Dissociating effect of salivary gland extract from Ixodes ricinus on human fibroblasts: Potential impact on Borrelia transmission

Abstract

Understanding the mechanism of pathogen transmission is essential for the development of strategies to reduce arthropod-borne diseases. The pharmaco- and immunomodulatory properties of insect and acarine saliva play an essential role in the efficiency of pathogen transmission. The skin as the site where arthropod saliva and pathogens are inoculated - represents the key interface in vector-borne diseases. We identified tick molecules potentially involved in pathogen transmission, using micro-HPLC and mass spectrometry, followed by in vitro assays on human skin cells. Histone H4 isolated from Ixodes ricinus salivary gland extract was identified as a molecule with a dissociating effect on human primary fibroblasts. This histone might be involved in the formation of the feeding pool formed around the tick mouthparts and responsible of tissue necrosis in the vertebrate host. Thanks to its selective antimicrobial activity, it may also sterilize the feeding pool and facilitate transmission of pathogens such as Borrelia burgdorferi sensu lato.

Introduction

Hard ticks of the Ixodidae family are involved in numerous tick-borne diseases caused by diverse pathogens including bacteria, viruses or parasites. Certain Ixodes species are obligate hematophagous vectors for bacteria including Borrelia burgdorferi sensu lato, Rickettsia, Anaplasma, the parasite Babesia or the tick-borne encephalitis virus (de la Fuente et al., 2008). These pathogens are transmitted when a tick attaches to a host through its sophisticated mouthparts, the hypostome and the chelicerae. The skin injury eventually develops to a feeding pool (Sonenshine and Anderson, 2014). During a tick blood meal which typically lasts for several days, the hard tick concurrently inoculates saliva to secure its blood meal by forming a cement layer around the mouthparts and by the secretion of pharmacologically and immunologically active molecules (Kazimírová and Stibrániová, 2013). The salivary glands modify their structure during the blood meal and degenerate after the blood meal in some tick species (Alarcon-Chaidez, 2014; Kahl et al., 1990) and the tick detaches from the host to either molt (nymphs) or to die after laying eggs (female adults). The tissue damage very often develops to a skin necrosis after the tick blood meal (Krause et al., 2009).

Saliva plays a crucial role during the feeding process: it allows the tick to neutralize host hemostasis (vasoconstriction, platelet aggregation, blood clotting), innate immunity (inflammatory response, complement activation) and acquired immunity. For that purpose, tick saliva contains a wide range of pharmacologically active molecules (Francischetti et al., 2009; Hovius et al., 2008; Ribeiro et al., 1985) with anti-clotting (Chmelar et al., 2012; Francischetti et al., 2004; Narasimhan et al., 2004; Prevot et al., 2006), vasodilatory (Bowman et al., 1996; Dickinson et al., 1976) and anti-inflammatory properties (Beaufays et al., 2008; Paesen et al., 1999; Ribeiro and Mather, 1998; Sangamnatdej et al., 2002). In addition, tick saliva is able to target vertebrate host immune responses such as the complement cascade (Couvreur et al., 2008; Daix et al., 2007; Schroeder et al., 2007; Valenzuela et al., 2000), antimicrobial peptides (Kern et al., 2011; Marchal et al., 2011), B-cells (Hannier et al., 2004) and the T-cells (Anguita et al., 2002; Garg et al., 2006; Leboulle et al., 2002). In the context of tick-borne diseases, this cocktail of active molecules renders the infection of a host more efficient.

The skin of the vertebrate host is the key interface where the vector bites and inoculates its saliva containing the pathogens (Bernard et al., 2015, 2014). The epidermis with its keratinocytes represents the first barrier encountered by ticks. They overcome this barrier using their biting pieces to penetrate deeply into the skin and reach the dermis, where the saliva interacts with resident cells like dermal dendritic cells, mast cells or fibroblasts. Some pathogens already multiply in the dermis before disseminating to the target organs. Investigating these initial steps is essential to understand the further development of most of the vector-borne diseases and to develop new strategies to control these diseases (de la Fuente et al., 2017).

We have selected the causative agent of Lyme disease, Borrelia burgdorferi sensu stricto (ss), which is transmitted by certain Ixodes species in Europe to study these transmission mechanisms. In a previous study, we have shown the antialarmin effect of tick saliva on human keratinocytes (Marchal et al., 2011). We observed also that crude salivary gland extract induced a dissociating effect on fibroblast monolayer adherent to tissue culture plate (Schramm et al., 2012). Identification of the tick molecules responsible of this specific effect could help to understand the formation of the feeding pool, which is essential for the tick blood meal process and thus for the transmission of pathogens. Therefore, we performed a fractionation of tick salivary gland extract by micro RP-HPLC and tested the different chromatographic fractions for a potential dissociating effect on human dermal fibroblasts. A tick-borne histone H4 was identified in the active fractions by enzymatic digestion and nanoLC-MS/MS. This protein was further characterized by mass spectrometry. The biological significance of this protein is discussed in the context of B. burgdorferi sl transmission.