A triple-sugar regulated Salmonella vaccine protects against Clostridium perfringens-induced necrotic enteritis in broiler chickens.

Gram-positive Clostridium perfringens type G, the causative agent of necrotic enteritis (NE), has gained more attention in the poultry industry due to governmental restrictions on the use of growth-promoting antibiotics in poultry feed. Our previous work has proved that regulated delayed lysis Salmonella vaccines delivering a plasmid encoding an operon fusion of the nontoxic C-terminal adhesive part of alpha toxin and a GST-NetB toxin fusion were able to elicit significant protective immunity in broilers against C. perfringens challenge. We recently improved our S. Typhimurium antigen delivery vaccine strain by integrating a rhamnose-regulated O-antigen synthesis gene enabling a triple-sugar regulation system to control virulence, antigen-synthesis and lysis in vivo traits. The strain also includes a ΔsifA mutation that was previously shown to increase the immunogenicity of and level of protective immunity induced by Salmonella vectored influenza and Eimeria antigens. The new antigen-delivery vaccine vector system confers on the vaccine strain a safe profile and improved protection against C. perfringens challenge. The strain with the triple-sugar regulation system delivering a regulated lysis plasmid pG8R220 encoding the PlcC and GST-NetB antigens protected chickens at a similar level observed in antibiotic-treated chickens. Feed conversion and growth performance were also similar to antibiotic-treated chickens. These studies made use of a severe C. perfringens challenge with lesion formation and mortality enhanced by pre-exposure to Eimeria maxima oocysts. The vaccine achieved effectiveness through three different immunization routes, oral, spray and in drinking water. The vaccine has a potential for application in commercial hatcher and broiler-rearing conditions.

Correction to: characterizing active transportation mechanisms for free fatty acids and antibiotics in Synechocystis sp. PCC 6803.

Following publication of the original article [1], the author reported that the gene/protein names of slr2131 and sll0180 were swapped in the Discussion section. The details of the correction are mentioned below.

Characterizing active transportation mechanisms for free fatty acids and antibiotics in Synechocystis sp. PCC 6803.

BACKGROUND: Synechocystis sp. PCC 6803 is a photosynthetic bacterium that has been genetically modified to produce industrially relevant chemicals, yet efflux mechanisms have not been well elucidated. These photosynthetic organisms live in environments that are often nutrient limited; therefore, the genome of these organisms encodes far fewer proteins used for efflux of chemicals when compared to members of the Enterobacteriaceae family. Understanding efflux mechanisms can lead to a greater efficiency of chemical production within the cyanobacterial cell. RESULTS: Both sll0180 and slr2131 genes that encode the Sll0180 and Slr2131 proteins, respectively, were removed from Synechocystis sp. PCC 6803 and SD277, a high fatty acid-producing Synechocystis-based strain, to test the hypothesis that Sll0180 and Slr2131 contribute to the efflux of chemicals out of Synechocystis sp. PCC 6803 and SD277. The mutant Synechocystis sp. PCC 6803 and SD277 strains with either sll0180 or slr2131 removed from the chromosome had significantly decreased half maximal inhibitory concentrations to various antibiotics. The free fatty acid (FFA) concentration of the SD277 mutant strains increased intracellularly yet decreased extracellularly indicating that Sll0180 and Slr2131 have a role in FFA efflux. E. coli wild-type gene acrA (a homolog to sll0180) was added on a plasmid to the respective mutant strains lacking the sll0180 gene. Similarly, the E. coli wild-type gene acrB (a homolog to slr2131) was added to the respective mutant strains lacking the slr2131 gene. The tolerance to chloramphenicol of each mutant strain containing the wild-type E. coli gene was restored when compared to the parent stains. The extracellular FFA concentration of SD277 Δslr2131 with E. coli acrB increased significantly compared to both SD277 and SD277 Δslr2131. CONCLUSIONS: Two proteins involved in the transportation of antibiotics and FFAs out of the Synechocystis sp. PCC 6803 cell were identified. In an effort to alleviate costs associated with mechanically or chemically separating the cells from the FFAs, the combination of genome editing of SD277 and the addition of exogenous transport gene increased extracellular concentrations of FFAs. This understanding of active transportation is critical to improving the production efficiency for all industrially relevant chemicals produced in Synechocystis sp. PCC 6803.

