Recombinant Truncated Flagellin of Burkholderia pseudomallei as a Molecular Probe for Diagnosis of Melioidosis

Current serological tests for melioidosis, using impure or uncharacterizedcell antigens from Burkholderia pseudomallei, have problemsin detection sensitivity and specificity. Therefore, we designedand expressed the recombinant flagellin (truncated at both theN- and C-terminal ends), and used the antigen to develop anindirect enzyme-linked immunosorbent assay (ELISA) to diagnosemelioidosis. Comparison of the immunoreactivities of the full-lengthand truncated flagellins reveals that the truncated flagellinperformed much better in detection specificity and sensitivity.Only the full-length flagellin was recognized by other bacterialcausing septicemia and gave a false-positive result in Westernanalysis, indicating that the cross-reactive epitopes were locatedon the more highly conserved N- and C-terminal regions of flagellin.The indirect ELISA using recombinant truncated flagellin asthe antigen achieved 93.8% sensitivity and 96.3% specificityand offered a more efficient serodiagnosis of melioidosis.

INTRODUCTION

Top

Introduction

Melioidosis, caused by Burkholderia pseudomallei, is endemicin Southeast Asia and northern Australia in particular (8, 12).The clinical manifestations of meliodosis are protean, and thedisease usually leads to death from acute septicemia (8, 13).Thus, the efficient diagnostic methods are very imperative.Several serological methods including the indirect hemagglutinationtest (11), immunofluorescence assay (2, 3, 15), and enzyme-linkedimmunosorbent assay (ELISA) (1, 4, 7) had been developed todiagnose the early B. pseudomallei-specific antibodies. However,the use of living bacteria as antigens in these assays createsa risk of laboratory-acquired infections (16). In addition,impure or uncharacterized cell antigens of B. pseudomallei usuallycross-react with other bacterial in the sera, making the methodsless specific. To avoid the above problems, we developed a verysafe, rapid, and efficient method to identify B. pseudomallei-specificantibodies by using the recombinant truncated flagellin as theantigen.

Flagellin (flagellar protein) elicits early specific antibodiesagainst B. pseudomallei infection in mice. It is therefore anideal antigen for detecting melioidosis (5, 6). The N- and C-terminalregions of flagellin in B. pseudomallei have higher homologyto those of other bacteria and may carry cross-reactive epitopes(9). Therefore, we designed a recombinant flagellin strippedof the original ends and developed an indirect ELISA to detectthe B. pseudomallei-specific immunoglobulin G (IgG) by usingthe truncated flagellin as the antigen.

MATERIALS AND METHODS

Top

Materials and Methods

Bacterial strains. B. pseudomallei VGH07 was isolated from a patient admitted tothe Kaohsiung Veterans General Hospital in southern Taiwan.The isolate was confirmed by standard biochemical tests andthe ID 32 GN profile (API system; BioMérieux, Mercy l'Etoile,France) and kept in Luria-Bertani (LB) broth at 37°C.

Amplification of flagellin genes. The chromosomal DNA of B. pseudomallei was isolated by usinga modified proteinase K digestion technique (10). Primers5'-TTTTGGATCCATGCTCGGAATCAACAGCAACATTAAC-3' (forwardprimer)and 5'-TTTTGCGGCCGCTTATTGCAGGAGCTTCAGCACTTGC-3 (reverse primer)(9) were designed to amplify the full-length flagellin gene.The nucleotide sequence of the PCR-amplified DNA fragment hadbeen confirmed to be the flagellin gene sequence (GenBank accessionnumber U73848). The amino acid sequence of the flagellin proteinhas 33 to 46% identity to that of Pseudomonas aeruginosa, Salmonellaenterica serovar Typhimurium, Proteus mirabilis, and Escherichiacoli. However, the N- and C-terminal amino acid sequences (aminoacids 1 to 40 and 300 to 387) of flagellin from the above bacteriahave approximately 67 and 50% homology, respectively. Thus,primers 5'-AAAAGGATCCGCGTCGGCGCTGCAACAGGAACTCG-3' and 5'-AAAAGCGGCCGCTTACATCGCCTGGTACGCGCCCGTCTGC-3' were de-signedto amplify the truncated flagellin gene (encoding amino acids41 to 299 of the flagellin). The PCR mixtures contained 20 µlof DNA extract, 2 µl of each primer, 5 µl of 10xamplification buffer (Promega, Madison, Wis.), a 10 µMconcentration of each deoxyribonucleotide, and 1 U of Taq polymerase(Promega) in a final volume of 50 µl. The PCR amplificationwas conducted for 25 cycles: denaturation at 95°C for 1min, annealing at 60°C for 30 s, and extension at 72°Cfor 1 min.

