Cell-penetrating bacterial E3-ubiqitin-ligases for use in immunotherapy

Abstract

The present invention relates to cell-penetrating effector proteins of type III secretion system (T3SS)-containing bacteria of the genus Salmonella or Shigella and variants, fragments and immunomodulatory domains thereof, for use in immunotherapy. The present invention further relates to cell-penetrating effector proteins of type III secretion system (T3SS)-containing bacteria of the genus Salmonella or Shigella and variants, fragments and immunomodulatory domains thereof, for delivering cargo molecules into eukaryotic cells.

Claims

1. A pharmaceutical composition, comprising an isolated effector protein of a type III secretion system (T3SS)-containing bacterium of the genus Salmonella or Shigella, or a variant, fragment, or immunomodulatory domain of the effector protein, wherein the effector protein or the variant, fragment, or immunomodulatory domain of the effector protein is covalently linked via a linker comprising a glycine-containing amino acid sequence to a cargo molecule selected from the group consisting of viruses, modified viruses, viral vectors, and antibodies wherein the effector protein or variant, fragment, or immunomodulatory domain of the effector protein: a) is recombinantly produced or chemically synthesized, b) comprises an E3 ubiquitin ligase domain, c) is a cell-penetrating protein which translocates into eukaryotic cells without the requirement of a bacterial T3SS, and d) optionally comprises at least one leucine-rich repeat.

2. The pharmaceutical composition of claim 1, wherein said type III secretion system (T3SS)-containing bacterium is classified as Salmonella bongori, Salmonella enterica, Salmonella subterranean, Salmonella typhi, Salmonella typhimurium, Salmonella enterica serovar typhimurium, Salmonella enteritidis, Salmonella pullorum, Salmonella dublin, Salmonella arizonae, Salmonella choleraesius, Shigella flexneri, Shigella dysenteriae, Shigella sonnei, or Shigella boydi.

3. The pharmaceutical composition of claim 1, wherein the E3 ubiquitin ligase domain is (a) classified as Novel E3 Ligase, (b) closer to the C-terminus of said effector protein than to the N-terminus of said effector protein, or both (a) and (b).

4. The pharmaceutical composition of claim 1, wherein the leucine-rich repeat(s) is/are (a) a leucine-rich repeat of the LPX-subtype, (b) closer to the N-terminus of said effector protein than to the C-terminus of said effector protein, or both (a) and (b).

5. The pharmaceutical composition of claim 1, wherein said effector protein is a bacterial effector protein of the LPX-Subytpe, or is selected from the group consisting of SspH1, SspH2, SlrP, IpaH1.4, IpaH2.5, IpaH3, IpaH4.5, IpaH7.8, and IpaH9.8.

6. The pharmaceutical composition of claim 1, wherein said effector protein has (a) an amino acid sequence as set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9, and (b) is encoded by a polynucleotide sequence as set forth in SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, or SEQ ID NO: 18.

7. The pharmaceutical composition of claim 1, wherein said effector protein, variant, fragment or immunomodulatory domain comprises an amino acid sequence as set forth in SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, or SEQ ID NO: 27.

8. The pharmaceutical composition of claim 1, wherein said effector protein comprises at least one Leucine-rich repeat, or at least one Leucine-rich repeat that is comprised in an amino acid sequence as set forth in SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, or SEQ ID NO: 36.

9. The pharmaceutical composition of claim 1, wherein said effector protein, or a variant, fragment or immunomodulatory domain thereof comprises at least one amino acid sequence as set forth in SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, or SEQ ID NO: 550; and/or at least one C-terminally or N-terminally truncated fragment of an amino acid sequence as set forth in SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, or SEQ ID NO: 550.

10. The pharmaceutical composition of claim 1, wherein the composition does not include: (a) an auxiliary agent which can cause the penetration of said effector protein into a eukaryotic cell, (b) an auxiliary agent selected from the group consisting of T3 SS-containing bacterium, and bacterium of the genus Salmonella or Shigella, (c) a cell-penetrating molecule, that is different from an effector protein according to claim 1, or (d) a cell-penetrating nanoparticle, or (e) any combination of (a)-(d).

11. The pharmaceutical composition of claim 1, wherein said cargo molecule has therapeutic activity in a subject and/or is useful in a diagnostic method.

12. The pharmaceutical composition of claim 1, wherein said effector protein or a variant, fragment or immunomodulatory domain thereof is capable of (a) ubiquinating itself and/or eukaryotic proteins after it has autonomously penetrated into a eukaryotic cell, (b) modulating cellular pathway(s) of the innate immune system of eukaryotic cells after it has autonomously penetrated into said eukaryotic cells, (c) modulating cytokines and/or cytokine receptors of eukaryotic cells and/or eukaryotic genes which respond to cytokines of eukaryotic cells after said effector protein or variant, fragment or immunomodulatory domain has autonomously penetrated into said eukaryotic cells, (d) downregulating the expression of cytokines and/or cytokine receptors of eukaryotic cells after it has autonomously penetrated into said eukaryotic cells, or any combination of (a)-(d).

13. A kit comprising the pharmaceutical composition of claim 1.

Description

DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 shows a schematic overview of the recombinant SspH1 and its derivative SspH1-Nter. SspH1-Nter lacks the C-terminal domain of SspH1 which encodes an ubiquitin ligase. Proteins were tagged with a C-terminal 6His-tag (not shown) for purification.

(2) FIG. 2 shows an immunoblot analysis of subcellular fractionations of HeLa cells incubated with 25 g/ml SspH1 and SspH1-Nter, respectively. In order to assess their purity, membrane and cytosolic fractions were analyzed using antibodies against transferrin receptor (TF-R) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH), respectively (left panel). Purity of cytoplasmic and nuclear fractions was determined using antibodies against tubulin and Lysine-specific demethylase 1 (LSD-1), respectively (right panel). SspH1 and SspH1-Nter partition was assessed using anti-FLAG antibody. CF: cytoplasmic fraction MF: membrane fraction NF: nuclear fraction.

(3) FIG. 3 shows an immunofluorescence microscopy of HeLa cells incubated with SspH1-Cy3 and SspH1-Nter-Cy3 1 h. DNA was stained with DAPI. Both fluorescence images were merged.

(4) FIG. 4 shows a quenched time lapse assay of HeLa cells incubated with YopM-ALEXA FLUOR 488, SspH1-ALEXA FLUOR 488 or Tat-GFP-GSK. HeLa cells were incubated with 20 g/ml protein. Samples were taken from the incubation at different time points. Extracellular fluorescence was quenched with trypan blue (final concentration: 0.2%) and intracellular fluorescence was measured using a flow cytometer. Data are expressed as geometric means of fluorescence intensities from at least 10,000 cells relative to background fluorescence of untreated cells, and are presented as meansstandard deviations from three independent experiments.

(5) FIGS. 1 to 4 show that SspH1 and SspH1-Nter translocate into the host cell cytoplasm independently of Salmonella's T3SS.

(6) FIG. 5 shows a Membrane integrity assay of HeLa cells incubated with SspH1, YopM and Tat, respectively. Cells were incubated with 20 g/ml protein and co-incubated with 1 g/ml propidium iodide (PI).

(7) FIG. 6 shows Recombinant SspH1 and SspH1-Nter do not have cytotoxic effects on Hela cells. Cells were incubated with SspH1 and SspH1-Nter, respectively, for 1, 6, and 24h and analysed using the CYTOTOX 96 Non-Radioactive Cytotoxicity Assay. LDH: lactate dehydrogensase.

(8) FIG. 7 shows in vitro ubiquitination assays which were performed in the presence of UbcH5c. Shown are synthesized unanchored polyubiquitin chains detected by anti-ubiquitin western blot. Upon incubation with E1, E2 and ubiquitin, SspH1 was able to remove ubiquitin from the E2 enzyme and perform self-ubiquitination

(9) FIG. 8 shows that recombinant SspH1 ubiquitinates PKN1 in vitro. Shown are immunoblot analysis using anti-HA, anti-PKN1, and anti-His antibodies of reactions performed in the presence of Ub-Ha, E1, E2, Ub-Ha, SspH1, and PKN1. Upon incubation with E1, E2 and ubiquitin, SspH1 was able to remove ubiquitin from the E2 enzyme and ubiquitinate PKN1.

(10) FIG. 9 shows that recombinant SspH1 co-immunoprecipitates with PKN1 from transfected cells and self-ubiquitinates. HeLa cells were transiently transfected with PKN1-Myc. 24 h later, cells were incubated with FLAG-tagged SspH1 for 3 h. Following SspH1 incubation, PKN1 was immunoprecipitated and the immunoprecipitates and supernatants were immunoblotted using anti-FLAG and anti-Ubiquitin antibodies.

(11) FIG. 10 shows the effect of SspH1 on IL-8 mRNA in A549 cells stimulated with IL1. A549 cells were incubated with SspH1 for 3 h and subsequently stimulated with 20 ng/ml IL1. After RNA isolation and cDNA synthesis, all qRT-PCR data were obtained using SYBR green and IL8-specific primers. The HPRT1 gene (hypoxanthine phosphoribosyl-transferase I), a low abundance housekeeping gene, was used as reference. Data represent means and standard deviations of at least three independent experiments each performed in duplicate*p<0.05.

(12) FIGS. 6 to 10 show that recombinant SspH1 possesses E3 ubiquitin ligase activity.

(13) FIG. 11a to 11e shows an uptake analysis of effector proteins of the LPX family. Upper panel: Confocal laser scanning immunofluorescence microscopy of HeLa cells incubated with IpaH1.4-Cy3, IpaH4.5-Cy3, IpaH7.8-Cy3, IpaH9.8-Cy3, and SlrP-cy3 for 1 h. Actin was counterstained with phalloidin ALEXA FLUOR 488 and nuclei with DRAQ5. All three fluorescence images were merged and confocal Z-stack projections are included in all images. The cross hairs show the position of the xy and yz planes. Scale bars: 10 m. Lower panel: Quenched time-lapse assay of HeLa cells incubated with different ALEXA FLUOR 488 labeled effector proteins (20 g/ml). Samples were taken from the incubation at different time points. Extracellular fluorescence was quenched with trypan blue (final concentration: 0.2%) and intracellular fluorescence was measured using a flow cytometer. Data are expressed as geometric means of fluorescence intensities from at least 10,000 cells relative to fluorescence of untreated cells, and are presented as meansstandard deviations from three independent experiments.

(14) FIG. 12 shows a schematic overview of an in-silico prediction analysis YopM, SspH1, SspH2, SlrP, IpaH1.4, IpaH2.5, IpaH3, IpaH4.5, IpaH7.8, and IpaH9.8. Boxes indicate sequence segments containing predicted PTD sequences (PTDs of maximal length).

(15) FIG. 13. FIG. 13a shows an in vitro ubiquitination assay of recombinant SspH1. Upon incubation with E1, E2 and ubiquitin, SspH1 is able to remove ubiquitin from the E2 enzyme and to self-ubiquitinate (see also FIG. 7). In the presence of PKN1, SspH1, but not SspH1-Nter, polyubiquitinates the kinase (FIG. 13a). FIG. 13b shows 118 mRNA expression in A549 cells following incubation with SspH1 and subsequent stimulation with TNF.

(16) FIGS. 14 to 22 shows amino acid sequences of SlrP, SspH1, SspH2, IpaH1.4, IpaH2.5, IpaH3, IpaH4.5, IpaH7.8 and IpaH9.8. leucine-rich repeats (called LRR stretch) are marked in light grey and are non-edged. E3 Ubiquitin ligase domains are marked in dark grey and are edged. Sequence sections within which the protein transduction domains (PTDs) are predicted are bold and underlined. Marked are the maximal sequence sections within the PTD is predicted. PTDs can correspond to the marked sequence sections or to C-terminally and/or N-terminally truncated fragments of the marked sequence sections.

(17) FIG. 23 shows a cell fractionation of HeLa cells incubated with recombinant IpaH1.4, IpaH2.5, IpaH4.5, IpaH7.8, and IpaH9.8, respectively.

(18) Western blot analysis after cell fractionation of HeLa cells which were incubated with the indicated LPX effector protein (25 g/ml) for 3 h. Proteins were separated by 12.5% SDS-PAGE and immobilized on a nitrocellulose membrane by Western blotting. Proteins were detected using an a FLAG-antibody and corresponding PO-conjugated secondary antibody. Purity of both the cytoplasmic (CF) and membrane fraction (MF) were assesses using an -GAPDH-antibody and a TF-R-antibody and corresponding PO-conjugated secondary antibodies.

(19) FIG. 24 shows a confocal fluorescence microscopy of HeLa cells incubated with recombinant IpaH1.4, IpaH4.5, IpaH7.8, IpaH9.8, or SlrP, concerning co-localization with the endocytic markers Rab5, Rab7, and CD63. HeLa cells were incubated with the indicated recombinant, Cy3-labeled (red) LPX effector proteins (25 g/ml) for 1 h (i), 3 h (ii), and 6 h (iii), respectively (A)-(F). The endocytic markers Rab5, Rab7, and CD63 were detected using appropriate primary antibodies and stained via incubation with Cy2-labelled (green) secondary antibodies. DNA was stained using Draq5 (blue). Overlays of all three channels are shown in large squares; pictures of the individual channels are shown on the right. Magnification 63.

(20) FIG. 25 shows a flow cytometry analysis of effector proteins of the LPX family. HeLa cells were with endocytic inhibitors cytochalasin D (2.5 M), amiloride (3 mM), filipin (7.5 M), nocodazole (20 M), dynasore (80 M), methyl--cyclodextrin (5 mM) for 1 h prior to the addition of recombinant LPX effectors. Three hours later the cells were washed with D-PBS (with Ca.sup.2+/Mg.sup.2+), trypsinized, resuspended in D-PBS (without Ca.sup.2+/Mg.sup.2+), diluted with trypan blue (final concentration 0.2%) and analyzed by flow cytometry

(21) FIG. 26 shows a membrane integrity assay of HeLa cells incubated with recombinant IpaH1.4, IpaH2.5, IpaH4.5, IpaH7.8, IpaH9.8, or SlrP. HeLa cells were co-incubated with both the indicated LPX effector protein (25 g/ml) and propidium iodide (PI) (1 g/ml) at 37 C. or 4 C. for a total duration of 6 h and 3 h, respectively, (A)-(F). Samples were taken from the ongoing incubation at given time points and subjected to FACS analysis. Fluorescence of PI was detected at 617 nm. All samples were measured twice and three independent experiments were carried out for each protein. (A)-(F): Diagrams show the fluorescence intensity of HeLa cells co-incubated with the indicated ALEXA FLUOR 488-labeled LPX effector protein and PI at different incubation times both at 37 C. and 4 C. in comparison to HeLa cells which were solely incubated with PI (dashed lines). The geometric mean fluorescence [arbitrary units] (normalized to negative/untreated control cells) is plotted against the incubation time [minutes].

(22) FIG. 27 shows the relative release of LDH of HeLa cells upon incubation with recombinant IpaH1.4, IpaH2.5, IpaH4.5, IpaH7.8, IpaH9.8, or SlrP. HeLa cells were incubated with the indicated LPX effector protein (25 g/ml) for a total duration of 24 h, 6 h, and 1 h, respectively, (A)-(F). The release of LDH was measured using the CYTOTOX 96 Non-Radioactive Cytotoxicity Assay. Diagrams show the relative amount of released LDH normalized to the amount releases by non-treated (Medium control) which was set equal to 1. Additionally, a LDH positive control was measured. LDH: Lactate dehydrogenase.

(23) FIG. 28 shows an in vitro ubiquitination assay using either recombinant IpaH1.4, IpaH2.5, IpaH4.5, IpaH7.8, IpaH9.8, or SlrP. Western blot analysis of in vitro ubiquitination assays which were performed in 40 l reaction mixtures containing (+) or not containing () the indicated protein (4 g), HA-tagged ubiquitin (2 g), E2 (UbcH5b)(2 g), and E1 (0.5 g). Reactions were stopped by the addition of 4SDS sample buffer without DTT. Samples were subjected to 10% SDS-PAGE and immobilized on a Nitrocellulose membrane by Western blotting. Ubiquitin was detected using an -HA antibody and a corresponding PO-conjugated secondary antibody. Different Western blots are separated by dashed lines.

