- Research article
- Open Access
Immunization of mice with YscF provides protection from Yersinia pestis infections
© Matson et al; licensee BioMed Central Ltd. 2005
- Received: 26 March 2005
- Accepted: 24 June 2005
- Published: 24 June 2005
Yersinia pestis, the causative agent of plague, is a pathogen with a tremendous ability to cause harm and panic in populations. Due to the severity of plague and its potential for use as a bioweapon, better preventatives and therapeutics for plague are desirable. Subunit vaccines directed against the F1 capsular antigen and the V antigen (also known as LcrV) of Y. pestis are under development. However, these new vaccine formulations have some possible limitations. The F1 antigen is not required for full virulence of Y. pestis and LcrV has a demonstrated immunosuppressive effect. These limitations could damper the ability of F1/LcrV based vaccines to protect against F1-minus Y. pestis strains and could lead to a high rate of undesired side effects in vaccinated populations. For these reasons, the use of other antigens in a plague vaccine formulation may be advantageous.
Desired features in vaccine candidates would be antigens that are conserved, essential for virulence and accessible to circulating antibody. Several of the proteins required for the construction or function of the type III secretion system (TTSS) complex could be ideal contenders to meet the desired features of a vaccine candidate. Accordingly, the TTSS needle complex protein, YscF, was selected to investigate its potential as a protective antigen. In this study we describe the overexpression, purification and use of YscF as a protective antigen. YscF immunization triggers a robust antibody response to YscF and that antibody response is able to afford significant protection to immunized mice following challenge with Y. pestis. Additionally, evidence is presented that suggests antibody to YscF is likely not protective by blocking the activity of the TTSS.
In this study we investigated YscF, a surface-expressed protein of the Yersinia pestis type III secretion complex, as a protective antigen against experimental plague infection. Immunization of mice with YscF resulted in a high anti-YscF titer and provided protection against i.v. challenge with Y. pestis. This is the first report to our knowledge utilizing a conserved protein from the type III secretion complex of a gram-negative pathogen as a candidate for vaccine development.
- Subunit Vaccine
- Protective Antigen
- Yersinia Pestis
- Strong Antibody Response
Yersinia pestis, the causative agent of plague, causes rapidly progressing disease in humans with a high mortality rate, especially in the pneumonic form of the disease. Due to the severe nature of plague, its ability for aerosol transmission, and the potential for human to human transmission plague is considered to be a disease of high concern as an agent of biological warfare or biological terrorism . For this reason, an improved vaccine for plague is desirable. Current efforts for vaccine development have focused on two proteins: LcrV (also known as the V antigen) and the capsular F1 antigen . The best results to date have been obtained by using a combination of recombinant LcrV and F1 subunits  separately or as a fusion protein [4, 5]. These subunit vaccines demonstrate very good protection against both pneumonic and systemic forms of plague  in mouse models. One of the potential limitations of these subunit vaccines is that F1 is not required for full virulence of Y. pestis, as F1-negative strains have the same LD50 value as F1-positive strains [6–9]. A second limitation that could result in undesired side-effects in immunized individuals is the demonstrated immunosuppressive effect of LcrV [10–13]. Additionally, serologic diversity of LcrV has been reported, in Yersinia species other than Y. pestis, that could theoretically limit the usefulness of an LcrV based vaccine. While the recombinant subunit vaccines are very effective in experimental animals and offer protection against F1 minus strains of Y. pestis , the inclusion of other antigens with the LcrV/F1 subunit vaccine candidates could improve the ability of the resulting vaccine to offer protection against multiple Y. pestis strains, or the new antigens could be developed as separate vaccine candidates.
