Identification and characterization of a salivary adenosine deaminase from the sand fly Phlebotomus duboscqi, the vector of Leishmania major in sub-Saharan Africa

Abstract

SUMMARY Two transcripts coding for an adenosine deaminase (ADA) were identified by sequencing a Phlebotomus duboscqi salivary gland cDNA library. Adenosine deaminase was previously reported in the saliva of the sand fly Lutzomyia longipalpis but it was not present in the saliva of the sand flies Phlebotomus papatasi, P. argentipes, P. perniciosus and P. ariasi, suggesting that this enzyme is only present in the saliva of sand flies from the genus Lutzomyia. In the present work, we tested the hypothesis that the salivary gland transcript coding for ADA in Phlebotomus duboscqi, a sister species of Phlebotomus papatasi, produces an active salivary ADA. Salivary gland homogenates of P. duboscqi converted adenosine to inosine, suggesting the presence of ADA activity in the saliva of this species of sand fly; furthermore, this enzymatic activity was significantly reduced when using either salivary glands of recently blood-fed sand flies or punctured salivary glands, suggesting that this enzyme is secreted in the saliva of this insect. This enzymatic activity was absent from the saliva of P. papatasi. In contrast to other Phlebotomus sand flies, we did not find AMP or adenosine in P. duboscqi salivary glands as measured by HPLC-photodiode array. To confirm that the transcript coding for ADA was responsible for the activity observed in the saliva of this sand fly,we cloned this transcript into a prokaryotic expression vector and produced a soluble and active recombinant protein of approximately 60 kDa that was able to convert adenosine to inosine. Extracts of bacteria transformed with control plasmids did not show this activity. These results suggest that P. duboscqi transcripts coding for ADA are responsible for the activity detected in the salivary glands of this sand fly and that P. duboscqiacquired this activity independently from other Phlebotomus sand flies. This is another example of a gene recruitment event in salivary genes of blood-feeding arthropods that may be relevant for blood feeding and,because of the role of ADA in immunity, it may also play a role in parasite transmission.

Las glándulas salivales de dos flebotominos vectores de Leishmania: Lutzomyia migonei (França) y Lutzomyia ovallesi (Ortiz) (Diptera: Psychodidae)

“…Other authors report that after being fed with blood or carbohydrates, the total protein content of sand fly saliva can decrease by half or more of its original quantity (7,39,45,46). These observations provide support for the differences in gland size described above.…”

The Immune Response to Sand Fly Salivary Proteins and Its Influence on Leishmania Immunity

“…The direct effect of the adenosine from the saliva on in vitro stimulation may overwhelm the acquired immune response to sand fly salivary proteins. Adenosine has been shown to be present in large quantities in P. papatasi and P. argentipes saliva (Ribeiro et al, 1999), but absent from L. longipalpis and P. duboscqi (Kato et al, 2007). This fact may explain the discrepancy between the results of stimulation with L. longipalpis and P. papatasi salivary glands in humans.…”

Ayadualin, a novel RGD peptide with dual antihemostatic activities from the sand fly Lutzomyia ayacuchensis, a vector of Andean-type cutaneous leishmaniasis

“…Although ayadualin also inhibited kallikrein, FIXa and FXa activities, the inhibition was lower than that of FXIIa. These and other insects [16,33,[45][46][47]. On the other hand, only a few structurally unique proteins identified from sand fly saliva have been functionally characterized; maxadillan as a strong vasodilator [48] and lufaxin as a factor Xa inhibitor [49], both from Lu.…”