Trypsin-Like Serine Proteases in Lutzomyia longipalpis – Expression, Activity and Possible Modulation by Leishmania infantum chagasi

Background: Midgut enzymatic activity is one of the obstacles that Leishmania must surpass to succeed in establishing infection. Trypsins are abundant digestive enzymes in most insects. We have previously described two trypsin cDNAs of L. longipalpis: one (Lltryp1) with a bloodmeal induced transcription pattern, the other (Lltryp2) with a constitutive transcription pattern. We have now characterized the expression and activity of trypsin-like proteases of Lutzomyia longipalpis, the main vector of visceral leishmaniasis in Brazil. Methodology and Principal Findings: In order to study trypsin expression profiles we produced antibodies against peptides specific for Lltryp1 and Lltryp2. The anti-Lltryp1-peptide antibody revealed a band of 28 kDa between 6 and 48 hours. The anti-Lltryp2 peptide antibody did not evidence any band. When proteinaceous substrates (gelatin, hemoglobin, casein or albumin) were co-polymerized in polyacrylamide gels, insect midguts obtained at 12 hours after feeding showed a unique proteolytic pattern for each substrate. All activity bands were strongly inhibited by TLCK, benzamidine and 4-amino-benzamidine, indicating that they are trypsin-like proteases. The trypsin-like activity was also measured in vitro at different time points after ingestion of blood or blood containing Leishmania infantum chagasi, using the chromogenic substrate BArNA. L. longipalpis females fed on blood infected with L. i. chagasi had lower levels of trypsin activity after 12 and 48 hours than non-infected insects, suggesting that the parasite may have a role in this modulation. Conclusions and Significance: Trypsins are important and abundant digestive enzymes in L. longipalpis. Protein production and enzymatic activity followed previously identified gene expression of a blood modulated trypsin gene. A decrease of enzymatic activity upon the parasite infection, previously detected mostly in Old World vectors, was detected for the first time in the natural vector-parasite pair L. longipalpis-L. i. chagasi

Zymography performed with gelatin co-polymerized gel, incubated in different pH defined buffers.

<p><b>4</b> to <b>12</b>: pH of buffers. Samples corresponding to 1/50 of one insect midgut obtained at 12 h ABF were loaded in lanes. Molecular mass standard values (kDa) are indicated by numbers and arrows on the left side of the figure.</p

Enzymatic assay performed in solution with trypsin synthetic substrate BAρNA.

<p>Horizontal axis indicates insect midgut samples obtained at different hours ABF. Vertical axis indicate enzyme units detected per midgut. <b>0</b>: sugar-fed females; <b>6</b> to <b>72</b>: females obtained at 6, 12, 24, 48 and 72 hours ABF. White bars indicate samples obtained from non infected insect midguts. Gray bars indicate samples obtained from infected insect midguts. Samples corresponding to half of one midgut were used in each assay. Statistically significant difference (<i>P</i><0.05) is marked with an asterisk.</p

Zymography performed with gelatin co-polymerized gel, incubated at pH 8 with different protease inhibitors, as indicated.

<p>Samples corresponding to 1/50 of 1 insect midgut obtained at 12 h ABF were loaded in the gel. Molecular mass standard values (kDa) are indicated by numbers and arrows on the left side of the figure.</p

Zymography performed with gels co-polymerized incubated at pH 8 with different protein substrates.

<p><b>G</b>: gelatin; <b>C</b>: casein; <b>H</b>: hemoglobin; <b>B</b>: BSA. Samples corresponding to 1/50 of 1 insect midgut obtained at 12 h ABF were loaded in lanes <b>G</b> and <b>C</b>, and 1/10 of 1 insect midgut obtained at 12 h ABF in lanes <b>H</b> and <b>B</b>. Molecular mass standard values (kDa) are indicated by numbers and arrows on the left side of the figure.</p

Western blot performed with anti-Lltryp1-peptide antibody.

<p><b>0</b>: non-fed female; <b>6</b> to <b>72</b>: females obtained at 6, 12, 24, 48 and 72 hours ABF; <b>B</b>: 2 µL of hamster blood. Samples corresponding to 2 insect midguts were loaded in lanes <b>0</b> to <b>72</b>. Molecular mass standard values (kDa) are indicated by numbers and arrows on the left side of the figure.</p

Zymography performed with gelatin co-polymerized gel incubated at pH 8 with samples obtained at different times ABF.

<p>M: males, <b>F</b>: females; <b>0</b>: sugar-fed females; <b>6</b> to <b>72</b>: females obtained at 6, 12, 24, 48 and 72 hours ABF; <b>B</b>: 2 µL of hamster blood. Samples corresponding to 1/50 of 1 insect midgut were loaded in lanes <b>M</b> and <b>0</b> to <b>72</b>. A control sample contained 1/50 of 2 µL of hamster blood. Molecular mass standard values (kDa) are indicated by numbers and arrows on the left side of the figure.</p

Alignment of deduced amino acid sequences of <i>L. longipalpis</i> trypsins (Lltryp1 and Lltryp2).

<p>Conserved residues are shown as white letter on black background. Non-conserved residues are shown as black letters on a white background. Black letters on gray background indicate conserved substitutions. The predicted secretory signal peptides for Lltryp1 (residues 2–17) and Lltryp2 (residues 1–19) are indicated by a traced line. Lltryp1 and Lltryp2-specific peptides used for immunization procedures are boxed (residues 89 to 111).</p