Association of the prtF1 Gene (Encoding Fibronectin-Binding Protein F1) and the sic Gene (Encoding the Streptococcal Inhibitor of Complement) with emm Types of Group A Streptococci Isolated from Japanese Children with Pharyngitis

Abstract

A total of 66 clinical isolates of group A streptococci (GAS) were obtained from 66 Japanese children with pharyngitis. The prtF1 gene (encoding fibronectin-binding protein F1) and the sic gene (encoding the streptococcal inhibitor of complement) were present in 51 (77.3%) and 48 (72.7%) of the 66 isolates, respectively. These results indicated that a high prevalence of two virulence genes, prtF1 and sic, is characteristic of GAS in Japan.

Group A streptococci (GAS; also referred to as Streptococcus pyogenes) are major etiological agents causing a wide variety of human diseases, ranging from pharyngitis to severe invasive diseases (3). The N-terminal region of the M protein is located distally to the surface of the bacterium and displays antigenic diversity. This heterogeneity provides the basis for identifying more than 100 different GAS serotypes (6). However, many GAS isolates are often nontypeable because of the lack of appropriate type-specific antisera or because of a loss of antigen expression under cultivation conditions. T typing has also been used as an alternative or supplement to M typing. The T antigen is a trypsin-resistant cell surface protein that exhibits extensive antigenic diversity but is not defined as a virulence factor (3). Recently, a genomic typing method based on the variable 5′ emm gene has become widely used, and more than 150 different types of the emm gene, encoding the N-terminal region of the M protein, have been characterized (13) (http://www.cdc.gov/ncidod/biotech/strep/strains.html). This methodology has enabled the recognition of several previously unknown GAS M types in different geographic areas. Additionally, emm typing has been shown to be useful for detecting genetic diversity among GAS isolates.

GAS cell surface proteins have been identified as virulence factors. More than 40 putative virulence-associated genes have also been identified (5). Some of these GAS cell surface proteins, such as fibronectin-binding proteins, function as adhesins. Adherence of GAS to pharyngeal or dermal epithelial cells is the first step in colonization of the host and development of infectious disease. Other GAS cell surface proteins, such as the streptococcal inhibitor of complement, are protective against the host defense system or trigger a severe nonspecific immunological response in the human host (3). The M protein, an important GAS virulence factor, has a dual role: it is a major antiphagocytic factor of GAS, and it is also an adhesin that binds fibronectin, fibrinogen, and albumin. Fibronectin-binding protein F1, encoded by the prtF1 gene, is one of the adhesins; it has been shown to be important for binding of the bacterium to the extracellular matrix of respiratory epithelial cells and for promoting entry into these cells (8). Intracellular localization of GAS has been suggested as an explanation for the failure of penicillin treatment of GAS infections (4). However, the relationship between the presence of the prtF1 gene and the failure of eradication is still controversial (2, 4, 11). The protein encoded by the sic gene is expressed extracellularly and inhibits in vitro the normal cytolytic function of the complement C5b-C9 membrane attack complex, which is responsible for target killing (1). The presence of the sic gene in GAS strains of 55 different M serotypes has been investigated; the genes have been reported to be present in M1- and M57-type strains but absent from other GAS strains (1). The purpose of this study was to determine the phenotypic (M and T types and azithromycin [AZM] sensitivity) and genotypic (emm type and the presence or absence of the prtF1 and sic genes) characteristics of 66 GAS isolates from 66 pediatric patients (2 to 12 years old) with pharyngitis treated at eight hospitals in Hokkaido, the northernmost island of Japan, during the period from August 2000 to March 2001.

