<p>Gram-positive pathogens, such as <italic>Streptococcus pyogenes</italic> and <italic>Staphylococcus aureus</italic>, are etiological agents of a large array of human diseases. Unfortunately, our ability to treat these infections is increasingly limited due to the development of bacterial resistance to many existing therapies. Thus, novel targets for antimicrobial development are urgently needed. An attractive candidate for a new class of anti-virulence chemotherapeutics is the sortase class of enzymes. Sortases are extracellular transpeptidases unique to Gram-positive bacteria. Their function is to covalently attach secreted virulence factors to the bacterial cell wall. Deletion or inhibition of sortases results in severe attenuation of bacteria for infection. In order to develop novel effective antimicrobial agents, a robust understanding of the biological and chemical mechanisms of the target are required. To this end, this dissertation endeavors to further illuminate the biochemical mechanism of sortase enzymes and to extend the current knowledge of the roles of sortases and their substrates during infection.</p><p>Through steady-state kinetics, active site reactivity measurements, three-dimensional structure determination via X-ray crystallography, and computational modeling of substrate binding, the basic enzyme mechanism of <italic>S. pyogenes</italic> sortase A (SrtA) has been revealed. In general, <italic>S. pyogenes</italic> SrtA displays many of the same mechanistic characteristics as previously studied sortases, including a reverse protonation mechanism, a conserved tertiary structure arrangement, and utilization of similar substrate binding interfaces and conserved active site residue functions. These findings suggest a general sortase mechanism, conserved among classes and species.</p><p>Initial steps have also been taken to characterize <italic>S. pyogenes</italic> sortase C (SrtC). SrtC enzymes are unique in that they covalently polymerize secreted proteins, rather than attach them to peptidoglycan. Full length and truncation mutant constructs of SrtC and its substrate, T3, and peptide substrate mimics have been produced in soluble form for use in kinetic assays. Additionally, initial crystallization conditions have been identified for <italic>S. pyogenes</italic> SrtC towards the goal of three-dimensional structure determination. A homology model of the structure has also been produced, displaying many of the general features observed for other sortase enzymes.</p><p>Additionally, a computational analysis of the mechanism of isopeptide bond formation in <italic>S. pyogenes</italic> SPy0128, a substrate of <italic>S. pyogenes</italic> SrtC, has been performed. Isopeptide bonds have previously been found in structural studies of Gram-positive bacterial adhesins in each domain of these multi-domain proteins. The bonds are typically formed between conserved lysine and asparagine residues, and formation is likely catalyzed by adjacent conserved glutamates. A direct nucleophilic attack mechanism, starting from an inverse protonation state, is supported in this study. Of note, there appears to be temporal regulation of isopeptide bond formation in the different domains of <italic>S. pyogenes</italic> SPy0128, with the C-terminal domain isopeptide bond forming prior to or simultaneously with the N-terminal domain isopeptide bond.</p><p>Previous studies suggest that SrtA activity is required for <italic>S. aureus</italic> to survive phagocytosis by a macrophage. The production of reactive oxygen species by professional phagocytes could lead to inhibition of SrtA via oxidation of a conserved nucleophilic cysteine residue in the active site. Through determination of inhibition kinetics, identification of oxidative modifications, reduction potential measurements, and analyses of SrtA in vivo activity in the presence of reactive oxygen species, it has been demonstrated that <italic>S. aureus</italic> SrtA is resistant to oxidative inhibition. These findings support SrtA activity inside the phagolysosome of a professional phagocyte and likely contribute to the ability of <italic>S. aureus</italic> to evade the innate immune system.</p><p>The roles of sortases and their substrates during <italic>S. aureus</italic> survival inside professional phagocytes have not been thoroughly investigated. Through analysis of the regulation of these surface proteins under phagolysosomal conditions and macrophage phagocytosis survival assays, initial characterization of the functions of sortases and their substrates in this environment has been completed. Previous studies have suggested a role for SrtA and its substrate, Protein A, and these genes and two other sortase-substrates were upregulated in response to phagolysosomal conditions. However, neither sortases nor their substrates demonstrated a direct function in phagocytosis survival. These findings imply a complex interplay between <italic>S. aureus</italic> and professional phagocytes. Further studies are necessary to delineate the direct activities of surface anchored proteins during phagocytosis of <italic>S. aureus</italic> by professional phagocytes.</p> / Dissertation

Thesis (Ph.D.)--University of Wollongong, 2006. / Typescript. Includes bibliographical references: leaf 148-160.

Thesis (Ph. D.)--University of Oklahoma. / Bibliography: leaves 143-155.

Thesis (honors)--Georgia State University, 2007. / Title from file title page. Under the direction of Dabney White Dixon. Electronic text (88 p. : col. ill.) : digital, PDF file. Description based on contents viewed Sept. 30, 2008. Includes bibliographical references (p. 46-47).

