INHIBITOR OF RNA POLYMERASE II

Abstract

An inhibitor of RNA polymerase II is described, wherein said inhibitor is selected a moiety which targets a protein selected from cyclin kinase 12 (CDK12) or its recruiting protein PAF1C. Particular examples of such inhibitors are polypeptides expressed by a gene selected from lldD, lldR, nlpD or rfaH of a bacterial species, such as a commensal bacteria or asymptomatic carrier, or a variant of said protein. Inhibitors may be based upon bacterial Sigma S or NplD proteins. These inhibitors are useful in therapies, to suppress protein expression. Thus they may be used as immunosuppressants, anti-inflammatory or anti-infection agents.

Claims

1. A pharmaceutical composition comprising an inhibitor of RNA polymerase II and a pharmaceutically acceptable carrier, wherein said inhibitor targets a protein selected from cyclin kinase 12 (CDK12) or its recruiting protein PAF1C.

2. The pharmaceutical composition according to claim 1 wherein the inhibitor is a polypeptide expressed by a gene selected from lldD, lldR, nlpD or rfaH of a bacterial species, or a variant of said protein.

3. The pharmaceutical composition according to claim 2 wherein the bacteria is a commensal bacteria or an asymptomatic carrier.

4. The pharmaceutical composition according to claim 3 wherein the bacteria is asymptomatic bacteriuria (ABU).

5. The pharmaceutical composition according to claim 2 wherein the bacteria is an E. coli strain.

6. The pharmaceutical composition according to claim 5 wherein the bacteria are E. coli 83972.

7. The pharmaceutical composition according to claim 1 wherein the inhibitor is a Sigma S protein or a variant thereof or an active fragment of either of these.

8. The pharmaceutical composition according to claim 7 wherein the Sigma S protein is e# SEQ ID NO 1 or SEQ ID NO 2 or SEQ ID NO 3.

9. The pharmaceutical composition according to claim 1 wherein the inhibitor is a bacterial NplD protein, or a variant thereof, or an active fragment of either of these.

10. The pharmaceutical composition according to claim 9 wherein the bacterial NplD protein is SEQ ID NO 4.

11. The pharmaceutical composition according to claim 2 wherein the inhibitor is secreted by the bacteria.

12. The pharmaceutical composition according to claim 9 wherein the inhibitor is of less than 3 kDa in molecular weight.

13. The pharmaceutical composition according to claim 2 wherein the inhibitor is synthetic.

14. (canceled)

15. A method for preparing an inhibitor of RNA polymerase II that is expressed by a bacterial species which comprises culturing suitable bacteria in a culture medium, and isolating a factor having RNA polymerase II inhibitor activity from the supernatant.

16. (canceled)

17. A method for providing immunosuppression, anti-inflammatory or anti-infection therapy to a patient in need therefore, the method comprising administering to the patient an effective amount of an inhibitor of RNA polymerase II.

18-27. (canceled)

28. The method of claim 17, wherein the inhibitor is a polypeptide expressed by a gene selected from lldD, lldR, nlpD or rfaH of a bacterial species, or a variant of said protein.

29. The method of claim 17, wherein the inhibitor is a bacterial NplD protein, or variant thereof, or active fragment of either of these.

30. The method of claim 29, wherein the bacterial NplD protein is SEQ ID NO 4.

31. The method of claim 17, wherein the inhibitor is a Sigma S protein, or a variant thereof, or an active fragment of either of these.

32. The method of claim 31, wherein the Sigma S protein is SEQ ID NO 1 or SEQ ID NO 2 or SEQ ID NO 3.

Description

[0046] The invention will now be particularly described by way of example with reference to the accompanying Figures in which:

