctr-infected cells occurred through a K-Ras- and Raf-1-independent mechanism. Infection by Ctr also led to the Akt1- and Akt2-dependent phosphorylation of Raf-1 at Ser²⁵⁹, a modification known to inactivate Raf-1 (Rommel et al., 1996; Zimmermann and Moelling, 1999). In addition, we showed that Raf-1 was recruited to the inclusion in an Akt- and 14-3-3β-dependent manner. These data suggest that infection with Ctr triggers a modular regulation of components of the Ras-Raf-MEK-ERK pathway to support growth of the pathogen.

In the present invention, by modulation of a polypeptide selected from Table 1, Akt1, Akt2, Akt and 14-3-3β, a chlamydial infection can be successfully treated. A polypeptide selected from Table 1, Akt1, Akt2, Akt and 14-3-3β is a suitable target for the prophylaxis or/and treatment of an infection with a microorganism selected from the family Chlamydiaceae. A polypeptide selected from Table 1, Akt1, Akt2, Akt and 14-3-3β may be used in a screening method, as described herein, for compounds suitable for the prophylaxis or/and treatment of an infection with a microorganism selected from the family Chlamydiaceae. Furthermore, a modulator of a polypeptide selected from Table 1, Akt1, Akt2, Akt and 14-3-3β may be used for the prophylaxis or/and treatment of an infection with a microorganism selected from the family Chlamydiaceae. The subject-matter of the present invention is further described by the claims disclosed herein.

A preferred embodiment of the present invention refers to guanosine monophosphate synthase GMPS.

In the present invention, modulation of the GMPS is in particular modulation of the activity of GMPS. Modulation of the GMPS refers in particular to the modulation of GMP synthesis by the GMPS. In the present invention, inhibition of the GMPS is in particular inhibition of the activity of GMPS. Inhibition of the GMPS refers in particular to the inhibition of GMP synthesis by the GMPS.

Modulation of GMPS includes modulation of the interaction of GMPS with HAUSP, such as inhibition of the interaction of GMPS with HAUSP. Modulation of GMPS also includes modulation of recruitment of GMPS to the chlamydial inclusion, such as inhibition of recruitment of GMPS to the chlamydial inclusion.

Another preferred embodiment of the present invention refers to Akt1, Akt2, or/and Akt.

Yet another preferred embodiment of the present invention refers to 14-3-3β.

In the present invention, a reference to Table 1 includes a reference to Table 1a and Table 1b.

Specific embodiments of the present invention refer to the specific nucleic acid sequences, the specific polypeptide sequences, and the specific targets disclosed in Table 1. Preferred embodiments refer to the specific targets disclosed in Table 1. In the present invention, a “target” is a target for a modulator for the prevention or/and treatment of a chlamydial infection. A “target”, as used herein, includes a nucleic acid describing a gene, or/and a polypeptide encoded by said gene. Table 1 discloses target nucleic acid sequences and target polypeptide sequences. A target nucleotide sequence can comprise the complete sequence of a gene, or a partial sequence thereof, such as an siRNA target sequence. In Table 1, target nucleic acid sequences and target polypeptide sequences are described for example by at least one selected from NCBI gene symbol, Entrez Gene Id, mRNA accession number, and EC number.

In the present invention, “modulation” includes inhibition and activation.

If not stated otherwise, fragments of polypeptides or partial sequences of polypeptides, as used herein, may have a length of at least 10 amino acid residues, at least 20 amino acid residues, at least 30 amino acid residues, at least 40 amino acid residues, at least 50 amino acid residues, at least 80 amino acid residues, at least 100 amino acid residues, or at least 150 amino acid residues, up to the total length of the polypeptide.

If not stated otherwise, fragments of nucleic acid molecules or partial sequences of nucleic acid molecules, as used herein, may have a length of at least 15 nucleic acid residues, at least 30 nucleic acid residues, at least 60 nucleic acid residues, at least 90 nucleic acid residues, at least 120 nucleic acid residues, at least 150 nucleic acid residues, at least 200 nucleic acid residues, at least 240 nucleic acid residues, at least 300 nucleic acid residues, or at least 450 nucleic acid residues, up to the total length of the nucleic acid molecule.

