Cloning and transfer of the Salmonella pathogenicity island 2 type III secretion system for studies of a range of gram-negative genera

Abstract

The engineering of bacterial strains with specific phenotypes frequently requires the use of blocks or "cassettes" of genes that act together to perform a desired function. The potential benefits of utilizing type III secretion systems in this regard are becoming increasingly realized since these systems can be used to direct interactions with host cells for beneficial purposes such as vaccine development, anticancer therapies, and targeted protein delivery. However, convenient methods to clone and transfer type III secretion systems for studies of a range of different types of bacteria are lacking. In addition to functional applications, such methods would also reveal important information about the evolution of a given type III secretion system, such as its ability to be expressed and functional outside of the strain of origin. We describe here the cloning of the Salmonella enterica serovar Typhimurium pathogenicity island 2 (SPI-2) type III secretion system onto a vector that can be easily transferred to a range of gram-negative bacterial genera. We found that expression of the cloned SPI-2 system in different Gammaproteobacteria and Alphaproteobacteria (as monitored by SseB protein levels) is dependent on the bacterial strain and growth medium. We also demonstrate that the cloned system is functional for secretion, can direct interactions with macrophages, and can be used as a novel tool to analyze the predicted interaction of SseB with host cells. This work provides a foundation for future applications where the cloned SPI-2 region (or other cloned type III systems) can provide a desired function to an engineered gram-negative strain.

FIG. 1.

S. enterica serovar Typhimurium SPI-2 region. A map of the S. enterica serovar Typhimurium SPI-2 region is depicted. The arrows indicate the genes of the region that have been cloned onto R995 using VEX-capture. The genes that comprise the SPI-2 genomic island and the type III secretion system within that island are also indicated.

FIG. 2.

Transfer of R995 + SPI-2 to different bacterial hosts as shown via PCR analysis. Plasmid DNA isolated from the indicated transconjugant strains containing either R995 or R995 + SPI-2 was used as a template in PCR analysis with primers hybridizing to portions of the indicated SPI-2 genes. The PCR products were run on DNA agarose gels and stained with ethidium bromide. Odd-numbered and even-numbered lanes indicate samples from strains containing R995 or R995 + SPI-2, respectively. The lane marked “+” indicates PCRs where chromosomal DNA from wild-type S. enterica serovar Typhimurium strain χ3339 was used as a template. Abbreviations: S.t., S. enterica serovar Typhimurium; E.c., E. coli; P.a., P. aeruginosa.

FIG. 3.

Western blot analysis of SseB expression in different bacterial strains containing R995 + SPI-2 grown in MgM 10 and MgM 8. (A) Total cell lysates were obtained from S. enterica serovar Typhimurium and E. coli strains containing either R995 (R) or R995 + SPI-2 (R + SPI-2) grown in the indicated medium, and equivalent amounts of these lysates were analyzed via SDS-PAGE, Western blotting, and probing with anti-SseB antisera. Lanes numbered 1 to 4 indicate four separate isolates of the strain TOP10 (R + SPI-2). MgM 10 and MgM 8 indicate media used for SPI-2 noninducing and inducing conditions, respectively. WT, wild type. (B) An analysis identical to that described for panel A was performed for other gram-negative strains containing R995 (R) or R995 + SPI-2 (R + SPI-2). The lane marked “+” contains a sample from the same batch of lysate obtained from the strain S. enterica serovar Typhimurium ΔSPI-2 (R + SPI-2) as a positive control. P.a., P. aeruginosa.

FIG. 4.

Western blot analysis of SseB expression in different bacterial strains containing R995 + SPI-2 grown in LB medium. (A) Equivalent amounts of total cell lysates from the indicated strains grown in LB medium were analyzed via Western blotting and probing with anti-SseB antisera. WT, wild type. (B) Equivalent amounts of total cell lysates from the indicated strains containing R995 (R) or R995 + SPI-2 (R + SPI-2) grown in LB medium were analyzed as described for panel A. P.a., P. aeruginosa.

FIG. 5.

Expression of SseB from the R995 + SPI-2::plac-ssrAB construct. Strains containing the plasmids R995 (R), R995 + SPI-2 (R + SPI-2), and R995 + SPI-2::plac-ssrAB (R + SPI-2::plac-ssrAB) were grown in the indicated media and assayed for SseB protein expression via Western blotting of equivalent amounts of cell lysate. Please refer to the text for additional details.

FIG. 6.

Protein secretion via the cloned SPI-2 type III secretion system. Preparations of secreted proteins were obtained from culture supernatants of the indicated strains containing either R995 (R), R995 + SPI-2 (R + SPI-2), or R995 + SPI-2 ssaV (R + SPI-2 ssaV) grown in MgM 8. (A) Coomassie blue staining of secretion preparations obtained from the indicated S. enterica serovar Typhimurium strains and run on an SDS-polyacrylamide gel. In this assay, the SPI-2-encoded SseD protein is detected as a prominent band running at approximately 20 kDa. (B) Samples of secreted proteins from the indicated strains were Western blotted and probed with anti-SseB antisera. Samples from secretion preparations (“Sec preps”) and cell lysates are indicated. WT, wild type.

FIG. 7.

Survival of strains containing the cloned SPI-2 region in RAW264.7 macrophages. Equivalent numbers of cells from cultures of the indicated bacterial strains containing either R995 (R), R995 + SPI-2 (R + SPI-2), or R995 + SPI-2 ssaV (R + SPI-2 ssaV) were incubated with RAW264.7 macrophages for 1 h. At this point, the infection medium was changed to contain gentamicin, and the infected-cell cultures were incubated for an additional 17 h (18-h total infection). Intracellular bacteria were obtained at this time and enumerated via plating for CFU. The results were replicated in four independent trials with each trial being performed in triplicate sample wells. WT, wild type.

FIG. 8.

Association of SseB with host macrophage cells. The eukaryotic host cell protein fraction from infections of J774 macrophages with the indicated bacterial strains was obtained as described in Materials and Methods and analyzed for the presence of SseB via Western blotting and probing with anti-SseB antisera. Samples of cell lysate from cultures of the bacterial strains used in the infections are also indicated. The protein control was performed by adding SseB protein from a cell lysate (equivalent to the number of bacterial cells used in the infection) to demonstrate that any extracellular SseB is washed away before macrophage lysis. WT, wild type.