pharyngeal mucosa were identified by biochemical means and were examined for. IgAl protease production. IgAl protease production was demonstrated in ...
INFECTION AND IMMUNITY, Mar. 1981, p. 868-873 0019-9567/81/030868-06$02.00/0
Vol. 31, No. 3
Ecology and Nature of Immunoglobulin Al ProteaseProducing Streptococci in the Human Oral Cavity and Pharynx MOGENS KILIAN* AND KIRSTEN HOLMGREN Department of Oral Biology, Royal Dental College, Vennelyst Boulevard, DK-8000 Aarhus C, Denmark
The identity and proportional distribution of immunoglobulin Al (IgAl) protease-producing streptococci in the oral and pharyngeal microflora were studied. A collection of 459 streptococcal strains, including reference strains of Streptococcus species, and fresh isolates from human dental plaque and buccal and pharyngeal mucosa were identified by biochemical means and were examined for IgAl protease production. IgAl protease production was demonstrated in some, but not all, strains of Streptococcus sanguis and Streptococcus mitior and in a group of strains of uncertain taxonomic affiliation. The property was not associated with particular biotypes within the two species. Strains of S. sanguis and S. mitior isolated from Macaca fascicularis also cleaved human IgAl. A significantly different proportion of streptococcal populations in different ecosystems produced IgAl protease. The enzyme was released by 62.7% of streptococcal isolates from buccal mucosa in contrast to only 7.8% from pharyngeal mucosa. In samples from minitial and mature dental plaque 38 to 40% of streptococcal isolates produced IgAl protease. This difference was largely a result of the frequency by which IgAl protease activity was present in S. mitior, the predominant streptococcal species in all samples. Among otherwise identical isolates of S. mitior, 67.8% from buccal mucosa in contrast to only 5.9% from pharyngeal mucosa produced IgAl protease. The results indicate that IgAl protease may confer an ecological advantage to streptococci colonizing surfaces exposed to a secretory IgA-mediated selection pressure.
Immunoglobulin A (IgA) is an importint mediator of specific immunity on mucosal surfaces lining the human respiratory, alimentary, and urogenital tracts. It is generally accepted that secretory IgA (S-IgA) as found in secretions helps maintain the integrity of the mucosal surfaces by reducing bacterial and viral colonization, by neutralizing microbial toxins, and by preventing the penetration of antigens and allergens through the mucosal barrier (7, 9, 22, 23). In view of this it is interesting that some strains of the species Streptococcus sanguis, which is a numerically important human oral microorganism (1), have been shown to release an endopeptidase capable of cleaving IgA in the hinge region (18). This enzyme, designated IgA protease, has a unique specificity for IgA subclass I (20), which is predominant in serum and secretions (6). More recent discoveries that Neisseria gonorrhoeae and the three leading causes of bacterial meningitis, N. meningitidis, Streptococcus pneumoniae, and Haemophilus influenzae, release similar, though not identical, enzymes have suggested that IgAl proteases may be important bacterial virulence factors (10, 11, 16, 17, 19). 868
S. sanguis only occasionally causes systemic infection, but is an important initiator of plaque formation on teeth (1, 12, 21). Since only some strains of this species seem to produce IgAl protease (5) it was considered of interest to study the ecology and nature of such strains in the oral cavity and the pharynx. Such information could add to the understanding of the significance of bacterial IgAl proteases. MATERIALS AND METHODS Bacterial strains. A total of 459 streptococcal strains were included in the study. (i) The 52 reference strains represented streptococcal species commonly encountered in the oral cavity and in the pharynx. The strains, which are listed in Table 1, were kindly provided by B. Perch, J. Henrichsen, and E. Kjems, Statens Seruminstitut, Copenhagen; R. R. Facklam, Center for Disease Control, Atlanta, Ga.; W. Liljemark, University of Minnesota, Minn.; or were obtained from the National Collection of Type Cultures (NCTC), Colindale, London, or the American Type Culture Collection (ATCC), Rockville, Md.
(ii) A total of 16 streptococcal strains were isolated from initial dental plaque in Macaca fascicularis monkeys (12). These included nine identified as S.
