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Salmonella enterica cause diarrheal and systemic diseases and are of considerable concern worldwide. Vaccines that are cross-protective against multiple serovars could provide effective control of Salmonella-mediated diseases. Bacteria-derived outer membrane vesicles (OMVs) are highly immunogenic and are capable of eliciting protective immune responses. Alterations in lipopolysaccharide (LPS) length can result in outer membrane remodeling and composition of outer membrane proteins (OMPs) changing. In this study, we investigated the impact of truncated LPS on both the production and immunogenicity of Salmonella OMVs, including the ability of OMVs to elicit cross-protection against challenge by heterologous Salmonella strains. We found that mutations in waaJ and rfbP enhanced vesiculation, while mutations in waaC, waaF and waaG inhibited this process. Animal experiments indicated that OMVs from waaC, rfaH and rfbP mutants induced stronger serum immune responses compared to OMVs from the parent strain, while all elicited protective responses against the wild-type S. Typhimurium challenge. Furthermore, intranasal or intraperitoneal immunization with OMVs derived from the waaC and rfbP mutants elicited significantly higher cross-reactive IgG responses and provided enhanced cross-protection against S. Choleraesuis and S. Enteritidis challenge than the wild-type OMVs. These results indicate that truncated-LPS OMVs are capable of conferring cross protection against multiple serotypes of Salmonella infection.

Salmonella Typhi is the causative agent of typhoid fever in humans, responsible for approximately 21 million infections and 222,000 deaths globally each year. The current licensed vaccines provide moderate protection to recipients aged >2 years. Prior work on typhoid vaccines has focused on injectable Vi capsular polysaccharide or Vi–protein conjugates and live, oral attenuated S. Typhi vaccines to induce humoral anti-Vi antibodies, while the value and importance of anti-O9 antibodies is less well established. In this study, we constructed a S. Typhimurium strain that synthesizes Vi capsular antigen in vivo and produces the immunodominant O9-antigen polysaccharide instead of its native O4-antigen. The live recombinant attenuated S. Typhimurium mutants were effective in stimulating anti-Vi and anti-O9 antibodies in a mouse model, and the surface Vi capsular expression did not affect the immune responses against the O9 O-antigen polysaccharide. Moreover, the resulting anti-Vi and anti-O9 antibodies were effective at killing S. Typhi and other Salmonella spp. expressing Vi or O9 antigen polysaccharides and provided efficient protection against lethal challenge by S. Typhimurium and S. Enteritidis. Our work highlights the strategy of developing live attenuated S. Typhimurium vaccines to prevent typhoid fever by targeting the both Vi capsular and O9 O-polysaccharide antigens simultaneously.

Salmonella enterica serovar Typhi causes the systemic disease typhoid fever. After ingestion, it adheres to and invades the host epithelium while evading the host innate immune response, causing little if any inflammation. Conversely, Salmonella enterica serovar Typhimurium causes gastroenteritis in humans and thrives in the inflamed gut. Upon entering the host, S . Typhimurium preferentially colonizes Peyer's patches, a lymphoid organ in which microfold cells (M cells) overlay an arrangement of B cells, T cells, and antigen-presenting cells. Both serovars can adhere to and invade M cells and enterocytes, and it has been assumed that S . Typhi also preferentially targets M cells. In this study, we present data supporting the alternative hypothesis that S . Typhi preferentially targets enterocytes. Using a tissue culture M cell model, we examined S . Typhi strains with a deletion in the stg fimbriae. The stg deletion resulted in increased adherence to M cells and, as expected, decreased adherence to Caco-2 cells. Adherence to M cells could be further enhanced by introduction of the long polar fimbriae (Lpf), which facilitate adherence of S . Typhimurium to M cells. Deletion of stg and/or introduction of lpf enhanced M cell invasion as well, leading to significant increases in secretion of interleukin 8. These results suggest that S . Typhi may preferentially target enterocytes in vivo .

Vesicle shedding from bacteria is a universal process in most Gram-negative bacteria and a few Gram-positive bacteria. In this report, we isolate extracellular membrane vesicles (MVs) from the supernatants of Gram-positive pathogen Clostridium perfringens (C. perfringens). We demonstrated vesicle production in a variety of virulent and nonvirulent type A strains. MVs did not contain alpha-toxin and NetB toxin demonstrated by negative reaction to specific antibody and absence of specific proteins identified by LC-MS/MS. C. perfringens MVs contained DNA components such as 16S ribosomal RNA gene (16S rRNA), alpha-toxin gene (plc) and the perfringolysin O gene (pfoA) demonstrated by PCR. We also identified a total of 431 proteins in vesicles by 1-D gel separation and LC-MS/MS analysis. In vitro studies demonstrated that vesicles could be internalized into murine macrophage RAW264.7 cells without direct cytotoxicity effects, causing release of inflammation cytokines including granulocyte colony stimulating factor (G-CSF), tumor necrosis factor-alpha (TNF-α) and interleukin-1 (IL-1), which could also be detected in mice injected with MVs through intraperitoneal (i.p.) route. Mice immunized with C. perfringens MVs produced high titer IgG, especially IgG1, antibodies against C. perfringens membrane proteins. However, this kind of antibody could not provide protection in mice following challenge, though it could slightly postpone the time of death. Our results indicate that release of MVs from C. perfringens could provide a previously unknown mechanism to induce release of inflammatory cytokines, especially TNF-α, these findings may contribute to a better understanding of the pathogenesis of C. perfringens infection.

