Master of Science / Department of Animal Sciences and Industry / Terry A. Houser / Nursery age pigs (n=198) were used to evaluate the difference in the occurrence of injection site abscesses between needle-free jet injection and conventional needle-and-syringe injection systems. Pigs were fed for 21 d prior to treatment administration to acclimate the pigs to the environment of the Kansas State University Segregated Early Weaning (SEW) unit. On d 21 each pig was injected with aluminum hydroxide adjuvant in the neck and ham with needle-free jet injection (Pulse Needle-Free Systems, Lenexa, KS) and conventional needle-and-syringe injection. Needle-free and conventional needle-and-syringe injections were randomly assigned to pig side yielding a total of 396 injections per treatment with a total of 792 injections sites. Immediately prior to injection, the external surface of the injection sites were contaminated with an inoculum of Arcanobacterium pyogenes, a bacterium commonly associated with livestock abscesses. The pigs were then fed for a period of 27 or 28 d. On d 27 or d 28 the pigs were humanely euthanized and sent to the Kansas State Veterinary Diagnostics Laboratory where necropsies were performed and the injection sites harvested for histopathological evaluation. The needle-free jet injection system was associated with more injection site abscesses than the conventional needle-and-syringe injection method for both neck (P=0.0625) and ham (P=0.0313) injection sites. Twelve abscesses were found at injection sites administered via needle-free jet injection method while only 1 abscess was found with the conventional needle-and-syringe injection method. 5 abscesses were found at the neck injection sites and 8 abscesses were found at ham injection sites. There were no significant differences seen in tissue granulation resulting from reaction to the adjuvant. In summary, the implementation of needle-free jet injection systems in market hog production will be beneficial to eliminate needles and needle fragments in meat products but, when in the presence of Arcanobacterium pyogenes, it may increase the occurrence of injection site abscesses in pork carcasses that will need to be trimmed in pork processing plants. Although more abscesses were associated with needle-free jet injection, their occurrence was observed at a very low rate given that all injection sites were intentionally contaminated prior to injection.

The pathogen Streptococcus pyogenes is a significant cause of human morbidity and mortality. Most of the work in this thesis is focused on streptococcal virulence factor IdeS, but the thesis also features work on SpeB, another streptococcal virulence factor. Both IdeS and SpeB are secreted cysteine proteases and both have previously been shown to degrade human IgG. IgG is the only known substrate for IdeS while SpeB is a more promiscuous protease with a larger number of identified substrates. A significant part of the data presented in this thesis is the result of designing and optimizing methods to detect and accurately measure the proteolytic degradation of IgG. Methods aimed at measuring the binding interactions between enzyme and substrate have also been frequently utilized. I show that IdeS is a monomeric protease, as opposed to previously published data that suggested it to be dimeric. IdeS cleaves the two heavy chains of IgG in a two-step reaction and I demonstrate that the first cleavage is magnitudes faster than the second one. This means that IdeS is a more efficient enzyme than previously thought. The difference in rate cannot completely be explained by a loss of affinity between IdeS and IgG after the cleavage of the first heavy chain. The velocity of IdeS is further increased by the presence of human Cystatin C, via an unknown mechanism. Cystatin C is normally a protease inhibitor and it having an opposite effect is puzzling.The synthesis and evaluation of novel inhibitors are also described. Peptide analogues mimicking the sequence surrounding the scissile bond on IgG - with an amino acid replaced with a more rigid motif - act as specific, but low-affinity, inhibitors of IdeS. The peptide analogues’ inhibitory capacity for SpeB and papain was also assayed.When it comes to SpeB, I show that it does not have IgG as a substrate under physiological conditions, in contrast to what was previously thought. This thesis does not only present findings on the IgG degrading capacity of IdeS and SpeB but also include data on fundamental enzymatic properties for these proteases.

The problem in this investigation consists of, first, the procurement of several strains of Micrococcus pyogenes var. aureus; second, the comparison of the degree and rate of development of resistance of these organisms to terramycin; and, third, to study the morphological and physiological changes which occur during the development of resistance.

