Iron-regulated Proteins in Outer Membranes of Campylobacter jejuni Diarrhoea Isolates and Immune Response to the Proteins in Patients
D. SCHWARTZ1,2, N. KONFORTI!, RACHEL PERRy2, H. GOOSSENS3, J. P. BUTZLER3 , P. WILLIAMS\ and JANINA GOLDHAR2*
1 Microbiology Laboratory, Sourasky (Ichilov Medical Center), Tel-Aviv, Israel 2 Department of Human Microbiology, Sackler School of Medicine, Tel-Aviv University,
Tel-Aviv, Israel 3 W.H.O. Collaborating Centre for Enteric Campylobacter St. Pieters University Hospital,
1000 Brussels, Belgium 4 Department of Genetics, University of Leicester, Leicester, U.K.
With 4 Figures· Received January 18, 1993 . Revision received March 23,1993· Accepted May 24,1993
Summary
The outer membrane protein (OMP) profiles from 8 Campylobaeter jejuni and 5 Campylobaeter coli fecal isolates grown under various conditions were compared by SDS-PAGE. The bacteria were grown under usual conditions, in iron-deficient medium (Dip) and on iron-supplemented medium (Fe). The OMP profiles of most bacterial strains grown under usual conditions, or in the Fe-supplemented medium, contained four major bands of approximately 31, 45, 63-66 and 97 kDa, and in addition, a number of minor bands. It was found that OMP from 10 of 13 strains tested and grown on iron deficient medium contained an intensive band of a protein in the molecular weight region of 76 kDa which was lacking in the OMP of bacteria grown in the presence of iron (iron-regulated protein). Sera from 11 children with C. jejuni infection analyzed by Western blot recognized the 76 kDa bands, in contrast to only one out of 10 control sera from healthy children. The Western-blot experiments demonstrated also vatious bands of other OMP components, both in OMP-Dip and OMP-Fe. The 45 kDa (porin protein) was recognized by all 11 serum samples from C. jejuni-infected patients and in 8 out of 10 control sera. The data suggested that the 76 kDa iron-regulated protein was expressed by bacteria during infection and it stimulated the immune response in children infected with C. jejuni.
* Corresponding author
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Zusammenfassung
Die Zusammensetzung der auBeren Membranproteine von 8 Campylabaeter jejuni- und 5 Campylabaeter eali-Isolaten aus Stuhlproben wurde nach Wachstum unter verschiedenen Kulturbedingungen mittels SDS-PAGE verglichen. Die Anziichtung erfolgte unter den iiblichen Kulturbedingungen sowie in einem Eisen-Mangelmedium (Dip) und in einem mit Eisen angereicherten Medium (Fe). Die OMP-Profile der meisten Stamme, die unter normalen Bedingungen oder im Eisen-angereicherten Medium gewachsen waren, zeigten vier Hauptbanden von ca.31, 45, 63-66 und 97 kDa sowie mehrere kleinere Banden. Bei 10 von 13 Stammen, die auf dem Eisen-Mangelmedium gewachsen waren, war eine intensive Proteinbande mit einer Molekularmasse urn 76 kDa im OMP-Prafil nachweisbar, die in den OMPProfilen der Bakterien, die in Gegenwart von Eisen gewachsen waren, nicht auftrat (Eisenreguliertes Protein).
In den Seren von 11 Kindem mit Campylabaeter jejuni-Infektion wurden mittels Western blot Technik Antikorper nachgewiesen, die mit der 76 kDa Bande reagierten, wahrend nur in einem von 10 Seren von gesunden Kindem eine entsprechende Reaktion auftrat. Die Western blot Untersuchung zeigte auch verschiedene Banden von anderen auBeren Membranbestandteilen sowohl im OMP-Dip als auch im OMP-Fe. Das 45 kDa Protein (PorinProtein) wurde von allen 11 Seren der Patienten mit Campylabaeter jejuni-Infektion erkannt, jedoch auch von 8 von 10 der Kontrollseren.
Die Ergebnisse deuten darauf hin, daB das 76 kDa Eisen-regulierte Protein durch die Bakterien wahrend der Infektion exprimiert wird und die Immunantwort von Kindem mit Campylabaeter jejuni-Infektion stimuliert.
Introduction
Campylobaeter jejunileoli (c. jejuni) are recognized as human enteric pathogens all over the world, particularly in children (5, 7, 16). The pathogenesis of the disease is still poorly understood (30, 34). Production of enterotoxins (cytotonic and cytotoxic) and the invasion capacity of strains isolated from diarrhoeic cases in various geographical areas was demonstrated (10,17-19,21). A number of authors (8, 23) drew attention to the possible role of outer membrane proteins (OMP) (molecular weight 23000-30000 and 43 000) in association with the invasion of epithelial cells (HEp-2). Blaser et a1. (4) analysed the OMP of C. jejuni strains by Sodium-dodecyl-sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting, and they found that both healthy and infected humans recognized the major protein (MOMP 43 kDa). The immune response to flagellar protein (63 kDa) and MOMP was stronger in sera from infected humans than in controls. The immune response to a variety of OMP components, including flagellar protein, was described by a number of authors (3, 24, 25,27,35). Figueroa et a1. (13) detected five antigenic components of C. jejuni (23, 42, 47,52 and 63 kDa) which reacted with sera of C. jejuni-infected children.
