Project description:A laboratory colony of Phlebotomus perniciosus sand flies was maintained. Sand flies were infected with cultured Leishmania infantum promastigotes in stationary phase. Ten infected sand flies were dissected after 5 days and promastigotes within the gut pooled. The cells were immediately washed in PBS once and lysed in TRIzol reagent (Life Technologies). RNA isolation was completed according to the manufacturer's instructions, obtaining 63ng. RNA-seq libraries were generated using the spliced leader sequence for second strand synthesis (Cuypers et al., 2017; Haydock et al., 2015), thus allowing for specific amplification of sequences from L. infantum promastigotes, thus avoiding contamination with material from the sand fly gut. Single-end sequencing was performed in an Illumina HiSeq2500 instrument and data analysis was conducted using bowtie2, samtools, featureCounts and Geneious. The main findings are: i) substantial differences in differential gene expression between sand fly-derived (sfPro) and cultured (acPro) promastigotes; and ii) over-expression of genes involved in metacyclogenesis in sfPro vs. acPro, including gp63 genes, autophagy genes, etc.
Project description:The debilitating disease kala-azar or visceral leishmaniasis (VL) is caused by the kinetoplastid protozoan parasite Leishmania donovani. The parasite is transmitted by the hematophagous sandfly vector of the genus Phlebotomus in the old world and Lutzomyia in the new world. The predominant Phlebotomine species associated with transmission of kala-azar are Phlebotomus papatasi and Phlebotomus argentipes. The infected female sandfly transmits the parasite when it takes a blood meal. Understanding the molecular interaction of the sand fly-Leishmania during the development of parasite within the gut of the sandfly is crucial to understanding parasite life cycle. The complete genome sequences of sandfly vectors (Phlebotomus and Lutzomyia) are currently not available and sequencing efforts are underway. Non-availability of genome sequence can hamper identification of proteins in the sandfly vector. In the present study we have carried out proteogenomic analysis of unsequenced sandfly vector P. paptasi cell line using high-resolution mass spectrometry and comparative homology-based searches using related dipteran protein data (mosquitoes and fruit fly). This study resulted in identification of 1,312 proteins from P. papatasi based on homology. Our study demonstrates the power of proteogenomic approaches in mapping the proteomes of unsequenced organisms.