DESCRIPTION OF THE SEQUENCE LISTING

(24) SEQ ID NO: 1: Amino acid sequence of SlrP from Salmonella enterica subsp. enterica serovar Typhimurium str. 14028S SEQ ID NO: 2: Amino acid sequence of SspH1 from Salmonella enterica subsp. enterica serovar Typhimurium str. 14028S SEQ ID NO: 3: Amino acid sequence of SspH2 from Salmonella enterica subsp. enterica serovar Typhimurium str. 14028S SEQ ID NO: 4: Amino acid sequence of IpaH1.4 from Shigella flexneri SEQ ID NO: 5: Amino acid sequence of IpaH2.5 from Shigella flexneri SEQ ID NO: 6: Amino acid sequence of IpaH3 from Shigella flexneri SEQ ID NO: 7: Amino acid sequence of IpaH4.5 from Shigella flexneri SEQ ID NO: 8: Amino acid sequence of IpaH7.8 from Shigella flexneri SEQ ID NO: 9: Amino acid sequence of IpaH9.8 from Shigella flexneri SEQ ID NO: 10: Nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 1 SEQ ID NO: 11: Nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 2 SEQ ID NO: 12: Nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 3 SEQ ID NO: 13: Nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 4 SEQ ID NO: 14: Nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 5 SEQ ID NO: 15: Nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 6 SEQ ID NO: 16: Nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 7 SEQ ID NO: 17: Nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 8 SEQ ID NO: 18: Nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 9 SEQ ID NOs: 19 to 27: Ubiquitin ligase domains of SEQ ID NOs: 1 to 9 as indicated in FIGS. 14-22 SEQ ID NOs: 28 to 36: Leucine rich repeat stretches or areas of comprising Leucine rich repeat stretches SEQ ID NOs: 1 to 9 as indicated in FIGS. 14-22 SEQ ID NOs: 37 to 65 and 550: Maximal protein-transduction domains of SEQ ID NOs: 1 to 9 as indicated in FIGS. 14-22 SEQ ID NOs: 66 to 529: Protein-transduction domains of SEQ ID NOs: 1 to 9 as determined in Example 5 and indicated in Example 9 SEQ ID NOs: 530 to 549: Oligonucleotide sequences as indicated in Table 3.3

EXAMPLES

(25) The following examples are for illustrative purposes only and are not intended to limit the scope of the claims.

Example 1

SspH1 can Translocate into the Host Cell Cytoplasm Independently of Salmonella's T3SS

(26) The present inventors constructed and recombinantly expressed SspH1, an LPX effector protein of Salmonella enterica serovar typhimurium. Potential T3SS-independent translocation of this effector protein was analyzed by cell fractionation of HeLa cells, immunofluorescence microscopy, and FACS.

(27) To enable the expression and purification of SspH1, recombinant proteins tagged with a C-terminal 6His-tag were constructed. The C-terminal region of SspH1 encodes an ubiquitin ligase that might interfere with potential T3SS-independent uptake of SspH1 due to size and structure. Therefore, a truncated derivative of SspH1 was constructed in addition to the full length SspH1 protein. The N-terminal construct SspH1-Nter comprises the N-terminal -helical domain as well as the Leucine-rich repeats (LRRs), lacking the enzymatic domain (FIG. 1). In order to initially analyse whether the recombinant proteins SspH1 and SspH1-Nter are able to translocate into the cytoplasm of eukaryotic host cells, cell fractionation of HeLa cells was performed.

(28) The results of the cell fractionation indicate that recombinant SspH1 is able to translocate across eukaryotic plasma membranes in a T3SS-independent manner and is taken up into the host cell cytoplasm. Furthermore, the N-terminal construct of SspH1 (SspH1-Nter), comprising the N-terminal -helical domain and the LRRs, was also detected in the cytoplasmic fraction after 1 h of incubation of HeLa cells with the recombinant protein (FIG. 2). Internalization of the N-terminal construct indicates that T3SS-independent uptake of recombinant SspH1 depends on the N-terminal domain of the protein. The enzymatic C-terminal domain of SspH1 does not seem to interfere with membrane penetration. During infection, T3SS-translocated SspH1 has been shown to localize to the host cell nucleus (Haraga & Miller, 2003). In order to analyze localization of recombinant SspH1 to the nucleus, nuclear fractionation was performed. SspH1 as well as SspH1-Nter were detected in the nuclear fraction of HeLa cells after 1 h of incubation (FIG. 2), indicating that recombinant SspH1 is able to translocate to the nucleus following internalization by a yet unknown mechanism.

(29) The uptake of recombinant SspH1 was further characterized by immunofluorescence microscopy of HeLa cells incubated with the recombinant protein. In order to allow detection of SspH1 and SspH1-Nter by immunofluorescence microscopy, the proteins were labeled with the fluorescent dye Cy3 (GE Healthcare, Braunschweig). Both proteins, Cy3-labeled SspH1 and SspH1-Nter are clearly detected in the HeLa cell cytoplasm indicating uptake of SspH1 (FIG. 3). The proteins show no obvious difference in their intracellular localization. Both proteins are equally distributed throughout the cytoplasm following internalization (FIG. 3).

(30) For quantitative analysis of cell-penetration by SspH1, a flow cytometry-based quenched time-lapse assay was performed and compared to uptake efficiencies of YopM and the Tat-GFP-GSK fusion protein (FIG. 4). The results indicate a significantly higher uptake of the bacterial proteins YopM and SspH1 compared to the Tat-GFP-GSK fusion protein. After 3 h of incubation, the relative fluorescence intensity of cells incubated with AF488-labeled SspH1 and YopM, respectively is about 3.5 to 4 times higher than of cells incubated with Tat-GFP-GSK. These results correlate with the data obtained from fluorescence microscopy, where less Tat-GFP-GSK was detected within the cells compared to YopM.

(31) Taken together, these experiments show that both bacterial effector proteins YopM and SspH1 are efficient cell-penetrating effector proteins (CPE), which share the ability to enter host cells independently of T3SS.

Example 2

Recombinant SspH1 Affects Membrane Integrity but is not Cytotoxic

(32) HeLa cells were incubated with the recombinant proteins SspH1-AF488, YopM-AF488 and Tat-GFP-GSK and co-incubated with 1 g/ml PI during the ongoing incubation (FIG. 5). Samples were taken from the culture at defined time points and directly subjected to FACS analysis. In addition to fluorescence of PI, the fluorescence of the recombinant proteins was measured to monitor uptake of the peptides. As a negative control, HeLa cells were incubated with PI but without addition of recombinant proteins. Untreated control cells were used to select viable cells for data acquisition before the samples were measured in triplicate with 10,000 events per measurement.

(33) In order to monitor potential peptide-induced membrane lysis, the fluorescence intensity of PI of cells incubated with the recombinant proteins YopM-AF488, SspH1-AF488 and Tat-GFP-GSK, respectively, was measured. Membrane disruption is indicated by an increase of PI fluorescence due to increasing permeability of the plasma membrane for PI. Cells incubated with the recombinant proteins were compared to control cells which were also incubated with PI but not with the proteins.

(34) Incubation of HeLa cells with SspH1 results in the highest increase of PI fluorescence indicating massive membrane disruption by the recombinant protein. The PI fluorescence intensity of cells incubated with SspH1 is 4 to 5 times higher during the course of incubation compared to the control cells. However, SspH1-induced membrane disruption does not seem to lead to cell lysis since only viable cells were detected for data acquisition. Cells incubated with the bacterial CPP YopM and the well described Tat peptide show an approximately 2-fold increase in PI fluorescence intensity compared to the control cells. These data indicate some effect on the membrane integrity by YopM and Tat though compared to SspH1, the effects are much lower. However, both proteins have been suggested to be internalised by endocytic uptake mechanisms which per se do not imply disruption of the plasma membrane. Thus, the present results indicate that accumulation of both proteins at the plasma membrane might cause destabilisation of the plasma membrane. Whether the observed membrane disruption leads to a potentially direct uptake of the peptides cannot be concluded from the present results. As for cells incubated with SspH1, effects of YopM and Tat-GFP-GSK do not lead to complete cell lysis as cells were still detected as viable.

(35) In order to further exclude that the observed effect of SspH1 on membrane integrity result from cell lysis, a cytotoxicity assay was performed (FIG. 6). Therefore, HeLa cells were incubated with 25-50 g/ml SspH1 and SspH1-Nter, respectively for 1, 6 and 24 h. Cytotoxicity was measured using the CYTOTOX 96 Non-Radioactive Cytotoxicity Assay (Promega). In this assay, the amount of lactate dehydrogenase (LDH) released by the cells into the supernatant is measured. An increase in LDH release indicates cytotoxic effects of the proteins. Following incubation with SspH1 and SspH1-Nter, no increase in LDH release was observed when compared to untreated control cells even after 24 h of incubation, indicating that the proteins do not have any cytotoxic effects on the HeLa cells.

(36) The strong effects of SspH1 on the membrane integrity of HeLa cells with no effects on cell viability, suggest a potential direct uptake mechanism by the formation of a transient membrane pore.

Example 3

Functionality of the Bacterial Effector Protein SspH1

(37) SspH1 belongs to the family of proteins containing the LRR motif and comprises 8 LRRs. The C-terminal domain of the protein resembles that of the type III secreted IpaH proteins from Shigella flexneri which possess E3 ubiquitin ligase activity. Ubiquitin ligases mediate the transfer of ubiquitin to target proteins. Ubiquitination is a process generally occurring in all eukaryotic cells that is involved in protein degradation, signal transduction as well as cell cycle regulation. It has been shown that the C-terminal domain of SspH1 is indeed an E3 ubiquitin ligase domain (Quezada et al, 2009) that uses ubiquitin as well as protein kinase 1 (PKN1) as substrates for ubiquitination (Rohde et al, 2007). Furthermore, it has been suggested that this interaction is involved in downregulation of expression of pro-inflammatory cytokines by SspH1.

(38) To test if recombinant SspH1 is also functional and could polyubiquitinate proteins, in vitro ubiquitination assays were carried out in a 40 l reaction mixture containing ubiquitination buffer (25 mM Tris*HCl [pH 7.5], 50 mM NaCl, 5 mM ATP, 10 mM MgCl2, 0.1 mM DTT), 2 g Ha-ubiquitin, 0.5 g E1, and 2 g E2 (UbcH5B) in the presence or absence of 1 g recombinant SspH1. Ubiquitination analysis of PKN1 was performed in the presence or absence of additional 0.4 g GST-tagged PKN1. Reactions were incubated at 37 C. for 1 h and stopped by the addition of Laemmli sample buffer with or without 100 mM DTT.

(39) Western blot analysis showed that also recombinant SspH1 has the ability to remove ubiquitin from ubiquitinated UbcH5B, to autoubiquitinate, and to polyubiquitinate Ha-tagged ubiquitin (FIG. 7). Additionally, the inventors also tested ubiquitination of PKN1 by SspH1. When the reaction was performed in the presence of SspH1 and PKN1 in vitro, anti-PKN1 antibodies detected an additional species migrating at a size >148 kDa (FIG. 8). Furthermore, the inventors wanted to confirm interaction of SspH1 with PKN1 and its Ubiquitination also in vivo (FIG. 9). Therefore, HeLa cells were transiently transfected with a PKN1 expression vector and subsequently incubated with recombinant FLAG-tagged SspH1 and Ssph1-Nter, respectively. The proteins were then immunoprecipitated with anti-PKN1-agarose antibody conjugate. As shown in FIG. 9, Ssph1-FLAG co-immunoprecipitated with PKN1. Furthermore, using anti-ubiquitin antibodies, the inventors could confirm self-ubiquitination of SspH1 in HeLa cells.

(40) To determine whether cytokine production is also down-regulated by recombinant SspH1, the levels of IL-8 mRNA of A549 cells after stimulation with IL1 in the presence or absence of SspH1 were analyzed. As shown in FIG. 10, cells which have been pre-incubated with SspH1 for 3 h produced significantly less IL-8 mRNA upon IL-1 stimulation than untreated cells.

(41) Taken together, the present inventors showed that the SspH1 effector protein of Salmonella enterica serovar typhimurium is able to translocate into eukaryotic cells without a requirement for additional factors. Furthermore, the inventors demonstrated that recombinant SspH1 is functional as an E3 ubiquitin ligase that uses PKN1 as substrate after penetrating the host cells and that is able to reduce the expression of Interleukin 8 in IL113 stimulated cells, but not in TNF-stimulated cells (FIG. 13b)

Example 4

Analysis of Further Effector Proteins of the LPX Family

(42) The inventors identified SspH1 as a bacterial cell-penetrating protein. Subsequently the inventors investigated whether there is a general concept of T3SS-independent translocation by LPX effector proteins. For this, the effector proteins of the IpaH subset from Shigella flexneri as well as SlrP from Salmonella enterica serovar typhimurium have been cloned with a C-terminal 6His-tag for purification. After purification, the different effector proteins were labelled with the fluorescent dyes Cy3 (IF) or ALEXA FLUOR 488 (FACS) for subsequent uptake analysis by immunofluorescence microscopy and FACS (FIG. 11a-e).

(43) Fluorescence microscopy analysis revealed that the effector proteins of the IpaH subset (IpaH1.4-Cy3, IpaH4.5-Cy3, IpaH7.8-Cy3, IpaH9.8-Cy3), as well as Cy3-labeled SlrP have entered cells after 1 h incubation (FIG. 11a-e, upper panel). Although the intracellular amount of the different effector proteins appeared to differ, all proteins are distributed throughout the cytoplasm and show only little difference in their intracellular localization (FIG. 11a-e, upper panel). Additionally, after the proteins have been conjugated to an ALEXA FLUOR 488 fluorescent dye, a flow cytometry based quenched time lapse assay was performed for quantitative analysis of uptake efficiency (FIG. 11a-e, lower panel). In all cases, an increase of relative fluorescence intensity was monitored for HeLa cells after incubation with the recombinant proteins, indicating efficient internalization. The increase of fluorescence intensities appeared different for the shown effector proteins. However, comparison of the curves over the entire time course shows a considerable increase of intracellular fluorescence intensity for incubation times of up to 180 min for IpaH1.4, IpaH4.5, IpaH7.8, IpaH9.8, and SlrP respectively, indicating an efficient internalization (FIG. 11a-e, lower panel).

(44) In summary, the experiments of the inventors demonstrate that the LPX-family from S. flexneri and S. enterica serovar typhimurium share the ability of YopM and SspH1 to enter the cytosol independently of the T3SS. These results strengthen the hypothesis of a new general concept for internalization of these effector proteins.

Example 5

In-Silico Prediction of Cell-Penetrating Properties of LPX Family Members

(45) To identify putative protein transduction domains (PTD) within the amino acid sequences of the LPX family members, the inventors used in-silico prediction of cell-penetrating properties based on a method developed by Stephen White's laboratory shareware (blanco.biomol.uci.edu; Jaysinghe S., 2009). This approach utilizes the Wimley-White hydrophobicity scales (White & Wimley, 1999), reflecting the ability of binding and insertion into lipid bilayers. Hence, available amino acid sequences of SspH1, SspH2, IpaH1.4, IpaH2.5, IpaH3, IpaH4.5, IpaH7.8, IpaH9.8, SlrP, YopM were screened for putative PTD with the help of CEPEP company (lo Langel, Schweden). The results of this screen are described in Example 5 and summarized in FIG. 12.

(46) The computational analysis of the YopM amino acid sequence from Y. enterocolitica (pYV8081) identified a PTD within the N-terminal domain of the protein (FIG. 12). This sequence overlaps with the previous experimentally identified transport domain of YopM (Rter el al., 2010), indicating that the in-silico prediction of cell-penetrating properties is a usable tool to identify potential PTDs within the amino acid sequences of CPEs. Several unknown PTDs have been predicted for the rest of the family members. Obviously, all effector proteins of the IpaH subset share two potential PTDs in their C-terminal region (FIG. 12). Furthermore, IpaH4.5 harbors two additional PTDs and IpaH7.8 as well as IpaH9.8 only one additional transport domain. Similarly to the IpaH proteins of Shigella, several PTDs were also identified within the sequence of the effector proteins from Samonella. While three unknown PTDs were identified within the sequence of SlrP, both SspH effector proteins (SspH1&2) showed in total twelve PTDs (six each) with different sequences to the previously reported.