Generation of influenza virus from avian cells infected by Salmonella carrying the viral genome.

Domestic poultry serve as intermediates for transmission of influenza A virus from the wild aquatic bird reservoir to humans, resulting in influenza outbreaks in poultry and potential epidemics/pandemics among human beings. To combat emerging avian influenza virus, an inexpensive, heat-stable, and orally administered influenza vaccine would be useful to vaccinate large commercial poultry flocks and even migratory birds. Our hypothesized vaccine is a recombinant attenuated bacterial strain able to mediate production of attenuated influenza virus in vivo to induce protective immunity against influenza. Here we report the feasibility and technical limitations toward such an ideal vaccine based on our exploratory study. Five 8-unit plasmids carrying a chloramphenicol resistance gene or free of an antibiotic resistance marker were constructed. Influenza virus was successfully generated in avian cells transfected by each of the plasmids. The Salmonella carrier was engineered to allow stable maintenance and conditional release of the 8-unit plasmid into the avian cells for recovery of influenza virus. Influenza A virus up to 107 50% tissue culture infective doses (TCID50)/ml were recovered from 11 out of 26 co-cultures of chicken embryonic fibroblasts (CEF) and Madin-Darby canine kidney (MDCK) cells upon infection by the recombinant Salmonella carrying the 8-unit plasmid. Our data prove that a bacterial carrier can mediate generation of influenza virus by delivering its DNA cargoes into permissive host cells. Although we have made progress in developing this Salmonella influenza virus vaccine delivery system, further improvements are necessary to achieve efficient virus production, especially in vivo.

The Asd(+)-DadB(+) dual-plasmid system offers a novel means to deliver multiple protective antigens by a recombinant attenuated Salmonella vaccine.

We developed means to deliver multiple heterologous antigens on dual plasmids with non-antibiotic-resistance markers in a single recombinant attenuated vaccine strain of Salmonella enterica serotype Typhimurium. The first component of this delivery system is a strain of S. Typhimurium carrying genomic deletions in alr, dadB, and asd, resulting in obligate requirements for diaminopimelic acid (DAP) and d-alanine for growth. The second component is the Asd(+)-DadB(+) plasmid pair carrying wild-type copies of asdA and dadB, respectively, to complement the mutations. To evaluate the protection efficacy of the dual-plasmid vaccine, S. Typhimurium strain χ9760 (a strain with multiple attenuating mutations: Δasd Δalr ΔdadB ΔrecF) was transformed with Asd(+) and DadB(+) plasmids specifying pneumococcal antigens PspA and PspC, respectively. Both plasmids were stable in χ9760 for 50 generations when grown in nonselective medium. This was significantly (P < 0.05) greater than the stability seen in its recF(+) counterpart χ9590 and could be attributed to reduced interplasmid recombination in χ9760. Oral immunization of BALB/c mice with 1 × 10(9) CFU of χ9760 (carrying Asd(+)-PspA and DadB(+)-PspC plasmids) elicited a dominant Th1-type serum IgG response against both antigens and protected mice against intraperitoneal challenge with 200 50% lethal doses (LD(50)s) of virulent Streptococcus pneumoniae strain WU2 or intravenous challenge with 100 LD(50)s of virulent S. pneumoniae strain L81905 or intranasal challenge with a lethal dose of S. pneumoniae A66.1 in a pneumonia model. Protection offered by χ9760 was superior to that offered by the mixture of two strains, χ9828 (Asd(+)-PspA) and χ11026 (DadB(+)-PspC). This novel dual-plasmid system marks a remarkable improvement in the development of live bacterial vaccines.

Comparative genome analysis of the high pathogenicity Salmonella Typhimurium strain UK-1.

