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. 2014 May;21(5):667–673. doi: 10.1128/CVI.00013-14

Direct Neutralization of Type III Effector Translocation by the Variable Region of a Monoclonal Antibody to Yersinia pestis LcrV

Maya I Ivanov a, Jim Hill b, James B Bliska a,✉
Editor: D L Burns
PMCID: PMC4018875  PMID: 24599533

Abstract

Plague is an acute infection caused by the Gram-negative bacterium Yersinia pestis. Antibodies that are protective against plague target LcrV, an essential virulence protein and component of a type III secretion system of Y. pestis. Secreted LcrV localizes to the tips of type III needles on the bacterial surface, and its function is necessary for the translocation of Yersinia outer proteins (Yops) into the cytosol of host cells infected by Y. pestis. Translocated Yops counteract macrophage functions, for example, by inhibiting phagocytosis (YopE) or inducing cytotoxicity (YopJ). Although LcrV is the best-characterized protective antigen of Y. pestis, the mechanism of protection by anti-LcrV antibodies is not fully understood. Antibodies bind to LcrV at needle tips, neutralize Yop translocation, and promote opsonophagocytosis of Y. pestis by macrophages in vitro. However, it is not clear if anti-LcrV antibodies neutralize Yop translocation directly or if they do so indirectly, by promoting opsonophagocytosis. To determine if the protective IgG1 monoclonal antibody (MAb) 7.3 is directly neutralizing, an IgG2a subclass variant, a deglycosylated variant, F(ab′)2, and Fab were tested for the ability to inhibit the translocation of Yops into Y. pestis-infected macrophages in vitro. Macrophage cytotoxicity and cellular fractionation assays show that the Fc of MAb 7.3 is not required for the neutralization of YopJ or YopE translocation. In addition, the use of Fc receptor-deficient macrophages, and the use of cytochalasin D to inhibit actin polymerization, confirmed that opsonophagocytosis is not required for MAb 7.3 to neutralize translocation. These data indicate that the binding of the variable region of MAb 7.3 to LcrV is sufficient to directly neutralize Yop translocation.

INTRODUCTION

Yersinia pestis is a Gram-negative bacterium and the agent of plague, an acute, often fatal infection that can manifest in three forms: bubonic, pneumonic, or septicemic (1, 2). Y. pestis has several characteristics that could facilitate its development as a biological weapon, resulting in its classification as a tier 1 select agent. These characteristics include the capacity for aerosol dissemination and the high fatality rate of pneumonic plague (3). In addition, Y. pestis remains a constant threat to public health because there are large enzootic reservoirs of plague in rodents in North and South America, Asia, and Africa, resulting in regular outbreaks of the disease in human populations (3).

It is important to develop new strategies to counteract Y. pestis infection. For example, there is a need for the development of immunotherapeutics to treat plague. Y. pestis secretes several proteins that have been studied as immunotherapeutic targets (2, 4, 5). The F1 protein is encoded on plasmid pMT1 and is assembled into an antiphagocytic capsule by a chaperone-usher pathway (1, 6). Mice passively immunized with an anti-F1 monoclonal antibody (MAb) (e.g., F1-04-A-G1) are protected against bubonic or pneumonic plague (7–9). However, F1− mutants of Y. pestis have been shown to retain full virulence in animal infection models, and therefore, F1 may not be an ideal immunotherapeutic target (1, 5).

LcrV is a multifunctional and essential virulence protein that is encoded together with other components of a type III section system (T3SS) on plasmid pCD1 (10, 11). LcrV is exported to the bacterial surface by the T3SS, localizes to the tip of the needle structure, and is secreted into the extracellular milieu (10–12). LcrV function is necessary for the T3SS to translocate a set of Yersinia outer protein (Yop) effectors, including YopJ and YopE, into host cells targeted by Y. pestis (11, 12). Delivery of effectors into host cells is thought to occur through a channel, or translocon, formed by the insertion of the YopB and YopD proteins into the plasma membrane (12).

Mice actively vaccinated with LcrV or passively immunized with anti-LcrV antibodies are protected against bubonic or pneumonic Y. pestis infection (4, 5). Anti-LcrV antibodies opsonize Yersinia by binding LcrV at the needle tip (13, 14). Protection by an anti-LcrV antibody in vivo correlates with reduced Yop translocation and cytotoxicity and increased opsonophagocytosis by macrophages in vitro (15, 16). Polyclonal F(ab′)2 to LcrV is as effective as intact IgG at inhibiting cytotoxicity in Y. pestis-infected macrophages (16). However, F(ab′)2 specific for LcrV was ineffective in promoting opsonophagocytosis (15, 16). These results suggest that the Fc region of the anti-LcrV antibody is not required for the neutralization of Yop translocation but is required for opsonophagocytosis. However, an anti-LcrV antibody did not neutralize the translocation of Yops into Y. pestis-infected macrophages that were treated with cytochalasin D (CD) to inhibit actin polymerization (15), suggesting that opsonophagocytosis neutralizes translocation indirectly, through internalization of the bacteria. Thus, the mechanism by which anti-LcrV antibodies neutralize the translocation of Yops into immune cells infected with Y. pestis remains unclear.

As reviewed in reference 17, several murine MAbs specific for LcrV have been shown to passively protect mice from bubonic or pneumonic plague (9, 18–21). The murine MAb 7.3 is potently protective; a single dose of 30 μg fully protects mice against intranasal challenge with 12 50% lethal doses (LD50) of Y. pestis (22). MAb 7.3 neutralizes Yop-dependent cytotoxicity and promotes opsonophagocytosis in macrophages infected with Y. pestis in vitro (16, 23).

