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Infection and Immunity, June 2006, p. 3687-3691, Vol. 74, No. 6
0019-9567/06/$08.00+0     doi:10.1128/IAI.01837-05
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

The ABC Transporter Protein OppA Provides Protection against Experimental Yersinia pestis Infection

Mikio Tanabe,1,2 Helen S. Atkins,3 David N. Harland,3 Stephen J. Elvin,3 Anthony J. Stagg,3 Osman Mirza,2 Richard W. Titball,3,4* Bernadette Byrne,2 and Katherine A. Brown1

Division of Cell and Molecular Biology, Centre for Molecular Microbiology and Infection, Imperial College London, London SW7 2AZ, United Kingdom,1 Division of Molecular Biosciences, Membrane Protein Crystallography Group, Imperial College London, London SW7 2AZ, United Kingdom,2 Defence Science and Technology Laboratory, Porton Down, Salisbury SP4 0JQ, United Kingdom,3 Department of Infectious and Tropical Disease, London School of Hygiene and Tropical Medicine, Keppel St., London, WC1E 7HT, United Kingdom4

Received 10 November 2005/ Returned for modification 21 December 2005/ Accepted 16 March 2006


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ABSTRACT
 
The identification of Yersinia pestis as a potential bioterrorism agent and the emergence of antibiotic-resistant strains have highlighted the need for improved vaccines and treatments for plague. The aim of this study was to evaluate the potential for ATP-binding cassette (ABC) transporter proteins to be exploited as novel vaccines against plague. Western blotting of ABC transporter proteins using sera from rabbits immunized with killed whole Y. pestis cells or human convalescent-phase sera identified four immunologically reactive proteins: OppA, PstS, YrbD, and PiuA. Mice immunized with these proteins developed antibody to the immunogen. When the immunized mice were challenged with Y. pestis, the OppA-immunized mice showed an increased time to death compared to other groups, and protection appeared to correlate with the level of immunoglobulin G antibody to OppA.


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TEXT
 
Yersinia pestis is the causative agent of plague, and globally between 1,000 and 3,000 cases of plague are reported to the World Health Organization each year (http://www.who.int/csr/disease/plague/en/). Most of these cases are the bubonic form of the disease, usually a consequence of the transmission of bacteria to humans via bites from fleas that have previously fed on infected rodents. More rarely, cases of pneumonic plague are reported that are characterized by a short incubation period of 2 to 3 days and a high rate of mortality, even if treated. Pneumonic plague can be transmitted person to person or animal to person via the inhalation of contaminated air droplets (16). Pneumonic plague is the likely consequence of the use of Y. pestis as a biological weapon (12). A number of antibiotics are active against Y. pestis, but their use to treat plague is dependent on early intervention, in particular for the treatment of pneumonic plague (3, 6).

Killed whole Y. pestis cells have been used in various plague vaccine formulations for many years. The evidence is that this type of vaccine provides protection against bubonic plague. However, the ability of killed whole-cell vaccines to provide protection against pneumonic plague is questionable (24). Furthermore, the vaccine is associated with a high incidence of side effects (15). Thus, an aim of research over the past decade has been to identify a subunit vaccine which provides protection against both bubonic and pneumonic plagues. A wide range of cell surface molecules have been evaluated as vaccine components, including lipopolysaccharide and components of the type III secretion system (22). To date, only the F1 and V antigens have been shown to induce protective immunity, and improved vaccines based on the F1 and V antigens (21, 26, 27) are being developed. However, since F1 antigen-negative strains of Y. pestis have been reported (28), the identification of an additional protective antigen against Y. pestis that may be included in a subunit vaccine is desirable.

ATP-binding cassette (ABC) transporters have previously been identified as targets for development of vaccines against pathogenic bacteria (7). The ABC transporter family belongs to the primary energy-dependent transporter group and is one of the largest transporter families responsible for diverse physiological processes, including drug efflux from cancer cells and bacterial nutrient uptake across the cell envelope using the free energy of hydrolysis of ATP (10). Conventional ABC transporters have two transmembrane domains and two ATP- or nucleotide-binding domains. In gram-negative bacteria, periplasmic binding proteins bind substrate in the periplasm and transfer it to the membrane-bound ABC transporter (11). In a recent study, we reported the cloning, expression, and preliminary crystallization of putative ABC transporter proteins and other related proteins from Y. pestis (20). In the present study, we have assessed the potential of ABC transporters to induce protective immunity against Y. pestis in a mouse model of infection.