Colanic Acid (CA) and lipopolysaccharide (LPS) are two major mannose-containing extracellular polysaccharides of Salmonella. Their presence on the bacterial surface can mask conserved protective outer membrane proteins (OMPs) from the host immune system. The mannose moiety in these molecules is derived from GDP-mannose, which is synthesized in several steps. The first two steps require the action of phosphomannose isomerase, encoded by pmi (manA), followed by phosphomannomutase, encoded by manB. There are two copies of manB present in the Salmonella chromosome, one located in the cps gene cluster (cpsG) responsible for CA synthesis, and the other in the rfb gene cluster (rfbK) involved in LPS O-antigen synthesis. In this study, it was demonstrated that the products of cpsG and rfbK are isozymes. To evaluate the impact of these genes on O-antigen synthesis, virulence and immunogenicity, single mutations (Δpmi, ΔrfbK or ΔcpsG) and a double mutation (ΔrfbK ΔcpsG) were introduced into both wild-type Salmonella enterica and an attenuated Δcya Δcrp vaccine strain. The Δpmi, ΔrfbK and ΔcpsG ΔrfbK mutants were defective in LPS synthesis and attenuated for virulence. In orally inoculated mice, strain S122 (Δcrp Δcya ΔcpsG ΔrfbK) and its parent S738 (Δcrp Δcya) were both avirulent and colonized internal tissues. Strain S122 elicited higher levels of anti-S. Typhimurium OMP serum IgG than its parent strain. Mice immunized with S122 were completely protected against challenge with wild-type virulent S. Typhimurium and partially protected against challenge with either wild-type virulent S. Choleraesuis or S. Enteritidis. These data indicate that deletions in rfbK and cpsG are useful mutations for inclusion in future attenuated Salmonella vaccine strains to induce cross-protective immunity.

Lipopolysaccharide (LPS) is a major virulence factor of Salmonella enterica serovar Typhimurium and is composed of lipid A, core oligosaccharide (C-OS), and O-antigen polysaccharide (O-PS). While the functions of the gene products involved in synthesis of core and O-antigen have been elucidated, the effect of removing O-antigen and core sugars on the virulence and immunogenicity of Salmonella enterica serovar Typhimurium has not been systematically studied. We introduced nonpolar, defined deletion mutations in waaG ( rfaG ), waaI ( rfaI ), rfaH , waaJ ( rfaJ ), wbaP ( rfbP ), waaL ( rfaL ), or wzy ( rfc ) into wild-type S. Typhimurium. The LPS structure was confirmed, and a number of in vitro and in vivo properties of each mutant were analyzed. All mutants were significantly attenuated compared to the wild-type parent when administered orally to BALB/c mice and were less invasive in host tissues. Strains with Δ waaG and Δ waaI mutations, in particular, were deficient in colonization of Peyer's patches and liver. This deficiency could be partially overcome in the Δ waaI mutant when it was administered intranasally. In the context of an attenuated vaccine strain delivering the pneumococcal antigen PspA, all of the mutations tested resulted in reduced immune responses against PspA and Salmonella antigens. Our results indicate that nonreversible truncation of the outer core is not a viable option for developing a live oral Salmonella vaccine, while a wzy mutant that retains one O-antigen unit is adequate for stimulating the optimal protective immunity to homologous or heterologous antigens by oral, intranasal, or intraperitoneal routes of administration.

Three great plague pandemics caused by the gram-negative bacterium Yersinia pestis have killed nearly 200 million people and it has been linked to biowarfare in the past. Plague is endemic in many parts of the world. In addition, the risk of plague as a bioweapon has prompted increased research to develop plague vaccines against this disease. Injectable subunit vaccines are being developed in the United States and United Kingdom. However, the live attenuated Y. pestis-EV NIIEG strain has been used as a vaccine for more than 70 years in the former Soviet Union and in some parts of Asia and provides a high degree of efficacy against plague. This vaccine has not gained general acceptance because of safety concerns. In recent years, modern molecular biological techniques have been applied to Y. pestis to construct strains with specific defined mutations designed to create safe, immunogenic vaccines with potential for use in humans and as bait vaccines to reduce the load of Y. pestis in the environment. In addition, a number of live, vectored vaccines have been reported using attenuated viral vectors or attenuated Salmonella strains to deliver plague antigens. Here we summarize the progress of live attenuated vaccines against plagu

