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 method of treatment of disease caused by autoimmunity, and/or treatment of acute inflammation, chronic inflammation, inflammatory disorder, or pathogenic inflammatory reaction of the immune system, and/or a method of suppressing the immune system in a subject, comprising administering a therapeutically effective amount of a pharmaceutical composition to said subject, wherein the pharmaceutical composition comprises 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 method 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 method 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 method 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 method of claim 1, wherein said effector protein is a bacterial effector protein of the LPX-Subtype, 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 method 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 method 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 method 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 method 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 method 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 method of claim 1, wherein said cargo molecule has therapeutic activity in a subject and/or is useful in a diagnostic method.

12. The method 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. The method of claim 1, wherein the pharmaceutical composition is in the form of a kit, prior to the administering.

14. The method of claim 1, wherein the disease caused by autoimmunity comprises acute disseminated encephalomyelitis (ADEM), Addison's disease, ankylosing spondylitis, antiphospholipid antibody syndrome (APS), aplastic anemia, autoimmune hepatitis, autoimmune Oophoritis, celiac disease, Crohn's disease (Morbus Crohn), diabetes mellitus type 1, gestational pemphigoid, goodpasture's syndrome, Graves' disease, Guillain-Barré syndrome (GBS), Hashimoto's disease, idiopathic thrombocytopenic purpura, Kawasaki's disease, lupus erythematosus, Mixed Connective Tissue Disease, multiple sclerosis, myasthenia gravis, opsoclonus myoclonus syndrome (OMS), optic neuritis, Ord's thyroiditis, pemphigus, pernicious anaemia, primary biliary cirrhosis, rheumatoid arthritis, Reiter's syndrome, Sjögren's syndrome, Takayasu's arteritis, temporal arteritis, Warm autoimmune hemolytic anemia or Wegener's granulomatosis.

15. The method of claim 1 wherein acute inflammation or chronic inflammation comprises asthma, chronic prostatitis, glomerulonephritis, hypersensitivities, inflammatory bowel disease, pelvic inflammatory disease, reperfusion injury, arthritis, osteoarthritis, (juvenile) chronic arthritis, rheumatoid arthritis, psoriatic arthritis, arthritis mutilans, septic arthritis, infectious arthritis and/or reactive arthritis, transplant rejection, vasculitis, inflammatory myopathy, atherosclerosis, ischaemic heart disease, gastroenteritis, chronic gastritis, colitis ulcerose, or psoriasis or psoriasis arthritis.

16. The method of claim 1, wherein treatment of diseases caused by autoimmunity, and/or treatment of acute inflammation, chronic inflammation, inflammatory disorders, or pathogenic inflammatory reaction of the immune system, and/or suppressing the immune system comprises treatment of gastroenteritis, chronic gastritis, inflammatory bowel diseases (IBD), colitis ulcerosa, psoriasis, allergic reaction, Morbus Crohn, arthritis, osteoarthritis, (juvenile) chronic arthritis, rheumatoid arthritis, psoriatic arthritis, arthritis mutilans, septic arthritis, infectious arthritis or reactive arthritis.

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 6× His-tag (not shown) for purification.

(2) FIG. 2 shows an immunoblot analysis of subcellular fractionations of HeLa cells incubated with 25 μg/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 μg/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 means±standard deviations from three independent experiments. FIGS. 1 to 4 show that SspH1 and SspH1-Nter translocate into the host cell cytoplasm independently of Salmonella's T3SS.

(5) FIG. 5 shows a Membrane integrity assay of HeLa cells incubated with SspH1, YopM and Tat, respectively. Cells were incubated with 20 μg/ml protein and co-incubated with 1 μg/ml propidium iodide (P1).

(6) 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 24 h and analysed using the CYTOTOX 96® Non-Radioactive Cytotoxicity Assay. LDH: lactate dehydrogensase.

(7) 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

(8) 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.

(9) 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.

(10) FIG. 10 shows the effect of SspH1 on IL-8 mRNA in A549 cells stimulated with IL1β. A549 cells were incubated with SspH1 for 3 h and subsequently stimulated with 20 ng/ml IL1β. 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.

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

(12) FIGS. 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 μm. Lower panel: Quenched time-lapse assay of HeLa cells incubated with different ALEXA FLUOR® 488 labeled effector proteins (20 μg/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 means±standard deviations from three independent experiments.

(13) 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).

(14) 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 II8 mRNA expression in A549 cells following incubation with SspH1 and subsequent stimulation with TNFα.

(15) 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.

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

(17) Western blot analysis after cell fractionation of HeLa cells which were incubated with the indicated LPX effector protein (25 μg/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 α-GAPDH-antibody and a TF-R-antibody and corresponding PO-conjugated secondary antibodies.

(18) 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 μg/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×.

(19) FIG. 25 shows a flow cytometry analysis of effector proteins of the LPX family. HeLa cells were with endocytic inhibitors cytochalasin D (2.5 μM), amiloride (3 mM), filipin (7.5 μM), nocodazole (20 μM), dynasore (80 μM), methyl-β-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

(20) 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 μg/ml) and propidium iodide (PI) (1 μg/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].

(21) 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 μg/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.

(22) 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 μl reaction mixtures containing (+) or not containing (−) the indicated protein (4 μg), HA-tagged ubiquitin (2 μg), E2 (UbcH5b)(2 μg), and E1 (0.5 μg). Reactions were stopped by the addition of 4× SDS 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 α-HA antibody and a corresponding PO-conjugated secondary antibody. Different Western blots are separated by dashed lines.

DESCRIPTION OF THE SEQUENCE LISTING

(23) SEQ ID NO: 1: Amino acid sequence of SlrP from Salmonella enterica subsp. enterica serovar Typhimurium str. 14028S

(24) SEQ ID NO: 2: Amino acid sequence of SspH1 from Salmonella enterica subsp. enterica serovar Typhimurium str. 14028S

(25) SEQ ID NO: 3: Amino acid sequence of SspH2 from Salmonella enterica subsp. enterica serovar Typhimurium str. 14028S

(26) SEQ ID NO: 4: Amino acid sequence of IpaH1.4 from Shigella flexneri

(27) SEQ ID NO: 5: Amino acid sequence of IpaH2.5 from Shigella flexneri

(28) SEQ ID NO: 6: Amino acid sequence of IpaH3 from Shigella flexneri

(29) SEQ ID NO: 7: Amino acid sequence of IpaH4.5 from Shigella flexneri

(30) SEQ ID NO: 8: Amino acid sequence of IpaH7.8 from Shigella flexneri

(31) SEQ ID NO: 9: Amino acid sequence of IpaH9.8 from Shigella flexneri

(32) SEQ ID NO: 10: Nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 1

(33) SEQ ID NO: 11: Nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 2

(34) SEQ ID NO: 12: Nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 3

(35) SEQ ID NO: 13: Nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 4

(36) SEQ ID NO: 14: Nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 5

(37) SEQ ID NO: 15: Nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 6

(38) SEQ ID NO: 16: Nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 7

(39) SEQ ID NO: 17: Nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 8

(40) SEQ ID NO: 18: Nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 9

(41) SEQ ID NOs: 19 to 27: Ubiquitin ligase domains of SEQ ID NOs: 1 to 9 as indicated in FIGS. 14-22

(42) 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

(43) 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

(44) 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

(45) SEQ ID NOs: 530 to 549: Oligonucleotide sequences as indicated in Table 3.3

EXAMPLES

(46) 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

(47) 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.

(48) To enable the expression and purification of SspH1, recombinant proteins tagged with a C-terminal 6× His-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 α-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.

(49) 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 α-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.

(50) 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).

(51) 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.

(52) 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

(53) HeLa cells were incubated with the recombinant proteins SspH1-AF488, YopM-AF488 and Tat-GFP-GSK and co-incubated with 1 μg/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.