869
STREPTOCOCCAL IgAl PROTEASE
VOL. 31, 1981
TABLE 1. Reference streptococcal strains IgAl proStrain
Species (group)
Source
duction
Streptococcus pyogenes (A)
NCTC" 8191 NCTC 8322 NCTC 8326 NCTC 100079 NCTC 100080
B. Perch B. Perch B. Perch B. Perch B. Perch
S. agalactiae (B)
NCTC 8197 NCTC 9993 ATCCb 12403 II 18RS 21
B. Perch B. Perch B. Perch B. Perch
S. pneumoniae
19FL, 1N, 19C, 34, 36 Fuller
J. Henrichsen
S. anginosus (F)
HF60R NCTC 8539
B. Perch B. Perch
NCTC 10708 MG 216 SS 14132/70 SS 6235/64 SS 1986/64
B. Perch B. Perch B. Perch B. Perch B. Perch
(G) S. milleri
(Group A antigen)
NCTC 7864 (S. sanguis type II) NCTC 8029 (group 0) 5119 (group 0) SS-429 (S. mitis) SS-1066 (S. sanguis type II)
B. Perch B. Perch B. Perch R. Facklam R. Facklam
NCTC 10449 6715 GS5
NCTC
S. salivarius
ATCC 25975 SK12
ATCC Own collection
S. sanguis (H)
NCTC 10231 ATCC 10556 ATCC 10558 ATCC 12396 Challis S7 804
B. Perch ATCC ATCC B. Perch E. Kjems W. Liljemark J. Kelstrup
S. faecalis (D)
Skadhauge OI 503, OII 783, OIII 3, OIV B. Perch 782, OV 567
S. faecium (D)
Skadhauge 7, 19, 20, 237, 244
B. Perch
Streptococcus sp. (G)
ATCC 12394 Harrison
B. Perch B. Perch
CN 1085 (M animalis)
B. Perch B. Perch
S. mitior
S. mutans
Streptococcus sp. (M)
tease pro-
from a collection of strains from a bacteriological study
+
+
+
B. Perch B. Perch
Lindstr0m (M humanis) a NCTC, National Collection of Type Cultures, London. b ATCC, American Type Culture Collection, Rockville, Md.
sanguis and seven identified as Streptococcus mitior. (iii) A total of 84 str9ins were randomly selected
-
of dental plaque of Tanzanian children (13). (iv) A total of 307 streptococcal strains were isolated from the oral cavity and the oropharynx of two male
870
KILIAN AND HOLMGREN
subjects. Samples were taken, by using cotton swabs, from the posterior wall of the oropharynx, from the cheek mucosa, and from the facial surface of one of the upper first molars. The latter sample was taken 4 h after thorough pumiceing of the teeth. The swabs were transferred to 1.5 ml of cold nutrient broth (Difco Laboratories, Detroit, Mich.) and were vigorously shaken on a Vortex mixer. Serial dilutions of the samples were plated on Mitis Salivarius Agar (Difco) and incubated for 2 days in 5% C02-95% N2. From each sample approximately 50 colonies representing all colonies in a certain section of one agar plate were transferred to nutrient broth containing 10% horse serum. After overnight incubation all isolates were subcultivated on blood agar until pure. Each isolate was examined for hemolysis on 5% bovine blood agar, hydrolysis of arginine and esculine, production of acetoin (Voges-Proskauer test), production of extracellular polysaccharides in 5% sucrose broth, production of hydrogen peroxide, optochin sensitivity, and ability to ferment lactose, raffinose, sorbitol, mannitol, salicin, and inulin by using standard methods (2-4, 8). Biochemical examination. Detailed biochemical examination of selected strains was performed by using the API ZYM and API System 50 (Analytab Products, Plainview, N.Y.). IgAl protease production. All 459 strains were examined for IgAl protease production. The substrate was a polymeric IgAl myeloma protein (Car) generously donated by J. Mestecky, University of Alabama in Birmingham. A small loopful of bacteria from a blood agar culture was suspended in 40 jl of a 2-mg/ ml solution of the IgAl paraprotein in phosphatebuffered saline, pH 7.0. After incubation for 4 h at 35°C cleavage of the IgA protein was detected by immunoelectrophoresis of samples in 2% Noble agar (Difco) in Veronal buffer. Antisera used to develop the immunoelectrophoresis were our own rabbit sera raised against purified colostral S-IgA and monospecific sera against heavy (a) and light (K) chains of IgA (DAKO-immunoglobulins, Copenhagen, Denmark). Identification of cleavage products was done by immunochemical means and by sodium dodecyl sulfatepolyacrylamide gel electrophoresis as described previously (11).