[0047] FIG. 1 illustrates inhibition of eukaryotic RNA Pol II phosphorylation by 83972 ABU strain: A. Cells were infected in suspension, labelled for Pol II-Ser2 with fluorescently labeled AB and run on cell flow cytometer. The distribution of phosphorylated Pol II fluorescence was analyzed. Infection with the AU strain decreases host Pol II phosphorylation; B Pol II phosphorylation in control and ABU infected cells, visualized by laser-scanning, slides were mounted and imaged. Nuclei were counterstained with DRAQS and fluorescence intensity was quantified by ImageJ software 1.46r. C. Mean value of Pol II phosphorylation in ABU infected cells and uninfected control cells. One representative experiment is shown, measured with flow cytometry as shown in A and one was quantified by confocal microscopy, using ImageJ software 1.46r. Means of two experiments. D-E shows how a particular re-isolate, designated SN25, has lost the inhibitory effect on RNA Pol II phosphorylation. Results show loss of Pol II phosphorylation repression by reisolate strain SN25 as compared to ancestor strain 83972 in A-C. Error bars show standard error of the mean. G. Hit map of gene expression after infection with both strains shows 465 genes upregulated and 385 genes downregulated in both ABU and SN25, 224 genes downregulated only by ABU and 2001 genes differentially regulated by SN25, but not changed by ABU. H. Moreover, host gene expression of the innate immune response genes was also activated more efficiently by SN25 than E. coli 83972. The expression of cytokines and cytokine receptors was enhanced in SN25-infected compared to ABU-infected human kidney cells. More highly regulated cytokines include CXCL1, 2, 3 and 8, CCLS and 10, IL1B as well as CSF2, LTB and WNTSA. The results identify SN25 as a loss of Pol II inhibition mutant, with stronger pro-inflammatory effects than the parent strain, consistent with the loss of Pol II inhibition.

[0048] FIG. 2 A. Schematic location of mutations in SN25 genome compared to ABU 83972, B List of specific mutations found. C. List of genes mutated in SN25 and their corresponding functions.

[0049] FIG. 3 illustrates the effect on Pol II phosphorylation of ABU deletion mutant reproducing the genomic changes in SN25. The following genes were deleted from 83972 ABU genome: lldD, lldR, nlpD, rfaH, cysE, rcsB, mdoH and lrhA. These mutants were studied with flow cytometry and fluorescence scanning microscopy for level of Pol II phosphorylation; A Cells were infected in suspension, stained for Pol II-Ser2 with fluorescently labeled AB and analyzed with flow cytometry. B. Mean value of Pol II phosphorylation in control, ABU infected sample and samples infected with single gene ABU mutants as measured by flow cytometry and C. by microscopy. It is shown that upon infection with some single gene ABU mutants, host Pol II phosphorylation becomes higher compared to ABU infection implying that these genes are involved in repression of host Pol II phosphorylation. Statistical difference in level of Pol II phosphorylation was measured with T test; D-E. Experimental design aimed to identify the Pol II inhibitor; Bacteria taken from agar plates and suspended in PBS did not show inhibitory activity; RPMI incubated with 10.sup.9 CFU/mL of bacteria for 4 hours was collected, centrifuged to remove bacterial cells and filtered through a 0.2 m filter. The filtrate contained significant inhibitory activity (p<0.001), suggesting that bacterial growth in cell culture medium (RPMI) induces the secretion of inhibitor(s). Screening of supernatant of mutants revealed that all single gene mutants, apart from lldD, lldR, nlpD and rfaH, secrete the substance with inhibitory activity.

[0050] FIG. 4 illustrates how the ABU strain inhibits the Pol II Ser2 CTD phosphorylating machinery by targeting cyclin kinase 12 and its recruiting protein PAF1C. A. Eukaryotic phosphorylation machinery consists of two cyclin-dependent kinases CDK9 and 12, that phosphorylate CTD of Pol II biggest subunit at Ser2 residue. PAF1C complex of proteins recruits CDK12 to promoter. B. Western blot of whole cell lysates after infection with ABU and SN25 showing decrease of expression of proteins required for Pol II phosphorylation. Table inset shows % inhibition of protein as mean value of 2 experiments. C. Confocal microscopy of cells infected with ABU strain and SN25 reisolate. Cells were treated with anti-PAF1c or anti-CDK12 primary antibody and corresponding fluorescently labeled secondary antibodies. Nuclei were counterstained with Draq5. D. Fluorescence intensity of staining in C was quantified by ImageJ software 1.46r. Mean values of two experiments are shown. E. To identify genes involved in PAF1c and CDK12 inhibition, cells were infected with single gene ABU mutants and level of PAF1C and CDK12 proteins was assessed with western blot. Table below specifies % of inhibition. NlpD was identified as gene that is primarily involved in suppression. F. Confirmation with confocal microscopy of the effect of attenuated PAF1C and CDK12 suppression by d NlpD mutant.