(54) 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.

(55) 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.

(56) 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 μg/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.

(57) 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

(58) 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.

(59) To test if recombinant SspH1 is also functional and could polyubiquitinate proteins, in vitro ubiquitination assays were carried out in a 40 μl 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 μg Ha-ubiquitin, 0.5 μg E1, and 2 μg E2 (UbcH5B) in the presence or absence of 1 μg recombinant SspH1 Ubiquitination analysis of PKN1 was performed in the presence or absence of additional 0.4 μg 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.

(60) 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.

(61) To determine whether cytokine production is also down-regulated by recombinant SspH1, the levels of IL-8 mRNA of A549 cells after stimulation with IL1β 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-1β stimulation than untreated cells.

(62) 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 IL1β stimulated cells, but not in TNFα-stimulated cells (FIG. 13b)

Example 4

Analysis of Further Effector Proteins of the LPX Family

(63) 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 6× His-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).

(64) 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).

(65) 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

(66) 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 (Ülo Langel, Schweden). The results of this screen are described in Example 5 and summarized in FIG. 12.

(67) 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 (Rüter et 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 Salmonella. 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.

(68) 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

(69) Construction of 6× His-Tagged Recombinant Proteins

(70) 1.1 Restriction Free (RF) Cloning by Linear Amplification

(71) 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 & Löwe, 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.

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

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

(74) 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

(75) 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.

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

(77) 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

(78) Following the amplification reaction, 0.2 U/μl DpnI were added for 2 h at 37° C. to eliminate parental plasmid DNA. 10 μl of the DpnI treated reaction mixture were then used for transformation of competent E. coli DH5α cells.

(79) 2. Expression and Purification of Recombinant Protein

(80) 2.1 Expression of Recombinant Protein in E. Coli

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

(82) 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 μg/μl 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 3,000×g and 4° C. for 15 min and the cell pellet was stored at −20° C. until further usage.

(83) 2.2 Preparation of Cleared E. Coli Lysates

(84) The bacterial pellets were thawed on ice and resuspended in 10 ml of lysis buffer. Cleared lysates were prepared by sonication (Branson Sonifier 250; 4×30 s, level 4, 50% cycle, on ice). Bursts were followed by 15 s breaks. The cellular debris was removed by centrifugation (7,200×g, 15 min, 4° C.) and the supernatant containing the recombinant protein was subjected to affinity chromatography.

(85) 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.

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

(87) 2.3 Purification of Recombinant Protein

(88) 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).

(89) To enable binding of the 6× His-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 (800×g, 2 min, 4° C.) and the supernatant was discarded. Three wash steps were carried out (800×g, 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 μl aliquots using 5 ml of elution buffer. All fractions were stored at 4° C. for SDS-PAGE analysis.

(90) 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) 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) 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)

(91) 2.4 Protein Dialysis and Concentration

(92) 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 500×g and 4° C.

(93) 2.5 Protein Labelling with Fluorescent Dyes

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

(95) Protein Labelling with ALEXA FLUOR® 488

(96) 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.

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

(98) Protein Labelling with Cy3

(99) 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.

(100) 3 Cell Fractionation of Eukaryotic Cells

(101) 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 and Finlay, 1997; Gauthier et al., 2000).

(102) The cells were cultured to confluency in a 10 cm culture dish and subsequently incubated with recombinant protein (25 μg/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, 4×1 s, level 4, 4° C.) followed by centrifugation (108,000×g, 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 (108,000×g, 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.

(103) 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 TRITON ™ Buffer Sonication Buffer 1 l TRITON ™ X-100 1% (v/v) Acid Buffer Wash Glycine 0.2 g D-PBS Add to 100 ml pH 2.0

(104) 4 Nuclear Fractionation of Eukaryotic Cells

(105) 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.

(106) Cells grown to confluency in a 10 cm culture dish were incubated with recombinant protein (25 μg/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 500×g for 5 min. The cells were washed with D-PBS, transferred to a microcentrifuge tube and centrifuged again (500×g, 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.

(107) Immunofluorescence Microscopy

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

(109) 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 μg/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 μl 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 permeabilised 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.

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

(111) 6 Fluorescence Activated Cell Sorting (FACS)

(112) 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).

(113) 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.

(114) 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 μg/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.

(115) 6.1 Quenched Time-Lapse Uptake and CPP-induced Membranolysis Assay

(116) 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 (Florén et al., 2011).

(117) 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.

(118) 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).

(119) 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 μg/ml PI was added to the medium.

(120) 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).

(121) 7 In Vitro Ubiquitination Assay

(122) In vitro ubiquitination experiments were performed in 40 μl reaction buffer (50 mM Tris-HCl, pH7.4, 10 mM MgCl.sub.2, 0.1 mM DTT and 2 mM ATP) containing 0.5 μg E1, 2 μg UbcH5b (E2), 2 μg HA-ubiquitin, 2 μg SspH1/SspH1-Nter in the presence or absence of 0.4 μg PKN1. Reactions were incubated at 37° C. for 1 h 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.

(123) 8 Immunoprecipitation (IP)

(124) 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 μg/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+) (3×5 min), before they were scraped from the surface and resuspended in 800 μl IP lysis buffer (supplemented with Complete EDTA-free Protease Inhibitor Cocktail (Roche Biochemicals, Mannheim) prior to use). The cells were permeabilised by sonication (3×20 s, 4° C.) and subsequently incubated on a rotary shaker for 30 min at 4° C. Lysates were cleared by centrifugation (16,000×g, 30 min, 4° C.) and a sample of the lysate was taken and stored at −20° C. until further usage. 30 μl protein A/G agarose beads (Santa Cruz, sc-2003) were incubated with 5 μg α-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 (1000×g, 3 min, 4° C.) and a sample of the supernatant was collected and stored until further usage. The beads were washed with 600 μl IP lysis buffer three times and centrifuged as mentioned above. The supernatant was discarded and 30 μl 4× SDS sample buffer were added to the beads, heated at 95° C. for 5 min and subsequently centrifuged at 16,000×g for 5 min. The supernatant along with the samples of the lysate and the unbound protein were subjected to immuno blot analysis.

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

(126) 9 Non-Radioactive Cytotoxicity Assay

(127) 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 μg/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.

(128) 10 Analysis of Eukaryotic Gene Expression

(129) 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 II1β, before RNA was isolated according to the manufacturer's recommendations using the RNeasy Mini Kit (Qiagen, Hilden).

(130) 10.1 cDNA Synthesis

(131) 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.

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

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

(134) 10.2 Quantitative Real Time PCR

(135) 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).

(136) 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.

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

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

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

(140) 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.