The protective epitope in LcrV that is recognized by MAb 7.3 is conformational and localizes to amino acids 135 to 275 (18, 24, 25). Determination of the 3-dimensional structure of LcrV (26) revealed that it has an overall dumbbell shape, with the “handle” composed of two helices (alpha 7 and alpha 12) that form a coiled-coil. The LcrV N terminus forms a globular domain at one end of the handle. A second globular domain that is formed by the region between alpha 7 and alpha 12 in LcrV is found at the other end of the handle. The protective epitope recognized by MAb 7.3 corresponds to alpha helix 7 and the globular domain between helices 7 and 12.

The goal of this study was to determine if MAb 7.3 neutralizes Yop translocation directly or indirectly, by promoting opsonophagocytosis. To achieve this goal, variants of the IgG1 MAb 7.3 were obtained, by either class switching (to IgG2a), deglycosylation, or removal of the Fc region [F(ab′)2 or Fab]. The resulting variants were tested for the ability to inhibit the translocation of Yops into macrophages infected with Y. pestis in vitro. In addition, the importance of opsonophagocytosis for MAb 7.3 to neutralize Yop translocation was tested using macrophages deficient in the Fc receptor (FcR) or macrophages treated with CD to inhibit actin polymerization.

MATERIALS AND METHODS

Bacterial strains and growth conditions.

All Y. pestis strains used lack the pigmentation locus (Δpgm) and are exempt from select agent guidelines. KIM5 and KIM5yopB contain the pCD1 and pPCP1 plasmids and have been described previously (27). To prepare bacteria for macrophage infection assays, Y. pestis cultures were grown in heart infusion (HI) supplemented with ampicillin at 25 μg/ml with aeration overnight at 26°C. Bacteria were subcultured into HI broth containing 2.5 mM CaCl2 to an optical density at 600 nm (OD600) of 0.1. Cultures were shaken at 37°C for 2 h. Bacteria were pelleted by centrifugation and were resuspended in warm (37°C) phosphate-buffered saline (PBS) solution to an OD600 of 1.0 (∼1 × 109 CFU per milliliter).

Mice and macrophage cultures.

Eight-week-old female C57BL/6 female mice were purchased from Jackson Laboratories. FcRγ−/− mice have been described previously (28). Bone marrow-derived macrophages (BMDMs) were isolated from C57BL/6 mice as described previously (29). Femurs from FcRγ−/− mice were provided by Stylianos Bournazos and Jeffrey Ravetch (The Rockefeller University) and were isolated as described above. BMDMs were grown in Dulbecco's modified Eagle medium (DMEM) containing 10% fetal bovine serum (FBS), 15% L-cell conditioned medium, and 1 mM sodium pyruvate (BMM-Low) as described previously (29). The mouse macrophage-like cell line RAW 264.7 was obtained from the ATCC (ATCC TIB-71) and was grown in DMEM containing 10% FBS and 1 mM sodium pyruvate (DMEM-10%). In some experiments, RAW 264.7 cells were cultured in DMEM containing 0.2% FBS and 1 mM sodium pyruvate (DMEM-0.2%).

Monoclonal antibodies.

Anti-LcrV MAbs 7.3 and 29.3 (both IgG1) were produced and provided by Jim Hill (Defence Science and Technology Laboratory [DSTL]). MAb 7.3 has been described previously (18). MAb 29.3 is protective against plague infection in mice but has not been extensively characterized (unpublished data). The nonprotective anti-LcrV MAb 5.28 (IgG1) was provided by Raymond Dattwyler and Maria Gomes-Solecki (New York Medical College) and was produced as described previously (30). The IgG2a isotype switch variant of MAb 7.3 was provided by Matthew Scharff (Albert Einstein College of Medicine) and was produced as described previously (31). The IgG2a MAb was purified from hybridoma supernatants by protein G-Sepharose (P-3296; Sigma) affinity chromatography. Peak fractions containing IgG were brought to neutral pH with 1.5 M Tris (pH 8.8) and were dialyzed against PBS. MAb 7.3 F(ab′)2 and Fab, produced by digestion with papain or pepsin, respectively, and MAb 7.3 deglycosylated by treatment with endoglycosidase S were provided by Stylianos Bournazos and Jeffrey Ravetch (The Rockefeller University). The digested Fab preparation was subjected to consecutive incubations with protein G immobilized on agarose (P-4691; Sigma) to remove Fc fragments. Fab (950 μg) in a total volume of 400 μl was incubated with a 100-μl volume of packed resin with rotation at 4°C overnight. The resin was pelleted by centrifugation, and the supernatant was removed and was incubated as described above with fresh resin. The procedure described above was then repeated, and a supernatant obtained from a final centrifugation step was collected. The mouse anti-YopE MAbs (MAbs 149 and 202) used for immunoblotting will be described elsewhere (unpublished data).

Macrophage infections.

For the lactate dehydrogenase (LDH) release assay, 1.5 × 105 BMDMs were seeded into the wells of a 24-well tissue culture plate in 1 ml of BMM-Low 24 h before infection. For the translocation assay, 2 × 106 RAW 264.7 cells were seeded into the wells of a 6-well tissue culture plate in 3 ml of DMEM-10% 24 h before infection. Wells incubated in 3 ml of DMEM-10% without RAW 264.7 cells but otherwise treated identically were used as controls for the translocation assay. Prior to addition to the tissue culture plates, bacteria prepared as described above were diluted into fresh tissue culture medium, either BMM Low for BMDMs or DMEM-0.2% for RAW 264.7 cells, containing the MAb, a MAb variant, or PBS as indicated in the figure legends. The tissue culture medium in the wells was aspirated; wells were washed twice with PBS; and tissue culture medium containing diluted bacteria was added to each well. After the addition of bacteria, the plate was centrifuged for 5 min at 50 × g and was then incubated at 37°C in a CO2 incubator.