The human pathogen Streptococcus pyogenes (Group A Streptococcus or GAS) are a versatile Gram-positive cocci that havw shown complex modes of regulation of its different virulence factors. Discoveries of a few small non-coding RNAs (sRNAs) in S. pyogenes and their influence on the expression of virulence factors revealed an important role of sRNAs on S. pyogenes virulence. The genome-wide analysis of bacterial genomes for the discovery of sRNAs through computational methods has become an effective way to discover new sRNAs. In this study we provided a computational scheme where three different algorithms (RNAz, eQRNA, and sRNAPredict) were combined to increase the probabilities of predicting putative sRNAs within S. pyogenes' intergenic regions (IGR). A total of 46 candidates were chosen based on our criteria, and through Northern blot we analyzed each candidate. We obtained hybridization signals from twelve newly discovered sRNAs in S. pyogenes. Subsequently, we analyzed their sequence and their location within the IGR to find a putative -10 promoter region and possible Rho-independent terminator site, and their possible targets through computational methods. We further expanded our analysis of the new sRNAs by using Real-Time RT-PCR to determine the expression of sRNAs during different phases of growth. Our results showed that our computational scheme and experimental method was effective in predicting sRNAs previously undiscovered in S. pyogenes, and that more sRNAs are yet to be discovered and characterized, helping to further understand the regulation of virulence factors in S. pyogenes

Streptococcus pyogenes infections can give rise to a diverse array of long-term secondary sequelae, including those in the brain characterized by both motor and neuropsychiatric disorders: Sydenham’s chorea and Pediatric Autoimmune Neuropsychiatric Disorders Associated with Streptococcus infections (PANDAS). These conditions are thought to be mediated by neuroinflammatory responses and autoantibody entry into the brain, but the mechanisms are not well understood.Previous work by our laboratory has demonstrated that recurrent intranasal S. pyogenes (Group A Streptococcus, or GAS) inoculations in mice cause infiltration of CD4 T cells into the anterior brain, disruption of the blood-brain barrier (BBB), increased numbers of activated myeloid cells and degradation of excitatory synapses leading to neural circuitry deficits. However, the molecular mechanisms underlying these phenotypes have not been fully explored. To understand how the neurovasculature and myeloid cells respond to recurrent GAS infections at the transcriptome level, I profiled cells from mouse olfactory bulb (OB) and nasal lymphoid tissue by single-cell RNA sequencing (scRNAseq). I found marked shifts in both endothelial cell and microglia populations at the transcriptome level after GAS infections, including downregulation of BBB-associated transcripts by endothelial cells (ECs), and increased production of inflammatory cytokines and chemokines, type I interferon response, and antigen presentation genes by microglia (Chapter 3). I validated several differentially expressed genes using flow cytometry, immunosorbant assays, RNA fluorescence in situ hybridization (FISH), and multiplexed error-robust FISH (MERFISH). Single-cell spatial transcriptomics of the OB revealed regional heterogeneity among microglial responses to GAS, possibly driven by proximity to infiltrating T cells. Analysis of transgenic CX3CR1/TMEM119 dual myeloid reporter mice confirmed that perivascular and meningeal macrophage numbers increase in response to GAS, but, unlike in other neuroinflammatory diseases, few macrophages infiltrate the brain parenchyma. Our laboratory has previously shown that Th17 cells are critical for BBB damage and activated microglia in response to repeated intranasal GAS infections, but the contribution of T helper (Th) 17 cell-derived cytokines in this process, as well as the transcriptional effects of Th17 cells on endothelial cells and microglia are unknown. To expand on these findings, I performed scRNAseq on retinoic acid-related orphan receptor γt (RORγt) mutant mice (Chapter 4) which showed a significant rescue in BBB-associated genes (e.g. Mfsd2a, Itm2a and Itih5) in endothelial cells. Chemokine production and type I interferon gene expression by microglia was also significantly rescued in RORγt mutants; surprisingly antigen presentation by microglia in response to GAS was exacerbated, at both the gene and protein level. Interleukin (IL)-17A is a major cytokine produced by Th17 cells. To examine the role of IL-17A in disease pathogenesis, I treated wild-type mice with an IL-17A neutralizing antibody during the course of GAS infections (Chapter 4). This treatment was sufficient to recapitulate the transcriptional effects on microglia and endothelial cells, as well as rescue BBB permeability previously found in RORγt mutants, indicating that IL-17A may play a critical role in transcriptional responses of endothelial cells and microglia to recurrent GAS infections in vivo. However, IL-17A did not disrupt tight junctions or induce transcytosis on ECs in vitro, suggesting that its effects on ECs in vivo are indirect. Th17 cells are capable of considerable phenotypic plasticity in response to chronic inflammation. To understand this process during recurrent GAS infections, I performed a time course analysis of CD4 T cell subsets after two, three, four and five infections (Chapter 5). This analysis revealed that proportions of “pathogenic” interferon γ-expressing Th17 cells increased over time, as did the number of CD4 T cells expressing granulocyte-macrophage colony stimulating factor (GM-CSF), a cytokine with pleiotropic effects on autoimmunity. Moreover, I determined that RORγt mutants have decreased proportions of GM-CSF+ CD4 T cells in their nasal mucosa, raising the question of whether GM-CSF may also contribute to CNS pathology (BBB permeability or microglial activation) in addition to IL-17A. To address this question, I generated mice deficient for GM-CSF in T cells and found that conditional deletion of GM-CSF in CD4+ cells partly rescued type I interferon and antigen presentation responses in microglia by scRNAseq, but did not rescue BBB leakage, suggesting that GM-CSF and IL-17A have distinct roles in the neurovascular and neuroinflammatory responses to GAS. To relate the findings in mice to the human disease, in Chapter 6 we performed cytokine profiling in sera from PANDAS/PANS patients at the acute phase of the disease using a multiplex bead-based immunoassay. We found that many chemokines and cytokines produced by activated microglia or macrophages in the mouse model were also highly elevated in the sera of PANDAS/PANS patients. These findings suggest an important link to the human disorder both to understand disease mechanisms in humans and to use them as future clinical biomarkers for diagnosis and treatment monitoring.

