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Table of Contents
RESEARCH ARTICLE
Year : 2022  |  Volume : 59  |  Issue : 1  |  Page : 63-69

The optimization of PpSP15 purification from salivary glands in Iranian wild Phlebotomus papatasi (Diptera: Psychodidae)


Molecular Systematics Laboratory, Parasitology Department, Pasteur Institute of Iran, Tehran, Iran

Date of Submission20-Feb-2021
Date of Acceptance22-Oct-2021
Date of Web Publication07-Jun-2022

Correspondence Address:
P Parvizi
Molecular Systematics Laboratory, Parasitology Department, Pasteur Institute of Iran, 1316943551, Tehran
Iran
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0972-9062.331405

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  Abstract 

Background & objectives: Sand fly saliva contains proteins that modulate the host immune system and it plays an important role in both blood feeding and the outcome of Leishmania infections. The profile of the salivary proteins was examined and analyzed from an endemic focus of zoonotic cutaneous leishmaniasis by wild P. papatasi to find local and suitable antigens as potential proteins for developing Leishmania vaccine alongside the development of a new extraction technique.
Methods: Specimens were caught from Bojnord, using funnel and CDC traps. Different methods of protein extraction were employed and a new technique was developed. The proteins were extracted from the salivary glands tissues with a lysis buffer. Purification was performed using RP-HPLC, with a linear gradient protocol from 0-60 % of acetonitrile. PpSP15 was characterized by SDS-PAGE.
Results: The concentration of extracted protein content was 0.5 and 0.03 μg/μl in chemical and physical methods, respectively. PpSP15 was isolated at a weight of 15kDa in 80–85 min of run time. SDS-PAGE was able to characterize PpSP15. The crude extract of the chemical method, revealed 15 separated bands, ranging from 11–100 KDa. Tajima D index was positive.
Interpretation & conclusion: PpSP15 was characterized from Iranian specimens; it is a very highly hydrophobic protein of salivary glands among SP15- like proteins. The chemical method of extraction was found to be more effective than physical methods (P < 0.05). For developing a vaccine against leishmaniasis, depending on the location, choosing suitable proteins should be considered and an efficient extraction method should be used.

Keywords: Phlebotomus papatasi; PpSP15; RP-HPLC; SDS-PAGE; Iran


How to cite this article:
Ghafari S M, Parvizi P. The optimization of PpSP15 purification from salivary glands in Iranian wild Phlebotomus papatasi (Diptera: Psychodidae). J Vector Borne Dis 2022;59:63-9

How to cite this URL:
Ghafari S M, Parvizi P. The optimization of PpSP15 purification from salivary glands in Iranian wild Phlebotomus papatasi (Diptera: Psychodidae). J Vector Borne Dis [serial online] 2022 [cited 2022 Jul 1];59:63-9. Available from: https://www.jvbd.org/text.asp?2022/59/1/63/331405


  Introduction Top


Leishmaniasis is increasing in the world[1],[2]. Reservoirs previously exposed to saliva by injection or unin-fected sand fly bites showed a cellular immune response that protected them against Leishmania major[3],[4],[5],[6]. So these antigens were considered as candidates for vaccines. Phlebotomus papatasi is a proven vector of zoonotic cutaneous leishmaniasis (ZCL) in Iran and was studied in Iran and the world[7],[8],[9],[10] but no investigation has been done on the purification of sand fly salivary proteins in Iran except by Ghafari and colleagues[11]. Besides, for vaccine production, every effort on non-endemic proteins leads to failure because these proteins are species specific[12],[13].

Immunity against P. papatasi saliva protects against L. major infection were obtained with long-term colonized female P. papatasi[14]. As is evident, most of the studies were performed by using long-term laboratory colonized sand flies mainly because of the difficulty of working with wild-caught flies. It was shown that pre-exposure to salivary gland homogenate (SGH) of long-term, colonized P. papatasi do not confer protection against infection with L. major inoculated with SGH of wild-caught P. papatasi[14],[15].