RESULTS Reference strains and strains from monkeys. Among the reference strains listed in Table 1 nine strains produced enzyme capable of cleaving IgAl myeloma protein. These nine strains included all five strains of S. pneumoniae and two strains each of the species S. sanguis and S. mitior (S. mitis) (Table 1). None of the representatives of the serological groups A, B, D, F, G, and M and the species Streptococcus mutans, S. salivarius, and S. milleri (S. intermedius) produced IgAl protease. Of the 16 streptococcal strains isolated from initial dental plaque in M. fascicularis monkeys, 6 cleaved IgAl paraprotein. These six strains included one out of nine strains of S. sanguis and five out of seven strains of S. mitior.
INFECT. IMMUN.
Freshly isolated strains. To study the distribution and identity of IgAl protease-producing streptococci in various ecological niches in the oral cavity and in the pharynx, 391 strains isolated from mature and initial (4-h) dental plaque, from buccal mucosa, and from the posterior wall of the oropharynx were examined for IgAl protease production. Of these streptococcal isolates, 146 cleaved IgAl paraprotein. Identity of IgAl protease-producing strains. Preliminary biochemical examination of the 391 isolates revealed that, with the exception of two strains of S. pneumoniae, all freshly isolated IgAl protease-producing strains had properties considered characteristic (2-4, 8) of the species S. sanguis or S. mitior (S. mitis). Since IgAl protease production was a property of only a fraction of the strains assigned to the two species S. sanguis and S. mitior, 28 strains were subjected to detailed biochemical characterization. Reference strains of the two species were included in this examination. The purpose of this was to study whether IgAl protease production is a property associated with a certain biotype within the two species. The results (Table 2) show that the IgAl protease-producing strains could be divided into four groups on the basis of their biochemical reactivities. Two of these groups, although differentiated by several biochemical reactions, would both be classified as S. sanguis by present standards. In Table 2 the two groups are referred to as S. sanguis A and B. Biotype A included the type strain ATCC 10556 and strain 804. The reference strains ATCC 10558 and Challis had the same properties as shown for biotype B. The third group could be directly equated with the species S. mitior (S. mitis) and encompassed both dextrannegative and dextran-positive strains ("S. sanguis II"). The fourth group, consisting of eight strains isolated from initial dental plaque and oropharyngeal mucosa, resembled S. mitior. However, since several biochemical reactions did differentiate these strains from S. mitior, their exact classification is pending a clarification of the taxonomy of viridans streptococci. All eight strains having the characteristics of this fourth group produced IgAl protease. The characteristics of the three other groups were shared by both IgAl protease-producing strains and strains lacking this property. Thus, within the species S. sanguis and S. mitior IgAl protease production was not an exclusive property of certain biotypes. Origin of IgAl protease-producing strains. The origin and identity of all 391 freshly isolated strains is presented in Table 3. The table demonstrates that IgAl protease production was associated exclusively with the species
STREPTOCOCCAL IgAl PROTEASE
VOL. 31, 1981
S. mitior, S. sanguis, S. pneumoniae, and the unclassified taxon. Among the strains identified as S. mitior and S. sanguis 42.1% and 69.7%, respectively, produced IgAl protease. S. mitior was the predominant streptococcal species in all samples from teeth and from buccal and pharyngeal mucosa. However, the proportion of S. mitior strains that produced IgAl protease in these three locations was significantly different (Table TABLE 2. Biochemical and physiological characteristics of IgAl protease-producing streptococci isolated from the oral cavity and the oropharynx Reaction of isolates
Test
mi- Strepto-
S. san-
S. san-
guis A
guis B
S.
