Yersinia Outer Proteins Research Articles

Pre-existing anti- Salmonella vector immunity prevents the development of protective antigen-specific CD8 T-cell frequencies against murine listeriosis

Our laboratory has focused its research on the use of the type III secretion system of Salmonella enterica serovar Typhimurium to translocate heterologous antigens directly into the cytosol of antigen-presenting cells. We have previously reported that the single oral immunization of mice with a recombinant Salmonella aroA/ sptP mutant strain expressing the translocated Yersinia outer protein E fused to the immunodominant antigen p60 from Listeria monocytogenes in a type III-mediated fashion results in the efficient induction of p60-specific CD8 T cells and confers protection against a lethal Listeria challenge infection. In the present study, we determined whether pre-existing anti- Salmonella vector immunity influences the induction of p60-specific CD8 T cells and modulates protective immunity against listeriosis after oral vaccination with recombinant Salmonella. After single oral immunization, the Salmonella aroA/ sptP double mutant strain was found to colonize spleens of mice for 21 days. In contrast, the period of colonization was significantly shortened to 6 days due to anti- Salmonella vector immunity after second oral immunization. The latter scenario led to the induction of low-level frequencies of antigen-specific CD8 T cells. Compared to the significantly higher numbers of p60-specific T lymphocytes elicited after single oral immunization, the low amount of Listeria-specific CD8 T cells did not confer protection against listeriosis.

Catalytically active Yersinia outer protein P induces cleavage of RIP and caspase-8 at the level of the DISC independently of death receptors in dendritic cells

Yersinia outer protein P (YopP) is injected by Y. enterocolitica into host cells thereby inducing apoptotic and necrosis-like cell death in dendritic cells (DC). Here we show the pathways involved in DC death caused by the catalytic activity of YopP. Infection with Yersinia enterocolitica, translocating catalytically active YopP into DC, triggered procaspase-8 cleavage and c-FLIPL degradation. YopP-dependent caspase-8 activation was, however, not mediated by tumor necrosis factor (TNF) receptor family members since the expression of both CD95/Fas/APO-1 and TRAIL-R2 on DC was low, and DC were resistant to apoptosis induced by agonistic anti-CD95 antibodies or TNF-related apoptosis-inducing ligand (TRAIL). Moreover, DC from TNF-Rp55-/- mice were not protected against YopP-induced cell death demonstrating that TNF-R1 is also not involved in this process. Activation of caspase-8 was further investigated by coimmunoprecitation of FADD from Yersinia-infected DC. We found that both cleaved caspase-8 and receptor interacting protein 1 (RIP1) were associated with the Fas-associated death domain (FADD) indicating the formation of an atypical death-inducing signaling complex (DISC). Furthermore, degradation of RIP mediated by the Hsp90 inhibitor geldanamycin significantly impaired YopP-induced cell death. Altogether our findings indicate that Yersinia-induced DC death is independent of death domain containing receptors, but mediated by RIP and caspase-8 at the level of DISC.

The Yersinia enterocolitica type three secretion chaperone SycO is integrated into the Yop regulatory network and binds to the Yop secretion protein YscM1

BackgroundPathogenic yersiniae (Y. pestis, Y. pseudotuberculosis, Y. enterocolitica) share a virulence plasmid encoding a type three secretion system (T3SS). This T3SS comprises more than 40 constituents. Among these are the transport substrates called Yops (Yersinia outer proteins), the specific Yop chaperones (Sycs), and the Ysc (Yop secretion) proteins which form the transport machinery. The effectors YopO and YopP are encoded on an operon together with SycO, the chaperone of YopO. The characterization of SycO is the focus of this study.ResultsWe have established the large-scale production of recombinant SycO in its outright form. We confirm that Y. enterocolitica SycO forms homodimers which is typical for Syc chaperones. SycO overproduction in Y. enterocolitica decreases secretion of Yops into the culture supernatant suggesting a regulatory role of SycO in type III secretion. We demonstrate that in vitro SycO interacts with YscM1, a negative regulator of Yop expression in Y. enterocolitica. However, the SycO overproduction phenotype was not mediated by YscM1, YscM2, YopO or YopP as revealed by analysis of isogenic deletion mutants.ConclusionWe present evidence that SycO is integrated into the regulatory network of the Yersinia T3SS. Our picture of the Yersinia T3SS interactome is supplemented by identification of the SycO/YscM1 interaction. Further, our results suggest that at least one additional interaction partner of SycO has to be identified.

