Comparative evaluation of salivary glands proteomes from wild Phlebotomus papatasi –proven vector of zoonotic cutaneous leishmaniasis in Iran

Abstract Background Zoonotic Cutaneous Leishmaniasis is increasing in the world and Phlebotomus papatasi as a proven vector was considered in different aspects for disease control. Sandfly saliva contains proteins which provoke host immune system. These proteins are candidates for developing vaccines. Objectives The main purpose of this research was comparing evaluation of salivary glands proteomes from wild P. papatasi. Extracting these proteins and purifying of original SP15 as inducer agent in vector salivary glands from endemic leishmaniasis foci were other objectives. Methods Adult sandflies were sampled using aspirators and funnel traps from three endemic foci in 2017–2018. Each pair of salivary glands of unfed females was dissected and proteins were extracted using thermal shocking and sonication methods. Purification was performed through RP‐HPLC. All equivalent fractions were added together in order to reach sufficient protein concentration. Protein content and profile determination were examined with SDS‐PAGE. Results The protein concentration of whole‐salivary glands of specimens was determined approximately 1.6 µg/µl (Isfahan) and 1 µg/µl (Varamin and Kashan). SDS‐PAGE revealed 10 distinct bands between 10 and 63 kDa. Analysis of proteomes showed some similarities and differences in the chromatograms of different foci. SDS‐PAGE of all collected fractions revealed SP15‐like proteins were isolated in 24 min from Varamin, 26 to 30 min from Kashan and 29.4 min from Isfahan and were around 15 kDa. Conclusions Isolation of salivary components of Iranian wild P. papatasi is very important for finding potential proteins in vaccine development and measuring control strategy of zoonotic cutaneous leishmaniasis in Iran and this could be concluded elsewhere in the world.


| INTRODUC TI ON
Phlebotomus papatasi (Scopoli, 1786) as only well-known proven vector of zoonotic cutaneous leishmaniasis (ZCL) in Iran has been under a lot of considerations in different aspects of researches (Killick-Kendrick, 1990;Parvizi & Ready, 2008). In Old World foci of ZCL, the blood-feeding females of P. papatasi (Diptera: Psychodidae: Phlebotominae) sandfly are incriminated to be the natural vector of protozoan Leishmania major, the causative agent of the neglected tropical disease ZCL. In Iran and many countries, ZCL is an endemic disease and is increasing in many foci.
ZCL originally is a disease of gerbils. Sandflies, which transmit Leishmania parasites, live and breed in gerbil burrows. The function of P. papatasi salivary proteins is provoke of human immune system in ZCL foci (Mahamdallie & Ready, 2012;Ready, 2013;Zahirnia et al., 2018). Few researches are available on Iranian wild P. papatasi salivary glands proteins. Biological and environmental conditions were studied previously about affecting on protein content of salivary glands (Hosseini-Vasoukolaei, Mahmoudi, et al., 2016). Differential expression of salivary protein genes was described under various physiological conditions . There is not any report on purification and characterization of salivary glands proteins in P. papatasi from Iranian foci. The most salivary glands proteins have been purified and characterized from colonies breeding in insectarium (Marzouki et al., 2012;Valenzuela et al., 2001;Vlkova et al., 2014). In this investigation, wild P. papatasi was considered and worked from endemic foci of ZCL in Iran. As we are aware, SP15 family of proteins is the most abundant in the majority of sandfly salivary glands but not in cDNA library (Abdeladhim et al., 2012). These proteins with unknown functions contain PpSP15 from P. papatasi saliva (Acc. no. AAL11047) could elicit specific humoral and cellular immunity.
PpSP15 is able to protect immunized mice against Leishmania major (Oliveira et al., 2008;Valenzuela et al., 2001;Vlkova et al., 2014). SP15 as an antigen in vaccine production could enhance protective efficacy against leishmaniasis (Zahedifard et al., 2014). Salivary gland proteins from colonized sandflies do not cause protection against infection with L. major in the case of pre-exposure to wild specimens (Ben Hadji Ahmed et al., 2010).
Nevertheless there are many endeavours to vaccine development against leishmaniasis, it has been failed because of some reasons. The origin of utilized SP15 as the inducer agent on human immunity system is important. Almost purification of salivary gland proteins has been performed on reared sandflies rather than wild-caught sandflies. Extracting salivary gland proteins from wild-caught P. papatasi was one of the purposes of this research.
Purifying of SP15 as an adjuvant in vaccine production and comparison between protein profiles from different ZCL foci were other objectives. Wild adult sandflies were collected using aspirators and funnel traps from domestic animal shelters and rodent borrows respectively. The collected specimens put in microtubes without any buffer and were frozen (−20°C). All collected specimens were kept on ice and transferred to Pasteur institute of Iran in Tehran and stored in −70°C. All adult sandflies were identified using morphological characters of the head and abdominal terminalia, following a dissection for salivary glands with sterilized forceps and micro-needles (Parvizi et al., 2003). After dissecting, each pair of salivary glands of unfed females P. papatasi was moved into a micro-tube without any buffer and flash frozen at −70°C for protein extraction.