LDH release assay.

The LDH assay measures cytotoxicity resulting from the activity of translocated YopJ and has been described previously (32). Samples of culture media from wells containing BMDMs were collected at 5 h after infection. Levels of LDH were assayed by using the CytoTox 96 assay kit (Promega) according to the manufacturer's instructions. After 30 min of incubation with the substrate, the reaction was stopped, and absorbance at 490 nm was determined using a VersaMax tunable microplate reader (Molecular Devices). The level of spontaneous LDH release was determined by assaying the supernatants of uninfected macrophages. The level of total LDH was determined by assaying supernatants from uninfected BMDMs that had been lysed by a freeze-thaw cycle. The percentage of LDH release was calculated according to the manufacturer's protocol, which includes a correction for spontaneous release.

Translocation assay.

The translocation of YopE into RAW 264.7 cells was measured by using detergent solubility and immunoblotting as described previously (33). After a 2-h incubation, the 6-well dishes were placed on ice and were washed twice with 3 ml of ice-cold Hanks balanced salt solution (HBSS). Fifty microliters of Triton X-100 lysis buffer (150 mM NaCl, 10 mM Tris [pH 7.5], 1% Triton X-100, 1 mM Na3VO4, 10 mM NaF, and an EDTA-free protease inhibitor cocktail [Roche]) was added to each well, and the plate was incubated for 15 min on ice with occasional rocking. The contents of the wells were scraped into microcentrifuge tubes and were centrifuged for 10 min at 12,000 × g and 4°C. The supernatants were transferred to new tubes. Protein samples were boiled for 5 min in Laemmli sample buffer containing 0.1 M dithiothreitol prior to electrophoresis. The proteins were separated on an SDS–10% polyacrylamide gel via electrophoresis and were transferred to nitrocellulose membranes for immunoblot analysis. The membranes were blocked in Tris-buffered saline containing 0.05% Tween 20 (TBST) and 1% bovine serum albumin. Membranes were incubated with anti-YopE MAbs 202 and 149 at a final concentration of 6 μg/ml in TBST, followed by a horseradish peroxidase (HRP)-conjugated anti-mouse secondary antibody (Jackson) diluted 1:50,000 in TBST. Band signals on the membranes were detected by chemiluminescence (PerkinElmer). To quantify band signals on the immunoblots, blocking was performed with 1% casein, secondary anti-mouse antibodies conjugated with IR800 were used, and signals were detected by an infrared imaging system (Odyssey; Li-Cor). The band intensities were calculated by using the software provided by the Odyssey system, and the values were plotted as arbitrary units.

Statistical analysis of data.

Statistical analysis of LDH release data was performed with Prism (GraphPad) software, version 4.0. The tests used are indicated in the figure legends. A P value of <0.05 was considered significant.

RESULTS

IgG1 and IgG2a subclasses of anti-LcrV MAb 7.3 inhibit cytotoxicity equally in Y. pestis-infected macrophages.

In cases where interaction with FcR is important for the neutralization of a bacterial virulence factor by a MAb, the subclass can influence the neutralizing activity (31). This results from differential Fc-FcR interactions; for example, IgG1 binds to the low-affinity receptor FcγRIII, while IgG2a binds the high-affinity receptor FcγRI (34). To determine if the IgG1 subclass of MAb 7.3 was important for the neutralization of Yop translocation by this MAb, a class switch variant belonging to IgG2a (7.3-IgG2a) was obtained. Y. pestis KIM5 opsonized with MAb 7.3 or 7.3-IgG2a was used to infect BMDMs in tissue culture wells, and the levels of cytotoxicity resulting from YopJ translocation were measured by an LDH release assay. The neutralizing activity of an additional protective MAb, 29.3, was characterized in parallel. As negative controls, the bacteria either were left nonopsonized or were opsonized with the nonprotective anti-LcrV MAb 5.28. As shown in Fig. 1, MAbs 7.3 and 7.3-IgG2a inhibited cytotoxicity equally, indicating that the subclass does not influence the neutralizing activity of this MAb. Additionally, MAb 29.3, but not MAb 5.28, had significant neutralizing activity.

FIG 1.

FIG 1

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Neutralizing activities of anti-LcrV MAbs as determined by cytotoxicity assays in BMDMs infected with Y. pestis. BMDMs from C57BL/6 mice were infected with Y. pestis KIM5 at a multiplicity of infection of 10 in 24-well tissue culture plates. Prior to infection, bacteria were suspended in 100 μl of tissue culture medium alone (no MAb) or tissue culture medium containing the indicated MAb at 25 μg/ml (final concentration). After 20 min at 37°C to allow for opsonization, the volume of each sample was increased to 500 μl, bringing each MAb to 5 μg/ml (final concentration); infection was then initiated. Cytotoxicity was monitored by the percentage of LDH released at 5 h postinfection. Supernatants were collected, and the percentage of LDH released was determined as described in Materials and Methods. The results shown are corrected for spontaneous LDH release and are averages from three independent experiments. Error bars represent standard deviations. Asterisks indicate results significantly different from those for the no-MAb condition (***, P < 0.001; **, P < 0.01) as determined by analysis of variance.

Anti-LcrV MAb 7.3 inhibits cytotoxicity in Y. pestis-infected macrophages in the absence of Fc-FcR interaction.