Metabolism, cytoskeleton and cellular signalling in the grip of protein Nϵ ‐ and O‐acetylation

Acetylation of the epsilon-amino group of lysine residues (N(epsilon)-acetylation) is a reversible post-translational modification with the potential to rival phosphorylation. In addition to histones and many transcription factors such as p53, regulators of DNA repair, replication and recombination are subject to N(epsilon)-acetylation. This modification is also important for governing the activities of various enzymes, including histone acetyltransferases, histone deacetylases, bacterial and mammalian acetyl-CoA synthases, kinases, phosphatases, the ubiquitin ligase murine double minute 2 and the chaperonin heat shock protein 90. Furthermore, lysine acetylation occurs in cellular structure proteins such as alpha-tubulin, actin, cortactin and p120 catenin. Strikingly, the Yersinia outer protein YopJ promotes O-acetylation of crucial serine and threonine residues that are required for activation of the MAPK/ERK kinase and IkappaB kinase families, which precludes their phosphorylation and blocks signal transduction. Thus, N(epsilon)- and O-acetylation are becoming recognized as two prominent mechanisms for regulating protein functions in diverse organisms.

Extracytoplasmic-Stress-Responsive Pathways Modulate Type III Secretion in Yersinia pseudotuberculosis

Three signal transduction pathways, the two-component systems CpxRA and BaeSR and the alternative sigma factor sigma(E), respond to extracytoplasmic stress that facilitates bacterial adaptation to changing environments. At least the CpxRA and sigma(E) pathways control the production of protein-folding and degradation factors that counter the effects of protein misfolding in the periplasm. This function also influences the biogenesis of multicomponent extracellular appendages that span the bacterial envelope, such as various forms of pili. Herein, we investigated whether any of these regulatory pathways in the enteropathogen Yersinia pseudotuberculosis affect the functionality of the Ysc-Yop type III secretion system. This is a multicomponent molecular syringe spanning the bacterial envelope used to inject effector proteins directly into eukaryotic cells. Disruption of individual components revealed that the Cpx and sigma(E) pathways are important for Y. pseudotuberculosis type III secretion of Yops (Yersinia outer proteins). In particular, a loss of CpxA, a sensor kinase, reduced levels of structural Ysc (Yersinia secretion) components in bacterial membranes, suggesting that these mutant bacteria are less able to assemble a functional secretion apparatus. Moreover, these bacteria were no longer capable of localizing Yops into the eukaryotic cell interior. In addition, a cpxA lcrQ double mutant engineered to overproduce and secrete Yops was still impaired in intoxicating cells. Thus, the Cpx pathway might mediate multiple influences on bacterium-target cell contact that modulate Yersinia type III secretion-dependent host cell cytotoxicity.

Identification of a Molecular Target for the Yersinia Protein Kinase A

Pathogenic bacteria of the genus Yersinia employ a type III secretion system to inject bacterial effector proteins directly into the host cytosol. One of these effectors, the Yersinia serine/threonine protein kinase YpkA, is an essential virulence determinant involved in host actin cytoskeletal rearrangements and in inhibition of phagocytosis. Here we report that YpkA inhibits multiple Galphaq signaling pathways. The kinase activity of YpkA is required for Galphaq inhibition. YpkA phosphorylates Ser47, a key residue located in the highly conserved diphosphate binding loop of the GTPase fold of Galphaq. YpkA-mediated phosphorylation of Ser47 impairs guanine nucleotide binding by Galphaq. Y. pseudotuberculosis expressing wild-type YpkA, but not a catalytically inactive YpkA mutant, interferes with Galphaq-mediated signaling pathways. Identification of a YpkA-mediated phosphorylation site in Galphaq sheds light on the contribution of the kinase activity of YpkA to Yersinia pathogenesis.

