Animal & Avian Sciences Theses and Dissertations
Permanent URI for this collectionhttp://hdl.handle.net/1903/2741
Browse
2 results
Search Results
Item Regulation of factors contributing to virulence in Escherichia coli(2008-04-25) Mitra, Arindam; Mukhopadhyay, Suman; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Extraintestinal pathogenic strains of Escherichia coli cause a wide range of diseases including colibacillosis in chickens and urinary tract infections in humans. Persistent infections in E. coli and other gram-negative species are associated with population-dependent physiological processes such as cell-cell signaling and biofilm formation. Such social behaviors require careful coordination and modulation of gene expression in response to environmental cues. Adaptive response of bacteria in new environment is predominantly achieved through a signaling cascade called two-component regulatory systems. The function of the BarA/UvrY two-component regulatory system and its downstream factors in controlling virulence associated processes, specifically regulation of AI-2 based signaling and biofilm formation was investigated. In E. coli, a type of cell-cell signaling termed Quorum Sensing involves release, detection, and response to small molecule called autoinducer (AI-2), synthesis of which is dependent on luxS gene products via methyl cycle. The BarA-UvrY and Csr system displayed dual regulation on luxS expression at the level of transcription and post-transcription. The uptake of AI-2 by the Lsr transporter is also modulated by the signaling cascade suggested a balance between AI-2 synthesis and uptake in the cell. The role of transcriptional regulator uvrY in biofilm formation in Uropathogenic Escherichia coli was also studied. Mutation of uvrY reduced expression of fimA and papA, major fimbrial subunit of Type 1 and Pap pilus respectively. Acidic exopolysaccharide accumulation and the ability to swarm are also being impaired. Finally, uvrY mutants demonstrated poor colonization in kidneys and bladders in an ascending model of UTI. Overall, the effect of uvrY on biofilm formation seems to be multi-factorial and might play a critical role in adaptation and colonization of UPEC. The fine tuning of processes associated with cell-cell communication and biofilm formation at the level of transcription and post-transcription by the BarA/UvrY/CsrA signaling cascade indicated that this system might be crucial for quick adaptation, social behavior, colonization and virulence attributes in Escherichia coli.Item FUNCTIONAL STUDIES OF INFECTIOUS PANCREATIC NECROSIS VIRUS PROTEINS AND MECHANISM OF VIRUS-INDUCED APOPTOSIS(2003-12-04) Song, Haichen; Vakharia, Vikram N; Animal SciencesInfectious pancreatic necrosis virus (IPNV) encodes a 12 or 15-kDa nonstructural protein, known as VP5. To study the function of VP5, we generated three recombinant viruses rNVI15, rNVI15-15K, and rNVI15-ΔVP5, which could encode either 12-kDa VP5, 15-kDa VP5 or be deficient in VP5, respectively. VP5 was detected in rNVI15 and rNVI-15K infected cells but not in the cells infected with rNVI15-ΔVP5. However, the opal stop codon at nucleotide position 427 in rNVI15 virus was read-through, giving rise to a 15-kDa VP5 that is expressed poorly than rNVI15-15K virus-infected cells. All three recombinant viruses show similar replication kinetics in both Chinook salmon embryo (CHSE-214) and rainbow trout gonad (RTG-2) cells. Moreover, in Sp strains, IPNV segment A could encode a novel, putative 25-kDa protein from another ORF. This 25-kDa protein could not be detected in virus-infected cells, however, we could recover a mutant virus lacking this ORF, indicating that it is not essential for virus replication. To assess the molecular basis of virus adaptation in the cell culture, virulent rNVI15 was serially passaged in CHSE cells nine times to obtain a tissue-culture adapted virus, rNVI15TC. Comparison of the deduced amino acid sequences showed only one amino acid substitution at position 221 (Ala → Thr) in VP2. However, this adaptation mutation is only acquired in CHSE cells but not in RTG-2 cells. Two chimeric viruses, rNVI15ΔVP2 and rNVI15-15KΔVP2 were also generated, in which the residues at positions 217 and 247 in VP2 of the rNVI15 and rNVI15-15K viruses were replaced by the corresponding residues of an attenuated strain, Sp103. These two viruses have similar replication kinetics as Sp103, which replicates faster than rNVI15 in vitro, indicating that residues at positions 217 and 247 of VP2 may be the important markers for virus adaptation and attenuation in vitro. By generating a reassortant virus between rNVI15-15K and Sp103, we also demonstrate that VP1 is not involved in virus cell adaptation. The signal pathways and nature of IPNV-induced apoptosis were investigated in RTG-2 cells. IPNV-induced apoptosis occurs at the late stage of viral life cycle. Caspase-3 is activated during virus infection, and inhibition of caspase-3 could partially inhibit virus-induced apoptosis. Moreover, NF-κB activation is essential for IPNV-induced apoptosis, and it is involved in interferon-induced antiviral state. Both the NF-κB inhibitor and the antioxidant could inhibit NF-κB activity and apoptosis induced by IPNV infection, but they do not affect viral replication.