(47) Taken together, the in-silico PTD-prediction analysis of SspH1, SspH2, SlrP, IpaH1.4, IpaH2.5, IpaH3, IpaH4.5, IpaH7.8, and IpaH9.8 in compare to YopM revealed that they harbor several unknown PTDs, which might be responsible for T3SS-independent uptake of the effector proteins into eukaryotic cells.

Example 6

Methods and Materials of Experiments 1 to 5 and FIGS. 1 to 22

(48) 1 Construction of 6His-Tagged Recombinant Proteins

(49) 1.1 Restriction Free (RF) Cloning by Linear Amplification

(50) Restriction Free (RF) cloning is an alternative method to insert a DNA fragment into a desired location within the vector (Chen et al. 2000, van den Ent & Lwe, 2006) Using primers that contain a 3 target-specific sequence and a 5 extension that is complementary to the desired insertion site within the vector, a double-stranded PCR product is generated, containing the target sequence and the sequence of the insertion site at both the 3 and 5 end. This PCR product is then used as a pair of mega-primers in a second amplification reaction. Both strands of the PCR product anneal to the insertion site of the vector and are extended in a linear amplification reaction resulting in a nicked double-stranded plasmid.

(51) The PCR reaction for target DNA amplification was carried out as listed in Tables 1 & 2.

(52) TABLE-US-00001 TABLE 1 PCR reaction RF cloning Template DNA ~1-200 ng dNTP mix (10 mM each) 1 l (final concentration 100 M) Oligonucleotide primers 0.1 g (each) Phusion Polymerase buffer 1x Phusion Polymerase 1 unit Add H.sub.20 to 50 l

(53) TABLE-US-00002 TABLE 2 PCR cycling conditions Reaction step Temperature Duration Cycles I Initial denaturation 98 C. 1 min 1x II Denaturation 98 C. 30 s III Annealing T.sub.m 30 s 35x IV Elongation 72 C. 15-30 s/kb V Final elongation 72 C. 10 min 1x

(54) Linear amplification of vector and insert using the double-stranded PCR product from the first PCR as megaprimers was performed according to Unger et al. (2010) using the reaction mixture listed in Table 3 and carried out in a PCR thermal cycler with the cycling conditions listed in Table 4.

(55) TABLE-US-00003 TABLE 3 PCR reaction linear amplification Template DNA 20 ng dNTP mix (10 mM each) 1 l (final concentration 200 M) PCR product (mega-primers) 100 ng Phusion Polymerase buffer 1x Phusion Polymerase 1 unit Add H.sub.20 to 50 l

(56) TABLE-US-00004 TABLE 4 PCR cycling conditions for linear amplification Reaction step Temperature Duration Cycles I Initial denaturation 98 C. 30 s 1x II Denaturation 98 C. 30 s III Annealing 60 C. 1 min 30x IV Elongation 72 C. 5 min V Final elongation 72 C. 7 min 1x

(57) Following the amplification reaction, 0.2 U/l DpnI were added for 2 h at 37 C. to eliminate parental plasmid DNA. 10 l of the DpnI treated reaction mixture were then used for transformation of competent E. coli DH5a cells.

(58) 2 Expression and Purification of Recombinant Protein

(59) 2.1 Expression of Recombinant Protein in E. coli

(60) For expression of recombinant proteins, the pET24b(+) expression vector was chosen which provides the coding sequence for a C-terminal 6His-tag. All plasmids used in this study are derivatives of pET24b(+).

(61) For protein expression, the recombinant plasmids carrying the respective coding sequences were transformed into E. coli BL21(DE3) cells. Expression of the target proteins was carried out in 500 ml of Standard I medium containing 50 g/l kanamycin. The culture was inoculated 1:100 with an overnight culture and incubated at 37 C. and 180 rpm until an OD.sub.600 of 0.6-0.8 was reached. Then expression of the recombinant proteins was induced by adding IPTG to a final concentration of 1 mM before the culture was incubated for an additional 4 h. Cells were harvested by centrifugation at 3,000g and 4 C. for 15 min and the cell pellet was stored at 20 C. until further usage.

(62) 2.2 Preparation of Cleared E. coli Lysates

(63) The bacterial pellets were thawed on ice and resuspended in 10 ml of lysis buffer. Cleared lysates were prepared by sonication (Branson Sonifier 250; 430 s, level 4, 50% cycle, on ice). Bursts were followed by 15 s breaks. The cellular debris was removed by centrifugation (7,200g, 15 min, 4 C.) and the supernatant containing the recombinant protein was subjected to affinity chromatography.

(64) Heterologous expression of recombinant proteins can lead to formation of inclusion bodies; aggregates of the overexpressed protein that remain insoluble. These can be solubilised by addition of anionic detergents such as N-lauroylsarcosine sodium salt. Where the desired protein was detected in the insoluble fraction, 2% (w/v) N-lauroylsarcosine sodium salt were added following sonication and incubated on a rotary shaker (15 rpm, 4 C.) for 1 h before centrifugation as mentioned above.

(65) TABLE-US-00005 Lysis buffer N-Lauroylsarcosine sodium salt Tris-HCl, pH 8.0 50 mM Tris-HCl, pH 8.0 25 mM NaCl 500 mM NaCl 100 mM Imidazole 10 mM N-Lauroylsarcosine 10% (w/v) Glycerol 10% (v/v) sodium salt TRITON X-100 0.1% (v/v)

(66) 2.3 Purification of Recombinant Protein

(67) Protein purification was performed by nickel-nitrilotriacetic acid (Ni-NTA) metal-affinity chromatography (Qiagen, Hilden) under native conditions according to The QIAexpressionist handbook (Qiagen, Hilden).

(68) To enable binding of the 6His-tagged recombinant protein to the Ni-NTA resin, 1 ml of Ni-NTA Agarose was added to the cleared lysate and mixed by shaking an a rotary shaker (15 rpm, 4 C.) for 1 h. The mixture was centrifuged (800g, 2 min, 4 C.) and the supernatant was discarded. Three wash steps were carried out (800g, 2 min, 4 C.) using 10 ml of wash buffer each before the mixture was loaded into a column. Generally, wash buffer I was used for purification. Wash buffer II was employed for purification of proteins that have previously been solubilised using N-lauroylsarcosine sodium salt. The protein was eluted in 500 l aliquots using 5 ml of elution buffer. All fractions were stored at 4 C. for SDS-PAGE analysis.

(69) TABLE-US-00006 Wash buffer I Tris-HCl, pH 8.0 50 mM NaCl 300 mM Imidazole 20 mM Glycerol 10% (v/v) TRITON X-100 0.1% (v/v) Elution buffer Tris-HCl, pH 8-0 50 mM NaCl 300 mM Imidazole 200 mM Glycerol 10% (v/v) TRITON X-100 0.1% (v/v) Wash buffer II Tris-HCl, pH 8.0 50 mM NaCl 300 mM Imidazole 20 mM Glycerol 10% (v/v) TRITON X-100 2% (v/v)

(70) 2.4 Protein Dialysis and Concentration

(71) After purification, fractions of highest purity were pooled and dialysed in PBS. The protein solution was applied to a dialysis tube (pore size: 6-10 kDa, Roth, Karlsruhe) and dialysed overnight in 2 l PBS at 4 C. with gentle stirring. After dialysis, the protein was concentrated to a final volume of 2 ml using AMICON centrifugal filters (Millipore, Eschborn) at 500g and 4 C.

(72) 2.5 Protein Labelling with Fluorescent Dyes

(73) In order to detect the recombinant proteins used in this study by fluorescence microscopy and FACS analysis, the proteins were labelled using fluorescent dyes.

(74) Protein Labelling with ALEXA FLUOR 488

(75) For FACS analysis, the recombinant proteins YopM and SspHI were labelled with the green fluorescent dye ALEXA FLUOR 488 using the ALEXA FLUOR 488 Protein labelling kit (Invitrogen, Karlsruhe). Fluorescence of proteins labelled with ALEXA FLUOR 488 can be excited at 494 nm and emission is detected at 519 nm.

(76) Labelling of recombinant YopM and SspHI with ALEXA FLUOR 488 was performed according to the manufacturer's recommendations.

(77) Protein Labelling with Cy3

(78) For subsequent fluorescence microscopy, recombinant proteins were labelled with the orange fluorescent cyanine dye Cy3 using the Cy3 Ab labelling kit (Amersham Biosciences, Freiburg). Fluorescence of Cy3-labelled proteins can be excited at 550 nm and emission is detected at 570 nm.

(79) 3 Cell Fractionation of Eukaryotic Cells

(80) Cell fractionation of eukaryotic cells allows separation of soluble cytoplasmic proteins from insoluble membrane proteins (Behrens, 1938). In this study, cell fractionation was used to check for autointegration of the recombinant proteins into the cytoplasm (Kenny und Finlay, 1997; Gauthier et al., 2000).

(81) The cells were cultured to confluency in a 10 cm culture dish and subsequently incubated with recombinant protein (25 g/ml) in 10 ml culture medium for 1 h. Cells were washed with ice cold D-PBS (with Ca.sup.2+ and Mg.sup.2+) 3 for 15 min before the cells were quenched with acid buffer wash for 5 min. After an additional wash step with D-PBS (with Ca.sup.2+ and Mg.sup.2+), the cells were scraped from the surface and resuspended in 1 ml sonication buffer (supplemented with Complete EDTA-free Protease Inhibitor Cocktail (Roche Biochemicals, Mannheim) prior to use). The cells were permeabilised by sonication (ultrasound water bath, 41 s, level 4, 4 C.) followed by centrifugation (108,000g, 15 min, 4 C.). The supernatant containing the cytoplasmic proteins was collected and saved as cytoplasmic fraction (CF) until further usage. The insoluble pellet was washed once in 1 ml sonication buffer before it was resuspended in 1 ml TRITON buffer (supplemented with Complete EDTA-free Protease Inhibitor Cocktail (Roche Biochemicals, Mannheim) prior to use) and incubated on a rotary shaker (15 rpm, 4 C.) for 30 min. The cell lysate was centrifuged (108,000g, 15 min, 4 C.) again and the supernatant containing membrane proteins soluble in TRITON X-100 was collected as the membrane fraction (MF). Cytoplasmic and membrane fraction were precipitated using trichloroacetic acid and subsequently analysed by Western blotting.

(82) TABLE-US-00007 Sonication Buffer Tris-HCl 50 mM NaCl 150 mM EDTA 1 mM EGTA 1 mM Glycerol 30% (v/v) NaVO.sub.4 0.4 mM NaF 1 mM Acid Buffer Wash Glycine 0.2 g D-PBS Add to 100 ml pH 2.0 TRITON Buffer Sonication Buffer 1 l TRITON X-100 1% (v/v)

(83) 4 Nuclear Fractionation of Eukaryotic Cells

(84) Nuclear fractionation allows separation of cytoplasmic and nuclear protein fractions. In this study nuclear fractionation was performed to verify cell penetration by recombinant proteins and to check for a potential nuclear localisation of these recombinant proteins. Therefore, the NE-PER Nuclear and Cytoplasmic Extraction Kit (Thermo Scientific, Rockford, USA) was used which is based on stepwise cell lysis and centrifugal separation of cytoplasmic and nuclear protein fractions.

(85) Cells grown to confluency in a 10 cm culture dish were incubated with recombinant protein (25 g/ml) for 1 h. Following incubation, the cells were washed with D-PBS (without Ca.sup.2+ and Mg.sup.2+) and subsequently detached with trypsin and centrifuged at 500g for 5 min. The cells were washed with D-PBS, transferred to a microcentrifuge tube and centrifuged again (500g, 3 min). All buffers were included in the NE-PER Nuclear and Cytoplasmic Extraction Kit (Thermo Scientific, Rockford, USA) and used at the recommended volumes. Buffers CER I and NER were supplemented with Complete EDTA-free Protease Inhibitor Cocktail (Roche Biochemicals, Mannheim) prior to use.

(86) 5 Immunofluorescence Microscopy

(87) Recombinant proteins used for immunofluorescence analyses in this study were either GFP fusion proteins or labelled with the fluorescent dye Cy3.

(88) In order to detect proteins by immunofluorescence analysis, cells were cultured on cover slips in 24-well plates and subsequently incubated with recombinant protein (25 g/ml). Cells were washed with D-PBS (with Ca.sup.2+ and Mg.sup.2+) three times to remove non-integrated proteins. A different experimental setup included trypsinization after HeLa cells were incubated with the proteins. This procedure is supposed to be even more efficient in removing cell-surface bound proteins (Richard et al., 2003). After trypsinization, HeLa cells were seeded on cover slips in 24-well plates and incubated overnight to allow cell recovery. Afterwards cells were fixed using 500 l 4% PFA (w/v) per well and washed with D-PBS for 5 min. Subsequently, the cells were quenched with 0.2% glycine for 20 min and washed again. Cells were permeabilzed using 0.2% TRITON X-100 for 4 min followed by an additional wash step. DNA was selectively stained using Hoechst 33 258 (DAPI) diluted 1:1000 in D-PBS for 7.5 min followed by three wash steps with D-PBS for 5 min each. The preparations were embedded in Moviol/DABCO and analyzed by fluorescence microscopy using appropriate filters.

(89) TABLE-US-00008 0.2% TRITON X-100 TRITON X-100 200 l D-PBS Add to 100 ml 4% PFA Paraformaldehyde 4 g D-PBS Add to 100 ml 0.2% Glycine Glycine 0.2 g D-PBS Add to 100 ml

(90) 6 Fluorescence Activated Cell Sorting (FACS)

(91) Internalisation of YopM, SspHI and Tat-GFP-GSK into HeLa cells was monitored by fluorescence activated cell sorting (FACS). FACS is a specialised type of flow cytometry which allows separation of cells labelled with fluorescent markers and measurement of fluorescence intensity (Bonner et al., 1972).

(92) In this study, FACS analyses were performed to confirm and further investigate uptake of YopM and SspH1 in comparison to the Tat-GFP-GSK construct, a derivative of the well characterised CPP Tat. Uptake of the recombinant proteins was examined by determining the fluorescence intensity of HeLa cells. Therefore recombinant YopM and SspH1 were labelled with the fluorescent dye ALEXA FLUOR 488.

(93) HeLa cells were grown to confluency in 175 cm.sup.2 culture flasks and subsequently detached from the surface by adding trypsin. The cells were centrifuged and resuspended in D-PBS (without Ca.sup.2+ and Mg.sup.2+) and preheated to 37 C. prior to incubation with the recombinant proteins (20 g/ml). To assure that only viable cells are detected, the FACS cytometer was adjusted using control cells that were not incubated with protein. Dead cells and cellular debris can be excluded from acquisition by measuring forward and side scatter which display cell size and granularity, respectively. Dead cells have lower forward scatter and higher side scatter which allows distinction from living cells. First, the control cells were applied to the cytometer and viable cells were gated due to side and forward scatter and thus selected for acquisition. The cytometer (BD Biosciences, Heidelberg) was set to acquire 10,000 events from the gated cell population per run, every event being a single cell. In case of internalisation of the proteins tagged with the fluorescent markers GFP and ALEXA FLUOR 488, respectively, fluorescence of the cells can be measured. However, even cells without labelling show fluorescence, so-called autofluorescence which is caused by components of the cell like pyridinic (NADPH) and flavin coenzymes (Monici, 2005). To subtract the autofluorescence from the total fluorescence intensity, the fluorescence intensity of HeLa cells which were not incubated with the fluorescent labelled proteins was measured.

(94) 6.1 Quenched Time-Lapse Uptake and CPP-Induced Membranolysis Assay

(95) In this study, the uptake kinetics of the recombinant proteins YopM, SspHI and Tat-GFP-GSK were monitored in two parallel experiments that are based on an ongoing incubation and allow measurements from the same incubation at different time points (Florn et al., 2011).

