Serum IgG response to an outer membrane porin protein of Burkholderia cepacia in patients with cystic fibrosis

Authors


  • Laboratory work was carried out at Aston Universty, Department of Pharmaceutical Sciences.

Corresponding author. Tel: +44 (151) 228 4811; fax: +44 (151) 2280328.

Abstract

Early and accurate diagnosis of Burkholderia cepacia infection is important, particularly if segregation is to prevent patient-to-patient transmission. We have examined the serum response to a B. cepacia-specific 80-kDa outer membrane protein. 21 patients colonised with B. cepacia and Pseudomonas aeruginosa for 2–51 months (mean 11 months) were age- and sex-matched with 21 patients colonised with P. aeruginosa but not B. cepacia. The 80-kDa protein was recovered by electroelution from outer membrane proteins, separated by SDS-PAGE, coated onto ELISA plates, reacted with patient sera diluted 1:200, protein A-peroxidase and chromogenic substrate. We found that 19/24 patients colonised with B. cepacia and P. aeruginosa had high values, 2/24 patients had intermediate values, and 2/24 patients had a low value. 20/21 patients colonised with P. aeruginosa alone had low values and 1/21 had an intermediate value. We found that in the longitudinal serum samples studied from four patients only one patient developed high values after the first isolation of B. cepacia suggesting that seroconversion does not occur immediately after the first sputum culture of B. cepacia. We conclude that an ELISA test using B. cepacia-specific 80-kDa outer membrane protein can distinguish B. cepacia colonised and non-colonised patients and may be useful in the early diagnosis of B. cepacia infection.

1Introduction

Burkholderia cepacia is a major pulmonary pathogen in patients with cystic fibrosis. The isolation of B. cepacia from blood cultures accompanied by clinical evidence of systemic infection provides evidence of a pathogenic role for this organism [1]. There is growing evidence that the transmission of B. cepacia can occur by patient to patient contact [2–4]. Segregation of patients in hospital and clinic, and advising patients to avoid social contact has resulted in a reduction in the number of new cases in some CF centres [5]. Early identification of B. cepacia colonisation may be important, especially if segregation of colonised and non-colonised patients is to be practiced.

However, early detection of B. cepacia may be difficult using routine sputum culture alone, despite the use of selective medium [6]. Some clinical isolates may take up to seven days to grow, and confusion may occur with Xanthomonas maltophilia, Pseudomonas acidovorans[7]and Burkholderia gladioli[8].

The diagnosis of B. cepacia currently depends on initial isolation using a selective medium such as MAST, UK, and biochemical identification, e.g., API 20 NE System, and further identification by ribotyping. Serological diagnosis may have a role in providing an early diagnosis of B. cepacia infection prior to confirmation by culture. An ELISA could be performed within 24 h, whereas culture may take between four and seven days; ribotyping may take longer and is currently only performed at a small number of centres. Furthermore serological testing may be useful in younger children who are unable to expectorate sputum.

Previous studies have indicated that there is a rise in antibody titre to B. cepacia whole cells, outer membrane, and lipopolysaccharide prior to isolation in the sputum [9], suggesting that a rise in antibody levels may be an early indicator of infection. By measuring a specific anti-B. cepacia serum antibody it may be possible to diagnose B. cepacia infection early and accurately.

To avoid possible cross-reaction with P. aeruginosa antigens we have used a B. cepacia-specific 80-kDa outer membrane protein. The 80-kDa outer membrane protein is thought to be a porin molecule and has been previously purified and characterised [10, 11]. In a previous study of serum IgG response to B cepacia outer membrane, the 80-kDa outer membrane protein appeared to be B. cepacia-specific [12].

