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Vaccines rank amongst the most effective public health tools for lowering the incidence of infectious diseases. It has recently been shown that administration of nucleic acids, typically DNA, to animals can elicit both humoral and cytotoxic T-lymphocyte responses to a variety of antigens (4). A chief aim of vaccine researchers is to expand this technique of DNA immunization to the prevention of mucosal infections. Diseases of the mucosadefined as the epithelial surfaces of the gastrointestinal, respiratory, and urogenital tractsare responsible for a substantial portion of the world's infectious disease morbidity and mortality. For example, respiratory infections and diarrheal diseases alone currently claim more than 10 million lives each year (17). Protection against diseases of the mucosal surface is thought to occur by a process that involves local antigen exposure followed by induction of humoral antibodies, particularly secretory immunoglobulin A (15). However, little work on how to induce mucosal immune responses by DNA-based vaccination has been done.

An attractive concept for delivering DNA vaccines to the mucosal epithelium is the use of live attenuated bacterial vectors. Sizemore et al. (18) first reported the successful use of an attenuated Shigella flexneri mutant as a delivery system for DNA. More recently, Dietrich et al. (3) have explored the use of Listeria monocytogenes for the same application. Because both of these organisms have an inherent ability to invade the cells of the mucosal epithelium, they are attractive delivery vectors; however, concerns about reactogenicity (i.e., adverse events in vaccinees) also make both organisms problematic for human use (13). In contrast, live, attenuated vaccines derived from Vibrio cholerae and Salmonella species represent promising mucosal vaccines that have presented far fewer reactogenicity problems in humans (1, 11, 12). These organisms have the ability to enter the M cells of the gut epithelium and induce mucosal immune responses (15) but have not been explored as DNA delivery vectors.

A key step required for this application is to devise methods that allow the programmed lysis of these organisms in a way that would allow bacterial cells to deliver DNA constructs to the eukaryotic cell cytosol. Here we have investigated whether phage lysis proteins, the S and R gene products of phage lambda (9), can be used to lyse V. cholerae or Salmonella enterica serovar Typhimurium cells.

Construction and testing of inducible lysis plasmid in different strain backgrounds. Phage lambda encodes two proteins (S and R) which are sufficient to cause bacterial cell lysis (8, 9, 20). In Escherichia coli, the protein product of the S gene is termed "holin" and oligomerizes to form a channel in the bacterial inner membrane. The R gene encodes a transglycosylase which gains access to the periplasm via this channel and then proceeds to degrade the cell wall, causing lysis. The R_Z gene product has no known function but is part of most E. coli "SR lysis cassettes" that have been studied previously (20). To test the effect of these lysis proteins on various bacterial species, we constructed a plasmid (pVJ4) that expressed the S, R, and R_Z genes under the control of the arabinose-inducible promoter P_BAD (10). This plasmid was constructed by cloning the 1.5-kbp EcoRI-HindII fragment of plasmid pRY100 (generously provided by Ry Young, Texas A&M University, College Station, Tex.), containing S, R, and R_Z genes, into plasmid pBAD18 (10). Plasmid pVJ4 and control plasmid pBAD18 or pBAD24 were introduced into E. coli DH5, V. cholerae O395, and Salmonella serovar Typhimurium CDC 6516-60 (Table 1), and the resultant six strains were tested for arabinose-induced cell lysis.

View larger version (22K): [in this window] [in a new window]   FIG. 1.   Effects of induced phage lysin expression on growth of bacterial cells. E. coli DH5 cells carrying either pBAD18 (A) or pVJ4 (B) were grown in the absence (circles) or presence (triangles) of 0.02% arabinose. Growth was monitored by optical density measurements. Growth of Salmonella serovar Typhimurium CDC 6516-60 (C and D) and V. cholerae O395 (E and F) were similarly quantitated.

Measurement of DNA released by cell lysis. Given that arabinose induction of the three different strains carrying pVJ4 caused the apparent cell lysis, we next addressed whether the lysis process mediated by S and R was compatible with the release of intact plasmid DNA molecules into the surrounding media. Towards this end, nucleic acids were purified from the extracellular media of these lysed cell cultures. Southern blot analysis confirmed that intact plasmid was being released by the SR-mediated lysis process (data not shown). To better assess how much plasmid DNA was being released by these three different bacterial species, DNA from cell free culture supernatant fluids was purified and then transformed into E. coli under standard conditions. The number of transformants per CFU per milliliter provided a quantitative assessment of the level of intact plasmid DNA that was released by arabinose induction of control (pBAD18) and pVJ4-carrying cells.

View larger version (18K): [in this window] [in a new window]   FIG. 2.   (A) Quantitation of transformation efficiency of released plasmids. Plasmid purified from culture supernatants was transformed into electrocompetent E. coli, and the resulting numbers of transformants were counted. Ratios of transformants to CFU of the culture per milliliter at the time of lysis were calculated for the three strains under study. (B) Analysis of the effects of DNase mutations on efficiency of plasmid release. Ratios of transformants to CFU of cultures of V754 and V751 as well as their isogenic wild-type strains per milliliter were determined as before. Values represent means of triplicate experiments; error bars represent standard errors of the means.

Effects of V. cholerae DNases on the levels of DNA release. To prove that the degradative action of DNase produced by V. cholerae O395 was reducing the level of intact plasmid in our lysis experiments, we repeated these with two V. cholerae strains that carried mutations that affect the production of DNase (kindly provided by Paul Manning). Strain V754 carries a mutation in the dns gene, one of the two genes implicated in V. cholerae DNase secretion (7), while strain V751 is a double-knockout strain lacking both dns and xds, the structural gene for a major V. cholerae DNase (16). The parental strains of V754 and V751 are strains V33 and V62, respectively. Plasmid pVJ4 or control plasmid pBAD18 was introduced into these four strains, and release of plasmid DNA was monitored with and without arabinose inductions by quantitative transformation assays. As shown in Fig. 2B, approximately 24-fold more plasmid was released by strain V754 than by its parental strain, V33, suggesting that the dns mutation does increase the yield of intact plasmid released by arabinose-induced lysis. Furthermore, strain V751 released approximately 240 times more plasmid after arabinose induction than its parental strain V62 (Fig. 2B). Because strain V751 has a more severe defect in the secretion of extracellular DNase (due to its double mutations in both dns and xds (6, 16), these results indicate that the presence of extracellular DNases is detrimental to the recovery of DNA released by arabinose induction of lysis plasmid pJV4. Interestingly, the peak ratio of transformants to number of CFU per milliliter achieved with the double DNase-knockout strain V751 was the highest for all V. cholerae strains tested and higher than that seen with Salmonella serovar Typhimurium. It is not known whether the Salmonella strains examined here make DNases, but our results strongly suggest that deletion of DNase genes might improve the utility of live, bacterial DNA delivery vectors derived from this or other bacterial species.