To determine if FcRs for IgG are important for the neutralization of Yop translocation by MAb 7.3, the abilities of the MAb to inhibit Yop translocation in BMDMs from wild-type and FcRγ−/− mice were compared. The γ chain is required for signal transduction by all activating FcRs in mice (34). The cytotoxicity resulting from Y. pestis infection was equally inhibited by MAb 7.3 in BMDMs from wild-type mice and in BMDMs from FcRγ−/− mice (Fig. 2).

FIG 2.

FIG 2

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Neutralizing activity of MAb 7.3 in wild-type (wt) or FcγR-deficient macrophages by cytotoxicity assays. BMDMs from C57BL/6 mice (wt) or congenic FcγR−/− mice (FcR KO) were infected with KIM5 in the absence or presence of MAb 7.3, and the percentage of LDH release was determined as described in the legend to Fig. 1. The results shown are averages from three independent experiments. Error bars represent standard deviations. The values obtained for corresponding wt and FcR KO conditions did not differ significantly by analysis of variance.

IgG molecules are glycosylated primarily at Asn-297 of the CH2 domain within the Fc region. This modification is essential for the FcR binding of IgG (34). A preparation of MAb 7.3 that was deglycosylated by treatment with endoglycosidase S was compared with the native MAb over a concentration range for the ability to inhibit cytotoxicity in Y. pestis-infected BMDMs. Deglycosylated MAb 7.3 had slightly lower neutralizing activity than native MAb 7.3, especially at lower MAb concentrations, but this difference was not significant (Fig. 3).

FIG 3.

FIG 3

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Comparison of neutralizing activities of MAb 7.3, deglycosylated MAb 7.3, and F(ab′)2 of MAb 7.3 by a cytotoxicity assay. BMDMs were infected with KIM5, and the percentage of LDH release was determined as described in the legend to Fig. 1, except that MAb 7.3, deglycosylated MAb 7.3, or MAb 7.3 F(ab′)2 was used for opsonization. Final concentrations of MAb 7.3 and deglycosylated MAb 7.3 are shown, and the concentration of F(ab′)2 was adjusted to give molar equivalency. The results shown are averages from three independent experiments. Error bars represent standard deviations. The dashed line represents the average level of LDH release from BMDMs infected with KIM5 that was not opsonized. The differences between the values obtained with MAb 7.3, deglycosylated MAb 7.3, and MAb 7.3 F(ab′)2 were not significant at any concentration (P, >0.05) as determined by 2-way analysis of variance.

To directly examine the importance of the Fc region of MAb 7.3 for the neutralization of Yop translocation, macrophages were infected with Y. pestis in the presence of MAb 7.3 F(ab′)2, prepared by digestion of the MAb with pepsin. Like deglycosylated MAb 7.3, MAb 7.3 F(ab′)2 had slightly lower neutralizing activity than native MAb 7.3, especially at lower MAb concentrations, but this difference was not significant (Fig. 3). These results indicate that interaction with FcRs is not required for MAb 7.3 to neutralize the translocation of YopJ into macrophages infected with Y. pestis.

MAb 7.3 F(ab′)2 inhibits the translocation of YopE into Y. pestis-infected macrophages.

The cytotoxicity assay measures YopJ translocation indirectly. To extend the results presented above to another effector, YopE, and to measure translocation more directly, we used subcellular fractionation by detergent solubility (33). RAW 264.7 macrophage-like cells in tissue culture wells were infected with Y. pestis KIM5 in the presence or absence of F(ab′)2. KIM5yopB, a mutant defective in Yop translocation, was used as a control. Lysis buffer containing a nonionic detergent was added to wells containing infected RAW 264.7 cells, and the amount of YopE in the resulting soluble cytosolic fraction was determined by immunoblotting. Control wells containing bacteria or RAW 264.7 cells alone were treated identically. In the absence of F(ab′)2, substantially more YopE was detected in the soluble fraction of RAW 264.7 cells infected with KIM5 than in that for KIM5yopB-infected cells (Fig. 4A, compare lanes 3 and 6). The small amount of YopE detected in the cytosolic fraction of RAW 264.7 cells infected with KIM5yopB represents a background signal from nontranslocated YopE that is solubilized during detergent extraction. In addition, the amount of YopE in RAW 264.7 cells infected with KIM5 was reduced by ∼50% in the presence of F(ab′)2 (Fig. 4A, lane 9, and B), confirming that interaction with FcRs is not required for MAb 7.3 to neutralize Yop translocation.

FIG 4.

FIG 4

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Neutralizing activity of F(ab′)2 as determined by a YopE translocation assay. Six-well dishes containing either RAW 264.7 macrophage-like cells in tissue culture medium or tissue culture medium alone were either left untreated or infected (multiplicity of infection, 10) with KIM5 or KIM5yopB in the absence or presence of MAb 7.3 F(ab′)2 at 5 μg/ml. After 2 h of incubation, the tissue culture medium was removed, the wells were washed, and cold 1% Triton X-100 lysis buffer was added to the wells. The well contents were transferred to tubes and were centrifuged to obtain the soluble fraction. Samples of the soluble fraction were subjected to immunoblotting using anti-YopE MAbs as described in Materials and Methods. (A) Chemiluminescent signals from a single representative experiment, detected using HRP-conjugated secondary antibodies. The reduced migration of YopE in lane 9 compared to that in lane 6 is due to an edge effect of SDS-PAGE. (B) Quantification of the infrared signal by using an IR800-conjugated secondary antibody and an Odyssey imaging system. Data are intensities of YopE bands (expressed in arbitrary units) from KIM5-infected RAW 264.7 cells in the presence or absence of F(ab′)2, averaged from three independent experiments. Error bars represent standard deviations.

MAb 7.3 Fab inhibits the translocation of Yops into macrophages.