No / N-terminal lipidation is a major mechanism by which bacteria can tether proteins to membranes and one which is of particular importance to Gram-positive bacteria due to the absence of a retentive outer membrane. Lipidation is directed by the presence of a cysteine-containing `lipobox' within the lipoprotein signal peptide sequence and this feature has greatly facilitated the identification of putative lipoproteins by gene sequence analysis. The properties of lipoprotein signal peptides have been described previously by the Prosite pattern PS00013. Here, a dataset of 33 experimentally verified Gram-positive bacterial lipoproteins (excluding those from Mollicutes) has been identified by an extensive literature review. The signal peptide features of these lipoproteins have been analysed to create a refined pattern, G+LPP, which is more specific for the identification of Gram-positive bacterial lipoproteins. The ability of this pattern to identify probable lipoprotein sequences is demonstrated by a search of the genome of Streptococcus pyogenes, in comparison with sequences identified using PS00013. Greater discrimination against likely false-positives was evident from the use of G+LPP compared with PS00013. These data confirm the likely abundance of lipoproteins in Gram-positive bacterial genomes, with at least 25 probable lipoproteins identified in S. pyogenes

Pacientes com doença reumática cardíaca (DRC) desenvolvem lesões valvares mediadas por linfócitos T CD4+, capazes de reconhecer cruzadamente proteínas cardíacas e estreptocócicas pelo mecanismo de mimetismo molecular. Neste trabalho empregamos uma biblioteca peptídica de Phage Display para identificar auto-antígenos cardíacos capazes de serem reconhecidos por duas linhagens intralesionais de linfócitos T e um clone derivado de uma das linhagens isolados de pacientes com DRC. A análise dos peptídeos dos fagos em banco de dados de proteínas revelou novos epitopos da miosina cardíaca, laminina, vimentina e outras proteínas coiled-coil, provavelmente involvidos no processo auto-imune da DRC. Outras moléculas inflamatórias como citocinas, integrinas e fatores de crescimento também foram identificadas / Rheumatic heart disease (RHD) patients develop valvar lesions with CD4+ T lymphocytes infiltrating the heart. Molecular mimicry between streptococcal and cardiac proteins recognized by these T cells may explain these auto-aggressive lesions. In the present work we used a Phage Display peptide library to identify cardiac antigens which could be recognized by two heart infiltrate T cell lines and by a T cell clone derived from one of the lines which were isolated from RHD patients. Using the protein data bank to analyse the phage peptides, we observed that many sequences showed homology with cardiac myosin, laminin, vimentin and other coiled-coil proteins, suggesting the involvement of these proteins in the autoimmune process of RHD. Other inflammatory molecules such as cytokines and integrins were also identified