[0051] FIG. 5. Sigma S as an effector molecule suppressing host gene expression. A. NlpD gene is located upstream of rpoS, which encodes Sigma S; the DNA binding subunit of bacterial RNA Polymerase. NlpD regulates Sigma S expression through internal promoter. B. An rpoS deletion mutant was constructed in E. coli 83972. The loss of Sigma S protein is demonstrated by Western blot analysis of bacterial lysates of strains SN25, d nlpD and d rpoS. C. The rpoS deletion mutant was used to infect human kidney cells. Confocal microscopy shows the reduction in Pol II Ser2, Pafl c and CDK12 levels was attenuated compared to the ABU strain. D. Quantification of data in C. E. Comparison of bacterial and eukaryotic type II RNA polymerase. Homologous subunits are similarly shaded. F. A schematic illustration of the hypothesis for Sigma S binding to eukaryotic promoter DNA and competitive inhibition of TBP binding. G. Testing of hypothesis in E. Human and bacterial TATA box oligo-nucleotides were incubated with synthetic peptide covering the DNA-binding domain of Sigma S. Sigma S is shown to bind prokaryotic and human TATA box oligonucleotides, creating band shifts with similar mobility. Specificity was confirmed by inhibition of binding with Sigma S-specific antibodies. H. Adding of TBP with whole cell lysate to IRF3 promoter DNA leads to TBP-DNA complex. Sigma S competes with TBP for binding to IRF3 promoter as visualized by concentration dependent decrease in intensity of shifted band.

[0052] FIG. 6. Interactions of Sigma S and NlpD with the Pol II complex A. Confocal imaging of infected human kidney cells after staining with antibodies specific for Pol II Ser2 and Sigma S. A parallel loss of nuclear Pol II staining and accumulation of RpoS in nuclear aggregates was detected. B. Detection by Western blots of Sigma S in whole cell extracts and the nuclear fraction of cells infected with the ABU strain. Sigma S was not detected in cells infected with the SN25 reisolate or nlpD or rpoS deletion mutants. C. Binding of Sigma S and NlpD to the Pol II complex is illustrated and detected by western blot after co-immunoprecipitation of whole cell lysates with antibodies specific for total Pol II. D. Confocal imaging of human kidney cells, infected with E. coli 83972WT and stained with antibodies specific for Sigma S or Pol II, with nuclear DRAQ5 counterstaining.

[0053] FIG. 7. Functional relevance confirmed with in vivo data A. Asymptomatic bacteriuria in C57BL/6 WT mice. Mice were inoculated with 210.sup.5 CFU/mL of ABU 83972, SN25, delta-nlpD or delta-rpoS. Mice were sacrificed after 24 hours and bladder tissues were stained with a-Pol IIp Ab. B. Loss Pol II phosphorylation is apparent after infection with ABU, this is rescued after infection with SN25, d nlpD and d rpoS mutants. This confirms in vitro data on NlpD and RpoS being involved in inhibition of Pol II phosphorylation. C. Neutrophil counts of urine of mice infected with ABU, SN25, d nlpD and d rpoS mutants. Functional relevance of Pol II de-repression in SN25 infected mutant is further suggested by higher neutrophil counts.

EXAMPLE 1

Inhibition of Eukaryotic RNA Pol II Phosphorylation by 83972 ABU Strain

[0054] The productive mRNA elongation step is generally marked by the phosphorylation of Pol II carboxy terminal domain on Serine-2 residues, consequently, Ser2 phosphorylation of Pol II is a good indicator of its activation. As a starting point, flow cytometry was developed as a technology to quantify Pol II phosphorylation (FIG. 1). Human kidney epithelial A498 cells were infected with ABU 83972 strain for 4 hours and after fixing and permeabilization, cells were stained with antibodies against phosphorylated Ser-2 in Pol II and goat anti-rabbit secondary antibodies labelled with Alexa-fluor 488. A marked reduction in Ser 2 phosphorylation was detected, compared to uninfected cells (FIG. 1A).

[0055] This effect was also confirmed using confocal microscopy (FIG. 1B). Infection with E. coli 83972 induced a change in the magnitude and distribution of phosphorylation, compared to uninfected cells. The loss of Pol II staining was visible as a loss in total fluorescence (mean value of 158, 493 AU compared to 588,307 AU, p<0.001), (FIG. 1C). In addition, a change in distribution resulted in the emergence of two peaks, with intensities of 5,522 and 256,779 AU. Uninfected cells showed one single peak with higher fluorescence intensity at 605,108 AU. Pol II inhibition was also clearly visible using confocal microscopy (FIG. 1B). The results confirm the inhibition of eukaryotic RNA Pol II phosphorylation by E. coli 83972.