Expression of flagellin. The amplicons of the flagellin gene with the full-length andtruncated forms (central region) were cloned in frame into thepGEX4T-2 plasmid (Amersham Pharmarcia Biotech, Aslington Heights,Ill.) at the BamHI-NotI site behind the junction with glutathiononeS-transferase. Approximately 10⁶ CFU of E. coli BL21 [hsdS gal( ${lambda}$ cI ts857 ind-1 Sam7 nin-5 lacUV5-T7 gene 1)] per ml, carryingthe full-length or truncated form of the flagellin gene, wasgrown in LB broth containing 50 µg of ampicillin per mland 500 µM isopropyl-ß-D-thiogalactoside (IPTG;Fluka, Buchs, Switzerland) until the optical density at 600nm (OD₆₆₀) reached 0.8. After a 2 h-induction with 500 µMIPTG, the recombinant protein was extracted with 200 µlof B-PRE solution (Pierce, Rockford, Ill.). The lysate was loadedonto as immobilized glutathione column (Clontech), and the fusionproteins were eluted with 50 mM reduced gluthathione by centrifugation.The purified flagellin was identified by sodium dodecyl sulfate-polyacrylamidegel electrophoresis (10% polyacylamide gel) and confirmed byWestern blot analysis (with the rabbit anti-flagellin antiserum,produced by Genesis Biotech Inc.).

Serum specimens. A total of 32 serum samples from 16 patients with septicemiadue to melioidosis, admitted to Kaohsiung Veterans General Hospital,Chang Gang Memorial Hospital—Kaohsiung, and Jiarren Hospital,Kaoshiung, Taiwan, were collected at different times as listedin Table 1. Patients in Kaoshiung Veteran General Hospitalwith septicemia caused by other infectious bacteria were usedas disease controls. The serum specimens contained 100 samplesfrom disease controls infected with bacteria including Pseudomonasgroup (P. aeruginosa [n = 18] and P. putida [n = 4]), Klebsiellagroup (K. pneumoniae [n = 12] and K. oxytoca [n = 3]), E. coli(n = 11), Salmonella group (n = 11), Enterobacter cloacae (n= 11), other gram-negative bacteria (Citrobacter freundii [n= 3], Serratia marcescens [n = 5], Moraxella spp. [n = 5] Chromobacteriumsp. [n = 1] and Aeromonas spp. [n = 4]), and other gram-positivebacteria group (Staphylococcus spp. [n = 7] and Streptococcusspp. [n = 5]). The etiology of the septicemia in these patientswas identified by biochemical and morphological characterizationof clinical isolates from blood culture. The sera collectedfrom 60 healthy adults served as the negative control group.

View this table:
[in this window]
[in a new window]
TABLE 1. Antiflagellin antibody levels in sera from patients with melioidosis at different collection times after the onset of melioidosis

Evaluation of ELISA for detecting B. pseudomallei-specific antibody. The 96-well polystyrene microtiter plates were coated with theflagellin antigens (full-length form, 0.5 µg/ml; truncatedform, 1 µg/ml) in coating buffer (50 mM carbonate/bicarbonatebuffer [pH 9.6]) at 4°C overnight. The plates were blockedfor 2 h using 100 µl of bovine serum albumin (1 mg/ml;GIBCO, Grand Island, N.Y.). After being washed with saline-Tweensolution (0.9% [wt/vol] NaCl and 0.05% [vol/vol] Tween 20 inphosphate-buffered saline [PBS]) three times, the wells wereincubated at 37°C for 1 h with twofold serial dilutionsof various sera in PBS. Then the wells were washed with saline-Tweensolution and incubated with diluted (1:1,000) anti-human IgGconjugated with peroxidase (Zymed, South San Francisco, Calif.)at 37°C for 1 h. The wells were washed with PBS three times,and 100-µl volumes of 1-Step Turbo TMB-ELISA substrate(Pierce) were added. The OD₄₅₀ of the reactions products weredetermined using a microplate reader (Anthos 2010). The meanand standard deviations of six repeated measurements were calculatedfor each tested specimen. On each plate, sera from six healthyindividuals were used as the negative controls. In addition,rabbit anti-flagellin antiserum and anti-rabbit IgG conjugatedwith peroxidase were used as positive groups for detecting thepresence of flagellin antigen coating in the plate. When theaverage of the OD readings of the tested sample was greaterthan that of the negative controls plus 2 standard deviations,the tested sample was considered positive for specific antibody.The highest dilution of the tested sample which still gave apositive result was considered the endpoint titer and listedon the data sheet.

