The antibacterial role of Hirudo verbana RNASET2: evaluation in vivo and in vitro

Recent data obtained in the medicinal leech Hirudo verbana revealed that the enzyme RNASET2, belonging to T2 ribonucleases family, took part in the regulation and modulation of the innate immune system, by the direct recruitment of immunocompetent cells during bacterial infection. Indeed, the injection of LPS (lipopolysaccharides) and LTA (lipoteichoic acid), principal components of the bacterial cell wall of Gram-negative and Gram-positive respectively, into the leech body wall, triggered an increased expression of the endogenous leech enzyme HvRNASET2 in both macrophages and granulocytes. These results suggested a possible antibacterial role for this enzyme, as observed for the RNase 3 and the RNase 7 in vertebrates. In order to better clarify this aspect, here we conducted in vitro experiments by treating Escherichia coli and Staphylococcus aureus bacterial strains with the recombinant protein HvRNASET2. The effect on microorganisms was analyzed by light, transmission (TEM) and scanning (SEM) electron microscopy, after 3 and 24 h from the incubation. The images showed an evident agglutination in both cases and immunogold assays indicated a direct interaction between HvRNASET2 and the bacterial cell walls. Moreover, experiments performed both in vivo and by using the Matrigel biomatrix (MG), confirmed the presence of S. aureus bacteria aggregates in the leech body wall, supporting the idea that, during leech innate immune response, HvRNASET2 triggers bacterial clumps formation to promote a more rapid phagocytosis by macrophages and to elicit a rapid and effective eradication of the infecting microorganisms from inoculated area recruiting phagocytic cells.