The acquisition of iron by bacteria from the environment is necessary for bacterial survival (26). The ability to acquire iron in iron limiting environments is considered to be an important factor in the virulence of certain bacterial invasive pathogens like Salmonella (11). The nature of the iron uptake systems of C. jejuni and its role in pathogenesis have not been clearly defined (1). Field et a1. (12) reported that tested c. jejuni strains produced three OMP (82, 74 and 76 kDa) when the bacteria were grown in low-iron medium. Recently, Pickett et a!. (29) reported about the expression of ironregulated proteins in the 70-75 kDa region in OMP of C. jejuni strains grown under conditions of iron limitation. We found (14) that growth under iron-limiting condi-
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tions induced a protein of an apparent molecular weight of 75 kDa in OMP preparations of 25 strains of C. fetus and one strain of C. coli. The iron-regulated proteins may play an important role in the ability of C. jejuni to invade and cause infection in the human host.
It was the aim of the present study to see whether the iron-regulated proteins were expressed by C. jejuni during human infections, by looking for the immune response in patients. We compared the profiles of OMPs from C. jejuni strains grown in ironlimiting medium to those grown in iron-supplemented medium and found that a protein band of 76 kDa appeared in most of the strains grown on iron-deficient medium, while it was lacking in most OMPs from bacteria grown on iron-supplemented media. This iron-regulated proteins reacted in the Western blot with sera from children with confirmed C. jejuni enteritis.
Materials and Methods
Bacterial strains and culture conditions. The C. jejuni/Coli strains used in this study were isolated from children with diarrhea (age 0-12 years) in the Tel-Aviv area (32) and biotyped according to the scheme of Lior (22). A total of 13 isolates was tested: 7 C. jejuni I (strains 666,782/90, 196/88,884/89,50/89,863/89,748/89), one C. jejuni II (865), and 5 strains of C. coli 1(73,819/89,618,390/89,521). Before initiating the experiments, the bacteria were inoculated onto Mueller-Hinton agar (M.H. DIFCO) supplemented with 5% sheep blood (MH-S) and incubated at 42°C for 24 h under microaerophilic conditions. The organisms grown on MH-S served as the inoculum for subsequent culturing on Brucella broth (B.B.). For testing the influence of iron, the organisms grown in B.B. were used to inoculate a) medium-M.H. supplemented with 50 IlM 2,2-dipyridil (Sigma) - (Dip), b) M.H. supplemented with 10!lM FeCh (Fe), and c) M.H. (U) as controls. The media were incubated at 37°C for 24 h under microaerophilic conditions.
Serum samples. Eleven patients' serum samples collected (one from each patient, 1-10 days after admission) were included in the experiments (1/83, 6/87, 322, 345, 2/87,11,279, 863/89,22/87, 157, 819/89). Ten control serum samples (Al, Al, A3, A4, AS, A6, 2C, 3C, 4C, 9C) were collected from healthy children, matched according to age and sex, who had been selected from those not having had diarrhea during the last two weeks (32).
Preparation of outer membrane proteins (OMP). The method of Schnaitman (31) as described by Huyer et ai. (15) was used. Briefly, the cultures from 20 Petri dishes (M.H.-140 mm diameter) were harvested with "envelope buffer" (1M TRIS-HCI pH 7.8) and washed twice (10000 X g, 10 min). The bacterial pellet was suspended in "envelope buffer" (1 g bacterial wet mass/10 ml) and submitted to sonication three times for 30 sec, which 30 sec intervals. After centrifugation at 15000 x g for 5 min, the supernatant containing membranes was centrifuged at 100 000 x g for 40 min. The pellet was suspended in 1 ml" envelope buffer" containing 2% Triton-X 100 (Sigma) and centrifuged again at 100000 x g for 40 min. The resulting pellet was suspended in 1 ml "envelope buffer" with 2% Triton-X-100 and after incubation at room temperature for 30 min under continous mixing, the suspension was centrifuged at 100000 x g for 90 min. The final pellet (OMP) was suspended in 0.5 ml1M Tris-HCL pH 6.8 and stored at -20°C until use.