RESULTS AND DISCUSSION

Top

Results and Discussion

The cutoff dilutions in the indirect ELISA with full-lengthflagellin were determined to be 1:1,024, which was greater thanthe mean endpoint titer plus 2 standard deviations for the healthycontrol group (n = 60). The results indicated that 24 (75%)of 32 sera from patients with acute septicemic melioidosis werediagnosed as positive (Table 2). In the same manner, 5 (23%)of 22 from the Pseudomonas group, 4 (27%) of 15 from the Klebsiellagroup, 3 (27%) of 11 from the E. coli group 3 (27%) of 11 fromthe Salmonella group, 1 (9%) of 11 from the Enterobacter cloacaegroup, 4 (22%) of 18 from the other gram-negative bacteria group,0 (0%) of 12 from the other gram-positive bacteria group, and8 (13%) of 60 from the healthy group were recognized as false-positivereactions to full-length flagellin. In brief, with the full-lengthflagellin as antigen, this assay showed a moderate sensitivity(75%; 24 of 32) and specificity (82.5%; 132 of 160) based on192 serum specimens (melioidosis group, n = 32; control diseasegroup, n = 100; healthy group, n = 60). The profile was significantlydifferent when the truncated flagellin was used as the antigenicmaterial, since 30 (93.8%) of 32 sera from patients with melioidosiscould be distinguished from those from patients in the healthygroups at a 1:256 cutoff dilution, which was the maximum endpointtiter in sera from all the healthy individuals in the indirectELISA using the truncated flagellin as antigen (Table 3). Wethen used this 1:256 cutoff dilution to evaluate the truncatedflagellin and observed that six sera from the group of controldiseases (P. aeruginosa, n = 1; E. coli, n = 1; Salmonella groupD, n = 1; E. cloacae, n = 2; S. marcescens, n = 1) were positivefor IgG (Table 3). All six sera but one cross-reacted simultaneouslywith both the full-length and truncated flagellin. The interactionsof various sera with the full-length and truncated flagellinswere also examined by Western blot analysis. The results werethe same irrespective of the use of full-length or truncatedflagellin in Western blot analysis or indirect ELISA. (datanot shown). Taken together, the present results clearly indicatethat the cross-reactive epitopes were in the N- and C-terminalregions of flagellin. Use of the truncated flagellin as theantigen in the indirect ELISA for detection of antibodies specificto B. pseudomallei has greatly enhanced the sensitivity to 93.8%and specificity to 96.3%.

View this table:
[in this window]
[in a new window]
TABLE 2. IgG results with full-length flagellin antigen in an indirect ELISA

View this table:
[in this window]
[in a new window]
TABLE 3. IgG results with truncated flagellin antigen in an indirect ELISA

The specificity and sensitivity of assays using various antigensand the same panel of sera for serodiagnosis of meliodosis hadseldom been above 90% (14). One of the recently improved methodsusing partially purified culture filtrate as antigen claimedrather high sensitivity (96%) and specificity (94%) in detectingspecific IgG in patients with melodosis (7); however, the possiblelaboratory-acquired infection of B. pseudomallei in the preparationof culture filtrate antigens with living materials is stilla serious drawback. Attempts to use recombinant antigen havebeen only partially successful. One successful example whichused the recombinant Bps-1 protein as antigen achieved a moderatesensitivity (69.7%) based on Western blot analysis (16).

In conclusion, we expressed the recombinant truncated flagellinencoding the immunodominant epitopes and used it as the antigento detect B. pseudomallei-specific antibodies. The indirectELISA method using the recombinant truncated flagellin achieved93.8% sensitivity and 96.3% specificity and offered safer andmore rapid and efficient means of serodiagnosis of melioidosisin areas such as Taiwan, where the disease is not endemic. However,this assay still needs to be tested while evaluating a largenumber of sera collected from patients with melioidosis in anarea of endemic infection, such as Thailand, Australia, or Malaysia;before it can be widely used for serodiagnosis.

ACKNOWLEDGMENTS

This work was supported by grant NSC 91-2626-B-242-002 fromthe National Science Council, ROC.

We thank Chang Gang Memorial Hospital—Kaohsiung and JiarrenHospital for supplying the melioidosis sera, and we thank Hui-HuaLu for collection of these tested sera.

FOOTNOTES

* Corresponding author. Mailing address: Department of Medical Technology, Fooyin University, 151 Chin-Hsuen Rd., Ta-Liao, Kaohsiung 83101, Taiwan, Republic of China, Phone: 886-07-7811151, ext. 627. Fax: 886-07-7827162. E-mail: dan1001{at}ms31.hinet.net.

REFERENCES

Top