Eight Novel Capsular Polysaccharide Synthesis Gene Loci Identified in Nontypeable Streptococcus suis Isolates

Abstract

cStreptococcus suis is an important pathogen of pigs and may cause serious disease in humans. Serotyping is one of the important diagnostic tools and is used for the epidemiological study of S. suis. Nontypeable S. suis strains have been reported in many studies; however, the capsular polysaccharide (CPS) synthesis cps loci of nontypeable strains have not been analyzed. In this study, we investigated the genetic characteristics of cps loci in 78 nontypeable strains isolated from healthy pigs. Eight novel cps loci (NCLs) were found, and all of them were located between the orfZ-orfX region and the glf gene. All NCLs possess the wzy and wzx genes, strongly suggesting that the CPSs of these NCLs were synthesized using the Wzx/Wzy-dependent pathway. The cps genes found in the 78 isolates were assigned to 96 homology groups (HGs), 55 of which were NCL specific. The encapsulation of the 78 isolates was also examined using transmission electron microscopy. Fifty-three isolates were found to have a capsule, and these were of varied thicknesses. Our data enhance our understanding of the cps gene cluster diversity of nontypeable S. suis strains and provide insight into the evolution of the S. suis capsular genes. Streptococcus suis is an important pathogen of pigs (1) and may cause serious disease in humans (2-4). Serotyping is one of the important tools for diagnosis and is used in epidemiological studies of S. suis. A total of 35 S. suis serotypes (serotypes 1 through 34 and 1/2) are known on the basis of the antigenic differences in their capsular polysaccharides (CPSs) (5-8) and the coagglutination test. The S. suis cps gene clusters of the 35 serotypes have been sequenced, and the cps gene clusters were shown to be diverse among different serotypes (9). The predicted products of the cps genes found in the 35 serotypes were assigned to 291 homology groups (HGs). The precise function of many cps genes is still unknown.The synthesis and export of bacterial polysaccharides are mediated by three pathways known as the Wzx/Wzy-dependent pathway, the synthase-dependent pathway, and the ABC transporter-dependent pathway. The Wzx/Wzy-dependent pathway is most commonly used in Streptococcus pneumoniae capsular biosynthesis (10). Although the precise function of most of the cps genes of S. suis is still unknown, the CPSs of all serotypes of S. suis are also thought to be synthesized by the Wzx/Wzy-dependent pathway. This pathway involves the synthesis of the polysaccharide repeat units, which are initially built on the inner face of the cytoplasmic membrane; transport of the repeat units to the outer face of the membrane by Wzx flippase; and then polymerization of the repeat units by Wzy polymerase. Wzy-dependent polymers usually contain various sugars and glycosidic linkages. The specificity of the Wzy polymerase determines the linkage that it catalyzes between sugars on the growing chain and the next repeat unit. Therefore, the wzy gene is serotype specific. Hence, the serotype-specific wzy gene is an ideal target to discrimin...

Novel Capsular Polysaccharide Loci and New Diagnostic Tools for High-Throughput Capsular Gene Typing in Streptococcus suis

“…Capsular gene typing methods developed in our laboratory were used to assign cps loci of 486 isolates (13,16,17,20). Known subtypes of NCL1, NCL2, NCL3, NCL7, and NCL8 were determined based on the variable presence of 13 genes and 4 transposase genes found in our previous study by PCR amplification (16) (Tables 1 and 2).…”

“…Each cps locus sequence was extracted from the draft genome sequence, and open reading frames (ORFs) were identified and annotated based on methods reported previously (22). cps loci were analyzed by using the same bioinformatics methods as those described in previous studies (16,22). The cps genes were assigned to the 361 known homology groups (HGs) of 35 serotype reference strains (22) and NCL1 to -8 (16) if the length of the match was Ͼ50% of the sequence and the identity was also Ͼ50%.…”

“…The presence or absence of the capsule was examined by electron microscopy using the same method as the one described in our previous study (16).…”

“…All evidence from genetic analyses and phenotypes strongly supported that they represented novel serotypes of S. suis. Based on the variable presence of 13 genes and 4 transposase genes, multiple subtypes of NCL1, -2, -3, -7, and -8 were found (16). Those studies showed the high level of diversity within the same NCL and underscore the importance of investigating the subtypes of different NCLs.…”

“…Recently, 9 novel cps loci (NCLs) were identified in nonserotypeable isolates and were designated NCL1 to -8 (16) and Chz (17). These loci possessed the specific polysaccharide polymerase gene wzy, the flippase gene wzx, and glycosyltransferase (GT) and acetyltransferase genes, which were obviously different from those of reference serotypes.…”