WO2021077231A1 - Système de vibrio cholerae mutant pour l'administration de protéines - Google Patents

Système de vibrio cholerae mutant pour l'administration de protéines Download PDF

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WO2021077231A1
WO2021077231A1 PCT/CA2020/051429 CA2020051429W WO2021077231A1 WO 2021077231 A1 WO2021077231 A1 WO 2021077231A1 CA 2020051429 W CA2020051429 W CA 2020051429W WO 2021077231 A1 WO2021077231 A1 WO 2021077231A1
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vasx
gene
mutation
tsel
vgrg3
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English (en)
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Tao Dong
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UTI LP
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UTI LP
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/28—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Vibrionaceae (F)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/36—Adaptation or attenuation of cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/90—Stable introduction of foreign DNA into chromosome

Definitions

  • T6SS secretion 33, 34. It is not known whether the T6SS requirement for effectors is dependent on their activities or physical structures.
  • the T6SS of Acinetobacter requires a membrane-associated peptidoglycan hydrolase TagXto facilitate formation of the trans-envelope TssJLM complex of the T6SS
  • V. cholerae lacks a TagX homolog
  • how the T6SS of V. cholerae assembles its trans-envelope complex across the cell wall remains elusive.
  • TseL and VgrG3 are capable of reaching the periplasm when expressed in the cytosol
  • said mutation in said tseL gene consist of comprises tseL D425A .
  • said mutant effector consists or comprises of: a mutation in the tseL gene is the region that encodes the TseL polypeptide from amino acid V381 to W547, a mutation in the vgrG3 gene is in the region that encodes the VgrG3 polypeptide from amino acid K889 to K1278, and the mutation in the vasX gene in in the region that encodes the VasX polypeptide from amino acid R972 to 11156.
  • polynucleotide molecule comprising or consisting of a nucleic acid sequence that encodes a first polypeptide -cargo fusion polypeptide.
  • the mutation in the tseL gene comprises or consists of a mutation in the catalytic domain of the tseL gene.
  • the mutant effector may be a portion of or fragment of tseL, VgrG3, or VgrG3.
  • T6SS assembly and secretion is energetically costly. Assembling a 1- ⁇ m long sheath-needle (average length in V. cholerae) requires 260 rings of hexameric VipA/B-Hcp subunits (16). Assuming sheath contraction ejects the whole Hep needle out, each contraction will cost 1560 molecules of Hep to accompany the delivery of a few molecules of effectors (Fig. 6A). Therefore, how could cells prevent futile delivery of T6SS, that is T6SS secretion without any effectors loaded? Our results demonstrate an on-board checking mechanism by which the physical presence of effectors is required for T6SS assembly in V. cholerae, thereby ensuring effectors are loaded for each T6SS ejection (Fig. 6B).
  • V. cholerae and E. coli strains and plasmids are listed in Table 1. Chromosomal deletions and site-directed mutations in V. cholerae were constructed using homologous recombination (57). Cultures were grown in LB medium (1% [w/v] tryptone, 0.5% [w/v] yeast extract, 0.5% [w/v] NaCI) aerobically. Antibiotics were as follows: ampicillin (100 ⁇ g/ml), streptomycin (100 ⁇ g/ml), chloramphenicol (25 ⁇ g/ml for Escherichia coli, 2.5 ⁇ g/ml for V52), and kanamycin (50 ⁇ g/ml). Expression vectors were constructed as previously described (31). All constructs were verified by sequencing. [00161] Bacterial killing assay
  • Hood RD et al. (2010) A Type VI Secretion System of Pseudomonas aeruginosa Targets a Toxin to Bacteria. Cell Host Microbe 7(1):25-37.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biotechnology (AREA)
  • Biomedical Technology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Biophysics (AREA)
  • Microbiology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Virology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Cell Biology (AREA)
  • Physics & Mathematics (AREA)
  • Plant Pathology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

L'invention concerne un système de Vibrio cholerae mutant pour l'administration de protéines.
PCT/CA2020/051429 2019-10-25 2020-10-23 Système de vibrio cholerae mutant pour l'administration de protéines Ceased WO2021077231A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US201962926070P 2019-10-25 2019-10-25
US62/926,070 2019-10-25

Publications (1)

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WO2021077231A1 true WO2021077231A1 (fr) 2021-04-29

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116103217A (zh) * 2023-03-21 2023-05-12 集美大学 一株杀香鱼假单胞菌vgrG基因敲除菌株及其构建与应用
CN116254217A (zh) * 2023-03-21 2023-06-13 集美大学 一株变形假单胞菌vgrG基因回补菌株及其构建与应用
CN116492476A (zh) * 2022-12-06 2023-07-28 上海交通大学 细菌T6SS核心组分VgrG作为药物递送载体的构建方法

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
DONG TG ET AL.: "Identification of T6SS-dependent effector and immunity proteins by Tn-seq in Vibrio cholerae", PNAS, vol. 110, no. 7, 12 February 2013 (2013-02-12), pages 2623 - 2628, XP055368864, ISSN: 1091-6490, DOI: 10.1073/pnas.1222783110 *
LIANG XIAOYE, KAMAL FATIMA, PEI TONG-TONG, XU PING, MEKALANOS JOHN J., DONG TAO G.: "An onboard checking mechanism ensures effector delivery of the type VI secretion system in Vibrio cholerae", PNAS, vol. 116, no. 46, 12 November 2019 (2019-11-12), pages 23292 - 23298, XP055813947, ISSN: 1091-6490 *
SARAH T. MIYATA,DANIEL UNTERWEGER,SYDNEY P. RUDKO,STEFAN PUKATZKI: "Dual Expression Profile of Type VI Secretion System Immunity Genes Protects Pandemic Vibrio cholerae", PLOS PATHOG, vol. 9, no. 12, 2013, pages e1003752, XP055813946, ISSN: 1553-7374 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116492476A (zh) * 2022-12-06 2023-07-28 上海交通大学 细菌T6SS核心组分VgrG作为药物递送载体的构建方法
CN116103217A (zh) * 2023-03-21 2023-05-12 集美大学 一株杀香鱼假单胞菌vgrG基因敲除菌株及其构建与应用
CN116254217A (zh) * 2023-03-21 2023-06-13 集美大学 一株变形假单胞菌vgrG基因回补菌株及其构建与应用

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