The type III secretion apparatus encoded on the low-calcium response (LCR) virulence plasmid, pCD1 in strain KIM , of Y. pestis is a conserved virulence mechanism that is absolutely required for virulence of Y. pestis . YscF is a surface localized protein that is required both to secrete Yops and to translocate toxins into eukaryotic cells [16–19]. One report speculates that YscF polymerization is required for a YscF needle to puncture eukaryotic cell membranes . Other researchers suggest that YscF and its homologs function to provide a base that a translocon complex is built upon, or that YscF builds a conduit from the bacterium to the eukaryotic membrane . This suggestion seems more likely given that other proteins such as YopB, YopD, and LcrV are also required for translocation into eukaryotic cells [21–28]. Additionally, YscF needle producing Y. enterocolitica deficient in production of the translocators (LcrV, YopB, and YopD) do not translocate Yops into macrophages, demonstrating that the YscF-needle is not sufficient for translocation .
Most currently described pathogenesis-related type III secretion systems possess homologs to YscF. In pathogenic Salmonella and Shigella, the YscF homologs (PrgI and MxiH, respectively) have been demonstrated to form a needle structure that protrudes from the surface of bacterial cells [29–31]. The best-characterized homolog of YscF is EscF of enteropathogenic E. coli (EPEC). EscF is required for "attaching and effacing" (A/E) lesion formation on the intestinal mucosa and for type III secretion of effector proteins [32–34]. EscF is thought to be a structural component of the needle complex on the bacterial surface as it binds EspA, the major component of a filamentous surface organelle, and is required for formation of the EspA filaments. [32–34] However, this surface localization has never been visualized directly, as the only EscF antiserum generated was unable to recognize the native protein .
Based on the fact that YscF is thought to be a surface-expressed protein in Y. pestis and is required for virulence, we sought to determine if YscF could serve as a protective antigen against experimental plague infection. Immunization of mice with His-tagged YscF resulted in a high anti-YscF titer and significant protection against i.v. challenge with Y. pestis. The findings of this study suggest that YscF may be a potential plague vaccine candidate that could be used in conjunction with other plague antigens, or possibly alone if its efficacy can be improved by alternative delivery methods.
Expression and purification of HT-YscF
Specificity of the antibody response to YscF
Active immunization of outbred mice followed by challenge with Y. pestis KIM5
IgG response to HT-YscF vaccination and LD50 determination.
Fold increase in survival
Characterization of the antibody response to HT-YscF
Antibody isotype titers from mice* immunized with HT-YscF.
Ability of α-YscF to effect Yop translocation
To examine one possible method that anti-YscF could be functioning to provide protection in immunized animals the ability to translocate Yops in the presence of anti-YscF antiserum was examined. Antibody to the surface-localized LcrV has been shown to block the ability of the TTSS in Y. pestis to translocate Yops into cultured macrophages [41, 42] but anti-LcrV was unable to block translocation into HeLa cells .
However, anti-LcrV was able to block Yops translocation by Y. pseduotuberculosis into HeLa cells . Since YscF is also surface-localized the ability of anti-HT-YscF to block Yop translocation into HeLa cells was tested. Day et al have described an elegant methodology to follow the translocation of Yop effector by fusing them to a Elk reporter . Elk, a eukaryotic transcriptional activator, becomes phosphorylated only after entering the nucleus, providing a reporter for translocation into eukaryotic cells [43, 44]. This methodology has the advantage of not requiring cell fractionation and protease protection assays to establish the intracellular localization of translocated proteins. To test the ability of anti-YscF to block translocation Y. pestis KIM8-3002 was transformed with plasmid pYopE129-Elk . HeLa cells were infected at an MOI of 10 and infection was allowed to progress for 4 h. After the 4 h incubation infected HeLa cells were harvested and immunoblotted to analyze YopE, Elk and PO4-Elk production. Y. pestis KIM8-3002 (wt; ) and an isogenic translocation defective strain containing a yopD deletion (KIM8-3002.2, ΔyopD; ) both containing pYopE129-Elk were used as positive and negative translocation controls, respectively. Immunoblots of HeLa cells infected with the wt and the ΔyopD strains showed that only the wt strain elicited the production of PO4-Elk while the ΔyopD strain had no production of PO4-Elk. The wt and the ΔyopD strains were used to infect HeLa cells in the presence of a 1:10 or a 1:25 dilution of anti-YscF (titer for HT-YscF, 1:100,000) or in the presence of anti-PcrG (a Yersinia non-reactive antibody control, titer for PcrG, 1:20,000). The wt strain was capable of translocating YopE-Elk in presence of both anti-sera, demonstrating that anti-YscF was not capable of blocking Yops translocation and expectedly the ΔyopD strain was still defective for translocation. The experiment likely contained sufficient antibody against YscF to block translocation. In a previous report Pettersson et al used as low as a 1:100 dilution of an anti-LcrV anti-sera with a titer of 1:20,000 for LcrV and in that experiment translocation of Yops into HeLa cells was blocked . These results suggest that antibody to YscF may not exert its protective effect by blocking Yops translocation. The results also suggest that while YscF is surface-exposed in the yersinae, antibody directed against YscF, unlike, anti-LcrV cannot block translocation. This may imply that YscF activity is shielded from neutralization by antibody, unlike LcrV activity that is blocked by antibody in some cases. However, since anti-LcrV was unable to block Yops translocation into by Y. pestis into HeLa cells  but could block translocation into cultured macrophages [41, 42], the possibility remains that anti-YscF also display this type of differential blockage.