tior
coccus
Hemolysis Arginine hydrolysis Esculine hydrolysis Voges-Proskauer H202 production Dextran production Glucose Fructose Galactose Cellobiose Lactose Threhalose Raffinose Amygdalin Salicin Mannitol Sorbitol
a + -
a + +
a
sp. a
-
-
+ + + +
+ + + + + + + +
+
+
+ + + -
+
+ + + + +
+ +
±
+
-
-
+
+
-
+
N-acetyl glucosa-
-
-
+
+
+ + +
+ + +
-
+
+
mine a-Galactosidase B-Galactosidase a-Glucosidase B6-Glucosidase Acid phosphatase a W, Weak reaction.
± wa W
+
+-
±
871
3); 67.8% of S. mitior isolates from buccal mucosa, in contrast to only 5.9% ofS. mitior isolates from pharyngeal mucosa, produced this enzyme. Among S. mitior isolates from both mature and initial dental plaque 38% were capable of cleaving IgAl. The percentage of the total streptococcal populations that produced IgAl protease in mature and initial dental plaque and on buccal and pharyngeal mucosa was 40.5, 38.2, 62.7, and 7.8%, respectively. The corresponding individual figures for the two subjects from which the bacteria were isolated are presented in Table 4. Properties of cleavage products. Previously published results have demonstrated that IgAl proteases from S. sanguis and S. pneumoniae are identical with respect to site of attack in the IgAl molecule (10). Immunochemical examination of fragments produced by incubation of substrate IgAl with representative strains of S. mitior indicated that this organism cleaved the IgAl molecule into Fab and Fc fragments as do S. sanguis and S. pneumoniae. This was indicated by the immunoelectrophoretic pattern and antigenic reactivity of the cleavage products (Fig. 1). The fragments derived from IgAl paraprotein cleaved by S. sanguis and S. mitior were indistinguishable by immunoelectrophoresis (Fig. 1). The similarity of the fragments obtained with the two species was further supported by identical molecular weight as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Bacteria of both species cleaved the polymeric IgAl paraprotein (Car) into Fab and Fc fragments with apparent molecular weights of 127.000 and 65.000, respectively (unreduced samples). Representative strains of S. mitior partially cleaved colostral S-IgA, whereas myeloma IgA2 and IgG were resistant to cleavage.
TABLE 3. IgAI protease production and identity of 391 streptococcal isolates from dental plaque and buccal and pharyngeal mucosa
Species
Mature dental
plaque
S. mitior (S. mitis) S. sanguis S. salivarius S. intennedius (S. milleri) S. mutans S. pneumoniae Streptococcus sp. (unclassified) Unidentified strains
19/50 (38.0) 15/20 (75.0) 0/2 0/7 0/5 0/0 0/0
Total
34/84 (40.5)
0/0
a Figures indicate the number of strains
percentage share).
No. of IgAl protease-producing strains from:' Buccal mucosa Tooth surface Pharyngeal (4-h plaque) mucosa
29/75 (38.7) 5/5 (100) 0/13 0/1 0/0 2/2 3/3 (100) 0/3
.39/102 (38.2)
61/90 (67.8) 3/3 (100) 0/4 0/4 0/1 0/0 0/0 0/0
64/102 (62.7)
3/51 (5.9) 0/5 0/26 0/12 0/0 0/0 5/5 (100) 0/4
Total
112/266 (42.1) 23/33 (69.7) 0/45 0/24 0/6 2/2 (100) 8/8 (100) 0/7
8/103 (7.8)
producing IgAl protease/number of strains examined (and their
872
*.,'_:.-3iN,s:e._o' KILIAN AND HOLMGREN
INFECT. IMMUN.
TABLE 4. Percentages of oral and pharyngeal streptococcal isolates that produce IgA1 protease % of IgAl proteaseproducing isolates from subject:
Site of sample
Tooth surface (4-h plaque) Buccal mucosa Pharyngeal mucosa
.^
. -i_
i'
A
B
34.7 69.4 9.1
44.6 60.4 6.3
.
?1!__y
