Study of Predominant Bacterial Antigens Triggering Antibody ...

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... Lacoste, Diego Esteban Cargnelutti, Héctor Tamashiro1 and María Silvia Di Genaro* ..... Rodriguez-Morales, O., Fernández-Mora, M., Hernández-Lucas, I., et.

Jpn. J. Infect. Dis., 60, 220-224, 2007

Short Communication

Study of Predominant Bacterial Antigens Triggering Antibody Response in Salmonella Reactive Arthritis: Apropos of a Case María Gabriela Lacoste, Diego Esteban Cargnelutti, Héctor Tamashiro1 and María Silvia Di Genaro* Laboratory of Immunology, Chemistry, Biochemistry and Pharmacy Faculty, National University of San Luis, and 1Public Hospital of San Luis, San Luis, Argentina (Received July 27, 2006. Accepted April 6, 2007) SUMMARY: Reactive arthritis (ReA) is a sterile arthritis triggered by distal mucosal infection, which suggests a contribution from bacterial products. The pathogenesis of ReA is unclear. There are no international standards for the serological methods used to confirm ReA. In the present work, we analyzed the predominant bacterial component that triggered an immune response in a 24-year-old woman with acute ReA. The candidate bacterial trigger was investigated by measuring the antibacterial antibodies (all immunoglobulin classes and IgA) to Salmonella enteritidis, Shigella flexneri and Yersinia enterocolitica. ELISA for Salmonella gave a positive result. To identify the bacterial component triggering ReA, antibodies to crude lysate, outer membrane proteins (OMP), cytosolic fraction, supernatant proteins and lipopolysaccharide of S. enteritidis were analyzed in sera and synovial fluid (SF) by ELISA, dot blot and Western blot. Among the antigen preparations, the antibody response to OMP was dominant in both serum and SF; a strong reaction to seven OMP bands (50 - 21 kDa) was observed. We concluded that OMP were the main bacterial antigens that trigged ReA in the reported case. Determining the triggering bacterial components in each case can help elucidate the precise causes of ReA and will contribute to the designing of a specific serological diagnostic method for this arthritis. pose of the present work was to report the case of a 24-yearold woman with ReA and to identify the bacterial components triggering ReA by assessing the antibody response to different S. enteritidis antigens. A 24-year-old woman was admitted to the Emergency Department of the Hospital of San Luis in Argentina with complaints of diarrhea, fever, nausea, vomiting and abdominal cramping. She reported that the onset of the gastrointestinal symptoms occurred 3 days after she visited a free-fork restaurant, where she ate various foods, some of them with mayonnaise dressing. Physical examination revealed that the patient had abdominal pain and a temperature of 38.2°C, blood pressure of 110/70 mm Hg, and a pulse of 104/min. Results of the laboratory investigation showed increased erythrocyte sedimentation rate (ESR) at 47 mm/h (Table 1). After this initial diagnostic approach, empirical broad-spectrum antibiotic therapy with ciprofloxacin was started and continued for 7 days. Four weeks later, she was admitted to the Rheumatology Section of the Hospital, and reported a history of progressive pain and swelling in the left knee and ankle, associated with morning stiffness. Physical examination revealed that the patient was unable to put weight on her left leg, and revealed florid synovitis of the left knee. She had tenderness at the insertion of the left Achilles tendon, but not in the lower back or buttocks. There was no recent history of conjunctivitis. The patient reported fatigue and vague abdominal pain for 2 weeks prior to her admission. The results of the laboratory tests are shown in Table 1. Synovial fluid (SF) was aspirated from the left knee and revealed severe inflammation, but there was no evidence of infection, as a culture of SF had a negative result, and no bacteria were observed by means of Gram’s staining (Table 1). Radiographs of the knee joint showed only soft tissue swelling, and neither enthesopathic lesions nor erosive joint damage were visualized. Considering the clinical picture and the preceding symptomatic enteritis, a presumptive diagnosis of enteric ReA was