Cloning of yscF for overexpression and HT-YscF purification
Plasmid pJM119 was constructed by cloning a Bam HI-and Xho I-cleaved PCR product into pET24b (Novagen, Madison, WI). The primers used to amplify yscF were HT-YscF Start (5' CGG GAT CCG ATG AGT AAC TTC TCT GGA TTT 3') and HT-YscF Stop (5' CCG CTC GAG TGG GAA CTT CTG TAG GAT GCC 3'). E. coli BL21(DE3) (Novagen) harboring pJM119 was used for HT-YscF overpexpression according to the manufacturer's suggestions. HT-YscF was purified using Talon resin (BD Clontech, Palo Alto, CA) according the manufacturer's directions.
Immunization of mice and infection with Y. pestis KIM5
For primary immunization 6-to 8-week-old female Swiss-Webster mice were immunized i.p. with 40 μg/mouse His-tagged YscF or phosphate-buffered saline  PBS (control mice) emulsified 1:1 with complete Freund's adjuvant (CFA). Booster immunizations were performed the same as the primary immunization with the substitution of Incomplete Freund's Adjuvant for CFA. Mice were challenged with Y. pestis via the retro-orbital sinus using blunt-feeding needles. Y. pestis used to infect mice was grown overnight in HIB broth, sub-cultured to an A620 of 0.2 absorbance units and grown with shaking to an A620 of 1.0 absorbance unit. Y. pestis cells for infection were harvested by centrifugation and resuspended in PBS. Plate counts were performed to verify CFUs for the infectious doses. Infected animals were monitored for death for up to 19 days, after which survivors were euthanized by CO2 inhalation, according to the guidelines of the Panel on Euthanasia of the American Veterinary Medical Association. All animal work for this project was reviewed and approved by UND's IACUC.
Bacterial strains, growth and fractionation
Bacterial strains used were KIM8-3002 , ΔyscF an isogenic in-frame deletion of yscF (G. Plano, University of Miami), and KIM8.3002.2 ΔyopD . Y. pestis strains were grown in heart infusion broth or on tryptose blood agar base medium (Difco Laboratories, Detroit, MI) at 26°C for genetic manipulations. For physiological studies, growth of Y. pestis was conducted in a defined medium, TMH, as previously described . Bacterial cells were fractionated as previously described . Briefly, bacterial cells were chilled on ice after growth, harvested by centrifugation, and washed in cold phosphate-buffered saline (PBS; ). Bacterial whole cell fractions were prepared by resuspending the washed cells in cold PBS and precipitating total proteins with 10% (vol/vol) trichloroacetic acid (TCA) on ice overnight. Secreted proteins were recovered from the bacterial growth medium by centrifuging the spent medium a second time, transferring the supernatant to a clean tube, and precipitating with 10% (vol/vol) TCA on ice overnight. The TCA-precipitated proteins were pelleted by centrifugation (20,800 × g at 4°C) for 20 min and resuspended in 2X sodium dodecyl sulfate (SDS) sample buffer .