[0056] To refine the Pol II phosphorylation data for middle-size events, Pol II phosphorylation was measured for events within gate R2. A higher number of cells fell into gate R2 for control compared to ABU infected sample (76.2 and 3 5.2%). ABU infection causes formation of smaller cells or broken cells (nuclei) when compared to uninfected sample as was seen from the number of small-size events. When R2-gated events are taken into consideration, lower-intensity peak of Pol II fluorescence in ABU sample becomes much less prominent. Mean value of Pol II phosphorylation in control and ABU infected sample for the representative experiment was 510,668 and 222,972 AU, respectively.

EXAMPLE 2

Identification of an ABU 83972 Variant SN25 with Loss of Pol II Inhibitory Activity and its Genome Sequencing

[0057] Patients were inoculated with therapeutic doses of ABU 83972. The protocol for therapeutic bladder inoculation of patients with E. coli 83972 has been described previously (Agace, J Clin Invest, 1993; Wullt, Mol Microbiol, 2000; Sunden, J Urol, 2010). Briefly, after antibiotic treatment to remove prior infection, patients were inoculated with E. coli 83972 through a catheter (30 ml, 10.sup.5 cfu/ml in saline). Blood and urine samples were obtained before and repeatedly after inoculation. Throughout the colonization period, viable bacterial counts in urine were determined, monthly urine samples were collected and analyzed for IL-6 and IL-8 as well as neutrophil infiltration. Bacteria from each urine sample were verified by PCR for presence of a kryptic plasmid unique for strain 83972 and one chromosomal marker (4.7-kb deletion in strain 83972 in the type 1 fimbrial gene cluster). For further analysis, five independent colonies per urine sample were used.

[0058] Re-isolates from inoculated patients were then screened for Pol II inhibitory activity as described in Example 1. One re-isolate, designated SN25, had lost Pol II inhibitory activity (FIG. 1D). A498 cells were infected with ABU or SN25 E. coli strain and labelled for phosphorylated Pol II as described above. Mean values of RNA Pol II phosphorylation in SN25-infected samples compared to uninfected control cells and obtained with flow cytometry and confocal microscopy, are shown in (FIGS. 1E and 1F, respectively). The ABU strain suppressed Pol II phosphorylation by about 73%, while SN25 by only 19%, suggesting that some genes in SN25 genome, responsible for suppression of Pol II phosphorylation, might have been lost or inactivated.

[0059] Genome of SN25 was sequenced (FIG. 2A) to identify responsible mutations, and 36 genomic changes were found compared to E. coli 83972 wt; 25 of them were in coding region with 8 resulting in amino acid change (FIG. 2B). Among the affected genes are those, responsible for L-lactate metabolism (lldD and lldR), regulation of motility and chemotaxis (lrhA), cell wall formation (nlpD) and biofilm formation (rfaH and cysE) (FIG. 2C). RfaH is a transcriptional anti-terminator, required for efficient expression of long chain LPS expression and hemolysin, its loss attenuates virulence of UPEC. CysE is a serine acetyltransferase, catalyzes the conversion of L-serine to O-acetyl-L-serine. Inactivation of mdoH leads to increased expression of colanic acid capsular polysaccharide. RcsB is a positive response regulator for colanic capsule biosynthesis.

EXAMPLE 3

Screening of Single-Gene Mutants to Identify Genes Responsible for Pol II Inhibition

[0060] To identify genetic determinants of Pol II phosphorylation, genes comprising the identified variant sequences were replaced in E. coli 83972 chromosome by homologous recombination with chloramphenicol resistance cassette. Deletions were validated (Uli). The mutants were subsequently screened for effects on Pol II (FIG. 3A). Single deletions of lldD, lldR, nlpD, rfaH and cysE reduced the inhibitory effect of E. coli 83972 wt, as shown by flow cytometry and confocal microscopy (FIG. 3B-C). Statistical difference in level of Pol II phosphorylation was measured with t test.