(96) The first approach is based on addition of trypan blue (TB) to the samples prior to FACS analysis to measure the protein uptake rate. TB is a quencher that is not able to enter the cells and efficiently eliminates fluorescence (Sahlin et al., 1983, Hed et al., 1987). Thus extracellular fluorescence can be excluded from the measurement, ensuring that only intracellularly located proteins are detected which are inaccessible to TB.

(97) The second assay allows monitoring of potential lytic effects of the recombinant proteins on membrane integrity. Protein-induced membranolysis can be assessed by the addition of propidium iodide (PI). PI is a fluorescent DNA intercalating agent that is not able to cross membranes of intact cells. PI can only enter cells with disrupted membranes and hence accumulates in dead cells. Thus, protein-induced membrane lysis can be monitored by the detection of PI fluorescence (Radosevic et al., 1990).

(98) In order to measure the fluorescence intensity of the samples, the detectors were tuned to the appropriate wavelength of emission of the used fluorescent marker. As mentioned above, YopM and SspHI were labelled with ALEXA FLUOR 488 which can be excited at 494 nm and emits light at 519 nm. Fluorescence intensity of cells incubated with Tat-GFP-GSK depends on fluorescence of GFP which is excited at 488 nm and emission was detected at 510 nm. For monitoring membranolytic effects of the proteins, 1 g/ml PI was added to the medium. Fluorescence of PI is excited at 536 nm and can be detected at 617 nm. Samples for measuring intracellular accumulation of the proteins were diluted 1:1 with TB (0.4% (w/v)) prior to FACS analysis. The samples were applied to the cytometer and fluorescence emitted by the cells was measured. In addition, side and forward scatter were measured for each cell crossing the laser beam. The obtained data were analysed using CellQuest Pro (BD Biosciences, Heidelberg).

(99) 7 In Vitro Ubiquitination Assay

(100) In vitro ubiquitination experiments were performed in 40 l reaction buffer (50 mM Tris-HCl, pH7.4, 10 mM MgCl.sub.2, 0.1 mM DTT and 2 mM ATP) containing 0.5 g E1, 2 g UbcH5b (E2), 2 g HA-ubiquitin, 2 g SspH1/SspH1-Nter in the presence or absence of 0.4 g PKN1. Reactions were incubated at 37 C. for 1h and stopped by addition of SDS sample buffer, with or without 100 mM DTT. Samples were separated by SDS-PAGE and subjected to western blotting using anti-ubiquitin, anti-His and anti-PKN1 antibodies.

(101) 8 Immunoprecipitation (IP)

(102) HeLa cells were grown to 80% confluency in 10 cm dishes and subsequently transfected with pCMVEntry-PKN1-Myc. 24 h post transfection, cells were incubated with 25 g/ml FLAG-tagged SspH1 and SspH1-Nter, respectively for 3 h. Cells were washed with D-PBS (with Ca.sup.2+ and Mg.sup.2+) (35 min), before they were scraped from the surface and resuspended in 800 l IP lysis buffer (supplemented with Complete EDTA-free Protease Inhibitor Cocktail (Roche Biochemicals, Mannheim) prior to use). The cells were permeabilised by sonication (320 s, 4 C.) and subsequently incubated on a rotary shaker for 30 min at 4 C. Lysates were cleared by centrifugation (16,000g, 30 min, 4 C.) and a sample of the lysate was taken and stored at 20 C. until further usage. 30 l protein A/G agarose beads (Santa Cruz, sc-2003) were incubated with 5 g -PKN1 antibody (BD Biosciences) on ice for 5 min before the lysate was added to the mix and incubated on a rotary shaker overnight at 4 C. The beads were pelleted by centrifugation (1000g, 3 min, 4 C.) and a sample of the supernatant was collected and stored until further usage. The beads were washed with 600 l IP lysis buffer three times and centrifuged as mentioned above. The supernatant was discarded and 30 l 4SDS sample buffer were added to the beads, heated at 95 C. for 5 min and subsequently centrifuged at 16,000g for 5 min. The supernatant along with the samples of the lysate and the unbound protein were subjected to immuno blot analysis.

(103) TABLE-US-00009 IP lysis Buffer Tris-HCl pH 7.4 50 mM NaCl 150 mM EDTA 2 mM

(104) 9 Non-Radioactive Cytotoxicity Assay

(105) In order to analyse a potential cytotoxic effect of the recombinant proteins on the eukaryotic cells, the CYTOTOX 96 Non-Radioactive Cytotoxicity Assay (Promega) was used. HeLa cells were grown in 96 well plates and incubated with 25-50 g/ml SspH1 and SspH1-Nter, respectively for different time points (1, 6 & 24 h). Following incubation, the assay was performed according to the manufacturer's recommendations.

(106) 10 Analysis of Eukaryotic Gene Expression

(107) In order to analyse the effect of recombinant SspH1 on cytokine and chemokine expression, qRT-PCR was performed. Therefore, A459 cells were grown in 6 well plates to confluency and incubated with SspH1 for 3 h. The cells were subsequently stimulated with 20 ng/ml 11113, before RNA was isolated according to the manufacturer's recommendations using the RNeasy Mini Kit (Qiagen, Hilden).

(108) 10.1 cDNA Synthesis

(109) The RNA was then used for cDNA synthesis using the Transcriptor Reverse Transcriptase-Kit (Roche, Mannheim) according to the manufacturer's recommendations. First, primers were annealed to the RNA, before the cDNA synthesis was initiated by addition of the RT mix.

(110) TABLE-US-00010 Step Reaction mixture Concentration Program I. RNA 3-5 g 10 min, T7 Oligo (dT).sub.24 Primer 10 pmol 65 C. DNA/RNA free H.sub.2O ad 13 l >4 C. II. 5 x RT-Puffer 4 l 30 min, dNTPS 2 l 55 C. RNase Inhibitor (40 U/l) 0.5 l 5 min, Reverse Transcriptase (20 U/l) 0.5 l 85 C. >4 C.

(111) cDNA was synthesised in a PCR thermo cycler and stored at 20 C. until further usage.

(112) 10.2 Quantitative Real Time PCR

(113) The qRT-PCR enables quantification of PCR products by measuring the fluorescent intensities of a DNA-intercalating fluorescent dye. In this study, qRT-PCR was performed using the LightCycler1.5 (Roche, Mannheim). Data was analysed using the LIGHTCYCLER Data Analysis 5.3.2 software (Roche, Mannheim). Values for each sample were normalised for a low abundance Housekeeping gene (here HPRT: Hypoxanthin-Phosphoribosyl-Transferase I) (Vandesompele et al., 2002). qRT-PCR was performed using the LIGHTCYCLER Fast Start DNA Master.sup.PLUS SYBR Green I kit (Roche, Mannheim) which contains the reaction buffer, the dNTP mix, the SYBR Green I dye and MgCl.sub.2. The hot start Taq polymerase is added to the mix and heat-activated at 95 C. for 15 min before the PCR reaction. Different dilutions of the cDNA were prepared (1:10, 1:100, 1:1000) in sterile dH.sub.2O and used as a template for the qRT-PCR.

(114) TABLE-US-00011 2 l cDNA 2 l LightCycler Fast Start DNA Master.sup.PLUS SYBR Green I 2 l Primer Mix 4 l Sterile dH.sub.2O

(115) qRT-PCR was performed using the following cycling conditions.

(116) TABLE-US-00012 Reaction Step Temperature Time Cycles I. Denaturation and DNA 95 C. 15 min 1x Polymerase Activation II. 1. Denaturation 95 C. 12 s 2. Annealing 60 C. 10 s 35-55x 3. Elongation 72 C. 12 s III. Melting 60-95 C. stepwise 1x

(117) Statistical analysis of the obtained data was performed using Prism 4 (GraphPad Software). The statistical significance of differences in gene expression was analysed using the unpaired student t-test and values p<0.05 were regarded as significant.

(118) 3 Material

(119) 3.1 Bacterial Strains

(120) Bacterial strains used in this study are listed in Table 3.1.

(121) TABLE-US-00013 TABLE 3.1 Bacterial strains Strain Relevant Characteristics Reference E. coli DH5 F.sup., endA1, recA1, hsdR17(r.sub.K.sup. M.sub.K.sup.+) deoR, thi-1, supE44, Hanahan et al., gyrA96, (lacZYA-argF) U169 (80dlacZM15) 1991 E. coli BL21 (DE3) F.sup., hsdS.sub.B (r.sub.B.sup.m.sub.B.sup.), dcm, gal, ompT, (DE3) Studier & Moffatt, 1986

(122) 3.4 Plasmids and Oligonucleotides

(123) Plasmids used in this study are listed in Table 3.3.

(124) TABLE-US-00014 TABLE 3.2 Plasmids Plasmid Relevant Characteristics Reference pET24b(+) Expression vector, Kan.sup.R Novagen pET-YopM Nucleotides 1-1101 of yopM from Y. enterocolitica O:8 Heusipp et al., JB580v (Nhel/Xhol) in pET24b(+) 2006 pET-SspHI Nucleotides 1-2103 of sspHI of S. enterica subspec. Lubos, M.-L. enterica serovar Typhimurium 14928S (Nhel/Xhol) in pET24b(+) pET-SspHI-Nter Nucleotides 1-1161 of sspHI of S. enterica subspec. Lubos, M.-L. enterica serovar Typhimurium 14928S (Nhel/Xhol) in pET24b(+) pET:Tat-GFP-GSK Coding sequence of the Tat CPP with 3 GFP coding Lubos, M.-L. sequence and GSK-3 tag coding sequence in pET24b(+) pET:IpaH1.4 ipaH1.4 of S. flexneri in pET24b+ S. Norkowski pET:IpaH2.5 ipaH2.5 of S. flexneri in pET24b+ S. Norkowski pET:IpaH3 ipaH3 of S. flexneri in pET24b+ S. Norkowski pET:IpaH4.5 ipaH4.5 of S. flexneri in pET24b+ S. Norkowski pET:IpaH7.8 ipaH7.8 of S. flexneri in pET24b+ S. Norkowski pET:IpaH9.8 ipaH9.8 of S. flexneri in pET24b+ S. Norkowski pET:SspH2 SspH2 of S. typhimurium in pET24b+ S. Norkowski pET:SlrP slrP of S. typhimurium in pET24b+ S. Norkowski pCMVEntry- Myc-DDK-tagged Human pkn1 transcript variant 1 in Origene PKN1 pCMV6-Entry, RC215735

(125) Synthetic oligonucleotides used for DNA amplification are listed in Table 3.4. All primers were purchased from MWG Biotech AG (Ebersberg).

(126) TABLE-US-00015 TABLE3.3 OligonucleotidesequencesforDNAamplificationbyPCR(restrictionsitesare underlined) Oligonucleotide Sequence(5-3) F-SspHI(NheI) CTAGCTAGCGTTACCGATAAATAATAACTT SEQIDNO:530 R-SspHI(XhoI) CGCCTCGAGTGAATGGTGCAGTTGTGAGCC SEQIDNO:531 R-SspHI-Nter(XhoI) CCGCTCGAGCCGTGGGCCGTGGTAGTCCGG SEQIDNO:532 F-Tat(NdeI) TATGATGTGCGGCCGTAAGAAACGTCGCCAGCG SEQIDNO:533 TCGCCGTCCGCCGCAATGCG R-Tat(NheI) CTAGCGCATTGCGGCGGACGGCGACGCTGG SEQIDNO:534 CGACGTTTCTTACGGCCGCACAGCA F-IpaH1.4 GTTTAACTTTAAGAAGGAGATATACATATGATT SEQIDNO:535 AAATCAACCAATATACAG R-IpaH1.4 CTTATCGTCGTCATCCTTGTAATCGCTAGCTGC SEQIDNO:536 GATATGATTTGAGCCGTTTTCAGACAA F-IpaH2.5/IpaH4.5 GTTTAACTTTAAGAAGGAGATATACATATGATT SEQIDNO:537 AAATCAACAAATATACAGGTAATCGGT R-IpaH2.5 CTTATCGTCGTCATCCTTGTAATCGCTAGCGGC SEQIDNO:538 CAGTACCTCGTCAGTCAACTGACGGTA F-IpaH3 GTTTAACTTTAAGAAGGAGATATACATATGTTA SEQIDNO:539 CCGATAAATAATAACTTTTCATTGTCC R-IpaH3 CTTATCGTCGTCATCCTTGTAATCGCTAGCGTC SEQIDNO:540 AGCTGACGGTAAATCTGCTGTTACAGT F-IpaH4.5 GTTTAACTTTAAGAAGGAGATATACATATGAAA SEQIDNO:541 CCGATCAACAATCATTCTTTTTTTCGT F-IpaH7.8 GTTTAACTTTAAGAAGGAGATATACATATGTTC SEQIDNO:542 TCTGTAAATAATACACACTCATCAGTT R-IpaH7.8 CTTATCGTCGTCATCCTTGTAATCGCTAGCTGA SEQIDNO:543 ATGGTGCAGTCGTGAGCCGTTTTCAGA F-IpaH9.8 GTTTAACTTTAAGAAGGAGATATACATATGTTA SEQIDNO:544 CCGATAAATAATAACTTTTCATTGCCC R-IpaH9.8 CTTATCGTCGTCATCCTTGTAATCGCTAGCTGA SEQIDNO:545 ATGGTGCAGTTGTGAGCCGTTTTCAAA F-SspH2 GTTTAACTTTAAGAAGGAGATATACATATGCCC SEQIDNO:546 TTTCATATTGGAAGCGGATGTCTTCCC R-SspH2 CTTATCGTCGTCATCCTTGTAATCGCTAGCGTT SEQIDNO:547 ACGACGCCACTGAACGTTCAGATAGCT F-SlrP GTTTAACTTTAAGAAGGAGATATACATATGTTT SEQIDNO:548 AATATTACTAATATACAATCTACGGCA R-SlrP CTTATCGTCGTCATCCTTGTAATCGCTAGCTCG SEQIDNO:549 CCAGTAGGCGCTCATGAGCGAGCTCAC

(127) 3.6 Antibodies

(128) Primary and secondary antibodies used for Western blot analysis and fluorescent dyes for immunofluorescence studies are listed in Tables 3.6 and 3.7.

(129) TABLE-US-00016 TABLE 3.4 Primary antibodies used for Western blot analyses Antibody Dilution Characteristics Reference -Penta-His 1:1000 Mouse monoclonal antibody Quiagen against His.sub.6-epitope (Hilden) (SEQ ID NO: 555), isotype IgG Anti--Tubulin 1:5000 Mouse monoclonal antiserum Sigma-Aldrich against human -tubulin (Mnchen) -LSD1 1:1000 Rabbit monoclonal antibody Cell Signaling (C69G12) against human lysine-specific (Danvers, USA) demethylase 1 (LSD1) -GAPDH 1:200 Rabbit polyclonal antibody Santa Cruz against glyceraldehyde Biotechnology 3-phosphate dehydrogenase (Heidelberg) (GAPDH) -Transferrin 1:500 Mouse monoclonal antibody Invitrogen receptor against human transferrin (Karlsruhe) receptor -FLAG 1:1000 Mouse monoclonal antibody Sigma-Aldrich against DYKDDDDK-tag (Mnchen) epitope (SEQ ID NO: 556), Clone M2 -PKN1 1:1000 Monoclonal mouse antibody BD Bioscience against human protein (Heidelberg) kinase N1

(130) TABLE-US-00017 TABLE 3.5 Secondary antibodies used for Western blot (WB) analyses and fluorescent dyes for immunofluorescence (IF) analyses Antibody Dilution Characteristics Reference GAM-PO (WB) 1:10000 Peroxidase (PO) conjugated goat Dianova monoclonal antibody against mouse-IgG (Hamburg) GAR-PO (WB) 1:10000 Peroxidase (PO) conjugated goat Dianova monoclonal antibody against rabbit-IgG (Hamburg) Hoechst (IF) 1:1000 Selective DNA dye Sigma Aldrich 33258 (DAPI) (Taufkirchen)

(131) 3.7 Kits

(132) Kits used in this study are listed in Table 3.6.