Protein electrophoresis, visualization and immunodetection
Proteins were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), using 12.5 % or 15 % (wt/vol) polyacrylamide gels according to the method of Laemmli . Samples were boiled 3–5 min before loading on the gels. Samples were loaded such that lanes containing different culture fractions represented equivalent amounts of the original cultures. Proteins were visualized in gels using GelCode Blue stain (Pierce Chemical, Rockford, IL) according to directions. For immunoblots, proteins resolved by SDS-PAGE were transferred to Immobilon-P membranes (Millipore Corp., Bedford, MA) using carbonate transfer buffer (pH 9.9) . Specific proteins were visualized using mouse or rabbit polyclonal antibodies specific for YopE (rabbit α-YopE; gift from G. Plano, University of Miami, Miami, FL), YscF (mouse α-YscF, this study), Elk (rabbit α-Elk, Cell Signaling Technology, Beverley, MA) and PO4-Elk (rabbit α-PO4-Elk, Cell Signaling Technology). Hexahistidine tagged YscF was visualized using a penta-histidine specific antibody (mouse α-Penta-His, Qiagen, Valencia, CA) Alkaline phosphatase-conjugated secondary antibody (goat anti-rabbit immunoglobulin G or goat anti-mouse immunoglobulin G; Pierce) was used to visualize proteins by development with nitroblue tetrazolium and 5-bromo-4-chloro-3-indolylphosphate (NBT-BCIP; Fisher Scientific, Fair Lawn, NJ).
Antibody characterization and isotyping
Flat-bottom, 96-well Nunc Maxisorp immunoplates (Fisher Scientific, Pittsburgh, PA) were coated with 100 μl of HT-YscF solution (4 μg/ml in Binding solution (0.1 M NaH2PO4, ph 9.0) at room temperature for 2 h (or overnight at 4°C). The wells were blocked with 200 μl/well blocking buffer (1% bovine serum albumin in TTBS (tris-buffered saline  + 0.5% Tween 20) and washed with TTBS. Test sera were serially diluted in blocking buffer and 100 μl of each dilution was added to duplicate wells that were incubated for 2 h at RT (or overnight at 4°C). The plates were washed and incubated for 2 h at RT with alkaline-phosphatase-conjugated anti-mouse secondary antibody. For quantitation of YscF-specific immunoglobulin isotypes and subclasses the plates were coated with alkaline-phosphatase-labeled anti-mouse isotype-specific antibody (1:400 in blocking buffer; Southern Biotech, Birmingham, AL). The wells were washed and 75 μl 3 mM para-nitro phenyl phosphate (p-NPP) was added to each well. The plates were incubated for 15 min at RT and the reaction was stopped by the addition of 50 μl of 1.5 M NaOH to each well. A405 was measured to monitor the cleavage of p-NPP. Antibody titers were determined as reciprocal numbers of the highest serum dilution that displayed values for optical density twofold higher than the value of the control serum.
Infection of eukaryotic cells was performed as described previously . Prior to infection, eukaryotic cells were subcultured into 35-mm-diameter six-well tissue culture plates in RPMI-FBS and incubated at 37°C under 5% CO2 for 48 to 72 h to a density of 5 × 105 to 8 × 105 cells per well. Cells were washed twice with warm L15 lacking FBS immediately prior to infection. Bacteria were cultivated at 26°C in HIB and used at an OD620 of ~1.0 for tissue culture infections. Bacteria were added (at a multiplicity of infection (MOI) of 5 to 10) directly to prewarmed medium in the wells of the six-well plates. Plates were then centrifuged at 200 × g at RT for 5 min to achieve contact between the bacteria and the target cells and incubated at 37°C for 4 h.
