Genomic and proteomic profiling of Streptococcus agalactiae serotypes Ia, Ib and III to reveal novel surface antigens as cross protective vaccine candidates

Summary

Aquaculture is the fastest-growing animal food production sector globally having a key role in food security. Tilapia (Oreochromis sp.) is the second most predominant farmed fish species globally and a major source of income to middle-income countries (LMIC). Intensification of tilapia farming has promoted severe disease outbreaks, resulting in high mortalities and economic with Streptococcus agalactiae being a major pathogen in tilapia culture. Three serotypes of the bacterium, i.e.,Ia, Ib and III, exist. Although commercial S.agalactiae vaccines exist, they do not cross-protect between serotypes, making vaccine development a challenge. This project brings together scientists from India, Norway and UK, to develop a vaccine that will cross-protective against different S.agalactiae serotypes found in tilapia farming systems in Southeast Asia. Conserved surface proteins are among the most promising candidates for the development of new and effective vaccines. Herein, we propose to target the major S.agalactiae serotypes (1a, 1b and III)affecting tilapia by “shaving” the live bacterial cells with trypsin, followed by LC-MS/MS analysis isolate, to identify common and unique proteins to use to develop a universal vaccine against the bacterium in future studies. We also plan to use whole genome sequencing (WGS), followed by pangenome analysis, to identify common immune genes shared by all three S. agalactiae serotypes for vaccine design. The information gathered from this project is paramount for future vaccine-related studies to ultimately lead to the development of more effective strategies to develop a universal streptococcus vaccine for tilapia aquaculture.

Project Outcomes:

Animal husbandry in LMICs is threatened by the increase in anti- microbial resistance and, if not properly managed, would drive increases in zoonotic infections. Streptococcosis is one of the major bacterial diseases resulting in severe economic losses for tilapia farmers.  There is no commercial vaccine against Streptococcus infection to date and so farmers resort to using antibiotics to control this disease. Although commercial S. agalactiae vaccines exist, they do not cross-protect between different serotypes rendering current vaccines less effective overall. This provides the impetus for continued vaccine development by encompassing all major serotypes.. Conserved surface proteins are among the most promising candidates for the development of new and effective vaccines. The present study focused on the major S. agalactiae serotypes (1a, 1b and III) affecting tilapia aquaculture and to develop a vaccine that will cross-protective against different S. agalactiae serotypes found in tilapia farming systems in Southeast Asia. The present study aims to: i) To identify common and unique proteins to develop a universal vaccine against the bacterium by “shaving” live bacterial cells with trypsin followed by LC-MS/MS analysis ii) To identify common surface antigens shared by all three S. agalactiae serotypes for vaccine design and iii) To identify immunogenic (and potentially protective) vaccine candidates by immunoproteomics that are common to all three serotypes (Ia, Ib and III) using archived serum from infected and convalescent fish (i.e. fish that have survived an infection).

The present study identified four identical proteins in the 3 serotypes of S.agalactiae by 2D gel electrophoresis followed by LC-MS/MS analysis. Briefly, bacterial surface proteins from S. agalactiae 1a, 1b and III were prepared, quantified and subjected 2D gel electrophoresis followed by Coomassie staining. The identical spots corresponding to protein were excised and digested with trypsin for the LC-MS/MS analysis. The identical proteins corresponding to the spots in the gels were identified by Q-Exactive Plus / UltiMate 3000 RSLCnano system (Thermo Fisher Scientific, Hemel Hempstead, UK). Indirect ELISA assays were performed to assess specific antibody responses in serum from fish challenged with each serotype using protein extracts from each serotype as bait.  Archived serum from tilapia infected with each serotype was used for immunoproteomic studies to identify immunogenic vaccine candidates common to all three serotypes. Pan-genome analysis was also undertaken to identify virulence genes and proteins common to all three serotypes (Ia, Ib, and III).

Our findings will provide new insights for developing vaccines based on representative antigens common to all three S. agalactiae serotypes increasing the potential to develop a cross protective vaccine against S. agalactiae in farmed tilapia and other fish species. Methods described in this study can be used in subsequent studies as a general approach for identifying common surface antigens in other fish bacterial pathogens where cross protection across a range of serotypes is required.