The invention is further illustrated by the following Figures and Examples.

Table 1: (a) Results of the screening for genes or/and polypeptides involved in chlamydial infection, (b) Results of the screening for genes or/and polypeptides involved in host cell nucleotide metabolism, which genes or/and polypeptides are essential for Chlamydia growth, propagation or/and infection.

1a) Primary Screen

To identify host cell factors that might have crucial functions during Ctr infection and the progression of the pathogen's developmental cycle (FIG. 1A), we established a two-step assay that enabled us to determine (i) the number of EBs that infected cells or/and differentiated into RBs inside host cells (termed infection), or/and (ii) the resulting infectious progeny (termed infectivity). We used fluorescence microscopy as a read-out system (FIG. 1B). One day prior to transfection with small interfering RNAs (siRNAs), HeLa cells were seeded in three 96-well plates. The cells in one plate were fixed 72 hours post-transfection to exclude possible effects of gene knockdown on cell number. At the same time, cells in both of the remaining plates were infected with Ctr. Cells in one of the plates were used to monitor the infection rate 24 hours-post infection, whereas cells in the other plate were lysed 48 hours post-infection, and dilutions of the lysates were used to infect nontransfected HeLa cells, which were fixed 24 hours post-infection to monitor the infectivity rate of Ctr. All of the plates were then processed for immunofluorescence microscopy by staining the cell nuclei with Hoechst dye whereas bacterial inclusions were detected with an antibody against the major outer membrane protein (MOMP) of Ctr. The number of inclusions per cell and sizes of these inclusions were determined by automated microscopic readout.

To test the reliability of the functional assay, we used siRNAs specific for the small GTPase adenosine diphosphate (ADP)-ribosylation factor (ARF1) (siARF1), and a combination of siRNAs specific for the light-chain subunits of the microtubule-associated proteins MAP1 LC3A and MAP1 LC3B (siLC3). Transfection of cells with siARF1 prior to infection with Ctr resulted in larger inclusions and higher infectivity than occurred when cells were transfected with an siRNA (siLuci) against luciferase (thus, siARF1 was considered an activating control), whereas siLC3-mediated knockdown of MAP1 LC3A and MAP1 LC3B prior to infection resulted in the formation of smaller inclusions and almost no infectivity (FIG. 1C); thus, siLC3 was considered an inhibitory control Three siRNA libraries were screened: A kinase library that targeted 646 kinases and kinase-binding proteins, an apoptosis library directed against 418 apoptosis-related genes, and a custom library that targeted 471 genes with a broad range of cellular functions. Altogether, 1,289 unique genes were targeted with two pooled siRNAs per gene. Each pooled siRNA was tested a minimum of three times in 96-well plates. Only plates in which the controls showed increased or decreased infectivity rates of at least two-fold were analyzed further.

For quality control, a plate-wise correlation coefficient matrix was generated for each of the tested parameters in the assay, based on all samples. Data were normalized by B-Score and percent-of-control (POC) analyses (FIG. 2A), and targeted genes were designated as primary hits according to defined thresholds, as described in the Materials and Methods. With this approach, we identified 204 and 203 primary hits from the B-Score and POC analyses, respectively. For further analyses, we focused on the 80 genes common to both methods, in addition to 26 genes that were identified exclusively from either the B Score or POC methods, giving a total of 132 primary hits.

To validate the initial 132 hits, we performed a second round of screening that used four independent, newly designed siRNAs for each target gene (FIG. 2B). Data were normalized by POC. Validated hits that showed a minimum change in infectivity of two-fold with at least three siRNAs were classified as strong, those that exhibited a 1.5-fold effect with at least three siRNAs were classified as medium hits, and those that exhibited a 1.5-fold effect with two siRNAs were categorized as weak hits. Primary hits that did not meet the validation criteria or that showed opposing phenotypes were grouped as “not validated.” With these stringent criteria, of the 132 primary hits subjected to hit validation, 30 qualified as weak, 15 as medium, and 14 as strong hits (FIG. 2B, Table 1a). Of the primary hits that were exclusively derived from the B-Score and POC methods, we achieved a validation rate of 35% and 46%, respectively; a validation rate of 48% was achieved by combining both methods (FIG. 2C). These validation rates indicate that control-based normalization of RNAi screening data may be more reliable than sample-based normalization.