Yersinia outer proteins E, H, P, and T differentially target the cytoskeleton and inhibit phagocytic capacity of dendritic cells

Through Yersinia outer proteins (Yops) Yersinia disrupt the actin cytoskeleton of epithelial cells and macrophages, and this leads to a decreased capability of these cells to internalize bacteria. We examined the effects of different Yops of Y. enterocolitica serotype O8 on the cytoskeleton and phagocytic capacity of murine dendritic cells (DCs). DCs were infected with several Yersinia mutant strains deficient in one Yop or translocating only a single Yop. Analyses of infected DCs by microscopy showed that YopE, YopH and YopT cooperate to rapidly damage the actin cytoskeleton of DCs. Furthermore, microscopic analyses and gentamicin killing assays revealed that the maximum reduction of bacterial uptake was achieved by Yersinia mutant strains translocating only a single Yop (YopE or YopH) indicating that these Yops enable Yersinia to inhibit the phagocytic function of DCs.

Effector functions of pathogenic Yersinia species

Pathogenic species of the genus Yersinia suppress and reorient the immune system to infect lymphatic tissues, inner organs and at times also the vasculature. For this purpose yersiniae employ a type III secretion system to translocate effector proteins (Yersinia outer proteins; Yops) into immune cells. Yops often exert unique biochemical activities for modulating the activity of Rho GTP-binding proteins, focal adhesion proteins, inflammatory pathways and cell survival/apoptosis. In this review we will put emphasis on the biochemistry, cell- and infection biology of Yersinia effector Yops.

Protective Immunity against Respiratory Tract Challenge withYersinia pestisin Mice Immunized with an Adenovirus‐Based Vaccine Vector Expressing V Antigen

The aerosol form of the bacterium Yersinia pestis causes the pneumonic plague, a rapidly fatal disease. At present, no plague vaccines are available for use in the United States. One candidate for the development of a subunit vaccine is the Y. pestis virulence (V) antigen, a protein that mediates the function of the Yersinia outer protein virulence factors and suppresses inflammatory responses in the host. On the basis of the knowledge that adenovirus (Ad) gene-transfer vectors act as adjuvants in eliciting host immunity against the transgene they carry, we tested the hypothesis that a single administration of a replication-defective Ad gene-transfer vector encoding the Y. pestis V antigen (AdsecV) could stimulate strong protective immune responses without a requirement for repeat administration. AdsecV elicited specific T cell responses and high IgG titers in serum within 2 weeks after a single intramuscular immunization. Importantly, the mice were protected from a lethal intranasal challenge of Y. pestis CO92 from 4 weeks up to 6 months after immunization with a single intramuscular dose of AdsecV. These observations suggest that an Ad gene-transfer vector expressing V antigen is a candidate for development of an effective anti-plague vaccine

Yersinia YopP-induced apoptotic cell death in murine dendritic cells is partially independent from action of caspases and exhibits necrosis-like features

