Project description:In addressing R. microplus - A. marginale interactions, we propose and test three linked hypotheses. The first is that the tick gene response is organ specific: the midgut gene regulation is unique during feeding and during acquisition of A. marginale as compared to the salivary gland. This distinction is relevant as the two organs serve very different roles in the transmission biology of A. marginale with early survival and replication within the midgut epithelium, composed of highly phagocytic cells, required for initial colonization while a second round of replication in the salivary gland acini, composed of highly secretory cells, is required for transmission of an infectious dose in the saliva. Importantly, both the midgut epithelium and salivary glands have been identified as separate and distinct barriers for transmission of A. marginale and thus represent two potential sites where transmission could be blocked. The second hypothesis to be tested is that the salivary gland transcriptome is temporally dynamic. Initiation of tick attachment and feeding involves secretion of a virtual pharmacopeia including lytic enzymes, anticoagulants, and inhibitors of the mammalian innate immune and nocioceptive systems. Concomitantly, the acini provide an environment where A. marginale replicates >100 fold and are secreted into the saliva. Prior studies show that duration of feeding is a critical component of transmission efficiency, with increased efficiency positively correlated with time of tick feeding. The third hypothesis to be tested is that A. marginale colonization does not significantly modulate the tick midgut and salivary gland transcriptome. This hypothesis is based on observations by ourselves and others that tick infection does not impart a significant fitness cost on the vector. This is in contrast to other bacterial and protozoal pathogens that have dramatic effects on success of tick attachment, engorgement, and survival. A. marginale, similar to other tick-borne pathogens in the Family Anaplasmataceeae, is believed to have evolved from an arthropod-specific bacterium with relatively late adaptation to specific niches in mammalian hosts. Consequently, we predict that A. marginale is well adapted to its tick vector and utilizes the normal signaling pathways of the feeding tick with few, if any, effects on the midgut and salivary gland transcriptome. In this manuscript, we report the testing of these three hypotheses and present the results in context of the vector-pathogen-mammalian host interaction at the time of transmission. A Roche NimbleGen high-density gene expression microarray was custom designed based on the expressed sequence tag (EST) database, B. microplus Gene Index Version 2 (BmiGI V2) for R. microplus. The expression level of 14,447 R. microplus genes was analyzed from total RNA extracted from 10 different tick tissue samples; 30 arrays were included since triplicates of each different sample were analyzed as follow: unfed (midgut and salivary glands), blood feeding (2 days midgut and 2, 6 and 9 days salivary glands), A. marginale-infected blood feeding (2 days midgut and 2, 6 and 9 days salivary glands).
Project description:In addressing R. microplus - A. marginale interactions, we propose and test three linked hypotheses. The first is that the tick gene response is organ specific: the midgut gene regulation is unique during feeding and during acquisition of A. marginale as compared to the salivary gland. This distinction is relevant as the two organs serve very different roles in the transmission biology of A. marginale with early survival and replication within the midgut epithelium, composed of highly phagocytic cells, required for initial colonization while a second round of replication in the salivary gland acini, composed of highly secretory cells, is required for transmission of an infectious dose in the saliva. Importantly, both the midgut epithelium and salivary glands have been identified as separate and distinct barriers for transmission of A. marginale and thus represent two potential sites where transmission could be blocked. The second hypothesis to be tested is that the salivary gland transcriptome is temporally dynamic. Initiation of tick attachment and feeding involves secretion of a virtual pharmacopeia including lytic enzymes, anticoagulants, and inhibitors of the mammalian innate immune and nocioceptive systems. Concomitantly, the acini provide an environment where A. marginale replicates >100 fold and are secreted into the saliva. Prior studies show that duration of feeding is a critical component of transmission efficiency, with increased efficiency positively correlated with time of tick feeding. The third hypothesis to be tested is that A. marginale colonization does not significantly modulate the tick midgut and salivary gland transcriptome. This hypothesis is based on observations by ourselves and others that tick infection does not impart a significant fitness cost on the vector. This is in contrast to other bacterial and protozoal pathogens that have dramatic effects on success of tick attachment, engorgement, and survival. A. marginale, similar to other tick-borne pathogens in the Family Anaplasmataceeae, is believed to have evolved from an arthropod-specific bacterium with relatively late adaptation to specific niches in mammalian hosts. Consequently, we predict that A. marginale is well adapted to its tick vector and utilizes the normal signaling pathways of the feeding tick with few, if any, effects on the midgut and salivary gland transcriptome. In this manuscript, we report the testing of these three hypotheses and present the results in context of the vector-pathogen-mammalian host interaction at the time of transmission.
