PheMaDB: a solution for storage, retrieval, and analysis of high throughput phenotype data.

BACKGROUND: OmniLog™ phenotype microarrays (PMs) have the capability to measure and compare the growth responses of biological samples upon exposure to hundreds of growth conditions such as different metabolites and antibiotics over a time course of hours to days. In order to manage the large amount of data produced from the OmniLog™ instrument, PheMaDB (Phenotype Microarray DataBase), a web-based relational database, was designed. PheMaDB enables efficient storage, retrieval and rapid analysis of the OmniLog™ PM data. DESCRIPTION: PheMaDB allows the user to quickly identify records of interest for data analysis by filtering with a hierarchical ordering of Project, Strain, Phenotype, Replicate, and Temperature. PheMaDB then provides various statistical analysis options to identify specific growth pattern characteristics of the experimental strains, such as: outlier analysis, negative controls analysis (signal/background calibration), bar plots, pearson's correlation matrix, growth curve profile search, k-means clustering, and a heat map plot. This web-based database management system allows for both easy data sharing among multiple users and robust tools to phenotype organisms of interest. CONCLUSIONS: PheMaDB is an open source system standardized for OmniLog™ PM data. PheMaDB could facilitate the banking and sharing of phenotype data. The source code is available for download at http://phemadb.sourceforge.net.

In vitro intracellular trafficking of virulence antigen during infection by Yersinia pestis.

Yersinia pestis, the causative agent of plague, encodes several essential virulence factors on a 70 kb plasmid, including the Yersinia outer proteins (Yops) and a multifunctional virulence antigen (V). V is uniquely able to inhibit the host immune response; aid in the expression, secretion, and injection of the cytotoxic Yops via a type III secretion system (T3SS)-dependent mechanism; be secreted extracellularly; and enter the host cell by a T3SS-independent mechanism, where its activity is unknown. To elucidate the intracellular trafficking and target(s) of V, time-course experiments were performed with macrophages (MPhis) infected with Y. pestis or Y. pseudotuberculosis at intervals from 5 min to 6 h. The trafficking pattern was discerned from results of parallel microscopy, immunoblotting, and flow cytometry experiments. The MPhis were incubated with fluorescent or gold conjugated primary or secondary anti-V (antibodies [Abs]) in conjunction with organelle-associated Abs or dyes. The samples were observed for co-localization by immuno-fluorescence and electron microscopy. For fractionation studies, uninfected and infected MPhis were lysed and subjected to density gradient centrifugation coupled with immunoblotting with Abs to V or to organelles. Samples were also analyzed by flow cytometry after lysis and dual-staining with anti-V and anti-organelle Abs. Our findings indicate a co-localization of V with (1) endosomal proteins between 10-45 min of infection, (2) lysosomal protein(s) between 1-2 h of infection, (3) mitochondrial proteins between 2.5-3 h infection, and (4) Golgi protein(s) between 4-6 h of infection. Further studies are being performed to determine the specific intracellular interactions and role in pathogenesis of intracellularly localized V.