Sodium Dodecyl Sulfate-Polyacrylamide Gel Typing System for Characterization of Neisseria meningitidis Isolates

Transcription

1 JOURNAL OF CLINICAL MICROBIOLOGY, Aug. 1982, p Vol. 16, No /82/ $02.00/0 Sodium Dodecyl Sulfate-Polyacrylamide Gel Typing System for Characterization of Neisseria meningitidis Isolates LOUIS F. MOCCA AND CARL E. FRASCH* Division of Bacterial Products, Bureau of Biologics, Food and Drug Administration, Bethesda, Maryland Received 10 December 1981/Accepted 10 May 1982 Thirty to fifty percent of group B and group C Neisseria meningitidis carrier isolates are not serotypable with existing outer membrane protein typing sera. A typing system based on differences in the outer membrane protein profiles after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was therefore developed as an adjunct to existing serotyping methods. Although most N. meningitidis strains contain several outer membrane proteins visible by SDS- PAGE, there are only one to three predominant proteins. The SDS-PAGE profiles of these major proteins were used to establish 10 different PAGE types. Greater than 95% of all meningococcal isolates, regardless of serogroup, fit into 1 of the 10 PAGE types. The outer membrane protein profile of individual strains after SDS- PAGE was constant when outer membrane fractions were prepared from the same strain on several different days. A comparison of gel profiles of meningococcal isolates obtained from different sites of the same patient revealed no significant differences among both major and minor proteins for isolate sets thus far examined. Characterization of strains by PAGE type can be a valuable epidemiological tool in addition to serotyping and in the absence of specific serotype antisera. A single serogroup is often responsible for most meningococcal diseases occurring in a given geographic region. Epidemiological studies on transmission and carriage of Neisseria meningitidis within defined populations need therefore employ both serogrouping and serotyping to clearly identify meningococcal isolates. Serogroups within N. meningitidis are defined by chemically and immunologically distinct capsular polysaccharides (3), whereas serotypes are based upon immunologically distinct outer membrane protein (11) and lipopolysaccharide antigens (17). Strains of N. meningitidis can be serogrouped by bacterial slide agglutination (7) or by the antiserum-agar technique (3). Serotypes have been identified by agar gel double diffusion (10), bactericidal (9) and radioactive antigen binding assays (25), and by coagglutination (6). The 18 characterized serotyping sera of Frasch (10) and Zollinger (25) do not cover the full range of antigenic differences found among the major protein surface antigens of N. meningitidis. Many group B and group C N. meningitidis isolates, especially those from carriers, fail to react with the presently available meningococcal serotyping antisera (4). Although new typing sera can be made and characterized, another typing procedure was required to alleviate the immediate problem of nonserotypable 240 strains. Large differences in protein band patterns were observed when outer membrane preparations from different N. meningitidis strains were compared after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (12). Similar differences have been observed among Neisseria gonorrhoeae (14). These differences can be used to differentiate among epidemiologically related meningococcal and gonococcal strains and have been used to identify nosocomially transmitted N. meningitidis (2, 4, 5, 18, 21). An advantage of such a typing system would be rapid distinction among epidemiologically associated strains without the necessity of production and standardization of typing sera; however, both methods used in concert can provide the most useful information. This paper will show that most serotypable and nonserotypable meningococcal isolates can be differentiated into 1 of 10 PAGE types based upon their outer membrane protein profiles after SDS-PAGE. MATERIALS AND METHODS Strains and growth conditions. The prototype strains were described previously (9). Strains with S-prefixes, and B-prefixes were obtained from Harry Feldman (Upstate Medical Center, Syracuse, N.Y.) and S. DeMaeyer-Cleempoel (Institute of Hygiene and Epidemiology, Brussels, Belgium), respectively. Oth-