Induction of strong immune responses against a vectored antigen in hosts immunized with live attenuated Salmonella vaccines is related in part to the amount of antigen delivered and the overall fitness of the Salmonella vector in relation to its ability to stimulate the host immune system. Constitutive high-level antigen synthesis causes a metabolic burden to the vaccine vector strain that can reduce the vaccine strain's ability to interact with host lymphoid tissues, resulting in a compromised immune response. A solution to this problem is the use of systems that regulate antigen gene expression, permitting high levels of antigen synthesis only after the vaccine strain has reached its target tissues. In vivo -inducible promoters (IVIPs) are often used to accomplish this. We recently developed an alternative strategy, a regulated delayed antigen synthesis (RDAS) system, in which the LacI-repressible P trc promoter controls antigen gene expression by adding arabinose. In this paper, we compared the RDAS system with two commonly used IVIPs, P ssaG and P pagC . Three nearly identical plasmids, differing only in the promoter used to direct transcription of the pneumococcal pspA gene, P trc , P ssaG , or P pagC , were constructed and introduced into isogenic Salmonella vaccine strains with or without arabinose-inducible LacI synthesis. Mice immunized with the RDAS strain developed slightly higher titers of mucosal and serum anti-PspA antibodies than P pagC -immunized mice, while titers in mice immunized with the P ssaG strain were 100-fold lower. Both the RDAS and P pagC strains conferred similar levels of protection against Streptococcus pneumoniae challenge, significantly greater than those for the P ssaG strain or controls. Thus, RDAS provides another choice for inclusion in the live vaccine design to increase immunogenicity.

Pneumococcal surface protein A (PspA) is highly immunogenic and can induce a protective immune response against pneumococcal infection. PspA is divided into two major families based on serological variability: family 1 and family 2. To provide broad protection, PspA proteins from pneumococcal strains Rx1 (family 1) and EF5668 (family 2) were combined to form two PspA fusion proteins, PspA/Rx1-EF5668 and PspA/EF5668-Rx1. Each protein was fused to a type II secretion signal and delivered by a recombinant attenuatedSalmonellavaccine (RASV). Both PspA/Rx1-EF5668 and PspA/EF5668-Rx1 were synthesized in the RASV and secreted into the periplasm and supernatant. The fusion proteins reacted strongly with both anti-PspA/Rx1 and anti-PspA/EF5668 antisera. Oral immunization of BALB/c mice with RASV synthesizing either PspA fusion protein elicited serum immunoglobulin G (IgG) and mucosal IgA responses against both families of PspA. Analysis of IgG isotypes (IgG2a and IgG1) indicated a strong Th1 bias to the immune responses to both proteins. Sera from mice immunized with RASV synthesizing PspA/Rx1-EF5668 bound to the surface and directed C3 complement deposition on representative strains from all five PspA clades. Immunization with RASV synthesizing either protein protected mice against intraperitoneal challenge withStreptococcus pneumoniaeWU2 strain (family 1), intravenous challenge withS. pneumoniae3JYP2670 strain (family 2), and intranasal challenge withS. pneumoniaeA66.1 (family 1). The PspA/Rx1-EF5668 protein elicited significantly greater protection than PspA/EF5668-Rx1, PspA/Rx1, or PspA/EF5668. These results indicate an RASV synthesizing a PspA fusion protein representing both PspA families constitutes an effective antipneumococcal vaccine, extending and enhancing protection against multiple strains ofS. pneumoniae.

The use of live recombinant attenuated Salmonella vaccines (RASV) synthesizing Yersinia proteins is a promising approach for controlling infection by Yersinia species. In this study, we constructed attenuated Salmonella strains which synthesize a truncated form of LcrV, LcrV196 and evaluated the immune response and protective efficacy elicited by these strains in mice against two other major species of Yersinia: Yersinia pseudotuberculosis and Yersinia enterocolitica. Surprisingly, we found that the RASV strain alone was sufficient to afford nearly full protection against challenge with Y. pseudotuberculosis, indicating the likelihood that Salmonella produces immunogenic cross-protective antigens. In contrast, lcrV196 expression was required for protection against challenge with Y. enterocolitica strain 8081, but was not sufficient to achieve significant protection against challenge with Y. enterocolitica strain WA, which expressed a divergent form of lcrV. Nevertheless, we are encouraged by these findings to continue pursuing our long-term goal of developing a single vaccine to protect against all three human pathogenic species of Yersinia.

The synthesis of Vi polysaccharide, a major virulence determinant in Salmonella enterica serotype Typhi (S. Typhi), is under the control of two regulatory systems, ompR-envZ and rscB-rscC, which respond to changes in osmolarity. Some S. Typhi isolates exhibit over-expression of Vi polysaccharide, which masks clinical detection of LPS O-antigen. This variation in Vi polysaccharide and O-antigen display (VW variation) has been observed since the initial studies of S. Typhi. We have reported that the status of the rpoS gene is responsible for this phenomenon. We review the regulatory network of the Vi polysaccharide, linking osmolarity and RpoS expression. Also, we discuss how this may impact live attenuated Salmonella vaccine development.