Materials. Antibodies were obtained from Sigma-Aldrich and Serotec. All other chemicals were obtained from Sigma-Aldrich and BDH Biosciences.

Identification, cloning, expression, and purification of His-tagged antigen candidate proteins. The strategy for cloning, expression, and purification of CysP, LolC, OppA, PiuA, PotF, PstS, TolC, UgpB, YfeA, and YrbD (Table 1) was as described previously (20). Briefly, the Y. pestis proteins were expressed by using a pET vector system in an Escherichia coli host and purified by using Ni2+-nitrilotriacetic acid (NTA) affinity chromatography. The purity of the proteins was ascertained by sodium dodecyl sulfate-polyacrylamide gel electrophoresis after they were stained with Coomassie brilliant blue R250, and the concentrations of the proteins were measured by the Bradford method (Bio-Rad), with bovine serum albumin as a protein standard. Truncated CysP, LolC, PiuA, and YfeA and full-length OppA, PotF, PstS, TolC, UgpB, and YrbD were used for further analysis (see Table 1).


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  		TABLE 1. Vaccine candidate proteins from Y. pestis

Western blot and enzyme-linked immunosorbent assay (ELISA) analyses of serum samples. For Western blot analysis, purified Y. pestis proteins were separated in 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels, transferred onto polyvinylidene difluoride membrane, and then probed with the appropriate antiserum according to standard protocols (18). Either rabbit antiserum from animals exposed to heat-killed Y. pestis strain JAVA9 or serum from humans who had recovered from a Y. pestis infection (1:2,000 dilution) was used as the primary antibody, and goat anti-rabbit or goat anti-human horseradish peroxidase-conjugated immunoglobulin G (IgG; 1:10,000 dilution), as appropriate, was used as the secondary antibody. F1 antigen was used as a positive control. Labeled proteins were detected by using an ECL kit (Amersham Biosciences).

ELISAs were used to quantify the antibody responses against individual proteins in immunized mice. Each individual protein sample (~50 µg) was applied to 48 wells of a 96-well plate. Serum from the test mice (n = 6) was added at a 1:500, 1:1,000, 1:2,000, 1:4,000, 1:8,000, 1:16,000, 1:32,000, or 1:64,000 dilution. Bound antibodies in the serum samples were detected by using horseradish peroxidase-conjugated anti-mouse IgG (1:10,000) or, where relevant, the individual IgG subclasses (IgG1, IgG2a, IgG2b, or IgG3; 1:2,000), as the secondary antibody. 1,2-Phenylethidiamine dihydrochloride and hydrogen peroxide were added as the substrate, and the plates were incubated at room temperature for 10 min. Endpoint total IgG antibody titers were expressed as the maximum dilution of sample giving an A490 of more than 0.1 unit after subtraction of the absorbance due to nonspecific binding measured by using negative control sera. Alternatively, the concentrations of the different IgG subclasses were estimated by using His-tagged OppA as a standard in which the mean ± standard error for each test point was calculated from ELISA data, using the results for four mice after exclusion of the highest and lowest values from the data.

Immunization and protection experiments. Purified His-tagged proteins were evaluated as candidate antigens in mouse immunization experiments. The proteins were prepared for immunization at a 100-µg/ml concentration in phosphate-buffered saline together with one of the following adjuvants: 25% (vol/vol) Alhydrogel or 2% Alhydrogel (Superfos Biosector a/s, Vedback, Denmark), 50% (vol/vol) MPL-plus-TDM adjuvant (Sigma-Aldrich Co., Ltd., Poole, United Kingdom), or Freund incomplete adjuvant (1:1 protein-adjuvant; Sigma-Aldrich). Six female BALB/c mice per group (8 to 12 weeks old) were used for all immunization experiments. Mice were administered 10 µg of each protein preparation by intramuscular injection (for Alhydrogel- or MPL+TDM-adjuvanted proteins) or by intraperitoneal injection (for Freund incomplete-adjuvanted proteins) on days 0, 14, and 28. Sera were collected from mice by retro-orbital bleeding on day 40. On day 57 the mice were subcutaneously challenged with approximately 25 CFU of Y. pestis GB, known to have a median lethal dose of ca. 1 CFU in BALB/c mice by the subcutaneous route (17), and then mice were observed daily until 15 days after challenge. One-way analysis of variance (ANOVA) with Tukey's multiple comparison post-analysis test and statistical analysis of survival using the Mantel-Haenszel Logrank test were performed using GraphPad Prism version 3.02 for Windows (GraphPad Software, San Diego, CA).

