Article Figures & Data
Figures
- Fig. 1.
Structural organization of emm genes. (A) Location of the high-affinity plasminogen-binding site and corresponding nucleotide coding sequence within the Emm53 (PAM or M53) protein. Schematic organization of the Emm53 protein: S, signal sequence; C, conserved (C repeat region) repeats; W, cell wall (peptidoglycan)-spanning domain; M, membrane anchor. The a repeats (a1 and a2) are 13 amino acids in length, and each a repeat binds plasminogen with high affinity. The nucleotide sequence of the a2 repeat is given. An oligonucleotide (PAM-F) corresponding to the underlined sequence or its complimentary sequence (PAM-R) was paired with a SF-specific primer for PCR-based genotype mapping. (B) Arrangement of emm SF genes and position of SF-specific oligonucleotide hybridization sites. The emm andemm-like genes are represented by four emm gene SF forms that are based on nucleotide sequence differences in the 3′-end portion encoding for the peptidoglycan-spanning domain (8). Most GAS strains have one of the five emmpatterns (designated A through E), which are defined by the number ofemm genes, their SF content, and their relative arrangement on the chromosome. For strains with multiple emm oremm-like genes, each gene differs at its 5′ end, and genes are usually separated from one another by 0.2 to 0.3 kb. The centrally positioned gene is used for emm sequence typing (3). Alternative nomenclature for emm andemm-like genes is indicated (bottom). Arrows depict the hybridization sites for oligonucleotide primers. The emm53gene, depicted in panel A, represents the central emm gene (SF1) of an emm pattern D strain.
- Fig. 2.
Binding of Glu plasminogen to select strains and recombinant Emm proteins. (A) Inset, top: Human plasma (0.5 ml) was mixed with approximately 1010 CFUs of GAS (lane 1, strain AP53; lane 2, strain AP52), cells were washed, and bound proteins were eluted with 0.1 M glycine-HCl (pH 2.0). Blots of eluate were incubated with MAb directed to Glu plasminogen. Purified human plasminogen was analyzed by SDS-PAGE and Western blotting. The gel was stained with Coomassie brilliant blue (lane 3), and the blot was incubated with a MAb directed to the NH2-terminal peptide specific for Glu plasminogen (lane 4). Purified, 125I-labeled human plasminogen was analyzed by autoradiography (lane 5). Purified125I-labeled human plasminogen (0.25 ng) was mixed with approximately 1010 CFUs of strain AP53 in 0.5 ml of PBS containing 2% bovine serum albumin. Following incubation, cells were washed extensively, and bound material was eluted with glycine and subjected to autoradiography (lane 6). (A) Inset, middle: A schematic representation of plasminogen. The five kringle domains and the activation cleavage sites for tissue-type plasminogen activator and urokinase in the serine protease domain (open arrow) are indicated. Also depicted is the cleavage site for plasmin, resulting in a 77-amino-acid residue NH2-terminal peptide and conversion of plasminogen from the Glu to the Lys form (closed arrow). (A) Bottom: Bacteria-bound 125I-labeled plasminogen was measured by an adsorption assay, except that 0.1 ng per 0.25 ml of125I-labeled plasminogen was incubated with GAS present over a wide range of concentrations, expressed as CFU/ml. Shown are strain 1RP144 (M5, emm pattern A–C; ▴) and emmpattern D strains D617 (■) and AP53 (●) (Table 2). (B) Inset, top: Recombinant emm gene products (rM5, derived from M5 Manfredo, emm pattern A–C, lanes 1 and 3; rEmm52, derived from AP52, lanes 2 and 4) were separated by SDS-PAGE and stained with Coomassie blue (lanes 1 and 2); a replica of the gel was blotted and incubated with 125I-labeled human plasminogen (lanes 3 and 4). (B) Bottom: Purified rM5 (▴) and rEmm52 (■) proteins were immobilized in microtiter plate wells and incubated with125I-labeled plasminogen. The amount of radio-iodinated material bound to various concentrations of recombinant proteins was measured in a γ counter.
- Fig. 3.
Binding of human plasminogen by intact GAS. Each symbol represents a different GAS isolate (n = 83) that is defined according to its emm pattern. Intact GAS were measured for the percentage of 125I-labeled human plasminogen bound in an adsorption assay. Approximately 2.0 × 109 CFUs were incubated with 125I-labeled plasminogen (∼10,000 cpm, corresponding to ∼1 ng of plasminogen) in a total reaction volume of 0.25 ml. The values for the percentage of plasminogen bound are rounded to the nearest 5% interval. Also, shown is the PAM genotype for each isolate, as determined by PCR amplification of GAS-derived DNA using PAM-specific oligonucleotide primers.
Tables
- Table 1.
Epidemiologic features of group A streptococcal strains under study
emm pattern No. of strains represented No. of unique M or emm types No. (%) isolated from: Nasopharynx Impetigo Unknown site A, B, or C 35 15 32 (91) 2 (6) 1 (3) D 28 17 5 (18) 19 (68) 4 (14) E 20 13 10 (50) 10 (50) 0 (0) - Table 2.
Properties associated with plasminogen-binding activity among emm pattern D strains
Strain emmtypea Tissue site of isolationb Isolation date Placeb PCR product (PAM primers) % of bound 125I-labeled plasminogen 13RS60 33 NP 1941 New York + ≥50 A910 33 NP 1966 ND + ≥50 A982 33 Imp 1968 New York + ≥50 3732-S 33 Imp 1969 Alabama + ≥50 6010-S 33 Imp 1971 Alabama + ≥50 D735 33 Imp 1973 New York + ≥50 29487 33 Imp 1987 Czech Republic + ≥50 AP52c 52 Imp 1967 Minnesota + ≥50 D617 52 Imp 1972 Trinidad + ≥50 AP53c 53 Imp 1967 Minnesota + ≥50 ALAB49 53 Imp 1986 Alabama + ≥50 29689 pt2110 Imp 1988 Czech Republic + ≥50 ALAB55 pt2110 Imp 1986 Alabama + 25 D964 pt2631 Imp 1976 Trinidad + ≥50 D821 pt5757 Imp 1975 Trinidad + ≥50 29486 st2370 Imp 1987 Czech Republic + ≥50 D432 stD432 ND 1971 Egypt + ≥50 D631 stD631 Imp 1972 Trinidad + ≥50 D633 stD633 Imp 1972 Trinidad + ≥50 D680 stD633 NP 1972 Trinidad + ≥50 D641 stNS5 Imp 1972 Trinidad + ≥50 D466 potter41 ND 1971 Egypt + 25 D502 potter41 Imp 1971 Trinidad + 10 D998 70 ND 1976 Japan + 20 10RS101 32 NP 1942 New York − 15 1RS79 42 NP 1941 New York − 10 A457 36 ND 1961 Former Yugoslavia − ≤5 D626 st88/31 Imp 1972 Trinidad − ≤5 ↵a For several isolates, previous reports (5, 8) indicated M serological types that are discordant with the emm sequence types presented here.
↵b Imp, impetigo; NP, nasopharyngeal site; ND, not determined.
↵c AP52 and AP53 are also designated CV686 and CV265, respectively (31). AP53 is the strain from which Emm53 (PAM) was originally derived (4).