2a) Knockdown of K-Ras and Raf-1 leads to increased Ctr infectivity

The Ras-Raf-MEK-ERK pathway is activated after infection with Ctr, which leads to the phosphorylation and activation of cPLA₂ by ERK (Su et al., 2004). Blocking the Ras-Raf-MEK-ERK pathway with chemical inhibitors, for example the MEK inhibitor U0126, decreases the infectivity of Ctr and reduces the extent of phosphorylation of cPLA₂ (Su et al., 2004). In contrast, our screening results showed that knockdown of K-Ras and Raf-1 led to increased Ctr infectivity (Table 1a). Knockdown of the other Raf and Ras family members failed to elicit equivalent increases in Ctr infectivity. To further elucidate the mechanism of by which the Ras-Raf-MEK-ERK pathway was activated during Ctr infection, we compared the cellular outcomes generated by chemical inhibitors with those caused by siRNA-mediated knockdown of gene expression. Western blotting analysis revealed that ERK and cPLA₂ were strongly phosphorylated 30 hours post-infection, whereas the MEK inhibitor U0126 repressed the phosphorylation of ERK and cPLA₂ in response to infection (FIG. 3A). Knockdown of MEK also hampered the phosphorylation of ERK after infection with Ctr (FIG. 3B), whereas ERK and cPLA₂ were still phosphorylated when K-Ras and Raf-1 were knocked down (FIG. 3C). Consistently, U0126 decreased the infectivity of Ctr (FIG. 3D), whereas knockdown of K-Ras and Raf-1 led to increased infectivity (FIG. 3E). These data strongly suggest that the phosphorylation of ERK and the phosphorylation and activation of cPLA₂ during Ctr infection require MEK but not K-Ras or Raf-1. In addition, both the activation of ERK and the depletion of K-Ras and Raf-1 supported the growth of Chlamydia within host cells. Thus, we further investigated the fate of Raf-1 during Ctr infection.

2B) Raf-1 is Phosphorylated at Ser²⁵⁹ after Ctr Infection

Because knockdown of Raf-1 supported the growth of Chlamydia, we investigated whether the phosphorylation of Raf-1 was influenced by Ctr infection. Previous studies showed that Raf-1 is inactivated when it is phosphorylated at Ser²⁵⁹ by Akt (Wu et al., 2008; Zimmermann and Moelling, 1999). Our Western blotting analysis revealed the increased abundance of Raf-1 phosphorylated at Ser²⁵⁹ in Ctr-infected cells compared to that in uninfected cells, and that knockdown of Akt inhibited this infection-dependent phosphorylation event (FIG. 4). These findings strongly suggested that Raf-1 was inactivated by Akt-dependent phosphorylation at Ser²⁵⁹ in response to infection by Ctr. Thus, by inhibition of Akt1, Akt2 or/and Akt, a chlamydial infection can be successfully treated. Akt1, Akt2 or/and Akt are suitable targets for the prophylaxis or/and treatment of an infection with a microorganism selected from the family Chlamydiaceae. Akt1, Akt2 or/and Akt may be used in a screening method, as described herein, for compounds suitable for the prophylaxis or/and treatment of an infection with a microorganism selected from the family Chlamydiaceae. Furthermore, an inhibitor of Akt1, Akt2 or/and Akt may be used for the prophylaxis or/and treatment of an infection with a microorganism selected from the family Chlamydiaceae.

Preferably, inhibition of Akt1, Akt2 or/and Akt includes inhibition of the interaction of Akt1, Akt2 or/and Akt with Raf-1.