Reactive arthritis (ReA) is a sterile synovitis that develops after gastrointestinal or urogenital infections, often with some latency, at joints distant from the site of the primary infection. This genesis suggests that there is a contribution from bacterial products in the induction of ReA (1). Because the pathogenesis of ReA is incompletely understood, appropriate treatments are not available. Infections by Salmonella, Shigella, Yersinia and Campylobacter spp. are implicated in triggering enteric ReA (2). At the time of arthritis, stool cultures are usually negative, and the background of ReA has usually been confirmed by serological methods (2). However, there are no international standards for the tests, and the techniques vary greatly (3). Secreted proteins called Yersina outer proteins (Yops) are unique antigens of pathogenic yersiniae, and the detection of specific IgG and IgA by Western blot has proved to be the best diagnostic tool for Yersinia enterocolitica-associated ReA (4). In contrast, although antibody responses are important for achieving protection against Salmonella infection (5), there are no studies that show the relevant antibody response in human Salmonella ReA. Determining this reponse could help to define a specific diagnostic test for this arthritis. The incidence of ReA following Salmonella infection strongly fluctuates according to various factors, such as the rate of this infection in a given area, from 1.2 to 14% (3). Salmonella enteritidis is a major cause of human food-borne illness. It is the most frequently detected cause of outbreaks of human salmonellosis (6), and a high frequency of ReA has been observed after S. enteritidis outbreaks (3,7). To date, there have been no reported studies of ReA in Argentina. The pur*Corresponding author: Mailing address: Immunology, Laboratory of Microbiology, Chemistry, Biochemistry and Pharmacy Faculty, National University of San Luis, Chacabuco y Pedernera, 5700, San Luis, Argentina. Tel: +54-2652-423789, Fax: +54-2652431301, E-mail: [email protected] 220

Table 1. Clinical course and laboratory tests Time (month) Clinical symptoms and physical examination

Laboratory tests ESR (mm/h) Hemoglobin (g/dl) WBC (cell /mm3) PMN leukocytes (%) MN leukocytes (%) Platelets/mm3 CRP (mg/dl) C3 (mg/dl) C4 (mg/dl) Stool culture Blood culture Antinuclear antibodies Chlamydia trachomatis IgG HLA-B27 by PCR in blood Serum antibacterial IgA to -Salmonella antigens -Yersinia antigens -Shigella antigens Blood MN proliferation with: -Salmonella antigens -Yersinia antigens -Shigella antigens SF analysis Cell number (cell/mm3) PMN leukocytes (%) MN leukocytes (%) Protein (g/dl) Glucose (mg/dl) Culture Gram staining Crystals C. trachomatis DNA by PCR SF antibacterial IgA to: -Salmonella antigens -Yersinia antigens -Shigella antigens

0

1

4

10 - 14

Gastrointestinal symptoms: diarrhea, fever, nausea, vomiting, abdominal cramping. Abdominal pain Temperature 38.2°C Basal pressure 110/70 mm Hg Pulse 104/min

Arthritis onset: morning stiffness, pain and swelling in the left knee and ankle. Fatigue and vague abdominal pain. Florid synovitis of left knee, unable to put weight in the left leg. Tenderness at the insertion of the left Achilles tendon. Radiographic study of knee showed soft tissue swelling.

