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Saliva is an important biological fluid that reflects human’s health. Its main function is protection of the oral cavity from pathogens. Antimicrobial peptides (AMPs) of the innate immunity may play an important role in anti-infectious defense of the oral cavity, but their relative amount in saliva is low. It’s major component is Proline-rich peptides (PRPs), whose impact in antimicrobial protection remains poorly understood. We suggest that salivary PRPs may reveal their defensive functions upon interaction with other molecules, in particular with AMPs. The aim of this work is an investigation of the combined antibacterial action of salivary PRPs (fragments of Basic salivary proline-rich protein 1: P-H (37-51), IB6 (98-116), p1932) with antimicrobial peptides (histatin 5 and cathelicidin LL-37 and beta-defensin hBD3). Listed PRPs have been obtained by chemical solid-phase synthesis. The method of broth microdilutions was used to compare minimal inhibitory concentrations (MICs) of individual fractions of AMPs and their MICs in the presence of salivary peptides. It was found that in the presence of peptides IB6 (98-116) or P-H (37-51) the activity of defensin hBD3 was increased (reduction of MICs by 2 times) against Staphylococcus aureus SG511. In the presence of IB6 (98-116) or p1932 the activity of this defensin against E. coli ML35p was also improved (MICs of hBD3 was lowered by 2 times). For other combinations of the peptides, this effect was not observed. The obtained data confirm the assumption that the combined action of varied salivary peptides, including cationic Proline-rich peptides, plays an important role in anti-infectious protection of the oral cavity.

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Introduction. Nowadays an interest to saliva as a promising diagnostics tool is rising. The content of this biological fluent perfectly reflects human’s health. The main function of saliva is kipping integrity of an upper region of digestive tract mucus and protection from pathogens [1]. Among salivary components there are varied antimicrobial peptides (AMPs): alpha- and beta-defensins, cathelicidins, histatins, but their concentration is relatively low [2]. The major protein fraction of saliva includes Proline-Rich proteins and their proteolytic fragments - Proline-Rich Peptides (PRPs). The biological role of these substances is still poorly understood [3]. We suggested that PRPs may contribute to antibacterial defense working together with AMPs. The aim of the present study was an investigation of a combined action of salivary PRPs (P-H (37-51), IB6 (98-116), p1932) and AMPs (histatin 5, cathelicidin LL-37, beta-defensin hBD3). Material and methods. The listed PRPs have been produced by a solid phase synthesis using Fmoc/tBu-strategy on a 2-chlorotrityl chloride resin [4]. The peptide sequence was built up in the automatic peptide synthesizer Symphony X (Protein Technologies, Inc.), USA), and also by use of manual and automatic peptide synthesis on the equipment of Institute of Chemistry of Molecular Recognition (Milan, Italy). Synthesized peptides have been purified by reversed-phase high-performance liquid chromatography (RF-HPLC) at the Gold System chromatograph (Beckman, USA). Their purity confirmed by means of analytical RF-HPLC and mass spectrometric analysis (MALDI-TOF MS) was not less than 95%. Chemically synthesized peptides histatin 5 and cathelicidin LL-37 were purchased from Anaspec Inc., USA. Antimicrobial activity of peptides was determined by broth microdilution assay [5] against gram-negative bacteria Escherichia coli ML35р and gram-positive bacteria Staphylococcus aureus SG511. To determine the minimum inhibitory concentrations of the peptides we performed their serial twofold dilutions in sterile Terasaki micro-chambers in 0.01 M sodium-phosphate buffer containing 0.1% bovine serum albumin (5 µl/well). Suspension of microorganisms in 2.1% Mueller-Hinton broth M391 (HiMedia, India) was added to wells of Terasaki chambers (5 µl/well). Micro-chambers were incubated for 20 h at 37 °C. The results are presented as medians obtained from 3-5 independent experiments (each sample was done in triplicates). Results and discussion. Antimicrobial activity of individual fractions of PRPs (P-H (37-51), IB6 (98-116), p1932) against Escherichia coli ML35p and Staphylococcus aureus SG 511 was insignificant in the range of concentrations of the tested peptides from 1 to 1000 µM. Because of the lack of MIC values for all these salivary peptides it was not possible to use a standard method of “Checkerboard titration” for characterization of their combined antimicrobial action with AMPs. We studied effects of salivary PRPs, taken in single concentration - 64 µM, on the antimicrobial activity of AMPs (histatin 5, cathelicidin LL-37, beta-defensin hBD3) applied in serial (twofold) dilutions. Comparative analysis of MICs of AMPs alone or in the presence of salivary PRPs was carried out. It was shown that the antibacterial activity of defensin hBD3 towards S. aureus SG511 is enhancing in the presence of IB6 (98-116) or Р-Н (37-51): MIC of hBD3 alone was 1 µM, MIC of hBD3 with the IB6 (98-116) or Р-Н (37-51) - 0.5 µM. The peptide р1932 did not affect the activity of defensin against this strain of staphylococci. The antimicrobial activity of hBD3 towards E. coli ML35p raised in the presence of salivary PRPs IB6 (98-116) or р1932 (MIC of the defensin was 30 µM, MIC of hBD3 with IB6 (98-16) or p1932 - 15 µM). In the case of other PRPs this effect was not observed. The antimicrobial activity of AMPs histatin 5 and LL-37 was not changed in the presence of 64 µM of salivary peptides IB6 (98-116), p1932 or P-H (37-51). Conclusion. The obtained data confirm the suggestion on the biological importance of the combined action of human salivary peptides of varied structures, in particular cationic proline-rich peptides and antimicrobial peptides, in implementation of anti-infectious defense of the oral cavity. The work was supported by RFFR grant No. 17-04-02177 and No. 18-315-00333.

About the authors

M S Sukhareva

Institute of Experimental Medicine, Saint Petersburg; Peter the Great Saint Petersburg Polytechnic University, Saint Petersburg

P M Kopeykin

Institute of Experimental Medicine, Saint Petersburg

M S Zharkova

Institute of Experimental Medicine, Saint Petersburg

O V Shamova

Institute of Experimental Medicine, Saint Petersburg


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