The results presented above indicate that binding of the bivalent fragment F(ab′)2 to LcrV is sufficient to neutralize Yop translocation. To determine if monovalent binding of the MAb 7.3 variable region to LcrV can inhibit translocation, Fab prepared by digestion of MAb 7.3 with papain was compared with F(ab′)2 over a concentration range for the ability to inhibit cytotoxicity in Y. pestis-infected BMDMs. As shown in Fig. 5A, the Fab and F(ab′)2 preparations inhibited cytotoxicity equally. A Fab preparation depleted of Fc fragments by affinity chromatography with protein G also inhibited cytotoxicity in Y. pestis-infected BMDMs in a dose-dependent manner (Fig. 5B). Therefore, binding of the monovalent variable region of MAb 7.3 to LcrV is sufficient to directly neutralize Yop translocation.

FIG 5.

FIG 5

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Comparison of neutralizing activities of F(ab′)2 and Fab by a cytotoxicity assay. The percentage of LDH release from KIM5-infected BMDMs was determined as described in the legend to Fig. 1, except that in panel B, the results are from two independent experiments. (A) BMDMs were infected with KIM5 in the absence or presence of a F(ab′)2 or Fab preparation of MAb 7.3 at the final concentrations indicated (adjusted for the difference in molecular weight between the variants). There was no significant difference between the values obtained with the Fab and F(ab′)2 preparations at any concentration (P, >0.05), as determined by 2-way analysis of variance. Relative to the results for the no-MAb control, the values obtained were significantly lower for F(ab′)2 at 2.5 μg/ml (P, <0.05), F(ab′)2 at 5 μg/ml (P, <0.001), Fab at 2.5 μg/ml (P, <0.05), and Fab at 5 μg/ml (P, <0.001), as determined by 1-way analysis of variance. (B) BMDMs were infected with KIM5 in the absence or presence of the indicated concentrations of Fab depleted of Fc fragments by affinity chromatography with protein G. The value obtained at 5 μg/ml was significantly lower (P, <0.05) than that for the no-MAb control, as determined by 1-way analysis of variance.

A previous study found that a polyclonal anti-LcrV antibody did not inhibit the translocation of Yops into Y. pestis-infected macrophages that were treated with CD to inhibit actin polymerization and phagocytosis (15). To determine if actin polymerization is required for the neutralization of Yop translocation by a MAb, RAW 264.7 cells were infected with Y. pestis in the presence or absence of CD or Fab. The results of a detergent solubility assay show that CD treatment by itself reduced YopE translocation (Fig. 6A, compare lanes 2 and 4, and B). In the presence of both CD and Fab, YopE translocation was reduced to the level obtained by Fab treatment alone (Fig. 6A, lane 5, and B). These data indicate that actin polymerization is not required for the inhibition of Yop translocation by a Fab to LcrV, and they provide additional evidence that neutralization occurs directly rather than as a result of opsonophagocytosis.

FIG 6.

FIG 6

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Neutralizing activity of Fab in the presence of cytochalasin D (CD) as determined by a YopE translocation assay. RAW 264.7 macrophage-like cells were either left untreated or treated with CD, followed by infection with KIM5 or KIM5yopB in the absence or presence of MAb 7.3 Fab at 5 μg/ml. After 2 h of incubation, YopE translocation was measured as described in the legend to Fig. 4. (A) Chemiluminescent signals from a single representative experiment. (B) Quantified signals from four independent experiments were averaged. Error bars represent standard deviations.

DISCUSSION

LcrV can be considered a prototype for the class of proteins that are found at the tips of T3SSs in Gram-negative pathogens (35). Tip proteins orthologous to LcrV in other bacterial species include Pseudomonas PcrV, Shigella IpaD, and Salmonella SipD (36). Anti-tip protein antibodies have been shown to inhibit the function of the corresponding T3SS for LcrV (37), PcrV (38), IpaD (39, 40), or SipD (41). A better understanding of how antibodies to LcrV neutralize Yop translocation thus has important implications for understanding how antibodies inhibit other tip proteins.

Three lines of evidence obtained here indicate that MAb 7.3 neutralizes Yop translocation directly, while opsonophagocytosis via FcRs is dispensable for this activity. First, changing the subclass of MAb 7.3 from IgG1 to IgG2a did not affect neutralizing activity significantly (Fig. 1). Second, preventing the interaction of the MAb with FcRs by deglycosylation of the Fc or removal of the Fc to generate F(ab′)2 did not significantly reduce neutralizing activity (Fig. 3 and 4). In addition, monovalent Fab with one variable region was neutralizing (Fig. 5). Third, preventing opsonophagocytosis by use of FcR-deficient macrophages or by use of CD to inhibit phagocytosis did not reduce neutralizing activity (Fig. 2 and 6).

Studies showing that anti-LcrV antibodies can inhibit the translocation of Yops into HeLa cells infected with Y. pestis (19) could also be taken as evidence that interaction with FcRs is dispensable for neutralizing activity. However, HeLa cells can express FcγIII (42), and blocking of FcRs on HeLa cells infected with Y. pestis has been reported to reduce the neutralizing activity of an anti-LcrV antibody (15).

Weeks et al. showed that polyclonal anti-LcrV F(ab′)2 prevented cytotoxicity in Y. pestis-infected macrophages (16), findings that support the direct neutralization mechanism. However, the study of Cowan et al. reported that polyclonal anti-LcrV did not inhibit the translocation of Yops into Y. pestis-infected macrophages treated with CD (15), a finding that is inconsistent with direct neutralization. Weeks et al. and Cowan et al. both used J774A.1 cells, but different assays (cytotoxicity versus detergent solubility) and different preparations of polyclonal anti-LcrV, suggesting that one of the latter two variables resulted in different findings. Experiments that measure the translocation of Yops into Y. pestis-infected macrophages in the presence of CD need to be carefully controlled and quantified, because CD treatment by itself can reduce Yop translocation (43) (Fig. 6).

