Abstract
The innate immune response represents a first-line defense against pathogen infection that has been widely conserved throughout evolution. Using the invertebrate Hirudo verbana (Annelida, Hirudinea) as an experimental model, we show here that the RNASET2 ribonuclease is directly involved in the immune response against Gram-positive bacteria. Injection of lipoteichoic acid (LTA), a key component of Gram-positive bacteria cell wall, into the leech body wall induced a massive migration of granulocytes and macrophages expressing TLR2 (the key receptor involved in the response to Gram-positive bacteria) toward the challenged/inoculated area. We hypothesized that the endogenous leech RNASET2 protein (HvRNASET2) might be involved in the antimicrobial response, as already described for other vertebrate ribonucleases, such as RNase3 and RNase7. In support of our hypothesis, HvRNASET2 was mainly localized in the granules of granulocytes, and its release in the extracellular matrix triggered the recruitment of macrophages toward the area stimulated with LTA. The activity of HvRNASET2 was also evaluated on Staphylococcus aureus living cells by means of light, transmission, and scanning electron microscopy analysis. HvRNASET2 injection triggered the formation of S. aureus clumps following a direct interaction with the bacterial cell wall, as demonstrated by immunogold assay. Taken together, our data support the notion that, during the early phase of leech immune response, granulocyte-released HvRNASET2 triggers bacterial clumps formation and, at the same time, actively recruits phagocytic macrophages in order to elicit a rapid and effective eradication of the infecting microorganisms from inoculated area.
Highlights
The RNase T2 family is represented by extracellular ribonucleolytic enzymes that act at an optimal pH of about 4.0–5.0 and have been found variously distributed throughout taxa, from viruses to higher eukaryotes [1]
Based on our previous results, showing that HvRNASET2 was both detected in leech granulocytes and able to actively recruit macrophages in a bacterially infected area [17], we investigated the HvRNASET2 antimicrobial action toward Gram-positive bacteria
In the last decades, several studies have demonstrated that members of the human RNase A superfamily play a crucial role in the defense against bacterial infection [50], very little is known about the potential antibacterial activity of T2 RNase family members and their possible involvement in microbial recognition
Summary
The RNase T2 family is represented by extracellular ribonucleolytic enzymes that act at an optimal pH of about 4.0–5.0 and have been found variously distributed throughout taxa, from viruses to higher eukaryotes [1]. Despite their highly conserved biochemical and structural features, T2 RNases from different organisms show a marked pleiotropic nature, being involved in an impressive range of biological functions, often related to stress response and host defense [1, 2]. In keeping with such pleiotropic role, besides extracellular compartments, T2 RNases have been reported in cytoplasmic vacuoles, lysosomes, P-bodies, and mitochondria [6, 12,13,14], further arguing in support to their involvement in a wide range of key biological processes
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