After treatment: less pain and swelling in the left knee

Asymptomatic period: without articular inflammation

47 12.8 6,020 68 32 321,000 ND ND ND ND ND ND ND ND

59 14 11,180 63 37 316,000 26 245 44.9 Negative Negative Negative Positive (1/16) ND

13 ND ND ND ND ND ND 194 35.1 ND ND ND Positive (1/16) ND

ND ND ND ND ND ND ND ND ND ND ND ND ND ND

Positive, OMP titer: 1/200 Negative Negative ND

Negative Negative Negative

Negative Negative Negative

ND

ND

Positive Negative Negative ND

ND

ND

590 64 36 5.37 72 Negative No bacteria Absent Negative Positive, OMP titer: 1/400 Negative Negative

ND, not done; WBC, white blood cell count; PMN, polymorphonuclear; MN, mononuclear; ESR, erythrocyte sedimentation rate; CRP, C-reactive protein; C3 and C4, complement 3 and 4 components; SF, synovial fluid; OMP, outer membrane proteins.

proposed. The patient was treated initially with nonsteroidal anti-inflammatory drugs (NSAIDs): diclofenac 50 mg every 8 h for 2 weeks, and then 50 mg twice daily for another 2 weeks. She initially responded well to the treatment. However, articular inflammation returned 1 month later, and she was then treated with sulfasalazine 2 g daily for 4 months. During this time she gradually recovered, and the ESR returned to normal. Follow-up after 6 months showed that she was asymptomatic. The clinical course of the patient including the results of the laboratory tests is shown in Table 1. The puncturing of a vein or joint was done only when necessary for diagnostic or therapeutic reasons. Table 1 shows the time when clinical specimens were taken. Blood and SF were taken on arthritis onset and a new sample of blood was taken after

3 months of evolution. With the written consent of the patient (Declaration of Helsinki, 2000), her serum and SF samples were remitted to the Microbiology Laboratory of the National University of San Luis, where the candidate bacterial trigger was investigated. Bacterial antigens were prepared from S. enteritidis, Shigella flexneri (both isolated from clinical samples) and Y. enterocolitica O:8 WAP (kindly provided by Dr. Kapperud, Department of Bacteriology, National Institute of Public Health, Oslo, Norway). Lipopolysaccharide (LPS) was obtained by extraction with hot phenol-water as previously described (8). Crude lysate (CL) was prepared from whole bacteria disrupted by sonication from washed bacterium pellets (9). Cytoplasmic fraction (CF) and outer membrane proteins 221

(OMP) were isolated from the CL (9). Culture supernatant proteins (SN) were obtained by precipitation with ammonium sulphate (9). The protein concentrations of the antigens were determined by Lowry’s procedure. LPS had less than 2% protein, and 3 - 4% 2-keto-3-deoxyoctulosonic acid (8). Total immunoglobulins and IgA responses were studied by enzymelinked immunosorbent assay (ELISA) (10). Single 1:50 or serial 1:50 - 12,800 dilutions of sera and SF were tested in a plate coated with 10 μg/ml of each antigen in 0.15 M phosphate buffered saline, pH 7.2. Bound antibodies were demonstrated by reaction with horseradish peroxidase-conjugated goat anti-human IgM, IgG and IgA (Sigma, St. Louis, Mo., USA) or goat anti-human IgA and peroxidase-conjugated rabbit anti-goat IgG (Sigma). The antibody response was considered to be positive if the serum or SF had an optical density (OD) exceeding the mean + 2 SD of the healthy control group values. Control sera were obtained from healthy subjects with no symptoms of infection, who required routine laboratory tests. For SF analysis, since osteoarthritis (OA) is not associated with infectious disease, we used SF from OA patients as a control for the SF cut-off value calculation. To compare the reactivity to the bacterial antigens, the antibody titer to each antigen was determined as the last dilution with an OD over the cut-off value. Yops were prepared from culture supernatants as described previously (11), and released proteins were precipitated with trichloroacetic acid (11). The Yops were dissolved in SDS sample buffer supplemented with 5 mM PMSF, electrophoresed in 12% polyacrylamide gels-SDS and transferred to nitrocellulose membranes by electro-blotting. Western blot for Yops was performed in sera and SF. In addition, since ReA could also occur subsequent to an asymptomatic genitourinary tract infection, especially an infection with Chlamydia trachomatis, we also investigated serum IgG specific for this bacteria using a microimmuno-fluorescence test (MIF) (Focus Diagnostics, Cypress, Calif., USA) (12) and searched for C. trachomatis DNA in SF using in-house polymerase chain reaction (PCR) (13). Dot blot was performed by seeding Salmonella antigens on nitrocellulose strips, incubating them with the serum or SF (dilution 1:50) and then with horseradish peroxidaseconjugated goat anti-human IgM, IgG and IgA (Sigma). The reaction was developed using 4-chloro-1-naphtol (BioRad, Hercules, Md., USA) as chromogen. The membrane strip with the seeded antigens was stained with Ponceau S for protein detection. Moreover, Western blot of IgM, IgG and IgA for all Salmonella antigens was carried out (6). Four months after the gastrointestinal symptoms, blood was obtained and proliferation assays of peripheral blood mononuclear cells (PBMC) were performed. Cells were cultured at 5 × 105 viable cells per well in flat-bottom 96well tissue plates, and duplicate wells were stimulated with Salmonella, Yersinia and Shigella CL (2.5 μg/ml). After incubation at 37°C in 5% CO2 for 3 days, cell proliferation was measured by MTT reduction with the colorimetric assay described by Mosmann (14). Results were expressed in terms of the stimulation index (SI), defined as the ratio of proliferation (OD) induced by the antigen to that in medium alone, and an SI ≥ 2 was considered to be a positive response. Because HLA-B27 positivity is linked to ReA, the presence of HLA-B27 antigen was determined using PCR (12). Briefly, blood-extracted DNA was amplified with the primers B27/ E136 as 5´-CGGCGGTCCAGGAGCT-3´ and B37/E91 s+ 5´GGGTCTCACACCCTCCAGAAT-3´. HLA-B27 product

