Cell Biology & Molecular Genetics Theses and Dissertations
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Item Characterization of the TrxSR Two-Component Signal Transduction System of Streptococcus pyogenes and its Role in Virulence Regulation(2011) Gold, Kathryn; McIver, Kevin; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The Gram-positive group A streptococcus (GAS) is a strict human pathogen, which causes a wide variety of infections, ranging in severity from minor to life threatening. In order to cause such a diverse array of diseases, GAS utilizes two-component signal transduction systems (TCS) to coordinately regulate sets of virulence genes in response to changing host conditions. The present study investigates the role of the TrxSR TCS in the regulation of virulence of the GAS. Using an insertional inactivation mutation in TrxR in serotype M1 MGAS5005, transcriptome studies established that TrxR activates transcription of Mga-regulated virulence genes, a separate non-TCS regulatory pathway controlling factors important for immune evasion and colonization. Transcriptional reporter fusions of Pmga to firefly luciferase revealed that the TrxR regulation occurs through the Pmga promoter. Additionally, electrophoretic mobility shift assays using purified His-MBP tagged TrxR established specific binding of TrxR to Pmga, although the interaction appeared to be transient. To determine the importance of signal transduction for TrxR-mediated regulation of the Mga regulon and virulence, an in vitro reconstitution assay was performed with purified TrxR and TrxS. Using both wild type and mutated forms of the TrxSR proteins, we demonstrated that TrxSR is a functional two-component phosphorelay system. Interestingly, phosphorylation of TrxR did not appear to be critical for DNA binding and regulation, since a TrxR D55A mutation did not change the expression of TrxR regulated genes in GAS based on EMSA and qPCR. In order to investigate whether there is a functional conservation of TrxR's involvement in GAS virulence regulation, mutations were made in serotype M4 and M49 strains representing either throat only or generalist strains. We have determined that TrxR regulates mga and Mga-regulated genes (emm, arp) in the M4 and M49 backgrounds, suggesting conservation of TrxR's role in virulence regulation. Overall, TrxSR represents a functional TCS that appears to directly regulate the Mga virulence regulon independent of phosphorelay. Furthermore, the functional conservation of TrxR regulation of Mga in other serotypes suggests a conserved role for its involvement in virulence regulation in GAS.Item CHARACTERIZATION OF TWO HIGHLY CONSERVED POXVIRUS TRANSMEMBRANE PROTEINS OF UNKNOWN FUNCTION(2009) Sood, Cindy Leigh; Moss, Bernard; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The vaccinia virus I5L open reading frame encodes a 79-amino-acid protein, with two predicted transmembrane domains, conserved among all sequenced members of the chordopoxvirus subfamily. No nonpoxvirus homologs or functional motifs have been recognized, and the role of the I5 protein remains unknown. I5 synthesis was dependent on viral DNA replication and occurred exclusively at late times, consistent with a consensus late promoter motif adjacent to the start of the open reading frame. I5 was present in preparations of purified virions and could be extracted with nonionic detergent, suggesting membrane insertion. Transmission electron microscopy of immunogold-labeled thawed cryosections of infected cells revealed the association of an epitope-tagged I5 with the membranes of immature and mature virions. Viable I5L deletion and frameshift mutants were constructed and found to replicate like wild-type virus in a variety of cell lines, indicating that the protein was dispensable for in vitro cultivation. However, mouse intranasal challenge experiments indicated that a mutant virus with a frameshift resulting in a stop codon near the N terminus of I5 was attenuated compared to control virus. The attenuation correlated with clearance of mutant viruses from the respiratory tract and with less progression and earlier resolution of pathological changes. We suggest that I5 is involved in an aspect of host defense that is evolutionarily conserved although a role in cell tropism should also be considered. The vaccinia virus A43R open reading frame encodes a 168-amino acid protein with a predicted N-terminal signal sequence and a C-terminal transmembrane domain. Although A43R is conserved in all sequenced members of the orthopoxvirus genus, no non-orthopoxvirus homolog or functional motif was recognized. Biochemical and confocal microscopic studies indicated that A43 is expressed at late times following viral DNA synthesis and is a type-1 membrane protein with two N-linked oligosaccharide chains. Neither mature nor enveloped virions contained appreciable amounts of A43, which was detected in Golgi and plasma membranes. Loss of A43R expression had no discernible effect on plaque size or virus replication in cell culture and little effect on virulence in a mouse intranasal infection model. Although the A43 mutant produced significantly smaller lesions in the skin of mice than the control, the amounts of virus recovered from the lesions were similar.