Pradeep R. Dumpala

High-Throughput Bioluminescence-Based Mutant Screening Strategy for Identification of Bacterial Virulence Genes

ABSTRACT A high-throughput bioluminescence screening procedure for identification of virulence genes in bacteria was developed and applied to the fish pathogen Edwardsiella ictaluri. A random transposon mutant library expressing bioluminescence was constructed and robotically arrayed on 384-well plates. Mutants were cultivated and mixed with catfish serum and neutrophils in 96-well plates, and bioluminescence was used to detect mutants that are more susceptible to killing by these host factors. The virulence and vaccine efficacy of selected mutants were determined in channel catfish. Transposon insertion sites in 13 mutants attenuated in the natural host were mapped to the E. ictaluri genome. Ten unique genes were mutated, including genes encoding a negative regulator of sigmaE activity, a glycine cleavage system protein, tricarboxylic acid cycle enzymes, an O polysaccharide biosynthesis enzyme, proteins encoded on the native plasmid pEI1, and a fimbrial chaperon protein. Three of these mutants were found to have potential as live attenuated vaccines. This study demonstrates a novel application of bioluminescence to identify bacterial genes required for host resistance; as a result, efficacious and genetically defined live attenuated vaccine candidates were developed.

Proteomic analysis of the fish pathogen Flavobacterium columnare

BackgroundFlavobacterium columnare causes columnaris disease in cultured and wild fish populations worldwide. Columnaris is the second most prevalent bacterial disease of commercial channel catfish industry in the United States. Despite its economic importance, little is known about the expressed proteins and virulence mechanisms of F. columnare. Here, we report the first high throughput proteomic analysis of F. columnare using 2-D LC ESI MS/MS and 2-DE MALDI TOF/TOF MS.ResultsProteins identified in this study and predicted from the draft F. columnare genome were clustered into functional groups using clusters of orthologous groups (COGs), and their subcellular locations were predicted. Possible functional relations among the identified proteins were determined using pathway analysis. The total number of unique F. columnare proteins identified using both 2-D LC and 2-DE approaches was 621, of which 10.95% (68) were identified by both methods, while 77.29% (480) and 11.76% (73) were unique in 2-D LC and 2-DE, respectively. COG groupings and subcellular localizations were similar between our data set and proteins predicted from the whole genome. Twenty eight pathways were significantly represented in our dataset (P < 0.05).ConclusionResults from this study provide experimental evidence for many proteins that were predicted from the F. columnare genome annotation, and they should accelerate functional and comparative studies aimed at understanding virulence mechanisms of this important pathogen.

Proteome analysis of Edwardsiella ictaluri

Edwardsiella ictaluri is a facultative intracellular Gram‐negative bacterium causing enteric septicemia of catfish (ESC), the most prevalent disease affecting farm‐raised channel catfish in the United States. Despite its economic importance, studies addressing high‐throughput proteomics were not possible because of lack of comprehensive protein database. Here, we report the first high‐throughput proteomics analysis of E. ictaluri using 2‐D LC ESI MS/MS and 2‐DE MALDI TOF/TOF MS. Proteins identified in this study and predicted from the whole E. ictaluri genome were clustered into functional groups using clusters of orthologous groups (COG), and their subcellular locations were predicted. Possible functional relationships among proteins were determined using pathway analysis. The total number of unique E. ictaluri proteins identified using both 2‐D LC and 2‐DE approaches was 788, of which 15.48% (122) were identified by both methods while 78.43% (618) and 6.09% (48) were unique in 2‐D LC and 2‐DE, respectively. COG groupings and subcellular localizations were quite similar between our data set and proteins predicted from the whole genome. Twelve pathways were significantly represented in our dataset (p <0.05). Results from this study provided experimental evidence for many proteins that were predicted from the E. ictaluri genome annotation, and they should accelerate future functional and comparative studies aimed at understanding virulence mechanisms of this important pathogen.

Identification of Differentially Abundant Proteins of Edwardsiella ictaluri during Iron Restriction

Edwardsiella ictaluri is a Gram-negative facultative anaerobe intracellular bacterium that causes enteric septicemia in channel catfish. Iron is an essential inorganic nutrient of bacteria and is crucial for bacterial invasion. Reduced availability of iron by the host may cause significant stress for bacterial pathogens and is considered a signal that leads to significant alteration in virulence gene expression. However, the precise effect of iron-restriction on E. ictaluri protein abundance is unknown. The purpose of this study was to identify differentially abundant proteins of E. ictaluri during in vitro iron-restricted conditions. We applied two-dimensional difference in gel electrophoresis (2D-DIGE) for determining differentially abundant proteins and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI TOF/TOF MS) for protein identification. Gene ontology and pathway-based functional modeling of differentially abundant proteins was also conducted. A total of 50 unique differentially abundant proteins at a minimum of 2-fold (p ≤ 0.05) difference in abundance due to iron-restriction were detected. The numbers of up- and down-regulated proteins were 37 and 13, respectively. We noted several proteins, including EsrB, LamB, MalM, MalE, FdaA, and TonB-dependent heme/hemoglobin receptor family proteins responded to iron restriction in E. ictaluri.