[0061] The lldD gene is responsible for aerobic L-lactate metabolism, whose product catalyzes the interconversion of L-lactate and pyruvate, while lldR is a regulator of the lldPRD operon. It was concluded from these data that products of both lldD and lldR genes are responsible for suppression of RNA Polymerase II phosphorylation. The low-intensity Pol II phosphorylation peak is less prominent after infection with SN25, ABU lldD and ABU lldR, which is in agreement with higher mean values of Pol II phosphorylation for cells infected with SN25 and ABU mutants compared to ABU. As shown, mean Pol II phosphorylation for ungated events for control cells and cells infected with ABU, SN25, ABU lldD and ABU lldR is 100, 78, 79, 50 and 50%, respectively. Thus, the that products of lldD (p<0.4) and lldR genes lead to inhibition of Pol II phosphorylation, with effect of lldR being statistically significant (p<0.05).

[0062] Several other mutants, which had significant effect on de-repression of Pol II phosphorylation, are 83972nlpD (p<0.01), 83972rfaH (p<0.01) and 83972cysE (p<0.05) (FIGS. 3B and C). Gene nlpD encodes a lipoprotein with a potential function in cell wall formation. Gene rfaH encodes for a transcriptional anti-terminator, required for efficient expression of long chain LPS, hemolysin and affects biofilm formation. Gene cysE encodes for serine acetyltransferase, which catalyzes the conversion of L-serine to O-acetyl-L-serine that is the first step of L-cysteine biosynthesis from L-serine; cysE product also affects biofilm formation in E. coli K-12.

EXAMPLE 4

Secretion of Bacterial Inhibitors of Pol II Phosphorylation

[0063] In parallel with the genetic studies, a biochemical approach was taken to identify the compound responsible for the Pol II inhibitory activity. Bacteria were incubated for 4 hours in tissue culture medium (RPMI supplemented with 1 mM pyruvate). The medium was harvested after 4 hours, centrifuged at 4,000g for 10 min and sterile filtered to remove remaining bacteria (0.2 m filter), before addition to human kidney cells (FIG. 3D). E. coli are typically 2 m long and 0.5 m in diameter, and filtration through 0.2 m syringe filters removes bacterial cells. Significant Pol II inhibitory activity (p=0.023) was identified in the ABU culture supernatants compared to uninfected, substituted RPMI, suggesting that growth in cell-free culture medium induced the secretion of the inhibitor(s).

[0064] As it was shown that supernatant of ABU bacteria have similar inhibitory effect on Pol II as ABU bacteria per se, we questioned if this effect is lost in the SN25 supernatant. Phosphorylation of Pol II was significantly higher (p<0.05) after incubation with SN25 supernatant compared to ABU supernatant, suggesting that the strain has lost the ability secrete inhibitors. Mutants of SN25 were therefore were grown in RPMI and their supernatants were harvested. Supernatants of lldD, lldR, nlpD and rfaH mutants had lost Pol II inhibitory activity (p<0.02 and p<0.05, respectively) compared to ABU supernatant, (FIG. 3E) indicating that genes lldD, lldR and nldD achieve their effect via effector molecules that are secreted by the bacteria.

[0065] The inhibitory activity of the supernatant was shown to be heat sensitive (100 C., 30 min) but Proteinase K resistant. The molecular size of the inhibitory component was found to be <3 kDa, by centrifugal ultrafiltration with a 3 kDa filter. Elevated levels of acetic acid and formic acid were detected in the filtrate of the ABU supernatant, using ion exchange chromatography. A rapid increase in formic-, succinic- and acetic acids was also detected by Mass spectrometry analysis of metabolites secreted by ABU upon growth in urine. Finally, high concentrations of formic and acetic acids were shown to inhibit Pol II phosphorylation (.sup.2 test for independence compared to medium control).

EXAMPLE 5

The ABU Strain Inhibits the Pol II Ser2 CTD Phosphorylating Machinery by Targeting Cyclin Kinase 12 and its Recruiting Protein PAF1C

[0066] The Pol II phosphorylation complex required to phosphorylate Ser2 is assembled in several steps (FIG. 4A). The preinitiation complex containing the TATA box binding protein (TBP), binds to DNA upstream of the transcription start site and the activated complex then recruits transcription factor IID, and the N-terminal Zink ribbon domain of TFII b is required to recruit Pol II. following the binding of TATA box binding to DNA, TBP recruits Pol II and the DNA-binding. The DNA binding subunit of Pol II anchors the preformed complex to DNA recruits Pol II to different eukaryotic promoters and the beta subunit is phosphorylated by the C-terminal domain (CTD) phosphorylation machinery. Cyclin-dependent kinase 9 (CDK9) brings the adaptor PAF1C into close proximity with Pol II and the PAF1C subunit CDC73 recruits CDK12 to the complex. CDK9 and CDK12 then phosphorylate the Pol II CTD domain, at Ser 2 (FIG. 4A).

