Comparative analysis of pathogenicity factors of closely related Mitis group streptococci and surface protein-containing antigens isolated from them

Relevance. Mitis group streptococci (SMG) include 9 species of streptococci most frequently isolated from patients, including Streptococcus oralis and Streptococcus pneumoniae. The study of SMG shows not only differences but also overlaps in the genes encoding pathogenicity factors of S. pneumoniae and S. oralis. Earlier it was shown that antigens from S. pneumoniae serotype 6B induced cross-protective effect against virulent strains of S. pneumoniae. It was found that the experimental surface protein-containing fraction with molecular weight 30-100 kDa (PCF) from S. oralis has protective activity, stimulates in mice indicators of innate and adaptive immunity.
Objective: to carry out a comparative analysis of pathogenicity factors of S. oralis and S. pneumoniae strains and protective activity of surface protein-containing antigens isolated from them.
Materials and methods. The strains of S. oralis and S. pneumoniae serotype 6B Nos. 3353 and 1121 of different virulence were used. Experimental protein-containing antigens, in particular, PCF, were prepared from these strains. The protective activity of the obtained PCFs was evaluated in active protection experiments during intraperitoneal immunization and infection of BALB/c mice. Mass spectrometric study of PCF was carried out using UniProtKB database, taxon S. pneumoniae serotype 4 (strain ATCC BAA-334 / TIGR4.) Statistical processing of the results was carried out using parametric and nonparametric methods of comparison (p<0.05).
Results and discussion. PCF from S. oralis has cross-species protective activity against virulent strains of S. pneumoniae of different serotypes, while PCF from S. pneumoniae strain 6B No. 3353 protects only against a virulent strain of the homologous serotype. Comparative analysis of genes encoding pathogenicity factors revealed differences: only in S. oralis genes encoding the synthesis of proteins promoting adhesion and biofilm formation (SspA, GtfD RlrA), and in S. pneumoniae strains - pore-forming toxin (Ply) and choline-binding protein (LytA) were identified. In the proteomic analysis of S. oralis and S. pneumoniae 6B No. 3353 PCFs, out of 673 identified proteins, 57 common proteins of most interest and 21 and 35 unique proteins, respectively, were selected for each PCF: in S. oralis PCFs, adhesion proteins were identified, as well as proteins related to metabolic systems and mediating the development of the infection process, while in S. pneumoniae PCFs, adhesion proteins were predominant those indirectly involved in the development of the infectious process, while the S. pneumoniae PCF is dominated by proteins related to the main pathogenicity factors.
Conclusions. The surface protein-containing fraction 30 -100 kDa (PCF) isolated from a weakly virulent strain of S. oralis, belonging to the Mitis group streptococci, has interspecies protective activity against strains of closely related species - S. pneumoniae of different serotypes. PCF is capable of both preventing adhesion and colonization of pneumococcus and possibly playing the role of a natural adjuvant. In addition, it may be of interest to determine the protective effect of this antigen against other closely related SMG representatives. Both different and common genes encoding the synthesis of the main pneumococcal pathogenicity factors were identified in the strains, and unique proteins for each of them were determined by proteomic analysis of PCF.

1. Pichichero ME. Pneumococcal whole-cell and protein –based vaccines: Changing the paradigm. Expert. Rev. Vaccines. 2017;6(12):1181–1190. doi: 10.1080/14760584.2017.1393335

2. Principi N, Esposito S. Development of pneumococcal vaccines over the last 10 years. Expert Opin. Biol. Ther. 2018;18(1):7–17. doi: 10.1080/14712598.2018.1384462

3. Masomian M, Ahmad Z, Gew LT, et al. Development of Next Generation Streptococcus pneumoniae Vaccines Conferring Broad Protection. Vaccines (Basel). 2020;8(1):132. doi: 10.3390/vaccines8010132

4. Gruber I.M., Kukina O.M., Egorova, N.B., Zhigunova O.V. Different Technologies for Obtaining Pneumococcal Immunogens. Epidemiology and Vaccinal Prevention. 2021;20(1):76–91 (In Russ.). doi: 10.31631/2073-3046-2021-20-1-76-91