SGH was acquired from unfed female P. papatasi in order to protein extraction following PpSP15 purification. SGH of long-term colonized flies is observed different from wild-caught or recently colonized flies. Colonization of insects reduces natural genetic variability. It could suppress certain traits in field populations. Wild-caught P. papatasi compared with colonized populations were shown the highest genetic variation in SP-15. Moreover, analysis of genetic variation at 17 enzyme loci of one colonized and five field populations of P. papatasi showed that polymorphism of the examined loci in colonized (23.5%) and in field populations (76.6%)[16]. SP15 is able to immunize people against P. papatasi bites. Only SP15 protein originated from P. papatasi contains immunization specificity and this trait was confirmed. Impressive action of SP15 in vaccine development and originated protein from wild caught specimens rather than colonized specimens are very important. In this investigation, specimens were sampled from endemic areas of ZCL. Extraction and purification of PpSP15 from natural specimens was one of the important objectives of this research.


  Material & Methods Top


Collection, identification, and preparation of salivary glands of adult sand flies

The sand fly collections were carried out from June to August 2016, during the main season of activity of adult sand flies[17],[18]. North Khorasan province is known as the endemic focus of ZCL and visceral leishmaniasis (VL) in Iran. P. papatasi is proven vector and rodents are reservoir hosts of ZCL. Larrossius sand flies are potential vectors and dogs are reservoir hosts of VL. ZCL is important in epidemiological aspects because of spreading in many villages recently. Most collections were made at the edges of seven villages (900–1729 m.a.s.l.) near the city of Bojnord: 37°28′33.511″ N, 57°19′55.157″ E (www. gps-coordinates.net/) North Khorasan province, namely Peygho, Malkesh, Sisab, Qare bashlo, Mehnan, Dartom, and Baba aman [Figure 1].
Figure 1: Geographical location of Bojnord, in North Khorasan province, North East of Iran where sand flies were sampled.

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Adult sand flies were collected with CDC miniature light traps (with the white light bulb 1–2m above ground level) were set overnight to sample sand flies in domestic animal shelters and inside houses in the morning. Besides, funnel traps were used to sampling from rodent borrows. The collected specimens were put in microtubes without ethanol and then were frozen. All adult sand flies were identified using the external and internal morphological characters of the head and abdominal terminalia [Figure 2], following a dissection with sterilized forceps and microneedles[16],[17].
Figure 2: A. P. papatasi after dissection. B. Abdominal terminalia with a pair of spermatheca capsules and their ducts. C. Head of P. papatasi and a pair of salivary glands; the photo prepared using stereo microscope (Nikon SMZ645) at 5X magnification. The spermatheca was observed with a microscope (Bell, photonic) at 40X magnification and all images were documented using a digital camera (Canon G12 – Japan).

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Protein extraction

Different methods of protein extraction (sonication, homogenizing, freeze-thawing, centrifuge, and lysis buffer) were tried to obtain a sufficient amount of protein from the salivary glands of the female P. papatasi. The physical methods of extraction were examined (physical methods such as freeze- thawing, centrifuge, filtration, sonication and homogenizing are contrary to chemical methods which use of chemical materials in order to extraction process), For the physical methods, after the dissection and isolation of the salivary glands from 20 unfed female P. papatasi sand flies, they had been frozen at -70°C to procure the salivary gland protein (SGSs) for three cycles of rapid freeze-thaw (-70°C for 2 min, then 37°C water bath for 1 min), centrifuged for one minute at 14000 rpm, sonicated in a room temperature water bath for one minute at 45%, and for five minutes at 90% power using an ultrasonic processor (Hielscher), then filtersterilized by centrifugation at 14,000 rpm for one minute through a 0.22-μm Ultrafree centrifugal filter unit (Millipore, Billerica, MA)[19],[20]. The chemical extraction with lysis buffer was prepared according to a modified protocol including SDS, five microtubes contain five pairs of P. papatasi salivary glands that were chosen for the consecutive protein extraction processes. The extraction method was followed as described before with some modifications[21]. The salivary glands were dissolved in a solvent buffer (Tris-HCl 0.175M, pH = 8.8, SDS 5%, Glycerol 15%, DTT 0.3 M) and were squashed by a tip (size 100μl) completely. The sample was centrifuged at 10000 rpm for five minutes, and the supernatant was filter-sterilized by centrifugation at 10000 rpm through a 0.22-μm Ultrafree centrifugal filter unit (Millipore, Billerica, MA). For removing undesirable substances, cold acetone (four times, -20°C) was added for protein precipitation. The tube was vortexed and incubated for 60 min at -20°C. Then, the supernatant containing the interfering substance was decanted, and the pellet was properly disposed of so as not to dislodge the protein pellet[21]. Finally, the protein pellet was re-dissolved in double distilled water (DDW) that is compatible with the downstream application.

