Streptococcus pneumoniae est une bactérie à la fois commensale et pathogène opportuniste chez l’humain. Elle est responsable de nombreuses infections telles que la pneumonie, la méningite, l’otite moyenne et la sinusite. En maladie infectieuse, S. pneumoniae occupe une place importante en tant que l’une des principales causes de morbidité et de mortalité dans le monde. Elle est dotée de plusieurs capacités fascinantes, comme la compétence naturelle pour l’aider à résister aux antibiotiques et la grande diversité des sérotypes capsulaires pour contourner la vaccination. Puisque la résistance aux antibiotiques ne cesse de menacer l’efficacité des thérapies standards, la thérapie par phage est maintenant reconsidérée comme une des alternatives thérapeutiques. La réévaluation des phages fait renaitre l’espoir thérapeutique, mais sans élucider leur mécanisme d’interaction et décortiquer leur mystère cet espoir restera modeste. Ce projet de doctorat consiste à mieux comprendre les phages infectant S. pneumoniae et les interactions phage-hôte. Dans un premier temps, le potentiel des pneumophages à infecter Streptococcus mitis, une espèce phylogénétiquement proche de S. pneumoniae, a été mis en évidence. Deux pneumophages se sont avérés les premiers phages virulents capables d’infecter S. mitis, bactérie pathogène responsable d’endocardite. Les deux phages pouvaient non seulement se répliquer dans S. mitis mais également produisent des plages de lyses plus visibles. Ensuite, la comparaison du génome des phages a confirmé que le changement de l’hôte n’induit aucune variation aux génomes des phages testés. Cependant, plusieurs mutations ont été observées dans la séquence génomique du podophage sauvage et il a fait ensuite l’objet d’une nouvelle annotation. Dans un deuxième temps, l’étude des interactions phage-hôte chez S. pneumoniae a été approfondie. Pour ce faire, l’implication de plusieurs facteurs de l’hôte dans la réplication des pneumophages a été étudiée. Plusieurs gènes pneumococciques se sont avérés nécessaires ou impliqués pour assurer l'efficacité de la réplication des phages seuls ou en cocktail. D’un autre côté, en étudiant ces facteurs de l’hôte, des gènes/ protéines potentiellement essentiels à la viabilité de S. pneumoniae ont été identifiés. Cette étude a aussi permis d’identifier de nouvelles cibles thérapeutiques et donne un nouvel aperçu du réseau complexe des interactions phage-hôte. / Streptococcus pneumoniae is a commensal and opportunistic pathogen bacterium, exclusively found in humans. It is the main agent of many infections such as pneumonia, meningitis, otitis media and sinusitis. S. pneumoniae infections are a major cause of morbidity and mortality worldwide. S. pneumoniae has several fascinating abilities, such as natural competence to facilitate the acquisition of antibiotic resistance genes and diversity of capsular serotypes to circumvent the vaccination. The rise of antibiotic resistant bacteria continues to threaten the effectiveness of standard therapies and as such phage therapy is now reconsidered as a therapeutic alternative. The reevaluation of phages as therapeutic agents must go through a better understanding of phage-bacterium interactions. This PhD thesis aims to better understand S. pneumoniae virulent phages and phage-host interactions. First, the ability of pneumophages to infect Streptococcus mitis, a species phylogenetically related to S. pneumoniae, was demonstrated. The pneumophages are the first two virulent phages able to infect this pathogenic bacterium, the common cause of bacterial endocarditis. Both pneumophages could not only replicate in S. mitis but also produced more visible plaques on this host. The comparison of the genomes of each phage grown on both hosts produced identical nucleotide sequences, confirming that S. mitis as a host does not induce any nucleotide variation. However, the genomic sequence of wild-type podophage was different than the previously reported sequence and it was the subject of a new annotation. In addition, S. pneumoniae phage-host interactions were investigated. The involvement of several host factors in replication of both pneumophages was observed. Indeed, several pneumococcal genes were found to be necessary or involved to ensure efficient phage replication. Moreover, the study of these host factors has led to the identification of new genes that appear to be essential for viability and normal growth of S. pneumoniae. This project led to identify new potential therapeutic targets and provided new insight into the complex network of phage-host interactions.