2c) Phosphorylated Raf-1 is Recruited to the Inclusion in an Akt- and 14-3-3β-dependent manner

During Ctr infection, 14-3-3β is recruited to the inclusion by Inclusion protein G (IncG) (Scidmore and Hackstadt, 2001) and interacts with other host cell proteins, such as BAD (Verbeke et al., 2006). Phosphorylation of Raf-1 at Ser²⁵⁹ results in the binding of Raf-1 to 14-3-3β, a negative regulator of Raf-1 (Zimmermann and Moelling, 1999), and Raf-1 is redistributed within Chlamydia-infected cells (Chu et al., 2008). Thus, we speculated that Raf-1 might also be recruited to the inclusion upon infection in a 14-3-3β- and Akt-dependent manner. Uninfected and Ctr-infected HeLa cells were fixed 30 hours post-infection and incubated with antibodies against 14-3-3β, Raf-1, or phosphorylated Raf-1 (pRaf-1). Confocal images revealed that Raf-1 and pRaf-1 colocalized with 14-3-3β at the membranes of inclusions in infected cells, whereas in uninfected cells, Raf-1 and pRaf-1 were dispersed throughout the cytoplasm (FIG. 5, A and B). Additionally, ectopic expression of wild-type Raf-1 or a Ser²⁵⁹→Ala mutant of Raf-1 (S259A) revealed that only the wild-type protein localized to the inclusions, whereas the mutant form remained in the cytoplasm of infected cells (FIG. 5, C and D). These data confirmed the phosphorylation-dependent recruitment of Raf-1 to the inclusion.

To corroborate these observations, we performed fractionation experiments. Uninfected and Ctr-infected cells transfected with siRNAs specific for luciferase (a negative control), Akt, or 14-3-3β were lysed 30 hours post-infection, separated into subcellular fractions, and subjected to Western blotting analysis to detect Raf-1 and chlamydial heat shock protein 60 kD (Hsp60), as a marker for Chlamydia. As expected, chlamydial Hsp60 was found mainly in the membrane- and organelle-containing fraction of infected cells (FIG. 6, A to C). Consistent with our confocal results, Raf-1 was distributed between the cytosolic and the membrane- and organelle-containing fractions in uninfected, control cells transfected with an siRNA against luciferase. In contrast, Raf-1 was predominantly localized to the membrane- and organelle-containing fraction in infected cells (FIG. 6A). However, in Akt-knockdown cells, we observed a strong increase in the abundance of Raf-1 in the cytosolic fractions of both uninfected and Ctr-infected cells (FIG. 6B). A similar scenario was observed when cells were depleted of 14-3-3β (FIG. 6C). To investigate whether Raf-1 directly interacted with 14-3-3β at the inclusion, an in situ proximity ligation assay was performed, which enabled us to visualize protein-protein interactions. In Ctr-infected cells we clearly observed a strong accumulation of signals at the inclusion (FIG. 6D). Thus, our findings demonstrate pRaf-1 was recruited to the inclusion in a manner that was dependent on Akt and a direct interaction with 14-3-3β. Thus, by inhibition of 14-3-3β, a chlamydial infection can be successfully treated. 14-3-3β is a suitable target for the prophylaxis or/and treatment of an infection with a microorganism selected from the family Chlamydiaceae. 14-3-3β may be used in a screening method, as described herein, for compounds suitable for the prophylaxis or/and treatment of an infection with a microorganism selected from the family Chlamydiaceae. Furthermore, an inhibitor of 14-3-3β may be used for the prophylaxis or/and treatment of an infection with a microorganism selected from the family Chlamydiaceae.

Preferably, inhibition of 14-3-3β includes inhibition of the interaction of 14-3-3β with Raf-1, in particular phosphorylated Raf-1.

Here, we present an siRNA-based, loss-of-function screen in human epithelial cells that identified 59 targets that positively or negatively regulated C. trachomatis infectivity. Network and gene-enrichment analyses pointed towards K-Ras and Raf-1 as central players involved in several signaling networks engaged during Ctr infection. To validate this observation, we dissected the functions of K-Ras and Raf-1 during infection. We found that ERK was activated even when Raf-1 was depleted; that Raf-1 was phosphorylated at Ser', a known inactivating modification of Raf-1, in an Akt-dependent manner; and that phosphorylated Raf-1 was recruited to the inclusion, in a manner that was dependent on Akt and a direct interaction with 14-3-3β. These findings have revealed an unexpected Ras- and Raf-independent MEK-ERK signaling pathway during Ctr infection.