Salmonella enterica serovar Typhimurium, a gram-negative facultative rod-shaped bacterium causing salmonellosis and foodborne disease, is one of the most common isolated Salmonella serovars in both developed and developing nations. Several S. Typhimurium genomes have been completed and many more genome-sequencing projects are underway. Comparative genome analysis of the multiple strains leads to a better understanding of the evolution of S. Typhimurium and its pathogenesis. S. Typhimurium strain UK-1 (belongs to phage type 1) is highly virulent when orally administered to mice and chickens and efficiently colonizes lymphoid tissues of these species. These characteristics make this strain a good choice for use in vaccine development. In fact, UK-1 has been used as the parent strain for a number of nonrecombinant and recombinant vaccine strains, including several commercial vaccines for poultry. In this study, we conducted a thorough comparative genome analysis of the UK-1 strain with other S. Typhimurium strains and examined the phenotypic impact of several genomic differences. Whole genomic comparison highlights an extremely close relationship between the UK-1 strain and other S. Typhimurium strains; however, many interesting genetic and genomic variations specific to UK-1 were explored. In particular, the deletion of a UK-1-specific gene that is highly similar to the gene encoding the T3SS effector protein NleC exhibited a significant decrease in oral virulence in BALB/c mice. The complete genetic complements in UK-1, especially those elements that contribute to virulence or aid in determining the diversity within bacterial species, provide key information in evaluating the functional characterization of important genetic determinants and for development of vaccines.

Fur-regulated iron uptake system of Edwardsiella ictaluri and its influence on pathogenesis and immunogenicity in the catfish host.

The ability of bacterial pathogens to take up iron from the host during infection is necessary for their multiplication within the host. However, host high-affinity iron binding proteins limit levels of free iron in fluids and tissues. To overcome this deficiency of iron during infection, bacterial pathogens have developed iron uptake systems that are upregulated in the absence of iron, typically tightly controlled by the ferric uptake regulator (Fur) protein. The iron uptake system of Edwardsiella ictaluri, a host-restricted pathogen of channel catfish (Ictalurus punctatus) and the main pathogen of this fish in aquaculture, is unknown. Here we describe the E. ictaluri Fur protein, the iron uptake machinery controlled by Fur, and the effects of fur gene deletion on virulence and immunogenicity in the fish host. Analysis of the E. ictaluri Fur protein shows that it lacks the N-terminal region found in the majority of pathogen-encoded Fur proteins. However, it is fully functional in regulated genes encoding iron uptake proteins. E. ictaluri grown under iron-limited conditions upregulates an outer membrane protein (HemR) that shows heme-hemoglobin transport activity and is tightly regulated by Fur. In vivo studies showed that an E. ictaluri Δfur mutant is attenuated and immune protective in zebrafish (Danio rerio) and catfish (Ictalurus punctatus), triggering systemic immunity. We conclude that an E. ictaluri Δfur mutant could be an effective component of an immersion-oral vaccine for the catfish industry.

Complete genome sequence of the universal killer Salmonella enterica Serovar Typhimurium UK-1 (ATCC 68169).

The Salmonella enterica serovar Typhimurium strain UK-1 exhibits the highest invasion and virulence attributes among the most frequently studied strains. S. Typhimurium UK-1 has been used as the foundation for developing recombinant vaccines and has been used extensively on virulence and colonization studies in chickens and mice. We describe here the complete genome sequence of S. Typhimurium UK-1. Comparative genomics of Salmonella Typhimurium will provide insight into factors that determine virulence and invasion.

Improving Salmonella vector with rec mutation to stabilize the DNA cargoes.

BACKGROUND: Salmonella has been employed to deliver therapeutic molecules against cancer and infectious diseases. As the carrier for target gene(s), the cargo plasmid should be stable in the bacterial vector. Plasmid recombination has been reduced in E. coli by mutating several genes including the recA, recE, recF and recJ. However, to our knowledge, there have been no published studies of the effect of these or any other genes that play a role in plasmid recombination in Salmonella enterica. RESULTS: The effect of recA, recF and recJ deletions on DNA recombination was examined in three serotypes of Salmonella enterica. We found that (1) intraplasmid recombination between direct duplications was RecF-independent in Typhimurium and Paratyphi A, but could be significantly reduced in Typhi by a ΔrecA or ΔrecF mutation; (2) in all three Salmonella serotypes, both ΔrecA and ΔrecF mutations reduced intraplasmid recombination when a 1041 bp intervening sequence was present between the duplications; (3) ΔrecA and ΔrecF mutations resulted in lower frequencies of interplasmid recombination in Typhimurium and Paratyphi A, but not in Typhi; (4) in some cases, a ΔrecJ mutation could reduce plasmid recombination but was less effective than ΔrecA and ΔrecF mutations. We also examined chromosome-related recombination. The frequencies of intrachromosomal recombination and plasmid integration into the chromosome were 2 and 3 logs lower than plasmid recombination frequencies in Rec+ strains. A ΔrecA mutation reduced both intrachromosomal recombination and plasmid integration frequencies. CONCLUSIONS: The ΔrecA and ΔrecF mutations can reduce plasmid recombination frequencies in Salmonella enterica, but the effect can vary between serovars. This information will be useful for developing Salmonella delivery vectors able to stably maintain plasmid cargoes for vaccine development and gene therapy.