Translocation of YopE
Detection of Elk-tagged YopE from pYopE129-Elk was performed as described . Y. pestis strains carrying plasmid pYopE129-Elk were used to infect HeLa cells. After 4 h, the culture supernatants were removed, and the infected adherent cells were lysed by the addition of 100 μl of 2X SDS-PAGE lysis buffer containing Pefabloc (Roche Molecular Biochemicals, Indianapoli, IN) and phosphatase inhibitor (P-2850) cocktail (Sigma, St. Louis, MO). Samples were boiled for 5 min and loaded onto 12.5 % SDS-PAGE gels, immunoblotted to PVDF membranes and probed with Elk-1 (#9182) or phosphospecific Elk-1 (#9181) antibody preparations (Cell Signaling Technology). Anti-sera specific for HT-YscF (titer of 1:100,000, this study) or the Pseudomonas aeruginosa LcrG homolog, PcrG (titer of 1:20,000, Matson and Nilles, unpublished), were used at dilutions of 1:10 or 1:25 in the infection medium to assess the ability of α-YscF to effect YopE translocation.
The author's would like to thank Deanna O'Bryant and Jennifer Lamoureux for assistance with mouse experiments, Jennifer Miller for help with antibody ELISAs and Gregory Plano (University of Miami, Miami, FL) for YopE antiserum and the ΔyscF strain of Y. pestis. This work was supported by the UND Faculty Research Seed Money program. J. S. M was supported by a pre-doctoral fellowship from ND-EPSCoR. Work in M. L. N.'s laboratory is supported by NIAID grants R01-AI051520 and U01-AI54815.
- Inglesby TV, Dennis DT, Henderson DA, Bartlett JG, Ascher MS, Eitzen E, Fine AD, Friedlander AM, Hauer J, Koerner JF, Layton M, McDade J, Osterholm MT, O'Toole T, Parker G, Perl TM, Russell PK, Schoch-Spana M, Tonat K: Plague as a biological weapon: medical and public health management. Working Group on Civilian Biodefense. Jama. 2000, 283 (17): 2281-2290. 10.1001/jama.283.17.2281.View ArticlePubMedGoogle Scholar
- Titball RW, Williamson ED: Yersinia pestis (plague) vaccines. Expert Opin Biol Ther. 2004, 4 (6): 965-973. 10.1517/147125220.127.116.115.View ArticlePubMedGoogle Scholar
- Titball RW, Williamson ED: Vaccination against bubonic and pneumonic plague. Vaccine. 2001, 19 (30): 4175-4184. 10.1016/S0264-410X(01)00163-3.View ArticlePubMedGoogle Scholar
- Anderson GWJ, Heath DG, Bolt CR, Welkos SL, Friedlander AM: Short- and long-term efficacy of single-dose subunit vaccines against Yersinia pestis in mice. Am J Trop Med Hyg. 1998, 58 (6): 793-799.PubMedGoogle Scholar
- Heath DG, Anderson GWJ, Mauro JM, Welkos SL, Andrews GP, Adamovicz J, Friedlander AM: Protection against experimental bubonic and pneumonic plague by a recombinant capsular F1-V antigen fusion protein vaccine. Vaccine. 1998, 16 (11-12): 1131-1137. 10.1016/S0264-410X(98)80110-2.View ArticlePubMedGoogle Scholar
- Davis KJ, Fritz DL, Pitt ML, Welkos SL, Worsham PL, Friedlander AM: Pathology of experimental pneumonic plague produced by fraction 1-positive and fraction 1-negative Yersinia pestis in African green monkeys (Cercopithecus aethiops). Arch Pathol Lab Med. 1996, 120 (2): 156-163.PubMedGoogle Scholar
- Drozdov IG, Anisimov AP, Samoilova SV, Yezhov IN, Yeremin SA, Karlyshev AV, Krasilnikova VM, Kravchenko VI: Virulent non-capsulate Yersinia pestis variants constructed by insertion mutagenesis. J Med Microbiol. 1995, 42 (4): 264-268.View ArticlePubMedGoogle Scholar