Protein concentration determination and SDS-PAGE

A Picodrop (PICOPET01) fulfilled the protein concentration directly. The concentration of protein was determined in 280nm. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS–PAGE) was carried out according to standard protocol[22]. The samples from the same source and the same number were loaded on to two 15% polyacrylamide gels under non reducing conditions, separately and monomer concentrations were: Acrylamide (30% W/V) /Bis- acrylamide (0.8% W/V), specific buffers used: Tris/ Glysine buffer 0.2%, the electrophoresis equipment used: BioRad mini PROTEIN-tetra cell, running voltage:100 V for stacking and 150 for resolving, thickness of gel was 1.5 mm, stacking was 4mm and resolving was 8mm length, and run time as appropriate 75 min. The gels stained one with silver nitrate and another with Coomassie brilliant blue. In silver nitrate staining, the apparent molecular masses of the purified proteins were estimated by comparison with a mixture of molecular protein markers (CinnaGen corporation, catalog number: SL7001 (PR901641), 11–100 kDa). A single 29 kDa protein (SNAP29: Synaptosomal-associated protein 29: catalog number: OAAN01135, Aviva Systems Biology Corporation) was used as a marker in Coomassie brilliant blue staining.

Protein purification with RP-HPLC

The extract of five pairs of salivary glands (0.5 μg/μl) was dissolved in PBS (100 μl), and the fractions were separated using an HPLC instrument (Knauer- Germany). Then 10 μl of the prepared extract (5μg) was manually injected into C18 column (5μm 100Å - 250 × 4.6mm, Beckman, USA) and eluted in a linear gradient of acetonitrile containing 0.05% TFA (solution A) and 0.05% TFA in water (solution B) at a flow rate of 0.2 ml/ min. In order to isolate the proteins, the column was eluted by a linear gradient of solution A from 0 to 60 percent for 95 minutes at 0.2 ml/min. The eluted peaks were monitored at 214nm and 280nm and collected manually[23]. 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.

Statistical analysis

Tajima’s D index was used as a statistical test analyses data. For this research it is able to find differences between protein bands numbers in physical and chemical methods. Chi-square test was applied for computing the qualitative data. In addition, t-test and ANOVA test were performed for the analysis of mean differences via PASW®statistics (formerly SPSS Statistics) version 18.


  Results Top


Sand fly species collected and subsampling of P. papatasi for protein analysis

The P. papatasi was the abundant sand fly in the collections, followed by the Sergentomyia (Sergentomyia) sintoni Pringle and Phlebotomus species in the subgenus Paraphlebotomus Theodor. From 1400 trapped specimens by CDC traps and funnel traps (the collected specimens by funnel traps were more than CDC miniature light traps) in the Northern Khorasan province in 2016, 440 numbers including eleven species were mounted, identified and salivary glands were dissected. Males and females of different sand flies were caught (males was two times more than females); including 105 unfed female sand flies (different species) [Table 1]. 44 out of 105 were P. papatasi. 20 out of 44 specimens were extracted for protein processes in physical methods. Each specimens contains two salivary glands; in total 40 salivary glands. Five specimens (ten salivary glands) were obtained for chemical extraction method.
Table 1: The number of unfed female sand flies which were dissected, identified and from them, P. papatasi considered for protein extraction process.

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Improvement and modification of protein extraction methods

