Identification, Characterization, and Molecular Application of a Virulence-Associated Autotransporter from a Pathogenic Pseudomonas fluorescens Strain

doi:10.1128/AEM.00159-09

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	Identification, Characterization, and Molecular Application of a Virulence-Associated Autotransporter from a Pathogenic Pseudomonas fluorescens Strain
	Hu, Yong-hua 1,2; Liu, Chun-sheng 1,2; Hou, Jin-hui 3; Sun, Li 1; Sun, L, Chinese Acad Sci, Inst Oceanol, 7 Nanhai Rd, Qingdao 266071, Peoples R China
	2009-07-01
发表期刊	APPLIED AND ENVIRONMENTAL MICROBIOLOGY
ISSN	0099-2240
卷号	75 期号:13 页码:4333-4340
文章类型	Article
摘要	A gene, pfa1, encoding an autotransporter was cloned from a pathogenic Pseudomonas fluorescens strain, TSS, isolated from diseased fish. The expression of pfa1 is enhanced during infection and is regulated by growth phase and growth conditions. Mutation of pfa1 significantly attenuates the overall bacterial virulence of TSS and impairs the abilities of TSS in biofilm production, interaction with host cells, modulation of host immune responses, and dissemination in host blood. The putative protein encoded by pfa1 is 1,242 amino acids in length and characterized by the presence of three functional domains that are typical for autotransporters. The passenger domain of PfaI contains a putative serine protease (Pap) that exhibits apparent proteolytic activity when expressed in and purified from Escherichia coli as a recombinant protein. Consistent with the important role played by PfaI in bacterial virulence, purified recombinant Pap has a profound cytotoxic effect on cultured fish cells. Enzymatic analysis showed that recombinant Pap is relatively heat stable and has an optimal temperature and pH of 50 degrees C and pH 8.0. The domains of PfaI that are essential to autotransporting activity were localized, and on the basis of this, a PfaI-based autodisplay system (named AT1) was engineered to facilitate the insertion and transport of heterologous proteins. When expressed in E. coli, AT1 was able to deliver an integrated Edwardsiella tarda immunogen (Et18) onto the surface of bacterial cells. Compared to purified recombinant Et18, Et18 displayed by E. coli via AT1 induced significantly enhanced immunoprotection.; A gene, pfa1, encoding an autotransporter was cloned from a pathogenic Pseudomonas fluorescens strain, TSS, isolated from diseased fish. The expression of pfa1 is enhanced during infection and is regulated by growth phase and growth conditions. Mutation of pfa1 significantly attenuates the overall bacterial virulence of TSS and impairs the abilities of TSS in biofilm production, interaction with host cells, modulation of host immune responses, and dissemination in host blood. The putative protein encoded by pfa1 is 1,242 amino acids in length and characterized by the presence of three functional domains that are typical for autotransporters. The passenger domain of PfaI contains a putative serine protease (Pap) that exhibits apparent proteolytic activity when expressed in and purified from Escherichia coli as a recombinant protein. Consistent with the important role played by PfaI in bacterial virulence, purified recombinant Pap has a profound cytotoxic effect on cultured fish cells. Enzymatic analysis showed that recombinant Pap is relatively heat stable and has an optimal temperature and pH of 50 degrees C and pH 8.0. The domains of PfaI that are essential to autotransporting activity were localized, and on the basis of this, a PfaI-based autodisplay system (named AT1) was engineered to facilitate the insertion and transport of heterologous proteins. When expressed in E. coli, AT1 was able to deliver an integrated Edwardsiella tarda immunogen (Et18) onto the surface of bacterial cells. Compared to purified recombinant Et18, Et18 displayed by E. coli via AT1 induced significantly enhanced immunoprotection.
关键词	Periplasmic Chaperone Skp Cell-surface Presentation Gram-negative Bacteria v Protein Secretion Escherichia-coli Trimeric Autotransporter Serine-protease Yersinia-enterocolitica Recombinant Proteins Translocator Domain
学科领域	Biotechnology & Applied Microbiology ; Microbiology
DOI	10.1128/AEM.00159-09
URL	查看原文
收录类别	SCI
语种	英语
WOS记录号	WOS:000267373000013
引用统计	被引频次：47[WOS] [WOS记录] [WOS相关记录]
文献类型	期刊论文
条目标识符	http://ir.qdio.ac.cn/handle/337002/1605
专题	实验海洋生物学重点实验室
通讯作者	Sun, L, Chinese Acad Sci, Inst Oceanol, 7 Nanhai Rd, Qingdao 266071, Peoples R China
作者单位	1.Chinese Acad Sci, Inst Oceanol, Qingdao 266071, Peoples R China 2.Chinese Acad Sci, Grad Univ, Beijing, Peoples R China 3.Xuzhou Inst Technol, Xuzhou, Peoples R China
第一作者单位	中国科学院海洋研究所
推荐引用方式 GB/T 7714	Hu, Yong-hua,Liu, Chun-sheng,Hou, Jin-hui,et al. Identification, Characterization, and Molecular Application of a Virulence-Associated Autotransporter from a Pathogenic Pseudomonas fluorescens Strain[J]. APPLIED AND ENVIRONMENTAL MICROBIOLOGY,2009,75(13):4333-4340.
APA	Hu, Yong-hua,Liu, Chun-sheng,Hou, Jin-hui,Sun, Li,&Sun, L, Chinese Acad Sci, Inst Oceanol, 7 Nanhai Rd, Qingdao 266071, Peoples R China.(2009).Identification, Characterization, and Molecular Application of a Virulence-Associated Autotransporter from a Pathogenic Pseudomonas fluorescens Strain.APPLIED AND ENVIRONMENTAL MICROBIOLOGY,75(13),4333-4340.
MLA	Hu, Yong-hua,et al."Identification, Characterization, and Molecular Application of a Virulence-Associated Autotransporter from a Pathogenic Pseudomonas fluorescens Strain".APPLIED AND ENVIRONMENTAL MICROBIOLOGY 75.13(2009):4333-4340.

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