Stephen R. Attridge

Regulation of Salmonella typhimurium lipopolysaccharide O antigen chain length is required for virulence; identification of FepE as a second Wzz

Wzz proteins regulate the degree of polymerization of the O antigen (Oag) subunits in lipopolysaccharide (LPS) biosynthesis. Although the pathogenic relevance of Oag is well recognized, the significance of Oag chain length regulation is not well defined. In this report, Salmonella typhimurium was shown to possess two functional wzz genes resulting in a bimodal Oag length distribution. In addition to the previously described wzzST that results in long (L) modal length LPS with 16–35 Oag repeat units (RUs), we now report that wzzfepE, a homologue of Escherichia coli fepE, is responsible for the production of very long (VL) modal length LPS Oag, estimated to contain> 100 Oag RUs. Analysis of a series of isogenic S. typhimurium C5 mutants found that the presence of either wzz gene (and hence either modal length) was sufficient for complement resistance and virulence in the mouse model of infection, suggesting a degree of redundancy in the role of these two wzz genes and their respective Oag modal lengths. In contrast, the wzzST/wzzfepE double mutant, with relatively short, random‐length Oag, displayed enhanced susceptibility to complement and was highly attenuated in the mouse. This clearly demonstrates the molecular genetic basis for the longer LPS Oag chains previously identified as the basis of complement resistance in Salmonella. The presence of wzzfepE homologues in the genomic sequences of strains of Escherichia coli, Shigella flexneri and multiple serovars of Salmonella suggests that bimodality of LPS Oag is a common phenomenon in the Enterobacteriaceae.

A chromosomal integration system for stabilization of heterologous genes in Salmonella based vaccine strains

We have developed a system whereby heterologous DNA encoding an antigen from an enteropathogen may be recombined into the chromosome of an attenuated Salmonella carrier strain. The system involves two steps: (i) integration of a hisOG deletion mutation into the chromosome; (ii) replacement of the hisOG deletion by the complete hisOG region and the segment of heterologous DNA which encodes the antigen of interest. Recombinants may be selected (his+). The system was used to integrate the genes encoding K88 fimbriae from enterotoxigenic Escherichia coli into the chromosome of a galE mutant of Salmonella typhimurium (LT2H1). Recombinants were detected at a frequency of between 1.0 x 10(-3) and 1.5 x 10(-3). A variety of tests confirmed that the K88 genes were integrated into the chromosome of LT2H1 and were expressed. The stability of the recombinant was tested both in vivo and in vitro. When administered orally to mice, the recombinant elicited a serum antibody response to K88, and retained the Salmonella vaccine potential of the vector strain.

Oral delivery of foreign antigens by attenuated Salmonella: Consequences of prior exposure to the vector strain

Several strains of Salmonella have been used as vectors for the delivery of Escherichia coli fimbrial proteins to the gut-associated lymphoid tissue (GALT) of the mouse. Plasmids carrying a complementing thyA+ gene, together with genes specifying synthesis of K88 or K99, were introduced into non-reverting thyA Salmonella mutants. The resulting constructs expressed the foreign pilin protein on their surfaces and, provided the vector was able to colonize the GALT, elicited strong serum responses to K88 or K99. These responses were dramatically impaired however, in recipients with pre-existing immunity to the vector strain. Mice initially infected with Salmonella stanley ca 4, 10 or 20 weeks prior to oral administration of S. stanley-K88 showed greatly reduced serum responses to K88 as determined by ELISA. The hypo-responsiveness seen in vector-primed mice could be largely overcome by changing the serotype of the strain subsequently used to deliver the foreign protein.

Significance of toxin-coregulated pili as protective antigens of vibrio cholerae in the infant mouse model

The infant mouse cholera model has been used to evaluate the relative importance of toxin-coregulated pili (TCP) as protective antigens of Vibrio cholerae 01. Electron microscopic and immunoblotting analyses revealed that, under the cultural conditions examined, TCP were only expressed by strains of classical biotype. Antibodies to TCP were sufficient to confer protection against two such strains, and were more efficient if the challenge vibrios were cultured for TCP expression. In contrast, such antibodies did not protect mice against challenge with any of four strains of El Tor biotype. Since two of the latter have previously been shown to possess non-lipopolysaccharide protective antigens, these results suggest that TCP are not the only such antigen in this model.

Characterization and immunogenicity of Vibrio cholerae ghosts expressing toxin-coregulated pili

Bacterial ghosts are attractive for use as non-living vaccines and as carriers of heterologous antigens of vaccine relevance. Ghosts were prepared from Vibrio cholerae strains of O1 or O139 serogroup after growth under culture conditions, which favor or repress the production of toxin-coregulated pili (TCP). Immunoblotting confirmed the TCP status of these V. cholerae ghosts (VCG), which retained the cellular morphology and envelope sub-component profile of viable bacteria. Rabbits were immunized with VCGs prepared from O139 bacteria with TCP-positive or TCP-negative phenotypes and the resulting sera assayed for antibodies to lipopolysaccharide (LPS) and to TCP. Regardless of the TCP status of the VCG preparations used for immunization, all animals produced antibodies to LPS as demonstrated in bactericidal assays. These antibodies were probably responsible for the capacity of the antisera to confer passive immunity to challenge with the homologous O139 strain in the infant mouse cholera model (IMCM). Only following immunization with TCP-positive VCG, however, were antibodies to TCP generated, as judged by the potential of antisera to mediate protection against a challenge strain of heterologous serogroup.