(133) TABLE-US-00018 TABLE 3.6 Kits Kit Supplier ZYPPY Plasmid Miniprep Kit Zymo Research (Irvine, USA) ZYMOCLEAN Gel DNA Recovery Kit Zymo Research (Irvine, USA) Cy3 Ab Labelling Kit PA 33000 GE Healthcare (Braunschweig) ALEXA FLUOR 488 Protein Labelling Kit Invitrogen (Karlsruhe) NE-PER Nuclear and Cytoplasmic Extraction Thermo Scientific Reagents (Rockford, USA) CYTOTOX 96 Non-Radioactive Promega Cytotoxicity Assay

References (Materials and Methods of Examples 1 to 5)

(134) Behrens, M. (1938), Hoppe-Seylers Z, 253, Pflgers Archiv-European Journal of Physiology, 185. Bonner, W. A., Hulett, H. R., Sweet, R. G. and Herzenberg, L. A. (1972), Fluorescence activated cell sorting. Rev. Sci. Instrum., 43(3), 404-409. Chen, G. J., Qiu, N., Karrer, C., Caspers, P. and Page, M. G. (2000), Restriction site-free insertion of PCR products directionally into vectors. BioTechniques, 28(3), 498-500, 504-5. Gauthier, A., de Grado, M. and Finlay, B. B. (2000), Mechanical fractionation reveals structural requirements for enteropathogenic Escherichia coli Tir insertion into host membranes. Infect. mmun., 68(7), 4344-4348. Hed, J., Hallden, G., Johansson, S. G. and Larsson, P. (1987), The use of fluorescence quenching in flow cytofluorometry to measure the attachment and ingestion phases in phagocytosis in peripheral blood without prior cell separation. J. Immunol. Methods, 101(1), 119-125. Kenny, B. and Finlay, B. B. (1997), Intimin-dependent binding of enteropathogenic Escherichia coli to host cells triggers novel signaling events, including tyrosine phosphorylation of phospholipase C-gamma1. Infect. Immun., 65(7), 2528-2536. Radosevic, K., Garritsen, H. S., Van Graft, M., De Grooth, B. G. and Greve, J. (1990), A simple and sensitive flow cytometric assay for the determination of the cytotoxic activity of human natural killer cells. J. Immunol. Methods, 135(1-2), 81-89. Sahlin, S., Hed, J. and Rundquist, I. (1983), Differentiation between attached and ingested immune complexes by a fluorescence quenching cytofluorometric assay. J. Immunol. Methods, 60(1-2), 115-124. Unger, T., Jacobovitch, Y., Dantes, A., Bernheim, R. and Peleg, Y. (2010), Applications of the Restriction Free (RF) cloning procedure for molecular manipulations and protein expression. J. Struct. Biol., 172(1), 34-44. van den Ent, F. and Lowe, J. (2006), RF cloning: a restriction-free method for inserting target genes into plasmids. J. Biochem. Biophys. Methods, 67(1), 67-74.

Methods of Examples 7 to 20

(135) The experiments described in Examples 7 to 20 and shown in FIGS. 23 to 28 may be carried out in various ways well-known to one skilled in the art. For example, according to the methods indicated above or described in Examples 7 to 20.

(136) Selected methods are described below in more detail. Methods well-known to one skilled in the art have not been described in detail in order to not unnecessarily obscure the present invention.

(137) Cell Fractionation of Eukaryotic Cells

(138) Cellular uptake of proteins was assessed by cell fractionation of eukaryotic cells. This method allows separation of soluble cytoplasmic proteins from insoluble membrane proteins (Behrens, 1938).

(139) Therefore, HeLa cells were seeded in 10 cm cell-culture dishes and grown to 80% confluence. Upon protein treatment, the dishes were transferred to ice and the cells were washed twice with ice-cold D-PBS (with Ca2+/Mg2+). An additional wash step with acid buffer for 5 min was used to remove any residual surface-bound proteins (Langel, . (ed) (2005). Handbook of cell-penetrating peptides. CRC Press, Taylor and Francis Group). After washing the cells again with D-PBS (with Ca2+/Mg2+), cells were collected using a cell scraper, transferred to a reaction tube on ice and resuspended in 1 ml sonication buffer (supplemented with Complete EDTA-free Protease Inhibitor Cocktail). The suspension was applied to sonication (ultrasound water bath, 41 sec, level 4, 4 C.) in order to permeabilize the cells. Subsequently, the suspension was centrifuged (108,000g, 15 min, 4 C.) and the supernatant was taken as the cytoplasmic fraction (CF). After the insoluble pellet was washed once with 1 ml sonication buffer (108,000g, 15 min, 4 C.), it was resuspended in 1 ml TRITON buffer (supplemented with Complete EDTA-free Protease Inhibitor Cocktail) and incubated on a rotary shaker at 15 rpm and 4 C. for 1 h or overnight. Subsequently, the cell lysate was centrifuged (108,000g, 30 min, 4 C.) and the supernatant was taken as the membrane fraction (MF).

(140) Both the cytoplasmic and the membrane fraction were precipitated using trichloracetic acid. Subsequently, the samples were subjected to SDS-PAGE and analyzed by Western blotting.

(141) TABLE-US-00019 Acid Buffer Glycine 62.5 mM in PBS, pH 2.0 TRITON buffer TRITON X-100 1% (v/v) in Sonication buffer Sonication buffer 1 mM Tris-HCl, pH 7.8 50 mM NaCl 150 mM EDTA 1 mM EGTA 1 mM Glycerol 30% (v/v) NaVO4 0.4 mM NaF 1 mM

(142) Membranolysis Assay

(143) For analysis of effects on membrane integrity induced by LPX effector proteins, a FACS-based membranolysis assay following the CPP-induced Membranolysis Assay (Florn et al., 2011) was performed.

(144) HeLa cells were cultured and prepared as described above. For monitoring membranolytic effects of the proteins, HeLa cells were incubated with the respective protein and co-incubated with 1 g/ml PI. After defined time points samples were taken and applied to the FACS analysis. Each sample was measured in duplicates. Fluorescence of PI is excited at 536 nm and can be detected at 617 nm. The obtained data were analyzed using the CELLQUEST Pro software.

(145) Lactate Dehydrogenase Assay

(146) In order to assess cytotoxicity and potential lytic effects of recombinant proteins, the release of lactate dehydrogenase (LDH) can be measured and used as a parameter for membrane integrity.

(147) Cytotoxicity and potential lytic effects of recombinant proteins were measured using CYTOTOX 96 Non-Radioactive Cytotoxicity Assay according to the manufacturer's instructions.

(148) HeLa cells were seeded in 96-well plates and grown to 80% confluence. Upon incubation with recombinant proteins for 24, 6, and 1 h in 100 l culture medium, the plate was centrifuged (400g, 4 min, RT) and 50 l of the supernatant from each well of the assay plate were transferred to the corresponding well of a new 96-well plate. In addition, 50 l of a LDH positive control were added to separate wells in order to verify that the assay is functioning properly. HeLa cells contained in the remaining 50 l were lysed by adding 5.5 l Lysis Solutions (10) for 30 min. Afterwards, 50 l of the reconstituted Substrate Mix were added to each well of the two plates and both plates were incubated for 30 min at RT, protected from light. Finally, 50 l of the Stop Solution were added to each well of the plates and the absorbance at 490 nm was recorded. All buffers used for this procedure were provided by the CYTOTOX 96 Non-Radioactive Cytotoxicity Assay-Kit.

(149) In Vitro Ubiquitination Assay

(150) In order to verify the functionality of recombinant LPX effectors as proposed E3 ubiquitin ligases, in vitro ubiquitination assays were performed. Upon incubation with ubiquitin-activating enzymes E1, ubiquitin-conjugating enzymes E2, and ubiquitin, LPX effector proteins were tested whether they were able to remove ubiquitin from the E2 enzyme and catalyze the formation of poly-ubiquitin chains.

(151) In vitro ubiquitination assays were performed in a volume of 40 l at 37 C. for 1 h. The reaction mixture was composed as shown below. The reaction was stopped by adding of 10 l of 4SDS sample buffer without dithiothreitol (DTT). The samples were prepared for subsequent SDS-PAGE analysis by incubation at 95 C. for 10 min Reaction mixture of in vitro ubiquitination assay:

(152) TABLE-US-00020 Component Amount E1 0.5 g E2 (UbcH5b) 2 g Ubiquitin-HA 2 g Putative E3 4 g Ubiquitin reaction buffer ad to 40 l

(153) TABLE-US-00021 Ubiquitination reaction buffer 4 x SDS sample buffer without DTT Tris-HCl, pH 7.5 25 mM Tris-HCl, pH 6.8 30 mM NaCl 50 mM Glycerol 10% (v/v) ATP 5 mM SDS 1.5% (v/v) MgCl2 10 mM Bromophenol blue Spatula tip DTT 0.1 mM

Example 7

Functional Domains of LPX Family Members

(154) Functional domains of SlrP, SspH1, SspH2, IpaH1.4, IpaH2.5, IpaH3, IpaH4.5, IpaH7.8 and IpaH9.8 are shown in FIGS. 14 to 22.

(155) Sequence segment comprising leucine-rich repeats (called LRR stretch) are marked in light grey and are non-edged. E3 Ubiquitin ligase domains are marked in dark grey and are edged. Sequence sections within which the protein transduction domains (PTDs) are predicted are bold and underlined. Marked are the maximal sequence sections within the PTD is predicted. PTDs can correspond to the marked sequence sections or to c-terminally and/or N-terminally truncated fragments of the marked sequence sections.

Example 8

Corresponding DNA Sequences of the Proteins of the Invention Analyzed in the Examples Above

(156) SlrP

(157) SEQ ID NO: 10

(158) >gb|CP001363.11:867285-869582 Salmonella enterica subsp. enterica serovar Typhimurium str. 14028S, complete genome

(159) SspH1

(160) SEQ ID NO: 11

(161) >gb|CP001363.11:1332051-1334153 Salmonella enterica subsp. enterica serovar Typhimurium str. 14028S

(162) SspH2

(163) SEQ ID NO: 12

(164) >gb|CP001363.1|:2392438-2394804 Salmonella enterica subsp. enterica serovar Typhimurium str. 14028S, complete genome

(165) IpaH1.4

(166) SEQ ID NO: 13

(167) >gi|12329037:206811-208538 Shigella flexneri virulence plasmid pWR100: from 1 to 213494

(168) IpaH2.5

(169) SEQ ID NO: 14

(170) >gi|12329037:43257-44948 Shigella flexneri virulence plasmid pWR100: from 1 to 213494

(171) IpaH3

(172) SEQ ID NO: 15

(173) >ENA|EID62303|EID62303.1 Shigella flexneri 5a str. M90T invasion plasmid antigen: Location: 1 . . . 1752

(174) IpaH4.5

(175) SEQ ID NO: 16

(176) >gi|12329037:66187-67911 Shigella flexneri virulence plasmid pWR100: from 1 to 213494

(177) IpaH7.8

(178) SEQ ID NO: 17

(179) >gi|12329037:64062-65759 Shigella flexneri virulence plasmid pWR100: from 1 to 213494

(180) IpaH9.8

(181) SEQ ID NO: 18

(182) >gi|12329037:174343-175980 Shigella flexneri virulence plasmid pWR100: from 1 to 213494

Example 9

Protein Transduction Domains Determined in Example 5

(183) IpaH1.4

(184) SEQ ID NO: 4

(185) >tr|Q9AJU5|Q9AJU5_SHIFL Putative uncharacterized protein ipaH1.4 OSShigella flexneri GN=ipaH1.4 PE=4 SV=1

(186) Predicted PTD Sequences:

(187) TABLE-US-00022 SEQIDNO:361 519SQRVADRLKA SEQIDNO:362 520QRVADRLKAS SEQIDNO:363 521RVADRLKASG SEQIDNO:364 520QRVADRLKASG SEQIDNO:365 521RVADRLKASGL SEQIDNO:366 520QRVADRLKASGL SEQIDNO:367 371RVALTWNNLRKTL SEQIDNO:368 371RVALTWNNLRKTLL SEQIDNO:369 370DRVALTWNNLRKTLL SEQIDNO:370 371RVALTWNNLRKTLLV

(188) IpaH2.5

(189) SEQ ID NO: 5

(190) >gi|12329051|emb|CAC05782.1| IpaH2.5, member of the IpaH family, probably secreted by the Mxi-Spa machinery, function unknown [Shigella flexneri 5a str. M90T]

(191) Predicted PTD Sequences:

(192) TABLE-US-00023 SEQIDNO:371 519SQRVADRLKA SEQIDNO:372 520QRVADRLKAS SEQIDNO:373 521RVADRLKASG SEQIDNO:374 520QRVADRLKASG SEQIDNO:375 521RVADRLKASGL SEQIDNO:376 520QRVADRLKASGL SEQIDNO:377 371RVALTWNNLRKTL SEQIDNO:378 371RVALTWNNLRKTLL SEQIDNO:379 370DRVALTWNNLRKTLL SEQIDNO:380 371RVALTWNNLRKTLLV

(193) IpaH3

(194) SEQ ID NO: 6

(195) >tr|I0VDT7|I0VDT7_SHIFL Invasion plasmid antigen OS=Shigella flexneri 5a str. M90T GN=ipaH_3 PE=4 SV=1

(196) Predicted PTD Sequences:

(197) TABLE-US-00024 SEQIDNO:381 511PQRVADRLKA SEQIDNO:382 512QRVADRLKAS SEQIDNO:383 513RVADRLKASG SEQIDNO:384 511PQRVADRLKAS SEQIDNO:385 512QRVADRLKASG SEQIDNO:386 513RVADRLKASGL SEQIDNO:387 363RVALIWNNLRKTL SEQIDNO:388 511PQRVADRLKASGL SEQIDNO:389 363RVALIWNNLRKTLL SEQIDNO:390 362DRVALIWNNLRKTLL SEQIDNO:391 363RVALIWNNLRKTLLV

(198) IpaH4.5

(199) SEQ ID NO: 7

(200) >gi|12329057|emb|CAC05788.1| IpaH4.5, member of the IpaH family, probably secreted by the Mxi-Spa secretion machinery, function unknown [Shigella flexneri 5a str. M90T]

(201) Predicted PTD Sequences:

(202) TABLE-US-00025 SEQIDNO:392 48NRIQAVRLLK SEQIDNO:393 49RIQAVRLLKI SEQIDNO:394 50IQAVRLLKIC SEQIDNO:395 51QAVRLLKICL SEQIDNO:396 54RLLKICLDTR SEQIDNO:397 208LKVGENQLRR SEQIDNO:398 209KVGENQLRRL SEQIDNO:399 530SQRVADRLKA SEQIDNO:400 531QRVADRLKAS SEQIDNO:401 532RVADRLKASG SEQIDNO:402 47ENRIQAVRLLK SEQIDNO:403 48NRIQAVRLLKI SEQIDNO:404 49RIQAVRLLKIC SEQIDNO:405 208LKVGENQLRRL SEQIDNO:406 531QRVADRLKASG SEQIDNO:407 532RVADRLKASGL SEQIDNO:408 46TENRIQAVRLLK SEQIDNO:409 47ENRIQAVRLLKI SEQIDNO:410 48NRIQAVRLLKIC SEQIDNO:411 49RIQAVRLLKICL SEQIDNO:412 52AVRLLKICLDTR SEQIDNO:413 216RRLSRLPQELLA SEQIDNO:414 531QRVADRLKASGL SEQIDNO:415 46TENRIQAVRLLKI SEQIDNO:416 47ENRIQAVRLLKIC SEQIDNO:417 48NRIQAVRLLKICL SEQIDNO:418 49RIQAVRLLKICLD SEQIDNO:419 51QAVRLLKICLDTR SEQIDNO:420 208LKVGENQLRRLSR SEQIDNO:421 209KVGENQLRRLSRL SEQIDNO:422 215LRRLSRLPQELLA SEQIDNO:423 216RRLSRLPQELLAL SEQIDNO:424 382RVALIWNNLRKTL SEQIDNO:425 47ENRIQAVRLLKICL SEQIDNO:426 48NRIQAVRLLKICLD SEQIDNO:427 49RIQAVRLLKICLDT SEQIDNO:428 50IQAVRLLKICLDTR SEQIDNO:429 205LKYLKVGENQLRRL SEQIDNO:430 208LKVGENQLRRLSRL SEQIDNO:431 214QLRRLSRLPQELLA SEQIDNO:432 215LRRLSRLPQELLAL SEQIDNO:433 382RVALTWNNLRKTLL SEQIDNO:434 46TENRIQAVRLLKICL SEQIDNO:435 48NRIQAVRLLKICLDT SEQIDNO:436 49RIQAVRLLKICLDTR SEQIDNO:437 204SLKYLKVGENQLRRL SEQIDNO:438 205LKYLKVGENQLRRLS SEQIDNO:439 208LKVGENQLRRLSRLP SEQIDNO:440 204SLKYLKVGENQLRRL SEQIDNO:441 205LKYLKVGENQLRRLS SEQIDNO:442 208LKVGENQLRRLSRLP SEQIDNO:443 214QLRRLSRLPQELLAL SEQIDNO:444 381DRVALIWNNLRKTLL SEQIDNO:445 382RVALTWNNLRKTLLV SEQIDNO:446 48NRIQAVRLLKICLDTR SEQIDNO:447 49RIQAVRLLKICLDTRE SEQIDNO:448 203QSLKYLKVGENQLRRL SEQIDNO:449 208LKVGENQLRRLSRLPQ SEQIDNO:450 213NQLRRLSRLPQELLAL SEQIDNO:451 47ENRIQAVRLLKICLDTR SEQIDNO:452 48NRIQAVRLLKICLDTRE SEQIDNO:453 205LKYLKVGENQLRRLSRL SEQIDNO:454 204SLKYLKVGENQLRRLSRL SEQIDNO:455 39WAREGTTTENRIQAVRLLK SEQIDNO:456 203QSLKYLKVGENQLRRLSRL SEQIDNO:457 208LKVGENQLRRLSRLPQELL SEQIDNO:458 209KVGENQLRRLSRLPQELLA SEQIDNO:459 39WAREGTTTENRIQAVRLLKI SEQIDNO:460 208LKVGENQLRRLSRLPQELLA SEQIDNO:461 209KVGENQLRRLSRLPQELLAL SEQIDNO:462 205LKYLKVGENQLRRLSRLPQEL SEQIDNO:463 206KYLKVGENQLRRLSRLPQELL SEQIDNO:464 208LKVGENQLRRLSRLPQELLAL SEQIDNO:465 54RLLKICLDTREPVLNLSLLKLR SEQIDNO:466 205LKYLKVGENQLRRLSRLPQELL SEQIDNO:467 206KYLKVGENQLRRLSRLPQELLA SEQIDNO:468 207YLKVGENQLRRLSRLPQELLAL SEQIDNO:469 205LKYLKVGENQLRRLSRLPQELLA SEQIDNO:470 206KYLKVGENQLRRLSRLPQELLAL SEQIDNO:471 204SLKYLKVGENQLRRLSRLPQELLA SEQIDNO:472 205LKYLKVGENQLRRLSRLPQELLAL SEQIDNO:473 206KYLKVGENQLRRLSRLPQELLALD SEQIDNO:474 51QAVRLLKICLDTREPVLNLSLLKLR SEQIDNO:475 203QSLKYLKVGENQLRRLSRLPQELLA SEQIDNO:476 204SLKYLKVGENQLRRLSRLPQELLAL SEQIDNO:477 205LKYLKVGENQLRRLSRLPQELLALD SEQIDNO:478 49RIQAVRLLKICLDTREPVLNLSLLKL SEQIDNO:479 50IQAVRLLKICLDTREPVLNLSLLKLR SEQIDNO:480 203QSLKYLKVGENQLRRLSRLPQELLAL SEQIDNO:481 49RIQAVRLLKICLDTREPVLNLSLLKLR SEQIDNO:482 48NRIQAVRLLKICLDTREPVLNLSLLKLR SEQIDNO:483 49RIQAVRLLKICLDTREPVLNLSLLKLRS SEQIDNO:484 47ENRIQAVRLLKICLDTREPVLNLSLLKLR SEQIDNO:485 49RIQAVRLLKICLDTREPVLNLSLLKLRSL SEQIDNO:486 48NRIQAVRLLKICLDTREPVLNLSLLKLRSL

(203) IpaH7.8

(204) SEQ ID NO: 8

(205) >gi|12329056|emb|CAC05787.1| IpaH7.8, member of the IpaH family, secreted by the Mxi-Spa secretion machinery, function unknown [Shigella flexneri 5a str. M90T]

(206) Predicted PTD Sequences:

(207) TABLE-US-00026 SEQIDNO:487 238TRVLQSLQRL SEQIDNO:488 239RVLQSLQRLT SEQIDNO:489 508SQRVADRLKA SEQIDNO:490 509QRVADRLKAS SEQIDNO:491 510RVADRLKASG SEQIDNO:492 509QRVADRLKASG SEQIDNO:493 510RVADRLKASGL SEQIDNO:494 509QRVADRLKASGL SEQIDNO:495 360RVALTWNNLRKTL SEQIDNO:496 360RVALTWNNLRKTLL SEQIDNO:497 359DRVALTWNNLRKTLL SEQIDNO:498 360RVALTWNNLRKTLLV

(208) IpaH9.8

(209) SEQ ID NO: 9

(210) >gi|12329122|emb|CAC05853.1| IpaH9.8, secreted by the Mxi-Spa secretion machinery, function unknown [Shigella flexneri 5a str. M90T]

(211) Predicted PTD Sequences:

(212) TABLE-US-00027 SEQIDNO:499 155LPQALKNLRA SEQIDNO:500 157QALKNLRATR SEQIDNO:501 158ALKNLRATRN SEQIDNO:502 488PQRVADRLKA SEQIDNO:503 489QRVADRLKAS SEQIDNO:504 490RVADRLKASG SEQIDNO:505 156PQALKNLRATR SEQIDNO:506 488PQRVADRLKAS SEQIDNO:507 489QRVADRLKASG SEQIDNO:508 490RVADRLKASGL SEQIDNO:509 155LPQALKNLRATR SEQIDNO:510 489QRVADRLKASGL SEQIDNO:511 151SLPSLPQALKNLRATR SEQIDNO:512 152LPSLPQALKNLRATRN SEQIDNO:513 154SLPQALKNLRATRNFL SEQIDNO:514 155LPQALKNLRATRNFLT SEQIDNO:515 154SLPQALKNLRATR SEQIDNO:516 155LPQALKNLRATRN SEQIDNO:517 158ALKNLRATRNFLT SEQIDNO:518 340RVALTWNNLRKTL SEQIDNO:519 488PQRVADRLKASGL SEQIDNO:520 153PSLPQALKNLRATR SEQIDNO:521 154SLPQALKNLRATRN SEQIDNO:522 157QALKNLRATRNFLT SEQIDNO:523 340RVALTWNNLRKTLL SEQIDNO:524 152LPSLPQALKNLRATR SEQIDNO:525 153PSLPQALKNLRATRN SEQIDNO:526 155LPQALKNLRATRNFL SEQIDNO:527 156PQALKNLRATRNFLT SEQIDNO:528 339DRVALTWNNLRKTLL SEQIDNO:529 340RVALTWNNLRKTLLV

(213) SspH1

(214) SEQ ID NO: 2

(215) >gi|267993082|gb|ACY87967.1| SspH1 [Salmonella enterica subsp. enterica serovar Typhimurium str. 14028S]

(216) Predicted PTD Sequences:

(217) TABLE-US-00028 SEQIDNO:152 372LSVRTLQALR SEQIDNO:153 70ARLKALTFPA SEQIDNO:154 626RFNALREKQI SEQIDNO:155 693LTARWRLN SEQIDNO:156 319LQKLWAYNNRL SEQIDNO:157 476ALRAKTFAMAT

(218) SspH2

(219) SEQ ID NO: 3

(220) >gi|267994325|gb|ACY89210.1| leucine-rich repeat-containing protein [Salmonella enterica subsp. enterica serovar Typhimurium str. 14028S]

(221) Predicted PTD Sequences:

(222) TABLE-US-00029 SEQIDNO:158 185SRGRAAVVQK SEQIDNO:159 186RGRAAVVQKM SEQIDNO:160 187GRAAVVQKMR SEQIDNO:161 188RAAVVQKMRA SEQIDNO:162 187GRAAVVQKMRA SEQIDNO:163 188RAAVVQKMRAC SEQIDNO:164 620FRLGKLEQIAR SEQIDNO:165 624KLEQIAREKVR SEQIDNO:166 627QIAREKVRTLA SEQIDNO:167 628IAREKVRTLAL SEQIDNO:168 629AREKVRTLALV SEQIDNO:169 185SRGRAAVVQKMR SEQIDNO:170 186RGRAAVVQKMRA SEQIDNO:171 188RAAVVQKMRACL SEQIDNO:172 177RRAAPAEESRGRA SEQIDNO:173 185SRGRAAVVQKMRA SEQIDNO:174 186RGRAAVVQKMRAC SEQIDNO:175 188RAAVVQKMRACLN SEQIDNO:176 620FRLGKLEQIAREK SEQIDNO:177 621RLGKLEQIAREKV SEQIDNO:178 622LGKLEQIAREKVR SEQIDNO:179 627QIAREKVRTLALV SEQIDNO:180 645WLAYQNKLKKSLG SEQIDNO:181 646LAYQNKLKKSLGL SEQIDNO:182 705RVLERKAPERVNAL SEQIDNO:183 706VLERKAPERVNALR SEQIDNO:184 710KAPERVNALREKQI SEQIDNO:185 751RTIGARAMESAKKT SEQIDNO:186 616GREMFRLGKLEQIAR SEQIDNO:187 619MFRLGKLEQIAREKV SEQIDNO:188 620FRLGKLEQIAREKVR SEQIDNO:189 751RTIGARAMESAKKTF SEQIDNO:190 621RLGKLEQIAREKVRT SEQIDNO:191 644VWLAYQNKLKKSLGL SEQIDNO:192 645WLAYQNKLKKSLGLT SEQIDNO:193 706VLERKAPERVNALREK SEQIDNO:194 751RTIGARAMESAKKTFL SEQIDNO:195 176WRRAAPAEESRGRAAVV SEQIDNO:196 177RRAAPAEESRGRAAVVQ SEQIDNO:197 178RAAPAEESRGRAAVVQK SEQIDNO:198 706VLERKAPERVNALREKQ SEQIDNO:199 176WRRAAPAEESRGRAAVVQ SEQIDNO:200 177RRAAPAEESRGRAAVVQK SEQIDNO:201 615TGREMFRLGKLEQIAREK SEQIDNO:202 619MFRLGKLEQIAREKVRTL SEQIDNO:203 620FRLGKLEQIAREKVRTLA SEQIDNO:204 621RLGKLEQIAREKVRTLAL SEQIDNO:205 175AWRRAAPAEESRGRAAVVQ SEQIDNO:206 176WRRAAPAEESRGRAAVVQK SEQIDNO:207 177RRAAPAEESRGRAAVVQKM SEQIDNO:208 178RAAPAEESRGRAAVVQKMR SEQIDNO:209 175AWRRAAPAEESRGRAAVVQK SEQIDNO:210 176WRRAAPAEESRGRAAVVQKM SEQIDNO:211 177RRAAPAEESRGRAAVVQKMR SEQIDNO:212 178RAAPAEESRGRAAVVQKMRA SEQIDNO:213 705RVLERKAPERVNALREKQIS SEQIDNO:214 751RTIGARAMESAKKTFLDGLR SEQIDNO:215 174SAWRRAAPAEESRGRAAVVQK SEQIDNO:216 175AWRRAAPAEESRGRAAVVQKM SEQIDNO:217 176WRRAAPAEESRGRAAVVQKMR SEQIDNO:218 177RRAAPAEESRGRAAVVQKMRA SEQIDNO:219 610AALVATGREMFRLGKLEQIAR SEQIDNO:220 612LVATGREMFRLGKLEQIAREK SEQIDNO:221 614ATGREMFRLGKLEQIAREKVR SEQIDNO:222 616GREMFRLGKLEQIAREKVRTL SEQIDNO:223 617REMFRLGKLEQIAREKVRTLA SEQIDNO:224 618EMFRLGKLEQIAREKVRTLAL SEQIDNO:225 619MFRLGKLEQIAREKVRTLALV SEQIDNO:226 620FRLGKLEQIAREKVRTLALVD SEQIDNO:227 621RLGKLEQIAREKVRTLALVD SEQIDNO:228 175AWRRAAPAEESRGRAAVVQKMR SEQIDNO:229 176WRRAAPAEESRGRAAVVQKMRA SEQIDNO:230 177RRAAPAEESRGRAAVVQKMRAC SEQIDNO:231 609LAALVATGREMFRLGKLEQIAR SEQIDNO:232 611ALVATGREMFRLGKLEQIAREK SEQIDNO:233 614ATGREMFRLGKLEQIAREKVRT SEQIDNO:234 615TGREMFRLGKLEQIAREKVRTL SEQIDNO:235 616GREMFRLGKLEQIAREKVRTLA SEQIDNO:236 617REMFRLGKLEQIAREKVRTLAL SEQIDNO:237 618EMFRLGKLEQIAREKVRTLALV SEQIDNO:238 619MFRLGKLEQIAREKVRTLALVD SEQIDNO:239 173WSAWRRAAPAEESRGRAAVVQKMR SEQIDNO:240 174SAWRRAAPAEESRGRAAVVQKMRA SEQIDNO:241 176WRRAAPAEESRGRAAVVQKMRACL SEQIDNO:242 177RRAAPAEESRGRAAVVQKMRACLN SEQIDNO:243 609LAALVATGREMFRLGKLEQIAREK SEQIDNO:244 610AALVATGREMFRLGKLEQIAREKV SEQIDNO:245 611ALVATGREMFRLGKLEQIAREKVR SEQIDNO:246 614ATGREMFRLGKLEQIAREKVRTLA SEQIDNO:247 615TGREMFRLGKLEQIAREKVRTLAL SEQIDNO:248 616GREMFRLGKLEQIAREKVRTLALV SEQIDNO:249 617REMFRLGKLEQIAREKVRTLALVD SEQIDNO:250 608DLAALVATGREMFRLGKLEQIAREKVR SEQIDNO:251 609LAALVATGREMFRLGKLEQIAREKVRT SEQIDNO:252 610AALVATGREMFRLGKLEQIAREKVRTL SEQIDNO:253 611ALVATGREMFRLGKLEQIAREKVRTLA SEQIDNO:254 612LVATGREMFRLGKLEQIAREKVRTLAL SEQIDNO:255 614ATGREMFRLGKLEQIAREKVRTLALVD SEQIDNO:256 607NDLAALVATGREMFRLGKLEQIAREKVRTL SEQIDNO:257 608DLAALVATGREMFRLGKLEQIAREKVRTLA SEQIDNO:258 609LAALVATGREMFRLGKLEQIAREKVRTLAL SEQIDNO:259 610AALVATGREMFRLGKLEQIAREKVRTLALV SEQIDNO:260 627QIAREKVRTL SEQIDNO:261 628IAREKVRTLA SEQIDNO:262 629AREKVRTLAL SEQIDNO:263 630REKVRTLALV SEQIDNO:264 648YQNKLKKSLG SEQIDNO:265 649QNKLKKSLGL SEQIDNO:266 709RKAPERVNAL SEQIDNO:267 710KAPERVNALR SEQIDNO:268 646LAYQNKLKKSL SEQIDNO:269 647AYQNKLKKSLG SEQIDNO:270 714RVNALREKQIS SEQIDNO:271 12ATISNRRIYRIA SEQIDNO:272 619MFRLGKLEQIAR SEQIDNO:273 621RLGKLEQIAREK SEQIDNO:274 623GKLEQIAREKVR SEQIDNO:275 627QIAREKVRTLAL SEQIDNO:276 628IAREKVRTLALV SEQIDNO:277 646LAYQNKLKKSLG SEQIDNO:278 647AYQNKLKKSLGL SEQIDNO:279 751RTIGARAMESAKK SEQIDNO:280 177RRAAPAEESRGRAA SEQIDNO:281 184ESRGRAAVVQKMRA SEQIDNO:282 186RGRAAVVQKMRACL SEQIDNO:283 619MFRLGKLEQIAREK SEQIDNO:284 620FRLGKLEQIAREKV SEQIDNO:285 621RLGKLEQIAREKVR SEQIDNO:286 644VWLAYQNKLKKSLG SEQIDNO:287 645WLAYQNKLKKSLGL SEQIDNO:288 177RRAAPAEESRGRAAV SEQIDNO:289 185SRGRAAVVQKMRACL SEQIDNO:290 186RGRAAVVQKMRACLN SEQIDNO:291 176WRRAAPAEESRGRAAV SEQIDNO:292 177RRAAPAEESRGRAAVV SEQIDNO:293 182AEESRGRAAVVQKMRA SEQIDNO:294 186RGRAAVVQKMRACLNN SEQIDNO:295 619MFRLGKLEQIAREKVR SEQIDNO:296 620FRLGKLEQIAREKVRT SEQIDNO:297 621RLGKLEQIAREKVRTL SEQIDNO:298 609LAALVATGREMFRLGKL SEQIDNO:299 614ATGREMFRLGKLEQIAR SEQIDNO:300 616GREMFRLGKLEQIAREK SEQIDNO:301 617REMFRLGKLEQIAREKV SEQIDNO:302 618EMFRLGKLEQIAREKVR SEQIDNO:303 619MFRLGKLEQIAREKVRT SEQIDNO:304 620FRLGKLEQIAREKVRTL SEQIDNO:305 621RLGKLEQIAREKVRTLA SEQIDNO:306 706VLERKAPERVNALREKQI SEQIDNO:307 707LERKAPERVNALREKQIS SEQIDNO:308 617REMFRLGKLEQIAREKVRT SEQIDNO:309 618EMFRLGKLEQIAREKVRTL SEQIDNO:310 619MFRLGKLEQIAREKVRTLA SEQIDNO:311 620FRLGKLEQIAREKVRTLAL SEQIDNO:312 621RLGKLEQIAREKVRTLALV SEQIDNO:313 611ALVATGREMFRLGKLEQIAR SEQIDNO:314 615TGREMFRLGKLEQIAREKVR SEQIDNO:315 616GREMFRLGKLEQIAREKVRT SEQIDNO:316 617REMFRLGKLEQIAREKVRTL SEQIDNO:317 618EMFRLGKLEQIAREKVRTLA SEQIDNO:318 619MFRLGKLEQIAREKVRTLAL SEQIDNO:319 620FRLGKLEQIAREKVRTLALV SEQIDNO:320 621RLGKLEQIAREKVRTLALVD SEQIDNO:321 705RVLERKAPERVNALREKQIS SEQIDNO:322 751RTIGARAMESAKKTFLDGLR SEQIDNO:323 174SAWRRAAPAEESRGRAAVVQK SEQIDNO:324 175AWRRAAPAEESRGRAAVVQKM SEQIDNO:325 176WRRAAPAEESRGRAAVVQKMR SEQIDNO:326 177RRAAPAEESRGRAAVVQKMRA SEQIDNO:327 610AALVATGREMFRLGKLEQIAR SEQIDNO:328 612LVATGREMFRLGKLEQIAREK SEQIDNO:329 614ATGREMFRLGKLEQIAREKVR SEQIDNO:330 614ATGREMFRLGKLEQIAREKVR SEQIDNO:331 616GREMFRLGKLEQIAREKVRTL SEQIDNO:332 617REMFRLGKLEQIAREKVRTLA SEQIDNO:333 618EMFRLGKLEQIAREKVRTLAL SEQIDNO:334 619MFRLGKLEQIAREKVRTLALV SEQIDNO:335 620FRLGKLEQIAREKVRTLALVD SEQIDNO:336 172VWSAWRRAAPAEESRGRAAVVQK SEQIDNO:337 174SAWRRAAPAEESRGRAAVVQKMR SEQIDNO:338 175AWRRAAPAEESRGRAAVVQKMRA SEQIDNO:339 176WRRAAPAEESRGRAAVVQKMRAC SEQIDNO:340 177RRAAPAEESRGRAAVVQKMRACL SEQIDNO:341 610AALVATGREMFRLGKLEQIAREK SEQIDNO:342 612LVATGREMFRLGKLEQIAREKVR SEQIDNO:343 614ATGREMFRLGKLEQIAREKVRTL SEQIDNO:344 615TGREMFRLGKLEQIAREKVRTLA SEQIDNO:345 616GREMFRLGKLEQIAREKVRTLAL SEQIDNO:346 617REMFRLGKLEQIAREKVRTLALV SEQIDNO:347 171AVWSAWRRAAPAEESRGRAAVVQKMR SEQIDNO:348 172VWSAWRRAAPAEESRGRAAVVQKMRA SEQIDNO:349 609LAALVATGREMFRLGKLEQIAREKVR SEQIDNO:350 610AALVATGREMFRLGKLEQIAREKVRT SEQIDNO:351 611ALVATGREMFRLGKLEQIAREKVRTL SEQIDNO:352 612LVATGREMFRLGKLEQIAREKVRTLA SEQIDNO:353 613VATGREMFRLGKLEQIAREKVRTLAL SEQIDNO:354 614ATGREMFRLGKLEQIAREKVRTLALV SEQIDNO:355 171AVWSAWRRAAPAEESRGRAAVVQKMRA SEQIDNO:356 607NDLAALVATGREMFRLGKLEQIAREKVR SEQIDNO:357 608DLAALVATGREMFRLGKLEQIAREKVRT SEQIDNO:358 609LAALVATGREMFRLGKLEQIAREKVRTL SEQIDNO:359 610AALVATGREMFRLGKLEQIAREKVRTLA SEQIDNO:360 611ALVATGREMFRLGKLEQIAREKVRTLAL