Yersinia outer protein P (YopP) is a virulence factor of Yersinia enterocolitica that is injected into the cytosol of host cells where it targets MAP kinase kinases (MKKs) and inhibitor of kappaB kinase (IKK)-beta resulting in inhibition of cytokine production as well as induction of apoptosis in murine macrophages and dendritic cells (DC). Here we show that DC death was only partially prevented by the broad spectrum caspase inhibitor zVAD-fmk, indicating simultaneous caspase-dependent and caspase-independent mechanisms of cell death induction by YopP. Microscopic analyses and measurement of cell size demonstrated necrosis-like morphology of caspase-independent cell death. Application of zVAD-fmk prevented cleavage of procaspases and Bid, decrease of the inner transmembrane mitochondrial potential DeltaPsi(m) and mitochondrial release of cytochrome c. From these data we conclude that YopP-induced activation of the mitochondrial death pathway is mediated upstream via caspases. In conclusion, our results suggest that YopP simultaneously induces caspase-dependent apoptotic and caspase-independent necrosis-like death in DC. However, it has to be resolved if necrosis-like DC death occurs independently from apoptotic events or as an apoptotic epiphenomenon.

Yersinia enterocolitica YopP inhibits MAP kinase-mediated antigen uptake in dendritic cells

Yersinia enterocolitica (Ye) targets mouse dendritic cells (DCs) and inhibits their ability to trigger T cell activation. Here we have investigated whether Ye might interfere with antigen presentation in DCs. Infection of DCs with the Ye wild-type strain reduced OVA uptake by DCs as demonstrated by flow cytometry and confocal laser scan microscopy. In contrast, DCs infected with Yersinia outer protein P (YopP)-deficient mutant strain rapidly internalized OVA. Furthermore, transfection of DCs with YopP, but not with a cysteine protease deficient YopP-C172A mutant, reduced uptake of OVA. This finding suggests that YopP, a virulence factor of Ye, inhibits OVA uptake by DCs. By the use of MAPK inhibitors we provide evidence that YopP mediates reduction of OVA uptake by its ability to block MAPK signalling pathways in host cells. Using transferrin (Tf) as specific marker for clathrin-mediated endocytosis (CME) and lucifer yellow (LY) as specific marker for macropinocytosis (MP) we could show that YopP inhibits CME, whereas other Yops inhibit MP. In keeping with these data, activation and proliferation of OVA-specific T cells was reduced when DCs were treated with MAPK inhibitors. Together, our data demonstrate that (i) MAPK play an important role in antigen uptake by CME in DCs, and (ii) that YopP inhibits this pathway of antigen uptake in DCs, which might contribute to evasion of adaptive immunity.

The Proteasome Pathway Destabilizes Yersinia Outer Protein E and Represses Its Antihost Cell Activities

Pathogenic Yersinia spp. neutralize host defense mechanisms by engaging a type III protein secretion system that translocates several Yersinia outer proteins (Yops) into the host cell. Although the modulation of the cellular responses by individual Yops has been intensively studied, little is known about the fate of the translocated Yops inside the cell. In this study, we investigated involvement of the proteasome, the major nonlysosomal proteolytic system in eukaryotic cells, in Yop destabilization and repression. Our data show that inhibition of the proteasome in Yersinia enterocolitica-infected cells selectively stabilized the level of YopE, but not of YopH or YopP. In addition, YopE was found to be modified by ubiquitination. This suggests that the cytotoxin YopE is physiologically subjected to degradation via the ubiquitin-proteasome pathway inside the host cell. Importantly, the increased levels of YopE upon proteasome inhibition were associated with decreased activity of its cellular target Rac. Thus, the GTPase-down-regulating function of YopE is enhanced when the proteasome is inhibited. The stabilization of YopE by proteasome inhibitor treatment furthermore led to aggravation of the cytotoxic YopE effects on the actin cytoskeleton and on host cell morphology. Together, these data show that the host cell proteasome functions to destabilize and inactivate the Yersinia effector protein YopE. This implies the proteasome as integral part of the cellular host immune response against the immunomodulatory activities of a translocated bacterial virulence protein.

