Identification of novel immunogenic proteins against Streptococcus parauberis in a zebrafish model by reverse vaccinology
Introduction
Streptococcus parauberis and S. iniae are pathogenic bacteria that cause streptococcosis in the olive flounder (Paralichthys olivaceus), characterised by dark colouration, nocturnal haemorrhage, abnormal swimming, eating disorder, and death due to cachexia [1]. The mortality rate is above 50%, resulting in severe economic losses [[2], [3], [4]]. The S. iniae infection rate is 43% in the growth season (summer), and that of S. parauberis is 57% in the harvest season (autumn) in Korea [5].
In Korea, S. parauberis, a gram-positive spherical bacterium, is classified as serotype I or II based on the capsule layer thickness, which is related to pathogenicity [6]. Formalin-killed cell vaccines, feed-based adjuvant vaccines, and antibiotics are used to prevent S. parauberis infection [[7], [8], [9], [10]]. S. parauberis-derived protein-based subunit vaccines have not been reported, although recombinant enolase from S. iniae was tested in a zebrafish model for protection against S. iniae and S. parauberis [11].
Reverse vaccinology approach, first described by Rino Rappuoli [12], predict the potential antigen candidates from the whole genome sequence of the pathogen. Selected candidate proteins can further be identified through in vitro and in vivo experiments. The reverse vaccinology was first used against Serogroup B meningococcus [13] and has been used in the development of many other vaccines [14,15]. The genome of S. parauberis KCTC11537 (GenBank No. CP002471), isolated from Jeju Island, South Korea, includes a single circular chromosome containing 1829 predicted coding sequences (CDS) [16]. The S. parauberis NCFD2020 genome (GenBank No. AEUT02000001), isolated in the USA, includes a single circular chromosome containing 2056 CDS. For S. parauberis KCTC11537, two-dimension electrophoresis (2-DE) has been used to screen immunogenic proteins [16].
In this study, vaccine candidates against S. parauberis were screened by reverse vaccinology. The selected candidates were cloned and expressed in Escherichia coli, and the expressed proteins were tested for immunogenicity. We used streptococcal challenges to evaluate the protective efficacies of the candidate immunogenic proteins as subunit vaccines against streptococcosis in a zebrafish model.
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Bacterial strains and culture conditions
E. coli BL21 (DE3) [F− ompT gal dcm lon hsdSB (rB− mB-) λ (DE3)] were used as host strains for cloning, maintenance of plasmids, and protein expression. S. parauberis P2 (serotype I) and S. parauberis YSFST02-111 (serotype II) were isolated from an olive flounder infected with streptococcosis in Jeju, Republic of Korea. Both S. parauberis strains were cultured on trypticase soy broth with 5% defibrinated sheep blood at 37 °C to mid-log exponential phase for use in the challenge test.
Selection of vaccine candidates by in silico analysis
The genome
Results and discussion
Reverse vaccinology was used to identify novel immunogenic proteins against S. parauberis (Fig. 1). Extracellular or cell surface-localized proteins as vaccine candidates were screened from S. parauberis KCTC 11537 and NCFD 2020 genomes. We also considered transmembrane helix number (≤1) and previously reported immunogenic proteins [16]. In total, 8 extracellular (EC), 7 cell wall (CW), and 18 cytoplasmic membrane (CM) proteins were selected from 1868 annotated proteins of KCTC 11537 and 5 EC,
Conflicts of interest
The authors declare that they have no competing interests.
Acknowledgements
This research was supported by a grant from the KRIBB Research Initiative Program.
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