| Protein extraction
Isolated salivary glands of the female P. papatasi, encompassed a rapid freeze-thaw process (−70°C for 2 min then 37°C water bath for 1 min), following sonication on ice for 1 min at 45% and for 5 min at 90% power using an ultrasonic processor (Hielscher; Ben Hadji Ahmed et al., 2010; F I G U R E 1 Geographical locations of ZCL foci where sandflies were sampled Hosseini-Vasoukolaei, Mahmoudi, et al., 2016). Extraction product was centrifuged for 1 min at 14,000 rpm. Supernatant was filter sterilized with centrifugation at 14,000 rpm for 30 min through a 0.22-µm Ultrafree centrifugal filter unit (Millipore; Geraci et al., 2014).
For removing undesirable substances, cold acetone (four times, −20°C) was added to protein precipitation. The tube was incubated at −20°C for 60 min. The supernatant containing the interfering substance was decanted and the pellet contains protein stored until usage (Fic et al., 2010;Peach et al., 2015). For every experimental test, the protein pellet was re-dissolved in phosphate buffer saline (pH = 7) that is compatible with the downstream applications.

| Protein concentration determination and SDS-PAGE
A microplate reader spectrophotometer (Synergy-HTX, Biotek) fulfilled the protein concentration directly. The concentration of protein was determined in 280 nm.
Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) was carried out according to standard method (Laemmli, 1970). The samples were loaded onto a 15% polyacrylamide gel and stained with silver nitrate. The apparent molecular masses of the proteins were estimated by comparison with a mixture of molecular protein markers (10-75 kDa).

| HPLC purification of proteins and peptides
The extract of 50 pairs of salivary glands (about 50 µg) from Kashan, Isfahan and Varamin was dissolved in PBS (500 μl) separately and purification was performed using an HPLC instrument (Knauer-Germany).
In order to isolate the proteins, the column was eluted by a linear gradient of solution B from 0% to 60% for 45 min at 0.3 ml/min. The eluted peaks were monitored at 280 nm and collected manually (Belkaid et al., 2000;Boulanger et al., 2004;Ribeiro, 1992). The isolated fractions were lyophilized by a freeze dryer (Christ, Alpha 1-2 LD plus-Germany). The purity of the isolated proteins was subsequently checked by SDS-PAGE.

| Protein extraction process and determination of protein profiles
After protein extraction, the equivalent concentration of 50 pairs of whole salivary glands was considered approximately 1.6 µg/µl from Isfahan and 1 µg/µl from both of Varamin and Kashan (Figure 2). SDS-PAGE showed separated bands with their molecular weights. These profiles revealed 10 proteins or peptides. Minor and major protein bands observed between 10 and 63 kDa. There were significant differences between extracted protein profiles from different ZCL foci (Figure 3).

| Purification of PpSP15-like proteins
Ten fractions of salivary gland proteins were yielded using RP-HPLC on C18 column with a gradient protocol. Chromatograms of salivary F I G U R E 2 Salivary glands of Phlebotomus papatasi were exploited for protein extraction using stereo microscope (Nikon SMZ645) at 5× magnification and a digital camera (Canon G12 -Japan).
Correlation between vertebrate host immune response and salivary glands proteins was defined. These proteins are recommending as a potential antigen for vaccine production against Leishmania infection because they can cause host immune response (Valenzuela et al., 2004). Salivary glands of P. papatasi with five members of small  Abbreviations: aa, number of amino acid residues; Acc. No, accession number; MW, predicted molecular weight; ND, not done; pI, predicted isoelectric point.
weight of these proteins has been predicted from 13.9 to 14.9 kDa and isoelectric point 8.0 to 9.2 (Vlkova et al., 2014).
PpSP15-like proteins belong to OBP family, but so far the exact function of these proteins remains unknown in sandflies. However, SP15 protein (accession number: AAL11047) was shown to elicit specific humoral and cellular immunity. These caused the protection in immunized mice against L. major infection (Oliveira et al., 2008;Valenzuela et al., 2001).
In addition, a delayed type hypersensitivity (DTH) reaction was also observed in immunized mice inoculated of DNA plasmid coding for SP15-like salivary protein in P. ariasi (accession number: AAX56359; Oliveira et al., 2006). In general, collecting wild sandflies from different ZCL foci is important for protein extraction and analysis. However, caching sandflies sometimes with long distances spends more time and high cost of budget. A lot of sandflies were collected and transported to the laboratory but after identifying and separating, different genera and species were found. Several times, collecting was tried until suitable numbers of female P. papatasi were separated and identified.
Moreover, average extracted protein content per a pair of glands in each P. papatasi was a little and is not enough so much more samples were required (Hosseini-Vasoukolaei, Mahmoudi, et al., 2016).
Comparison between HPLC chromatograms of extracted proteomes from occupied lands, Palestine specimens as standards and our specimens from different regions revealed similarity in four major peaks from all regions except Kashan that exposed some differences ( Figure 4; Belkaid et al., 2000).
There were some differences between Isfahan and Kashan chromatogram profiles and similarity in proteome analysis between Varamin and standard specimens (Belkaid et al., 2000). Moreover, evaluation of SDS-PAGE showed distinct differences among all samples from different locations (Figure 3). There were some varieties in number and weight of specified peptides from separate foci of ZCL (Geraci et al., 2014). Some differences in number and weight of protein bands were found among Iranian specimens and the world.

| CON CLUS ION
Now we can conclude that investigation on salivary gland proteins of natural wild specimens from local and endemic ZCL foci should be intended separately for each area, in order to vaccine production. To innovate vector-based and novel pharmacoactive proteins, study on salivary gland proteins of natural wild specimens is essential.

ACK N OWLED G EM ENTS
We acknowledge our colleagues in molecular systematics laboratory, Parasitology department, Pasteur institute of Iran. This project was made possible with financial support from Pasteur institute of Iran, grant 973 awarded to Prof. Parvizi.

CO N FLI C T O F I NTE R E S T
The authors have no conflict of interest to declare.