Distribution and genetic diversity of suilysin in Streptococcus suis isolated from different diseases of pigs and characterization of the genetic basis of suilysin absence

Abstract

Streptococcus suis is an economically important pathogen of pigs responsible for a variety of diseases including meningitis, septicemia, arthritis, and pneumonia, although little is known about the mechanisms of pathogenesis or virulence factors associated with this organism. Here, we report on the distribution and genetic diversity of the putative virulence factor suilysin, a member of the thiol-activated toxin family of gram-positive bacteria. On the basis of PCR analysis of over 300 isolates of S. suis, the suilysin-encoding gene, sly, was detected in 69.4% of isolates. However, sly was present in a considerably higher proportion of isolates obtained from cases of meningitis, septicemia, and arthritis (>80%) and isolates obtained from asymptomatic tonsillar carriage (>90%) than lung isolates associated with pneumonia (44%). With the exception of serotypes 1, 14, and 1/14, there was no strong correlation between the presence of suilysin and serotype. Analysis of the genetic diversity of suilysin by restriction fragment length polymorphism and sequence analysis found that the suilysin gene, where present, is highly conserved with a maximum of 1.79% diversity at the nucleotide level seen between sly alleles. Assays of hemolytic activity and hybridization analysis provided no evidence for a second member of the thiol-activated toxin family in S. suis. Inverse PCR was used to characterize regions flanking sly, which in turn allowed the first characterization of the equivalent region in a strain lacking sly. Sequence comparison of these regions from sly-positive (P1/7) and sly-negative (DH5) strains indicated that two alternative arrangements are both flanked by genes with highest similarity to haloacid dehalogenase-like hydrolases (5' end) and putative N-acetylmannosamine-6-phosphate epimerases (3' end). However, sly appears to be completely absent from the alternative arrangement, and a gene of unknown function is located in the equivalent position. Finally, PCR analysis of multiple sly-positive and -negative strains indicated that these two alternative genetic arrangements are conserved among many S. suis isolates.

FIG. 1

Alignments of the variable sites identified by sequencing sly PCR products from a representative strain of each distinct profile identified by RFLP (left) and the published sequences from isolates P1/7 and 1933 (33, 38). Panel A illustrates the nucleotide diversity of the 16 alleles identified, while panel B illustrates alterations within the predicted amino acid sequence. Identical sites are represented by periods (.) and numbering above the residues corresponds to their position within the sequence relative to P1/7.

FIG. 2

Schematic representation of the sly genomic region and corresponding sequence in strains lacking the gene. The small arrows represent the approximate location of primers used in mapping this region. (A) Diagrammatic representation of the region containing sly and the corresponding region in strains lacking the gene. (i) The arrangement of the region in sly-positive isolates, as determined by the amalgamation of our sequencing and the published sequence (P1/7), is shown at the top. (ii) The sequence determined from a sly-negative S. suis isolate (DH5) is illustrated. The white blocks represent regions of essentially identical sequence while the grey and black areas indicate regions specific to sly-negative and sly-positive strains, respectively. (B) Sequence of the boundaries between regions of sequence conserved between the sly-positive strain P1/7 and sly-negative strain DH5 and those containing diverged sequences. The 3′ region of orfB (i) and the 5′ region of orfD (ii) are shown. In each case, the top line represents the predicted amino acid sequence of P1/7, while the lower amino acid sequence refers to DH5. The top line of the nucleotide sequence refers to P1/7, and the lower line refers to DH5. Identical residues are represented by periods (.) and gaps in the sequence are represented by dashes (-).

FIG. 3

Dot blot analyses to determine to presence of sly and orfC in the 32 S. suis isolates described in Table 4. (A) Isolate numbers with allele designations (based on RFLP analysis) shown in parentheses. The boxed isolates represent those determined by PCR to be sly negative. (B) 5′ sly probe; (C) 3′ sly probe; (D) orfC probe.