(141) 3 Material

(142) 3.1 Bacterial Strains

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

(144) TABLE-US-00013 TABLE 3.1 Bacterial strains Strain Relevant Characteristics Reference E. coli DH5α F.sup.−, endA1, recA1, hsdR17(r.sub.K.sup.− M.sub.K.sup.+) deoR, Hanahan et al., thi-1, supE44, gyrA96, Δ(lacZYA-argF) U169 1991 (Φ80dlacZΔM15) E. coli BL21 F.sup.−, hsdS.sub.B (r.sub.B.sup.−m.sub.B.sup.−), dcm, gal, ompT, Studier & (DE3) (λDE3) Moffatt, 1986

(145) 3.4 Plasmids and Oligonucleotides

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

(147) 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-3β 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

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

(149) TABLE-US-00015 TABLE 3.3 Oligonucleotide sequences for DNA amplification by PCR (restriction sites are underlined) Oligonucleotide Sequence (5′.fwdarw.3′) F-SspHI (NheI) CTA GCT AGC GTT ACC GAT AAA TAA TAA CTT SEQ ID NO: 530 R-SspHI (XhoI) CCC CTC GAG TGA ATG GTG CAG TTG TGA GCC SEQ ID NO: 531 R-SspHI-Nter (XhoI) CCG CTC GAG CCG TGG GCC GTG GTA GTC CGG SEQ ID NO: 532 F-Tat (NdeI) TAT GAT GTG CGG CCG TAA GAA ACG TCG CCA GCG SEQ ID NO: 533 TCG CCG TCC GCC GCA ATG CG R-Tat (NheI) CTA GCG CAT TGC GGC GGA CGG CGA CGC TGG SEQ ID NO: 534 CGA CGT TTC TTA CGG CCG CAC AGC A F-IpaH1.4 GTT TAA CTT TAA GAA GGA GAT ATA CAT ATG_ATT SEQ ID NO: 535 AAA TCA ACC AAT ATA CAG R-IpaH1.4 CTT ATC GTC GTC ATC CTT GTA ATC GCT AGC TGC SEQ ID NO: 536 GAT ATG ATT TGA GCC GTT TTC AGA CAA F-IpaH2.5/IpaH4.5 GTT TAA CTT TAA GAA GGA GAT ATA CAT ATG ATT SEQ ID NO: 537 AAA TCA ACA AAT ATA CAG GTA ATC GGT R-IpaH2.5 CTT ATC GTC GTC ATC CTT GTA ATC GCT AGC GGC SEQ ID NO: 538 CAG TAC CTC GTC AGT CAA CTG ACG GTA F-IpaH3 GTT TAA CTT TAA GAA GGA GAT ATA CAT ATG TTA SEQ ID NO: 539 CCG ATA AAT AAT AAC TTT TCA TTG TCC R-IpaH3 CTT ATC GTC GTC ATC CTT GTA ATC GCT AGC GTC SEQ ID NO: 540 AGC TGA CGG TAA ATC TGC TGT TAC AGT F-IpaH4.5 GTT TAA CTT TAA GAA GGA GAT ATA CAT ATG AAA SEQ ID NO: 541 CCG ATC AAC AAT CAT TCT TTT TTT CGT F-IpaH7.8 GTT TAA CTT TAA GAA GGA GAT ATA CAT ATG TTC SEQ ID NO: 542 TCT GTA AAT AAT ACA CAC TCA TCA GTT R-IpaH7.8 CTT ATC GTC GTC ATC CTT GTA ATC GCT AGC TGA SEQ ID NO: 543 ATG GTG CAG TCG TGA GCC GTT TTC AGA F-IpaH9.8 GTT TAA CTT TAA GAA GGA GAT ATA CAT ATG TTA SEQ ID NO: 544 CCG ATA AAT AAT AAC TTT TCA TTG CCC R-IpaH9.8 CTT ATC GTC GTC ATC CTT GTA ATC GCT AGC TGA SEQ ID NO: 545 ATG GTG CAG TTG TGA GCC GTT TTC AAA F-SspH2 GTT TAA CTT TAA GAA GGA GAT ATA CAT ATG CCC SEQ ID NO: 546 TTT CAT ATT GGA AGC GGA TGT CTT CCC R-SspH2 CTT ATC GTC GTC ATC CTT GTA ATC GCT AGC GTT SEQ ID NO: 547 ACG ACG CCA CTG AAC GTT CAG ATA GCT F-SlrP GTT TAA CTT TAA GAA GGA GAT ATA CAT ATG TTT SEQ ID NO: 548 AAT ATT ACT AAT ATA CAA TCT ACG GCA R-SlrP CTT ATC GTC GTC ATC CTT GTA ATC GCT AGC TCG SEQ ID NO: 549 CCA GTA GGC GCT CAT GAG CGA GCT CAC

(150) 3.6 Antibodies

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

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

(153) 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) Peroxidase (PO) conjugated Dianova 1:10000 goat monoclonal antibody (Hamburg) against mouse-IgG GAR-PO (WB) Peroxidase (PO) conjugated Dianova 1:10000 goat monoclonal antibody (Hamburg) against rabbit-IgG Hoechst (IF) Selective DNA dye Sigma Aldrich 33258 (DAPI) 1:1000 (Taufkirchen)

(154) 3.7 Kits

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

(156) 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 Reagents Thermo Scientific (Rockford, USA) CYTOTOX 96 ® Non-Radioactive Cytotoxicity Assay Promega

(157) References (Materials and Methods of Examples 1 to 5)

(158) Behrens, M. (1938), Hoppe-Seylers Z, 253, Pflügers Archly—European Journal of Physiology, 185.

(159) 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.

(160) 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.

(161) 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. Immun., 68(7), 4344-4348.

(162) 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.

(163) 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.

(164) 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.

(165) 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.

(166) 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.

(167) 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

(168) 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.

(169) 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.

(170) Cell Fractionation of Eukaryotic Cells

(171) 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).

(172) 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, Ü. (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, 4×1 sec, level 4, 4° C.) in order to permeabilize the cells. Subsequently, the suspension was centrifuged (108,000×g, 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 (108,000×g, 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 (108,000×g, 30 min, 4° C.) and the supernatant was taken as the membrane fraction (MF).

(173) Both the cytoplasmic and the membrane fraction were precipitated using trichloracetic acid.

(174) Subsequently, the samples were subjected to SDS-PAGE and analyzed by Western blotting.

(175) TABLE-US-00019 Acid Buffer Glycine in PBS, pH 2.0 62.5 mM 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

(176) Membranolysis Assay

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

(178) 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 μg/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.

(179) Lactate Dehydrogenase Assay

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

(181) 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 μl culture medium, the plate was centrifuged (400×g, 4 min, RT) and 50 μl 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 μl 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 μl were lysed by adding 5.5 μl Lysis Solutions (10×) for 30 min. Afterwards, 50 μl 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 μl 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.

(182) In Vitro Ubiquitination Assay

(183) 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.

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

(185) Reaction Mixture of In Vitro Ubiquitination Assay:

(186) TABLE-US-00020 Component Amount E1 0.5 μg E2 (UbcH5b) 2 μg Ubiquitin-HA 2 μg Putative E3 4 μg Ubiquitin reaction buffer ad to 40 μl

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

Example 7

Functional Domains of LPX Family Members

(188) 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.

(189) 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.

(190) 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

(191) SlrP

(192) SEQ ID NO: 10

(193) >gb|CP001363.1|:867285-869582 Salmonella enterica subsp. enterica serovar Typhimurium str. 14028S, complete genome

(194) SspH1

(195) SEQ ID NO: 11

(196) >gb|CP001363.1|:1332051-1334153 Salmonella enterica subsp. enterica serovar Typhimurium str. 14028S

(197) SspH2

(198) SEQ ID NO: 12

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

(200) IpaH1.4

(201) SEQ ID NO: 13

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

(203) IpaH2.5

(204) SEQ ID NO: 14

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

(206) IpaH3

(207) SEQ ID NO: 15

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

(209) IpaH4.5

(210) SEQ ID NO: 16

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

(212) IpaH7.8

(213) SEQ ID NO: 17

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

(215) IpaH9.8

(216) SEQ ID NO: 18

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

Example 9

Protein Transduction Domains Determined in Example 5

(218) IpaH1.4

(219) SEQ ID NO: 4

(220) >tr|Q9AJU5|Q9AJU5_SHIFL Putative uncharacterized protein ipaH1.4 OS=Shigella flexneri GN=ipaH1.4 PE=4 SV=1

(221) Predicted PTD Sequences:

(222) TABLE-US-00022 519 SQRVADRLKA SEQ ID NO: 361 371 RVALTWNNLRKTL SEQ ID NO: 367 520 QRVADRLKAS SEQ ID NO: 362 371 RVALTWNNLRKTLL SEQ ID NO: 368 521 RVADRLKASG SEQ ID NO: 363 370 DRVALTWNNLRKTLL SEQ ID NO: 369 520 QRVADRLKASG SEQ ID NO: 364 371 RVALTWNNLRKTLLV SEQ ID NO: 370 521 RVADRLKASGL SEQ ID NO: 365 520 QRVADRLKASGL SEQ ID NO: 366

(223) IpaH2.5

(224) SEQ ID NO: 5

(225) >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]

(226) Predicted PTD Sequences:

(227) TABLE-US-00023 519 SQRVADRLKA SEQ ID NO: 371 371 RVALTWNNLRKTL SEQ ID NO: 377 520 QRVADRLKAS SEQ ID NO: 372 371 RVALTWNNLRKTLL SEQ ID NO: 378 521 RVADRLKASG SEQ ID NO: 373 370 DRVALTWNNLRKTLL SEQ ID NO: 379 520 QRVADRLKASG SEQ ID NO: 374 371 RVALTWNNLRKTLLV SEQ ID NO: 380 521 RVADRLKASGL SEQ ID NO: 375 520 QRVADRLKASGL SEQ ID NO: 376

(228) IpaH3

(229) SEQ ID NO: 6

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

(231) Predicted PTD Sequences:

(232) TABLE-US-00024 511 PQRVADRLKA SEQ ID NO: 381 513 RVADRLKASGL SEQ ID NO: 386 512 QRVADRLKAS SEQ ID NO: 382 363 RVALTWNNLRKTL SEQ ID NO: 387 513 RVADRLKASG SEQ ID NO: 383 511 PQRVADRLKASGL SEQ ID NO: 388 511 PQRVADRLKAS SEQ ID NO: 384 363 RVALTWNNLRKTLL SEQ ID NO: 389 512 QRVADRLKASG SEQ ID NO: 385 362 DRVALTWNNLRKTLL SEQ ID NO: 390 363 RVALTWNNLRKTLLV SEQ ID NO: 391

(233) IpaH4.5

(234) SEQ ID NO: 7

(235) >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]

(236) Predicted PTD Sequences:

(237) TABLE-US-00025  48 NRIQAVRLLK SEQ ID NO: 392 204 SLKYLKVGENQLRRL SEQ ID NO: 437  49 RIQAVRLLKI SEQ ID NO: 393 205 LKYLKVGENQLRRLS SEQ ID NO: 438  50 IQAVRLLKIC SEQ ID NO: 394 208 LKVGENQLRRLSRLP SEQ ID NO: 439  51 QAVRLLKICL SEQ ID NO: 395 204 SLKYLKVGENQLRRL SEQ ID NO: 440  54 RLLKICLDTR SEQ ID NO: 396 205 LKYLKVGENQLRRLS SEQ ID NO: 441 208 LKVGENQLRR SEQ ID NO: 397 208 LKVGENQLRRLSRLP SEQ ID NO: 442 209 KVGENQLRRL SEQ ID NO: 398 214 QLRRLSRLPQELLAL SEQ ID NO: 443 530 SQRVADRLKA SEQ ID NO: 399 381 DRVALTWNNLRKTLL SEQ ID NO: 444 531 QRVADRLKAS SEQ ID NO: 400 382 RVALTWNNLRKTLLV SEQ ID NO: 445 532 RVADRLKASG SEQ ID NO: 401  48 NRIQAVRLLKICLDTR SEQ ID NO: 446  47 ENRIQAVRLLK SEQ ID NO: 402  49 RIQAVRLLKICLDTRE SEQ ID NO: 447  48 NRIQAVRLLKI SEQ ID NO: 403 203 QSLKYLKVGENQLRRL SEQ ID NO: 448  49 RIQAVRLLKIC SEQ ID NO: 404 208 LKVGENQLRRLSRLPQ SEQ ID NO: 449 208 LKVGENQLRRL SEQ ID NO: 405 213 NQLRRLSRLPQELLAL SEQ ID NO: 450 531 QRVADRLKASG SEQ ID NO: 406  47 ENRIQAVRLLKICLDTR SEQ ID NO: 451 532 RVADRLKASGL SEQ ID NO: 407  48 NRIQAVRLLKICLDTRE SEQ ID NO: 452  46 TENRIQAVRLLK SEQ ID NO: 408 205 LKYLKVGENQLRRLSRL SEQ ID NO: 453  47 ENRIQAVRLLKI SEQ ID NO: 409 204 SLKYLKVGENQLRRLSRL SEQ ID NO: 454  48 NRIQAVRLLKIC SEQ ID NO: 410  39 WAREGTTTENRIQAVRLLK SEQ ID NO: 455  49 RIQAVRLLKICL SEQ ID NO: 411 203 QSLKYLKVGENQLRRLSRL SEQ ID NO: 456  52 AVRLLKICLDTR SEQ ID NO: 412 208 LKVGENQLRRLSRLPQELL SEQ ID NO: 457 216 RRLSRLPQELLA SEQ ID NO: 413 209 KVGENQLRRLSRLPQELLA SEQ ID NO: 458 531 QRVADRLKASGL SEQ ID NO: 414  39 WAREGTTTENRIQAVRLLKI SEQ ID NO: 459  46 TENRIQAVRLLKI SEQ ID NO: 415 208 LKVGENQLRRLSRLPQELLA SEQ ID NO: 460  47 ENRIQAVRLLKIC SEQ ID NO: 416 209 KVGENQLRRLSRLPQELLAL SEQ ID NO: 461  48 NRIQAVRLLKICL SEQ ID NO: 417 205 LKYLKVGENQLRRLSRLPQEL SEQ ID NO: 462  49 RIQAVRLLKICLD SEQ ID NO: 418 206 KYLKVGENQLRRLSRLPQELL SEQ ID NO: 463  51 QAVRLLKICLDTR SEQ ID NO: 419 208 LKVGENQLRRLSRLPQELLAL SEQ ID NO: 464 208 LKVGENQLRRLSR SEQ ID NO: 420  54 RLLKICLDTREPVLNLSLLKLR SEQ ID NO: 465 209 KVGENQLRRLSRL SEQ ID NO: 421 205 LKYLKVGENQLRRLSRLPQELL SEQ ID NO: 466 215 LRRLSRLPQELLA SEQ ID NO: 422 206 KYLKVGENQLRRLSRLPQELLA SEQ ID NO: 467 216 RRLSRLPQELLAL SEQ ID NO: 423 207 YLKVGENQLRRLSRLPQELLAL SEQ ID NO: 468 382 RVALTWNNLRKTL SEQ ID NO: 424 205 LKYLKVGENQLRRLSRLPQELLA SEQ ID NO: 469  47 ENRIQAVRLLKICL SEQ ID NO: 425 206 KYLKVGENQLRRLSRLPQELLAL SEQ ID NO: 470  48 NRIQAVRLLKICLD SEQ ID NO: 426 204 SLKYLKVGENQLRRLSRLPQELLA SEQ ID NO: 471  49 RIQAVRLLKICLDT SEQ ID NO: 427 205 LKYLKVGENQLRRLSRLPQELLAL SEQ ID NO: 472  50 IQAVRLLKICLDTR SEQ ID NO: 428 206 KYLKVGENQLRRLSRLPQELLALD SEQ ID NO: 473 205 LKYLKVGENQLRRL SEQ ID NO: 429  51 QAVRLLKICLDTREPVLNLSLLKLR SEQ ID NO: 474 208 LKVGENQLRRLSRL SEQ ID NO: 430 203 QSLKYLKVGENQLRRLSRLPQELLA SEQ ID NO: 475 214 QLRRLSRLPQELLA SEQ ID NO: 431 204 SLKYLKVGENQLRRLSRLPQELLAL SEQ ID NO: 476 215 LRRLSRLPQELLAL SEQ ID NO: 432 205 LKYLKVGENQLRRLSRLPQELLALD SEQ ID NO: 477 382 RVALTWNNLRKTLL SEQ ID NO: 433  49 RIQAVRLLKICLDTREPVLNLSLLKL SEQ ID NO: 478  46 TENRIQAVRLLKICL SEQ ID NO: 434  50 IQAVRLLKICLDTREPVLNLSLLKLR SEQ ID NO: 479  48 NRIQAVRLLKICLDT SEQ ID NO: 435 203 QSLKYLKVGENQLRRLSRLPQELLAL SEQ ID NO: 480  49 RIQAVRLLKICLDTR SEQ ID NO: 436  49 RIQAVRLLKICLDTREPVLNLSLLKLR SEQ ID NO: 481  48 NRIQAVRLLKICLDTREPVLNLSLLKLR SEQ ID NO: 482  49 RIQAVRLLKICLDTREPVLNLSLLKLRS SEQ ID NO: 483  47 ENRIQAVRLLKICLDTREPVLNLSLLKLR SEQ ID NO: 484  49 RIQAVRLLKICLDTREPVLNLSLLKLRSL SEQ ID NO: 485  48 NRIQAVRLLKICLDTREPVLNLSLLKLRSL SEQ ID NO: 486