Pacientes com doença reumática cardíaca (DRC) desenvolvem lesões valvares mediadas por linfócitos T CD4+, capazes de reconhecer cruzadamente proteínas cardíacas e estreptocócicas pelo mecanismo de mimetismo molecular. Neste trabalho empregamos uma biblioteca peptídica de Phage Display para identificar auto-antígenos cardíacos capazes de serem reconhecidos por duas linhagens intralesionais de linfócitos T e um clone derivado de uma das linhagens isolados de pacientes com DRC. A análise dos peptídeos dos fagos em banco de dados de proteínas revelou novos epitopos da miosina cardíaca, laminina, vimentina e outras proteínas coiled-coil, provavelmente involvidos no processo auto-imune da DRC. Outras moléculas inflamatórias como citocinas, integrinas e fatores de crescimento também foram identificadas / Rheumatic heart disease (RHD) patients develop valvar lesions with CD4+ T lymphocytes infiltrating the heart. Molecular mimicry between streptococcal and cardiac proteins recognized by these T cells may explain these auto-aggressive lesions. In the present work we used a Phage Display peptide library to identify cardiac antigens which could be recognized by two heart infiltrate T cell lines and by a T cell clone derived from one of the lines which were isolated from RHD patients. Using the protein data bank to analyse the phage peptides, we observed that many sequences showed homology with cardiac myosin, laminin, vimentin and other coiled-coil proteins, suggesting the involvement of these proteins in the autoimmune process of RHD. Other inflammatory molecules such as cytokines and integrins were also identified

La tagatose-1,6-biphosphate aldolase de Streptococcus pyogenes est une aldolase de classe I qui fait montre d'un remarquable manque de spécificité vis à vis de ses substrats. En effet, elle catalyse le clivage réversible du tagatose-1,6-biphosphate (TBP), mais également du fructose-1,6-biphosphate (FBP), du sorbose-1,6-biphosphate et du psicose-1,6-biphosphate, quatre stéréoisomères, en dihydroxyacétone phosphate (DHAP) et en glycéraldéhyde-3-phosphate (G3P). Afin de mettre à jour les caractéristiques du mécanisme enzymatique, une étude structurale de la TBP aldolase de S. pyogenes, un pathogène humain extrêmement versatile, a été entreprise. Elle a permis la résolution de la structure native et en complexe avec le DHAP, a respectivement 1.87 et 1.92 Å de résolution. Ces mêmes structures ont permis de se représenter plus clairement le site actif de l'enzyme en général, et les résidus catalytiques en particulier. Le trempage des cristaux de TBP aldolase dans une solution saturante de DHAP a en outre permis de piéger un authentique intermédiaire iminium, ainsi que sa géométrie particulière en atteste. Des expériences d'échange de proton, entreprises afin d'évaluer le stéréoisomérisme du transfert de proton catalytique, ont également permis de faire une intéressante découverte : la TBP aldolase ne peut déprotoner le coté pro-R du C3 du DHAP, mais peut le protonner. Ce résultat, ainsi que la comparaison de la structure du complexe TBP aldolase-DHAP avec la structure du complexe FBP aldolase de muscle de lapin- DHAP, pointe vers un isomérisme cis-trans autour du lien C2-C3 de la base de Schiff formée avec le DHAP. De plus, la résolution de ces deux structures a permis de mettre en évidence trois régions très mobiles de la protéine, ce qui pourrait être relié au rôle postulé de son isozyme chez S. pyogenes dans la régulation de l’expression génétique et de la virulence de la bactérie. La cristallographie par rayons X et la cinétique enzymatique ont ainsi permis d'avancer dans l'élucidation du mécanisme et des propriétés structurales de cette enzyme aux caractéristiques particulières. / Tagatose-1,6-biphosphate aldolase from Streptococcus pyogenes is a class I aldolase that shows a lack of stereospecificity that is rare in enzymes in general, and in aldolases in particular. This aldolase catalyzes the reversible cleavage of tagatose-1,6-biphosphate (TBP), fructose-1,6-biphosphate (FBP), sorbose-1,6-biphosphate and psicose-1,6-biphosphate, four stereoisomers, in dihydroxyacetone phosphate and glyceraldehyde-3-phosphate (DHAP). In order to understand its mechanism, a structural study of TBP aldolase from S. pyogenes, one of the most versatile and virulent human pathogen, was initiated and high resolution crystallographic structures of native and DHAP-liganded TBP aldolase were solved. These structures allowed us to gain informations regarding active site residues implicated in catalysis and that give rise to the apparent lack of specificity. Soaking of TBP aldolase crystals in saturating DHAP solution specifically trapped the iminium intermediate, as demonstrated by its geometry. Furthermore, proton transfer studies uncovered an interesting phenomenon: TBP aldolase from S. pyogenes is unable to detritiate pro-R labelled hydrogen position at C3 of DHAP, yet it is able to tritiate both the pro-R and the pro-S position. These results, taken together with the superposition of the DHAP-TBP aldolase with the DHAP-FBP aldolase from rabbit muscle, suggest a cis-trans isomerism about the Schiff base C2-C3 bond. The resolution of both the native and the liganded structure also proved useful in identifying three very mobile regions in the protein. This trend could be linked to the putative metabolic sensor and genetic expression regulator role of LacD.1 in S. pyogenes. X-rays crystallography and traditional enzymatic kinetics allowed us to gain insights into the catalytic mechanism and others structural properties of this important metabolic enzyme.