An important question that remains is how anti-LcrV antibodies directly neutralize translocation. One possibility is that antibody binding induces a conformational change in LcrV. From structural analysis and modeling of LcrV and the T3SS needle, it has been suggested that LcrV forms a homopentamer complex at the tip (44, 45). Binding of neutralizing antibodies could change the conformation of this complex, thereby inactivating the key function of LcrV in translocation. Interestingly, models of the tip complex indicate that the LcrV central globular domain that contains neutralizing epitopes is oriented away from the needle, potentially allowing access to antibodies. Determination of the structure of LcrV bound to MAb 7.3 Fab to allow comparison with the unbound monomer might provide evidence for a conformational change. A second possibility is that neutralizing antibodies prevent LcrV from interacting with another Yersinia protein required for translocation. LcrV is proposed to act as a platform for the insertion of YopB and YopD into the plasma membrane to form the translocon (12). Anti-LcrV antibodies reduce the amounts of translocator proteins that are inserted into the membranes of red blood cells infected with Yersinia (46). Therefore, neutralizing antibodies could prevent the interaction of LcrV with YopD or YopB. Neutralizing antibodies could also prevent LcrV from interacting with an unidentified host cell receptor.

A previous study reported that to be fully protective, anti-LcrV MAbs have to neutralize the translocation function of LcrV and promote opsonophagocytosis (20). This conclusion was based on the finding that MAb AH1 inhibited cytotoxicity, but did not promote opsonophagocytosis, in Y. pestis-infected macrophages and was not protective (20). However, it will be important to test more directly the importance of Fc for protection against plague by an anti-LcrV MAb in vivo.

A protective MAb specific for Pseudomonas aeruginosa PcrV, Mab166, recognizes a conformational epitope between amino acids 144 and 257 (38, 47). This corresponds to the needle-distal globular domain and is analogous to the domain recognized by protective anti-LcrV antibodies. Interestingly, Mab166 F(ab′)2 has been shown to be protective against P. aeruginosa in animal infection models (36, 47), indicating that Fc receptor function is dispensable for protection against P. aeruginosa. If MAb 7.3 Fc is dispensable for neutralizing activity in vivo, this could lead to the development of novel immunotherapeutics for plague, such as synthetic variants of protective antibodies, including single-chain variable fragments. Detailed understanding of the LcrV–MAb 7.3 binding relationship might also provide new opportunities for antiplague drug discovery.

ACKNOWLEDGMENTS

We thank Matthew Scharff, Ray Dattwyler, Maria Gomes-Solecki, Stylianos Bournazos, and Jeffrey Ravetch for providing reagents, Galina Romanov for preparing macrophage cultures, and Matthew Scharff and Hana Fukuto for reviewing the manuscript. We also thank the Hybridoma Facility of the Albert Einstein College of Medicine and Susan Buhl for generating the isotype-switched IgG2a MAb 7.3.

This research was supported by awards from the NIAID (R01AI099222 and U54AI057158-Lipkin).

