CS5 pilus biosynthesis genes from enterotoxigenic Escherichia coli O115:H40.

We have sequenced the entire region of DNA required for the biosynthesis of CS5 pili from enterotoxigenic Escherichia coli O115:H40 downstream of the major subunit gene, designated csfA (for coli surface factor five A). Five more open reading frames (ORFs) (csfB, csfC, csfE, csfF, and csfD) which are transcribed in the same direction as the major subunit and are flanked by a number of insertion sequence regions have been identified. T7 polymerase-mediated overexpression of the cloned csf ORFs confirmed protein sizes based on the DNA sequences that encode them. The expression of only the csf region in E. coli K-12 resulted in the hemagglutination of human erythrocytes and the cell surface expression of CS5 pili, suggesting that the cluster contains all necessary information for CS5 pilus biogenesis and function.

Salmonella vaccine carrier strains: effective delivery system to trigger anti-tumor immunity by oral route.

Recombinant Salmonella strains expressing heterologous antigens can be delivered by oral route triggering the elicitation of efficient antigen-specific humoral, T helper and cytotoxic responses. The potential of attenuated Salmonella spp. to trigger anti-tumor immunity was evaluated for the first time by using beta-galactosidase (beta-gal) as a model tumor-associated antigen (TAA). Beta-gal was expressed in a Salmonella typhimurium aroA vaccine carrier strain either constitutively or under the control of a promoter activated upon infection. Oral immunization with both vaccine prototypes resulted in the elicitation of beta-gal-specific humoral and cell-mediated immunity. Although both strains were able to trigger antigen-specific CTL, responses were more efficient when the expression was controlled by the promoter activated upon infection. The anti-tumor efficacy of the stimulated response was validated by challenging vaccinated animals with an aggressive fibrosarcoma transfected with beta-gal, which operationally acts as a TAA. Both groups of vaccinated mice exhibited a significant reduction in tumor take and growth with respect to animals vaccinated with plasmidless carrier (p < 0.05). However, the overall efficiency was better in the group in which beta-gal was controlled by the in vivo-activated promoter (85% versus 54%; p < 0.05).

Green fluorescent protein-based reporter systems for genetic analysis of bacteria including monocopy applications.

The green fluorescent protein (GFP) gene, gfp, was used to develop versatile reporter systems for genetic analysis in, and monitoring of bacteria. This reporter system is available on a plasmid and on a mini-transposon located in a suicide delivery plasmid for generation of chromosomal fusions. To achieve sensitivity levels necessary for use in monocopy applications and for detection of single cells, the 3'-end of gfp was replaced by that of a modified gfp gene characterized by a 45-fold stronger fluorescence signal than that exhibited by the natural GFP. This modified gfp gene was also equipped with the strong translation signals of the atpE gene. Transfer of the mini-transposon into two different Pseudomonas spp. and Alcaligenes eutrophus produced random chromosomal fusions, some 5% of which exhibited fluorescence detectable by eye. Individual GFP+ cells were readily observed by fluorescence microscopy.

Stable expression of pertussis toxin in Bordetella bronchiseptica under the control of a tightly regulated promoter.

Pertussis toxin (PT) is an essential component of accellular vaccines against whooping cough. However, the industrial production of PT from Bordetella pertussis is impaired by slow growth and poor yields. To overcome these problems, we have constructed a minitransposon containing the tox operon under the control of a tightly regulated promoter responsive to an aromatic inducer. The expression cassettes have been integrated into the chromosome of Bordetella bronchiseptica 5376 and ATCC 10580 bvg. Five recombinant clones containing the tox operon under the control of the Psal promoter, which is activated by the product of nahR, were further characterized. The recombinant clones expressed PT after only 3 h of induction with sodium salicylate at levels similar to those of B. pertussis grown for 24 h. The stability of the engineered phenotype was 100% after 72 h of growth without selective pressure. The growth pattern was not modified either under noninducing conditions or in the presence of the inducer at low concentrations, suggesting that strain performance would not be affected in bioreactors when uncoupled from gene expression. Recombinant PT, which was localized mainly in the periplasm, was purified by affinity chromatography. The recombinant protein was immunologically indistinguishable from wild-type PT and retained its biological activity as determined by the CHO cell-clustering test. These recombinant clones appear to be useful tools for the cost-effective production of PT under conditions of improved biosafety, as demonstrated by the inducible expression of PT uncoupled from the bacterial biomass in a nonvirulent and fast-growing B. bronchiseptica background.

Identification of Salmonella typhi promoters activated by invasion of eukaryotic cells.

The interaction of pathogenic microorganisms with host tissues, and the underlying genetic events which regulate these interactions, are difficult to analyse where no suitable animal model exists. The approach described here, for obtaining information on the genes involved in these interactions, employs an infection system based on the invasion of Henle cells by Salmonella typhi to select promoter-containing DNA sequences able to activate gene expression inside eukaryotic cells. Several DNA fragments exhibiting different promoter strengths and extent of selective activation within eukaryotic cells were identified. Three were selected and characterized according to the expression level of the reporter gene, the polynucleotide sequence, the transcription start, and the dependence upon regulatory proteins. All fragments gave much stronger expression of the reporter gene when the recombinant S. typhi carrier strains invaded cells compared with the expression measured in growth medium. One promoter-containing region exhibited sequence homology to sigma 54-dependent promoters, whereas another appears to be dependent on the stationary-phase RNA polymerase subunit sigma s. S. typhi containing the S1 subunit gene of pertussis toxin cloned under the control of these promoters, selectively expressed the S1 subunit following infection of different phagocytic and non-phagocytic cell lines of human or murine origin. Deletion and point mutant derivatives of two promoters enabled the identification of the main motif required for promoter activity. This method may be helpful for the analysis of pathogenesis in organisms previously difficult to study because of the lack of a convenient animal model, and could provide insights into the chronology and topology of gene expression during infection, including a possible genetic basis for tissue tropism.

Export of Bordetella pertussis serotype 2 and 3 fimbrial subunits by Escherichia coli.

Bordetella pertussis serotype 2 and 3 fimbrial subunits were expressed and exported in Escherichia coli using the recently described expression/secretion vector pCGV1. Two protease deficient E. coli strains (CAG629 and EC538) and two periplasmic-leaky mutants (AE84064 and A593) were transformed with the different constructs and, after thermal induction, proteins present in the various cellular compartments were analyzed by Western blot. The results obtained with the two types of fimbrial subunits were generally the same: a recombinant protein of the expected molecular mass (19.2 kDa) was present in the periplasm of the leaky mutants and of CAG629 strain (Ion protease- and heat shock protease-deficient). Only the expression of the recombinant fimbrial subunits by the tolB A593 mutant resulted in protein release into the extracellular medium. These results indicate that the use of hybrid plasmids based on pCGV1 in combination with the tolB mutant constitute an efficient system for the export of recombinant proteins.