Group A Streptococcal S Protein Utilizes Red Blood Cells as Immune Camouflage and Is a Critical Determinant for Immune Evasion.

Group A Streptococcus (GAS) is a human-specific pathogen that evades the host immune response through the elaboration of multiple virulence factors. Although many of these factors have been studied, numerous proteins encoded by the GAS genome are of unknown function. Herein, we characterize a biomimetic red blood cell (RBC)-captured protein of unknown function-annotated subsequently as S protein-in GAS pathophysiology. S protein maintains the hydrophobic properties of GAS, and its absence reduces survival in human blood. S protein facilitates GAS coating with lysed RBCs to promote molecular mimicry, which increases virulence in vitro and in vivo. Proteomic profiling reveals that the removal of S protein from GAS alters cellular and extracellular protein landscapes and is accompanied by a decrease in the abundance of several key GAS virulence determinants. In vivo, the absence of S protein results in a striking attenuation of virulence and promotes a robust immune response and immunological memory

Practical Approaches to Biological Network Discovery

This dissertation addresses a current outstanding problem in the field of systems biology, which is to identify the structure of a transcriptional network from high-throughput experimental data. Understanding of the connectivity of a transcriptional network is an important piece of the puzzle, which relates the genotype of an organism to its phenotypes. An overwhelming number of computational approaches have been proposed to perform integrative analyses on large collections of high-throughput gene expression datasets to infer the structure of transcriptional networks. I put forth a methodology by which these tools can be evaluated and compared against one another to better understand their strengths and weaknesses. Next I undertake the task of utilizing high-throughput datasets to learn new and interesting network biology in the pathogenic fungus Cryptococcus neoformans. Finally I propose a novel computational method for mapping out transcriptional networks that unifies two orthogonal strategies for network inference. I apply this method to map out the transcriptional network of Saccharomyces cerevisiae and demonstrate how network inference results can complement chromatin immunoprecipitation: ChIP) experiments, which directly probe the binding events of transcriptional regulators. Collectively, my contributions improve both the accessibility and practicality of network inference methods

Computational approaches to predict protein functional families and functional sites.

Understanding the mechanisms of protein function is indispensable for many biological applications, such as protein engineering and drug design. However, experimental annotations are sparse, and therefore, theoretical strategies are needed to fill the gap. Here, we present the latest developments in building functional subclassifications of protein superfamilies and using evolutionary conservation to detect functional determinants, for example, catalytic-, binding- and specificity-determining residues important for delineating the functional families. We also briefly review other features exploited for functional site detection and new machine learning strategies for combining multiple features

Importance of protein Ser/Thr/Tyr phosphorylation for bacterial pathogenesis

Protein phosphorylation regulates a large variety of biological processes in all living cells. In pathogenic bacteria, the study of serine, threonine, and tyrosine (Ser/Thr/Tyr) phosphorylation has shed light on the course of infectious diseases, from adherence to host cells to pathogen virulence, replication, and persistence. Mass spectrometry (MS)-based phosphoproteomics has provided global maps of Ser/Thr/Tyr phosphosites in bacterial pathogens. Despite recent developments, a quantitative and dynamic view of phosphorylation events that occur during bacterial pathogenesis is currently lacking. Temporal, spatial, and subpopulation resolution of phosphorylation data is required to identify key regulatory nodes underlying bacterial pathogenesis. Herein, we discuss how technological improvements in sample handling, MS instrumentation, data processing, and machine learning should improve bacterial phosphoproteomic datasets and the information extracted from them. Such information is expected to significantly extend the current knowledge of Ser/Thr/Tyr phosphorylation in pathogenic bacteria and should ultimately contribute to the design of novel strategies to combat bacterial infections

Genomic and experimental investigations into pneumococcal bacteriocins and their role in competition

Streptococcus pneumoniae ('the pneumococcus') is a frequent asymptomatic coloniser of the nasopharynx, from where it may disseminate to cause life-threatening infections including pneumonia, bacteraemia, and meningitis. Pneumococcal disease remains a leading cause of global mortality despite the use of safe and effective pneumococcal conjugate vaccines (PCVs). Bacteriocins are antimicrobial peptides that are produced by bacteria to target competitor bacteria within the ecological niche. Twenty pneumococcal bacteriocins have been characterised in silico, but their role in competition within the nasopharynx is not yet understood. In the first part of this project, I studied the distribution of bacteriocin genes in two large genomic datasets (>5,000 pneumococcal genomes in total) sampled from Iceland and Kenya. The distribution of some bacteriocins differed by location, between pneumococci recovered from carriage and disease, and between pneumococci recovered before and after the introduction of PCVs. These observations were largely explained by the association of bacteriocins with clonal complexes and suggested that there were different competition dynamics among pneumococci. A functional model of the streptococcin bacteriocins was generated using structural predictions. This informed further genomic studies, which observed genetic heterogeneity in the streptococcins. A dataset of >1,800 genomes from non-pneumococcal streptococci was screened for streptococcins, which were commonly harboured by viridans streptococci. There was evidence that the streptococcin diversification was driven by horizontal exchange between pneumococci and non-pneumococcal streptococci. In the final part of the project, the streptococcins were studied experimentally. A streptococcin toxin was isolated for the first time using a recombinant expression and purification method. The streptococcin was used in susceptibility assays against a panel of pneumococci and non-pneumococcal streptococci. Preliminary results suggested that the streptococcin had activity against some of the test strains. Results presented in this thesis expand our understanding of pneumococcal bacteriocins and will be used to inform further genomic and experimental studies.Open Acces