Recombinant attenuated Salmonella vaccines (RASVs) have been used to induce mucosal and systemic immunity either against itself or to a vectored heterologous antigen (30, 31). It is clear from previous studies that the mode of attenuation (10), antigen burden (12), and the expression system (11, 19, 21) can have a profound effect on the immune response to a vectored antigen. Many attenuating mutations were found to reduce Salmonella survival due to host-induced stresses and/or reduce colonization of lymphoid effector tissues leading to less than ideal immunogenicity (16, 48). An ideal Salmonella vaccine strain should exhibit wild-type abilities to withstand all stresses (enzymatic, acid, osmotic, ionic, etc.) and host defenses (bile, antibacterial peptides, etc.) encountered following oral or intranasal immunization and should exhibit wild-type abilities to colonize and invade host lymphoid tissues while remaining avirulent. Achieving maximal immune responses to a foreign antigen is directly correlated with the amount of the antigen produced, and thus, it is important that the immunizing strain produce adequate levels of antigen. However, for RASVs, this need must be weighed against the fact that high-level antigen production can be a drain on the energy resources of the cell, leading to reduced growth rates and a compromised ability to colonize and stimulate effector lymphoid tissues (55). We have addressed these problems by exporting the antigen outside of the attenuated Salmonella (19), by delaying expression of the attenuated phenotype (6; unpublished results), and by heterologous antigen synthesis (53) until the vaccine strain has colonized host lymphoid tissues. In addition, we have developed the Asd+-balanced lethal system for plasmid maintenance, obviating the need for antibiotic resistance markers (32). The SopB protein in Salmonella, encoded by SPI-5, is secreted and translocated into the cytosol through the SPI-1-encoded type III secretion system (60). SopB has an inositol phosphate phosphatase activity which affects cytoskeletal rearrangement, host-cell invasion and chloride homeostasis (34). sopB mutants of Salmonella enterica serovar Typhimurium are attenuated in their ability to cause gastroenteritis in calves (59), but not for invasion of host tissues (39). In addition, sopB mutants of both Salmonella enterica serovars Typhimurium and Dublin are similarly attenuated in their ability to induce inflammatory responses and fluid secretion in bovine ileal loops (13, 42). In the context of a vaccine, these features may be beneficial in reducing or eliminating reactogenicity and diarrhea sometimes observed in clinical trial volunteers receiving live attenuated bacterial vaccines (17, 47, 49-51). The SopB protein can also protect infected cells from apoptosis by sustained activation of Akt, which may be helpful for Salmonella survival in host tissues but will necessarily affect host immune responses (22). A recent study showed that introduction of a sopB mutation into several different attenuated Salmonella strains led to enhanced immune responses in mice to a vectored test antigen, β-galactosidase (27). Based on these observations, we evaluated the effects of a sopB deletion mutation on the performance of our Salmonella vaccine strains. Streptococcus pneumoniae is the principle cause of bacterial pneumonia in children and adults and is also a major cause of otitis media, bacterial meningitis, and septicemia (2). Developing an inexpensive, safe, and effective vaccine against this pathogen is still in urgent demand. Pneumococcal surface protein A (PspA) is an important virulence factor found on the surface of all pneumococci (29). It is a protective antigen in animal models and is immunogenic in humans (2, 58). Our previous work has demonstrated that mice immunized with an RASV expressing PspA are protected against challenge with virulent S. pneumoniae WU2 (20). In this work, we evaluated the effects of a sopB mutation on two different RASV strains with different modes of attenuation on their ability to deliver PspA. We analyzed humoral, mucosal, and cellular immune responses against both the bacterial vector and PspA and protective immunity against challenge with S. pneumoniae WU2 in immunized mice.

Enterotoxigenic Escherichia coli (ETEC) is the leading cause of traveler's diarrhea. The heat-labile (LT) and heat-stable (ST) toxins mediate ETEC induced diarrhea. ETEC strains may express LT, ST, or both LT and ST, with LT-expressing strains accounting for approximately 50-60% of ETEC-related traveler's diarrhea. Cholera toxin (CT) is80% homologous to LT and vaccination with CT-B subunit (CT-B) -based vaccines elicit a protective immune response against LT-producing ETEC strains. Peru-15 is an oral, single-dose, live-attenuated cholera vaccine candidate that has been investigated in several clinical trials (n400 subjects) and was proven well tolerated, immunogenic, and efficacious. Peru-15 was genetically engineered to express and secrete high levels of CT-B by cloning ctxB onto a glnA balanced-lethal plasmid under the transcriptional control of a strong constitutive promoter, resulting in Peru-15pCTB. In vitro characterization demonstrated that Peru-15pCTB secreted approximately 30-fold more CT-B than Peru-15 and CT-B was stably produced after 40 generations of growth and throughout simulated Seed Bank and FDP (Final Drug Product) production conditions. In preclinical studies, the geometric mean anti-CT-B IgG titer in the sera of mice inoculated intranasally with two doses of Peru-15pCTB was32-fold higher than in mice inoculated with Peru-15. Similarly, rabbits orally inoculated with a single dose of Peru-15pCTB developed titers that were approximately 30-fold higher than rabbits inoculated with a single dose of Peru-15. Sera from Peru-15pCTB vaccinated mice and rabbits neutralized LT toxicity in an in vitro assay. Peru-15pCTB has several promising characteristics of an oral, single-dose, bivalent cholera/ETEC vaccine and is advancing towards a Phase 1 clinical trial.