The extraction methods of sonication, homogenizing, and freeze-thawing or physical extraction methods were employed for the protein extraction of sand fly salivary glands. In this study, physical methods could extract only heavy proteins (more than 29 kDa). For all proteins of the sand fly salivary glands, a new method of protein extraction using lysis buffer was designed for the first time (until now as we are aware). On the other hand, because of low protein content from the low number of samples, these methods had to be discarded, and a more potential technique was designed to maintain the quantity and quality of proteins[8],[24],[25]. By employing SDS-PAGE, there were some significant differences between extractions using physical or chemical methods. For protein extraction, 40 salivary glands were used by employing all three physical methods, but only four proteins (more than 29 kDa) were revealed [Figure 3]A. However, by using a chemical method (lysis buffer), 15 separated bands were shown for ten salivary glands [Figure 3]B.
Figure 3: Electrophoretic profile of Iranian P. papatasi salivary glands protein. It was analyzed by SDS- PAGE using 15% poly-acrylamide gel under non reducing conditions. A. Extracted protein of 40 salivary glands lead to 0.03μg/μl protein which 0.75μg was loaded in a well and stained with Coomassie brilliant blue. S: Salivary gland protein profile of P. papatasi, M: Molecular weight marker (SNAP29: Synaptosomal-associat-ed protein 29, catalog number: OAAN01135, 29kDa, Aviva Systems Biology Corporation). B. Extracted protein of 10 salivary glands lead to 50μg protein which 12.5 μg was loaded in a well stained with silver nitrate. S: Salivary gland protein profile of P. papatasi. M: Molecular weight marker (Cin-naGen corporation, catalog number: SL7001 (PR901641), 11–100 kDa). The arrows show protein bands.

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Determination of protein concentration and analysis

According to physical methods of extraction, the protein content of 20 pairs of whole-salivary glands was achieved approximately 0.03 μg/μl protein based on estimating complete lobe pairs from female P. papatasi. It was extracted in 100 μl PBS so the protein content was 3 μg. In the chemical method, the protein content of five pairs of salivary glands was attained about 0.5 μg/μl then it was 50 μg in 100 μl PBS as extraction buffer. After the SDS-PAGE analysis, an electrophoretic separation profile of all salivary gland proteins was done, which showed separated bands with their molecular weights. This profile revealed 15 proteins or peptides and minor and major protein bands observed between 11 to 100 kDa [Figure 3]B.

Purification of proteins by RP-HPLC and isolation of PpSP15

15 fractions of salivary gland proteins were yielded using RP-HPLC on C18 column with a gradient protocol. The absorbance of the isolated proteins was read at 280 and 214 nm. The maximum optical density of the fractions was 400 mAU (milli Absorbance Unit). SDS-PAGE of all collected fractions illustrated that the fraction 15, which was isolated from 80 to 85 minutes was 15 kDa and PpSP15 was confirmed [Figure 4]. This fraction was presented from 80 to 85 minutes with 63.185% area under the curve [Figure 5].
Figure 4: Electrophoretic profile of PpSP15. It was analyzed by SDS-PAGE using 15% polyacrylamide gel and stained with silver nitrate. M: Molecular weight marker (Fermentas, catalog number: SM0431, 14.4–116 kDa).

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Figure 5: HPLC chromatogram of salivary gland proteins of P. papatasi revealed 15 peaks in 95 min. The numbers over the curve describe the area under curve percentage. It is 63.185 % for PpSP15 and refers to protein concentration.

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Statistical analysis

Tajima D index was positive. Applying Chi-square test as well as t-test and ANOVA test for the analysis of mean differences between protein bands number in physical and chemical methods of extraction revealed P-value was -0.01 < 0.05 which was considered statistically significant.


  Discussion Top


In this work, we present the purification and isolation of PpSP15 from Iranian P. papatasi salivary glands as well as a new technique of protein extraction from sand fly salivary glands. The objective of this investigation was characterization of the main proteins in the salivary glands of P. papatasi, also identifying an effective protein band from SDS-PAGE presenting SP15 which provides protection against co-inoculation of L. major plus SGH[26]. These data can help for further understanding of immunity of sand fly salivary gland proteins and may confer protection to subsequent Leishmania infection[24]. Only P. papatasi is judged as proven vector of ZCL in Iran[9]. The salivary gland proteins’ profiles of P. papatasi in different ZCL foci were considered, selected and analyzed [Table 2].
Table 2: Protein extraction methods of the sand flies salivary glands. Different protein extraction methods and protein content were compared from the salivary glands of P. papatasi between various endemic foci of the old world and this investigation.

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The secreted salivary gland proteins in the P. papatasi are estimated to contain between 30 and 35 different protein molecules[12]. Some of these molecules are related to exacerbation such as yellow-related proteins, and some are related to protection; one of those molecules is the PpSP15. The protection against L. major by PpSP15 in animals has been confirmed[26]. Also, PpSP15 was shown to produce delayed type hypersensitivity (DTH) response in animals and humans[4],[12]. Under laboratory conditions, this immune response helps the sand fly to probe and feed DTH sites in human skin faster than normal sites[4]. The strategy of sand fly SGH enhances Leishmania transmission when co-inoculated with parasites in naive mice but not in mice pre-exposed to sand fly bites and also sand fly saliva has immunomodulatory components that are possible in selecting vaccine candidates from the salivary glands of sand flies[26].