All 66 isolates were examined for M and T types by standard M protein precipitation in gel agar and by the T agglutination method, respectively (9). M-type-specific antisera were available for types 1, 2, 3, 4, 5, 6, 8, 9, 11, 12, 13, 14, 18, 19, 22, 23, 27, 28, 29, 39, 41, 49, 53, 56, and Matsuyama 2166 (a provisional type). The variable 5′ emm type was examined by sequence analysis of emm gene-specific PCR products (13). The primer sequences used were 5′-TATT(C/G)GCTTAGAAAATTAA-3′ and 5′-GCAAGTTCTTCAGCTTGTTT-3′. The PCR-amplified products were sequenced directly by using a BigDye dye terminator cycle sequencing ready-reaction kit (Perkin-Elmer Applied Biosystems, Tokyo, Japan) with an ABI PRISM 310 genetic analyzer (Perkin-Elmer Applied Biosystems). Primer seq2, 5′-TATTCGCTTAGAAAATTAAAAACAGG-3′, was used for sequencing. Nucleotide sequences of at least 160 bases were subjected to homology searches in the GenBank (http://www.ncbi.nlm.nih.gov/BLAST) and the Centers for Disease Control and Prevention (http://www.cdc.gov/ncidod/biotech/infotech_hp.html) databases. Table 1 summarizes the association of emm type with T type, M type, and the presence or absence of the prtF1 and sic genes. The 66 isolates were of 10 T types and 6 M types, including one provisional T B3264 type and one provisional M Matsuyama 2166 type. Although all of the isolates were T typeable, six exhibited complex forms of T antigens. T types of most GAS isolates were associated with specific M and emm types. Commonly isolated strains among the 66 isolates were M4 and M12 types. Ten emm types were found among the 66 isolates. The prevalent emm types were emm12 (18 isolates) and emm4 (16 isolates). No new emm type was detected. M Matsuyama 2166-type GAS has been found in Japan, Thailand, and Malaysia, but the emm type(s) and its worldwide prevalence are not known (9). The M Matsuyama 2166-type strain was type T2 and type emm75. The T B3264 type was correlated with either emm89 or emm94. The MIC of AZM was determined by the agar dilution method according to the National Committee for Clinical Laboratory Standard guidelines. AZM-resistant strains were defined as those for which the MIC of AZM was more than 2 μg/ml. Only 3 (4.5%) of the 66 isolates were resistant to AZM.

TABLE 1.

T Types, M types, and emm types of the isolates, and presence of sic and prtF1 genes

emm typea	M/T typea,b	No. of strains with:
		sic present	sic absent	prtF1 present						prtF1 absent
				Total	With RD2 repeat no. of:
					1	2	3	4	6
1 (1)	1/1 (1)	1								1
2 (4)	2-28/2-28 (4)	4								4
4 (17)	4/4 (16)	14	2	16	11	5
	4/4-28 (1)	1		1	1
12 (18)	12/12 (18)	13	5	18				18
28 (5)	28/28 (4)	1	3	4		4
	nt/28 (1)	1		1		1
75 (4)	Mastuyama 2166/2 (3)	2	1	3			3
	nt/6 (1)	1		1					1
77 (2)	nt/28 (2)		2	2			2
89 (4)	nt/B3265 (4)	2	2	4				4
94 (10)	nt/B3264 (10)	7	3							10
112 (1)	nt/25-5-4 (1)	1		1	1

^aNumbers in parentheses are numbers of isolates.

^bnt, nontypeable strain.

The presence of two virulence genes, prtF1 and sic, was determined by PCR. The primer sequences used to amplify the prtF1 gene and sequence the PCR-amplified products were 5′-TTTTCAGGAAATATGGTTGAGACA-3′ and 5′-TCGCCGTTTCACTGAAACCACTCA-3′. PCR-amplified products were subjected to electrophoresis through a 2.0% agarose gel. The prtF1 gene was present in 51 (77.3%) of the 66 isolates, which were of various emm types, a somewhat higher percentage than previously reported for that gene (2, 7, 10). The prtF1 gene was not detected in emm94-, emm2-, and emm1-type isolates. The association between the presence of the prtF1 gene and the emm type of the isolates in this study is in agreement with results of previous studies of the distribution of the prtF1 gene in GAS isolates of various emm types (2, 10). Gel electrophoresis revealed that the PCR-amplified products were of five different molecular sizes, ranging from approximately 100 to 750 bp. Repeat domain type 2 (RD2) is composed of 37 amino acid residues repeated fully four times and partially a fifth time (12). PCR-amplified products of 126, 237, 348, 459, and 745 bp corresponded to RD2 repeat numbers of 1, 2, 3, 4, and 6, respectively. The number of RD2 repeats was confirmed by direct sequencing of several representative PCR-amplified products and was constant among individual emm-type isolates except for those of type emm4 or emm75. The primer sequences used to amplify the sic gene and sequence the PCR-amplified products were 5′-GATGAGACAGAAGATAAAC-3′ and 5′-TTACGTTGCTGATGGTGTAT-3′. The presence of the sic gene was confirmed by Southern blot hybridization and direct sequencing of two representative PCR-amplified products. The sic gene was detected in GAS of various emm types, and 48 (72.7%) of the 66 isolates were sic gene positive.

In the present study, we have determined the phenotypic and genotypic characteristics of GAS isolates from patients with pharyngitis in Japan. Unexpectedly, the majority of GAS isolates possessed two virulence genes, the prtF1 and sic genes. The high prevalence of these genes may be due to horizontal gene transfer and recombination. The results of this study should contribute to a better understanding of the pathogenesis of GAS as well as the epidemiology of GAS-associated disease and to the establishment of methods for prevention of diseases caused by GAS in Japan.