Yersinia enterocolitica type III secretion chaperone SycD: Recombinant expression, purification and characterization of a homodimer

Yersinia species pathogenic to human benefit from a protein transport machinery, a type three secretion system (T3SS), which enables the bacteria to inject effector proteins into host cells. Several of the transport substrates of the Yersinia T3SS, called Yops ( Yersinia outer proteins), are assisted by specific chaperones (Syc for specific Yop chaperone) prior to transport. Yersinia enterocolitica SycD (LcrH in Yersinia pestis and Yersinia pseudotuberculosis) is a chaperone dedicated to the assistance of the translocator proteins YopB and YopD, which are assumed to form a pore in the host cell membrane. In an attempt to make SycD amenable to structural investigations we recombinantly expressed SycD with a hexahistidine tag in Escherichia coli. Combining immobilized nickel affinity chromatography and gel filtration we obtained purified SycD with an exceptional yield of 120 mg per liter of culture and homogeneity above 95%. Analytical gel filtration and cross-linking experiments revealed the formation of homodimers in solution. Secondary structure analysis based on circular dichroism suggests that SycD is mainly composed of α-helical elements. To prove functionality of purified SycD previously suggested interactions of SycD with Yop secretion protein M2 (YscM2), and low calcium response protein V (LcrV), respectively, were reinvestigated.

605. Long-Term Protection Afforded by a Single Administration of an Ad-Based Anti-Y. pestis Vaccine Against a Lethal Respiratory Challenge with Y. pestis

The aerosol form of the bacterium Yersinia pestis causes the pneumonic plague, a virulent and rapidly fatal disease that develops within days post-infection. In the context of a bioterror threat, an ideal anti-plague vaccine should elicit rapid, robust and long-acting protective immunity after a single dose. At present, no plague vaccines are available for use in the USA. One candidate for developing a subunit vaccine is the Yersinia pestis V antigen, a protein that influences the function of the Yersinia outer membrane proteins (Yops) virulence factors and has local anti-inflammatory effects in the host.

Seroprevalence of Brucellosis, Tularemia, and Yersiniosis in Wild Boars (Sus scrofa) from North‐Eastern Germany

Brucellosis and tularemia are classical zoonotic diseases transmitted from an animal reservoir to humans. Both, wildlife and domestic animals, contribute to the spreading of these zoonoses. The surveillance of the animal health status is strictly regulated for domestic animals, whereas systematic disease monitoring in wildlife does not exist. The aim of the present study was to provide data on the prevalence of anti-Brucella, anti-Francisella and anti-Yersinia antibodies in wild boars from North-Eastern Germany to assess public health risks. A total of 763 sera of wild boars from Mecklenburg-Western Pomerania hunted in 1995/1996 were tested using a commercially available Brucella suis ELISA, an in-house lipopolysaccharide (LPS)-based Francisella ELISA, and commercially available Western blot kits for the detection of anti-Francisella and anti-Yersinia antibodies. The Yersinia enterocolitica O:9 LPS is able to induce serological cross-reactions indistinguishable from brucellosis due to a similar immunodominant epitope in the Brucella O-polysaccharide. The Yersinia Western blot assay was, therefore, based on five recombinant Yersinia outer proteins which have been proved to be specific for the serodiagnosis of yersiniosis. Anti-Brucella, anti-Francisella and anti-Yersinia antibodies were detected in 22.0%, 3.1%, and 62.6% of the wild boars, respectively. The high seroprevalence of tularemia and brucellosis in wild boars indicates that natural foci of these zoonoses are present in wildlife in Germany. However, the impact of transmission of zoonotic pathogens from wildlife to livestock is unknown. Only careful and systematic monitoring will help to prevent the (re)emergence of these zoonotic diseases in domestic animals and consequently human infection.

