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Item Cortisol acetate-induced wasting disease in germfree and conventionally reared mice(Montana State University - Bozeman, College of Agriculture, 1966) Reed, Norman DuaneItem Contributions of pneumococcal virulence factors to secondary Streptococcus pneumoniae infection following influenza infection(Montana State University - Bozeman, College of Agriculture, 2009) King, Quinton Oliver; Chairperson, Graduate Committee: Allen G. Harmsen.Influenza infection increases susceptibility to secondary infection with Streptococcus pneumoniae resulting in significantly increased morbidity and mortality. Whereas viral contributions to this synergism have been explored, little is known concerning contributions of the bacterium, specifically those provided through bacterial virulence factors. To assess the contributions of the known pneumococcal virulence factors hyaluronidase (Hyl), neuraminidase (NanA) and pneumococcal surface protein A (PspA) to secondary S. pneumoniae infection following influenza infection, mutants lacking these proteins were administered with wildtype pneumococci in a competitive growth model. Whereas mutants lacking the Hyl and NanA proteins did not exhibit attenuation, mutants lacking PspA were severely attenuated in mice without influenza infection and significantly more so in mice with a prior influenza infection. Additionally, mice received intranasal immunization with recombinant PspA protein and subsequently received primary and secondary challenges with serotypes 2, 3 and 4 pneumococci. Immunization with PspA significantly reduced bacterial burdens of all three challenge serotypes in primary and secondary pneumococcal infection and significantly reduced lung damage markers in mice receiving secondary pneumococcal challenges. In addition to known virulence factors, two surface-exposed proteins, Spr0075 and Spr1345, were assessed for virulence contributions to primary and secondary pneumococcal infections. Mutants lacking Spr0075 or Spr1345 were found to be severely attenuated in both primary and secondary pneumococcal challenges. Whereas immunization with either recombinant Spr0075 or Spr1345 significantly reduced primary pneumococcal burdens, only immunization with Spr0075 significantly reduced secondary pneumococcal burdens. Together these results indicate virulence contributions to both primary and secondary pneumococcal challenges for the PspA, Spr0075 and Spr1345 proteins. However, whereas immunization with PspA and Spr0075 significantly reduced both primary and secondary pneumococcal burdens, immunization with Spr1345 did not significantly impact secondary pneumococcal burdens. This result illustrates that a virulence contribution and/or an ability to protect against primary infection does not necessarily translate into a protein's capacity to protect against secondary infection. The results presented here are the first experimental evidence demonstrating virulence roles for the Spr0075 and Spr1345 proteins and are the first reports of immunization with pneumococcal proteins, specifically PspA and Spr0075, providing protection against secondary pneumococcal infection following influenza.Item The mechanism of heme transfer from the strepcoccal cell surface protein Shp to HtsA of the HtsABC transporter(Montana State University - Bozeman, College of Agriculture, 2010) Ran, Yanchao; Chairperson, Graduate Committee: Benfang LeiGroup A Streptococcus relies on heme as a source of iron. The proteins Shp and HtsA are part of the heme acquisition machinery of Group A Streptococcus. Shp rapidly transfers its heme to HtsA; however, the mechanism of the Shp/HtsA reaction is unknown. This project was conducted to elucidate the structural basis and molecular mechanism of this rapid heme transfer reaction. Site-directed mutagenesis was used to identify the axial ligands of the heme iron in Shp and HtsA, and kinetic and spectroscopic analyses and animal infection were used to assess the effects of the elimination of the axial ligands on coordination and spin state of the heme iron, kinetic mechanism of the heme transfer, autoxidation of the Shp heme iron, and GAS virulence. The axial ligands of the heme iron in Shp and HtsA were found to be Met66/Met153 and Met79/His229, respectively. The Met153 Shp and His229 HtsA residues are critical for the affinity of the proteins for heme, and the other axial side of the heme irons is more accessible to solvent. The Met66Ala and Met153Ala replacements of Shp alter the kinetic mechanism of Shpto- HtsA heme transfer and unexpectedly slow down heme transfer, which allows detection of transfer intermediates. The HtsA Met79Ala and HtsA His229Ala mutant proteins cannot acquire heme from ferrous Shp but induce rapid autoxidation of ferrous Shp. The significance of these findings is three-fold. Firstly, the structural basis of the heme binding in Shp and HtsA and the spectral properties of their axial ligand mutants enable the interpretation of the kinetics and spectral changes of the heme transfer reactions. Secondly, HtsA axial mutant-induced autoxidation of ferrous Shp provides evidence for the activated heme transfer mechanism and the formation of a Shp-HtsA complex that weakens the heme binding in Shp. Thirdly, the findings allow us to propose a reaction model in which the side chains of the axial residues from HtsA are inserted into the axial positions of the heme in Shp to extract it from the surface protein and pull it into the transporter active site. The project significantly advances the understanding of how heme is rapidly transferred from one protein to another in heme acquisition.Item Effects of a primary influenza infection on susceptibility to a secondary Streptococcus pneumoniae infection(Montana State University - Bozeman, College of Agriculture, 2006) McNamee, Lynnelle Ann; Chairperson, Graduate Committee: Allen G. Harmsen.Influenza infections result in increased susceptibility to a secondary Streptococcus pneumoniae infection. The aim of the present studies was to determine the mechanism(s) responsible for this increase in susceptibility. Using an in vivo co-infection model, we found that susceptibility to S. pneumoniae was significantly increased at 6 days but not 3 days after an influenza infection. We depleted mice of neutrophils and found that neutrophils were important in the response to S. pneumoniae in mice infected with bacteria only or those infected with influenza for 3 days prior to a S. pneumoniae infection. However, at 6 days, neutrophil depletion did not alter the response to bacterial growth, indicating that neutrophil function was altered. We measured reactive oxygen species (ROS) generation and phagocytosis of S. pneumoniae by lung and bone marrow neutrophils isolated from mice infected with influenza for 3 or 6 days and compared these to neutrophils from either mice stimulated with LPS to induce neutrophil migration or from uninfected mice.Item Studies of peptide mimicry of the group B Streptococcus type III capsular polysaccharide antigen(Montana State University - Bozeman, College of Letters & Science, 2007) Pomwised, Rattanaruji; Chairperson, Graduate Committee: Mark Young; Seth Pincus (co-chair)Capsular polysaccharide (CPS) of Streptococcus group B (GBS) is a poor immunogen and functions as T cell independent antigen, eliciting low IgG antibody with deficient immunologic memory. We previously identified a peptide, S9, which mimics CPS of type III GBS. Here we have taken steps to develop the mimetic peptide as a vaccine against GBS group III. We enhanced the immunogenicity of the peptide by presenting it on the coat protein of Cowpea Chlorotic Mottle Virus (CCMV). And we searched for better mimetic peptides by constructing a secondary phage display library. To accomplish the first goal, DNA encoding S9 was cloned into five constructions CCMV coat protein loops using recombinant DNA techniques. The results indicated that inserting the S9 peptide sequence into CCMV coat protein loops disrupted virus and virus-like particle assembly. Therefore the S9 peptide was conjugated to CCMV coat protein using chemical linkers. The CCMV-S9 conjugation products remained intact as monomer virions.