(223) SlrP

(224) SEQ ID NO: 1

(225) >gi|267992540|gb|ACY87425.1| leucine-rich repeat-containing protein [Salmonella enterica subsp. enterica serovar Typhimurium str. 14028S]

(226) Predicted PTD Sequences:

(227) TABLE-US-00030 SEQIDNO:66 593SLAREKVKRL SEQIDNO:67 175CLKNNKTELRLKI SEQIDNO:68 176LKNNKTELRLKIL SEQIDNO:69 177KNNKTELRLKILG SEQIDNO:70 175CLKNNKTELRLKIL SEQIDNO:71 176LKNNKTELRLKILG SEQIDNO:72 177KNNKTELRLKILGL SEQIDNO:73 389ALQIMQASRNNLVR SEQIDNO:74 388AALQIMQASRNNLVR SEQIDNO:75 389ALQIMQASRNNLVRL SEQIDNO:76 175CLKNNKTELRLKILGL SEQIDNO:77 176LKNNKTELRLKILGLT SEQIDNO:78 388AALQIMQASRNNLVRL SEQIDNO:79 587RLEQIESLAREKVKRL SEQIDNO:80 581AGREIFRLEQIESLAREKVKR SEQIDNO:81 582GREIFRLEQIESLAREKVKRL SEQIDNO:82 167EAVQRMRDCLKNNKTELRLKIL SEQIDNO:83 168AVQRMRDCLKNNKTELRLKILG SEQIDNO:84 169VQRMRDCLKNNKTELRLKILGL SEQIDNO:85 170QRMRDCLKNNKTELRLKILGLT SEQIDNO:86 171RMRDCLKNNKTELRLKILGLTT SEQIDNO:87 580MAGREIFRLEQIESLAREKVKR SEQIDNO:88 581AGREIFRLEQIESLAREKVKRL SEQIDNO:89 167EAVQRMRDCLKNNKTELRLKILGL SEQIDNO:90 168AVQRMRDCLKNNKTELRLKILGLT SEQIDNO:91 170QRMRDCLKNNKTELRLKILGLTTI SEQIDNO:92 578LIMAGREIFRLEQIESLAREKVKR SEQIDNO:93 579IMAGREIFRLEQIESLAREKVKRL SEQIDNO:94 578LIMAGREIFRLEQIESLAREKVKRL SEQIDNO:95 164NREEAVQRMRDCLKNNKTELRLKILG SEQIDNO:96 165REEAVQRMRDCLKNNKTELRLKILGL SEQIDNO:97 168AVQRMRDCLKNNKTELRLKILGLTTI SEQIDNO:98 161EAANREEAVQRMRDCLKNNKTELRLKIL SEQIDNO:99 162AANREEAVQRMRDCLKNNKTELRLKILG SEQIDNO:100 163ANREEAVQRMRDCLKNNKTELRLKILGL SEQIDNO:101 164NREEAVQRMRDCLKNNKTELRLKILGLT SEQIDNO:102 165REEAVQRMRDCLKNNKTELRLKILGLTT SEQIDNO:103 168AVQRMRDCLKNNKTELRLKILGLTTIPA SEQIDNO:104 157APAKEAANREEAVQRMRDCLKNNKTELRL SEQIDNO:105 159AKEAANREEAVQRMRDCLKNNKTELRLKI SEQIDNO:106 161EAANREEAVQRMRDCLKNNKTELRLKILG SEQIDNO:107 162AANREEAVQRMRDCLKNNKTELRLKILGL SEQIDNO:108 163ANREEAVQRMRDCLKNNKTELRLKILGLT SEQIDNO:109 165REEAVQRMRDCLKNNKTELRLKILGLTTI SEQIDNO:110 173RDCLKNNKTELRLKILG SEQIDNO:111 175CLKNNKTELRLKILGLT SEQIDNO:112 172MRDCLKNNKTELRLKILG SEQIDNO:113 173RDCLKNNKTELRLKILGL SEQIDNO:114 386LPAALQIMQASRNNLVRL SEQIDNO:115 585IFRLEQIESLAREKVKRL SEQIDNO:116 171RMRDCLKNNKTELRLKILG SEQIDNO:117 172MRDCLKNNKTELRLKILGL SEQIDNO:118 173RDCLKNNKTELRLKILGLT SEQIDNO:119 171RMRDCLKNNKTELRLKILGL SEQIDNO:120 172MRDCLKNNKTELRLKILGLT SEQIDNO:121 173RDCLKNNKTELRLKILGLTT SEQIDNO:122 168AVQRMRDCLKNNKTELRLKIL SEQIDNO:123 169VQRMRDCLKNNKTELRLKILG SEQIDNO:124 170QRMRDCLKNNKTELRLKILGL SEQIDNO:125 171RMRDCLKNNKTELRLKILGLT SEQIDNO:126 173RDCLKNNKTELRLKILGLTTI SEQIDNO:127 167EAVQRMRDCLKNNKTELRLKILG SEQIDNO:128 168AVQRMRDCLKNNKTELRLKILGL SEQIDNO:129 169VQRMRDCLKNNKTELRLKILGLT SEQIDNO:130 170QRMRDCLKNNKTELRLKILGLTT SEQIDNO:131 171RMRDCLKNNKTELRLKILGLTTI SEQIDNO:132 579IMAGREIFRLEQIESLAREKVKR SEQIDNO:133 580MAGREIFRLEQIESLAREKVKRL SEQIDNO:134 581AGREIFRLEQIESLAREKVKRLF SEQIDNO:135 167EAVQRMRDCLKNNKTELRLKILGL SEQIDNO:136 168AVQRMRDCLKNNKTELRLKILGLT SEQIDNO:137 165REEAVQRMRDCLKNNKTELRLKILG SEQIDNO:138 167EAVQRMRDCLKNNKTELRLKILGLT SEQIDNO:139 168AVQRMRDCLKNNKTELRLKILGLTT SEQIDNO:140 171RMRDCLKNNKTELRLKILGLTTIPA SEQIDNO:141 162AANREEAVQRMRDCLKNNKTELRLKIL SEQIDNO:142 163ANREEAVQRMRDCLKNNKTELRLKILG SEQIDNO:143 164NREEAVQRMRDCLKNNKTELRLKILGL SEQIDNO:144 165REEAVQRMRDCLKNNKTELRLKILGLT SEQIDNO:145 157APAKEAANREEAVQRMRDCLKNNKTELRLK SEQIDNO:146 158PAKEAANREEAVQRMRDCLKNNKTELRLKI SEQIDNO:147 159AKEAANREEAVQRMRDCLKNNKTELRLKIL SEQIDNO:148 160KEAANREEAVQRMRDCLKNNKTELRLKILG SEQIDNO:149 161EAANREEAVQRMRDCLKNNKTELRLKILGL SEQIDNO:150 162AANREEAVQRMRDCLKNNKTELRLKILGLT SEQIDNO:151 163ANREEAVQRMRDCLKNNKTELRLKILGLTT

Example 13

Analysis of T3SS-Independent Internalisation of Bacterial Effector Proteins of the LPX Subtype

(228) Bacterial pathogens including Salmonella, Yersinia and Shigella spp. express effector proteins of the LPX subtype of leucine-rich repeat (LRR) proteins that are translocated into the host cell via a type three secretion system (T3SS) during infection.

(229) Previous studies identified the LPX effector protein YopM of Yersinia enterocolitica as a novel bacterial cell-penetrating protein. YopM's ability to translocate across the host cell plasma membrane independently of Yersinia's T3SS is mediated by it's two N-terminal -Helices.

(230) The inventors therefore constructed and recombinantly expressed LPX effector proteins of Shigella flexneri and Salmonella typhimurium. Potential T3SS-independent translocation of these proteins was analysed by cell fractionation of HeLa cells, immunofluorescence microscopy and FACS analyses. Functionality of the recombinant proteins was assessed by in vitro ubiquitination assays. Additionally, an effect of the recombinant proteins on the expression of pro-inflammatory cytokines was analysed by quantitative real time PCR.

(231) The inventors could show that the SspH1 effector protein of Salmonella typhimiurium is able to translocate into eukaryotic cells without a requirement for additional factors. Furthermore the inventors could show that recombinant SspH1 is a functional E3 ubiquitin ligase that is able to reduce the expression of Interleukin 8 in IL1l stimulated cells.

(232) These results show that SspH1 is a novel bacterial cell-penetrating protein and, together with YopM, a hint for a general concept of T3SS-independent translocation by LPX effector proteins.

Example 14

(233) Group 1:

(234) Leucine-rich repeats of SspH1, wherein SspH1 has the amino acid sequence indicated as SEQ ID NO: 2.

(235) LRR1 (identification number 1): AA217-238 of SEQ ID NO: 2.

(236) LRR2 (identification number 2): AA239-257 of SEQ ID NO: 2.

(237) LRR3 (identification number 3): AA258-279 of SEQ ID NO: 2.

(238) LRR4 (identification number 4): AA280-297 of SEQ ID NO: 2.

(239) LRR5 (identification number 5): AA 298-319 of SEQ ID NO: 2.

(240) LRR6 (identification number 6): AA320-337 of SEQ ID NO: 2.

(241) LRR7 (identification number 7): AA338-360 of SEQ ID NO: 2.

(242) LRR8 (identification number 8): AA361-381 of SEQ ID NO: 2.

(243) Group 2:

(244) Leucine-rich repeats of SspH2, wherein SspH2 has the amino acid sequence indicated as SEQ ID NO: 3.

(245) LRR1 (identification number 1): AA223-242 of SEQ ID NO: 3.

(246) LRR2 (identification number 2): AA243-264 of SEQ ID NO: 3.

(247) LRR3 (identification number 3): AA265-282 of SEQ ID NO: 3.

(248) LRR4 (identification number 4): AA283-302 of SEQ ID NO: 3.

(249) LRR5 (identification number 5): AA303-324 of SEQ ID NO: 3.

(250) LRR6 (identification number 6): AA325-342 of SEQ ID NO: 3.

(251) LRR7 (identification number 7): AA343-364 of SEQ ID NO: 3.

(252) LRR8 (identification number 8): AA365-382 of SEQ ID NO: 3.