[0067] To examine how the ABU strain suppresses phosphorylation of host RNA polymerase II CTD at Ser2 residue (FIG. 5E), we further analyzed potential protein targets in the Pol II phosphorylation pathway. Two cyclin dependent kinases are responsible for Pol II phosphorylation, CDK9 and CDK12 (FIG. 4a). CDK9 is involved as well, bringing PAF1C in close proximity to the Pol II promoter complex. PAF1C acts as a recruitment protein for CDK12.

[0068] To address how E. coli 83972 inhibits the Pol II phosphorylation machinery, CDK12, CDC73 (PAF1C subunit) and CDK9 in host cells, after infection with ABU and SN25 was quantified by Western Blot analysis (FIG. 4B). CDK12 and PAF1 decreased drastically after ABU infection but remained at control levels after SN25 infection. CDK9 levels were less strongly affected (40%). This effect on PAF1C and CDK12 was confirmed by confocal microscopy (FIG. 4C-D). SN25, in contrast, did not significantly alter PAF1C, CDK12 or CDK9 protein levels compared to uninfected cells.

[0069] To address if the inhibitory activity was a secreted bacterial product, A498 cells were treated with supernatants of cells infected with the single gene mutants of the ABU strain, and CDK12 and CDC73 levels were subsequently tested in WB. The results are shown in FIG. 4E. The nlpD deletion mutant had lost the inhibitory activity against PAF1 and CDK12.

[0070] This was confirmed by confocal microscopy (FIG. 4F). NlpD gene is located in one gene cluster with rpoS, encoding one of sigma subunits of bacterial RNA polymerase (FIG. 5A). Remarkably, the list of genes related to CDK12 and CDC73 suppression showed a direct association with the list of genes with effect on Pol II phosphorylation. In contrast, nlpD, lldD, lldR and rfaH mutations did not affect CDK12 or CDC73 and the cysE, lrhA, mdoH and rcsB deletion mutants resembled SN25.

[0071] Overall, these results indicate that nlpD and rpoS suppress Pol II phosphorylation by targeting the CDK12 and PAF arm of host phosphorylating machinery.

EXAMPLE 6

Investigation into NlpD and RpoS

[0072] In E. coli 83972, NlpD is located upstream of rpoS, which encodes Sigma S; the DNA binding subunit of bacterial RNA Polymerase (FIG. 5A). NlpD regulates Sigma S expression and may facilitate its release by activating cell wall hydrolases. To address if the effects of NlpD on Pol II and PAF are executed through rpoS, we constructed an rpoS deletion mutant in E. coli 83972. The deletion was confirmed by DNA sequencing and the loss of Sigma S protein was verified by Western blot analysis of bacterial cell extracts (FIG. 5B). Sigma S was present in extracts from E. coli 83972WT bacteria but was absent in extracts from SN25, nlpD and rpoS. In bacterial supernatants, the loss of Sigma S in these strains was confirmed (FIG. 5B).

[0073] The applicants subsequently examined the effect of the rpoS mutant on Pol II phosphorylation (FIG. 5C). The rpoS mutant had lost the ability to inhibit Pol II phosphorylation, and Pol II staining in infected cells was comparable to SN25 and the delta NlpD mutant (n.s.). A similar effect was observed for PAF1C, CDK12 as the rpoS mutant had lost the ability to inhibit PAF1C and CDK12 (FIGS. 6C-D). The results suggest that Sigma S acts as bacterial effector molecules in human cells, responsible for the inhibition of Pol II phosphorylation through PAF1C. The results suggest that the effects of NlpD on RpoS account for the attenuation of Pol IISer 2 phosphorylation.

EXAMPLE 7

Subcellular Distribution of nlpD and rpoS in Infected Human Cells

[0074] To address if these molecular interactions may be relevant in infected cells, the applicants subsequently examined, if Sigma S is internalized into human cells, after infection with E. coli 83972WT. By Western blot analysis of whole cell extracts, a single band of 40 kDa was detected, after staining with Sigma S specific antibodies. A band with similar mobility was detected in nuclear extracts, suggesting nuclear translocation of Sigma S (FIG. 6B). The Sigma S band was not detected in cells infected with SN25 or the AnlpD- or rpoS deletion mutants.