2 VOL. 16, 1982 er strains with BB-prefixes were obtained from the Washington D.C. area by the authors. The strains were grown on brain heart infusion agar (Difco Laboratories, Detroit, Mich.) containing 1% normal horse serum and stored as described previously (3). The strains were serogrouped by the antiserum-agar technique (3). Serotype antigen preparation. A 6- to 8-h growth on brain heart infusion agar containing horse serum was inoculated into 200 ml of tryptic soy broth (Difco) and grown overnight at 36 C on a gyratory shaker at 150 rpm. The cells were harvested by centrifugation at 10,000 x g for 15 min and then suspended without washing in 5 ml of 0.2 M lithium chloride-0.1 M sodium acetate (ph 6.0). The cells were extracted at 45 C for 2 h by vigorous shaking with 4-mm glass beads. The cell-free extracts were recovered by centrifugation at 10,000 x g for 15 min. The serotype antigen (STA) was pelleted from the extract by centrifugation at 100,000 x g for 2 h, suspended in water, repelleted, and finally taken up in 0.5 ml of distilled water containing 0.02% (wt/vol) sodium azide. The total protein yield was approximately 1 to 2 mg/ml as determined by the Lowry method (16). Serotype identification. The STAs were examined by double diffusion in agar gel (10), using typing sera prepared against the purified prototype STAs (11). PAGE typing. The STAs were examined by SDS- PAGE, using the Weber-Osborn neutral phosphate buffer system in a slab gel apparatus (12, 24). Briefly, 20-,ul samples containing 10,ug (±5,ug) of protein were mixed with 20,ll of 2% (wt/vol) SDS-2% (vol/vol) 2- mercaptoethanol in 8 M urea and heated at 100 C for 2 min. The samples were added to individual wells and electrophoresed on gels containing 10% (wt/vol) acrylamide and 0.3% (wt/vol) bisacrylamide. The protein bands were stained with Coomassie brilliant blue R- 250 (Bio-Rad Laboratories, Richmond, Calif.). Photographs were made with Polaroid type 57 film. Strains were typed by comparing the photographs to those of reference PAGE type patterns of STAs containing approximately 10,ug of protein as determined by Lowry (16) and by use of internal standards consisting of known reference strains. RESULTS Based on SDS-PAGE analysis of outer membrane preparations (STA) from over 400 meningococcal strains (primarily serogroups B and C), 10 distinct protein profiles were found. These are shown in Fig. 1, which is arranged by relatedness of pattern. The 10 different gel patterns, or PAGE types, are denoted by Roman numerals. For PAGE type determination only the one to three heavily staining bands between approximately 28,000 and 46,000 (28K and 46K) molecular weight were considered. The class 1, 2, 3, and 5 proteins described by Tsai and Frasch (23) are of major importance for PAGE type determination. PAGE types I, II, III, and V have class 2 (41K) proteins. Type II has an equally intense class 1 (46K) protein, and types I and III have class 1 proteins of 46K and 48K, respectively, of lesser intensity than their class 2 PAGE TYPING OF MENINGOCOCCI 241 VII V I I lmwi AnX x IX FIG. 1. SDS-PAGE of group B meningococcal serotype antigens. The different PAGE types are identified by Roman numerals and are arranged by relatedness of patterns. The arabic numerals show the class 1 (46K ± 1K), and class 5 (28K ± 1K) proteins. These major classes of proteins were compared when the 10 PAGE types were established, but the PAGE types are determined mostly by the class 1, 2, and 3 proteins. proteins. PAGE types IV and X each have the same molecular class 1 protein (46K), whereas the class 1 protein of type IX is 47K. Types IV and IX have class 3 and 5 proteins of 38K and 28K, respectively, and type X has 36K and 30K class 3 and 5 proteins. The small differences in apparent molecular weight between the major outer membrane proteins of some PAGE types necessitated the use of slab gels rather than individual tubular gels. To determine the correlation between serological typing and PAGE typing, 471 meningococcal strains representing serogroups B, C, Y, Z, W135, and 29E (approximately 65% B and C strains) were independently serotyped and PAGE typed (Table 1). Serotypes having PAGE types I, II, III, and V show some serological cross-reactivity, but there is