Footnotes

Published ahead of print 5 March 2014

REFERENCES

  • 1. Perry RD, Fetherston JD. 1997. Yersinia pestis—etiologic agent of plague. Clin. Microbiol. Rev. 10:35–66 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2. Prentice MB, Rahalison L. 2007. Plague. Lancet 369:1196–1207. 10.1016/S0140-6736(07)60566-2 [DOI] [PubMed] [Google Scholar]
  • 3. 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. 2000. Plague as a biological weapon: medical and public health management. Working Group on Civilian Biodefense. JAMA 283:2281–2290. 10.1001/jama.283.17.2281 [DOI] [PubMed] [Google Scholar]
  • 4. Titball RW, Williamson ED. 2004. Yersinia pestis (plague) vaccines. Expert Opin. Biol. Ther. 4:965–973. 10.1517/14712598.4.6.965 [DOI] [PubMed] [Google Scholar]
  • 5. Smiley ST. 2008. Immune defense against pneumonic plague. Immunol. Rev. 225:256–271. 10.1111/j.1600-065X.2008.00674.x [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6. Runco LM, Myrczek S, Bliska JB, Thanassi DG. 2008. Biogenesis of the F1 capsule and analysis of the ultrastructure of Yersinia pestis. J. Bacteriol. 190:3381–3385. 10.1128/JB.01840-07 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7. Xiao X, Zhu Z, Dankmeyer JL, Wormald MM, Fast RL, Worsham PL, Cote CK, Amemiya K, Dimitrov DS. 2010. Human anti-plague monoclonal antibodies protect mice from Yersinia pestis in a bubonic plague model. PLoS One 5:e13047. 10.1371/journal.pone.0013047 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8. Hill J, Copse C, Leary S, Stagg AJ, Williamson ED, Titball RW. 2003. Synergistic protection of mice against plague with monoclonal antibodies specific for the F1 and V antigens of Yersinia pestis. Infect. Immun. 71:2234–2238. 10.1128/IAI.71.4.2234-2238.2003 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9. Hill J, Eyles JE, Elvin SJ, Healey GD, Lukaszewski RA, Titball RW. 2006. Administration of antibody to the lung protects mice against pneumonic plague. Infect. Immun. 74:3068–3070. 10.1128/IAI.74.5.3068-3070.2006 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10. Brubaker RR. 2003. Interleukin-10 and inhibition of innate immunity to yersiniae: roles of Yops and LcrV (V antigen). Infect. Immun. 71:3673–3681. 10.1128/IAI.71.7.3673-3681.2003 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11. Cornelis GR. 2006. The type III secretion injectisome. Nat. Rev. Microbiol. 4:811–825. 10.1038/nrmicro1526 [DOI] [PubMed] [Google Scholar]
  • 12. Mueller CA, Broz P, Cornelis GR. 2008. The type III secretion system tip complex and translocon. Mol. Microbiol. 68:1085–1095. 10.1111/j.1365-2958.2008.06237.x [DOI] [PubMed] [Google Scholar]
  • 13. Mueller CA, Broz P, Muller SA, Ringler P, Erne-Brand F, Sorg I, Kuhn M, Engel A, Cornelis GR. 2005. The V-antigen of Yersinia forms a distinct structure at the tip of injectisome needles. Science 310:674–676. 10.1126/science.1118476 [DOI] [PubMed] [Google Scholar]
  • 14. Fields KA, Nilles ML, Cowan C, Straley SC. 1999. Virulence role of V antigen of Yersinia pestis at the bacterial surface. Infect. Immun. 67:5395–5408 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15. Cowan C, Philipovskiy AV, Wulff-Strobel CR, Ye Z, Straley SC. 2005. Anti-LcrV antibody inhibits delivery of Yops by Yersinia pestis KIM5 by directly promoting phagocytosis. Infect. Immun. 73:6127–6137. 10.1128/IAI.73.9.6127-6137.2005 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16. Weeks S, Hill J, Friedlander A, Welkos S. 2002. Anti-V antigen antibody protects macrophages from Yersinia pestis-induced cell death and promotes phagocytosis. Microb. Pathog. 32:227–237. 10.1006/mpat.2002.0498 [DOI] [PubMed] [Google Scholar]
  • 17. Froude JW, Stiles B, Pelat T, Thullier P. 2011. Antibodies for biodefense. mAbs 3:517–527. 10.4161/mabs.3.6.17621 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18. Hill J, Leary SE, Griffin KF, Williamson ED, Titball RW. 1997. Regions of Yersinia pestis V antigen that contribute to protection against plague identified by passive and active immunization. Infect. Immun. 65:4476–4482 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19. Quenee LE, Berube BJ, Segal J, Elli D, Ciletti NA, Anderson D, Schneewind O. 2010. Amino acid residues 196–225 of LcrV represent a plague protective epitope. Vaccine 28:1870–1876. 10.1016/j.vaccine.2009.11.076 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20. Eisele NA, Anderson DM. 2009. Dual-function antibodies to Yersinia pestis LcrV required for pulmonary clearance of plague. Clin. Vaccine Immunol. 16:1720–1727. 10.1128/CVI.00333-09 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21. Sofer-Podesta C, Ang J, Hackett NR, Senina S, Perlin D, Crystal RG, Boyer JL. 2009. Adenovirus-mediated delivery of an anti-V antigen monoclonal antibody protects mice against a lethal Yersinia pestis challenge. Infect. Immun. 77:1561–1568. 10.1128/IAI.00856-08 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22. Lin JS, Park S, Adamovicz JJ, Hill J, Bliska JB, Cote CK, Perlin DS, Amemiya K, Smiley ST. 2010. TNFα and IFNγ contribute to F1/LcrV-targeted immune defense in mouse models of fully virulent pneumonic plague. Vaccine 29:357–362. 10.1016/j.vaccine.2010.08.099 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23. Noel BL, Lilo S, Capurso D, Hill J, Bliska JB. 2009. Yersinia pestis can bypass protective antibodies to LcrV and activation with gamma interferon to survive and induce apoptosis in murine macrophages. Clin. Vaccine Immunol. 16:1457–1466. 10.1128/CVI.00172-09 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24. Vernazza C, Lingard B, Flick-Smith HC, Baillie LW, Hill J, Atkins HS. 2009. Small protective fragments of the Yersinia pestis V antigen. Vaccine 27:2775–2780. 10.1016/j.vaccine.2009.03.011 [DOI] [PubMed] [Google Scholar]
  • 25. Hill J, Leary S, Smither S, Best A, Pettersson J, Forsberg A, Lingard B, Lipka A, Brown KA, Williamson ED, Titball RW. 2009. N255 is a key residue for recognition by a monoclonal antibody which protects against Yersinia pestis infection. Vaccine 27:7073–7079. 10.1016/j.vaccine.2009.09.061 [DOI] [PubMed] [Google Scholar]