2Materials and methods

2.1Patients

Serum samples were collected from 24 patients with CF, colonised by B. cepacia for 2 to 51 months (mean 11 months). These 24 patients were all also colonised by P. aeruginosa. Sputum culture at the time of the serum sample was positive for both B. cepacia and P. aeruginosa. An age and sex-matched CF control in whom B. cepacia had never been isolated, was selected for 21 of the 24 patients colonised by B. cepacia. The CF control patients had all been colonised by P. aeruginosa for more than 6 months. Clinical details are outlined in Table 1. Two of the 24 B. cepacia-positive patient's serum samples (males, age 27.8 and 27.1 yrs) were studied but not age and sex matched and serum from a further patient B. cepacia-positive was used as a positive control throughout. There were four patients in whom serum was available before and after the first sputum isolation of B. cepacia (ages 16.1, 14.3 [twins] and 13.5 years) and their results were analysed separately.

Table 1.  Clinical details of the B. cepacia-positive and -negative patients
 B. cepacia-positiveB. cepacia-negativep
  1. Values are means (range).

  2. aCole T.J., Donnet M.L. Weight for height indices to assess nutritional status: a new index on a slide rule (1981) Am. J. Clin. Nutr. 34, 1935.

Males:females13:813:8 
Mean age (years)22.8 (12.3–29.7)22.4 (12.5–31.8)nsd
Mean height (cm)165 (136–181)160 (134–186)<0.1
Mean weight (kg)51.8 (28.9–70)49.6 (28.0–76.3)nsd
Weight for height (%)a94.5 (81–121)94.6 (65–125)nsd
Mean % predicted FEV137.5 (14–95)41.3 (16–86)nsd
Mean % predicted FVC50.7 (16–103)58.8 (26–111)nsd

2.2Bacterial strains and growth conditions

Sputum from patients with cystic fibrosis was routinely cultured for Pseudomonas aeruginosa cefsoludin chocolate agar and MacConkeys agar.

Sputum was also inoculated onto B. cepacia selective media (MAST, UK) incorporating ticarcillin (1000 mg/l) and polymyxin B (30 000 units/l). Agar plates were incubated for 48 h at 37°C and then for five days at room temperature, B. cepacia was identified by colonial appearance, API 20NE strips, oxidase reaction, and resistance to polymyxin B.

A rough type lipopolysaccharide clinical isolate of B. cepacia was collected from an adult CF patient at The Heartlands Hospital, Birmingham. The B. cepacia strain was known to belong to a ribotype common to many cystic fibrosis centres throughout the UK (ribotype A). The strain was cultured in a modified iron-depleted chemically defined medium (CDM-Fe) consisting of: 40 mM glucose; 0.62 mM KCl; 40 mM (NH4)2SO4; 0.4 mM MgSO4; 50 mM 3-(N-morpholino) propane sulphonic acid (pH 7.4) supplemented with 0.1% casamino acids (Difco). Bacteria were grown to early stationary phase (E470 0.9) in an orbital shaking incubator at 37°C, harvested by centrifugation at 10 000 g and washed once with saline.

2.3Preparation of outer membranes

Outer membranes (OMs) were prepared by the method of Filip et al. [13]. The washed bacterial pellet was suspended in 20 ml distilled water and broken by passage through a french pressure cell (Aminco). Unbroken cells were removed by centrifugation at 5000 g for 5 min. Sarkosyl (sodium N-lauroyl sarcosinate, Sigma) was added to the supernate to 2% w/v. After 1 h at room temperature the mixture was centrifuged at 40000 g for 40 min. The OM pellets were washed in distilled water and stored at −20°C.

2.4Purification of the 80-kDa protein by electroelution

Preparative sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). OM preparations were electrophoresed on 12% w/v acrylamide gels in a Protean II xi Vertical Electrophoresis cell (Bio-Rad, 15cm×15cm×0.1cm) and 750 μg of outer membrane protein (OMP) loaded into the single well spanning the whole cell. Electrophoresis was performed at 50 V overnight until the dye front had reached the bottom. The required OM protein band was excised by comparing with molecular markers and the stained edges of the gel (separated OMPs were stained with Coomassie Blue R-250 0.1% w/v in methanol:acetic acid:water 50:10:40).

