Hyperblebbing <i>Shigella </i>strains

Abstract

Hyperblebbing Shigella strains are generated by disrupting one or more components of the Tol-Pal system. The blebs from these strains are useful immunogens for vaccination. The individual proteins found in these blebs can also be used as immunogens.

Claims

1. A Shigella bacterium which expresses TolA, TolB, TolQ, and Pal proteins, does not express TolR protein, and does not express a native Shigella lipopolysaccharide, wherein the bacterium is Shigella sonnei or Shigella flexneri, and, during growth in culture medium, releases greater quantities of outer membrane blebs into the medium than the same bacterium expressing all of TolA, TolB, TolQ, TolR, and Pal proteins.

2. The Shigella bacterium of claim 1, which is a ΔtolR strain of Shigella.

3. The Shigella bacterium of claim 2, wherein the bacterium does not express a native Shigella lipopolysaccharide and does not express a native Shigella O antigen.

4. The Shigella bacterium of claim 1, wherein the TolA, TolQ, and/or Pal protein is located in the bacterium's inner or outer membrane.

5. The Shigella bacterium of claim 1, wherein the Shigella bacterium additionally does not express a Shigella enteric toxin.

6. The Shigella bacterium of claim 1, which does not express the functional HtrB enzyme.

7. The Shigella bacterium of claim 6, which is a htrB knockout.

8. The Shigella bacterium of claim 2, wherein the bacterium is of a ΔtolRΔgalU strain.

9. The Shigella bacterium of claim 2, wherein a stxA and/or stxB gene is inactivated.

10. Culture medium comprising a bacterium of claim 5, which has been grown under conditions which permit the release of blebs into the medium by the bacterium.

11. A method of producing Shigella sonnei or Shigella flexneri blebs, comprising culturing the bacterium of claim 1 in a culture medium under conditions which permit the release of blebs into the medium by the bacterium and separating the released blebs from the culture medium.

12. The method of claim 11, wherein the bacterium has been grown under iron-limiting conditions.

Description

BRIEF DESCRIPTION OF DRAWINGS

(1) FIG. 1 shows an electron micrograph of blebs of the invention purified from culture.

(2) FIG. 2 shows 2D SDS-PAGE of blebs from S. sonnei ΔtolR ΔgalU.

(3) FIG. 3 shows LPS of blebs in various indicated strains, stained with anti-core antibody.

(4) FIG. 4 shows an immunoblot of 2D-separated proteins of blebs using serum from immunised mice.

(5) FIG. 5 shows SDS-PAGE of bacteria grown in different conditions.

(6) FIG. 6 shows adsorbed proteins in blebs. Lanes from left to right: 1) protein molecular weight marker, 2) Ss OMB 10 μg, 3) Ss OMB 2 μg, 4) Ss OMB 10 μg adsorbed on alum for 1 month.

(7) FIGS. 7A-F show FACS data for indicated strains. FIG. 7A, S. sonneiG53 wt; FIG. 7B, S. sonnei G53 DGalU/DTolR; FIG. 7C, S. flexneri2a DGalU; FIG. 7D, S. flexneriM90TDGalU; FIG. 7E, S. flexneri2a; FIG. 7F, S. flexneri2a.

MODES FOR CARRYING OUT THE INVENTION

(8) Preparation of Mutant of S. sonnei

(9) The tolR gene of wild-type S. sonnei 53G was deleted using the λ Red system [11, 102]. Competent cells transformed with the λ Red plasmids are prepared and then transformed with a linear fragment designed to swap the tolR gene for an antibiotic resistance gene by homologous recombination. Clones that have integrated the fragment into the chromosome are selected by resistance to the antibiotic and deletion of the tolR is verified by PCR or other techniques. The temperature sensitive λ Red plasmids can then be removed by growth of the new clones at 37° C.

(10) The lack of TolR expression in this ΔtolR mutant was confirmed and, compared to the original wild-type isolate, it was confirmed to release more blebs into culture medium during growth.