In conclusion, this is the first comprehensive, human cell-based, RNAi loss-of-function screen for host cell factors that either positively or negatively affect the developmental cycle of Ctr. Detailed investigation of two of these factors, Ras and Raf-1, demonstrated an uncoupled regulation of components of the canonical Ras-Raf-MEK-ERK signaling cascade by Chlamydia. Our study also provides evidence for the inactivation of Raf-1 during Ctr infection. The functional importance of this inactivation is currently under investigation; however, we hypothesize that Ctr specifically inactivates and sequesters Raf-1 to actively interfere with the downstream signaling events induced by Raf-1 independently of MEK and ERK. Our observations indicate that Ctr has evolved efficient strategies to uncouple individual modules from otherwise coherent signaling cascades and further advance our understanding of Chlamydia-host cell interactions.

HeLa cells (ATCC CCL-2) were grown in Hepes-buffered growth medium [RPMI (GibCo) supplemented with 10% fetal calf serum (FCS) (Biochrome), 2 mM glutamine, and 1 mM sodium pyruvate], at 37° C. in a humidified incubator containing 5% CO₂. Ctr serovar L2 (ATCC VR-902B) was propagated in HeLa cells in infection medium (RPMI medium supplemented with 5% FCS).

Ctr was propagated in HeLa cells grown in 150-cm² cell culture flasks in 24 ml of infection medium. The cells were detached 48 hours after infection with 3-mm glass beads and were centrifuged at 500 g, for 10 min at 4° C. The pelleted cells were resuspended in sucrose-phosphate-glutamate (SPG) buffer and ruptured by vortexing with glass beads. Cell lysates were then centrifuged as before to sediment nuclei and cell debris. The supernatant was further centrifuged at 20,000 g for 40 min at 4° C. and the resulting bacterial pellet was resuspended in 15 ml of SPG buffer with a 21- to 22-gauge injection needle. Suspensions of Chlamydia were stored in aliquots at −75° C. until required. HeLa cells were infected with Ctr at a multiplicity of infection (MOI) of 0.5 to 3 in infection medium. The medium was refreshed 2 hours p.i, and the cells were grown at 35° C. in 5% CO₂ until they were fixed or used lysed to be used for reinfections.

Transfection of Cells with siRNAs

All siRNAs were purchased from Qiagen. The siRNAs of the custom library were validated at the Max Planck Institute for Infection Biology, Berlin, for their ability to knockdown mRNA expression of target genes by more than 70% compared to control cells transfected with siRNA specific for luciferase, as described previously (Machuy et al., 2005). Transfection of cells in 96-well plates with siRNAs was performed with the BioRobot 8000 system (Qiagen). One day prior to transfection, 1.5×10³ HeLa cells were seeded in each well of a 96-well plate. For each well, 5 μl of the siRNA stock solution (0.2 μM) was resuspended in 15 μl of RPMI without serum and incubated at room temperature for 10 min, to which was added 10 μl of a 1:20 diluted solution of Hiperfect (Qiagen) and the mixture was incubated at room temperature for a further 10 min before 25 ml of growth medium was added. 50 μl of this transfection mixture was added to each well of the plate in addition to 50 μl of growth medium, which resulted in a final concentration of siRNA of 10 nM. Cells were incubated at 37° C. and 5% CO₂ for 72 hours. For the analysis of functional experiments by Western blotting, 1×10⁵ cells were seeded into each well of a 12-well plate 24 hours prior to transfection. Cells were then transfected with Hiperfect transfection reagent according to the manufacturer's guidelines. In brief, 150 ng of specific siRNA was added to RPMI without serum and incubated with 6 μl of Hiperfect in a total volume of 100 μl. After 10 to 15 min, the liposome-siRNA mixture was added to the cells with 1 ml of cell culture medium, which gave a final concentration of siRNA of 10 nM. After 1 day, cells were trypsinized and seeded into new cell culture plates, depending on the experiments. Three days post-transfection, the cells were infected and incubated as indicated above.