5. Kilian M, Tettelin H. Identification of Virulence-Associated Properties by Comparative Genome Analysis of Streptococcus pneumoniae, S. pseudopneumoniae, S. mitis, Three S. oralis Subspecies, and S. infantis. mBio. 2019;10(5):e01985–19. doi:10.1128/mBio.01985-19

6. Joyce LR, Youngblom MA, Cormaty H, et al. Comparative Genomics of Streptococcus oralis Identifies Large Scale Homologous Recombination and a Genetic Variant Associated with. mSphere. 2022;7(6):e0050922. doi:10.1128/msphere.00509-22

7. Gruber IM, Egorova NB, Astashkina EA, et al. Immunogenic properties of cellular and extracellular protein-containing antigens of Staphylococcus aureus. Zh. Mikrobiol. 2019;1(1): 29–36 (In Russ).

8. Egorova NB, Kurbatova EA, Vorobev DS, et al. Strains of Streptococcus pneumoniae (versions) and method of producing protective protein fraction, possessing intraspecific immunogenic activity. Patent RUS № 2601158. 27.10.2016. Byul. №30. Available at: https://rusneb.ru/catalog/000224_000128_0002601158_20161027_C1_RU (In Russ).

9. Kukina OM, Gruber IM, Akhmatova NK, et al. Experimental Protein-Containing Preparations Streptococcus pneumoniae, Obtained from Fresh Isolated Strains and Museum. Epidemiology and Vaccinal Prevention. 2020; 19(1): 35–42 (In Russ.). doi: 10.31631/2073-3046-2020-19-1-35-42

10. Kukina OM, Gruber IM, Akhmatova NK, et al. Study of the Immunobiological Properties of Surface Protein-Containing Antigens of Streptococcus pneumoniae Serotype 6B. Epidemiology and Vaccinal Prevention. 2020;19(3):21–27 (In Russ). doi: 10.31631/2073-3046-2020-19-3-21-27

11. Grischenko NV, Tokarskaya MM, Kalina NG, et al. Influence of nutrient medium composition on the production of capsule polysaccharide by Streptococcus pneumonia 19A serotype. Journal of Microbiology, Epidemiology and Immunobiology. 2012; 2: 12–7 (In Russ).

12. Gevorgiz. R.G., Zheleznova S.N., Nekhoroshev M.V.. Quantitative determination of the mass fraction of total proteins in dry biomass of microalgae by the Lowry method : educational and methodological manual / RAS, Kovalevsky Institute of Marine Biological Research. Sevastopol, 2017.

13. . Vorob’ev D. S., Sidorov A. V., Kaloshin A. A., et al. Preparation a recombinant form of pneumolysin protein from Streptococcus pneumoniae. Bulletin of Experimental Biology and Medicine. 2022;174(12):723-727. doi: 10.47056/0365-9615-2022-174-12-723-727

14. Afanasyeva OM, Gruber IM, Brzhozovskaya EA, Astashkina EA. Faktory patogennosti blizkorodstvennyh streptokokkov gruppy Mitis raznoj virulentnosti. Sbornik Trudov XI Mezhdunarodnoj nauchno-prakticheskoj konferencii «Molekulyarnaya diagnostika 2023». Moskva: AO «SAJENS MEDIA PRODZhEKTS», 2023. (In Russ).

15. Pérez-Dorado I, Galan-Bartual S, Hermoso JA. Pneumococcal surface proteins: when the whole is greater than the sum of its parts. Mol Oral Microbiol. 2012;27(4):221–245. doi: 10.1111/j.2041-1014.2012.00655.x

16. Gámez G, Castro A, Gómez-Mejia A, et al. The variome of pneumococcal virulence factors and regulators. BMC Genomics. 2018;19(1):10. Published 2018 Jan 3. doi:10.1186/s12864-017-4376-0

17. Weiser JN, Ferreira DM, Paton JC. Streptococcus pneumoniae: transmission, colonization and invasion. Nat Rev Microbiol. 2018;16(6):355–367. doi355–367. doi:10.1038/s41579-018-0001-8