Most pathogens gain access to their host through mucosal surfaces. It is therefore desirable to develop mucosal vaccines that elicit an immune response to prevent this crucial first step in infection. Current mucosal vaccines are live attenuated strains of pathogens. More recent efforts have focused on the use of recombinant non-pathogenic gram-positive bacteria as live vaccine delivery vectors. Here I have tested the potential of Lactococcus lactis to be used as a vaccine vector. A recombinant strain of L. lactis has been constructed which expresses and displays on its surface the C repeat region (CRR) of the M6 protein of Streptococcus pyogenes. I show that nasal vaccination of mice with this strain elicited strong salivary IgA and serum lgG response. These responses protected mice against a nasal challenge with S. pyogenes. Subcutaneous vaccination with the same strain of L. lactis produced a strong serum lgG response, but no salivary lgA response. Subcutaneous vaccination did not protect the mice against nasal infections when the mice were challenged with S. pyogenes. The immune response and protection afforded by concomitant vaccination by both nasal and subcutaneous routes were better that that seen in nasal vaccination alone. This study shows that an effective vaccine against S. pyogenes is possible using L. lactis as a vaccine vector. It also opens up the potential of L. lactis to be used in the development of vaccines to other mucosal infections. / Graduation date: 2004

Heme is the major iron source for the deadly human pathogen, Group A Streptococcus (GAS). During infection, GAS lyses host cells releasing hemoglobin and other hemoproteins. This dissertation aims to elucidate the general mechanism by which GAS obtains and utilizes heme as an iron source from the host hemoproteins. GAS encodes a heme relay system consisting of Shr, Shp and the SiaABC transporter. We specifically determine the role of Shr in the heme uptake process, by conducting a detailed functional characterization of its constituent domains. We also undertake to solve the long-standing mystery surrounding the catabolism of heme in streptococci. The studies presented herein established Shr as a prototype of a new family of NEAT-containing hemoproteins receptors. They demonstrate its importance in heme acquisition by GAS and provide a molecular model for heme scavenging and transfer by the protein. We show that Shr modulates heme uptake depending on heme availability by a mechanism where NEAT1 facilitates fast heme scavenging and delivery to Shp, whereas NEAT2 serves as a temporary storage for heme on the bacterial surface. Finally, we identified and characterized for the first time, a heme oxygenase (HO) in the Streptococcus genus which was named HupZ. Sequence comparison between HupZ and several HOs from different structural families indicates that this enzyme is unrelated to any of the previously characterized HOs. However, orthologs of the protein are found in other important pathogens. The structure and the catalytic mechanism of HupZ suggest that it is the representative of a new family of flavoenzymes capable of degrading heme using their reduced flavin cofactor as a source of electrons. Overall, this work contributes significant knowledge to the topic of heme utilization by pathogens and importantly, provides new direct evidence that associates flavins with heme metabolism in bacteria. Thus it sets a new direction in the field and lays the ground for future fundamental and applied discoveries.

Some pathogenic bacteria derive significant amounts of iron heme from their hosts. In this study we investigated SiaA, a heme binding protein from Streptococcus pyogenes. The wildtype methionine79 putative axial ligand was mutated to alanine. SiaA M79A was expressed in E. coli in three production runs, lysed by sonication or French press, and purified by fast protein liquid chromatography (FPLC). Nickel affinity FPLC was found to give much purer SiaA when 30 mM imidazole was added to the binding buffer. The protocol using extensive sonication resulted in SiaA weighing 30464 Da. The protocol using French press resulted in SiaA weighting 33358 Da. Despite the difference in masses, the two forms of SiaA interacted with heme similarly.

The protein SiaA (Streptococcal iron acquisition) is involved in heme uptake in the bacterium Streptococcus pyogenes. It is difficult to obtain this protein in its fully holo form (completely loaded with heme). To increase the concentration of heme in the growing cell, we added ä-aminolevulinic acid (ALA) and ferrous sulfate (FeSO4), precursors of heme, to the growth media. Neither increasing the concentration of heme in vivo, nor growth at lower temperature for longer times, increased the production of holoprotein. The classical method of measuring the concentration of heme in a newly discovered heme protein is cumbersome. We have developed an improved method, which gives a solution that is more stable and has a cleaner spectrum. With further development, this new technique may replace the classical assay. Background information on S. pyogenes, SiaA, ABC transporters, heme biosynthesis, and the pyridine hemochrome assay are described.

Group A Streptococcus (GAS) is a common pathogen of the human skin and mucosal surfaces and is capable of producing a variety of diseases. This dissertation investigates the function of a metalloregulator named MtsR in GAS physiology and disease process. An mtsR mutant was constructed and analyzed. Consistent with MtsR role in iron uptake regulation, the mtsR mutant accumulates more iron (80 ± 22.5%) than the wild type strain. Inactivation of mtsR results in constitutive transcription of the sia (Streptococcal Iron Acquisition) operon, which is negatively regulated by iron in the parent strain. We identified the promoter that controls the expression of the sia operon (Pshr) and used it as a model to study MtsR interaction with DNA. Electrophoretic mobility gel shift assays (EMSAs) demonstrated that MtsR binds to the shr upstream region specifically and in an iron and manganese dependent manner. DNase I footprint analysis revealed that MtsR protects a 69 bp segment in Pshr that includes 2 inverted repeats, overlapping the core promoter elements. A global transcriptional analysis determined that MtsR modulates the expression of 64 genes, of which 44 were upregulated and 20 were downregulated in the mtsR mutant. MtsR controls genes with diverse functions including immune evasion, colonization, dissemination, metal homeostasis, nucleic acid and amino acid metabolism, and protein stability. MtsR functions as a dual regulator as it binds to the promoters of the repressed genes ska, aroE, and nrdF.2, as well as the upstream region of the positively regulated genes mga, emm49, and pyrF. A 16 bp MtsR-binding consensus region was identified in all of the promoters that are directly regulated by MtsR. In conclusion, we have demonstrated that MtsR is a global regulator in GAS that controls the expression of vital virulence factors and genes involved in metal transport, virulence and metabolic pathways.