In 96-well plates, HeLa cells were infected as described above. At 2 days post-infection, with a BioRobot 8000 system, cells were lysed by adding Nonidet P40 (NP40) (Fluka) at a final concentration of 0.06% for 15 min at room temperature. HeLa cells in 6-well plates were infected with Ctr for 48 hours and then were scraped off the plates with a rubber policeman. The cells were collected in 15-ml tubes containing sterile glass beads and lysed by vortexing (at 2,500 rpm for 3 min). For both plate formats, lysates were then diluted 1:100 in infection medium before being transferred to fresh, untreated HeLa cells. After incubation at 35° C. and 5% CO₂ for 24 hours, the cells were fixed in ice-cold methanol overnight at 4° C. and then processed with the indirect immunofluorescence protocol described below.

Antibodies were obtained from the following sources: Rabbit antibodies against Raf-1, Ras, phosphorylated cPLA₂, total cPLA₂, total p44 MAPK (ERK1), phosphorylated Raf-1 at Ser²⁵⁹, LAMP-1, MEK1 and MEK2, Akt, calpain and mouse antibodies against phosphorylated p44 and p42 MAPK (ERK1 and ERK2) were purchased from Cell Signaling Technology. Goat and mouse antibodies against 14-3-3β and rabbit antibodies against Raf-1 (H-71), cytokeratin-8, and the HA eptiope (Y-11) were purchased from Santa Cruz Biotechnology. Mouse antibody against lamin-A/C was obtained from Chemicon, mouse antibody against Chlamydia Hsp60 was purchased from Alexa, mouse antibody against β-actin was from Sigma, and mouse antibody against Chlamydia MOMP KK12 was from the University of Washington. Secondary antibodies conjugated to horseradish peroxidase (HRP) were purchased from Amersham Biosciences and secondary antibodies labeled with the fluorochromes Cy2, Cy3, and Cy5 were from Jackson Immuno Research Laboratories.

Fixed cells (in 96-well and 6-well plates) were washed twice with phosphate-buffered saline (PBS) and blocked by incubating with 0.2% bovine serum albumin (BSA) in PBS (blocking buffer) for 30 min at room temperature. Primary mouse antibody against C. trachomatis MOMP KK12 (at a 1 in 10,000 dilution) was added to the cells for 1 hour at room temperature before washing twice with PBS. The Cy3-labeled goat antibody against mouse immunoglobulin G (IgG) was then added at a 1 in 100 dilution for 1 hour. Host cell nuclei were stained with Hoechst 33342 (Sigma) at a 1 in 2,000 dilution.

Double Labeling of Raf-1 or pRaf-1 and 14-3-3β and Confocal Microscopy

Infected cells were grown on coverslips, washed twice with PBS, and then fixed with ice-cold methanol overnight at 4° C. Cells were washed again with PBS two times and then incubated in blocking buffer as described earlier. The cells were then incubated for 1 hour at room temperature with antibody against 14 3-3β together with antibody against Raf-1 or pRaf-1 (Ser²⁵⁹) in 100 μl of blocking buffer. The cells were then incubated for 1 hour at room temperature with the appropriate fluorochrome-conjugated secondary antibodies at a 1 in 100 dilution. Between incubation steps, cells were washed with PBS three times. Coverslips were washed and mounted on glass microscopic slides with Moviol. The fluorochromes were visualized with Cy2 and Cy5 filters. A series of images with Z stacks were acquired with a laser scanning confocal microscope (Leica) and analyzed with Imaris Software (Bitplane) and further processed with Photoshop CS3 (Adobe Systems).

Treatment of Cells with U0126

Cells (1×10⁵) were seeded in each well of a 12-well plate one day prior to infection. Two hours after infection with Ctr (at an MOI of 3), 1 ml of fresh infection medium containing either 10 μM or 100 μM U0126 was added to the cells. Depending on the experiment cells were harvested for western blotting analysis or for determination of infectivity.