Cloning, expression, and purification of Y. pestis proteins. Initially, we identified 10 Y. pestis proteins (CysP, LolC, OppA, PiuA, PotF, PstS, TolC, UgpB, YfeA, and YrbD) for further study. These proteins included homologues of ABC transporter proteins, which have been shown to be protective antigens or to play a role in virulence in other pathogens (7). All of the encoding genes were previously expressed in Escherichia coli (20), and the proteins were isolated to 80 to 90% purity, a level suitable for both crystallization trials and immunological studies (Table 1).

OppA, PstS, YrbD, and PiuA are recognized by antisera to Y. pestis. The purified Y. pestis proteins were screened for reactivity with antisera by Western blotting. Antibodies were detected against OppA, PstS, and YrbD in sera from rabbits previously immunized with killed Y. pestis whole cells (Fig. 1A), and antibodies to PiuA were detected in sera from humans who had recovered from plague (Fig. 1B). Subsequently, the OppA, PstS, YrbD, and PiuA proteins were selected for further evaluation as candidate protective antigens against Y. pestis challenge in a mouse model.


Figure 1
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  		FIG. 1. Western blot analysis of Y. pestis antigen candidate proteins. The proteins were probed with sera from rabbits injected with killed Y. pestis cells (A) or humans convalescent from Y. pestis infection (B). The lanes are labeled as follows: 1, F1 antigen; 2, truncated CysP; 3, truncated LolC; 4, full-length OppA; 5, truncated PiuA; 6, full-length PotF; 7, full-length PstS; 8, full-length TolC; 9, full-length UgpB; 10, truncated YrbD; and 11, truncated YfeD. The molecular size is indicated in kilodaltons. Boxes indicate the immunoreactive proteins, open arrowheads indicate the approximate locations of the nonimmunoreactive proteins, and the F1 antigen is indicated by the asterisk. It is assumed that the F1 band corresponds to a higher oligomeric state of the protein. The strong signals observed for some low-molecular-weight bands on the gel in panel A may correspond to cytoplasmic proteins from E. coli carried over from the purification, since all of the Y. pestis proteins run in these lanes were isolated from the cytoplasmic fraction. It is possible that the rabbit serum cross-reacts with these E. coli proteins.

Mice immunized with OppA show an increased time to death. Groups of six mice were immunized with three doses of the individual proteins, together with Alhydrogel. Approximately 4 weeks after the final immunizing dose, sera were taken and analyzed for antibodies, and the mice were challenged subcutaneously with 25 CFU (ca. 25 LD50 doses) of a virulent strain of Y. pestis and monitored for survival. IgG subclass antibodies were present in pooled serum samples of animals immunized with PiuA, YrbD or OppA (endpoint titers of 32,000). In contrast, the IgG response to PstS was very low, with an endpoint titer of 1,000.

The mean time to death (MTD) of mice immunized against PstS, YrbD, or PiuA was similar to that of control mice given Alhydrogel only (Table 2). In contrast, mice immunized with OppA plus Alhydrogel showed a significant increase in time to death (P < 0.05) compared to naive controls (Table 2). In a repeat experiment, in which mice were immunized with OppA plus Alhydrogel, the titer of serum IgG antibody in individual mice prior to challenge was measured. The results showed that the time to death correlated with the level of IgG to OppA, with the highest titers corresponding to the longest survival times (Table 3). In this experiment, one of the immunized mice was alive at the termination of the experiment (15 days after challenge).


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  		TABLE 2. MTD of mice that had been immunized with PiuA, PstS, Oppa, or YrbD and challenged with 25 CFU of Y. pestis


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  		TABLE 3. Relationship of IgG responses to OppA in immunized mice and survival after challenge with 25 CFU of Y. pestis

Influence of adjuvant on antibody response and protection with OppA. To determine whether alternative adjuvants might enhance the protective response, mice were immunized with OppA adjuvanted with MPL plus TDM or with Freund incomplete adjuvant. When these mice were challenged with Y. pestis, there was no statistically significant increase in time to death compared to naive mice (data not shown).