Project description:In order to select genes that are differentially expressed in salivary glands during Ixodes ricinus infection by Bartonella henselae we compare the transcriptome of infected and non-infected salivary glands
Project description:In spite of the many recent developments in the field of vector sialomics, the salivary glands of larvalmosquitoes have been largely unexplored. We used whole-transcriptome microarray analysis to create a gene-expression profile of the salivary gland tissue of fourth-instar Anopheles gambiae larvae, and compare it to the gene-expression profile of a matching group of whole larvae. We identified a total of 221 probes with expression values that were (a) significantly enriched in the salivary glands, and (b)sufficiently annotated as to allow the prediction of the presence/absence of signal peptides in their corresponding gene products. Based on available annotation of the protein sequences associated with these probes, we propose that the main roles of larval salivary secretions include: (a) immune response, (b) mouthpart lubrication, (c) nutrient metabolism, and (d) xenobiotic detoxification. Other highlights of the study include the cloning of a transcript encoding a previously unknown salivary defensin (AgDef5), the confirmation of mucus secretion by the larval salivary glands, and the first report of salivary lipocalins in the Culicidae. Keywords: Anopheles gambiae, salivary gland, Diptera, gene expression, salivary defensin, transcriptome, salivary lipocalin
Project description:Purpose: Next-generation sequencing (NGS) has revolutionized systems-based analysis of cellular functions. The goal of this study is to compare NGS-derived salivary gland transcriptome profilings (RNA-seq) to better understand the molecular nature of the physiological differences in adult murine salivary glands. Methods: Major murine salivary gland mRNA profiles were generated by deep sequencing, in triplicate, using Illumina HiSeq 2000. The sequence reads that passed quality filters were analyzed at the gene level with STAR followed by Cufflinks. In vivo NaCl reabsorption measurements were performed for validation. Results: Using an optimized data analysis workflow, we mapped about 15 million sequence reads per sample to the mouse genome (build mm10) and identified 1991 genes that were differentially expressed across three major salivary glands. RNA-seq data provided valuable insights into the nature of the functional differences among the major salivary glands Conclusions: Our study represents the first detailed analysis of murine salivary gland transcriptomes, with biologic replicates, generated by RNA-seq technology. Our results confirm functions of many genes, identified using genetically modified mice. We conclude that RNA-seq-based transcriptome characterization would offer a comprehensive and sensitive evaluation of the gene expression.
Project description:Saliva aids in the predigestion of food and perception of taste. It helps to maintain the integrity of the mineralized tooth and epithelial surfaces in the mouth, and shields the oro-digestive tract from environmental hazards and invading pathogens. Although salivary glands and saliva fluid are biologically and functionally inseparable, they have thus far been investigated as separate entities. To bridge this gap, we performed an integrative analysis of the transcriptome of 27 samples collected from the major human adult and fetal major salivary glands - submandibular, sublingual, and parotid - along with mass-spectrometry-based saliva proteome data and immunohistochemical localization in glandular tissue. Our results suggest that functional maturation at the transcriptome level occurs late in gland development, and is driven mainly by the transcription of genes that code for secreted saliva proteins. We further provide evidence that protein dosage of the most abundant salivary proteins secreted by the salivary glands is predominantly regulated at the transcriptome level. Finally, we demonstrate distinct transcriptomic profiles of each major salivary gland type that reveal functional specialization and will aid in future clinical analyses. Our study provides the hitherto most comprehensive RNAseq dataset of healthy salivary glands in humans, thus establishing a robust framework for deeper studies of saliva and salivary gland biology, development, and evolution, ultimately paving the way for better understanding the importance of these craniofacial secretory organs in health and their malfunctions in disease.
Project description:We analyzed ORC2 ChIP-Seq from hand dissected salivary glands of wandering third instar larvae from OrR or SuUR Drosophila. Goals were to ascertain the difference in binding profile between salivary glands expressing and not expressing the Supressor of UnderReplication protein. One replicate is included for each of OrR (WT) or SuUR salivary glands.