Salmonella vaccine vectors displaying delayed antigen synthesis in vivo to enhance immunogenicity.

We have developed a regulated delayed antigen synthesis (RDAS) system for use in recombinant attenuated Salmonella vaccine (RASV) strains to enhance immune responses by reducing the adverse effects of high-level antigen synthesis. This system includes a chromosomal repressor gene, lacI, expressed from the arabinose-regulated araC PBAD promoter. LacI serves to regulate expression from a plasmid promoter, Ptrc, that directs antigen synthesis. In the presence of arabinose LacI is produced, which binds to Ptrc, blocking antigen synthesis. In vivo, an arabinose-poor environment, the concentration of LacI decreases with each cell division, allowing increased antigen synthesis. To optimize the system and for comparison, we altered the lacI ribosome-binding site, start codon, and/or codon content to construct RDAS strains chi9095, chi9959, and chi9241, synthesizing from low to high levels of LacI, respectively, and non-RDAS strain chi9555 as a control. We evaluated this system with two test antigens, the green fluorescent protein for initial in vitro assessment and the Streptococcus pneumoniae PspA protein for validation of our system in mice. All RASV strains expressing PspA generated high antilipopolysaccharide antibody titers, indicating that expression of lacI did not interfere with the capacity to induce an immune response. Strain chi9241 induced significantly higher anti-PspA IgG and IgA antibody titers than strain chi9555, which expressed PspA constitutively. Anti-PspA antibody titers were inversely correlated to the level of LacI synthesis. Strain chi9241 also induced significantly greater protective efficacy against challenge with virulent S. pneumoniae. These results suggest that regulated delayed antigen synthesis is useful for improving immunogenicity of RASV strains.

Live recombinant Salmonella Typhi vaccines constructed to investigate the role of rpoS in eliciting immunity to a heterologous antigen.

We hypothesized that the immunogenicity of live Salmonella enterica serovar Typhi vaccines expressing heterologous antigens depends, at least in part, on its rpoS status. As part of our project to develop a recombinant attenuated S. Typhi vaccine (RASTyV) to prevent pneumococcal diseases in infants and children, we constructed three RASTyV strains synthesizing the Streptococcus pneumoniae surface protein PspA to test this hypothesis. Each vector strain carried ten engineered mutations designed to optimize safety and immunogenicity. Two S. Typhi vector strains (chi9639 and chi9640) were derived from the rpoS mutant strain Ty2 and one (chi9633) from the RpoS(+) strain ISP1820. In chi9640, the nonfunctional rpoS gene was replaced with the functional rpoS gene from ISP1820. Plasmid pYA4088, encoding a secreted form of PspA, was moved into the three vector strains. The resulting RASTyV strains were evaluated for safety in vitro and for immunogenicity in mice. All three RASTyV strains were similar to the live attenuated typhoid vaccine Ty21a in their ability to survive in human blood and human monocytes. They were more sensitive to complement and were less able to survive and persist in sewage and surface water than their wild-type counterparts. Adult mice intranasally immunized with any of the RASTyV strains developed immune responses against PspA and Salmonella antigens. The RpoS(+) vaccines induced a balanced Th1/Th2 immune response while the RpoS(-) strain chi9639(pYA4088) induced a strong Th2 immune response. Immunization with any RASTyV provided protection against S. pneumoniae challenge; the RpoS(+) strain chi9640(pYA4088) provided significantly greater protection than the ISP1820 derivative, chi9633(pYA4088). In the pre-clinical setting, these strains exhibited a desirable balance between safety and immunogenicity and are currently being evaluated in a Phase 1 clinical trial to determine which of the three RASTyVs has the optimal safety and immunogenicity profile in human hosts.

