Construction, Characterization, and Use of TwoListeria monocytogenesSite-Specific Phage Integration Vectors

Abstract

Two site-specific shuttle integration vectors were developed with two different chromosomal bacteriophage integration sites to facilitate strain construction in Listeria monocytogenes. The first vector, pPL1, utilizes the listeriophage U153 integrase and attachment site within the comK gene for chromosomal insertion. pPL1 contains a useful polylinker, can be directly conjugated from Escherichia coli into L. monocytogenes, forms stable, single-copy integrants at a frequency of ϳ10 ؊4 per donor cell, and can be used in the L. monocytogenes 1/2 and 4b serogroups. Methods for curing endogenous prophages from the comK attachment site in 10403S-derived strains were developed. pPL1 was used to introduce the hly and actA genes at comK-attBB in deletion strains derived from 10403S and SLCC-5764. These strains were tested for second-site complementation in hemolysin assays, plaquing assays, and cell extract motility assays. Unlike plasmid-complemented strains, integrated pPL1-complemented strains were fully virulent in the mouse 50% lethal dose assay. Additionally, the PSA phage attachment site on the L. monocytogenes chromosome was characterized, and pPL1 was modified to integrate at this site. The listeriophage PSA integrates in the 3 end of an arginine tRNA gene. There are 17 bp of DNA identity between the bacterial and phage attachment sites. The PSA prophage DNA sequence reconstitutes a complete tRNA Arg gene. The modified vector, pPL2, was integration proficient at the same frequency as pPL1 in common laboratory serotype 1/2 strains as well as serotype 4b strains.

Listeria monocytogenes Is Resistant to Lysozyme through the Regulation, Not the Acquisition, of Cell Wall-Modifying Enzymes

“…rli31 complement strains were constructed by amplifying rli31 and the rli31 promoter with TB140 and TB141. This fragment was introduced into L. monocytogenes by using pPL2 or pAM401 as described previously (16,17). For lists of all of the…”

Listeria-based cancer vaccines that segregate immunogenicity from toxicity

“…The central rationale is that the intracellular lifecycle of Listeria enables effective stimulation of CD4 ϩ ͞CD8 ϩ T cell immunity. There are also numerous practical features of Listeria-based vaccines, including its anticipated ease of production in defined media, combined with relatively simple vaccine construction by using well developed techniques for bacterial engineering (5). Listeria is not hindered by constraints on the size of the heterologous sequence, a limitation common to viral-based vector systems.…”

Directed antigen delivery as a vaccine strategy for an intracellular bacterial pathogen

“…The hly gene encoding LLO but lacking its N-terminal secretion signal sequence (cytoLLO), was cloned into a Lm site-specific integration vector (14), maintaining the native hly transcriptional and translational control elements (Fig. 1A).…”

“…The PCR products were gel purified and used as templates in a second SOE PCR using primers 260 and 038. The PCR product was digested with SacI and BamHI and ligated with the site-specific integration vector pPL2 (14) digested with the same enzymes. The resulting cytoLLO expression vector, pN8-02, was introduced into the chromosome of LLO-negative Lm strain DH-L377 as described in ref.…”