Anthraquinone (AQ) derivatives with relatively high reduction potentials have been synthesized to afford good candidates for electron transfer studies in DNA. Electron withdrawing groups on the anthraquinone ring gave derivatives with less negative reduction potentials. The anthraquinone imide (AQI) derivatives had reduction potentials less negative than AQ derivatives. The AQI ring system was subject to base-induced hydrolysis. Water-soluble sulfonated tetraarylporphyrins have been studied in a wide variety of contexts. Herein, we report the first synthesis of a pentasulfonated porphyrin bearing an internal cyclic sulfone ring. Treatment of 5,10,15,20-tetrakis(4-sulfonatophenyl)porphyrin (TPPS4) with fuming H2SO4 gave a structure consistent with initial sulfonation followed by dehydration to give a sulfone bridge between an ortho-position of one of the phenyl groups and a β-pyrrole position on the porphine ring (TPPS4Sc). The structure was established by ESI-MS and 1HNMR. The Soret absorption is red shifted by about 32 nm compared to that of TPPS4. Streptococcus pyogenes obtains iron by taking up heme from the environment during infection. One of the heme uptake pathways is the Sia or Hts pathway. The initial protein in this pathway is Shr, which has two heme-binding NEAT domains, NEAT1 nearer the N-terminus, and NEAT2 nearer the C-terminus. We report biophysical characteristics of these two NEAT domains. To assess stability of this domain towards heme release, denaturation studies of the Fe(II) and Fe(III) forms were performed. For each domain, both the Fe(II) and the Fe(III) forms behave similarly in thermal denaturation and guanidinium denaturation. Overall, NEAT2 is more stable than NEAT1. Spectral signatures, sequence alignment and homology modeling for both domains suggest that one of the axial ligands is methionine. NEAT2 autoreduces as the pH increases and autooxidizes as the pH decreases. Heme uptake from the host environment is the only iron acquisition pathway in S. pyogenes; inhibition of this pathway might be an approach to infection control. Compounds that might inhibit the heme uptake pathway were selected via virtual screening.

In Streptococcus pyogenes, the protein SiaA (HtsA) is part of a heme uptake pathway system and involved in heme transfer from Shp to the ABC transporter. SiaA mutants, in which alanine replaces the axial histidine (H229) and methionine (M79) ligands, as well as a lysine (K61) and cysteine (C58) located near the heme propionates, are reported. Studies on a mutant of a cysteine expected to be at a distance from the propionates (C47A) are also reported. The coordination state and spin state of the selected mutants were determined via Resonance Raman studies. The pKa values of mutants ranged from 9.0 to 9.4, which were close to the pKa of the WT SiaA (9.7). The midpoint reduction potential of lysine (K61A) mutant was determined by spectroelectrochemical titration to be 61 ± 3 mV vs. SHE, similar to the WT protein (68 ± 3 mV). The addition of guanidinium hydrochloride resulted in protein denaturation that could show more than one process and occurred over days. The ease of protein unfolding was directly related to the extent of interaction of the residues with the heme: changes in the axial ligands resulted in far greater changes in heme protein stability than changes in the residues near the heme propionates. The causative agent of diphtheriae, Corynebacterium diphtheriae, imports heme via an ABC uptake transporter. In this research, two of the five proteins in the heme uptake pathway of C. diphtheriae were studied. These proteins were HmuT, lipoprotein component of the ABC transporter, and HtaA, the heme receptor. UV-visible spectroscopy and fluorescence spectroscopy showed that HmuT protein as isolated bound a porphyrin, rather than heme. Electrospray ionization mass spectrometry (ESI-MS) studies suggested that two tetrapyrroles were bound. To assess stability of this protein towards heme release, thermal denaturation studies were performed. For HtaA, UV-visible and fluorescence spectroscopy also showed the protein as isolated was also bound a porphyrin, rather than heme. Homology studies showed that HtaA protein is quiet distant from homologous heme uptake proteins and could be a member of novel heme binding domain family.