Role of glucose and CcpA in capsule expression and virulence of Streptococcus suis

Streptococcus suis is one of the most important pathogens in pigs and is also an emerging zoonotic agent. After crossing the epithelial barrier, S. suis causes bacteraemia, resulting in meningitis, endocarditis and bronchopneumonia. Since the host environment seems to be an important regulatory component for virulence, we related expression of virulence determinants of S. suis to glucose availability during growth and to the sugar metabolism regulator catabolite control protein A (CcpA). We found that expression of the virulence-associated genes arcB, representing arcABC operon expression, cps2A, representing capsular locus expression, as well as sly, ofs, sao and epf, differed significantly between exponential and early stationary growth of a highly virulent serotype 2 strain. Deletion of ccpA altered the expression of the surface-associated virulence factors arcB, sao and eno, as well as the two currently proven virulence factors in pigs, ofs and cps2A, in early exponential growth. Global expression analysis using a cDNA expression array revealed 259 differentially expressed genes in early exponential growth, of which 141 were more highly expressed in the CcpA mutant strain 10¿ccpA and 118 were expressed to a lower extent. Interestingly, among the latter genes, 18 could be related to capsule and cell wall synthesis. Correspondingly, electron microscopy characterization of strain 10¿ccpA revealed a markedly reduced thickness of the capsule. This phenotype correlated with enhanced binding to porcine plasma proteins and a reduced resistance to killing by porcine neutrophils. Taken together, our data demonstrate that CcpA has a significant effect on the capsule synthesis and virulence properties of S. suis

Phenotypic and Genotypic Models of Streptococcal Colonization of the Human Tonsil

Pharyngitis due to oropharyngeal infection with Streptococcus pyogenes is most commonly treated by using penicillin-derived antibiotics.While treatment failure in the 1950’s was reported in 4-8% of children, more recent studies have alarmingly found antibiotic failure as high as 20-40% providing the impetus to study this important pathogen. The contiguous mucosa along neighboring oropharyngeal surfaces is classically unaffected during Strep ”Throat” suggesting pathogen specificity for palatine tonsil epithelium. While recent studies are advancing the premise of pathogen-host microenvironment effects on streptococcal virulence, the specific interaction between S. pyogenes and the human tonsillar surface relative to neighboring tissues remains insufficiently understood. This thesis investigates the unique interaction between the Group A Streptococcus, GrAS, and the human tonsil surface. We were particularly interested in the transcriptional response exhibited by the bacterium during coculture with this tissue epithelium compared to epithelium from an anatomically neighboring sight to determine if phenotype might be informed by the genotypic profile. During the course of this work, we introduce new palatine tonsil tumorderived cell lines for their novel usefulness as a model for studying pharyngitis. Using primary tonsil epithelial cells from non-malignant patient samples to confirm our studies with the cancer cell line, we were successful in demonstrating a tissue-specific phenotype and have begun the process of elucidating the genotypic nuances that an M1 strain exhibited in direct response to the tonsillar environment. These and further studies should allow us to better understand the pathogenic adaptions exhibited by this bacterium at its preferred target niche for infection

Environmental Implications of Francisella Tularensis Biofilms

Francisella tularensis survives in one of the widest environmental ranges of any pathogen. Numerous mammals and arthropod vectors are infected by this highly virulent organism. How this zoonotic pathogen persists outside of its many hosts remains unexplored. We aimed to examine how F. tularensis interacts with environmental surfaces, and hypothesized that biofilm formation may enable survival of this organism in nature. By understanding the role these surface-attached bacterial communities play in F. tularensis ecology, we hope to gain insight into the mechanisms of environmental persistence and transmission of this pathogen. We identify chitin as a potential non-host niche for F. tularensis in nature using genetic, microscopic, and biochemical techniques. This abundant polysaccharide supported F. tularensis biofilm formation in the absence of an exogenous carbon source. This interaction was dependent on putative chitinase enzymes which hydrolyze the glycosidic bonds that connect GlcNAc monomers. Using a genetic screen, we identified adherence factors, including FTN_0308 and FTN0714 that promote attachment to chitin and colonization of chitin surfaces. We propose that biofilm formation on chitin surfaces in nature enables nutrient scavenging in oligotrophic environments allowing this pathogen to replicate and seed disease transmission. We found that the effect of nutrient limitation on F. tularensis biofilm formation extended beyond chitin utilization. Genetic studies indicated that nutrient starvation triggers a biofilm stress response. We identified static growth and nutrient deprivation as cues for enhanced biofilm formation. Microarray expression studies identified genes highiy expressed under these conditions, including F. tularensis biofilm determinants. Expression of nutrient transporters further indicated that biofilm formation promotes environmental persistence. We finally examined statically grown F. tularensis microscopically to determine if altered morphology explained the enhanced biofilm phenotype of these cultures. We discovered a novel F. tularensis appendage conserved between subspecies and structurally homologous to the Caulobacter crescentus stalk. These structures were observed in association with surfaces during both biofilm formation and during intracellular infection. A genetic screen for mutants in stalk formation revealed that stalk biosynthetic components are essential. We predict this structure aids in environmental persistence by facilitating surface attachment and nutrient uptake. Through this collective work we define evidence that surface association via biofilm formation promotes survival during nutrient limitation

07. Environmental Implications of Francisella Tularensis Biofilms

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