  • 26. Derewenda U, Mateja A, Devedjiev Y, Routzahn KM, Evdokimov AG, Derewenda ZS, Waugh DS. 2004. The structure of Yersinia pestis V-antigen, an essential virulence factor and mediator of immunity against plague. Structure (Camb.) 12:301–306. 10.1016/j.str.2004.01.010 [DOI] [PubMed] [Google Scholar]
  • 27. Lilo S, Zheng Y, Bliska JB. 2008. Caspase-1 activation in macrophages infected with Yersinia pestis KIM requires the type III secretion system effector YopJ. Infect. Immun. 76:3911–3923. 10.1128/IAI.01695-07 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28. Nimmerjahn F, Ravetch JV. 2005. Divergent immunoglobulin G subclass activity through selective Fc receptor binding. Science 310:1510–1512. 10.1126/science.1118948 [DOI] [PubMed] [Google Scholar]
  • 29. Pujol C, Bliska JB. 2003. The ability to replicate in macrophages is conserved between Yersinia pestis and Yersinia pseudotuberculosis. Infect. Immun. 71:5892–5899. 10.1128/IAI.71.10.5892-5899.2003 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30. Gomes-Solecki MJ, Savitt AG, Rowehl R, Glass JD, Bliska JB, Dattwyler RJ. 2005. LcrV capture enzyme-linked immunosorbent assay for detection of Yersinia pestis from human samples. Clin. Diagn. Lab. Immunol. 12:339–346. 10.1128/CDLI.12.2.339-346.2005 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31. Abboud N, Chow SK, Saylor C, Janda A, Ravetch JV, Scharff MD, Casadevall A. 2010. A requirement for FcγR in antibody-mediated bacterial toxin neutralization. J. Exp. Med. 207:2395–2405. 10.1084/jem.20100995 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32. Viboud GI, Bliska JB. 2002. Measurement of pore formation by contact-dependent type III protein secretion systems. Methods Enzymol. 358:345–350. 10.1016/S0076-6879(02)58100-3 [DOI] [PubMed] [Google Scholar]
  • 33. Palmer LE, Pancetti AR, Greenberg S, Bliska JB. 1999. YopJ of Yersinia spp. is sufficient to cause downregulation of multiple mitogen-activated protein kinases in eukaryotic cells. Infect. Immun. 67:708–716 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34. Nimmerjahn F, Ravetch JV. 2008. Fcγ receptors as regulators of immune responses. Nat. Rev. Immunol. 8:34–47. 10.1038/nri2206 [DOI] [PubMed] [Google Scholar]
  • 35. Thanassi DG, Bliska JB, Christie PJ. 2012. Surface organelles assembled by secretion systems of Gram-negative bacteria: diversity in structure and function. FEMS Microbiol. Rev. 36:1046–1082. 10.1111/j.1574-6976.2012.00342.x [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36. Sato H, Frank DW. 2011. Multi-functional characteristics of the Pseudomonas aeruginosa type III needle-tip protein, PcrV; comparison to orthologs in other Gram-negative bacteria. Front. Microbiol. 2:142. 10.3389/fmicb.2011.00142 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37. Pettersson J, Holmstrom A, Hill J, Leary S, Frithz-Lindsten E, von Euler-Matell A, Carlsson E, Titball R, Forsberg A, Wolf-Watz H. 1999. The V-antigen of Yersinia is surface exposed before target cell contact and involved in virulence protein translocation. Mol. Microbiol. 32:961–976. 10.1046/j.1365-2958.1999.01408.x [DOI] [PubMed] [Google Scholar]
  • 38. Sawa T, Yahr TL, Ohara M, Kurahashi K, Gropper MA, Wiener-Kronish JP, Frank DW. 1999. Active and passive immunization with the Pseudomonas V antigen protects against type III intoxication and lung injury. Nat. Med. 5:392–398. 10.1038/7391 [DOI] [PubMed] [Google Scholar]
  • 39. Espina M, Olive AJ, Kenjale R, Moore DS, Ausar SF, Kaminski RW, Oaks EV, Middaugh CR, Picking WD, Picking WL. 2006. IpaD localizes to the tip of the type III secretion system needle of Shigella flexneri. Infect. Immun. 74:4391–4400. 10.1128/IAI.00440-06 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40. Sani M, Botteaux A, Parsot C, Sansonetti P, Boekema EJ, Allaoui A. 2007. IpaD is localized at the tip of the Shigella flexneri type III secretion apparatus. Biochim. Biophys. Acta 1770:307–311. 10.1016/j.bbagen.2006.10.007 [DOI] [PubMed] [Google Scholar]
  • 41. Desin TS, Mickael CS, Lam PK, Potter AA, Koster W. 2010. Protection of epithelial cells from Salmonella enterica serovar Enteritidis invasion by antibodies against the SPI-1 type III secretion system. Can. J. Microbiol. 56:522–526. 10.1139/W10-034 [DOI] [PubMed] [Google Scholar]
  • 42. Su H, Spangrude GJ, Caldwell HD. 1991. Expression of FcγRIII on HeLa 229 cells: possible effect on in vitro neutralization of Chlamydia trachomatis. Infect. Immun. 59:3811–3814 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 43. Mejia E, Bliska JB, Viboud GI. 2008. Yersinia controls type III effector delivery into host cells by modulating Rho activity. PLoS Pathog. 4:e3. 10.1371/journal.ppat.0040003 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 44. Broz P, Mueller CA, Muller SA, Philippsen A, Sorg I, Engel A, Cornelis GR. 2007. Function and molecular architecture of the Yersinia injectisome tip complex. Mol. Microbiol. 65:1311–1320. 10.1111/j.1365-2958.2007.05871.x [DOI] [PubMed] [Google Scholar]
  • 45. Deane JE, Roversi P, Cordes FS, Johnson S, Kenjale R, Daniell S, Booy F, Picking WD, Picking WL, Blocker AJ, Lea SM. 2006. Molecular model of a type III secretion system needle: implications for host-cell sensing. Proc. Natl. Acad. Sci. U. S. A. 103:12529–12533. 10.1073/pnas.0602689103 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 46. Goure J, Broz P, Attree O, Cornelis GR, Attree I. 2005. Protective anti-V antibodies inhibit Pseudomonas and Yersinia translocon assembly within host membranes. J. Infect. Dis. 192:218–225. 10.1086/430932 [DOI] [PubMed] [Google Scholar]
  • 47. Frank DW, Vallis A, Wiener-Kronish JP, Roy-Burman A, Spack EG, Mullaney BP, Megdoud M, Marks JD, Fritz R, Sawa T. 2002. Generation and characterization of a protective monoclonal antibody to Pseudomonas aeruginosa PcrV. J. Infect. Dis. 186:64–73. 10.1086/341069 [DOI] [PubMed] [Google Scholar]

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