little or no serological cross-reactivity with serotypes having PAGE types IV, IX, or X. Generally each serotype has predominantly one PAGE type, i.e., serotype 2 strains almost always have a PAGE type I pattern, and a few (5%) will have the related PAGE type II, III, or V patterns, possibly due to subpopulations (2a, 2b, and 2c) within this serotype (20). Two or more serotypes can have the same PAGE type; for example, the related serotypes 1, 8, and 15 all have PAGE type IV patterns. Over 95% of meningococcal isolates tested, regardless of serogroup, fell within 1 of the 10 PAGE types. The consistency of strain pattern is demonstrated in Fig. 2. Three group B isolates (BB-

3 242 MOCCA AND FRASCH TABLE 1. Correlation of serotypes with PAGE types among 471 strains of N. meningitidis Sero- PAGE typea (no. of strains) type I II III IV V VI ViI ix x NTb a Only two PAGE type VIII strains have been found, one group B, one group Y, both nonserotypable ḃ NT, Nonserotypable. 107, BB-130, and BB-138) were removed from frozen storage, grown, and extracted on four separate occasions, and their outer membrane proteins were examined by SDS-PAGE. The relative amounts of protein varied among preparations, but their PAGE patterns remained the same, with only small variations seen in minor low-molecular-weight proteins. The PAGE patterns of isolates obtained from different sites from individual patients are shown in Fig. 3. Each isolate from the same patient had the same PAGE pattern. Minor variations were occasionally observed in the class 5 proteins (27 to 30K) (Fig. 3, patient 1). To examine whether prolonged carriage of N. meningitidis of a single serogroup was necessarily due to the same strain, we observed a chronic = ;;g> FIG. 2. SDS-PAGE of outer membrane proteins of three group B meningococcal isolates: strain 1, BB- 107; strain 2, BB-130; and strain 3, BB-138. The strains were grown and their outer membrane complexes extracted on four separate occasions. For the meaning of the arabic numerals on the right of figure, see Fig J. CLIN. MICROBIOL. FIG. 3. SDS-PAGE of outer membrane proteins of multiple isolates from three patients. Patient 1, (A) BB-37 (cerebral spinal fluid) and (B) BB-38 (nasopharynx); patient 2, (A) S-6056 (cerebral spinal fluid), (B) S-6057 (blood), and (C) S-6058 (nasopharynx); patient 3, (A) B-30 (nasopharynx) and (B) B-34 (cerebrospinal fluid). For the meaning of the arabic numerals on the right of the figure, see Fig. 1. group B carrier for a period of 34 months. Thirteen throat isolates, all group B, were obtained during this period and frozen. All of the isolates were then grown concurrently for PAGE type analysis (Fig. 4). Three distinct group B strains were demonstrated among the 13 isolates and were carried for at least 2, 5, and 18 months, respectively. Strains 1, 2, and 3 were later shown to be serotypes 14, 5, and 15, respectively. DISCUSSION To obtain a more complete understanding of the epidemiology of endemic and epidemic meningococcal disease, both serogrouping and serotyping should be utilized. Thirty percent or more of group B and group C meningococcal isolates and a higher percentage of other serogroups do not fit into any of the 18 serotypes characterized by Frasch and Chapman (10) and Zollinger and Mandrell (25). Although new serotypes are being characterized, an adjunct procedure was required to distinguish among the many nonserotypable isolates. The wide variation in the outer membrane protein profiles among meningococcal strains seen by SDS- PAGE provided a means to discriminate between nonserotypable isolates (4, 5, 14, 15, 21). We initially used the SDS-PAGE method to compare the outer membrane protein profiles of 15 prototype strains, with isolates submitted for serotyping as a check on our serotyping results. Sera were determined to be cross-reactive and were adsorbed if they reacted with the STAs from strains of more than one PAGE type. SDS-