(238) IpaH7.8

(239) SEQ ID NO: 8

(240) >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]

(241) Predicted PTD Sequences:

(242) TABLE-US-00026 238 TRVLQSLQRL SEQ ID NO: 487 510 RVADRLKASGL SEQ ID NO: 493 239 RVLQSLQRLT SEQ ID NO: 488 509 QRVADRLKASGL SEQ ID NO: 494 508 SQRVADRLKA SEQ ID NO: 489 360 RVALTWNNLRKTL SEQ ID NO: 495 509 QRVADRLKAS SEQ ID NO: 490 360 RVALTWNNLRKTLL SEQ ID NO: 496 510 RVADRLKASG SEQ ID NO: 491 359 DRVALTWNNLRKTLL SEQ ID NO: 497 509 QRVADRLKASG SEQ ID NO: 492 360 RVALTWNNLRKTLLV SEQ ID NO: 498

(243) IpaH9.8

(244) SEQ ID NO: 9

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

(246) Predicted PTD Sequences:

(247) TABLE-US-00027 155 LPQALKNLRA SEQ ID NO: 499 154 SLPQALKNLRATR SEQ ID NO: 515 157 QALKNLRATR SEQ ID NO: 500 155 LPQALKNLRATRN SEQ ID NO: 516 158 ALKNLRATRN SEQ ID NO: 501 158 ALKNLRATRNFLT SEQ ID NO: 517 488 PQRVADRLKA SEQ ID NO: 502 340 RVALTWNNLRKTL SEQ ID NO: 518 489 QRVADRLKAS SEQ ID NO: 503 488 PQRVADRLKASGL SEQ ID NO: 519 490 RVADRLKASG SEQ ID NO: 504 153 PSLPQALKNLRATR SEQ ID NO: 520 156 PQALKNLRATR SEQ ID NO: 505 154 SLPQALKNLRATRN SEQ ID NO: 521 488 PQRVADRLKAS SEQ ID NO: 506 157 QALKNLRATRNFLT SEQ ID NO: 522 489 QRVADRLKASG SEQ ID NO: 507 340 RVALTWNNLRKTLL SEQ ID NO: 523 490 RVADRLKASGL SEQ ID NO: 508 152 LPSLPQALKNLRATR SEQ ID NO: 524 155 LPQALKNLRATR SEQ ID NO: 509 153 PSLPQALKNLRATRN SEQ ID NO: 525 489 QRVADRLKASGL SEQ ID NO: 510 155 LPQALKNLRATRNFL SEQ ID NO: 526 151 SLPSLPQALKNLRATR SEQ ID NO: 511 156 PQALKNLRATRNFLT SEQ ID NO: 527 152 LPSLPQALKNLRATRN SEQ ID NO: 512 339 DRVALTWNNLRKTLL SEQ ID NO: 528 154 SLPQALKNLRATRNFL SEQ ID NO: 513 340 RVALTWNNLRKTLLV SEQ ID NO: 529 155 LPQALKNLRATRNFLT SEQ ID NO: 514

(248) SspH1

(249) SEQ ID NO: 2

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

(251) Predicted PTD Sequences:

(252) TABLE-US-00028 372 LSVRTLQALR SEQ ID NO: 152 693 LTARWRLN SEQ ID NO: 155 70 ARLKALTFPA SEQ ID NO: 153 319 LQKLWAYNNRL SEQ ID NO: 156 626 RFNALREKQI SEQ ID NO: 154 476 ALRAKTFAMAT SEQ ID NO: 157

(253) SspH2

(254) SEQ ID NO: 3

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

(256) Predicted PTD Sequences:

(257) TABLE-US-00029 185 SRGRAAVVQK SEQ ID NO: 158 627 QIAREKVRTL SEQ ID NO: 260 186 RGRAAVVQKM SEQ ID NO: 159 628 IAREKVRTLA SEQ ID NO: 261 187 GRAAVVQKMR SEQ ID NO: 160 629 AREKVRTLAL SEQ ID NO: 262 188 RAAVVQKMRA SEQ ID NO: 161 630 REKVRTLALV SEQ ID NO: 263 187 GRAAVVQKMRA SEQ ID NO: 162 648 YQNKLKKSLG SEQ ID NO: 264 188 RAAVVQKMRAC SEQ ID NO: 163 649 QNKLKKSLGL SEQ ID NO: 265 620 FRLGKLEQIAR SEQ ID NO: 164 709 RKAPERVNAL SEQ ID NO: 266 624 KLEQIAREKVR SEQ ID NO: 165 710 KAPERVNALR SEQ ID NO: 267 627 QIAREKVRTLA SEQ ID NO: 166 646 LAYQNKLKKSL SEQ ID NO: 268 628 IAREKVRTLAL SEQ ID NO: 167 647 AYQNKLKKSLG SEQ ID NO: 269 629 AREKVRTLALV SEQ ID NO: 168 714 RVNALREKQIS SEQ ID NO: 270 185 SRGRAAVVQKMR SEQ ID NO: 169  12 ATISNRRIYRIA SEQ ID NO: 271 186 RGRAAVVQKMRA SEQ ID NO: 170 619 MFRLGKLEQIAR SEQ ID NO: 272 188 RAAVVQKMRACL SEQ ID NO: 171 621 RLGKLEQIAREK SEQ ID NO: 273 177 RRAAPAEESRGRA SEQ ID NO: 172 623 GKLEQIAREKVR SEQ ID NO: 274 185 SRGRAAVVQKMRA SEQ ID NO: 173 627 QIAREKVRTLAL SEQ ID NO: 275 186 RGRAAVVQKMRAC SEQ ID NO: 174 628 IAREKVRTLALV SEQ ID NO: 276 188 RAAVVQKMRACLN SEQ ID NO: 175 646 LAYQNKLKKSLG SEQ ID NO: 277 620 FRLGKLEQIAREK SEQ ID NO: 176 647 AYQNKLKKSLGL SEQ ID NO: 278 621 RLGKLEQIAREKV SEQ ID NO: 177 751 RTIGARAMESAKK SEQ ID NO: 279 622 LGKLEQIAREKVR SEQ ID NO: 178 177 RRAAPAEESRGRAA SEQ ID NO: 280 627 QIAREKVRTLALV SEQ ID NO: 179 184 ESRGRAAVVQKMRA SEQ ID NO: 281 645 WLAYQNKLKKSLG SEQ ID NO: 180 186 RGRAAVVQKMRACL SEQ ID NO: 282 646 LAYQNKLKKSLGL SEQ ID NO: 181 619 MFRLGKLEQIAREK SEQ ID NO: 283 705 RVLERKAPERVNAL SEQ ID NO: 182 620 FRLGKLEQIAREKV SEQ ID NO: 284 706 VLERKAPERVNALR SEQ ID NO: 183 621 RLGKLEQIAREKVR SEQ ID NO: 285 710 KAPERVNALREKQI SEQ ID NO: 184 644 VWLAYQNKLKKSLG SEQ ID NO: 286 751 RTIGARAMESAKKT SEQ ID NO: 185 645 WLAYQNKLKKSLGL SEQ ID NO: 287 616 GREMFRLGKLEQIAR SEQ ID NO: 186 177 RRAAPAEESRGRAAV SEQ ID NO: 288 619 MFRLGKLEQIAREKV SEQ ID NO: 187 185 SRGRAAVVQKMRACL SEQ ID NO: 289 620 FRLGKLEQIAREKVR SEQ ID NO: 188 186 RGRAAVVQKMRACLN SEQ ID NO: 290 751 RTIGARAMESAKKTF SEQ ID NO: 189 176 WRRAAPAEESRGRAAV SEQ ID NO: 291 621 RLGKLEQIAREKVRT SEQ ID NO: 190 177 RRAAPAEESRGRAAVV SEQ ID NO: 292 644 VWLAYQNKLKKSLGL SEQ ID NO: 191 182 AEESRGRAAVVQKMRA SEQ ID NO: 293 645 WLAYQNKLKKSLGLT SEQ ID NO: 192 186 RGRAAVVQKMRACLNN SEQ ID NO: 294 706 VLERKAPERVNALREK SEQ ID NO: 193 619 MFRLGKLEQIAREKVR SEQ ID NO: 295 751 RTIGARAMESAKKTFL SEQ ID NO: 194 620 FRLGKLEQIAREKVRT SEQ ID NO: 296 176 WRRAAPAEESRGRAAVV SEQ ID NO: 195 621 RLGKLEQIAREKVRTL SEQ ID NO: 297 177 RRAAPAEESRGRAAVVQ SEQ ID NO: 196 609 LAALVATGREMFRLGKL SEQ ID NO: 298 178 RAAPAEESRGRAAVVQK SEQ ID NO: 197 614 ATGREMFRLGKLEQIAR SEQ ID NO: 299 706 VLERKAPERVNALREKQ SEQ ID NO: 198 616 GREMFRLGKLEQIAREK SEQ ID NO: 300 176 WRRAAPAEESRGRAAVVQ SEQ ID NO: 199 617 REMFRLGKLEQIAREKV SEQ ID NO: 301 177 RRAAPAEESRGRAAVVQK SEQ ID NO: 200 618 EMFRLGKLEQIAREKVR SEQ ID NO: 302 615 TGREMFRLGKLEQIAREK SEQ ID NO: 201 619 MFRLGKLEQIAREKVRT SEQ ID NO: 303 619 MFRLGKLEQIAREKVRTL SEQ ID NO: 202 620 FRLGKLEQIAREKVRTL SEQ ID NO: 304 620 FRLGKLEQIAREKVRTLA SEQ ID NO: 203 621 RLGKLEQIAREKVRTLA SEQ ID NO: 305 621 RLGKLEQIAREKVRTLAL SEQ ID NO: 204 706 VLERKAPERVNALREKQI SEQ ID NO: 306 175 AWRRAAPAEESRGRAAVVQ SEQ ID NO: 205 707 LERKAPERVNALREKQIS SEQ ID NO: 307 176 WRRAAPAEESRGRAAVVQK SEQ ID NO: 206 617 REMFRLGKLEQIAREKVRT SEQ ID NO: 308 177 RRAAPAEESRGRAAVVQKM SEQ ID NO: 207 618 EMFRLGKLEQIAREKVRTL SEQ ID NO: 309 178 RAAPAEESRGRAAVVQKMR SEQ ID NO: 208 619 MFRLGKLEQIAREKVRTLA SEQ ID NO: 310 175 AWRRAAPAEESRGRAAVVQK SEQ ID NO: 209 620 FRLGKLEQIAREKVRTLAL SEQ ID NO: 311 176 WRRAAPAEESRGRAAVVQKM SEQ ID NO: 210 621 RLGKLEQIAREKVRTLALV SEQ ID NO: 312 177 RRAAPAEESRGRAAVVQKMR SEQ ID NO: 211 611 ALVATGREMFRLGKLEQIAR SEQ ID NO: 313 178 RAAPAEESRGRAAVVQKMRA SEQ ID NO: 212 615 TGREMFRLGKLEQIAREKVR SEQ ID NO: 314 705 RVLERKAPERVNALREKQIS SEQ ID NO: 213 616 GREMFRLGKLEQIAREKVRT SEQ ID NO: 315 751 RTIGARAMESAKKTFLDGLR