YERSINIA OUTER PROTEINS: Role in Modulation of Host Cell Signaling Responses and Pathogenesis

A type III secretion system (TTSS) is encoded on a virulence plasmid that is common to three pathogenic Yersinia species: Y. enterocolitica, Y. pseudotuberculosis, and Y. pestis. Pathogenic Yersinia species require this TTSS to survive and replicate within lymphoid tissues of their animal or human hosts. A set of pathogenicity factors, including those known as Yersinia outer proteins (Yops), is exported by this system upon bacterial infection of host cells. Two translocator Yops (YopB and YopD) insert into the host plasma membrane and function to transport six effector Yops (YopO, YopH, YopM, YopT, YopJ, and YopE) into the cytosol of the host cell. Effector Yops function to counteract multiple signaling responses in the infected host cell. The signaling responses counteracted by Yops are initiated by phagocytic receptors, Toll-like receptors, translocator Yops, and additional mechanisms. Innate and adaptive immune responses are thwarted as a consequence of Yop activities. A biochemical function for each effector Yop has been established, and the importance of these proteins for the pathogenesis process is being elucidated. This review focuses on the biochemical functions of Yops, the signaling pathways they modulate, and the role of these proteins in Yersinia virulence.

Involvement of the virulence gene products of Yersinia enterocolitica in the immune response of infected mice

The virulence of Yersinia enterocolitica is known to be highly dependent on its virulence plasmid. However, it remains unclear whether the virulence plasmid is engaged also in the induction of cell-mediated immunity that is essential for protective immunity in the host. In this study, we have compared the induction of type 1 helper T cell immunity against Y. enterocolitica using a virulent strain (P+) harboring the pYV plasmid and an avirulent strain (P−) harboring no pYV. Spleen cells from both groups of mice immunized with 1/10 LD 50 of P+ strain and those with 1/10 LD 50 of P− strain produced a high level of gamma interferon (IFN-γ) upon stimulation with heat-killed bacteria, and CD4 + T cells were exclusively responsible for IFN-γ production. When crude Yersinia outer proteins (Yops) were used for antigenic stimulation, IFN-γ response of immune spleen cells against crude Yops was observed only in mice immunized with P+ strain. Flowcytometric analysis revealed a significant level of increase in IFN-γ-producing CD8 + T cells as well as the increase in IFN-γ-producing CD4 + T cells against crude Yops. These results suggest that the virulence plasmid of Y. enterocolitica is involved in the induction of Th1-type of possibly protective T cells in infected mice.

Inhibition of MAPK and NF-κB Pathways Is Necessary for Rapid Apoptosis in Macrophages Infected with Yersinia

Macrophages respond to infection with pathogenic Yersinia species by activating MAPK- and NF-kappaB-signaling pathways. To counteract this response, Yersiniae secrete a protease (Yersinia outer protein J (YopJ)) that is delivered into macrophages, deactivates MAPK- and NF-kappaB-signaling pathways, and induces apoptosis. NF-kappaB promotes cell survival by up-regulating expression of several apoptosis inhibitor genes. Previous studies show that deactivation of the NF-kappaB pathway by YopJ is important for Yersinia-induced apoptosis. To determine whether deactivation of the NF-kappaB pathway is sufficient for Yersinia-induced apoptosis, two inhibitors of the NF-kappaB pathway, IkappaBalpha superrepressor or A20, were expressed in macrophages. Macrophages expressing these proteins were infected with Yersinia pseudotuberculosis strains that secrete functionally active or inactive forms of YopJ. Apoptosis levels were substantially higher (5- to 10-fold) when active YopJ was delivered into macrophages expressing IkappaBalpha superrepressor or A20, suggesting that deactivation of the NF-kappaB pathway is not sufficient for rapid Yersinia-induced apoptosis. When macrophages expressing A20 were treated with specific inhibitors of MAPKs, similar levels of apoptosis (within approximately 2-fold) were observed when active or inactive YopJ were delivered during infection. These results suggest that MAPK and NF-kappaB pathways function together to up-regulate apoptosis inhibitor gene expression in macrophages in response to Yersinia infection and that YopJ deactivates both pathways to promote rapid apoptosis. In addition, treating macrophages with a proteasome inhibitor results in higher levels of infection-induced apoptosis than can be achieved by blocking NF-kappaB function alone, suggesting that proapoptotic proteins are stabilized when proteasome function is blocked in macrophages.