(11) The galU gene was also deleted in a similar way, to provide a ΔgalU single mutant and a ΔtolRΔgalU double mutant. Blebs released by mutants are confirmed to have a defective LPS lacking O antigen.

(12) A ΔtolRΔmsbB double mutant strain with modified LPS is prepared in the same way.

(13) The virulence plasmid has also been removed from the ΔtolR and ΔtolRΔmsbB strains.

(14) Preparation of Mutant of S. flexneri

(15) The tolR gene of S. flexneri was deleted using the λ Red system as described above for S. sonnei. O antigen biosynthesis in S. flexneri was abolished by deletion of a chromosomal fragment comprising the complete rfbG gene and as well as parts of rfbF and rfc, resulting in activation of all three genes. The deletion was generated using the λ Red system and is abbreviated as ΔrfbG.

(16) A ΔtolRΔrfbG double mutant has been generated in the same way.

(17) A ΔtolRΔhtrB double mutant containing modified LPS has been generated in the same way.

(18) The virulence plasmid has also been removed from these strains.

(19) Purification of Blebs

(20) Fermentation of the double mutant ΔtolRΔgalU strain was run under the following conditions: pH 7.1, 37° C., dissolved oxygen maintained at 30% saturation by controlling agitation and setting maximum aeration. The pH was controlled by addition of 4M ammonium hydroxide. The foam was controlled by addition of 10% PPG during the run. The medium consisted of the following components: KH.sub.2PO.sub.4 5 g/l, K.sub.2HPO.sub.4 20 g/l and yeast extract 30 g/l. After the medium was sterilized by autoclaving, glycerol 15 g/l and MgSO.sub.4 2 mM were added prior to inoculation. The culture inoculum was 5% of the fermentor volume. The fermentation process took approximately 13 hours and cell concentration was measured as optical density at 600 nm.

(21) The fermentation process of the S. sonnei ΔtolRΔmsbB double mutant strain was performed with defined medium: glycerol 30 g/l, KH.sub.2PO.sub.4 13.3 g/l, (NH.sub.4).sub.2HPO.sub.4 4 g/l, MgSO.sub.4.7H.sub.2O (1M) 2 ml, citric acid 1.7 g/l, CoCl.sub.2.6H.sub.2O 2.5 mg/l, MnCl.sub.2.4H.sub.2O 15 mg/l, CuCl.sub.2.2H.sub.2O 1.5 mg/l, H.sub.3BO.sub.3 3 mg/l, Na.sub.2MoO.sub.4.2H.sub.2O 2.5 mg/l, Zn(CH.sub.3COO).sub.2.2H.sub.2O 13 mg/l, ferric citrate 2 μM, thiamine 50 mg/l, nicotinic acid 10 mg/l, L-acid aspartic 2.5 g/l.

(22) Vesicles produced in the fermentation broth were purified using two consecutive TFF (tangential flow filtration) steps: micro-filtration at 0.22 μm and then a second micro-filtration at 0.1 μm. During the first filtration step the vesicles were separated from biomass by TFF through a 0.22 μm pore size cassette. The biomass was first concentrated 4-fold and, after five diafiltration steps against PBS, the vesicles were collected in the filtrate. In the second filtration step the filtrate from the 0.22 μm TFF was further micro-filtered trough a 0.1 μm cut-off cassette, in order to purify the vesicles from soluble proteins. The vesicles could not pass through the filter cassette. After five diafiltration steps, the retentate containing the vesicles was collected.

(23) The final purified product was observed with TEM (FIG. 1). The blebs have a homogenous size of about 50 nm in diameter.

(24) Blebs from S. flexneri mutants were purified in the same way after growing the various strains in yeast extract medium as used for S. sonnei ΔtolRΔgalU.

(25) Bleb Characterisation

(26) A proteomic approach confirmed that the blebs are essentially pure outer membranes. Unlike conventional outer membrane vesicles (OMV) derived by disruption of the outer membrane, the blebs conserve lipophilic proteins and are essentially free of cytoplasmic and inner membrane components.

