Functional cloning of Vibrio parahaemolyticus type III secretion system 1 in Escherichia coli K-12 strain as a molecular syringe

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Abstract

The type III secretion system (T3SS) of gram-negative bacteria involves dedicated protein translocation machinery that directly injects proteins into target cells. Pathogenic bacteria already benefit from this unique system. The successful functional cloning of this useful tool into non-pathogenic bacteria would help establish novel clinical and basic biotechnology strategies in areas such as vaccine administration, the development of screening systems for anti-T3SS drugs and the target-specific delivery of bioactive compounds. In this study, we successfully cloned the Vibrio parahaemolyticus T3SS1 genetic locus into a non-pathogenic Escherichia coli K-12 strain. Assays performed here revealed that the T3SS1 cloned into the E. coli K-12 strain has the ability to translocate V. parahaemolyticus T3SS1 secreted proteins. Importantly, we also observed this system to allow the E. coli K-12 strain to inject foreign protein, as well as the V. parahaemolyticus T3SS effector, into cultured cells. These results demonstrate a prospective useful tool with experimental and therapeutic applications.

Highlights

► We cloned functional Vibrio parahaemolytics T3SS1 in E. coli K-12. ► Cloned Vp T3SS1 in Escherichia coli K-12 injects foreign proteins into host cells. ► Cloned Vp T3SS1 has potential to be used in experimental and therapeutic application.

Introduction

Targeting bioactive molecules to specific human cells or organs is quite fascinating in many ways [1]. Vaccine development, anti-cancer therapy and other clinical treatments are all amenable to such systems. Various targeting systems have been developed. However, no perfect systems exist that are applicable to all conditions, and the development of novel technology has been anticipated. One of the potential systems for this purpose is a bacterial type III secretion system (T3SS) [2]. The T3SS is composed of the sophisticated protein translocation machinery found in gram-negative bacteria that allows them to inject virulence factors, or so-called ‘effectors’, directly into host cells. The T3SS thus contributes substantially to the establishment of a bacterial niche in the host. The details of the protein secretion mechanism and structures have been extensively studied, and at present, the minimal components required to establish the functional T3SS are known. The T3SS consists of two components: the needle and the basal body. The needle is the conduit for proteins secreted from the inside to the outside of the bacteria, whereas the basal body is the platform required to prepare the proteins for the secretion [3]. The establishment of this complicated system requires the expression of more than 20 genes. However, successful functional cloning of this useful tool, especially in non-pathogenic bacteria, is beneficial for many researchers seeking to develop novel clinical and basic application strategies in areas such as vaccine administration, the development of a screening system for anti-T3SS drugs and the target-specific delivery of bioactive compounds [4], [5], [6]. In this study, we successfully constructed the cloned Vibrio parahaemolyticus T3SS1 genetic locus into a non-pathogenic E. coli K-12 strain. A translocation assay revealed that the cloned T3SS had the ability to inject foreign proteins tagged with the region essential for secretion of the T3SS1 effector, VepA, as well as that of the proteins secreted by V. parahaemolyticus T3SS1, providing a prospective molecular syringe.

Section snippets

Bacterial strains and growth conditions

V. parahaemolyticus RIMD2210633 [7] and its derivative strains were employed as genomic DNA sources of the T3SS1-related gene-coding region (Fig. 1) [8], [9]. A non-pathogenic E. coli K12 derivative strain, VCS257 (Agilent Technologies, Santa Clara, CA) was used for the cosmid transduction and as a host strain of the cloned T3SS. Bacterial cultures were grown at 37 °C in 3% LB medium (1% bacto tryptone, 0.5% yeast extract, 3% NaCl) for V. parahaemolyticus and LB medium (1% bacto tryptone, 0.5%

Cloning of the T3SS1 genetic locus of V. parahaemolyticus

To construct non-pathogenic E. coli expressing T3SS, there are many candidates as a source for T3SS genetic locus. This locus can be constructed using any T3SS from a variety of pathogenic bacteria. However, the locus should be harmless and have no adverse effects in target cells when this molecular syringe is employed as a tool. From this point of view, the T3SS genetic locus must at the minimum establish a protein secretory apparatus. Our previous studies revealed that the genome of

Discussion

T3SS is a sophisticated system to deliver proteins directly into host cells. A number of studies about T3SS have been reported over the recent couple of decades [19]. T3SS has been found in pathogenic bacteria and has been utilized to establish a niche of infection in host cells. This approach implies that much potential exists for the usage of T3SS as a molecular tool. Previous studies have shown some promising results for the establishment of a “molecular syringe” [20], [21], [22], [23].

Acknowledgments

This work was supported by Grants-in-Aid for Young Scientists and Scientific Research in Priority Areas from the Ministry of Education, Culture, Sports, Science and Technology of Japan and from the Takeda Science Foundation. The plasmid pCX340 was generously provided by Dr. Eric Oswald of the Institut National de la Recherche Agronomique, Ecole Nationale Veterinaire de Toulouse, France.

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