[0075] The internalization and nuclear translocation of Sigma S was confirmed, by co-immunoprecipitation, using Pol II specific antibodies. A band corresponding to Sigma S was detected in extracts from cells infected with ABU but not SN25. In a further Pol II co-ip involving whole cell extracts from cells infected with the NlpD or RpoS mutants, Sigma S and NLPD bands were detected in ABU infected cells but not in the single gene mutants. Low Sigma S in SN25, which does not carry a deletion.

[0076] In addition, human kidney cells were infected with E. coli 83972WT and stained with antibodies specific for Sigma S or Pol II, with nuclear DRAQ5 counterstaining. A parallel loss of nuclear Pol II staining and accumulation of RpoS in nuclear aggregates was detected. In contrast, Sigma S was not detected in cells infected with SN25, rpoS or AnlpD (FIG. 6D).

EXAMPLE 8

Competitive Inhibition of TBP Binding by Sigma S

[0077] The Pol II phosphorylation complex required to phosphorylate Ser2 is assembled in several steps (FIG. 4A). The preinitiation complex containing the TATA box binding protein (TBP), binds to DNA upstream of the transcription start site and the activated complex then recruits transcription factor IID, and the N-terminal Zink ribbon domain of TFII b is required to recruit Pol II. following the binding of TATA box binding to DNA, TBP recruits Pol II.

[0078] Like the TATA-box binding protein in eukaryotes, Sigma S binds to DNA and is the TATA box binding protein of the bacterial RNA Pol II complex (FIG. 5E). We therefore tested the hypothesis that Sigma S may bind to eukaryotic promoter DNA and competitively inhibit TBP binding (FIG. 5F). Human and bacterial TATA box oligonucleotides were incubated with a synthetic peptide comprising the Sigma S DNA-binding domain (aa 149-183-SEQ ID NO 2) in an electro-mobility shift assay (EMSA). Sigma S was shown to bind prokaryotic and human TATA box oligonucleotides, creating band shifts with similar mobility (FIG. 5G). Specificity was confirmed by inhibition of the band shift with Sigma S-specific antibodies. The Sigma S peptide was subsequently shown to competitively inhibit TBP binding to the IRF3 promoter, in a concentration-dependent manner (FIG. 5H).

[0079] To examine if E. coli 83972 affects PIC formation in infected cells, we quantified the TATA box binding protein (TBP) in total cell extracts from uninfected and infected kidney cells (FIG. 5B-D). E. coli 83972 reduced the TBP and TF2b protein levels. By confocal microscopy, nuclear transcription factor II b (TF2b) staining was reduced. SN25, in contrast, did not affect the PIC components (FIGS. 5C and D) and the nlpD deletion mutant reproduced the SN25 phenotype (FIG. 5F).

EXAMPLE 9

In Vivo Relevance

[0080] The effects on Pol II phosphorylation were confirmed in vivo, in the murine urinary tract infection model. C57BL/6 WT mice were inoculated with 210.sup.5 CFU/mL of ABU 83972, SN25, delta-nlpD or delta-rpoS and sacrificed after 24 hours. Tissue levels of RNA Pol II were quantified by staining of frozen tissue sections after staining with specific antibodies (FIG. 7A). E. coli 83972 inhibited mucosal Pol II staining in the urinary bladder mucosa, confirming the cellular studies (p<0.05, FIG. 7B). This inhibitory effect was not detected in mice infected with the SN25 mutant strain or the delta NlpD or Sigma S mutant strains. Neutrophil counts in urine of infected mice were measured (FIG. 7C) and higher counts were found in SN25 infected mice, indicating functional relevance of Pol II de-repression. Furthermore, it was found that the SN25 infection led to higher level of bacteria in bladder, suggesting higher virulence.

Materials and Methods

Bacterial Strains

[0081] Asymptomatic bacteriuria (ABU) strain was isolated during a study of childhood UTI in Goteborg, Sweden (Lindberg et al., 1978). Bacteria were cultured on tryptic soy agar (TSA, 16 h, 37 C.) and harvested in phosphate-buffered saline (PBS, pH 7.2). For the course of infection, bacteria were diluted to reach final concentration in medium 110.sup.8 cfu/ml.

Cell Culture

[0082] Human kidney carcinoma (A498, ATCC HTB44) were cultured in RPMI-1640 (Thermo Scientific) supplemented with 1 mM sodium pyruvate, 1 mM non-essential amino acids (GE Healthcare) and 10% heat-inactivated FBS at 37 C. with 5% CO.sub.2.