Electroelution was performed in the Electroeluter Model 422 (Bio-Rad) at 8 mA per tube for 4 h [14]. The electroeluted protein was stored at −20°C. The presence of the electroeluted protein was confirmed by further SDS-PAGE, on this occassion using the Mini-Protean apparatus (Bio-Rad) (see Fig. 1). Immmunoblotting was performed on proteins separated by SDS-PAGE by a modification of the method of Towbin et al. [15].

Figure 1.

SDS-PAGE analysis of the whole outer membrane of B. cepacia (Lane 2) and the electroeluted proteins from the 80-kDa outer membrane band that had been excised from the preparative gel (Lane 3). Molecular weight markers in Lane 1.

2.5ELISA

Immulon 2 microELISA plates (Dynatech, USA) were coated overnight with 100 μl of the selected protein at 1.5 μg/ml in 50 mM carbonate/bicarbonate buffer, pH 9.6. Plates were washed in isotonic phosphate buffered saline (PBS) and then blocked for 4 h at 37°C with PBS containing 0.1% (w/v) bovine serum albumin and 0.1% Tween 20 (PBSTB). The plates were incubated at 37°C for 1 h with 100 μl volumes of patients serum, diluted 1:200 in PBSTB. Plates were washed three times in PBS and 100 μl of 0.25 μg/ml protein A peroxidase conjugate in PBSTB was added to each well and incubated at 37°C for 1 h. Plates were washed and developed with 100 μl 0.0058% (w/v) 3,3′,5,5′-tetra-methylbenzidine in 0.1 M acetate buffer, pH 5.2 until a colour reaction developed after 5–10 min; the reaction was stopped with 50 μl 2 M sulphuric acid. The A450 was measured using a Anthos 2001 reader (Labtec).

2.6Preadsorption of serum

A volume of 0.4 ml serum from a patient colonised with B. cepacia was diluted 1 in 1000 with PBSTB and was mixed with 0.1 ml of 0.3 mg/ml 80-kDa protein and left overnight at 4°C. Immune complexes were separated by centrifugation. Preadsorption with B. cepacia LPS was performed in a similar manner using 0.1 ml of LPS (10 mg/ml in water). LPS was prepared as previously described [12].

2.7Calculations

On each plate three control sera were used; a high and a low optical density (OD) reading serum, and a further high OD reading serum from a B. cepacia-positive patient which was used as a reference. To allow direct comparison between different assays the OD readings were multiplied by the reference high OD reading divided by the high OD result for that assay and the value expressed in ELISA units (EU).

2.8Data analysis

Because the data were not normally distributed the results were expressed as means and ranges. A paired non-parametric test was performed using the Wilcoxon signed rank test (Instat, GraphPad Software, San Diego, USA). A P value of <0.05 was regrded as statistically significant.

3Results

3.1SDS-PAGE and immunoblotting of the 80-kDa protein

When grown in the CDM-Fe medium the B. cepacia expressed six major OMPs (Fig. 1, lane 2). The 80-kDa protein was easily identified with another band at the 36-kDa position. After recovery from preparative gels by electroelution the band at the 80-kDa position was diminished but the band at the 36-kDa site was increased (Fig. 1, lane 3). No protein was detected at the 27.5-kDa position. IgG antibodies were detected at the 36-kDa site but not at the 80-kDa after immunoblotting and probing the immunoblot with serum from a CF patient colonised with B. cepacia.

3.2ELISA

The B. cepacia-colonised patients showed a high EU (median 1.40, range 0.37–1.88, 95% CI 1.21–1.54), whereas the B. cepacia-negative patients showed a lower EU (median 0.54, range 0.34–0.92, 95% CI 0.50–0.63 (Welch's alternative t-test P<0.0001; Fig. 2). Two B. cepacia-positive patients not matched with B. cepacia-negative patients gave EU results of 0.77 and 1.02.

Figure 2.

IgG response to the 80-kDa antigen in patients colonised with both Burkholderia cepacia and Pseudomonas aeruginosa (positives), patients colonised with Pseudomonas aeruginosa alone (negatives). IgG measured in ELISA units at a serum dilution of 1:200.