(136 bp) was detected on agarose electrophoresis and visualized with ethidium bromide. ELISA with Salmonella antigens had a positive result in SF upon arthritis onset (Table 1, Fig. 1A). After this result, as in other previous studies (10), Salmonella was considered to be the triggering bacterium that was the probable cause of the ReA. In addition, after 3 months of arthritis evolution, PBMC showed a positive proliferative response with Salmonella CL (Fig. 1A), which indicated again that this bacterium was a possible trigger of the ReA. In addition, Yops Western blot had a negative result, which excluded Yersinia as the cause of ReA. In addition, C. trachomatis IgG titer was 1/16. However, this bacterium was also excluded as the trigger of the ReA since this titer was too low to be associated with the arthritis. Thus, another study (12) concluded that titers of ≥512 in the IgG fraction indicate ongoing infection, and IgG titers of 1/32 - 1/256 indicate previous infection. The unchanging 1/16 titer of our patient in both serum samples (at arthritis onset and in month 4) could indicate anti-Chlamydia antibody persistence. A fourfold increase in paired sera, or a single endpoint of ≥1/512 is considered to indicate a possible C. trachomatis acute infection in the MIF assay that was used; however, this assay has not been established for diagnosing chronic infections. Moreover, cross-reactivity may also occur due to exposure to more than one Chlamydia spp. as described in the performance characteristics of the Chlamydia MIF IgG assay that was used. Furthermore, since several reports have demonstrated that C. trachomatis can be detected in the SF obtained from ReA patients (15), and the PCR test may be superior to MIF with respect to its sensitivity and specificity for the diagnosis of C. trachomatis, we investigated Chlamydia DNA in the SF of our patient by PCR. This PCR had a negative result. In addition, our patient had no pertinent sexual history; however, gastrointestinal symptoms preceded the arthritic symptoms by 4 weeks. Moreover, the strong immune response to Salmonella detected in serum and SF upon the arthritis onset became negative when the arthritic symptoms reduced. Based on the history of enteric symptoms and the results of laboratory tests, we considered that the ReA of our patient was associated with a Salmonella infection. When all class antibodies to different Salmonella antigens were studied by ELISA, we detected positive responses to CL, OMP and LPS in serum and to all Salmonella antigen preparations in SF with titers of 1/100 - 1/200 (Fig. 1B). This result could suggest different kinetics between the systemic and local clearance of the arthritogenic bacteria, and differences between the systemic and local immune responses. The highest antibody responses were responses against OMP in serum and responses to both OMP and LPS in SF (Fig. 1B). In the joint, OMP and LPS could trigger the synthesis of pro-inflammatory cytokines, thus contributing to the recruitment of leukocytes into the synovium and articular inflammation. In addition, a higher Salmonella-specific IgA response was detected in SF with titers of 1/100 - 1/400 (Fig. 1B), which could indicate a pathogenic link between gut inflammation and ReA. By ELISA, we observed only a slight IgA antibody response to CF in SF, indicating that Salmonella cytosolic antigens could not play a central role as an ReA trigger. In contrast, we found that the IgA antibody response was stronger to OMP, indicating that they were the major antigenic target in the reported case of Salmonella-induced ReA. The serum OMP-specific IgA response decreased with the reduction of the arthritis symptoms (from 1/200 at onset to negative in month 4) indicating that this response may reflect 222

