Comparative transcriptome profiling of human foreskin fibroblasts infected with the Sylvio and Y strains of Trypanosoma cruzi

Trypanosoma cruzi, the causative agent of Chagas Disease, is phylogenetically distributed into nearly identical genetic strains which show divergent clinical presentations including differences in rates of cardiomyopathy in humans, different vector species and transmission cycles, and differential congenital transmission in a mouse model. The population structure of these strains divides into two groups, which are geographically and clinically distinct. The aim of this study was to compare the transcriptome of two strains of T. cruzi, Sylvio vs. Y to identify differences in expression that could account for clinical and biochemical differences. We collected and sequenced RNA from T. cruzi-infected and control Human Foreskin Fibroblasts at three timepoints. Differential expression analysis identified gene expression profiles at different timepoints in Sylvio infections, and between Sylvio and Y infections in both parasite and host. The Sylvio strain parasite and the host response to Sylvio infection largely mirrored the host-pathogen interaction seen in our previous Y strain work. IL-8 was more highly expressed in Sylvio-infected HFFs than in Y-infected HFFs.

Mycobacteriosis in Chesapeake Bay Striped Bass (Morone saxatilis): The Interaction of Nutrition and Disease

Field and laboratory studies were conducted from 1998 - 2005 to examine the relationship between nutritional status and mycobacteriosis in Chesapeake Bay striped bass (Morone saxatilis). A review of DNA from archived tissue blocks indicated that the disease has been present since at least 1984. Field surveys and feeding trials were conducted from 1998-1999 to determine the nutritional condition of striped bass and the association with disease state. Proximate composition revealed elevated moisture (~ 80%) and low storage lipids (< 0.5% ww), characteristic of a poorly nourished population. These findings were not consistent with data collected in 1990-1991, or with experimentally fed fish. Mycobacteriosis explained little of the variance in chemical composition (p > 0.2); however elevated moisture and low lipid concentration were associated with fish with ulcerative lesions (p < 0.05). This suggests that age 3 and 4 striped bass were in poor nutritional health in 1998-1999, which may be independent from the disease process. Challenge studies were performed to address the hypothesis that disease progression and severity may be altered by nutritional status of the host. Intraperitoneal inoculation of 104 CFU M. marinum resulted in high mortality, elevated bacterial density, and poor granuloma formation in low ration (0.15% bw/d) groups while adequately fed fish (1% bw/d) followed a normal course of granulomatous inflammation with low mortality to a steady, equilibrium state. Further, we demonstrated that an active inflammatory state could be reactivated in fish through reductions in total diet. The energetic demand of mycobacteriosis, was insignificant in comparison to sham inoculated controls in adequately fed fish (p > 0.05). Declines in total body energy were only apparent during active, inflammatory stages of disease. Overall, these findings suggest that: 1) mycobacteriosis is not a new disease of Chesapeake Bay striped bass, 2) the disease has little energetic demand in the normal, chronic progression, and 3) poor nutritional health can greatly enhance the progression and severity, and reactivation of disease. The implications of this research are that management strategies focused on enhancing the nutritional state of striped bass could potentially alter the disease dynamics in Chesapeake Bay.

CHARACTERIZATION OF TOBACCO MOSAIC VIRUS DIRECTED REPROGRAMMING OF PHLOEM GENE EXPRESSION TO PROMOTE VIRAL PHLOEM LOADING AND SYSTMEIC MOVEMENT

Vascular phloem loading has long been recognized as an essential step in the establishment of a systemic virus infection. Yet little is known about this process and the mechanisms that control it. In this study, an interaction between the replication protein of Tobacco mosaic virus (TMV) and phloem specific auxin/indole acetic acid (Aux/IAA) transcriptional regulators was found to modulate virus phloem loading. Promoter expression studies show TMV 126/183 kDa interacting Aux/IAAs predominantly express and accumulate within the nuclei of phloem companion cells (CC). Furthermore, CC Aux/IAA nuclear localization is disrupted upon infection with an interacting virus but not during infection with a non-interacting virus. In situ analysis of virus spread shows the inability of TMV variants to disrupt Aux/IAA CC nuclear localization correlates with a reduced ability to load into the vascular tissue. Subsequent systemic movement assays also demonstrate that a virus capable of disrupting Aux/IAA localization is significantly more competitive at systemic movement than a non-interacting virus. Similarly, CC expression and over-accumulation of a degradation-resistant-interacting Aux/IAA protein was found to selectively inhibit TMV accumulation and phloem loading. Transcriptional expression studies demonstrate a role for interacting Aux/IAA proteins in the regulation of salicylic acid and jasmonic acid dependent host defense responses as well as virus specific movement factors including pectin methylesterase that are involved in regulating plasmodesmata size exclusion limits and promoting virus cell-to-cell movement. Further characterization of the phloem environment was done using two phloem specific promoters (pSUC2 and pSULTR2;2) to generate epitope-tagged polysomal-RNA complexes. Immuno-purification using the epitope tag allowed us to obtain mRNAs bound to polysomes (the translatome) specifically in phloem tissue. We found the phloem translatome is uniquely altered during TMV infection with 90% and 88% of genes down regulated in the pSUC2 and pSULTR2;2 phloem translatomes, compared to 31% of genes down regulated in the whole plant p35S translatome. Transcripts down regulated in phloem include genes involved in callose deposition at plasmodesmata, host defense responses, and RNA silencing. Combined, these findings indicate TMV reprograms gene expression within the vascular phloem as a means to enhance phloem loading and systemic spread.