Clostridium perfringens is an important Gram-positive pathogen responsible for food poisoning, necrotic enteritis, gas gangrene, and even death. Escherichia coli Nissle 1917 (EcN) is a well-characterized probiotic strain with demonstrated benefits. In this study, we evaluated the effects of EcN on growth, toxin production, biofilm formation, and inflammatory cytokine responses of C. perfringens. In vitro co-culture experiments demonstrated that EcN inhibited growth, gas production, and toxin production (α-toxin and NetB) of C. perfringens in a dose-dependent manner. The growth inhibition effect was not observed when C. perfringens was incubated with EcN cell-free supernatants (CFSE), suggesting that growth inhibition was caused by nutrition competition during co-incubation. In vitro studies demonstrated that pre-incubation with EcN did not inhibit C. perfringens attachment to Caco-2 cells, but did reduce C. perfringens total number, toxin production, and cytotoxicity after 24 h. The similar growth inhibition results were also observed during the formation of C. perfringens biofilm. Finally, pre-incubation of EcN with RAW264.7 cells significantly decreased the production of inflammatory cytokines caused by the introduction of C. perfringens. Our results indicate that EcN can inhibit many of the pathological effects of C. perfringens in vitro conditions.

Salmonella enterica serovar Gallinarum is the causative agent of fowl typhoid, an important systemic disease of poultry with economic consequences in developing nations. A live attenuated orally applied S. Gallinarum vaccine could provide a low cost method for controlling this disease. We constructed S. Gallinarum strains in which the expression of the crp, rfc and rfaH genes, important for virulence of Salmonella Typhimurium in mice, were under the control of an arabinose-regulated promoter. We evaluated the virulence of these strains compared to wild-type S. Gallinarum and to mutants carrying deletions in these genes. We found that rfc mutants were fully virulent, indicating that, unlike the S. Typhimurium mouse model, the rfc gene is dispensable in S. Gallinarum for virulence in birds. In the case of rfaH, the deletion mutant was attenuated and protective, while the strain with arabinose-regulated rfaH expression retained full virulence. The strain exhibiting arabinose-regulated crp expression was attenuated. Its virulence was not affected by the inclusion of 0.2% arabinose in the drinking water. Birds immunized with this strain were protected against a lethal S. Gallinarum challenge and against colonization with the human pathogen Salmonella Enteritidis. This work shows that an arabinose-regulated crp strain provides a basis for further development of a fowl typhoid vaccine.

Lipopolysaccharide (LPS), composed of lipid A, core, and O-antigen, is a major virulence factor of Salmonella enterica serovar Typhimurium, with lipid A being a major stimulator to induce the proinflammatory response via the Toll-like receptor 4 (TLR4)-MD2-CD14 pathway. While Salmonella msbB mutants lacking the myristate chain in lipid A were investigated widely as an anticancer vaccine, inclusion of the msbB mutation in a Salmonella vaccine to deliver heterologous antigens has not yet been investigated. We introduced the msbB mutation alone or in combination with mutations in other lipid A acyl chain modification genes encoding PagL, PagP, and LpxR into wild-type S. enterica serovar Typhimurium. The msbB mutation reduced virulence, while the pagL , pagP , and lpxR mutations did not affect virulence in the msbB mutant background when administered orally to BALB/c mice. Also, all mutants exhibited sensitivity to polymyxin B but did not display sensitivity to deoxycholate. LPS derived from msbB mutants induced less inflammatory responses in human Mono Mac 6 and murine macrophage RAW264.7 cells in vitro . However, an msbB mutant did not decrease the induction of inflammatory responses in mice compared to the levels induced by the wild-type strain, whereas an msbB pagP mutant induced less inflammatory responses in vivo . The mutations were moved to an attenuated Salmonella vaccine strain to evaluate their effects on immunogenicity. Lipid A modification caused by the msbB mutation alone and in combination with pagL , pagP , and lpxR mutations led to higher IgA production in the vaginal tract but still retained the same IgG titer level in serum to PspA, a test antigen from Streptococcus pneumoniae , and to outer membrane proteins (OMPs) from Salmonella .

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. Published version

Leucine-responsive regulatory protein (Lrp) is known to be an indirect activator of type 1 fimbriae synthesis in Salmonella enterica serovar Typhimurium via direct regulation of FimZ, a direct positive regulator for type 1 fimbriae production. Using RT-PCR, we have shown previously that fimA transcription is dramatically impaired in both lrp-deletion (Δlrp) and constitutive-lrp expression (lrp(C)) mutant strains. In this work, we used chromosomal P(fimA)-lacZ fusions and yeast agglutination assays to confirm and extend our previous results. Direct binding of Lrp to P(fimA) was shown by an electrophoretic mobility shift assay (EMSA) and DNA footprinting assay. Site-directed mutagenesis revealed that the Lrp-binding motifs in P(fimA) play a role in both activation and repression of type 1 fimbriae production. Overproduction of Lrp also abrogates fimZ expression. EMSA data showed that Lrp and FimZ proteins independently bind to P(fimA) without competitive exclusion. In addition, both Lrp and FimZ binding to P(fimA) caused a hyper retardation (supershift) of the DNA-protein complex compared to the shift when each protein was present alone. Nutrition-dependent cellular Lrp levels closely correlated with the amount of type 1 fimbriae production. These observations suggest that Lrp plays important roles in type 1 fimbriation by acting as both a positive and negative regulator and its effect depends, at least in part, on the cellular concentration of Lrp in response to the nutritional environment.