19/24 B. cepacia-positive patients gave EU values greater than 1.0, 2/24 B. cepacia-positive patients' values were just below 1.0 (0.95 and 0.96); 2/24 B. cepacia-positive patients had low results (0.77 and 0.37). 20/21 control patients that were colonised with P. aeruginosa but were not colonised with B. cepacia had very low EU values. However, one B. cepacia-negative patient had an intermediate value (0.92) (Fig. 2). Using a value of EU 1.0 to distinguish between B. cepacia-colonised and -non-colonised patients the test would have a specificty of 100%, a sensitivity of 83%, and a negative predictive value of 84%.

The results of the four patients who had serum samples before and after the first isolation of B. cepacia are summarised in Fig. 3.

Figure 3.

IgG response to the 80-kDa antigen in patients colonised with B. cepacia, before and after colonisation. Colonisation is represented by the short horizontal lines.

Inter-assay and intra-assay variation was determined from 96 and 5 measurements respectively. The inter-assay coefficient of variation was 6.0% and that for the intra-assay variation was 4.9%.

3.3Preadsorption

Preadsorption of the serum with the 80-kDa protein resulted in a marked reduction in the serum IgG response. However, the response was not diminished after preadsorption of the serum with LPS (Fig. 4).

Figure 4.

IgG response to the 80-kDa antigen before and after preadsorption of serum from a patient colonised with B. cepacia, with the 80-kDa and LPS antigens.

4Discussion

The immune reaction to the B. cepacia 80-kDa outer membrane protein was studied. A single protein was chosen because it is a more defined antigenic preparation than a system based on whole cells or whole outer membrane. The 80-kDa OMP was selected as a B. cepacia-specific antigen after initial SDS-PAGE studies and on the basis of previous immunoblotting studies [9, 11, 12]. The 80-kDa OMP is composed of either 36-kDa subunits alone or 36-kDa subunits in combination with 27-kDa subunits. Both the 36-kDa and 27-kDa subunits are thought to be specific for B. cepacia[9].

Parr et al. [10]purified the 80-kDa protein by gel filtration of proteins obtained from residues following SDS extraction of the outer membranes isolated by sucrose density gradient fractionation of B. cepacia whole cell isolates. They found that the protein has a molecular weight of 80 kDa on SDS-PAGE but is composed of a major subunit of 36 and a minor subunit of 27 kDa [10]. Their results also suggested that the 80 kDa protein was a peptidoglycan-associated protein.

To improve recovery of this porin protein Gotoh et al. [11]used total membranes (cell envelopes) and lithium dodecyl sulphate (LDS) instead of SDS to prevent crystallisation during chromatography. Using this protein Gotoh et al. [11], detected by SDS-PAGE a 140-kDa protein in addition to the 80-kDa protein and the subunits, the 36- and 27-kDa proteins. They purified the 36-kDa and 27-kDa subunits separately from the 80-kDa protein. Formation of the 80-kDa from a mixture of the purified 36- and 27-kDa subunits suggested that the 36- and 27-kDa subunits were not proteolytic fragments. The purified proteins were used for the production of murine antisera directed against each protein independently. Immunoblot assays with both murine antisera showed no cross reaction between the 36 and 27-kDa proteins, further supporting the conclusion that the 27-kDa protein is not a proteolytic fragment of the 36-kDa protein, and that the 36 and 27-kDa proteins are immunologically distinct components of the 80-kDa protein.

Gotoh et al. [11]also found that the 140-kDa and not the 80-kDa protein occurred in the protein preparation released from crude peptidoglycan and that the 140-kDa protein could be dissociated into the 80-kDa, which could then further dissociate in to the 36- and 27-kDa proteins. They suggested that the 140-kDa protein was composed of three molecules of 36-kDa protein and one or two molecules of the 27-kDa protein, but did not propose what the composition of the 80-kDa might be.

