Salivary gland hyaluronidase in various species of phlebotomine sand flies (Diptera: psychodidae)

https://doi.org/10.1016/S0965-1748(02)00109-1Get rights and content

Abstract

Hyaluronidase activity was detected and partially characterized in salivary gland extracts of females of six sand fly species. In Phlebotomus papatasi and Lutzomyia longipalpis the enzyme was active over a broad pH range; the pH optimum was 5.0. Besides high cleaving activity towards hyaluronic acid, it hydrolyzed chondroitin sulfates A and C. Hyaluronidases of various sand fly species differed in structure and sensitivity to reducing conditions. In the subgenera Phlebotomus (P. papatasi and P. duboscqi) and Adlerius (P. halepensis) the predominant active form of the enzyme was monomeric with the same apparent molecular weight under nonreducing and reducing conditions (around 65 kDa for P. papatasi and P. duboscqi and 110 kDa for P. halepensis). In P. sergenti the enzyme occurred as a putative homodimer but remained active under reducing conditions when separated into 60 kDa subunits. In L. longipalpis and P. perniciosus the activity was detectable under non-reducing conditions only. In P. duboscqi, low enzyme activity was found also in males. Salivary gland hyaluronidases of sand flies share characteristics with endo-N-acetyl-hexosaminidases of mammalian sperm cells and corresponding venom enzymes of Hymenoptera. Hypothetically, they facilitate blood meal acquisition but also may modulate immune reactions of the host and promote pathogen transmission.

Introduction

Sand flies (Diptera, Phlebotominae), similarly to other blood-sucking arthropods, possess an array of salivary compounds that modulate hemostasis and immunity of the host in order to facilitate blood meal acquisition without inducing an immediate hypersensitivity response (reviewed by Champagne and Valenzuela, 1996, Wikel et al., 1996. Sand fly salivary compounds also have a key role in transmission of parasitic protozoa of the genus Leishmania, serious human pathogens. The immunosuppressive effect of saliva decreases the infective dose of the parasite and has an enhancing effect on Leishmania infection in the vertebrate host (Titus and Ribeiro, 1988, Theodos et al., 1991). Studies carried out on two important vectors, L. longipalpis and P. papatasi, revealed that saliva inhibits activation of T-cells (Titus, 1998) and some macrophage functions, including the oxidative burst (Gillespie et al., 2000) and NO production (Hall and Titus, 1995, Katz et al., 2000). In natural conditions this allows the establishment of the infection from a low number of highly infective metacyclic forms ejected from the sand fly proboscis (Warburg and Schlein, 1986).

One of molecules which may participate in both, a blood meal acquisition and enhancement of Leishmania infection, is a hyaluronidase, an enzyme recently detected in saliva of L. longipalpis and P. papatasi (Charlab et al., 1999, Ribeiro et al., 2000). Hyluronidases cleave hyaluronic acid (HA) which is a high molecular weight glycosaminoglycan and a major component of the extracellular matrix in vertebrates. In addition, most of them also cleave other components of extracellular matrix, particularly chondroitin sulfates. There are, however, at least three types of hyaluronidases hydrolyzing HA via different mechanisms. Testicular hyaluronidases of mammals and venom hyaluronidases of Hymenoptera are endo-N-acetyl-hexosaminidases degrading high molecular weight substrate to tetrasaccharides. Hyaluronidases from leeches and nematodes belong to endo-glucuronidases that specifically hydrolyse hyaluronic acid (with tetrasaccharides as the final product), while bacterial hyaluronidases act via β-elimination, yielding disaccharides as the main end products (reviewed by Kreil (1995).

In venomous snakes and arthropods like spiders, scorpions and hymenopteran insects, hyaluronidases are frequently found in toxins. They are called “spreading factors” as they facilitate the spreading of toxic compounds by degradation of the extracellular matrix (see review by Kreil (1995)). In bloodsucking arthropods other than sand flies the hyaluronidase activity was detected in the tick Amblyomma hebraeum (Neitz et al., 1978) and the black fly Simulium vittatum (Ribeiro et al., 2000). The activity is thought to play an important role in blood-meal acquisition by increasing the permeability of host tissue for other pharmacological compounds present in saliva; by depolymerizing hyaluronic acid it may help diffusion of antihemostatic agents into the vicinity of the feeding lesion or help to enlarge the size of the feeding hematoma.

In this study, we present some physico-chemical properties of sand fly hyaluronidases and compare their activities among six species of the genera Lutzomyia and Phlebotomus.

