Streptococcus agalactiae 1a capsule (cpsE) and hemolysin (cylE) deletion mutants display attenuated virulence in Nile tilapia (Oreochromis niloticus).

Abstract

Streptococcus agalactiae (S. agalactiae), also known as Group B Streptococcus (GBS), is an increasingly concerning pathogen affecting humans and animals, including aquatic species. This bacterium significantly impacts aquaculture, causing disease outbreaks in various freshwater and marine fish, particularly in warm-water environments. This study examined the biological impact of two major virulence factors of S. agalactiae 1a and their roles in host pathogenesis. We attenuated a strain of S. agalactiae 1a (NAM) through targeted deletions of selected virulence genes, cpsE and cylE, involved in capsule formation and hemolysin production, respectively. In vitro experiments showed that the ΔcpsE strain lost antibody agglutination properties, whereas the ΔcylE strain lost the ability to lyse red blood cells. In vivo studies in Nile tilapia revealed reduced virulence of these mutant strains, with the double mutant (ΔcylE/ΔcpsE) showing the highest degree of attenuation, followed by the single capsule-deficient (ΔcpsE) and hemolysin-deficient (ΔcylE) strains. The brain appeared to be the primary site of bacterial establishment in fish challenged with viable bacteria. Unlike the wild-type strain, the capsule appeared to be a key component in localization, as capsule-deficient bacteria did not colonize the brain or spleen 10 days post-challenge. Deleting the cpsE gene completely prevented capsule formation, whereas deleting the cylE gene inhibited the hemolysin synthesis. The capsule proved to be a more potent virulence factor than hemolysin, with an additive attenuation effect in the double-mutant strain. These findings suggest that inhibiting hemolysin synthesis and capsule formation by gene deletion attenuates S. agalactiae 1a, reducing its virulence, and provide a basis for the development of a live-attenuated vaccine against the corresponding infection.