Unknown,Transcriptomics,Genomics,Proteomics

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Gene expression analysis of late exponential vs. stationary phase cultures using a custom designed Pseudo-nitzschia multiseries (Ps-n) cDNA microarray

ABSTRACT: Pseudo-nitzschia multiseries (Ps-n) is a toxigenic marine diatom that produces the neurotoxin, domoic acid. We screened for candidate genes that may be involved in domoic acid production by determining changes in transcript profiles in Ps-n cultures that were in late exponential (low-domoic-acid-producing) vs. stationary (high-domoic-acid-producing) growth states. We also identified a number of candidate reference genes for future RT-qPCR studies, based on their stability in this study. Ps-n RNA was extracted from late exponential and stationary phase cultures. Experiments included one axenic culture, and two non-axenic cultures. Experiments were dye-swapped to account for differences in dye labeling and detection efficiencies: a) the axenic culture experiment included 4 technical replicate arrays (i.e., 2 dye-swapped experiments = 4 total hybridizations), and b) the non-axenic culture experiments each included 6 technical replicate arrays (i.e., 3 dye-swapped experiments = 6 total hybridizations).

ORGANISM(S): Pseudo-nitzschia multiseries

SUBMITTER: Katie Rose Boissonneault

PROVIDER: E-GEOD-46845 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Protozoa of the genus Leishmania are the causative agents of leishmaniasis in humans. These parasites cycle between promastigotes in the sand fly mid-gut and amastigotes in phagolysosome of mammalian macrophages. During infection, host up-regulate nitric oxide synthase and parasite induce host arginase expression, both of which use arginine as a substrate. These elevated activities deplete macrophage arginine pools, a situation that invading Leishmania must overcome since it is an essential amino acid. Leishmania donovani imports exogenous arginine via a mono-specific amino acid transporter (AAP3) and utilizes it primarily through the polyamine pathway to provide precursors for trypanothione biosynthesis. Here we report the discovery of a pathway whereby promastigote and amastigote forms of the Leishmania sense the lack of environmental arginine and respond with rapid up-regulation in AAP3 expression and activity, as well as several other transporters. Significantly, this arginine deprivation response is also activated in parasites during macrophage infection. Phosphoproteomic analyses of L. donovani promastigotes have implicated a Mitogen-Activated Protein Kinase 2 (MPK2)-mediated signaling cascade in this response and L. mexicana mutants lacking MPK2 are unable to respond to arginine deprivation. In this study, we established that Leishmania cells sense the absence of arginine in their environment; both in culture (axenic promastigotes and amastigotes) and in macrophages during infection (amastigotes). This study describes the first amino acid deprivation sensing mechanism and the pathway that transduce this response, and reveals a novel host-pathogen metabolic interplay. Total RNA from Ten Leishmania donovani samples were analyzed using RNA-Seq. Cells from two life stages (promastigotes and amastigotes) were grown in axenic culture in the presence and absense of arginine. For each condition two biological replicates were grown and analyzed. In addition two macrophage grown amastigotes were analyzed.

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Gene expression analysis of late exponential vs. stationary phase cultures using a custom designed Pseudo-nitzschia multiseries (Ps-n) cDNA microarray

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