(27) Blebs from S. sonnei and S. flexneri strains were denatured with a detergent and proteins were identified with a LC-MS/MS approach. Alternatively, blebs were separated with SDS page or 2D gel electrophoresis (FIG. 2). Visible bands and spots were excised from the gel and proteins identified via protein mass fingerprint. The relative amount of different proteins was studied with densitometer analysis of SDS-PAGE bands or spots from the 2D gel.

(28) FIG. 3 shows a shift in LPS mobility in the ΔgalU mutant strain compared to wildtype Shigella and E. coli (all strains are in a ΔtolR background).

(29) A second proteomic approach, based on surface digestion, was used to characterize exposed portions of membrane proteins. A set of proteins was identified as reactive with sera from mice immunized with the blebs and many of these have been found to be conserved in a large panel of strains. Little is known about the structure of most integral outer membrane proteins. The surfome of blebs was investigated by treatment with a protease and recovery and identification via LC-MS/MS of released peptides. As blebs should represent the surface of the whole living bacterial cell, this map should be representative of exposed proteins on the surface of S. sonnei.

(30) By these and other approaches the 129 proteins listed in Table 1 have been seen in the blebs.

(31) Bleb Immunogenicity

(32) Mice immunised with the blebs from the ΔtolRΔgalU strain produce serum which reacts with a 2D gel of the blebs as shown in FIG. 4. Thus the blebs are immunogenic.

(33) Mice received 2 μg or 10 μg S. sonnei ΔtolRΔgalU blebs (measured as total protein), with or without adjuvant (aluminium hydroxide or Freund's complete). A classical ELISA method was performed to analyze IgG production in sera obtained from immunization studies. Sera from all groups of mice demonstrated a high level production of bleb-specific IgG. No significant differences in IgG production were detected when blebs were used alone or in combination with an adjuvant. The group immunized with the lower dose of 2 μg showed the same level of bleb-specific IgG as the group immunized with 10 μg, showing that a low dose vaccine may be achievable i.e. more doses per dollar. Blebs from other S. sonnei as well as S. flexneri strains were similarly immunogenic.

(34) Sera raised against the blebs were tested for reactivity with three different bacteria: S. sonnei G53, S. flexneri 2a 2457T or S. flexneri 5 M90T. The samples were than stained with a labeled secondary Ab and were analyzed by flow cytometry. As shown in FIG. 7, the S. sonnei and S. flexneri strains cross-react with the sera.

(35) Therefore the bleb approach has a strong potential to produce effective and low-cost vaccines and can be extended to different Shigella strains towards a broad spectrum vaccine.

(36) Bleb Adsorption

(37) Blebs were combined with aluminium hydroxide (2 mg/ml) for adsorption. The adsorbed material was stored at 4° C. for 1 day, 1 week or 1 month. The blebs were totally adsorbed after 1 day and there was no evidence of desorption even after 1 month (FIG. 6).

(38) Iron-Limiting Growth

(39) FIG. 5 shows proteins expressed by Shigella grown under various conditions. In lane 4 the bacteria were grown in the presence of 200 μM FeSO.sub.4 whereas in lane 5 the culture had 200 μM dypiridyl. The inset shows that three proteins are up-regulated under the iron-limiting conditions. These three proteins were identified as the FepA outer membrane receptor (GI 74311118), the colicin I receptor (GI 74312677), and the putative ferric siderophore receptor (GI 74313972). These proteins are well-conserved among Shigella spp. and enterobacteriaceae and are potentially highly immunogenic. Thus growth of Shigella under iron-limiting conditions can lead to the release of blebs which are, compared to normal growth conditions, enriched for these proteins.

(40) It will be understood that the invention has been described by way of example only and modifications may be made whilst remaining within the scope and spirit of the invention.