Section snippets

Materials and methods

Seven colonies of six sand fly species were used: Phlebotomus papatasi Scopoli, P. duboscqi Neveu-Lemaire, P. sergenti Parrot, P. perniciosus Newstead, P. halepensis Theodor and Lutzomyia longipalpis Lutz and Nieva. In P. papatasi, two colonies of different in geographical origin (Turkey and Cyprus) were compared. The flies used to establish the colonies were collected in Sanliurfa, Turkey (P. papatasi and P. sergenti) or were provided by Dr. M. Maroli (P. perniciosus), Prof. R. Ward (L.

Results

Protein content in SGEs differed between species. The highest protein level was observed in P. duboscqi - 0.78 μg per “optimal gland” (SD 0.071), followed by P. papatasi from Cyprus colony - 0.51 μg/gland (SD 0.053), P. halepensis - 0.41 μg/gland (SD 0.055), P. papatasi from Turkish colony - 0.33 μg/gland (SD 0.049) and P. sergenti - 0.23 μg/gland (SD 0.017). The lowest protein content was found in L. longipalpis - 0.18 μg/gland (SD 0.038).

The dot method on gels with copolymerized

Discussion

The pharmacological complexity of sand fly saliva was well-demonstrated by various authors in two sand flies, Lutzomyia longipalpis and P. papatasi, belonging to the New-World and Old-World species, respectively. Although considerable differences have been found in saliva composition of various species (Volf et al., 2000, Valenzuela et al., 2001, Ribeiro and Modi, 2001, Volf and Rohoušová, 2001), hyaluronidase activity is present in all species studied. This supports the hypothesis about the

Acknowledgements

We thank Vera Volfova for excellent dissection of salivary glands. The work was supported by the grants GUK 137/2001, GACR 310/00/0760 and J13/981131-B4.

References (29)

  • M. Gmachl et al.

    Bee venom hyaluronidase is homologous to a membrane protein of mammalian sperm

    Proceedings of the National Academy of Sciences of the United States of America

    (1993)
  • L.R. Hall et al.

    Sand fly vector saliva selectively modulates macrophage functions that inhibit killing of Leishmania major and nitric oxide production

    Journal of Immunology

    (1995)
  • R.A.P. Harrison

    Preliminary characterization of the multiple forms of ram sperm hyaluronidase

    Biochemical Journal

    (1988)
  • O. Katz et al.

    Adenosine, AMP and protein phosphatase activity in sandfly saliva

    American Journal of Tropical Medicine and Hygiene

    (2000)
  • Cited by (45)

    • The salivary hyaluronidase and apyrase of the sand fly Sergentomyia schwetzi (Diptera, Psychodidae)

      2018, Insect Biochemistry and Molecular Biology
      Citation Excerpt :

      Decreasing tissue viscosity and modulating the immune response, salivary hyaluronidases facilitate the diffusion of other salivary components, accelerate the blood-meal intake, and contribute to parasite transmission and establishment in the host (Martin-Martin et al., 2018; Volfova et al., 2008). In sand flies, hyaluronidase has been detected in L. longipalpis (Charlab et al., 1999) and eight Phlebotomus species studied to date (Cerna et al., 2002; Rohousova et al., 2012; Vlkova et al., 2014). The very first sand fly recombinant hyaluronidase from L. longipalpis saliva has been only recently functionally characterized by Martin-Martin et al. (2018).

    • A deep insight into the male and female sialotranscriptome of adult Culex tarsalis mosquitoes

      2018, Insect Biochemistry and Molecular Biology
      Citation Excerpt :

      Three nearly full length contigs coding for a female SG-enriched hyaluronidase were found following deep sequencing, all with EI values smaller than 0.7. This enzyme was not previously found in the limited C. tarsalis sialotranscriptome, but was found in the sialome of C. quinquefasciatus (Ribeiro et al., 2004) and is commonly found in sand fly sialotranscriptomes (Charlab et al., 1999; Hostomska et al., 2009; Rohousova et al., 2012; Vlkova et al., 2014), where the enzyme activity was determined (Cerna et al., 2002). It has been postulated that the combined action of endonucleases and hyaluronidases decrease the viscosity of the skin matrix facilitating diffusion of salivary anti-hemostatic components, as well as facilitating pathogen transmission (Calvo and Ribeiro, 2006; Chagas et al., 2014a; Volfova et al., 2008).

    • Basic and Translational Research on Sand Fly Saliva: Pharmacology, Biomarkers, and Vaccines

      2017, Arthropod Vector: Controller of Disease Transmission, Volume 2 Vector Saliva-Host-Pathogen Interactions
    View all citing articles on Scopus
    View full text