Preparation of Bacterial Supernatant

[0083] Bacteria were incubated for 4 hours in tissue culture medium, the medium was harvested, centrifuged at 4,000g for 10 min and filtered to remove remaining bacteria. Human kidney cells A498 were incubated with filtered supernatant for 4 hours.

Cell Flow Cytometry

[0084] Before infection, A498 cells were washed twice with RPMI medium without FCS. Cells were detached with Versene for 15 min, centrifuged at 400 g for 5 min and re-suspended in ice-cold PBS. 510.sup.5 cells were treated in suspension as follows: cells were infected, fixed (3.7% formaldehyde in PBS, 15 min), permeabilized (0.25% Triton X-100, 5% FBS in PBS, 10 min), blocked (5% FBS, 1 h at RT), incubated with primary antibodies in 5% FBS overnight at 4 C. (anti-RNA Polymerase II subunit B1 (phospho CTD Ser-2) 1:800, Merck) and with fluorescently labeled secondary antibody (Alexa Fluor 488 goat anti-rat IgG, A-11006, 1:600, Thermo Scientific) for 1 h at RT. After each step above apart from permeabilization and blocking, cells were washed twice with ice-cold PBS and centrifuged at 400 g for 5 min. After final wash, cells were re-suspended in flow cytometry buffer 0.02% EDTA 5% FCS in PBS. With BD Accuri C6 flow cytometer (BD Biosciences), 20,000 events were collected at 60 ul/min flow rate.

Confocal Microscopy

[0085] Cells were grown to 70-80% confluence on 8-well chamber Permanox slides (310.sup.4 cells/well, Thermo Fisher Scientific), and infected, fixed, permeabilized and treated with AB as for flow cytometry. After nuclear staining (15 min, DRAQS, Abcam), slides were mounted (Fluoromount, Sigma-Aldrich), imaged by laser-scanning microscopy (LSM800, Carl Zeiss) and quantified by ImageJ software 1.46r (NIH).

Ion Exchange Chromatography

[0086] Organic acids were analyzed on a Dionex anion chromatography system by the Swedish Environmental Research Institute. Potassium hydroxide was used as an eluent to separate ions in the sample. To obtain the best possible separation the concentration of the eluent was gradually changed during the process. After separation, a cation exchanger was used to reduce the conductivity of the eluent and to convert the anions into their respective acids.

Global Gene Expression

[0087] Total RNA was extracted from A498 cells in RLT buffer with 1% -Mercaptoethanol. 100 ng of RNA was amplified using GeneChip 3IVT Express Kit, 6 ng of fragmented and labeled aRNA was hybridized onto Human Genome array strips for 16 hours at 45 C., washed, stained and scanned using the GeneAtlas system (Affymetrix). All samples passed the internal quality controls included in the array strips (signal intensity by signal to noise ratio; hybridization and labeling controls; sample quality by GAPDH signal and 3-5 ratio<3).

[0088] Fold change was calculated by comparing cells treated with ABU or mutants to uninfected cells (PBS control) of the same genetic background. Significantly altered genes were sorted by relative expression (2-way ANOVA model using Method of Moments, P-values <0.05 and absolute fold change >1.41) (Eisenhart 1947). Heat-maps were constructed with Excel. Differentially expressed genes and regulated pathways were analyzed by Ingenuity Pathway Analysis software (IPA, Ingenuity Systems, Qiagen) and String and David open source software.

Western Blotting

[0089] Cells were lysed with RIPA lysis buffer, supplemented with protease and phosphatase inhibitors (both from Roche Diagnostics). Proteins were run on SDS-PAGE (3-8% or 4-12% Bis-Tris gels, Invitrogen), blotted onto PVDF membranes (GE Healthcare) blocked with 5% non-fat dry milk (NFDM), incubated with primary antibody: mouse anti-CDK12 (1:400 in 5% NFDM, ab9722, Abcam), mouse anti-Parafibromin (1:400 in 5% BSA, sc-22514-R, Santa Cruz), washed with PBS tween 0.1% and incubated with secondary antibodies in 5% NFDM (goat anti-mouse-HRP, Cell Signaling). Bands were imaged using ECL Plus detection reagent (GE Health Care) and quantified using ImageJ. GAPDH (1:1,000, sc-25778, Santa Cruz) was used as loading control.