Fig. 1. Immune response to Salmonella antigens. (A) ELISA of antibacterial antibodies in synovial fluid (SF) on arthritis onset. The response is represented as the optical density (OD) obtained for each bacterial antigen. Dotted line represents the cut-off value (OD mean + 2 SD of the control group). CL, crude lysate; OMP, outer membrane proteins; CF, cytosolic fraction; LPS, lipopolysaccharide; SN, supernatant proteins. Proliferative response of peripheral blood mononuclear cells to CL of Salmonella, Yersinia and Shigella after 3 months of arthritis onset. SI, stimulation index. (B) ELISA and Dot blot of antibody response to Salmonella antigens. (C) Western blot of IgM, IgG and IgA to Salmonella antigens. S, serum; SF, synovial fluid. (D) HLA-B27 PCR. MW, molecular weight marker; C(–): negative control without DNA; P, patient DNA; C(+), positive control DNA.

the disease activity (Table 1). Using dot blot, we analyzed the antigen preparations without dilution to avoid any contamination. Using Ponceau S-staining, we observed a higher protein concentration in CF. This result accords with the protein determination of each preparation by Lowry’s method (Fig. 1B). Among the antigen preparations, the strongest reaction in serum was again against OMP (Fig. 1B). Similar results were obtained in SF (data not shown). In addition, a reaction to Salmonella antigens (stronger for OMP) was also detected in the serum 3 months after the initial ReA symptoms (Fig. 1B). Because of its high sensitivity, Western blot showed IgM, IgG and IgA reactions with protein bands of all Salmonella antigens (Fig. 1C). However, these reactions were stronger to OMP. Reactions against flagellin (50 kDa), porins (38 - 36

kDa), OmpA (34 kDa) and unidentified OMP (32, 22, 21, 16 kDa) were observed and defined by their molecular weights as previously reported (6). Chronic Salmonella ReA (prolonged arthritis of >1 year extension) has been reported in only 20% of cases (3). In the present work, the patient showed higher anti-OMP antibody response, and she did not develop chronic ReA. In addition, HLA-B27 positiveness is associated with chronic and relapsing arthritis. In our patient, HLAB27 was negative (Fig. 1D), indicating the absence of a prognostic marker for chronicity (2). The frequency of HLA-B27 is not high (less than 50%) in Salmonella-induced ReA (3). On the other hand, porins play a role in Salmonella pathogenesis (6), and anti-porin antibodies, in contrast with antiLPS anti-bodies, have bactericidal activity (16). Therefore, fur223