HPr fait partie du système de transport phosphoénolpyruvate: sucre phosphotransferase (PTS). HPr peut être phosphorylée sur Hisis, par l'enzyme I (El) du PTS, et sur Sér46, par la HPr(Ser) kinase/phosphorylase (HprK/P). Finalement, HPr peut être phosphorylée sur les deux résidus, ce qui génère HPr(Ser-P)(His~P). L'objectif principal de l'étude présentée dans cette thèse était de déterminer par quelle(s) voie(s) HPr(Ser-P)(His~P) est synthétisée chez les streptocoques. Théoriquement, HPr(Ser-P)(His~P) peut être synthétisée via la phosphorylation de HPr(Ser-P) par El et/ou de HPr(His-P) par HprK/P. Nous avons étudié la cinétique de ces voies de synthèse chez Streptococcus salivarius. Les résultats de l'étude avec El ont montré que (i) la kcat/Km pour HPr(Ser-P) était ~5 OOOx plus faible que pour HPr à pH 7,9 et 37°C, (ii) aucun intermédiaire glycolytique ne stimulait la synthèse de HPr(Ser-P)(His~P), (iii) la synthèse de HPr(Ser-P)(His~P) était ~8x plus efficace à pH acide, des conditions retrouvées dans le cytoplasme des streptocoques en croissance, et (iv) la synthèse du HPr(Ser-P)(His~P) de Bacillus subtilis par El de cet organisme était aussi stimulée à pH acide. Les résultats suggèrent que la synthèse de HPr(Ser-P)(His~P) chez les streptocoques résulterait des concentrations élevées en HPr(Ser-P) et de la stimulation de la réaction à pH acide. L'absence de HPr(Ser-P)(His~P) chez B. subtilis cultivée en présence de glucose s'expliquerait en partie par le fait que cette bactérie maintient son pH intracellulaire constamment au-dessus de la neutralité. Les résultats de l'étude avec HprK/P ont montré que les kQJKm pour les HPr(H15D) et HPr(H15E) étaient ~13x plus faibles que pour HPr à pH 7,4 et 37°C. Dans des conditions où HPr(His~P) était stable (pH 8,6 et 15°C), les kcJKm pour HPr(H15D) et HPr(His~P) Ill étaient respectivement neuf et 26 fois plus faibles que pour HPr. Finalement, la phosphorylation de HPr(H15D) n'était que marginalement stimulée à pH acide et le FBP ne stimulait pas la synthèse de HPr(Ser-P)(His~P). Les résultats suggèrent que (i) l'inefficacité de la phosphorylation de HPr(His~P) résulterait de la présence de la charge négative en position 15 et d'autres éléments structuraux et (ii) la contribution de HprK/P à la synthèse de HPr(Ser-P)(His~P) chez les streptocoques serait marginale.

The bacterial pathogens Streptococcus agalactiae and S. iniae have the capacity to infect a wide range of fish species throughout the world, with Nile tilapia (Oreochromis niloticus) being particularly susceptible. Global tilapia aquaculture production was estimated to be 3.5 million tonnes in 2008, and has a significant contribution in the global farmed fish market. Due to their ability to adapt to a wide range of culture systems the commercialisation of tilapia production has occurred in more than 100 countries. However, countries such as China have suffered from severe and extensive outbreaks of streptococcosis in cultured tilapia continuously for many years. Such large-scale outbreaks in China have resulted in a loss of approximately US$0.4 billion in 2011. Fish are permanently exposed to a plethora of pathogens and natural disease outbreaks are complex host-pathogen interactions that seldom involve single pathogen infections. As a consequence, simultaneous infections, alternatively called concurrent or co-infections, are starting to receive interest from aquatic disease researchers. Streptococcus agalactiae and S. iniae infections can both occur in the same geographic area and both S. agalactiae and S. iniae have been found to be present on the same farm in a single disease outbreak. It has been found that a disease outbreak caused by one these pathogens can be followed by another outbreak from the other. These two pathogens have serious effects on the tilapia aquaculture industry yet there is no information regarding S. agalactiae and S. iniae co-infections. Such information would be valuable for understanding epidemiology and the development of improved treatment and control of aquatic streptococcosis infections. The overall aim of this study was to investigate the pathogenesis of S. agalactiae and S. iniae in Nile tilapia. One important aspect of investigating simultaneous infections was to examine if there was any competition or synergy between S. agalactiae and S. iniae in vitro or in vivo. It was found that competition between S. agalactiae and S. iniae in vitro was inconsistent between different experimental systems. Results indicated that there was either no interaction between bacterial species or they coexisted during in vitro competition assays. Whereas, an in vivo model utilising wax moth larvae (Galleria mellonella) suggested that during a simultaneous infection with S. agalactiae and S. iniae the total levels of larval mortality were lower than expected indicating that the pathogens may have interacted with one another in a competitive manner. Investigations were also conducted to identify the expression of virulence factors in vitro for S. agalactiae and S. iniae. Comparisons were then made to ascertain any inter- and intra-species variation. Results demonstrated that both S. agalactiae and S. iniae strains possessed a capsule but varied in their haemolytic activity, blood survival and resistance to complement-mediated killing. These variations suggested that the two bacterial species differed in their mechanisms of pathogenicity where aquatic S. agalactiae strains may initially have a more systemic spread of infection and aquatic S. iniae strains may utilise a more localised spread of infection within the host. This hypothesis was tested through the development of a robust and reliable challenge model for S. agalactiae and S. iniae in Nile tilapia. Through this work it was apparent that fish infected with S. iniae experienced an acute infection with morbidity/mortality occurring 1 – 3 days after exposure. Whereas, the S. agalactiae challenged fish showed a more chronic infection with morbidity/mortality occurring from 1 – 6 days after exposure. Findings clearly demonstrated a more systemic spread of infection during a S. agalactiae challenge with high bacterial loads in all the organs examined. Streptococcus iniae was observed in fewer organs of infected fish and bacterial numbers were substantially lower. Concurrent infections are complex in natural conditions and in experimental studies. As a result a substantial amount of research will be required to fully understand the nature of co-infection with these two streptococci. This study has provided a solid foundation upon which to base future work.

