Streptococcus suis Serotype 2 Capsule In Vivo

Many Streptococcus suis isolates from porcine endocarditis in slaughterhouses have lost their capsule and are considered avirulent. However, we retrieved capsule- and virulence-recovered S. suis after in vivo passages of a nonencapsulated strain in mice, suggesting that nonencapsulated S. suis are still potentially hazardous for persons in the swine industry.

S treptococcus suis is a gram-negative bacterium that infects pigs and causes severe economic losses to the swine industry. Moreover, it causes severe disease in persons in close contact with diseased pigs or their products (1). In Japan, S. suis has been frequently isolated from pigs with endocarditis in slaughterhouses; most of the isolates were expected to be sequence types (STs) that are potentially hazardous to humans (2). Many isolates from porcine endocarditis lost their capsule, and all the nonencapsulated isolates analyzed had mutations in the capsular polysaccharide synthesis (cps) genes (3,4). The capsule of S. suis is a major virulence factor (1). Although loss of the capsule gives S. suis some benefit in causing endocarditis by enhancing the ability of bacterial cells to adhere to porcine and human platelets, a major virulence determinant for infective endocarditis (3), nonencapsulated S. suis are generally considered avirulent (5). However, whether nonencapsulated S. suis lurking in porcine endocarditis poses a threat to persons working in the swine industry is unknown. To investigate whether nonencapsulated S. suis can restore the ability to express the capsule and become virulent again, we repeated in vitro or in vivo passages of nonencapsulated S. suis and attempted to retrieve capsule-recovered strains.

The Study
For the in vitro passages, we used 29 S. suis strains isolated from pigs with endocarditis. These isolates had the cps gene cluster of serotype 2 but had lost their capsule because of mutations in the cps genes (Table). We subcultured them twice in liquid media and separated the cells according to the buoyant density by Percoll density gradient centrifugation (online Technical Appendix, http://wwwnc. cdc.gov/EID/article/22/10/15-1640-Techapp1.pdf).
Because encapsulated cells show lower density than nonencapsulated cells (6,7), we investigated capsular expression of S. suis cells with low density by coagglutination tests using serotype 2 antiserum (online Technical Appendix). The retrieved S. suis was also used for the next subcultures. We repeated 4 cycles of this experiment (in total 8 subcultures) but obtained no encapsulated S. suis from any of the strains tested.
Although these results suggested that mutations in cps genes are not repaired easily, the conditions faced by S. suis in vivo could influence capsular expression. To investigate this possibility, we selected strain NL119 as a representative. NL119 is an ST1 strain, one of the types hazardous to humans, but one that has lost the capsule because of a point mutation that occurred at nt 490 (T490C, Cys164Arg) of a glycosyltransferase gene (cps2F) (Table; Figure 1, panel A) (4). We inoculated groups of 5 mice with 5 × 10 8 CFU of NL119 (online Technical Appendix). Bacteria persistent in mice were retrieved 36 h after infection from the blood, in which capsular expression works favorably for survival. We investigated capsular expression of the retrieved NL119 by coagglutination tests and used the colony giving the strongest reaction within 30 s for the subsequent in vivo passage.
As expected, the coagglutination test of the parental strain NL119 showed a negative result. Similarly, NL119 after the first and second passages (NL119 P1 and P2, respectively) reacted weakly, comparable to those of the parental strain, suggesting poor encapsulation. Meanwhile, NL119 after the third and fourth passages (NL119 P3 and P4, respectively) reacted strongly, suggesting recovery of the capsule. To confirm this finding, we further analyzed formalin-killed bacteria by dot-ELISA using monoclonal antibody Z3, which reacts with the sialic acid moiety of the serotype 2 capsule (8), and an anti-S. suis serotype 2 serum adsorbed with parental strain NL119 to select the capsule-specific antibodies (online Technical Appendix). In accordance with the coagglutination test, NL119 P1 and P2 gave weak reactions similar to those of NL119, whereas strong signals were detected in NL119 P3 and P4 with both the monoclonal antibody and serum ( Figure 1, panels B, C). Because NL119 P1-P4 were also ST1 as determined by multilocus sequence typing, these results suggested that NL119 had recovered the capsule during passages in animals. To find mutations that had contributed to the capsule recovery, we sequenced the cps2F gene of NL119 P1-P4. Although the cytosine residue at nt 490 was not changed in comparison with the parental strain, we found a further missense mutation at nt 491 (G491C, Arg164Pro) of the cps2F gene in NL119 P3 and P4 (Figure 1, panel A). To investigate whether this mutation was involved in the capsule recovery, we cloned cps2F of NL119 P4 into a gene expression vector pMX1 (9) and introduced it into the parental strain NL119 (online Technical Appendix). A coagglutination test using serotype 2 antiserum showed positive reactions in all transformants tested, demonstrating that the further missense mutation restored the function of cps2F, resulting in capsule recovery of NL119 P3 and P4, although how the Cps2F function was recovered by the amino acid substitution is unknown. Isolation of the capsule-recovered strains in vivo could have been the consequence of selection of encapsulated cells, which were already present as a subpopulation in the original nonencapsulated NL119 population, by resisting host immunity including phagocytosis. However, because NL119 was a nonencapsulated strain originally recovered from a single colony and well-isolated by repeated passages in vitro, and no encapsulated subpopulation was ever retrieved in vitro by the selection using Percoll density gradient centrifugation, the most plausible hypothesis would be that the capsule-recovered S. suis was generated in vivo.

Conclusions
Although capsule loss might contribute to S. suis infection by enhancing bacterial adherence to host cells and biofilm formation (3,(10)(11)(12), capsule loss makes S. suis cells susceptible to phagocytosis; therefore, the virulence of nonencapsulated mutants was attenuated when evaluated in animal models (5). In accordance with previous studies, nonencapsulated NL119 was avirulent. However, NL119 P4, which recovered its capsule in vivo, also recovered virulence. Because various mutations in cps genes, including large deletions and insertions, cause capsule loss in S. suis (3,4), not all mutations will be repaired like NL119. However, our results demonstrated the presence of a nonencapsulated mutant, which can recover the capsule and virulence in vivo. Hence, nonencapsulated S. suis strains can cause severe diseases to the next hosts by recovering the capsule, which indicates that some nonencapsulated S. suis lurking in pigs with endocarditis are still potentially hazardous to   (4). B, C) NL119 P1 and P2 retrieved from mice after the first and second in vivo passages, remained nonencapsulated, and their cps2F sequences were identical to that of NL119. In NL119 P3 and P4 retrieved after the third and fourth passages a further missense mutation at nt 491 (G491C, Arg164Pro) of cps2F restored the function of the gene, resulting in capsule recovery of the strains. Dot-ELISA of NL119 and strains retrieved from NL119-infected mice after each in vivo passage (NL119 P1-P4) using monoclonal antibody Z3 (B) and polyclonal anti-S. suis serotype 2 serum adsorbed with NL119 (C). Monoclonal antibody Z3 specifically recognizes the sialic acid moiety of the S. suis serotype 2 capsule. persons handling such pigs and their products. Further investigations using a variety of naturally occurring and laboratory-derived mutants are needed for a comprehensive understanding of the biological significance and mechanisms of this phenomenon.