The numbers and sizes of chlamydial inclusions and host cells were analyzed with an automated microscope (Olympus Soft Imaging Solutions). Images were taken with DAPI and Cy3 filtersets (AHF-Analysetechnik) at the same position. ScanR Analysis Software (Olympus Soft Imaging Solutions) was used to automatically identify and quantify inclusions and cells.

Subcellular fractionation was carried out with the ProteoExtract Subcellular Proteome Extraction kit (Calbiochem), according to the manufacturer's instructions.

Transfections with pcDNA3

HeLa cells were grown on coverslips in 12-well plates, transfected with 1 μg of plasmid DNA encoding HA-tagged WT Raf-1 (pcDNA3-Raf-1-WT) or the HA-tagged S259A mutant of Raf-1 (pcDNA3-Raf-1-S259A) with Lipofectamine 2000 (Invitrogen), as described by the manufacturer. Twenty-four hours later, cells were infected with Ctr at an MOI of 2. Thirty hours post-infection, cells were washed twice with PBS and fixed with ice-cold methanol overnight at 4° C. Cells were washed again in PBS two times and then incubated with blocking buffer as described earlier. The cells were then incubated with primary antibody against the HA tag for 1 hour at room temperature. Cells were then incubated with the secondary fluorochrome-conjugated antibody at a 1 in 100 dilution for 1 hour at room temperature. Between incubation steps, cells were washed with PBS three times. Coverslips were washed and mounted on glass microscopic slides with Moviol. Images were acquired with a fluorescent microscope (Leica) and processed with Photoshop CS3 (Adobe Systems).

HeLa cells grown on coverslips in 12-well plates, were infected with Ctr, 30 h post-infection washed twice with PBS 30 hours post-infection, and then fixed with ice-cold methanol overnight at 4° C. Incubation with antibodies against Raf-1 (H-71), or MEK1/2, or 14-3-3β (A-6) was performed with the Proximity Ligation Assay kit (OLINK) according to the manufacturer's instructions. A series of images with Z stacks were acquired with a laser scanning confocal microscope (Leica) and analyzed with Imaris Software (Bitplane) and further processed by Photoshop CS3 (Adobe Systems).

Depending on the experiment, untransfected or transfected HeLa cells were grown in six-well plates, infected with Ctr as described earlier, and then washed with PBS. To each well was added 200 μl of 1×SDS sample buffer (3% 2-mercaptoenthanol, 20% glycerin, 0.05% bromphenol blue, 3% SDS). Cell lysates were collected and boiled for 10 min. Samples were stored at −20° C. until required. Proteins from the cell lysates were resolved by SDS-PAGE, transferred to polyvinylidene difluoride (PVDF) membranes (PerkinElmer Life Sciences) and blocked with 3% milk powder in Tris-buffered saline (containing 0.5% Tween 20) for 30 min before incubation with the appropriate antibodies. The bound primary antibodies were incubated with the corresponding HRP-conjugated secondary antibodies. Immunoreactive proteins were detected on an X-ray film directly or with the AIDA Image Analyzer after addition of ECL reagent (Amersham Biosciences).

Screening data were corrected for plate-to-plate variability by normalizing compound measurements relative to controls with POC and B-score analyses (Malo et al., 2006). The resulting data from both methods were used for further analysis and hit classification. For the POC method, P values and log₂ ratios were calculated for each of the samples. Hits were then classified by defining P value (<0.05) and fold change (>2) for the primary screen, and fold change (>1.5) for the hit validation. In the B-Score method, hits were scored by transforming the normalized measurements into Z-scores. Hits were then classified by defining thresholds of the Z-score for both up-regulating and down-regulating phenotypes (3 and −1, respectively).

For gene enrichment analysis, we modified the R-script available from the Gaggle website at the following URL: http://gaggle.systemsbiology.net/svn/gaggle/PIPE2.0/trunk/PIPEletResource Dir/GOTableEnrichment/GOEnrichmentScript.R. This script applies the R-package GOstats developed by Falcon and Gentleman (Falcon and Gentleman, 2007) and is available at Bioconductor (http://www.bioconductor.org). Briefly, we defined a gene universe consisting of 1,289 genes targeted in our screen and processed different gene hit lists (strong, medium, and weak) against this universe with respect to molecular function (MF), cellular component (CC), and biological process (BP). For the significantly enriched gene ontology terms, we calculated the enrichment factors. Network analysis was carried out with Ingenuity Pathway Analysis (IPA) software (http://www.ingenuity.com/).