The toxin-coregulated pilus (TCP) of Vibrio cholerae: molecular cloning of genes involved in pilus biosynthesis and evaluation of TCP as a protective antigen in the infant mouse model

A serum containing antibodies to non-lipopolysaccharide (non-LPS) protective antigens of Vibrio cholerae has been used, after extensive absorption, to facilitate the cloning of genes involved in the synthesis of toxin-coregulated pili (TCP). A gene bank was constructed from V. cholerae Z17561 DNA using a mobilizable cosmid vector in Escherichia coli, and subsequently transferred by conjugation into V. cholerae O17. This strain does not produce TCP in vitro and lacks non-LPS protective antigens. Eight positive clones were isolated, and of these, four produced TCP as determined by electron microscopic and immunoblotting analyses. TCP-positive O17 clones were 70-fold more virulent than TCP-negative clones or O17 in the infant mouse cholera model. Only the former could remove protective antibodies from the clone-probing serum by absorption. As a corollary, serum containing antibodies to TCP protected mice from challenge with TCP-positive clones, but not with TCP-negative clones or O17. Our data indicate that TCP can function as both a virulence determinant and a protective antigen in the infant mouse model.

Comparison of the promoter proximal regions of the toxin-co-regulated tcp gene cluster in classical and E1 Tor strains of Vibrio cholerae O1

A physical map has been constructed of the 5-kb XbaI fragment encoding the promoter proximal of region the tcp gene cluster encoding the toxin-coregulated pilus (TCP) of Vibrio cholerae. This fragment contains the major regulatory regions for TCP. Comparison of the nucleotide (nt) sequences from strains of the classical and El Tor biotypes demonstrates that the regions are essentially identical, with several notable exceptions. The intergenic regions, between tcpI and tcpP, and between tcpH and tcpA, show significant sequence divergence which may account for the biotype-related differences in TCP, since this is the location of the major promoter sequences. The C-terminal coding regions of the major pilin subunit, TcpA, also differ. Southern hybridization analyses suggest that the tcpA nt sequence is conserved within a biotype, and Western blot analysis suggests that the two forms of TcpA are antigenically different, but related. Besides tcpA, tcpB, tcpH and tcpI, the genes encoding two additional proteins, TcpP and TcpQ, but not previously defined, were also identified. TcpH and TcpI have been previously suggested to be regulatory proteins but homology data imply that TcpI is a methyl-accepting chemotaxis protein (MCP), as recently reported [Harkey et al., Infect. Immun. 62 (1994) 2669-2678], and TcpH is predicted to be a periplasmic or exported protein. TcpP is thought to be a trans-cytoplasmic membrane (CM) protein which may have a regulatory role.

Impact of vector priming on the immunogenicity of recombinant Salmonella vaccines.

ABSTRACT There are conflicting reports concerning the impact of prior vector priming on the immunogenicity of recombinant-Salmonella-based vaccines. A comparison of experimental protocols identified two variables which might account for this inconsistency: the potential of the vector strain to colonize the murine gut-associated lymphoid tissue (GALT) and the nature of the foreign antigen subsequently delivered by the recombinant Salmonella construct. The former was investigated by constructing an aroA mutant of the Salmonella enterica serovar Stanley vector previously used in our laboratory. Although the introduction of an aroA mutation had surprisingly little effect on GALT colonization, it did reduce the strength of antilipopolysaccharide (anti-LPS) antibody responses and the impact of vector priming. Studies were also performed to ascertain the extent to which any observed hyporesponsiveness consequent upon vector priming might be determined by the characteristics of the foreign antigen. S. enterica serovar Stanley was used to deliver either of two Escherichia coli antigens, K88 pilus protein or the LT-B toxin subunit, to vector-primed mice. Both serum immunoglobulin G (IgG) and intestinal IgA responses to K88 were completely abolished, and those to LT-B were significantly reduced, as a consequence of vector priming. When similar experiments were performed with an aroA S. enterica serovar Dublin vector, responses to K88 were significantly reduced but those to LT-B were unaffected by vector priming. Paradoxically, a priming infection with this vector induced stronger anti-LPS antibody responses but was less likely to elicit a state of hyporesponsiveness to subsequently presented foreign antigen. The impact of vector priming thus depends on both the Salmonella strain used and the nature of the foreign antigen, but our present data strengthen concerns that preexisting antivector immunity represents a serious threat to the Salmonella-based vaccine strategy.

Construction of K88- and K99-expressing clones of Salmonella typhimurium G30: immunogenicity following oral administration to pigs

Salmonella typhimurium G30 was used as a vector to express the ETEC (enterotoxigenic Escherichia coli) fimbrial antigens K88 and K99. Two plasmids encoding K88 or K99 production and having a non-antibiotic selection marker (thyA+) were constructed. These were introduced into a thyA G30 derivative to give the vaccine strains EX841 and EX603, which were shown to express surface K88 or K99, respectively. When administered orally to adult pigs, a dose of 10(11) vaccine organisms elicited significant serum antibody responses to the respective fimbrial antigens. Two such immunizations with EX841 generated serum antibody levels comparable to those obtained with intramuscular injection of killed organisms. Attenuated salmonellae can thus be used to deliver ETEC fimbrial antigens to the porcine intestinal immune system.

Sensitive Microplate Assay for Detection of Bactericidal Antibodies to Vibrio cholerae O139

ABSTRACT A microplate assay for the detection of bactericidal antibodies to Vibrio cholerae O139 is described. The assay is sensitive, highly reproducible, specific, and convenient to perform. It has been used to demonstrate the induction of serum bactericidal antibodies in Vietnamese recipients of an oral, inactivated, bivalent O1/O139 vaccine, as well as in Bangladeshi patients with O139 disease. In both study groups there was a significant inverse correlation between the preexposure level of antibodies in serum and the magnitude of the subsequent bactericidal response. Although infection generated stronger responses than vaccination, the proportion of responders was similar among individuals with low background titers.