New technologies in using recombinant attenuated Salmonella vaccine vectors.

Recombinant attenuated Salmonella vaccines (RASVs) have been constructed to deliver antigens from other pathogens to induce immunity to those pathogens in vaccinated hosts. The attenuation means should ensure that the vaccine survives following vaccination to colonize lymphoid tissues without causing disease symptoms. This necessitates that attenuation and synthesis of recombinant gene encoded protective antigens do not diminish the ability of orally administered vaccines to survive stresses encountered in the gastrointestinal tract. We have eliminated these problems by using RASVs with regulated delayed expression of attenuation and regulated delayed synthesis of recombinant antigens. These changes result in RASVs that colonize effector lymphoid tissues efficiently to serve as "factories" to synthesize protective antigens that induce higher protective immune responses than achieved when using previously constructed RASVs. We have devised a biological containment system with regulated delayed lysis to preclude RASV persistence in vivo and survival if excreted. Attributes were added to reduce the mild diarrhea sometimes experienced with oral live RASVs and to ensure complete safety in newborns. These collective technologies have been used to develop a novel, low-cost, RASV-synthesizing, multiple-protective Streptococcus pneumoniae antigens that will be safe for newborns/infants and will induce protective immunity to diverse S. pneumoniae serotypes after oral immunization.

Construction of recombinant attenuated Salmonella enterica serovar typhimurium vaccine vector strains for safety in newborn and infant mice.

Recombinant bacterial vaccines must be safe, efficacious, and well tolerated, especially when administered to newborns and infants to prevent diseases of early childhood. Many means of attenuation have been shown to render vaccine strains susceptible to host defenses or unable to colonize lymphoid tissue effectively, thus decreasing their immunogenicity. We have constructed recombinant attenuated Salmonella vaccine strains that display high levels of attenuation while retaining the ability to induce high levels of immunogenicity and are well tolerated in high doses when administered to infant mice as young as 24 h old. The strains contain three means of regulated delayed attenuation, as well as a constellation of additional mutations that aid in enhancing safety, regulate antigen expression, and reduce disease symptoms commonly associated with Salmonella infection. The vaccine strains are well tolerated when orally administered to infant mice 24 h old at doses as high as 3.5 x 10(8) CFU.

Salmonella enterica serovar typhimurium strains with regulated delayed attenuation in vivo.

Recombinant bacterial vaccines must be fully attenuated for animal or human hosts to avoid inducing disease symptoms while exhibiting a high degree of immunogenicity. Unfortunately, many well-studied means for attenuating Salmonella render strains more susceptible to host defense stresses encountered following oral vaccination than wild-type virulent strains and/or impair their ability to effectively colonize the gut-associated and internal lymphoid tissues. This thus impairs the ability of recombinant vaccines to serve as factories to produce recombinant antigens to induce the desired protective immunity. To address these problems, we designed strains that display features of wild-type virulent strains of Salmonella at the time of immunization to enable strains first to effectively colonize lymphoid tissues and then to exhibit a regulated delayed attenuation in vivo to preclude inducing disease symptoms. We recently described one means to achieve this based on a reversible smooth-rough synthesis of lipopolysaccharide O antigen. We report here a second means to achieve regulated delayed attenuation in vivo that is based on the substitution of a tightly regulated araC P(BAD) cassette for the promoters of the fur, crp, phoPQ, and rpoS genes such that expression of these genes is dependent on arabinose provided during growth. Thus, following colonization of lymphoid tissues, the Fur, Crp, PhoPQ, and/or RpoS proteins cease to be synthesized due to the absence of arabinose such that attenuation is gradually manifest in vivo to preclude induction of diseases symptoms. Means for achieving regulated delayed attenuation can be combined with other mutations, which together may yield safe efficacious recombinant attenuated Salmonella vaccines.

