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. 2012;6(11):e1911.
doi: 10.1371/journal.pntd.0001911. Epub 2012 Nov 29.

Salivary antigen SP32 is the immunodominant target of the antibody response to Phlebotomus papatasi bites in humans

Soumaya Marzouki  1 , Maha Abdeladhim, Chaouki Ben Abdessalem, Fabiano Oliveira, Beya Ferjani, Dana Gilmore, Hechmi Louzir, Jesus G Valenzuela, Mélika Ben Ahmed
Affiliations

Salivary antigen SP32 is the immunodominant target of the antibody response to Phlebotomus papatasi bites in humans

Soumaya Marzouki et al. PLoS Negl Trop Dis. 2012.

Erratum in

Abstract

Background: Zoonotic cutaneous leishmaniasis (ZCL) due to Leishmania major is highly prevalent in Tunisia and is transmitted by a hematophagous vector Phlebotomus papatasi (P. papatasi). While probing for a blood meal, the sand fly injects saliva into the host's skin, which contains a variety of compounds that are highly immunogenic. We recently showed that the presence of anti-saliva antibodies was associated with an enhanced risk for leishmaniasis and identified the immunodominant salivary protein of Phlebotomus papatasi as a protein of approximately 30 kDa.

Methodology/principal findings: We cloned and expressed in mammalian cells two salivary proteins PpSP30 and PpSP32 with predicted molecular weights close to 30 kDa from the Tunisian strain of P. papatasi. The two recombinant salivary proteins were purified by two-step HPLC (High-Performance Liquid Chromatography) and tested if these proteins correspond to the immunodominant antigen of 30 kDa previously shown to be recognized by human sera from endemic areas for ZCL and exposed naturally to P. papatasi bites. While recombinant PpSP30 (rPpSP30) was poorly recognized by human sera from endemic areas for ZCL, rPpSP32 was strongly recognized by the tested sera. The binding of human IgG antibodies to native PpSP32 was inhibited by the addition of rPpSP32. Consistently, experiments in mice showed that PpSP32 induced the highest levels of antibodies compared to other P. papatasi salivary molecules while PpSP30 did not induce any detectable levels of antibodies.

Conclusions: Our findings demonstrate that PpSP32 is the immunodominant target of the antibody response to P. papatasi saliva. They also indicate that the recombinant form of PpSP32 is similar to the native one and represents a good candidate for large scale testing of human exposure to P. papatasi bites and perhaps for assessing the risk of contracting the disease.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Sequences of the Tunisian Phlebotomus papatasi 30 kDa salivary protein.
(A) mRNA sequence of PpSP30. (B) Protein sequence of PpSP30. Differences in the nucleotide and protein sequence PpSP30 described in the GenBank (GenBank AF335489.1) are highlighted in pink. Non coding sequence is highlighted in purple.
Figure 2
Figure 2. Sequences of the Tunisian Phlebotomus papatasi 32 kDa salivary protein.
(A) mRNA sequence of PpSP32. (B) Protein sequence of PpSP32. Differences in the nucleotide and protein sequence PpSP30 described in the GenBank (GenBank AF335490.1) are highlighted in pink. Non coding sequence is highlighted in purple.
Figure 3
Figure 3. Comparison of predicted B cell epitope between PpSP32 (GenBank AF335490.1) and PpSP32 (Tunisian strain).
Yellow shaded amino acids indicate lack of identity. Green shaded amino acids indicate a strong B cell epitope predicted by the bcepred software.
Figure 4
Figure 4. Staining of the recombinant proteins.
(A) Silver-stained SDS-PAGE gel of recombinant protein rPpSP30. (B) Commassie blue-stained SDS-PAGE gel of recombinant protein rPpSP32. (C) Western blot analysis of rPpSP32 using monoclonal anti-polyhistidine antibody.
Figure 5
Figure 5. Identification of the different rPpSP32 bands by mass spectrometry.
Recombinant PpSP32 (6 µg/well) was run on a 15% SDS- PAGE gel. Seven bands were cut from the gel after staining with Coomassie-blue and mass spectrometry was performed. The RPN800E molecular weight marker (Amersham) was used.
Figure 6
Figure 6. Correlation between the ELISA tests using rPpSP32 and total salivary extract.
Twenty-four negative (white circles) and forty-two positive sera (black circles) from donors living in areas with high prevalence of P. papatasi previously tested with the total salivary gland extract (SGE) were included.
Figure 7
Figure 7. Western blot analyses of native and recombinant salivary proteins.
(A) Salivary gland extract (SGE) as well as the recombinant forms of PpSP30 (rPpSP30) and PpSP32 (rPpSP32) were run on a 15% SDS-PAGE gel. Western blot analysis was performed with positive (S+) and negative (S−) human sera from donors living in areas with high prevalence of P. papatasi. Results are representative of three independent experiments. (B) Sera tested positive with IgG anti-SGE were pre-incubated with the recombinant proteins PpSP32 and/or rPpSP30 at 10 µg/ml and then tested in Western blot against SGE. Results are representative of three independent experiments. The arrows indicate the emplacement of the immunodominant protein.
Figure 8
Figure 8. Specificity of rPpSP32 ELISA test towards P. papatasi species.
Twenty sera obtained from donors living in the North regions of Tunisia where P. pernicious is prevalent and P. papatasi is absent were tested by ELISA using rPpSP32. Ten negative sera (C−) and fifteen positive sera (C+) from donors living in the regions where P. papatasi is prevalent were also included. The threshold of positivity was the mean optical density (OD) of negative controls plus 3 standard deviations.
Figure 9
Figure 9. ELISA and Western blot of sera of mice vaccinated with DNA plasmids.
(A) C57BL/6 mice were immunized three times at two weeks interval with their DNA plasmids coding for the most abundant P. papatasi salivary proteins and sera obtained 2 weeks after last immunization. Total anti-saliva IgG was measured by ELISA. Five mice were used in each group. Data are representative of three independent experiments. Bars represent mean ± SD. (B) Western blot showing recognition of P. papatasi salivary proteins by sera of mice injected with different DNA plasmid coding for P. papatasi salivary proteins. Mice sera of each group of plasmids were pooled before testing.
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