antibody memory response. Immunology, 117, 59-70. 6. Ochoa-Repáraz, J., Sesma, B., Alvarez, M., et al. (2004): Humoral immune response in hens naturally infected with Salmonella enteritidis against outer membrane proteins and other surface structural antigens. Vet. Res., 35, 291-298. 7. Locht, H., Molbak, K. and Krogfelt, K. (2002): High frequency of reactive joint symptoms after an outbreak of Salmonella enteritidis. J. Rheumatol., 29, 767-771. 8. Di Genaro, M., Muñoz, E., Aguilera, C., et al. (2000): Yersinia enterocolitica O:8 and O:5 lipopolysaccharide arthritogenicity in hamsters. Rheumatology, 39, 73-78. 9. Michiels, T., Wattieau, P., Brasseur, R., et al. (1990): Secretion of Yop proteins by Yersiniae. Infect. Immun., 58, 2840-2849. 10. Fendler, C., Leiko, S., Soerensen, H., et al. (2001): Frequency of triggering bacteria in patients with reactive arthritis and undifferentiated oligoarthritis and the relative importance of the tests used for diagnosis. Ann. Rheum. Dis., 60, 337-346. 11. Trˇcek, J., Wilharm, G., Jacovi, C., et al. (2002): Yersinia enterocolitica Yop Q: strain-dependent cytosolic accumulation and post-translational secretion. Microbiology, 148, 1457-1465. 12. Huhtinen, M., Laasila, K., Granfors, K., et al. (2002): Infectious background of patients with a history of acute anterior uveitis. Ann. Rheum. Dis., 61, 1012-1016. 13. Golijow, C.D., Abba, M.C., Mourón, S.A., et al. (2005): Chlamydia trachomatis and human papillomavirus infections in cervical disease in Argentina women. Gynecol. Oncol. 96:181-186. 14. Mosmann, T. (1983): Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J. Immunol. Methods, 65, 55-63. 15. Taylor-Robinson, D., Gilroy, C.B., Thomas, B.J., et al. (1992). Detection of Chlamydia trachomatis DNA on joints of reactive arthritis patients by polymerase chain reaction. Lancet, 340, 81-82. 16. Rodriguez-Morales, O., Fernández-Mora, M., Hernández-Lucas, I., et al. (2006): Salmonella enterica serovar Typhimurium ompS1 and ompS2 mutans are attenuated for virulence in mice. Infect. Immun., 74, 13981402.

ther studies could elucidate whether anti-OMP antibody response could play a role in the protection against chronic ReA. Our data stress that OMP are the immunodominant antigens to antibody response in the reported Salmonella ReA case. The results of our work could contribute to elucidating the precise causes of ReA and to the designing of a specific serological diagnostic method for this arthritis. ACKNOWLEDGMENTS This study was supported by Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET, PIP 6224) and Universidad Nacional de San Luis (Proj. 0401). Silvia Di Genaro is member career and Gabriela Lacoste fellow of CONICET. The technical assistance of Lic María Amelia Rodriguez in Chlamydia PCR is gratefully acknowledged.

REFERENCES 1. Granfors, K., Jalkanen, S., Lindberg, A., et al. (1990): Salmonella lipopolysaccharide in synovial cells from patients with reactive arthritis. Lancet, 335, 685-688. 2. Leirisalo-Repo, M. (2005): Reactive arthritis. Scand. J. Rheumatol., 34, 251-259. 3. Colmegna, I., Cuchacovich, R. and Espinoza, L. (2004): HLA-B27associated reactive arthritis: pathogenetic and clinical consideration. Clin. Microbiol. Rev., 17, 348-369. 4. Heesemann, J., Egger, C. and Shroeder, J. (1987): Serological diagnosis of yersiniosis by immunoblot technique using virulence-associated antigens of enteropathogenic yersiniae. Contrib. Microbiol. Immunol., 9, 285-289. 5. Secundino, I., López-Macías, C., Cervantes-Barragán, L., et al. (2005): Salmonella porins induce a sustained, lifelong specific bactericidal

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