ES2646320T3 - Salmonella enterica que presenta un N-glicano de C. Jejuni o derivados del mismo - Google Patents

Salmonella enterica que presenta un N-glicano de C. Jejuni o derivados del mismo Download PDF

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ES2646320T3
ES2646320T3 ES10715098.9T ES10715098T ES2646320T3 ES 2646320 T3 ES2646320 T3 ES 2646320T3 ES 10715098 T ES10715098 T ES 10715098T ES 2646320 T3 ES2646320 T3 ES 2646320T3
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glycan
jejuni
wbap
salmonella enterica
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Karin Ilg
Markus Aebi
Umesh Ahuja
Saba Amber
Flavio Schwarz
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Eidgenoessische Technische Hochschule Zurich ETHZ
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Abstract

Salmonella enterica, caracterizada por que comprende al menos un operón pgl (para la glicosilación de proteínas) de Campylobacter jejuni o un derivado funcional del mismo y presenta sobre su superficie celular al menos un N-glicano de Campylobacter jejuni o un derivado de N-glicano del mismo, en donde están inactivados uno más genes de la biosíntesis de bacilosamina por mutación y/o deleción parcial o completa, preferiblemente por deleción parcial y/o completa de los genes pgI, D, E, F.

Description

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Capacidad de colonización de ∆wbaP en un experimento de co-infección
La capacidad de colonización de S. Typhimurium ∆wbaP se probó en un experimento de co-infección en el cual los ratones se infectaron intragástricamente con el mutante ∆wbaP, así como con la cepa tipo silvestre. Los ratones 5 C57BL/6 (SPF, colonia del RCHCl, Zurich) se pre-trataron mediante sonda gástrica con 20 mg de estreptomicina. Veinticuatro horas después los ratones se inocularon con 5 x 107 UFC de la cepa serovar Typhimurium o las mezclas de las cepas como se indica. Las cargas bacterianas (UFC) en sedimentos fecales frescos, nodos linfoides mesentéricos (mLN), bazo, y contenido cecal se determinaron mediante la siembra en placas sobre placas de agar MacConkey (50 µg/ml de estreptomicina) como se ha descrito anteriormente (Barthel, Hapfelmeier et al. 2003). Los 10 índices competitivos (CI) se determinaron de acuerdo con la fórmula CI= (mutante/tipo silvestre) salida/(mutante/tipo silvestre) entrada después de la siembra en placa. Se realizó un experimento de co-infección de serovar Typhimurium tipo silvestre (M939) y la cepa ∆wbaP (SKI11). Cinco ratones tratados con estreptomicina se infectaron con una mezcla 1:2 (total 5 X 107 UFC) intragástricamente de la cepa ∆wbaP (SKI11) y la cepa tipo silvestre. La proporción de las 2 cepas (CI; índice competitivo, véase Materiales y Métodos) se determinó en las heces en el día 15 1, 2 y 3 p.i. Se detectó una disminución de los recuentos de ∆wbaP (SKI11) en comparación con el tipo silvestre (una escala logarítmica por día) y se probó que la cepa ∆wbaP (SKI11) de hecho tuvo un defecto competitivo importante en comparación con la cepa serovar Typhimurium tipo silvestre en el tracto intestinal (p>0,05; figura 4A). Además, el CI de las dos cepas en los sitios sistémicos (mLN, hígado, bazo) en el día 4 p.i. también demostró un defecto competitivo significativo de serovar Typhimurium ∆wbaP (SKI12). Sin embargo, el defecto era menos
20 pronunciado que en el intestino (Figura 4B).
Tabla 1: Cepas, plásmidos y cebadores para la deleción de wbaP utilizados en este trabajo
Cepas de Salmonella enterica sv Typhimurium
Cepa
Genotipo y fenotipo Fuente de la referencia
SL1344
tipo silvestre; strepR Hoiseth, S. K. and B. A. Stocker, Nature 291:238-239, 1981
SKI11
SL1344∆wbaP::cat; strepR , cam R este estudio
SKI12
SL1344∆wbaP; strepR este estudio
SKI34
SKI12::pKI14; strepR, tetR este estudio
SKI35
SKI12::pKI15; strepR, tetR este estudio
Cepas de Escherichia coli
DH5a
SupE44 ∆lacU169 (Φ80lacZ∆M15) hsdR17 recA1 endA1 gyrA96 thi-1 relA1 Hanahan, D., J. Mol. Biol., 5,166(4):557-80, 1983
CC118 λpir
∆(ara-leu), araD, ∆lacX74, galE, galK, phoA20, thi-1, rpsE, rpoB, argE(Am), recA, λpir Herrero, M., V. de Lorenzo, and K. N. Timmis. J Bacteriol 172:6557-6567.
Sm10λpir
thi thr leu tonA lacY supE recA::RP4 2-Tc::Mu λpir, kanR Miller, V. L. and J. J. Mekalanos. J. Bacteriol. 170:2575-2583, 1988.
Plásmidos
Plásmido
Genotipo Fuente de referencia
pSB377
tetR oriR6K Mirold et al., Proc. Natl. Acad. Sci. USA, 96:9845-9850, 1999.
pKD3
bla FRT cat FRT PS1 Datsenko, K. A. and B. L.
PS2 oriR6K
Wanner, Proc. Natl. Acad. Sci. USA, 97:6640-6645, 2000.
pKD46
bla PBAD gam bet exo pSC101 oriTS Datsenko, K. A., and B. L. Wanner, Proc. Natl. Acad. Sci. USA, 97:66406645, 2000.
pCP20
bla cat cl857 λPR flp pSC101 oriTS Datsenko, K. A., and B. L. Wanner,Proc. Natl. Acad. Sci. USA, 97:6640-6645, 2000
pACYC184
CmR, TcR, ori p15A New England Biolabs
pACYCpglmut
CmR, ori p15A; grupo C. jejuni pgl con pglBW458A,D459A clonado en pACYC184 Science, 298(5599):1790-3, 29. Nov. 2002
pACYCpgl3mut
CmR, ori p15A; grupo C. jejuni pgl con pglBW458A,D459A clonado en pACYC184, deleción en pglE, F, G y 3'-mitad de pglD Este estudio
pKI14
TetR, oriR6K, región de 500 pb 3' de PagC clonado en pSB377 Este estudio
pKI15
TetR, oriR6K, grupo C. jejuni pgl3mut con pglBW458A,D459A clonado en pKI15 Este estudio
Cebadores para la delación de wbaP
RfbP H1P1
12