Protein content was determined by the method of Bradford (6). Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The method
described by Laemmli (20) was used. The membrane proteins were separated by SDS-PAGE using 5% polyacrylamide as stacking gel and 10% polyacrylamide as separating gel by electrophoresis for 3-4 h at a constant cumint of 50 mAo The gels were stained with Coomassie Blue R (G.B.), (Sigma).
Western blotting. Proteins separated by SDS-PAGE gel electrophoresis were electrophoretically transferred onto nitrocellulose paper (0.45 !lffi) as described by Towbin et al.
Iron-regulated Proteins and Immune Response 341
(33). After blocking of nonspecific binding sites and washings, the blots were cut into strips and incubated with test serum (1: 30) in 0.1 % BSA-PBS-Tween 20 (PBS-T), with constant rocking at 25°C for 1 h. After washing three times (with PBS-T), the strips were incubated with the second antibody (1: 1000), peroxidase-conjugated rabbit anti-human IgG (DAKOPATIS), with constant rocking. After washings, the strips were developed with freshly prepared substrate solution (50 ml of peroxidase staining buffer acetate pH 5.5,2 ml of carbazole stock solution containing 1 % (w/v) 3 amino-9-ethyl carbazole acetone, 25 111 of 30% H20 2 ). The reaction was stopped by washing the strips with cold distilled water.
Results
The OMP profiles of 13 C. jejuni strains grown under iron-limiting conditions (OMP-Dip) were compared with those of C. jejuni grown in the presence of iron (OMP-Fe). The OMP profiles showed differences between the tested strains. However, a number of major bands (31,45,63-66, and 93 kDa) were shared by most of strains, both in OMP-Dip and OMP-Fe. Fig. 1 shows OMP profiles of nine out of total of 13
Fig.1. SDS-PAGE profiles of outer membrane proteins (OMP) from C. jejuni strains grown on iron-limiting and iron-supplemented medium (see methods). Lanes: 1, Low molecular weight standards (Bio-Rad Laboratories), soybean trypsin inhibitor 21.5 kDa, carbonic anhydrase 31 kDa, ovalbumin 45 kDa, bovine serum albumin 66.2 kDa and phosphorylase b 97.4 kDa. 20, High molecular weight standards (Bio-Rad, Laboratories), myosin 200 kDa, ~-galactosidase, 116 kDa, phosphorylase b 97.4 kDa, bovine serum albumin 66.2 kDa and ovalbumin 45 kDa. Lanes: evens (2, 4, 6, 8, 10, 12; 14, 16, 18)-OMP-Fe; unevens (3,5, 7, 9, 11, 13, 15, 17, 19)-OMP-Dip. Lanes 2, 3: C. jejuni I (782/90); Lanes 4, 5: C. jejuni II (865); Lanes 6, 7: C. jejuni 1(666); Lanes 8, 9: C. jejuni I (196/88); Lanes 10, 11: C. coli I (73); Lanes 12, 13: C. jejuni 1(884/ 89); Lanes 14, 15: C. jejuni I (50/89); Lanes 16, 17: C. jejuni I (863/89); Lanes 18, 19: C. coli 1(819/89).
342 D. Schwartz et al.
strains tested. It can be seen that iron limitation induced the appearance of an additional protein with an apparent molecular weight of 76 kDa in the outer membranes of seven of nine strains tested (Lanes: 3, 5, 7, 11, 13, 15, 17). This protein band was absent from the profiles of bacteria grown in iron-supplemented medium (Fe) (Lanes: 2, 4, 6, 8, 12, 14, 16). To confirm the appearance of the 76 kDa band in OMP of bacteria grown in a iron-limiting medium, the additional gels were run with double amounts of each of OMP-Dip and OMP-Fe loaded. The representative gel of one of 10 strains out of a total of 13 tested expressing iron-regulated protein is shown in Fig. 2. In some of the strains, multiple bands appeared in the 76 kDa region.
No difference was observed in the OMP profiles of bacteria grown under usual conditions (OMP-D) and in the medium supplemented with iron (OMP-Fe) (not shown).
To examine whether iron-regulated outer membrane protein is expressed by C. jejuni during natural infections, we looked for the presence of IgG antibodies against
Fig. 2. SDS-PAGE of OMP-Dip (lane 3) compared to OMP-Fe (lane 4) of C. jejuni 666 (50 fJ,g protein per lane). Lanes 1 and 2, high and low m. wt. standards, see legend to Fig. 1.
Iron-regulated Proteins and Immune Response 343
the 76 kDa proteins in the sera of children. Sera from 11 patients (from whom the C. jejuni strains had been isolated) were included into the study and their reactions (homologous and heterologous) with seven OMP-Dip and four OMP-Fe preparations were analysed using the Western blot technique. Representative immunoblots (heterologous reactions) are shown in Fig. 3 and the results are summarized in Table 1.