4 VOL. 16, ` _64 6oA tmj #mj. FIG. 4. SDS-PAGE of outer membrane proteins from 13 group B isolates recovered over 34 months from a chronic carrier. The three different strains which were isolated during this time are labeled 1, 2, and 3. PAGE was also used to determine the approximate quantity of protein in a STA preparation to avoid false-negative serotype reactions owing to inadequate amounts of extracted STA. PAGE typing does not add to the resolution of serotypable strains, but serves to confirm serotyping results. It is important to continue to look for new serotypes, especially among PAGE type IV, which accounts for approximately 60% of the nonserotypable strains. The present study shows that most serotypable and nonserotypable meningococcal isolates can be differentiated into 1 of 10 distinct PAGE types based on the relative molecular weights of their major outer membrane proteins. PAGE typing has been applied to all meningococcal serogroups, with the exception of group A, which has a single PAGE type (IV) (22). All other serogroups have at least 5 to 8 of the 10 PAGE types (8). The most common PAGE type is type IV, which accounts for approximately 40% of all meningococcal isolates thus far examined. PAGE type I is the second most common type. The degree of heterogeneity of PAGE types found among both serotypable and nonserotypable strains indicates that the potential number of serologically distinct meningococcal types is large. Our results show that the outer membrane PAGE types are stable. PAGE types did not change when frozen samples of the same strain were grown and extracted on several occasions or after several in vitro passages. The major proteins, with molecular weights between 38K and 46K, were quite stable and are present in most strains, whereas the lower-molecularweight proteins (30K and below) showed greater variability in molecular weight between strains PAGE TYPING OF MENINGOCOCCI 243 and were absent in some strains. Poolman et al. (19) state that meningococcal PAGE typing should be restricted to the higher-molecularweight major outer membrane proteins. Their data indicate that the class 5 heat-modifiable proteins are not suitable for PAGE typing because of their variability. Similarly, Loeb and Smith (15) demonstrated through multiple isolation of a single strain of Haemophilus influenzae that outer membrane protein differences, as seen by SDS-PAGE, were detected only in minor, lower-molecular-weight proteins. Barenkamp et al. also found proteins derived from H. influenzae type b to be stable under a variety of experimental manipulations (1). The PAGE typing system in conjunction with serotyping was used to identify three distinct strains harbored by chronic group B carrier, demonstrating the inaccuracy of reliance upon serogroup determination alone. Our ability to identify the same PAGE type organism on different occasions further demonstrates the accuracy and reliability of the system. One organism may be carried for long periods of time only to be cleared and replaced by a different organism of the same serogroup. Group B carriage does not apparently confer protection from recolonization with another group B strain. Craven et al. (4) found PAGE typing especially helpful in a study of carriage within a diseasefree military population in which 70% of the strains were nonserotypable. PAGE typing also proved useful in a study of familial contacts of patients with meningococcal disease (13). For epidemiological and public health reasons it becomes necessary to identify individual meningococcal strains isolated from patients and their contacts. Serological typing reagents are available only in a few laboratories, whereas PAGE typing can be performed in any laboratory experienced in PAGE analysis of proteins and which can be furnished with reference outer membrane preparations. LITERATURE CITED 1. Barenkamp, S. J., R. S. Munson, and D. M. Granoff Subtyping isolates of Haemophilus influenzae type b by outer-membrane protein profiles. J. Infect. Dis. 143: Centers for Disease Control Nosocomial meningococcemia-wisconsin. Morbid. Mortal. Weekly Rep. 27: Craven, D. E., C. E. Frasch, J. B. Robbins, and H. A. Feldman Serogroup identification of Neisseria meningitidis. Comparison of an antiserum agar method with bacterial slide agglutination. J. Clin. Microbiol. 7: Craven, D. E., C. E. Frasch, L. F. Mocca, F. B. Rose, and R. Gonzales Rapid serogroup identification of Neisseria meningitidis by using antiserum agar: prevalence of serotypes in a disease-free military population. J. Clin. Microbiol. 10: Craven, D. E., M. S. Peppler, C. E. Frasch, L. F. Mocca,

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