Differences in Heat Resistance among Pathogenic Yersinia enterocolitica Depended on Growth Temperature and Serotype

To gain a better understanding about the effect of growth temperature on heat resistance of Yersinia enterocolitica, we determined decimal reduction times at 60°C (D60-values) for O:3; O:5,27; O:8; and O:9 strains harboring virulence plasmid coding for Yersinia outer membrane protein and experimentally virulence plasmid–deleted strains after they were grown to stationary phase at 7, 25, or 37°C. Bacteria were inoculated into Trypticase soy broth and were incubated at several temperatures. D60-values of O:3; O:5,27; and O:8 strains were larger when they were grown at 37°C than at 7 or 25°C, despite the presence or absence of virulence plasmids. However, similar D60-values were observed in O:9 strains, despite growth at 7, 25, or 37°C. The results indicate two types of Y. enterocolitica strains, growth temperature–dependent and –independent, and a Yersinia outer membrane protein that is not directly involved in growth temperature–dependent heat resistance.

67. Protective Immunity Against Yersinia pestis in Mice Immunized with an Ad-Based Vaccine Vector Expressing the Y. pestis F1 and V Antigens

The aerosol form of the bacterium Yersinia pestis causes the pneumonic plague, an extremely virulent and rapidly fatal disease that develops within days post-infection. In the context of a bioterror threat, an effective anti-plague vaccine must elicit rapid, robust and long acting protective immunity after a single dose. At present, no plague vaccines are available for use in the USA. Two subunits of Y. pestis, fraction 1 (F1) and V antigen have been identified as vaccine candidates based on: (1) F1 antigen is a temperature-regulated capsular protein of Y. pestis with anti-phagocytic activity in the host; (2) V antigen has local anti-inflammatory effects and influences the function of important virulence factors such as the Yersinia outer membrane proteins (Yops). It is known that protective immunity against Y. pestis challenge can be induced by multiple immunizations with recombinant F1 and V proteins and immediate protection can be achieved by passive transfer of immune sera against these antigens. Based on the knowledge that adenovirus (Ad) gene transfer vectors act as adjuvants in eliciting host immunity against the transgene they carry, replication-defective Ad gene transfer vectors encoding either the Y. pestis V antigen or the F1 protein (AdsecV and AdsecF1, respectively) were constructed. The F1 and V antigen genes were synthesized by an overlap PCR strategy using mammalian preferred codons, and fused to the human Ig signal sequence for extracellular secretion. We tested the hypothesis that AdsecV and AdsecF1 can stimulate strong protective immune responses without a requirement for repeat administration. To test the efficacy of the vaccines, BALB/c mice were immunized with a single intramuscular administration of either vector at a dose of 108 to 1011 particles units (pu). Four wk after immunization, mice were challenged in a BSL3 experimental animal facility (n=10/group) by the intranasal route with 3103 cfu of the fully virulent Yersinia pestis strain, CO92. At challenge time, anti-V antigen IgG reciprocal titers from AdsecV–immunized mice (n=10/group) were 666137 (108 pu), 3045735 (109 pu), 97032193 (1010 pu), and 7611215914 (1011 pu). In contrast, anti-F1 antigen IgG reciprocal titers from AdsecF1-immunized mice (n=10/group) were much lower with 122 (108 pu), 9135 (109 pu), 1154138 (1010 pu), and 939157 (1011 pu). All (10/10) of the animals survived the Y. pestis challenge in the group vaccinated with 1011 pu AdsecV, and 40% (4/10) survived in the 1010 pu AdsecV dose, while in all the other experimental groups immunized with AdsecV and in the control groups the mice died within 2 to 5 days after challenge. No mice vaccinated with AdsecF1 survived the Y. pestis challenge. These observations suggest that an Ad-based vaccine encoding for the V antigen of Y. pestis (AdsecV) is an excellent candidate as a effective single dose vaccine against the plague.