Bacteriolytic activity in saliva of the hematophagous Triatoma infestans (Reduviidae) and novel characterization and expression site of a third lysozyme.

Abstract

Saliva of hematophagous insects contains many different compounds, mainly acting as anticoagulants. Investigating the bacteriolytic compounds of the saliva of the bloodsucking Triatoma infestans photometrically between pH 3 and pH 10 using unfed fifth instars and nymphs up to 15 days after feeding, we found bacteriolytic activity against lyophilized Micrococcus luteus was stronger at pH 4 and pH 6. After feeding, the activity level at pH 4 was unchanged, but at pH 6 more than doubled between 3 and 7 days after feeding. In zymographs of the saliva and after incubation at pH 4, bacteriolytic activity against Micrococcus luteus was present at eight lysis zones between 14.1 and 38.5 kDa, showing the strongest activity at 24.5 kDa. After incubation at pH 6, lysis zones only appeared at 15.3, 17, and 31.4 kDa. Comparing zymographs of the saliva of unfed and fed nymphs, bacteriolytic activity at 17 kDa increased after feeding. In total nine lysis bands appeared, also at >30 kDa, so far unreported in the saliva of triatomines. Reverse transcription polymerase chain reaction usingoligonucleotides based on the previously described lysozyme gene of T. infestans, TiLys1, verified expression of genes encoding TiLys1 and TiLys2 in the salivary glands, but also of an undescribed third lysozyme, TiLys3, of which the cloned cDNA shares characteristics with other c-type lysozymes of insects. While TiLys1 was expressed in the tissue of all three salivary glands, transcripts of TiLys2 and of TiLys3 seem to be present only in the gland G1 and G3, respectively.