A band in the 76 kDa region was observed in immunoblots of seven OMP-Dip prepared from various C. jejuni strains, reacting with all 11 serum samples derived from C. jejuni patients. A similar reaction at 76 kDa m. wt. band was observed with homologous as well as with heterologous serum samples. In contrast, the band did not appear in nine out of ten tested serum samples from the controls, as well as in control immunoblots with the OMP-Fe preparation with patient sera (Fig. 4 and Table 1).
Sera from both infected and healthy children displayed variable reactions with other OMP components in the ranges of 97.4,66.2,45 and 31 kDa of both OMP-Dip and OMP-Fe. The reaction with the 45 kDa band was observed in all 11 serum samples from infected children as well as in 8 out of 10 control samples.
Discussion
We examined outer membrane proteins profiles of 13 C. jejunilcoli strains growing in medium containing dipyridil (iron-limiting medium, OMP-Dip) as compared to the same bacterial strains cultured in iron-supplemented medium (OMP-Fe). We found that 10 out of the 13 tested strains grown in the absence of iron produced a strong
Fig. 3. Western blot of sera from four infected children reacting with OMP-Dip. compared to OMP-Fe (666). Lanes 1,4,6,8, OMP-Dip; Lanes 2,3,5,7, OMP-Fe. Lanes i, 2 - serum 1183, lanes 3, 4 - serum 100, lanes 5, 6 - serum 22/87, lanes 7, 8 - serum 345.
344 D. Schwartz et al.
Table 1. Summary of the results of Western blot of patients and control sera reating with OMP of C. jejuni1
Molecular weights OMP-Dip OMP-Fe OMP-Dip OMP-Fe of protein
Sera of patients2 Sera of controls2 components (kDa)
1 The outer membrane proteins of C.jejuni from an iron-deficient medium (OMP-Dip) and from an iron-supplemented medium (OMP-Fe) were tested by Western blot (see methods).
2 Numbers represent number of sera out of 11 tested ones reacting with respective bands.
protein band or multiple band in the region of 76 kDa m. wt. We cannot conclude whether the 76 kDa iron-regulated proteins differ from those of m. wt. of 71, 74, 75 described by other authors (12, 14), or if they are similar, merely for having shown a slightly different apparent m. wt. under variable experimental conditions.
In our previous experiments (14), we found antibodies against the iron-regulated proteins in sera from patients infected with C. fetus, c. jejuni and C. lari. In the present study, Western blot analysis showed antibody response (IgG) against 76 kDa OMP in sera from all 11 tested children with C. jejuni diarrhea. The reaction was observed both in homologous and heterologous antisera, suggesting that the proteins produced by various strains were antigenically similar. The one control sample which reacted with 76 kDa OMP-Dip was also positive in ELISA with glycine extract as antigen (32).
The specificity of the reaction with iron-regulated protein was confirmed by the absence of the reaction with the outer membrane extracted from C. jejuni growing in iron-supplemented medium (OMP-Fe). The findings suggest that the protein is synthesized by C. jejuni during natural infection. Whether such protein can be induced by iron starvation or other conditions, e. g. temperature, is not known.
The tested sera of infected children reacted also with other variable OMP components, as was also observed by other authors (2-4, 9, 16, 24, 25, 27). Recently, Panigrahi et al. (28) observed that C. jejuni growing in vivo in the rabbit intestine expressed a number of novel proteins, which were lacking in the bacteria grown in vitro. Some of these proteins reacted with sera of human volunteers challanged with C. jejuni.
According to our previous study (32), elevated levels of IgG were found in 43% of healthy children (age 0-12 y.) which may be explained by common subclinical infections among the population. It was shown that some C. jejuni infections in infant populations living in a non-tropical climate are asymptomatic but can trigger high levels of specific antibodies (13). Some of the OMP components, such as the 45 kDa (major outer membrane protein) may be shared not only by various C. jejuni strains, but also by other gram-negative bacteria. This also may explain the positive reaction found in control sera.
Iron-regulated Proteins and Immune Response 345
Fig. 4. Western blot of sera from three healthy children (controls) reacting with OMP-Dip and OMP-Fe (666). Lanes 1,3,5, OMP-Dip; lanes 2, 4, 6, OMP-Fe. Lanes 1,2 - serum A1, lanes 3, 4 - serum 2C, lanes 5, 6 - serum Ai.
In conclusion, the iron-regulated proteins were synthesized by most of the local C. jejunilcoli isolates, both in vitro under conditions of iron limitation and during natural infection. The protein(s) were recognized by all of the 11 tested sera obtained from patients, but only by one of ten control sera. This suggests that the reaction is specific of recent infection and may have a diagnostic potential.
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Prof. Janina Goldhar, M.D., Department of Human Microbiology, Sackler Faculty of Medicine, Tel-Aviv University, Ramat-Aviv, Tel-Aviv, Israel