(41) TABLE-US-00001 TABLE 1 SEQ ID NO: GI Gene name Definition 8 56480244 tolC outer membrane channel protein [Shigella flexneri 2a str. 301] 9 74312736 ompC outer membrane porin protein C [Shigella sonnei Ss046] 10 74311514 ompA outer membrane protein A [Shigella sonnei Ss046] 11 110807342 SFV_3519 hypothetical protein SFV_3519 [Shigella flexneri 5 str. 8401] 12 56479734 ompX outer membrane protein X [Shigella flexneri 2a str. 301] 13 24113033 slyB putative outer membrane protein [Shigella flexneri 2a str. 301] 14 24112608 lolB outer membrane lipoprotein LolB [Shigella flexneri 2a str. 301] 15 24111612 yaeT outer membrane protein assembly factor YaeT [Shigella flexneri 2a str. 301] 16 187733369 outer membrane protein C [Shigella boydii CDC 3083-94] 17 24113066 Lpp murein lipoprotein [Shigella flexneri 2a str. 301] 18 56479690 pal peptidoglycan-associated outer membrane lipoprotein [Shigella flexneri 2a str. 301] 19 24115506 ecnB entericidin B membrane lipoprotein [Shigella flexneri 2a str. 301] 20 30063370 yedD hypothetical protein S2067 [Shigella flexneri 2a str. 2457T] 21 30064374 ygiW hypothetical protein S3269 [Shigella flexneri 2a str. 2457T] 22 30065519 yjeI hypothetical protein S4565 [Shigella flexneri 2a str. 2457T] 23 24111837 ybaY hypothetical protein SF0398 [Shigella flexneri 2a str. 301] 24 24113773 SF2485 hypothetical protein SF2485 [Shigella flexneri 2a str. 301] 25 74313380 SSON_2966 hypothetical protein SSON_2966 [Shigella sonnei Ss046] 26 30063856 nlpB lipoprotein [Shigella flexneri 2a str. 2457T] 27 145294038 exc entry exclusion protein 2 [Shigella sonnei Ss046] 28 82775909 rlpB LPS-assembly lipoprotein RplB [Shigella dysenteriae Sd197] 29 74311310 ybhC putative pectinesterase [Shigella sonnei Ss046] 30 24114611 fkpA FKBP-type peptidyl-prolyl cis-trans isomerase [Shigella flexneri 2a str. 301] 31 74312826 hisJ histidine-binding periplasmic protein of high-affinity histidine transport system [Shigella sonnei Ss046] 32 24111599 htrA serine endoprotease [Shigella flexneri 2a str. 301] 33 30062097 tolB translocation protein TolB [Shigella flexneri 2a str. 2457T] 34 24111968 modA molybdate transporter periplasmic protein [Shigella flexneri 2a str. 301] 35 24114628 ppiA peptidyl-prolyl cis-trans isomerase A (rotamase A) [Shigella flexneri 2a str. 301] 36 24111499 surA peptidyl-prolyl cis-trans isomerase SurA [Shigella flexneri 2a str. 301] 37 30062764 oppA periplasmic oligopeptide binding protein [Shigella flexneri 2a str. 2457T] 38 30065614 osmY periplasmic protein [Shigella flexneri 2a str. 2457T] 39 74311404 artJ arginine 3rd transport system periplasmic binding protein [Shigella sonnei Ss046] 40 74311061 ushA bifunctional UDP-sugar hydrolase/5&apos;-nucleotidase periplasmic precursor [Shigella sonnei Ss046] 41 74311733 fliY cystine transporter subunit [Shigella sonnei Ss046] 42 110805056 mdoG glucan biosynthesis protein G [Shigella flexneri 5 str. 8401] 43 74312961 cysP thiosulfate transporter subunit [Shigella sonnei Ss046] 44 24114441 yraP hypothetical protein SF3191 [Shigella flexneri 2a str. 301] 45 74312191 SSON_1681 putative receptor [Shigella sonnei Ss046] 46 74312061 ydgA hypothetical protein SSON_1546 [Shigella sonnei Ss046] 47 24111764 proC pyrroline-5-carboxylate reductase [Shigella flexneri 2a str. 301] 48 24112431 SF1022 hypothetical protein SF1022 [Shigella flexneri 2a str. 301] 49 110806822 yggE hypothetical protein SFV_2968 [Shigella flexneri 5 str. 8401] 50 74312071 ydgH hypothetical protein SSON_1556 [Shigella sonnei Ss046] 51 74313729 yrbC hypothetical protein SSON_3340 [Shigella sonnei Ss046] 52 24115498 groEL chaperonin GroEL [Shigella flexneri 2a str. 301] 53 56479605 lpdA dihydrolipoamide dehydrogenase [Shigella flexneri 2a str. 301] 54 24112862 osmE DNA-binding transcriptional activator OsmE [Shigella flexneri 2a str. 301] 55 30065622 deoD purine nucleoside phosphorylase [Shigella flexneri 2a str. 2457T] 56 24111996 sucC succinyl-CoA synthetase subunit beta [Shigella flexneri 2a str. 301] 57 24113762 Crr glucose-specific PTS system component [Shigella flexneri 2a str. 301] 58 24111463 dnaK molecular chaperone DnaK [Shigella flexneri 2a str. 301] 59 74311033 glycoprotein-polysaccharide metabolism 60 30064444 yqjD hypothetical protein S3349 61 82777539 ycbO alkanesulfonate transporter substrate-binding 62 74313684 yraM putative glycosylase 63 24113841 SF2558 OM protein assembly complex subunit