DEVELOPMENT OF A METHOD FOR SELECTIVE ASSESSMENT OF COMPLEMENT MICROBICIDAL ACTIVITY IN HUMAN BLOOD SERUM

DOI: https://doi.org/10.29296/25877313-2021-01-07
Issue: 
1
Year: 
2021

S.V. Legkovoy Post-graduate Student, Faculty of Biology, Department of Biochemistry, Saint Petersburg State University (Saint Petersburg, Russia) E.S. Umnyakova Ph.D. (Biol.), Senior Research Scientist, Department of General Pathology and Pathophysiology, Institute of Experimental Medicine (Saint Petersburg, Russia) M.N. Berlov Ph.D. (Biol.), Senior Research Scientist, Department of General Pathology and Pathophysiology, Institute of Experimental Medicine (Saint Petersburg, Russia)

Background. The ability of blood serum to suppress viability of microorganisms is primarily implemented by the presence of cationic factors (e.g. lysozyme, β-lysine, etc.) and complement system proteins. The complement itself is only able to cause lysis of some gram-negative bacteria, while cationic factors are predominantly active against gram-positive ones. The study of the contribution of individual antimicrobial components of blood serum and their combined action to its microbicidal activity would help to expand the understanding of the molecular mechanisms of innate immunity. The aim of the study. Development of a method for selective depletion of cationic factors from serum while preserving the functional activity of the complement system. Materials and methods. Pooled blood serum from healthy donors was used as the starting material. The depletion of cationic molecules from blood serum was performed by its incubation with a suspension of carboxymethylcellulose. In order to find the optimal conditions for binding of cationic proteins and peptides, either the NaCl concentration in the sample or the volume of the applied polymer suspension were varied. Removal of cationic factors from the serum was evaluated by the residual activity of lysozyme. The retention of complement was evaluated by serum hemolytic activity against rabbit red blood cells. The bactericidal activity of native and depleted serum against the gram-positive bacterium Listeria monocytogenes EGD was also compared. Results. Incubation of serum with carboxymethylcellulose suspension taken in equal volume in the presence of 0.1 M NaCl allowed effective removal of cationic polypeptides without evident changes in complement functional activity. Antimicrobial activity of the serum depleted of cationic factors was remarkably lower compared with the native blood serum. Conclusions. A method for selective removal of cationic factors from blood serum was developed. The described procedure allows evaluation of the complement microbicidal activity independently on the action of antimicrobial serum cationic polypeptides.

Keywords: 
innate immunity
complement system
lysozyme
β-lysin

References: 
  1. Merle N.S., Church S.E., Fremeaux-Bacchi V., et al. Complement system. Part I. Molecular mechanisms of activation and regulation.Front. Immunol. 2015; 6: 262.
  2. Ricklin D., Hajishengallis G., Yang K., et al. Complement: a key system for immune surveillance and homeostasis. NatureImmunol. 2010; 11: 785–97.
  3. Kokrjakov V.N. Ocherki o vrozhdennom immunitete. SPb: Nauka, 2006; 262 s.
  4. Umnjakova E.S., Pashinskaja L.D., Krenev I.A. i dr. Zabolevanija, svjazannye s disreguljaciej sistemy komplementa, i perspektivy ih lechenija. Medicinskij akad-emicheskij zhurnal. 2018; 18: 7–16.
  5. Biolchi A., De Angelis G., Moschioni M., et al. Multicomponent meningococcal serogroup B vaccination elicits cross-reactive immunity in infants against genetically di-verse serogroup C, W and Y invasive disease isolates. Vaccine. 2020; 38: 7542–50.
  6. Liu D., Chen Z., Yuan Y., et al. Innate immune effectors play essential roles in acute respiratory infection caused by Klebsiellapneumonia. J. Immunol. Res. 2020; 2020: 291714.
  7. Chau N., Pérez-Morales D., Elhenawy W., et al. (p)ppGpp-dependent regulation of the nucleotide hydrolase PpnN confers complement resistance in Salmonella en-terica serovar Typhimurium. Infect. Immun. 2020; IAI.00639-20.
  8. Berends E.T., Mohan S., Miellet W.R., et al. Contribution of the complement Membrane Attack Complex to the bactericidal activity of human serum. Mol. Immunol. 2015; 65: 328–335.
  9. Ragland S.A., Criss A.K. From bacterial killing to immune modulation: Recent insights into the functions of lysozyme. PLOS Pathog. 2017; 13: e1006512.
  10. Donaldson D.M., Tew J.G. Beta-lysin of platelet origin. Bacteriol. Rev. 1977; 41: 501–513.
  11. Parry R.M. (Jr.), Chandan R.C., Shahani K.M. A rapid and sensitive assay of muramidase. Proc. Soc. Exp. Biol. Med. 1965; 119: 384–386.
  12. Umnyakova E.S., Gorbunov N.P., Zhakhov A.V., et al. Modulation of human complement system by antimicrobial peptide arenicin-1 from Arenicola marina. Mar. Drugs. 2018; 16: 480.
  13. Berlov M.N., Korableva E.S., Andreeva Ju.V. i dr. Laktoferrin iz nejtrofilov sobaki: vydelenie, fizi¬ko-himicheskie i antimikrobnye svojstva. Biohimija. 2007; 72: 551–559.