Culex quinquefasciatus Late Trypsin Biosynthesis Is Translationally Regulated by Trypsin Modulating Oostatic Factor

Abstract

Trypsin is a serine protease that is synthesized by the gut epithelial cells of female mosquitoes; it is the enzyme that digests the blood meal. To study its molecular regulation, Culex quinquefasciatus late trypsin was purified by diethylaminoethyl (DEAE), affinity, and C18 reverse-phase high performance liquid chromatography (HPLC) steps, and the N-terminal amino acid sequence was determined for molecular cloning. Five overlapping segments of the late trypsin cDNA were amplified by PCR, cloned, and the full sequence (855 bp) was characterized. Three-dimensional models of the pro-trypsin and activated trypsin were built and compared with other trypsin models. Trypsin modulating oostatic factor (TMOF) concentrations in the hemolymph were determined by ELISA and compared with trypsin activity in the gut after the blood meal. The results showed that there was an increase in TMOF concentrations circulating in the hemolymph which has correlated to the reduction of trypsin activity in the mosquito gut. Northern blot analysis of the trypsin transcripts after the blood meal indicated that trypsin activity also followed the increase and decrease of the trypsin transcript. Injections of different amounts of TMOF (0.025 to 50 μg) decreased the amounts of trypsin in the gut. However, Northern blot analysis showed that TMOF injections did not cause a decrease in trypsin transcript abundance, indicating that TMOF probably affected trypsin translation.

The Ribosome Is the Ultimate Receptor for Trypsin Modulating Oostatic Factor (TMOF)

“…We observed that AeaTMOF belongs to a family of proline-rich peptides that were shown to act as antimicrobial peptides by inhibiting the translation of proteins by bacterial ribosomes like Oncocin112 [13][14][15]. Therefore, we performed in vitro transcript/translation experiments using the E. coli 30S lysate extract system (Promega) with increasing concentrations of AeaTMOF or Oncocin112 (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13). The latter was shown by X-ray crystallography to occupy the ribosomal exit tunnel at its entrance, blocking tRNA movement, mRNA translation, and protein biosynthesis [14,15].…”

“…To test the effect of AeaTMOF on the translation of larval Ae. aegypti late trypsin, the TnT T7 insect cell extract protein expression system from S. frugiperda Sf21 cell line (Promega) was incubated with different concentrations of AeaTMOF in a reaction mixture containing TNT T7 ICE master mix of 10 µL, pFA25A ICE T7 flexi-late larval Trypsin (0.45 µg/mL) (see Section 2.2) 0.5 µL, [3,4,5 3 H]Leucine 0.5 µL, AeaTMOF (0.01 to 1000 µM), and nuclease-free water to a total volume of 12.5 µL. The incubation mixtures were incubated at 30 • C for 4 h. At the end of the incubation period, aliquots (10 µL) were removed and spotted on filter paper squares (1 × 1 cm) and washed in cold 10% TCA for 10 min, and twice in cold 5% TCA for 5 min each followed by 2 min with ethanol (100%).…”

“…The 96-well plate was incubated at 37 • C for 18 h and bacterial growth was followed at 630 nm using Biotech ElX808 absorbance microplate reader. Control wells contained bacterial cells growing without AeaTMOF or oncocin112 (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13). In a second experiment, E. coli mutant CGSC strain 8547 (2.7 × 10 4 cells) SbmAthat do not express the Oncocin112 importer SbmA but do express the ABC tmf A importer [7] were incubated in 100 µL of LB medium with AeaTMOF and oncocin112 (1-13) (3 mM and 10 mM) as described above for 18 h following bacterial growth.…”

“…In a second experiment, E. coli mutant CGSC strain 8547 (2.7 × 10 4 cells) SbmAthat do not express the Oncocin112 importer SbmA but do express the ABC tmf A importer [7] were incubated in 100 µL of LB medium with AeaTMOF and oncocin112 (1-13) (3 mM and 10 mM) as described above for 18 h following bacterial growth. After the incubations in the presence of Oncocin112 (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13) or AeaTMOF of 10 mM and 3 mM, aliquots (0.5 µL) were removed from each well and spread on LB agar plates containing 0.1 mM of ampicillin, and the plates were incubated overnight at 37 • C and viable colonies were counted. Incubations were done in triplicates and the experiments were repeated twice.…”

“…aegypti and Cx. quinquefasciatus after the blood meal and affects the translation of the late trypsin transcript in the gut epithelial cells [2,3]. AeaTMOF was also shown to inhibit the translation of the late trypsin transcript in Neobellieria [4] and in Heliothis virenscens.…”