To reach the overarching goal of this investigation, the extraction and illustration of all salivary gland proteins was inevitable. PpSP15 was isolated, purified, and identified from Iranian P. papatasi for the purpose of vaccine production against leishmaniasis. The collection of sand flies and storage were done under cold conditions and transported immediately to the laboratory to avoid protein damage. Regarding the low protein content from a low number of samples, different methods of extractions were employed and compared then a new extraction method was designed to maintain the quantity and quality of proteins[19],[20],[24]. The advantage of this new method is that the extracted proteins remain stable with no change in the conformation. Using the lysis-buffer, including the SDS reagent, does not affect the proteinacous nature of the extracted proteins[25]. SDS-PAGE confirmed 15 separated proteins or peptides, which were successfully extracted in the range of 11–100 kDa [Figure 3]B.

Using the freeze-thaw protocol, only proteins were extracted but not purified[13]. Using a small amount of salivary glands in our investigations (5 pairs), 15 separated bands were identified from wild-caught sand flies from 11 to 100 kDa. There is a significant difference of protein bands between the wild caught and reared P. papatasi (P < 0.05)[27]. Twenty specimens were extracted in this research using the freeze-thawing, homogenizing, and sonication methods and only four bands (more than 29 kDa) were revealed. But using the lysis buffer from five specimens, 15 separated bands were illustrated. The chemical technique using the lysis buffer is a more sufficient extracting protein method than the physical methods for salivary glands of sand flies. We found different number of protein bands with the same extraction method among P. papatasi specimens, this could be because of different protein composition of P. papatasi salivary glands i.e. loss of some protein or peptides in different ecologic and physiologic conditions [Table 2]. Tajima’s D Index was positive. The positive or negative variation for Tajima’s D Index in new modified chemical and physical protein extraction methods could be justified to compatibility in different circumstances[28],[29]. Salivary gland proteins including PpSP15 have specificity for the genus, species and subspecies[4],[24] because of differential expression and post-transcription modulations[10]. Also, various physiological, ecological, environmental conditions, and biotic or abiotic factors potentially influence sand fly capacity for parasite transmission as well as the outcome of diseases[13].

Using RP-HPLC, the percentage of area under the curve for PpSP15 was 63.185%, which was much more than other isolated proteins and this showed that the concentration of PpSP15 is more than other isolated proteins and peptides in the salivary extract composition [Figure 5][13]. Our research project is ongoing, and further investigation of salivary gland proteins is needed to submit the Iranian PpSP15 in the Protein Data Bank (www.rcsb.org). We can conclude that salivary gland proteins including PpSP15 are varied for sand fly species, origin, and where they were sampled. The specificity and variability of PpSP15 in ZCL foci have to be considered for each region in order to produce a vaccine. Also, an efficient protein extraction method is very important to have enough quantity and quality of salivary gland proteins.


  Conclusion Top


More than 80 sand fly species including P.papatasi are responsible in Leishmania transmission. It is important to describe local sand fly salivary proteins in the research for vaccine candidates and local markers of exposure. In this study, we prepared and analyzed the purification and isolation of PpSP15 from Iranian P. papatasi salivary glands and provide a new protein extraction technique. The salivary proteins of P. papatasi is homologous to compare with vectors of zoonotic cutaneous leishmaniasis (ZCL), human cutaneous leishmaniasis (ACL) or visceral cutaneous leishmaniasis (VCL)[19]. The divergence, diversity or amount of sand fly salivary proteins or non-proteinaceous components of the saliva could be correlated with different disease manifestations of the same species of Leishmania[30]. The purification and isolation of PpSP15 from Iranian P. papatasi salivary glands data can be used to as anti-Leishmania vaccines or in epidemiologic studies.


  Acknowledgements Top


The collections of sand flies were made possible by the assistance of Dr. Ali Bordbar and Sahar Ebrahimi who work in Molecular Systematics Laboratory.

Conflict of interest: None



 
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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]
 
 
    Tables

  [Table 1], [Table 2]



 

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