Gram-negative bacteria produce outer membrane vesicles (OMVs) that serve a variety of functions related to survival and pathogenicity. Periplasmic and outer membrane proteins are naturally captured during vesicle formation. This property has been exploited as a method to derive immunogenic vesicle preparations for use as vaccines. In this work, we constructed a Salmonella enterica serovar Typhimurium strain that synthesized a derivative of the pneumococcal protein PspA engineered to be secreted into the periplasmic space. Vesicles isolated from this strain contained PspA in the lumen. Mice intranasally immunized with the vesicle preparation developed serum antibody responses against vesicle components that included PspA and Salmonella -derived lipopolysaccharide and outer membrane proteins, while no detectable responses developed in mice immunized with an equivalent dose of purified PspA. Mucosal IgA responses developed against the Salmonella components, while the response to PspA was less apparent in most mice. Mice immunized with the vesicle preparation were completely protected against a 10× 50% lethal dose (LD 50 ) challenge of Streptococcus pneumoniae and significantly protected against a 200× LD 50 challenge, while control mice immunized with purified PspA or empty vesicles were not protected. These results establish that vesicles can be used to mucosally deliver an antigen from a Gram-positive organism and induce a protective immune response.

Yersinia pestis, the causative agent of plague, is a potential weapon of bioterrorism. Y. pestis evades the innate immune system by synthesizing tetra-acylated lipid A with poor Toll-like receptor 4 (TLR4)-stimulating activity at 37°C, whereas hexa-acylated lipid A, a potent TLR4 agonist, is made at lower temperatures. Synthesis of Escherichia coli LpxL, which transfers the secondary laurate chain to the 2'-position of lipid A, in Y. pestis results in production of hexa-acylated lipid A at 37°C, leading to significant attenuation of virulence. Previously, we described a Y. pestis vaccine strain in which crp expression is under the control of the arabinose-regulated araC P(BAD) promoter, resulting in a 4-5 log reduction in virulence. To reduce the virulence of the crp promoter mutant further, we introduced E. coli lpxL into the Y. pestis chromosome. The χ10030(pCD1Ap) (ΔlpxP32::P(lpxL)lpxL ΔP(crp21)::TT araC P(BAD)crp) construct likewise produced hexa-acylated lipid A at 37°C and was significantly more attenuated than strains harboring each individual mutation. The LD(50) of the mutant in mice, when administered subcutaneously or intranasally was >10(7)-times and >10(4)-times greater than wild type, respectively. Mice immunized subcutaneously with a single dose of the mutant were completely protected against a subcutaneous challenge of 3.6×10(7) wild-type Y. pestis and significantly protected (80% survival) against a pulmonary challenge of 1.2×10(4) live cells. Intranasal immunization also provided significant protection against challenges by both routes. This mutant is an immunogenic, highly attenuated live Y. pestis construct that merits further development as a vaccine candidate.

We evaluated the ability of Yersinia pestis antigens HmuR, Psn and modified forms of LcrV delivered by live attenuated Salmonella strains to stimulate a protective immune response against subcutaneous or intranasal challenge with Y. pestis CO92. LcrV196 is a previously described truncated protein that includes aa 131–326 of LcrV and LcrV5214 has been modified to replace five key amino acids required for interaction with the TLR2 receptor. Psn is the outer membrane receptor for the siderophore, yersiniabactin, and the bacteriocin, pesticin. Mice immunized with Salmonella synthesizing Psn, LcrV196 or LcrV5214 developed serum IgG responses to the respective Yersinia antigen and were protected against pneumonic challenge with Y. pestis. Immunization with Salmonella synthesizing Psn or LcrV196 was sufficient to afford nearly full protection against bubonic challenge, while immunization with the strain synthesizing LcrV5214 was not protective. Immunization with Salmonella synthesizing HmuR, an outer membrane protein involved in heme acquisition in Y. pestis, was poorly immunogenic and did not elicit a protective response against either challenge route. These findings indicate that both Psn and LcrV196 delivered by Salmonella provide protection against both bubonic and pneumonic plague.

We have developed a regulated delayed antigen synthesis (RDAS) system for use in recombinant attenuatedSalmonellavaccine (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-regulatedaraCPBADpromoter. 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 thelacIribosome-binding site, start codon, and/or codon content to construct RDAS strains χ9095, χ9959, and χ9241, synthesizing from low to high levels of LacI, respectively, and non-RDAS strain χ9555 as a control. We evaluated this system with two test antigens, the green fluorescent protein for initialin vitroassessment and theStreptococcus pneumoniaePspA protein for validation of our system in mice. All RASV strains expressing PspA generated high antilipopolysaccharide antibody titers, indicating that expression oflacIdid not interfere with the capacity to induce an immune response. Strain χ9241 induced significantly higher anti-PspA IgG and IgA antibody titers than strain χ9555, which expressed PspA constitutively. Anti-PspA antibody titers were inversely correlated to the level of LacI synthesis. Strain χ9241 also induced significantly greater protective efficacy against challenge with virulentS. pneumoniae. These results suggest that regulated delayed antigen synthesis is useful for improving immunogenicity of RASV strains.