The OppA-specific total IgG, IgG1, IgG2a, IgG2b, and IgG3 antibody responses in the sera from these OppA-immunized mice were measured by using ELISAs. These responses were compared to the responses in the mice which had been immunized with OppA plus Alhydrogel. (Table 4). Sera from naive mice, which did not receive OppA or adjuvant, did not contain measurable levels of antibody to IgG2a, IgG2b, or IgG3. However, a low level signal was obtained by using the IgG1 ELISA (0.03 µg/ml). The mice given OppA in MPL plus TDM developed low levels of all subclasses of IgG antibody. In comparison, mice given OppA in Alhydrogel or Freund incomplete adjuvant developed IgG antibodies. The level of IgG1 antibody in mice immunized with OppA in Alhydrogel (2.1 ± 0.3 mg/ml) was ~1.5 times higher than the level of IgG1 antibody in mice given OppA in Freund incomplete adjuvant (1.0 ± 0.2 mg/ml).


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  		TABLE 4. Subclasses of IgG raised against Y. pestis OppA using different adjuvants

The principal aim of the present study was to determine whether immunization with one or more of the ABC transporter proteins tested would protect against Y. pestis infection. Previous work had provided high-quality Y. pestis ABC transporter proteins in the quantities required for both immunological and structural studies (20). Initially, OppA, the periplasmic oligopeptide-binding protein (2), PstS, a periplasmic phosphate-binding protein (19), and YrbD, a putative toluene transport protein (13), were immunoreactive with the rabbit antisera, indicating that these proteins were expressed by Y. pestis when cultured in vitro. PiuA, an outer membrane iron uptake channel (4), was immunoreactive with convalescent human sera, indicating that this protein is recognized by the immune system during infection. The finding that PiuA was not protective in our model against Y. pestis infection is interesting since it has been shown that PiuA in Streptococcus pneumoniae is located on the surface and that recombinant PiuA protein is able to provide protection against systemic S. pneumoniae infection (5).

The results from the in vivo challenge experiments showed that mice immunized with OppA showed a significant increase in survival rate compared to controls. The correlation between serum IgG titers measured for the individual animals and survival after challenge suggests that antibody to OppA was responsible for protection. In addition, the protective effect of OppA seems to be adjuvant specific. Coadministration of OppA plus Freund incomplete adjuvant produced an immune response but no increase in time to death compared to controls. The IgG subclass profile stimulated by OppA given with Freund incomplete adjuvant differed from that of OppA delivered with Alhydrogel, perhaps reflecting the difference in protection afforded. OppA and Alhydrogel stimulated the highest titers of OppA-specific IgG1. Since studies of the F1 and V antigens have suggested an important role for the IgG1 subclass in protection against Y. pestis infection (25), the difference in OppA-specific IgG1 levels may reflect the difference in protection afforded by OppA in the various adjuvants.

OppA is a component of the oligopeptide (Opp) ABC transporter, binding substrate and delivering it to the membrane- bound complex OppBCDF for ATP-mediated transport across the membrane (1). Previous work with a range of bacteria has revealed that the Opp system plays a role in a variety of cellular processes, including nutrient acquisition (14), recycling of cell wall peptides for peptidoglycan synthesis (9), and virulence (8, 23). Y. pestis is predominantly an extracellular pathogen, which would allow antibody to bind to bacterial cells. However, OppA is generally considered to be located in the periplasm, a region of the bacterial cell with limited accessibility to the host immune system. Our data suggest that OppA is available to the immune system, and this finding is supported by other studies in which various ABC transporter components, including periplasmic binding proteins, have been shown to be immunogenic (7). It is possible that OppA-specific antibody could directly block the oligopeptide uptake system. Alternatively, it may have an opsonizing effect, promoting bacterial uptake into professional antigen-presenting cells. Further work is required to determine the role of OppA in virulence of Y. pestis and to elucidate how immunization with OppA provides protection against Y. pestis.

Taken together, these results indicate that OppA is a candidate for inclusion in future Y. pestis vaccines. It may be possible to improve the protection afforded by OppA against Y. pestis by using other delivery approaches or to include the protein in subunit vaccines together with other protective antigens. Studies to address these questions are under way.


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ACKNOWLEDGMENTS
 
This study was funded by the Defense Science and Technology Laboratory.

We are grateful to Joann Prior and Tim Webber for providing rabbit sera and to May Chu (World Health Organization, Geneva) for providing the human convalescent-phase sera used in this study.


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FOOTNOTES
 
* Corresponding author. Mailing address: Defence Science and Technology Laboratory, Porton Down, Salisbury SP4 0JQ, United Kingdom. Phone: 44 20 7594 3004. Fax: 44 1980 614307. E-mail: rtitball{at}dstl.gov.uk. Back

Editor: J. B. Bliska


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Infection and Immunity, June 2006, p. 3687-3691, Vol. 74, No. 6
0019-9567/06/$08.00+0     doi:10.1128/IAI.01837-05
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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