Evaluation of new generation Salmonella enterica serovar Typhimurium vaccines with regulated delayed attenuation to induce immune responses against PspA.

Increasing the immunogenicity to delivered antigens by recombinant attenuated Salmonella vaccines (RASV) has been the subject of intensive study. With this goal in mind, we have designed and constructed a new generation of RASV that exhibit regulated delayed attenuation. These vaccine strains are phenotypically wild type at the time of immunization and become attenuated after colonization of host tissues. The vaccine strains are grown under conditions that allow expression of genes required for optimal invasion and colonization of host tissues. Once established in the host, these virulence genes are turned off, fully attenuating the vaccine strain. In this study, we compared 2 of our newly developed regulated delayed attenuation Salmonella enterica serovar Typhimurium strains chi9088 and chi9558 with the Deltacya Deltacrp Deltaasd strain chi8133, for their abilities to express and present a secreted form of the alpha-helical region of pneumococcal surface protein A (PspA) to the mouse immune system. All 3 strains induced high levels of serum antibodies specific for PspA as well as to Salmonella antigens in orally immunized mice. However, both RASVs expressing delayed attenuation elicited significantly greater anti-PspA immune responses, including serum IgG and T cell secretion of IL-4 and IFN-gamma, than other groups. Also, vaccination with delayed attenuation strains resulted in a greater degree of protection against Streptococcus pneumoniae challenge than in mice vaccinated with chi8133 (71-86% vs. 21% survival, P

Regulated programmed lysis of recombinant Salmonella in host tissues to release protective antigens and confer biological containment.

We have devised and constructed a biological containment system designed to cause programmed bacterial cell lysis with no survivors. We have validated this system, using Salmonella enterica serovar Typhimurium vaccines for antigen delivery after colonization of host lymphoid tissues. The system is composed of two parts. The first component is Salmonella typhimurium strain chi8937, with deletions of asdA and arabinose-regulated expression of murA, two genes required for peptidoglycan synthesis and additional mutations to enhance complete lysis and antigen delivery. The second component is plasmid pYA3681, which encodes arabinose-regulated murA and asdA expression and C2-regulated synthesis of antisense asdA and murA mRNA transcribed from the P22 P(R) promoter. An arabinose-regulated c2 gene is present in the chromosome. chi8937(pYA3681) exhibits arabinose-dependent growth. Upon invasion of host tissues, an arabinose-free environment, transcription of asdA, murA, and c2 ceases, and concentrations of their gene products decrease because of cell division. The drop in C2 concentration results in activation of P(R), driving synthesis of antisense mRNA to block translation of any residual asdA and murA mRNA. A highly antigenic alpha-helical domain of Streptococcus pneumoniae Rx1 PspA was cloned into pYA3681, resulting in pYA3685 to test antigen delivery. Mice orally immunized with chi8937(pYA3685) developed antibody responses to PspA and Salmonella outer membrane proteins. No viable vaccine strain cells were detected in host tissues after 21 days. This system has potential applications with other Gram-negative bacteria in which biological containment would be desirable.

Analysis of type II secretion of recombinant pneumococcal PspA and PspC in a Salmonella enterica serovar Typhimurium vaccine with regulated delayed antigen synthesis.

Recombinant attenuated Salmonella vaccines (RASVs) have been used extensively to express and deliver heterologous antigens to host mucosal tissues. Immune responses can be enhanced greatly when the antigen is secreted to the periplasm or extracellular compartment. The most common method for accomplishing this is by fusion of the antigen to a secretion signal sequence. Finding an optimal signal sequence is typically done empirically. To facilitate this process, we constructed a series of plasmid expression vectors, each containing a different type II signal sequence. We evaluated the utilities of these vectors by fusing two different antigens, the alpha-helix domains of pneumococcal surface protein A (PspA) and pneumococcal surface protein C (PspC), to the signal sequences of beta-lactamase (bla SS), ompA, and phoA and the signal sequence and C-terminal peptide of beta-lactamase (bla SS+CT) on Asd(+) plasmids under the control of the P(trc) promoter. Strains were characterized for level of expression, subcellular antigen location, and the capacity to elicit antigen-specific immune responses and protection against challenge with Streptococcus pneumoniae in mice. The immune responses to each protein differed depending on the signal sequence used. Strains carrying the bla SS-pspA and bla SS+CT-pspC fusions yielded the largest amounts of secreted PspA and PspC, respectively, and induced the highest serum IgG titers, although all fusion proteins tested induced some level of antigen-specific IgG response. Consistent with the serum antibody responses, RASVs expressing the bla SS-pspA and bla SS+CT-pspC fusions induced the greatest protection against S. pneumoniae challenge.