Claims (1)

  1. imagen1
    imagen2
ES10715098.9T 2009-03-27 2010-03-25 Salmonella enterica que presenta un N-glicano de C. Jejuni o derivados del mismo Active ES2646320T3 (es)

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PCT/EP2010/001884 WO2010108682A1 (en) 2009-03-27 2010-03-25 Salmonella enterica presenting c. jejuni n-glycan or derivatives thereof

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KR101855380B1 (ko) * 2009-11-19 2018-05-08 글락소스미스클라인 바이오로지칼즈 에스.에이. 원핵세포에서 면역원성 폴리사카라이드를 제조하는 생합성 시스템
WO2013034664A1 (en) * 2011-09-06 2013-03-14 Glycovaxyn Ag Bioconjugate vaccines made in prokaryotic cells
EP2841559A4 (en) 2012-04-27 2015-12-16 Univ Alberta GLYCOMODIFIED EXTERNAL MEMBRANE VESICLES AND THEIR USE AS VACCINES
RU2671473C2 (ru) * 2013-04-05 2018-10-31 Де Гавенерс Оф Де Юниверсити Оф Альберта Вакцина против кампилобактериоза
CN103740632B (zh) * 2014-01-22 2016-02-10 山东大学 一株重组大肠杆菌及其在制备抗o157:h7的n-糖蛋白疫苗中的应用
CN110520153B (zh) * 2017-01-27 2024-04-02 佛罗里达大学研究基金公司 在禽类中控制肠道沙门氏菌和减少弯曲杆菌的食品安全疫苗
CA3054765A1 (en) 2017-03-15 2018-09-20 London School Of Hygiene And Tropical Medicine Whole cell vaccines
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US20200377620A1 (en) * 2017-12-19 2020-12-03 The Governors Of The University Of Alberta Clostridium perfringens surface glycans and uses thereof
US10973908B1 (en) 2020-05-14 2021-04-13 David Gordon Bermudes Expression of SARS-CoV-2 spike protein receptor binding domain in attenuated salmonella as a vaccine

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