YfgL 64 24112186 ybiS hypothetical protein SF0769 65 24111697 tauA taurine transporter substrate binding subunit 66 24115105 yifL putative outer membrane lipoprotein 67 24113718 vacJ lipoprotein precursor 68 1679580 phoN nonspecific phosphatase precursor [Shigella flexneri ] 69 13449092 mxiD Type III secretion protein 70 24112703 pspA phage shock protein PspA [Shigella flexneri 2a str. 301] 71 24112822 yeaF hypothetical protein SF1441 [Shigella flexneri 2a str. 301] 72 24113297 SF1963 cystine transporter subunit 73 24113931 SF2652 outer membrane protein assembly complex subunit YfiO [Shigella flexneri 2a str. 301] 74 24114232 sigA serine protease [S. flexneri 2a str. 301] 75 24115037 ATP- F0F1 ATP synthase subunit alpha synt_ab_C 76 24115158 glnA glutamine synthetase [Shigella flexneri 2a str. 301] 77 30061681 aceF dihydrolipoamide acetyltransferase 78 30062108 sucD succinyl-CoA synthetase subunit alpha 79 30062110 sucB dihydrolipoamide succinyltransferase 80 30062117 gltA type II citrate synthase 81 30062179 dacA D-alanyl-D-alanine carboxypeptidase fraction A 82 30062295 glnH glutamine ABC transporter periplasmic protein 83 30062539 agp Glucose-1-phosphate/inositol phosphatase 84 30062760 adhE bifunctional acetaldehyde-CoA/alcohol dehydrogenase 85 30062895 mdoD glucan biosynthesis protein D 86 30062959 gapA glyceraldehyde-3-phosphate dehydrogenase 87 30063091 rspA starvation sensing protein 88 30063194 S1842 bifunctional cysteine desulfurase/selenocysteine lyase 89 30063263 zwf glucose-6-phosphate 1-dehydrogenase 90 30063276 aspS aspartyl-tRNA synthetase 91 30063294 sitA Iron transport protein 92 30063449 yeeX hypothetical protein S2177 93 30063472 hisB imidazole glycerol-phosphate dehydratase/histidinol phosphatase 94 30063593 mglB Galactose-binding transport protein; receptor for galactose taxis 95 30064126 eno phosphopyruvate hydratase 96 30064248 tktA transketolase 97 30064278 ansB L-asparaginase II 98 30064289 S3169 superfamily I DNA helicase 99 30064503 yhbM lipoprotein NIpI 100 30064729 rpoC DNA-directed RNA polymerase subunit beta′ 101 30064730 rpoB DNA-directed RNA polymerase subunit beta 102 30064872 udp uridine phosphorylase 103 30064882 pldA phospholipase A 104 30064963 atpD F0F1 ATP synthase subunit beta 105 30065048 iutA putative ferric siderophore receptor 106 30065119 lldD L-lactate dehydrogenase 107 30065247 nikA Periplasmic binding proteins for nickel 108 30065291 glpD glycerol-3-phosphate dehydrogenase 109 30065404 rpoA DNA-directed RNA polymerase subunit alpha 110 30065544 hfq RNA-binding protein Hfq 111 56479788 yccZ exopolysacchande export protein [Shigella flexneri 2a str. 301] 112 56480532 lamB maltoporin [Shigella flexneri 2a str. 301] 113 58045130 sepA SepA [Shigella flexneri ] 114 74310732 aceE pyruvate dehydrogenase subunit E1 [Shigella sonnei Ss046] 115 74310771 fhuA ferrichrome outer membrane transporter [Shigella sonnei Ss046] 116 74311118 fepA outer membrane receptor FepA [Shigella sonnei Ss046] 117 74311859 prc Carboxy-terminal protease [Shigella sonni Ss046] 118 74312394 yciD outer membrane protein W [Shigella sonnei Ss046] 119 74312453 prsA ribose-phosphate pyrophosphokinase [Shigella sonnei Ss046] 120 74312677 cirA colicin I receptor [Shigella sonnei Ss046] 121 74312761 glpQ glycerophosphodiester phosphodiesterase [Shigella sonnei Ss046] 122 74312989 talA transaldolase A [Shigella sonnei Ss046] 123 74313764 degQ serine endoprotease [Shigella sonnei Ss046] 124 74314527 malE maltose ABC transporter periplasmic protein [Shigella sonnei Ss046] 125 82543910 SBO_1406 major capsid protein [Shigella boydii Sb227] 126 82544504 ycdO hypothetical protein SBO_2040 [Shigella boydii Sb227] 127 82545484 dsbC thiol:disulfide interchange protein DsbC [Shigella boydii Sb227] 128 82777619 ybjP putative lipoprotein [Shigella dysenteriae Sd197] 129 110807066 yhbN hypothetical protein SFV_3230 [Shigella flexneri 5 str. 8401] 130 161486535 yajG hypothetical protein S0385 131 187427808 tolC outer membrane protein TolC [Shigella boydii CDC 3083-94] 132 187731061 SbBS512_E3369 peptidase, M48B family [Shigella boydii CDC 3083-94] 133 187731375 SbBS512_E3904 outer membrane lipoprotein, Slp family [Shigella boydii CDC 3083-94] 134 187733898 osmY osmotically inducible protein Y [Shigella boydii CDC 3083-94] 135 187734005 bglX beta-glucosidase, periplasmic [Shigella boydii CDC 3083-94] 136 30065453 pepA leucyl aminopeptidase