We performed a genome-wide siRNA-based screen in human epithelial cells to identify host cell factors that are essential for Chlamydia infection using the Qiagen Hu_Genome Set V1.0 and the Human Druggable Genome siRNA Set V2.0. C. trachomatis L2 was used. In the primary screen we identified 60 sequences that target genes involved in nucleotide metabolism and that showed a strong inhibitory effect on the formation of Chlamydia infectious progeny. The results are summarized in Table 1b.

Our experiments using RNAi have shown that knockdown of human GMP synthase (GMPS) inhibits the intracellular replication of Chlamydia and the formation of infectious progeny.

Thus, by inhibition of GMPS, a chlamydial infection can be successfully treated. GMPS is a suitable target for the prophylaxis or/and treatment of an infection with a microorganism selected from the family Chlamydiaceae. GMPS may be used in a screening method, as described herein, for compounds suitable for the prophylaxis or/and treatment of an infection with a microorganism selected from the family Chlamydiaceae. Furthermore, an inhibitor of GMPS may be used for the prophylaxis or/and treatment of an infection with a microorganism selected from the family Chlamydiaceae.

The involvement of GMPS in Chlamydia infection has not been demonstrated so far. According to the state of the art, GMPS is required for the novo GMP synthesis but is also involved in transcriptional control, at least in part, through cooperation with USP7.

GMP synthase (GMPS, E.C. 6.3.5.2) is a glutamine amidotransferase involved in the de novo synthesis of purines. It catalyzes the conversion of xanthosine 5′-monophosphate to guanosine 5′-monophosphate in the presence of glutamine and ATP. GMPS is a bifunctional enzyme with two domains, an N-terminal glutaminase domain that generates ammonia from glutamine, and a C-terminal synthethase domain that aminates XMP to form GMP (Hirst et al., 1994, Nakamura et al., 1995).

It has been shown that GMPS has increased activity in highly proliferating cells and thus, it is a potential target for anticancer therapies. Glutamine analogs, like acivicin have been shown to inhibit GMPS (Chittur et al., 2001).

In Drosophila embryos GMPS is tightly associated with the ubiquitin-specific protease 7 (USP7) and contributes to epigenetic silencing of homeotic genes by Polycomb. The USP7-GMPS complex catalyzes the selective deubiquitylation of histone H2B. Indeed, USP7 binding to GMPS strongly augmented deubiquitylation of the human tumor suppressor p53 (Van der Knaap et al., 2005). Further, the GMPS-USP7 complex binds and regulates ecdysone target loci, implicating a complex of a biosynthetic enzyme and ubiquitin protease in gene control by hormone receptors (Van der Knaap et al., 2010).

Sarkari et al. (2009), has shown an interaction of USP7 with GMPS in human cells. After Epstein—Barr virus (EBV) infection, this interaction stimulates the ability of USP7 to cleave monoubiquitin from histone H2B. Here, the USP7-GMPS complex forms a quaternary complex with DNA-bound EBNA1 enabling the persistence of EBV genomes in infected cells.

The effect of chemical inhibitors of GMPS on Chlamydia infection was tested. Using acivicin we observed a complete block in Chlamydia replication. Decoyinine, an analogue of adenosine, which is used to block GMPS (Zhang et al., 2005), showed no inhibitory effect on Chlamydia replication.

We were able to recover Chlamydia replication by addition of the nucleotides GTP and GMP to GMPS knockdown cells and to cells to which acivicin was added, demonstrating GMPS to be essential for Chlamydia growth through its function in GMP synthesis.

In immunofluorescence staining studies we observed recruitment of GMPS to the Chlamydia inclusion. We applied the Proximity ligation Assay (PLA from OLink) to investigate a possible interaction of GMPS with HAUSP in Chlamydia infection and find interaction of GMPS and HAUSP.

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