Thesis (Ph. D.)--University of Alabama at Birmingham, 2008. / Title from first page of PDF file (viewed June 5, 2008). Includes bibliographical references.

Akademisk avhandling--Lund. / Extra t.p., with thesis statement, inserted. Bibliography: p. 11-14.

Akademisk avhandling--Lund. / Extra t.p., with thesis statement, inserted. Bibliography: p. 11-14.

Source: Masters Abstracts International, Volume: 46-04, page: 2101. Adviser: Carol J. Baker. Includes bibliographical references.

Thesis (doctoral)--Karolinska Institutet, Stockholm, 1990. / Extra t.p. with thesis statement inserted. Includes bibliographical references.

Thesis (doctoral)--Karolinska Institutet, Stockholm, 1990. / Extra t.p. with thesis statement inserted. Includes bibliographical references.

Autolysins are endogenous enzymes responsible for the cleavage of specific bonds in the bacterial sacculus resulting in damage to the integrity and protective properties of the cell wall. The true biological functions of these enzymes are largely unknown. However, they have been implicated in various important biological synthesis processes making their characterization important. Antibiotic susceptibility testing showed these streptococcal strains to have broad spectrum inhibitory concentrations. The major autolysins of selected streptococcal strains were detected and partially characterized by renaturing sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis with substrate-containing gels (zymograms). The autolysins were isolated from the specific culture supematants using 4% SDS precipitation and were shown to have apparent molecular masses ranging from 60kDa to 20kDa. Four major autolysins named A, B, C, and D from the Streptococcus milleri 77 strain were characterized. Lytic enzymes were blotted onto polyvinylidene difluoride (PVDF) membrane and N-terminally sequenced. Sequences showed between 100% and 80% similarity to that of a muramidase, glucosaminidase and a peptidase from S. mutans, S. pyogenes and S. pneumonia respectively. Biochemical characterization confirmed autolysin A to exhibit muramidase activity with both autolysin Band C exhibiting endopeptidase activity. Autolysin D showed an 80% N-terminal sequence similarity to Millericin B, a peptidoglycan hydrolase that is known to exhibit peptidase activity. Autolysis was determined using different buffers at two optimal pHs. Assaying for autolytic activity at different growth stages showed autolysis to be moderate during the lag and early exponential phases of the growth cycle. The activities of autolysins were the highest in the late exponential phase and the stationary phase of growth. Zymogram analysis showed that the Streptococcal milleri strains had moderate autolytic expression during the early and late exponential phases of the growth cycle. Control regulatory mechanisms of autolysins were determined in the presence or absence of specific charged groups, such as teichoic acids. In each case the absence of these charged groups inhibited the rate of autolysis, suggesting that the absence of teichoic acids could play a role in the regulation of the autolysins. Two-dimensional-SDS and zymographic-electrophoresis was used to determine total protein profiles for each strain. This is the first report using twodimensional zymography. Specific proteins which were either up- or down-regulated were identified. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2005.