Streptococcus pneumoniae is a leading cause of morbidity and mortality among children worldwide and particularly in developing countries. In this study, we evaluated PsaA, a conserved antigen important for S. pneumoniae adhesion to and invasion into nasopharynx epithelia, for its ability to induce protective immunity against S. pneumoniae challenge when delivered by recombinant attenuated Salmonella vaccine (RASVs) strains. RASVs were engineered to synthesize PsaA peptides of various lengths. Vaccination with an RASV synthesizing full-length PsaA induced high titers of anti-PsaA antibodies in both systemic (IgG in serum) and mucosal (IgA in vaginal washes, nasal washes, and lung homogenates) sites. BALB/c (haplotype H2 d ) or C57BL/6 (haplotype H2 b ) mice vaccinated either orally or intranasally exhibited a significant reduction in colonization of nasopharyngeal tissues after intranasal challenge with S. pneumoniae strains compared to controls, although protection was not observed with all challenge strains. None of the vaccine constructs provided protection against intraperitoneal challenge with S. pneumoniae strain WU2 (serotype 3). Immunization with RASVs synthesizing truncated PsaA generated lower titers of IgA and IgG and did not provide significant protection. Our results showed that RASVs synthesizing full-length PsaA can provide protection against nasal colonization by some S. pneumoniae strains. PsaA may be a useful addition to a multivalent vaccine, providing protection against pneumonia, otitis media, and other diseases caused by S. pneumoniae .

RfaH is a transcriptional antiterminator that reduces the polarity of long operons encoding secreted and surface-associated cell components of Salmonella enterica serovar Typhimurium, including O antigen and lipopolysaccharide core sugars. A rfaH mutant strain is attenuated in mice (50% lethal dose [LD50], >10 8 CFU). To examine the potential for using rfaH in conjunction with other attenuating mutations, we designed a series of strains in which we replaced the native rfaH promoter with the tightly regulated arabinosedependent araC PBAD promoter so that rfaH expression was dependent on exogenously supplied arabinose provided during in vitro growth. Following colonization of host lymphoid tissues, where arabinose was not available, the PBAD promoter was no longer active and rfaH was not expressed. In the absence of RfaH, O antigen and core sugars were not synthesized. We constructed three mutant strains that expressed different levels of RfaH by altering the ribosome-binding sequence and start codon. One mutation, PrfaH178, was introduced into the attenuated vaccine strain 9241 (pabA pabB asdA) expressing the pneumococcal surface protein PspA from an Asd balanced-lethal plasmid. Mice immunized with this strain and boosted 4 weeks later induced higher levels of serum immunoglobulin G specific for PspA and for outer membrane proteins from other enteric bacteria than either an isogenic rfaH derivative or the isogenic RfaH parent. Eight weeks after primary oral immunization, mice were challenged with 200 LD50 of virulent Streptococcus pneumoniae WU2. Immunization with PrfaH178 mutant strains led to increased levels of protection compared to that of the parent 9241 and of a rfaH derivative of 9241. When recombinant attenuated Salmonella enterica serovar Typhimurium vaccines (RASV) are used to deliver heterologous antigens, it may be advantageous to reduce the host immune response against the RASV carrier, thereby enhancing the immune response against the heterologous antigen. The dominant immunogen on the Salmonella cell surface is lipopolysaccharide (LPS) O antigen (41). However, strains with mutations that eliminate LPS O antigen may be less immunogenic due to their failure to colonize the intestinal tract and to invade intestinal mucosal cells (43, 44). We hypothesized that in vivo-programmed downregulation of O-antigen expression, occurring after colonization of host lymphoid tissues, would serve to reduce the immune response against the RASV carrier while triggering a strong immune response against heterologous antigens (10) and outer membrane proteins cross-reactive with other enteric bacteria (28). The genes for LPS core and O-antigen biosynthesis are clustered into long operons (37, 49) that cannot be fully transcribed if the native promoter is replaced by a heterologous promoter. RfaH, a transcriptional antiterminator, reduces the polarity of long operons by binding to the ops sequence, located in an untranslated 5 region of the transcript, and inter

Eight Salmonella enterica serovar Paratyphi A strains were screened as candidates to create a live attenuated paratyphoid vaccine. Based on biochemical and phenotypic criteria, four strains, RKS2900, MGN9772, MGN9773 and MGN9779, were selected as progenitors for the construction of Δ phoPQ mutant derivatives. All strains were evaluated in vitro for auxotrophic phenotypes and sensitivity to deoxycholate and polymyxin B. All Δ phoPQ mutants were more sensitive to deoxycholate and polymyxin B than their wild-type progenitors, however MGN10028, MGN10044 and MGN10048, required exogenous purine for optimal growth. Purine requiring strains had acquired point mutations in purB during strain construction. All four mutants were evaluated for reactogenicity and immunogenicity in an oral rabbit model. Three strains were reactogenic in a dose-dependent manner, while one strain, MGN10028, was well-tolerated at all doses administered. All Δ phoPQ strains were immunogenic following a single oral dose. The in vitro profile coupled with the favorable reactogenicity and immunogenicity profiles render MGN10028 a suitable live attenuated Paratyphi A vaccine candidate.