- Du Y, Galyov E, Forsberg A: Genetic analysis of virulence determinants unique to Yersinia pestis. Contrib Microbiol Immunol. 1995, 13: 321-324.PubMedGoogle Scholar
- Welkos SL, Davis KM, Pitt LM, Worsham PL, Freidlander AM: Studies on the contribution of the F1 capsule-associated plasmid pFra to the virulence of Yersinia pestis. Contrib Microbiol Immunol. 1995, 13: 299-305.PubMedGoogle Scholar
- Nakajima R, Brubaker RR: Association between virulence of Yersinia pestis and suppression of gamma interferon and tumor necrosis factor alpha. Infection and Immunity. 1993, 61: 23-31.PubMed CentralPubMedGoogle Scholar
- Nakajima R, Motin VL, Brubaker RR: Suppression of cytokines in mice by protein A-V antigen fusion peptide and restoration of synthesis by active immunization. Infection and Immunity. 1995, 63: 3021-3029.PubMed CentralPubMedGoogle Scholar
- Nedialkov YA, Motin VL, Brubaker RR: Resistance to lipopolysaccharide mediated by the Yersinia pestis V antigen-polyhistidine fusion peptide: amplification of interleukin-10. Infection and Immunity. 1997, 65: 1196-1203.PubMed CentralPubMedGoogle Scholar
- Sing A, Rost D, Tvardovskaia N, Roggenkamp A, Wiedemann A, Kirschning CJ, Aepfelbacher M, Heesemann J: Yersinia V-antigen exploits toll-like receptor 2 and CD14 for interleukin 10-mediated immunosuppression. J Exp Med. 2002, 196 (8): 1017-1024. 10.1084/jem.20020908.PubMed CentralView ArticlePubMedGoogle Scholar
- Perry RD, Straley SC, Fetherston JD, Rose DJ, Gregor J, Blattner FR: DNA sequencing and analysis of the low-Ca2+-response plasmid pCD1 of Yersinia pestis KIM5. Infection and Immunity. 1998, 66: 4611-4623.PubMed CentralPubMedGoogle Scholar
- Perry RD, Fetherson JD: Yersinia pestis — etiologic agent of plague. Clinical Microbiology Reviews. 1997, 10: 35-66.PubMed CentralPubMedGoogle Scholar
- Allaoui A, Schulte R, Cornelis GR: Mutational analysis of the Yersinia enterocolitica virC operon: characterization of yscE, F, G, I, J, K required for Yop secretion and yscH encoding YopR. Mol Microbiol. 1995, 18 (2): 343-355. 10.1111/j.1365-2958.1995.mmi_18020343.x.View ArticlePubMedGoogle Scholar
- Haddix PL, Straley SC: Structure and regulation of the Yersinia pestis yscBCDEF operon. J Bacteriol. 1992, 174 (14): 4820-4828.PubMed CentralPubMedGoogle Scholar
- Hoiczyk E, Blobel G: Polymerization of a single protein of the pathogen Yersinia enterocolitica into needles punctures eukaryotic cells. Proc Natl Acad Sci U S A. 2001, 98 (8): 4669-4674. 10.1073/pnas.071065798.PubMed CentralView ArticlePubMedGoogle Scholar
- Marenne MN, Journet L, Mota LJ, Cornelis GR: Genetic analysis of the formation of the Ysc-Yop translocation pore in macrophages by Yersinia enterocolitica: role of LcrV, YscF and YopN. Microb Pathog. 2003, 35 (6): 243-258. 10.1016/S0882-4010(03)00154-2.View ArticlePubMedGoogle Scholar
- Cornelis GR: The Yersinia Ysc-Yop 'type III' weaponry. Nat Rev Mol Cell Biol. 2002, 3 (10): 742-752. 10.1038/nrm932.View ArticlePubMedGoogle Scholar
- Fields KA, Nilles ML, Cowan C, Straley SC: Virulence role of V antigen of Yersinia pestis at the bacterial surface. Infection and Immunity. 1999, 67 (10): 5395-5408.PubMed CentralPubMedGoogle Scholar
- Holmström A, Olsson J, Cherepanov P, Maier E, Nordfelth R, Pettersson J, Benz R, Wolf-Watz H, Forsberg AA: LcrV is a channel size-determining component of the Yop effector translocon of Yersinia. Mol Microbiol. 2001, 39 (3): 620-632. 10.1046/j.1365-2958.2001.02259.x.View ArticlePubMedGoogle Scholar