To determine the molecular mass of a trimer of the 36-kDa protein, Gotoh et al. [11], compared the mobility of the 36-kDa protein with the mobility of Escherichia coli porin protein OmpF which is a 36-kDa trimer with a total molecular weight of 108 kDa. They found that after SDS-PAGE of the purified OmpF protein solubilised at 25°C for 10 minutes they detected a band at approximately 72 kDa; the purified B. cepacia 36-kDa also appeared as a band at 72 kDa under identical conditions. The authors therefore concluded that the 36-kDa trimer that appears as a band at 72-kDa actually has a molecular weight similar to the OmpF protein, of 108-kDa. It appears that the 80-kDa protein is probably composed of three 36-kDa subunits when the 27-kDa subunit is absent or alternatively a combination of the 36-kDa and 27-kDa subunits the ratio of which is not known.

The 80-kDa protein is thought to be an important porin molecule [10, 11]. As a porin protein it is likely to be surface-exposed and involved in early antigen reaction. The low permeability of B. cepacia outer membrane to nitrocefin is thought to be due to the small size of the 80-kDa porin protein, contributing to the beta-lactam resistance of B. cepacia[10, 11, 16]. Furthermore, in some clinical strains, failure to express the 27-kDa and reduced amounts of the 36-kDa polypeptide has been associated with increased resistance of B. cepacia to beta-lactam antibiotics [16]. In this study, the 27-kDa was not detected by SDS PAGE, suggesting that it was not expressed in this strain or that it was present in low levels. This is consistent with the fact that the clinical strains studied were resistant to beta-lactams but MICs were not measured.

By using electroeluted OM protein it was hoped that any residual LPS from the outer membrane protein would be removed, as LPS can associate tightly with protein and would elicit its own immune reaction [17]. Preadsorption of serum with purified LPS antigen did not diminish the immune reaction to OM protein, whereas preadsorption with the 80-kDa protein resulted in a significant reduction in the immune reaction confirming that the reaction was specific to the 80-kDa protein and not to LPS or non-specific antigens (Fig. 3). It can therefore be concluded that the antibody reaction is directed toward the 80-kDa antigen alone.

The 21 B. cepacia patients studied were matched as closely as possible with B. cepacia-negative patients (Table 1). Mean Forced Expiration in one second (FEV1) and Forced Vital Capacity (FVC) were slightly lower in the B. cepacia group, but this was not significant and therefore, it is unlikely that the increased antibody reaction seen in the B. cepacia patients is a reflection of the increased severity of the lung disease of the B. cepacia patients. Despite a wide age range of patients, and patients from two centres, the raised EUs were remarkably uniform for the B. cepacia-colonised patients.

Aronoff and Stern [18]have previously shown raised IgG antibody titres to B. cepacia whole outer membranes in patients colonised with B. cepacia. There was, however, considerable overlap with non-colonised patients. Nelson et al. [19]have reported raised IgG titres to a B. cepacia-specific lipopolysaccharide (LPS) core determinant. They found that an ELISA based on core LPS was better at distinguishing between B. cepacia-positive patients and B. cepacia-negative patients than whole cell ELISA. Three out of the nine B. cepacia colonised patients they studied were not colonised with P. aeruginosa whereas in this study all the patients were jointly colonised with P. aeruginosa and B. cepacia. Longitudinal analysis of their serum IgG reaction indicated that the rate of increase of the levels of anti-B. cepacia antibodies varied from patient to patient, but in some cases a 2–4-fold rise in titre preceded the first positive sputum culture for B. cepacia by several months.

In this study only four patients had longitudinal serum samples available for analysis. One patient had a two fold increase antibody titres whereas the other three patients showed no antibody response to B. cepacia. This suggests that antibody production to the 80-kDa porin does not always occur immediately after the first isolation of B. cepacia. Further longitudinal serum samples, and samples from other CF centres, are needed to assess the diagnostic applicability of this 80-kDa antibody assay for the early and accurate detection of B. cepacia in patients with CF.

Acknowledgements

D.E.L. was funded by the Cystic Fibrosis Trust, UK.

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