A model for determining an effective in vivo dose of transplanted islets based on in vitro insulin secretion

Allogeneic islet transplantation for the treatment of type 1 diabetes often requires multiple implant procedures, from as many as several human pancreas donors, to achieve lasting clinical benefit. Given the limited availability of human pancreases for islet isolation, porcine islets have long been considered a potential option for clinical use. Agarose‐encapsulated porcine islets (macrobeads) permit long‐term culture and thus a thorough evaluation of microbiological safety and daily insulin secretory capacity, prior to implantation. The goal of this study was the development of a method for determining an effective dose of encapsulated islets based on their measured in vitro insulin secretion in a preclinical model of type 1 diabetes.

Identification of Differentially Regulated Edwardsiella ictaluri Proteins During Catfish Serum Treatment

Edwardsiella ictaluri is a facultative, intracellular, gram-negative bacterium that causes enteric septicemia of catfish (ESC). Edwardsiella ictaluri is known to be resistant to defense mechanisms present in catfish serum, which might aid in its use of a hosts bloodstream to become septicemic. However, the precise mechanisms of the survival of E. ictaluri in host serum are not known. Analysis of the response of E. ictaluri to the host serum treatment at a proteomic level might aid in the elucidation of its adaptation mechanisms against defense mechanisms present in catfish serum. Thus, the objective of this study was to identify differentially regulated proteins of E. ictaluri upon exposure to naïve catfish serum. Two-dimensional difference gel electrophoresis (2D-DIGE) followed by in-gel trypsin digestion and MALDI-TOF/TOF analysis were used for identification of differentially expressed E. ictaluri proteins. A total of 19 differentially regulated proteins (7 up- and 12 downregulated) were identified. Among those were four putative immunogenic proteins, two chaperones and eight proteins involved in the translational process, two nucleic acid degradation and integration proteins, two intermediary metabolism proteins, and one iron-ion-binding protein. Further research focusing on the functions of these differentially expressed proteins may reveal their roles in host adaptation by E. ictaluri.

Abstract 2486: Docetaxel resistant cells display cancer stem cell properties and regulate growth via PI3/Akt signaling

Both normal organogenesis and tumor development follow a Gompertzian growth curve. An understanding of the mechanisms operating in these apparently discordant situations, in which growth rate slows as mass increases, may be useful for the treatment of cancer. We have previously shown that encapsulating murine renal adenocarcinoma (RENCA) cells in a double layer of agarose to form spherical macrobeads, undergo Gompertzian growth regulation as tumor colonies within the confines of the inner agarose matrix. As the colonies reach maximal size, the RENCA macrobeads (MBs) secrete factors that also inhibit the proliferation of freely growing target cells outside the MB. In the current study, we report the ability of the tumor colonies within the MB to re-form following the debulking of tumor mass with docetaxel (0.5 μg/ml or 5.0 μg/ml). Docetaxel-resistant cells were assessed for the stem cell marker OCT4 using immunohistochemistry (Abcam ab19857), RT-PCR (Qiagen Cancer Stem Cell PCR Array) and cell migration/invasion (Corning Cell Migration Chemotaxis and Invasion Assay). Docetaxel-resistant cells are shown to be a discrete population of cells that exhibit stem cell-like properties, including prolonged quiescence (>16 wk), OCT4 staining, stem cell gene expression, increased migration and invasion, and in vivo tumor induction. These cancer stem-like properties are dose and time post-treatment dependent with the greatest expression observed in the few surviving cells at 6 wk post-treatment with 5 μg/ml. Tumor growth is observed in syngeneic BALB/c mice following transplantation of a single docetaxel-resistant cell at higher frequencies (≥ 6 of 9) as compared to the grafting of single monolayer cells (0 of 8) at 5 wk when using cells recovered at 6 wk from the 5 μg/ml dose treated MBs. To understand the mechanism of colony growth regulation within the MB, we hypothesized that tumor cells external to the MB would use similar growth control signaling when exposed to RENCA MBs or MB conditioned media. We have previously shown an increase in the activity of the transcription factor MEF2 in cells exposed to RENCA MB conditioned media. To investigate PI3/Akt signaling, a known pathway for MEF2 regulation, in human DU145 prostate tumor cells exposed to MB conditioned media, we used In-Cell and Western blotting. Exposure to RENCA MBs resulted in Akt hyperphosphorylation (≥ 48 hours) and de-phosphorylation of MEF2D in DU145 target cells. These findings support the hypothesis that RENCA MBs, as biological cell systems with the in vitro capability of inhibiting cancer cell proliferation, achieve this effect, at least in part, by signaling through Akt to regulate MEF2D. This effect is being investigated in colorectal patients with progressive disease, following informed consent, who underwent laparoscopic intraperitoneal implantations of RENCA MBs as part of ongoing clinical trials. Citation Format: Prithy C. Martis, Melissa A. Laramore, Hunter L. Gazda, Atira Dudley, Pradeep R. Dumpala, Allyson J. Ocean, Nathaniel Berman, Tapan Parikh, Zoe P. Andrada, Angelica Nazarian, Joanne Thomas, Eugene Akahoho, George Stoms, Alex Yaroshinsky, Thomas J. Fahey, David J. Wolf, Lawrence S. Gazda, Barry H. Smith. Docetaxel resistant cells display cancer stem cell properties and regulate growth via PI3/Akt signaling. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2486.