(253) LRR9 (identification number 9): AA383-404 of SEQ ID NO: 3.

(254) LRR10 (identification number 10): AA405-422 of SEQ ID NO: 3.

(255) LRR11 (identification number 11): AA423-445 of SEQ ID NO: 3.

(256) LRR12 (identification number 12): AA446-466 of SEQ ID NO: 3.

(257) Group 3:

(258) Leucine-rich repeats of Slrp, wherein Slrp has the amino acid sequence indicated as SEQ ID NO: 1.

(259) LRR1 (identification number 1): AA200-219 of SEQ ID NO: 1.

(260) LRR2 (identification number 2): AA221-242 SEQ ID NO: 1.

(261) LRR3 (identification number 3): AA243-262 SEQ ID NO: 1.

(262) LRR4 (identification number 4): AA263-284 SEQ ID NO: 1.

(263) LRR5 (identification number 5): AA285-305 SEQ ID NO: 1.

(264) LRR6 (identification number 6): AA306-325 SEQ ID NO: 1.

(265) LRR7 (identification number 7): AA326-346 SEQ ID NO: 1.

(266) LRR8 (identification number 8): AA347-368 SEQ ID NO: 1.

(267) LRR9 (identification number 9): AA369-389 SEQ ID NO: 1.

(268) LRR10 (identification number 10): AA390-410 SEQ ID NO: 1.

(269) Group 4:

(270) Predicted leucine-rich repeats of IpaH1.4, wherein IpaH1.4 has the amino acid sequence indicated as SEQ ID NO: 4.

(271) LRR1 (identification number 1): AA92113 of SEQ ID NO: 4.

(272) LRR2 (identification number 2): AA132153 of SEQ ID NO: 4.

(273) LRR3 (identification number 3): AA172191 of SEQ ID NO: 4.

(274) LRR4 (identification number 4): AA192213 of SEQ ID NO: 4.

(275) Group 5:

(276) Predicted leucine-rich repeats of IpaH2.5, wherein IpaH2.5 has the amino acid sequence indicated as SEQ ID NO: 5.

(277) LRR1 (identification number 1): AA92113 of SEQ ID NO: 5

(278) LRR2 (identification number 2): AA132153 of SEQ ID NO: 5

(279) LRR3 (identification number 3): AA172191 of SEQ ID NO: 5

(280) LRR4 (identification number 4): AA192213 of SEQ ID NO: 5

(281) Group 6:

(282) Predicted leucine-rich repeats of IpaH3, wherein IpaH3 has the amino acid sequence indicated as

(283) SEQ ID NO: 6.

(284) LRR1 (identification number 1): AA8099 of SEQ ID NO: 6

(285) LRR2 (identification number 2): AA100121 of SEQ ID NO: 6

(286) LRR3 (identification number 3): AA140161 of SEQ ID NO: 6

(287) LRR4 (identification number 4): AA162179 of SEQ ID NO: 6

(288) LRR5 (identification number 5): AA180201 of SEQ ID NO: 6

(289) LRR6 (identification number 6): AA220241 of SEQ ID NO: 6

(290) Group 7:

(291) Leucine-rich repeats of IpaH4.5, wherein IpaH4.5 has the amino acid sequence indicated as SEQ ID NO: 7.

(292) LRR1 (identification number 1): AA63-82 of SEQ ID NO: 7.

(293) LRR2 (identification number 2): AA83-104 of SEQ ID NO: 7.

(294) LRR3 (identification number 3): AA105-122 of SEQ ID NO: 7.

(295) LRR4 (identification number 4): AA123-143 of SEQ ID NO: 7.

(296) LRR5 (identification number 5): AA144-165 of SEQ ID NO: 7.

(297) LRR6 (identification number 6): AA 166-183 of SEQ ID NO: 7.

(298) LRR7 (identification number 7): AA 184-205 of SEQ ID NO: 7.

(299) LRR8 (identification number 8): AA206-223 of SEQ ID NO: 7.

(300) LRR9 (identification number 9): AA224-246 of SEQ ID NO: 7.

(301) LRR10 (identification number 10): AA247-270 of SEQ ID NO: 7.

(302) Group 8:

(303) Leucine-rich repeats of IpaH7.8, wherein IpaH7.8 has the amino acid sequence indicated as SEQ ID NO: 8.

(304) LRR1 (identification number 1): AA58-79 of SEQ ID NO: 8.

(305) LRR2 (identification number 2): AA80-97 of SEQ ID NO: 8.

(306) LRR3 (identification number 3): AA98-119 of SEQ ID NO: 8.

(307) LRR4 (identification number 4): AA120-137 of SEQ ID NO: 8.

(308) LRR5 (identification number 5): AA138-157 of SEQ ID NO: 8.

(309) LRR6 (identification number 6): AA158-179 of SEQ ID NO: 8.

(310) LRR7 (identification number 7): AA180-199 of SEQ ID NO: 8.

(311) LRR8 (identification number 8): AA202-223 of SEQ ID NO: 8.

(312) LRR9 (identification number 9): AA225-248 of SEQ ID NO: 8.

(313) Group 9:

(314) Leucine-rich repeats of IpaH9.8, wherein IpaH9.8 has the amino acid sequence indicated as SEQ ID NO: 9.

(315) LRR1 (identification number 1): AA57-77 of SEQ ID NO: 9.

(316) LRR2 (identification number 1): 78-99 of SEQ ID NO: 9.

(317) LRR3 (identification number 1): 100-117 of SEQ ID NO: 9.

(318) LRR4 (identification number 1): 118-139 of SEQ ID NO: 9.

(319) LRR5 (identification number 1): 140-157 of SEQ ID NO: 9.

(320) LRR6 (identification number 1): 158-179 of SEQ ID NO: 9.

(321) LRR7 (identification number 1): 182-203 of SEQ ID NO: 9.

(322) LRR8 (identification number 1): 205-228 of SEQ ID NO: 9.

Example 15

(323) The uptake of recombinant LPX effector proteins was further analyzed by sub-cellular fractionation of eukaryotic HeLa cells. By this method, the internalization of putative CPPs can be assessed due to the separation of soluble cytoplasmic and insoluble membrane proteins (Behrens, 1938; Rter et al., 2010). HeLa cells which were grown to 80% confluence were incubated with the recombinant proteins (25 g/ml) for 3 h. After isolation of cytoplasmic and membrane fractions, proteins were separated by SDS-PAGE and subsequently immobilized on a nitrocellulose membrane by Western blotting. For detection of the recombinant protein, an -FLAG-antibody was used as a primary antibody. In case of internalization, proteins were expected to be detected in the cytoplasmic fraction. Since recombinant LPX effector proteins harboring only a single FLAG-tag were not detectable in the HeLa cell background at all (data not shown), constructs with 3FLAG-tags were chosen for this assay due to their improved detectability (Terpe, 2003).

(324) Recombinant IpaH1.4, IpaH2.5, IpaH4.5, IpaH7.8, and IpaH9.8 could be detected in the cytoplasmic fraction after 3 h incubation of HeLa cells, indicating successful internalization in a T3SS-independent manner (FIG. 23). However, all proteins were also detected in the membrane fraction in comparable amounts. Upon incubation with IpaH4.5 and IpaH7.8 the amounts found in the membrane fraction appear to be even slightly higher. Both the cytoplasmic and the membrane fraction appear to be free of contamination since the membrane marker transferring receptor (TF-R) is solely detectable in the membrane fraction, whereas a prominent band for GAPDH can be observed in all cytoplasmic but not in the membrane fractions.

Example 16

Analysis of Intracellular Trafficking of Recombinant LPX Effector Proteins by Co-Localization with Endocytic Markers

(325) For investigations of possible endocytic uptake mechanisms of recombinant LPX effector proteins, co-localization studies with endocytic markers were performed. Appropriate markers for this purpose are regulatory proteins which are involved in intracellular membrane trafficking between different sub-cellular compartments. These include both vesicle formation and movements as well as membrane fusion. Rab5 served as marker for early endosomes, whereas Rab7 was used as an indicator for maturation into late endosomes. In addition, CD63 served as marker for trafficking from late endosomes to lysosomes.

(326) HeLa cells were incubated with recombinant Cy3-labeled LPX effector proteins for 1 h, 3 h, and 6 h. Following incubation, cells were washed, fixed and permeabilized. Cell-compartment specific markers including Rab5, Rab7, and CD63 were stained using specific fluorescent antibodies. In addition, the nucleus was stained using Draq5. Finally, the co-localization with the Cy3-labeled LPX effector proteins was analyzed by confocal fluorescence microscopy. As depicted in FIG. 24 all tested LPX effector proteins were found to be partially co-localized with endosomal compartments. For each marker protein, representative pictures which revealed the highest amount of co-localization are shown; in case of Rab5, pictures taken after 1 h incubation of HeLa cells with labeled proteins show on average the highest degrees of co localization, whereas for Rab7 and CD63 maximal co-localizations were observed only after longer incubation times of 3 h and 6 h.

(327) All tested LPX effector proteins were found to be partially associated with early endosomes (i) since co-localization with the early endosomal marker Rab5 can be detected to some extent. After internalization, the proteins seem to remain in the endosomal compartments; so all shown LPX effector proteins co-localize with the late endosomal marker protein Rab7 (ii) after 3 h incubation. In addition, the overlay images reveal co-localization with CD63 (iii) after 6 h incubation.

(328) In summary, fluorescence microcopy studies reveal partial co-localization with the endosomal markers Rab5, Rab7, and CD63 indicating that endocytic mechanisms seem to be involved in the T3SS-independent uptake of the recombinant LPX effectors IpaH1.4, IpaH2.5, IpaH4.5, IpaH7.8, IpaH9.8, and SlrP into HeLa cells.

Example 16

Role of Different Endocytic Pathways in HeLa Cell Uptake of Recombinant LPX Effector Proteins

(329) HeLa cells were with endocytic inhibitors cytochalasin D (2.5 M), amiloride (3 mM), filipin (7.5 M), nocodazole (20 M), dynasore (80 M), methyl--cyclodextrin (MI3CD) (5 mM) for 1 h prior to the addition of recombinant LPX effectors. Three hours later the cells were washed with D-PBS (with Ca.sup.2+/Mg.sup.2+), trypsinized, resuspended in D-PBS (without Ca.sup.2+/Mg.sup.2+), diluted with trypan blue (final concentration 0.2%) and analyzed by flow cytometry. Fluorescence of ALEXA FLUOR 488 was detected at 510 nm. For exclusion of dead cells and cellular debris, forward and side scatter were set up appropriately and scaled to the living cell population before starting the incubation. Each sample was measured in duplicate counting 10,000 cells in every run. Three independent assays were performed for each protein.

(330) FIG. 25 summarizes the results of FACS-based uptake analysis of recombinant LPX effectors in the presence of different endocytic inhibitors. It can be recognized that the different LPX effectors act similar in response to a certain inhibitor. Especially the presence of cytochalasin D which effectively blocks actin-mediated endocytosis, of amiloride which inhibits macropinocytotic events as well as of methyl--cyclodextrin which serves as in inhibitor of lipid-raft-mediated endocytosis, leads to an enormous reduction of cellular internalization of all tested recombinant LPX effectors. These results indicate that especially the aforementioned endocytic pathways might act synergistically in the cellular transduction of LPX effectors.

Example 18

(331) For investigation of possible membranolytic effects of recombinant LPX effectors, the FACS-based membranolysis assay was performed (Florn et al., 2011).

(332) HeLa cells were co-incubated with 1 g/ml PI and different ALEXA FLUOR 488-labeled proteins. At given time points, samples were taken from the ongoing incubation and subjected to FACS analysis. Fluorescence of PI was detected at 617 nm. For exclusion of dead cells and cellular debris, forward and side scatter were set up appropriately and scaled to the living cell population before starting the incubation. Each sample was measured in duplicate counting 10,000 cells in every run. Three independent assays were performed for each protein. As a control, HeLa cells were incubated solely with PI under equal conditions.

(333) FIG. 26 summarizes the results of the membranolysis assays of HeLa cells which were incubated with recombinant LPX effector proteins. Disruption of the membrane leads to increased fluorescence intensities of PI due to increased permeability. Indeed, for all tested LPX effector proteins an increase of PI fluorescence intensity over the entire time is visible when HeLa cells were incubated at 37 C. However, this increase appears to slightly differ for the tested LPX effectors. In direct comparison to non-treated control cells (represented by dashed-lined curves), the strongest increase of PI fluorescence is induced by incubation with IpaH2.5, whereas upon incubation with IpaH9.8 almost no difference to non-treated cells is detectable. In contrast, co-incubation of HeLa cells with LPX effectors and PI at 4 C. does not result in increased levels of PI fluorescence intensity and is almost comparable to non-treated control cells.

Example 19

Quantification of Lactate Dehydrogenase (LDH) Release Induced by Recombinant LPX Effector Proteins

(334) For the evaluation of putative cytotoxic effects of recombinant LPX effector proteins on HeLa cells, the amount of released lactate dehydrogenase (LDH) was taken as a parameter for membrane integrity and measured colorimetrically using the CYTOTOX 96 Non-Radioactive Cytotoxicity Assay. For that, HeLa cells were cultured in 96-well plates and incubated with different recombinant LPX effector proteins (25 or 50 g/ml) for 24 h, 6 h, and 1 h. The amounts of released LDH of both culture supernatants and lysates were determined by measuring the absorbance at 490 nm. The quotient of both was calculated and normalized to non-treated cells.

(335) FIG. 27 shows the relative LDH release of HeLa cells upon incubation with recombinant IpaH1.4, IpaH2.5, IpaH4.5, IpaH7.8, IpaH9.8, or SlrP in comparison to non-treated cells and a LDH positive control. The measured amounts of LDH released upon incubation with the specific LPX effector proteins are comparable to basal LDH release displayed by HeLa cells incubated in culture medium (Medium Control). This appears to be independent of the protein concentration (25 g/ml or 50 g/ml) or the incubation time (24 h, 6 h, or 1 h). The large amounts of LDH measured for the positive control ensure that the assay was functional.

(336) Based on the amounts of LDH released by HeLa cells upon incubation with recombinant IpaH1.4, IpaH2.5, IpaH4.5, IpaH7.8, IpaH9.8, or SlrP potential cytotoxic effects of these LPX effector proteins can be excluded.

Example 20

(337) The proteins of the IpaH subset as well as the LPX effector SlrP and the SspH proteins of Salmonella harbor an enzymatic domain that was shown to possess an E3 ubiquitin ligase activity (Rohde et al., 2007; Singer et al., 2008; Zhu et al., 2008). In order to test whether recombinant LPX effector proteins are enzymatically functional as ubiquitin E3 ligases an in vitro ubiquitination assay was carried out. The assay was performed in a 40 l reaction mixture containing ubiquitin reaction buffer, 2 g of HA-tagged ubiquitin, 0.5 g of E1 and 2 mg of E2 (UbcH5b) in the presence or absence of 4 g of recombinant IpaH1.4, IpaH2.5, IpaH4.5, IpaH7.8, IpaH9.8, SlrP, or BSA as a negative control. As further controls, the mixtures were prepared without the E2 (UbcH5b) enzyme. Reactions were incubated for 1 h at 37 C. before they were stopped by the addition of 4SDS sample buffer without DTT.

(338) The results of Western blot analysis which are depicted in FIG. 28 reveal a ladder of ubiquitin chains from 40 to 200 kDa in reactions performed in the presence of LPX effectors. In reactions performed in the absence of E2 (UbcH5b), if anything, only ubiquitinated E1 at a size of >110 kDa was detected. In the presence of E2 (UbcH5b) but in the absence of any LPX effector proteins (see lane 1+2) both E1 (>110 kDa) and E2 (30 kDa) were found to be ubiquitinated.

(339) The results of the in vitro ubiquitination assay show that recombinant IpaH1.4, IpaH2.5, IpaH4.5, IpaH7.8, IpaH9.8, and SlrP have the ability to remove ubiquitin from ubiquitinated UbcH5b in order to form poly-ubiquitin chains of the HA-tagged ubiquitin. These findings confirm that the indicated LPX effector proteins are indeed enzymatically functional in vitro.