- Håkansson S, Schesser K, Persson C, Galyov EE, Rosqvist R, Homblé F, Wolf-Watz H: The YopB protein of Yersinia pseudotuberculosis is essential for the translocation of Yop effector proteins across the target cell plasma membrane and displays a contact-dependent membrane disrupting activity. EMBO Journal. 1996, 15: 5812-5823.PubMed CentralPubMedGoogle Scholar
- Nilles ML, Fields KA, Straley SC: The V antigen of Yersinia pestis regulates Yop vectorial targeting as well as Yop secretion through effects on YopB and LcrG. Journal of Bacteriology. 1998, 180 (13): 3410-3420.PubMed CentralPubMedGoogle Scholar
- Nordfelth R, Wolf-Watz H: YopB of Yersinia enterocolitica Is Essential for YopE Translocation. Infect Immun. 2001, 69 (5): 3516-3518. 10.1128/IAI.69.5.3516-3518.2001.PubMed CentralView ArticlePubMedGoogle Scholar
- Pettersson J, Holmström A, Hill J, Leary S, Frithz-Lindsten E, von Euler-Matell A, Carlsson E, Titball R, Forsberg , Wolf-Watz H: The V-antigen of Yersinia is surface-exposed before targent cell contact and involved in virulence protein translocation. Molecular Microbiology. 1999, 32 (5): 961-976. 10.1046/j.1365-2958.1999.01408.x.View ArticlePubMedGoogle Scholar
- Sory MP, Cornelis GR: Translocation of a hybrid YopE-adenylate cyclase from Yersinia enterocolitica into HeLa cells. Molecular Microbiology. 1994, 14: 583-594.View ArticlePubMedGoogle Scholar
- Tardy F, Homblé F, Neyt C, Wattiez R, Cornelis GR, Ruysschaert JM, Cabiaux V: Yersinia enterocolitica type III secretion-translocation system: channel formation by secreted Yops. EMBO Journal. 1999, 18 (23): 6793-6799. 10.1093/emboj/18.23.6793.PubMed CentralView ArticlePubMedGoogle Scholar
- Blocker A, Jouihri N, Larquet E, Gounon P, Ebel F, Parsot C, Sansonetti P, Allaoui A: Structure and composition of the Shigella flexneri "needle complex", a part of its type III secreton. Mol Microbiol. 2001, 39 (3): 652-663. 10.1046/j.1365-2958.2001.02200.x.View ArticlePubMedGoogle Scholar
- Kubori T, Sukhan A, Aizawa SI, Galan JE: Molecular characterization and assembly of the needle complex of the Salmonella typhimurium type III protein secretion system. Proc Natl Acad Sci U S A. 2000, 97 (18): 10225-10230. 10.1073/pnas.170128997.PubMed CentralView ArticlePubMedGoogle Scholar
- Kubori T, Matsushima Y, Nakamura D, Uralil J, Lara-Tejero M, Sukhan A, Galan JE, Aizawa SI: Supramolecular structure of the Salmonella typhimurium type III protein secretion system. Science. 1998, 280: 602-605. 10.1126/science.280.5363.602.View ArticlePubMedGoogle Scholar
- Sekiya K, Ohishi M, Ogino T, Tamano K, Sasakawa C, Abe A: Supermolecular structure of the enteropathogenic Escherichia coli type III secretion system and its direct interaction with the EspA-sheath-like structure. Proc Natl Acad Sci U S A. 2001, 98 (20): 11638-11643. 10.1073/pnas.191378598.PubMed CentralView ArticlePubMedGoogle Scholar
- Wilson RK, Shaw RK, Daniell S, Knutton S, Frankel G: Role of EscF, a putative needle complex protein, in the type III protein translocation system of enteropathogenic Escherichia coli. Cell Microbiol. 2001, 3 (11): 753-762. 10.1046/j.1462-5822.2001.00159.x.View ArticlePubMedGoogle Scholar
- Daniell SJ, Takahashi N, Wilson R, Friedberg D, Rosenshine I, Booy FP, Shaw RK, Knutton S, Frankel G, Aizawa S: The filamentous type III secretion translocon of enteropathogenic Escherichia coli. Cell Microbiol. 2001, 3 (12): 865-871. 10.1046/j.1462-5822.2001.00168.x.View ArticlePubMedGoogle Scholar
- Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA, Struhl K: Current Protocols in Molecular Biology. 1998, New York , John Wiley & SonsGoogle Scholar
- Cordes FS, Komoriya K, Larquet E, Yang S, Egelman EH, Blocker A, Lea SM: Helical structure of the needle of the type III secretion system of Shigella flexneri. J Biol Chem. 2003, 278 (19): 17103-17107. 10.1074/jbc.M300091200.View ArticlePubMedGoogle Scholar
- Coligan JE, Dunn BM, Speicher DW, Wingfield PT: Current protocols in protein science. 1998, New York , John Wiley & SonsGoogle Scholar
- Welkos S, O'Brien A: Determination of median lethal and infectious doses in animal model systems. Methods Enzymol. 1994, 235: 29-39.View ArticlePubMedGoogle Scholar
- Andrews GP, Strachan ST, Benner GE, Sample AK, Anderson GWJ, Adamovicz JJ, Welkos SL, Pullen JK, Friedlander AM: Protective efficacy of recombinant Yersinia outer proteins against bubonic plague caused by encapsulated and nonencapsulated Yersinia pestis. Infect Immun. 1999, 67 (3): 1533-1537.PubMed CentralPubMedGoogle Scholar
- Williamson ED, Vesey PM, Gillhespy KJ, Eley SM, Green M, Titball RW: An IgG1 titre to the F1 and V antigens correlates with protection against plague in the mouse model. Clin Exp Immunol. 1999, 116 (1): 107-114. 10.1046/j.1365-2249.1999.00859.x.PubMed CentralView ArticlePubMedGoogle Scholar
- Philipovskiy AV, Cowan C, Wulff-Strobel CR, Burnett SH, Kerschen EJ, Cohen DA, Kaplan AM, Straley SC: Antibody against V antigen prevents Yop-dependent growth of Yersinia pestis. Infect Immun. 2005, 73 (3): 1532-1542. 10.1128/IAI.73.3.1532-1542.2005.PubMed CentralView ArticlePubMedGoogle Scholar
- Weeks S, Hill J, Friedlander A, Welkos S: Anti-V antigen antibody protects macrophages from Yersinia pestis -induced cell death and promotes phagocytosis. Microb Pathog. 2002, 32 (5): 227-237. 10.1006/mpat.2002.0498.View ArticlePubMedGoogle Scholar
- Day JB, Ferracci F, Plano GV: Translocation of YopE and YopN into eukaryotic cells by Yersinia pestis yopN, tyeA, sycN, yscB and lcrG deletion mutants measured using a phosphorylatable peptide tag and phosphospecific antibodies. Mol Microbiol. 2003, 47 (3): 807-823. 10.1046/j.1365-2958.2003.03343.x.View ArticlePubMedGoogle Scholar
- Day JB, Lee CA: Secretion of the orgC gene product by Salmonella enterica serovar Typhimurium. Infect Immun. 2003, 71 (11): 6680-6685. 10.1128/IAI.71.11.6680-6685.2003.PubMed CentralView ArticlePubMedGoogle Scholar
- Williams AW, Straley SC: YopD of Yersinia pestis plays a role in the negative regulation of the low-calcium response in addition to its role in the translocation of Yops. Journal of Bacteriology. 1998, 180: 350-358.PubMed CentralPubMedGoogle Scholar
- Straley SC, Bowmer WS: Virulence genes regulated at the transcriptional level by Ca2+ in Yersinia pestis include structural genes for outer membrane proteins. Infection and Immunity. 1986, 51: 445-454.PubMed CentralPubMedGoogle Scholar
- Laemmli UK: Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970, 227: 680-685.View ArticlePubMedGoogle Scholar
- Skrzypek E, Straley SC: LcrG, a secreted protein involved in negative regulation of the low-calcium response in Yersinia pestis. Journal of Bacteriology. 1993, 175: 3520-3528.Google Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.