Role of RpoS in fine-tuning the synthesis of Vi capsular polysaccharide in Salmonella enterica serotype Typhi.

Regulation of the synthesis of Vi polysaccharide, a major virulence determinant in Salmonella enterica serotype Typhi, is under the control of two regulatory systems, ompR-envZ and rscB-rscC, which respond to changes in osmolarity. Some serotype Typhi strains exhibit overexpression of Vi polysaccharide, which masks clinical detection of lipopolysaccharide O antigen. This variation in Vi polysaccharide and O antigen display (VW variation) has been observed since the initial studies of serotype Typhi. In this study, we report that rpoS plays a role in this increased expression in Vi polysaccharide. We constructed a variety of isogenic serotype Typhi mutants that differed in their expression levels of RpoS and examined the role of the rpoS product in synthesis of Vi polysaccharide under different osmolarity conditions. Vi polysaccharide synthesis was also examined in serotype Typhi mutants in which the native promoter of the rpoS was replaced by an araCP(BAD) cassette, so that the expression of rpoS was arabinose dependent. The RpoS(-) strains showed increased syntheses of Vi polysaccharide, which at low and medium osmolarities masked O antigen detection. In contrast, RpoS(+) strains showed lower syntheses of Vi polysaccharide, and an increased detection of O antigen was observed. During exponential growth, when rpoS is unstable or present at low levels, serotype Typhi RpoS(+) strains overexpress the Vi polysaccharide at levels comparable to those for RpoS(-) strains. Our results show that RpoS is another regulator of Vi polysaccharide synthesis and contributes to VW variation in serotype Typhi, which has implications for the development of recombinant attenuated Salmonella vaccines in humans.

A recombinant attenuated Salmonella enterica serovar Typhimurium vaccine encoding Eimeria acervulina antigen offers protection against E. acervulina challenge.

Coccidiosis is a ubiquitous disease caused by intestinal protozoan parasites belonging to several distinct species of the genus Eimeria. Cell-mediated immunity (CMI) is critically important for protection against Eimeria; thus, our approach utilizes the bacterial type III secretion system (TTSS) to deliver an antigen directly into the cell cytoplasm of the immunized host and into the major histocompatibility complex class I antigen-processing pathway for induction of CMI and antigen-specific cytotoxic T-lymphocyte responses in particular. To accomplish this goal, Eimeria genes encoding the sporozoite antigen EASZ240 and the merozoite antigen EAMZ250 were fused to the Salmonella enterica serovar Typhimurium effector protein gene sptP in the parental pYA3653 vector, yielding pYA3657 and pYA3658, respectively. SptP protein is secreted by the TTSS of Salmonella and translocated into the cytoplasm of immunized host cells. The host strain chromosomal copy of the sptP gene was deleted and replaced by a reporter gene, xylE. The newly constructed vectors pYA3657 and pYA3658 were introduced into host strain chi8879 (DeltaphoP233 DeltasptP1033::xylEDelta asdA16). This strain is an attenuated derivative of the highly virulent strain UK-1. When strain chi8879(pYA3653) as the vector control and strain chi8879 harboring pYA3657 or pYA3658 were used to orally immunize day-of-hatch chicks, colonization of the bursa, spleen, and liver was observed, with peak titers 6 to 9 days postimmunization. In vitro experiments show that the EASZ240 antigen is secreted into the culture supernatant via the TTSS and that it is delivered into the cytoplasm of Int-407 cells by the TTSS. In vivo experiments indicate that both humoral and cell-mediated immune responses are induced in chickens vaccinated with a recombinant attenuated Salmonella serovar Typhimurium vaccine, which leads to significant protection against Eimeria challenge.