(42) SEQ ID NOs: 70, 71, 73, 74, 76, 111, 112, 114-129 & 131-135 were identified from S. sonnei ΔtolR blebs. SEQ ID NOs: 8-15 & 17-58 were identified from S. sonnei ΔtolRΔgalU blebs. SEQ ID NOs: 83, 94, 97 & 107 were identified from S. flexneri ΔtolR blebs. SEQ ID NOs: 68, 69, 72, 75, 77-82, 84-93, 95, 96, 98-106, 108-10, 113, 130 & 136 were identified from S. flexneri ΔtolRΔrfbG blebs. SEQ ID NOs: 60-67 were identified from surface digestion of S. sonnei.

(43) Subset 1:

(44) SEQ ID NOs: 68, 69, 72, 75, 77-110, 113, 130 & 136.

(45) Subset 2:

(46) SEQ ID NOs: 8-15, 17-58, 60-67, 70, 71, 73, 74, 76, 111, 112, 114-129 & 131-135.

(47) Subset 3:

(48) SEQ ID NOs: 1-60.

(49) NB: SEQ ID NO: 18 is the same as SEQ ID NO: 5; SEQ ID NO: 33 is the same as SEQ ID NO: 2; SEQ ID NOs: 9 & 16 are related (˜97% identity); SEQ ID NOs: 23 & 59 are related (˜98% identity).

REFERENCES

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