SEQ ID NO: 214 617 REMFRLGKLEQIAREKVRTL SEQ ID NO: 316 174 SAWRRAAPAEESRGRAAVVQK SEQ ID NO: 215 618 EMFRLGKLEQIAREKVRTLA SEQ ID NO: 317 175 AWRRAAPAEESRGRAAVVQKM SEQ ID NO: 216 619 MFRLGKLEQIAREKVRTLAL SEQ ID NO: 318 176 WRRAAPAEESRGRAAVVQKMR SEQ ID NO: 217 620 FRLGKLEQIAREKVRTLALV SEQ ID NO: 319 177 RRAAPAEESRGRAAVVQKMRA SEQ ID NO: 218 621 RLGKLEQIAREKVRTLALVD SEQ ID NO: 320 610 AALVATGREMFRLGKLEQIAR SEQ ID NO: 219 705 RVLERKAPERVNALREKQIS SEQ ID NO: 321 612 LVATGREMFRLGKLEQIAREK SEQ ID NO: 220 751 RTIGARAMESAKKTFLDGLR SEQ ID NO: 322 614 ATGREMFRLGKLEQIAREKVR SEQ ID NO: 221 174 SAWRRAAPAEESRGRAAVVQK SEQ ID NO: 323 616 GREMFRLGKLEQIAREKVRTL SEQ ID NO: 222 175 AWRRAAPAEESRGRAAVVQKM SEQ ID NO: 324 617 REMFRLGKLEQIAREKVRTLA SEQ ID NO: 223 176 WRRAAPAEESRGRAAVVQKMR SEQ ID NO: 325 618 EMFRLGKLEQIAREKVRTLAL SEQ ID NO: 224 177 RRAAPAEESRGRAAVVQKMRA SEQ ID NO: 326 619 MFRLGKLEQIAREKVRTLALV SEQ ID NO: 225 610 AALVATGREMFRLGKLEQIAR SEQ ID NO: 327 620 FRLGKLEQIAREKVRTLALVD SEQ ID NO: 226 612 LVATGREMFRLGKLEQIAREK SEQ ID NO: 328 621 RLGKLEQIAREKVRTLALVD SEQ ID NO: 227 614 ATGREMFRLGKLEQIAREKVR SEQ ID NO: 329 175 AWRRAAPAEESRGRAAVVQKMR SEQ ID NO: 228 614 ATGREMFRLGKLEQIAREKVR SEQ ID NO: 330 176 WRRAAPAEESRGRAAVVQKMRA SEQ ID NO: 229 616 GREMFRLGKLEQIAREKVRTL SEQ ID NO: 331 177 RRAAPAEESRGRAAVVQKMRAC SEQ ID NO: 230 617 REMFRLGKLEQIAREKVRTLA SEQ ID NO: 332 609 LAALVATGREMFRLGKLEQIAR SEQ ID NO: 231 618 EMFRLGKLEQIAREKVRTLAL SEQ ID NO: 333 611 ALVATGREMFRLGKLEQIAREK SEQ ID NO: 232 619 MFRLGKLEQIAREKVRTLALV SEQ ID NO: 334 614 ATGREMFRLGKLEQIAREKVRT SEQ ID NO: 233 620 FRLGKLEQIAREKVRTLALVD SEQ ID NO: 335 615 TGREMFRLGKLEQIAREKVRTL SEQ ID NO: 234 172 VWSAWRRAAPAEESRGRAAVVQK SEQ ID NO: 336 616 GREMFRLGKLEQIAREKVRTLA SEQ ID NO: 235 174 SAWRRAAPAEESRGRAAVVQKMR SEQ ID NO: 337 617 REMFRLGKLEQIAREKVRTLAL SEQ ID NO: 236 175 AWRRAAPAEESRGRAAVVQKMRA SEQ ID NO: 338 618 EMFRLGKLEQIAREKVRTLALV SEQ ID NO: 237 176 WRRAAPAEESRGRAAVVQKMRAC SEQ ID NO: 339 619 MFRLGKLEQIAREKVRTLALVD SEQ ID NO: 238 177 RRAAPAEESRGRAAVVQKMRACL SEQ ID NO: 340 173 WSAWRRAAPAEESRGRAAVVQKMR SEQ ID NO: 239 610 AALVATGREMFRLGKLEQIAREK SEQ ID NO: 341 174 SAWRRAAPAEESRGRAAVVQKMRA SEQ ID NO: 240 612 LVATGREMFRLGKLEQIAREKVR SEQ ID NO: 342 176 WRRAAPAEESRGRAAVVQKMRACL SEQ ID NO: 241 614 ATGREMFRLGKLEQIAREKVRTL SEQ ID NO: 343 177 RRAAPAEESRGRAAVVQKMRACLN SEQ ID NO: 242 615 TGREMFRLGKLEQIAREKVRTLA SEQ ID NO: 344 609 LAALVATGREMFRLGKLEQIAREK SEQ ID NO: 243 616 GREMFRLGKLEQIAREKVRTLAL SEQ ID NO: 345 610 AALVATGREMFRLGKLEQIAREKV SEQ ID NO: 244 617 REMFRLGKLEQIAREKVRTLALV SEQ ID NO: 346 611 ALVATGREMFRLGKLEQIAREKVR SEQ ID NO: 245 171 AVWSAWRRAAPAEESRGRAAVVQKMR SEQ ID NO: 347 614 ATGREMFRLGKLEQIAREKVRTLA SEQ ID NO: 246 172 VWSAWRRAAPAEESRGRAAVVQKMRA SEQ ID NO: 348 615 TGREMFRLGKLEQIAREKVRTLAL SEQ ID NO: 247 609 LAALVATGREMFRLGKLEQIAREKVR SEQ ID NO: 349 616 GREMFRLGKLEQIAREKVRTLALV SEQ ID NO: 248 610 AALVATGREMFRLGKLEQIAREKVRT SEQ ID NO: 350 617 REMFRLGKLEQIAREKVRTLALVD SEQ ID NO: 249 611 ALVATGREMFRLGKLEQIAREKVRTL SEQ ID NO: 351 608 DLAALVATGREMFRLGKLEQIAREKVR SEQ ID NO: 250 612 LVATGREMFRLGKLEQIAREKVRTLA SEQ ID NO: 352 609 LAALVATGREMFRLGKLEQIAREKVRT SEQ ID NO: 251 613 VATGREMFRLGKLEQIAREKVRTLAL SEQ ID NO: 353 610 AALVATGREMFRLGKLEQIAREKVRTL SEQ ID NO: 252 614 ATGREMFRLGKLEQIAREKVRTLALV SEQ ID NO: 354 611 ALVATGREMFRLGKLEQIAREKVRTLA SEQ ID NO: 253 171 AVWSAWRRAAPAEESRGRAAVVQKMRA SEQ ID NO: 355 612 LVATGREMFRLGKLEQIAREKVRTLAL SEQ ID NO: 254 607 NDLAALVATGREMFRLGKLEQIAREKVR SEQ ID NO: 356 614 ATGREMFRLGKLEQIAREKVRTLALVD SEQ ID NO: 255 608 DLAALVATGREMFRLGKLEQIAREKVRT SEQ ID NO: 357 607 NDLAALVATGREMFRLGKLEQIAREKVRTL SEQ ID NO: 256 609 LAALVATGREMFRLGKLEQIAREKVRTL SEQ ID NO: 358 608 DLAALVATGREMFRLGKLEQIAREKVRTLA SEQ ID NO: 257 610 AALVATGREMFRLGKLEQIAREKVRTLA SEQ ID NO: 359 609 LAALVATGREMFRLGKLEQIAREKVRTLAL SEQ ID NO: 258 611 ALVATGREMFRLGKLEQIAREKVRTLAL SEQ ID NO: 360 610 AALVATGREMFRLGKLEQIAREKVRTLALV SEQ ID NO: 259