A distinct pathovar of Salmonella enterica serovar Typhimurium, ST313, has emerged in sub-Saharan Africa as a major cause of fatal bacteremia in young children and HIV-infected adults. D23580, a multidrug resistant clinical isolate of ST313, was previously shown to have undergone genome reduction in a manner that resembles that of the more human-restricted pathogen, Salmonella enterica serovar Typhi. It has since been shown through tissue distribution studies that D23580 is able to establish an invasive infection in chickens. However, it remains unclear whether ST313 can cause lethal disease in a non-human host following a natural course of infection. Herein we report that D23580 causes lethal and invasive disease in a murine model of infection following peroral challenge. The LD50 of D23580 in female BALB/c mice was 4.7 x 105 CFU. Tissue distribution studies performed 3 and 5 days post-infection confirmed that D23580 was able to more rapidly colonize the spleen, mesenteric lymph nodes and gall bladder in mice when compared to the well-characterized S. Typhimurium strain SL1344. D23580 exhibited enhanced resistance to acid stress relative to SL1344, which may lend towards increased capability to survive passage through the gastrointestinal tract as well as during its intracellular lifecycle. Interestingly, D23580 also displayed higher swimming motility relative to SL1344, S. Typhi strain Ty2, and the ST313 strain A130. Biochemical tests revealed that D23580 shares many similar metabolic features with SL1344, with several notable differences in the Voges-Proskauer and catalase tests, as well alterations in melibiose, and inositol utilization. These results represent the first full duration infection study using an ST313 strain following the entire natural course of disease progression, and serve as a benchmark for ongoing and future studies into the pathogenesis of D23580., Author Summary A deadly form of non-typhoidal Salmonella has emerged as a major cause of invasive disease in sub-Saharan Africa. Initial genomic profiling of this novel Salmonella sequence type, ST313, indicated that although it is technically classified as S. Typhimurium (a serovar characterized by a broad host range), it may be evolving towards becoming a more human-specific, ‘typhoid-like’ pathogen. However, it was recently demonstrated that ST313 strains were indeed able to establish an invasive and damaging infection in chickens. Despite these important findings, it remains unclear whether ST313 is able to cause lethal disease in a non-human host, since no study has yet followed the entire natural course of disease progression. As such, there are no data available concerning the median lethal dose (LD50) of any ST313 strain. This is an important metric, as the LD50 value will serve as a benchmark for mechanistic studies focused on understanding the relationship between virulence and the phenotypic and molecular genetic attributes associated with ST313 infections. Here we report that D23580 causes lethal disease in BALB/c mice and determined the LD50 following peroral challenge. Phenotypic characterization revealed distinct differences in tissue distribution, acid stress resistance, and biochemical utilization between D23580 and the ‘classic’ Typhimurium strain SL1344.

The authors wish to thank Dr. C. Michael Reynolds for his valuable assistance in performing Mass spectra data (Fig. 2A and C), Dr. Susan traley for providing anti-YopM antibodies and Dr. Praveen Alamuri for his valuable assistance in performing animal experiments. Conflict of interest: All authors declare none. Funding: This work was supported by National Institutes of Health grant 5R01 AI057885 to .C. and by grant GM51310 to C.R.H.R. The mass spectrometry facility in the Department of Biochemistry of the Duke University Medical enter is supported by the LIPID MAPS Large Scale Collaborative Grant number GM-069338 from NIH.

Udgivelsesdato: 1981-Mar The potential of secretory immunoglobulin A (S-IgA) to interfere with the initial phase of dental plaque formation was studied by using an in vitro method which permits the quantitative determination of the sorption of radiolabeled oral bacterial cells to hydroxyapatite (HA) beads. The importance of specific S-IgA antibodies was evaluated by a comparison of the effect of pure preparations of colostral S-IgA, polymeric myeloma IgA, or preabsorbed S-IgA. Specific antibody molecules bound at the HA surface significantly enhanced the sorption of two Streptococcus sanguis strains. In contrast, HA-bound S-IgA antibodies inhibited the sorption of Streptococcus mitior and Streptococcus salivarius. The same was true for Streptococcus mutans cells, but only when they were propagated in the absence of sucrose. Suspended in saliva, cells of all streptococcal species adhered in significantly lower numbers to HA. Comparative experiments with bacteria suspended in solutions of various preparations of IgA or immunoglobulin-deficient salivas with S-IgA or myeloma IgA added indicated that the adherence inhibition seen with S. Sanguis, S. mitior, S. salivarius, and glucose-grown S. mutans was partly attributable to functions of S-IgA antibodies. Under the in vitro conditions of the study, S-IgA antibodies had no effect on the sorption of sucrose-grown S. mutans, Actinomyces viscosus, and Actinomyces naeslundii to HA. The results indicated that S-IgA can interfere with the sorption of some oral bacteria to HA by several different functions.