Project description:Cultures of Y. pestis Kimberley53 (Kim53) virulent strain and Kim53∆nlpD were grown in heart infusion broth supplemented with 0.2% xylose and 2.5 mM CaCl2 for 5 h at 37°C, 200rpm. Total RNA samples were extructed and used for cRNA synthesis and labelling (using Cy3-CTP and Cy5-CTP). cRNA of two independent samples from each strain were labeled and hybridized to custom made Agilent 8x15K slide. Goal: Identifing alterations in gene expression profile between the wild-type Kim53 virulent strain and Kim53∆nlpD. Those altered mRNA transcrips will be used for better understanding of the cause for Kim53∆nlpD attenuation.
Project description:transcriptomic analyses showed there was a very rapid proliferation of Y. pestis in assault on African green monkeys. The bacterial loads were enumerated in the blood at delayed time points supporting earlier observations (Layton et al., 2011). Interpretation of transcriptomic changes reveals that the host defense has been essentially shattered, and the process of multi-organ failure was initiated one day after inhalation exposure. At 24 h post-exposure, the diseased animals were possibly at the middle of their survival curves. Hence, such molecular indicators could have critical therapeutic values, particularly when therapies fail to intervene at the earliest opportunity. In the early phase of pathogenesis, systematic reprogramming of both adaptive and innate immunity programs is mediated, leading to sepsis. Results suggest the possibility of early molecular perturbations in the lungs and muscle tissues, and catabolic related activities, such as biosynthesis. Apoptosis ensued at the early stage, which potentially became rapid by recruiting an increasing number of apoptotic networks. Beyond 24 h, rapid apoptosis could have acted in concert with hypercytokinemia and hyperchemokinemia, resulting in failures of many peripheral organs. Following a published report (Layton et al., 2011), the present study was designed to expose the nonhuman primates with a target dose of 100 ± 50 ED50 aerosolized Y. pestis strain CO92 under ABSL-3 conditions. Seventy-two hours prior to aerosol exposure, all animals were moved into the ABSL-3 for acclimatization. As described in Figure S1, initial blood draws were carried out 24 h before Y. pestis aerosol challenge. The animals were challenged via aerosol with 1 x 106 virulent organisms while under anesthesia with 4 mg/kg Telazol (Fort Dodge Animal Health, Fort Dodge, IA). Post-exposure clinical illness and disseminated infection were evaluated by direct clinical observations. All animals within each group were exposed on the same day at 30 minute intervals, and subsequent blood draws were conducted at 45 m, 6 h, 9 h, 12 h, 18 h, 24 h, 32 h and 42 h.