(258) SlrP

(259) SEQ ID NO: 1

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

(261) Predicted PTD Sequences:

(262) TABLE-US-00030 593 SLAREKVKRL SEQ ID NO: 66 173 RDCLKNNKTELRLKILG SEQ ID NO: 110 175 CLKNNKTELRLKI SEQ ID NO: 67 175 CLKNNKTELRLKILGLT SEQ ID NO: 111 176 LKNNKTELRLKIL SEQ ID NO: 68 172 MRDCLKNNKTELRLKILG SEQ ID NO: 112 177 KNNKTELRLKILG SEQ ID NO: 69 173 RDCLKNNKTELRLKILGL SEQ ID NO: 113 175 CLKNNKTELRLKIL SEQ ID NO: 70 386 LPAALQIMQASRNNLVRL SEQ ID NO: 114 176 LKNNKTELRLKILG SEQ ID NO: 71 585 IFRLEQIESLAREKVKRL SEQ ID NO: 115 177 KNNKTELRLKILGL SEQ ID NO: 72 171 RMRDCLKNNKTELRLKILG SEQ ID NO: 116 389 ALQIMQASRNNLVR SEQ ID NO: 73 172 MRDCLKNNKTELRLKILGL SEQ ID NO: 117 388 AALQIMQASRNNLVR SEQ ID NO: 74 173 RDCLKNNKTELRLKILGLT SEQ ID NO: 118 389 ALQIMQASRNNLVRL SEQ ID NO: 75 171 RMRDCLKNNKTELRLKILGL SEQ ID NO: 119 175 CLKNNKTELRLKILGL SEQ ID NO: 76 172 MRDCLKNNKTELRLKILGLT SEQ ID NO: 120 176 LKNNKTELRLKILGLT SEQ ID NO: 77 173 RDCLKNNKTELRLKILGLTT SEQ ID NO: 121 388 AALQIMQASRNNLVRL SEQ ID NO: 78 168 AVQRMRDCLKNNKTELRLKIL SEQ ID NO: 122 587 RLEQIESLAREKVKRL SEQ ID NO: 79 169 VQRMRDCLKNNKTELRLKILG SEQ ID NO: 123 581 AGREIFRLEQIESLAREKVKR SEQ ID NO: 80 170 QRMRDCLKNNKTELRLKILGL SEQ ID NO: 124 582 GREIFRLEQIESLAREKVKRL SEQ ID NO: 81 171 RMRDCLKNNKTELRLKILGLT SEQ ID NO: 125 167 EAVQRMRDCLKNNKTELRLKIL SEQ ID NO: 82 173 RDCLKNNKTELRLKILGLTTI SEQ ID NO: 126 168 AVQRMRDCLKNNKTELRLKILG SEQ ID NO: 83 167 EAVQRMRDCLKNNKTELRLKILG SEQ ID NO: 127 169 VQRMRDCLKNNKTELRLKILGL SEQ ID NO: 84 168 AVQRMRDCLKNNKTELRLKILGL SEQ ID NO: 128 170 QRMRDCLKNNKTELRLKILGLT SEQ ID NO: 85 169 VQRMRDCLKNNKTELRLKILGLT SEQ ID NO: 129 171 RMRDCLKNNKTELRLKILGLTT SEQ ID NO: 86 170 QRMRDCLKNNKTELRLKILGLTT SEQ ID NO: 130 580 MAGREIFRLEQIESLAREKVKR SEQ ID NO: 87 171 RMRDCLKNNKTELRLKILGLTTI SEQ ID NO: 131 581 AGREIFRLEQIESLAREKVKRL SEQ ID NO: 88 579 IMAGREIFRLEQIESLAREKVKR SEQ ID NO: 132 167 EAVQRMRDCLKNNKTELRLKILGL SEQ ID NO: 89 580 MAGREIFRLEQIESLAREKVKRL SEQ ID NO: 133 168 AVQRMRDCLKNNKTELRLKILGLT SEQ ID NO: 90 581 AGREIFRLEQIESLAREKVKRLF SEQ ID NO: 134 170 QRMRDCLKNNKTELRLKILGLTTI SEQ ID NO: 91 167 EAVQRMRDCLKNNKTELRLKILGL SEQ ID NO: 135 578 LIMAGREIFRLEQIESLAREKVKR SEQ ID NO: 92 168 AVQRMRDCLKNNKTELRLKILGLT SEQ ID NO: 136 579 IMAGREIFRLEQIESLAREKVKRL SEQ ID NO: 93 165 REEAVQRMRDCLKNNKTELRLKILG SEQ ID NO: 137 578 LIMAGREIFRLEQIESLAREKVKRL SEQ ID NO: 94 167 EAVQRMRDCLKNNKTELRLKILGLT SEQ ID NO: 138 164 NREEAVQRMRDCLKNNKTELRLKILG SEQ ID NO: 95 168 AVQRMRDCLKNNKTELRLKILGLTT SEQ ID NO: 139 165 REEAVQRMRDCLKNNKTELRLKILGL SEQ ID NO: 96 171 RMRDCLKNNKTELRLKILGLTTIPA SEQ ID NO: 140 168 AVQRMRDCLKNNKTELRLKILGLTTI SEQ ID NO: 97 162 AANREEAVQRMRDCLKNNKTELRLKIL SEQ ID NO: 141 161 EAANREEAVQRMRDCLKNNKTELRLKIL SEQ ID NO: 98 163 ANREEAVQRMRDCLKNNKTELRLKILG SEQ ID NO: 142 162 AANREEAVQRMRDCLKNNKTELRLKILG SEQ ID NO: 99 164 NREEAVQRMRDCLKNNKTELRLKILGL SEQ ID NO: 143 163 ANREEAVQRMRDCLKNNKTELRLKILGL SEQ ID NO: 100 165 REEAVQRMRDCLKNNKTELRLKILGLT SEQ ID NO: 144 164 NREEAVQRMRDCLKNNKTELRLKILGLT SEQ ID NO: 101 157 APAKEAANREEAVQRMRDCLKNNKTELRLK SEQ ID NO: 145 165 REEAVQRMRDCLKNNKTELRLKILGLTT SEQ ID NO: 102 158 PAKEAANREEAVQRMRDCLKNNKTELRLKI SEQ ID NO: 146 168 AVQRMRDCLKNNKTELRLKILGLTTIPA SEQ ID NO: 103 159 AKEAANREEAVQRMRDCLKNNKTELRLKIL SEQ ID NO: 147 157 APAKEAANREEAVQRMRDCLKNNKTELRL SEQ ID NO: 104 160 KEAANREEAVQRMRDCLKNNKTELRLKILG SEQ ID NO: 148 159 AKEAANREEAVQRMRDCLKNNKTELRLKI SEQ ID NO: 105 161 EAANREEAVQRMRDCLKNNKTELRLKILGL SEQ ID NO: 149 161 EAANREEAVQRMRDCLKNNKTELRLKILG SEQ ID NO: 106 162 AANREEAVQRMRDCLKNNKTELRLKILGLT SEQ ID NO: 150 162 AANREEAVQRMRDCLKNNKTELRLKILGL SEQ ID NO: 107 163 ANREEAVQRMRDCLKNNKTELRLKILGLTT SEQ ID NO: 151 163 ANREEAVQRMRDCLKNNKTELRLKILGLT SEQ ID NO: 108 165 REEAVQRMRDCLKNNKTELRLKILGLTTI SEQ ID NO: 109

Example 13

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

(263) 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.

(264) 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 α-Helices.

(265) 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.

(266) The inventors could show that the SspH1 effector protein of Salmonella typhimurium 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 IL1β stimulated cells.

(267) 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

(268) Group 1:

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

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

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

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

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

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

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

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

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

(278) Group 2:

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

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

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

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

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

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

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

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

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

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

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

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

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

(292) Group 3:

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

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

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

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

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

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

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

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

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

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

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

(304) Group 4:

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

(306) LRR1 (identification number 1): AA92-113 of SEQ ID NO: 4.

(307) LRR2 (identification number 2): AA132-153 of SEQ ID NO: 4.

(308) LRR3 (identification number 3): AA172-191 of SEQ ID NO: 4.

(309) LRR4 (identification number 4): AA192-213 of SEQ ID NO: 4.

(310) Group 5:

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

(312) LRR1 (identification number 1): AA92-113 of SEQ ID NO: 5

(313) LRR2 (identification number 2): AA132-153 of SEQ ID NO: 5

(314) LRR3 (identification number 3): AA172-191 of SEQ ID NO: 5

(315) LRR4 (identification number 4): AA192-213 of SEQ ID NO: 5

(316) Group 6:

(317) Predicted leucine-rich repeats of IpaH3, wherein IpaH3 has the amino acid sequence indicated as SEQ ID NO: 6.

(318) LRR1 (identification number 1): AA80-99 of SEQ ID NO: 6

(319) LRR2 (identification number 2): AA100-121 of SEQ ID NO: 6

(320) LRR3 (identification number 3): AA140-161 of SEQ ID NO: 6

(321) LRR4 (identification number 4): AA162-179 of SEQ ID NO: 6

(322) LRR5 (identification number 5): AA180-201 of SEQ ID NO: 6

(323) LRR6 (identification number 6): AA220-241 of SEQ ID NO: 6

(324) Group 7:

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

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

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

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

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

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

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

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

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

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

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

(336) Group 8:

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

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

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

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

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

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

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

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

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

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

(347) Group 9:

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

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

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

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

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

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

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

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

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

Example 15

(357) 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; Rüter et al., 2010). HeLa cells which were grown to 80% confluence were incubated with the recombinant proteins (25 μg/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 α-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 3× FLAG-tags were chosen for this assay due to their improved detectability (Terpe, 2003).

(358) 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

(359) 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.

(360) 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.

(361) 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.

(362) 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

(363) HeLa cells were with endocytic inhibitors cytochalasin D (2.5 μM), amiloride (3 mM), filipin (7.5 μM), nocodazole (20 μM), dynasore (80 μM), methyl-β-cyclodextrin (MβCD) (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.

(364) 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-β-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

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

(366) HeLa cells were co-incubated with 1 μg/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.

(367) 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

(368) 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 μg/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.

(369) 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 μg/ml or 50 μg/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.

(370) 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

(371) 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 μl reaction mixture containing ubiquitin reaction buffer, 2 μg of HA-tagged ubiquitin, 0.5 μg of E1 and 2 mg of E2 (UbcH5b) in the presence or absence of 4 μg 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 4× SDS sample buffer without DTT.

(372) 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.

(373) 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.

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