京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22308号 / 医博第4549号 / 新制||医||1040(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 竹内 理, 教授 長尾 美紀, 教授 杉田 昌彦 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DGAM

京都大学 / 新制・課程博士 / 博士(医学) / 甲第23063号 / 医博第4690号 / 新制||医||1048(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 岩田 想, 教授 長尾 美紀, 教授 秋山 芳展 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 新制・課程博士 / 博士(医学) / 甲第23605号 / 医博第4792号 / 新制||医||1055(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 長尾 美紀, 教授 竹内 理, 教授 秋山 芳展 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Includes bibliographical references / Asymptomatic children can be a major reservoir of pharyngeal Group A Streptococcus (GAS) with reported figures ranging from <10% to >20% in developing countries. There is a need to document GAS carriage in school children, which, together with the molecular characterisation (M-typing) of strains harboured in the pharynx of carriers, will help to ascertain the extent to which disease strains are prevalent amongst carriers. This background information would serve to assist healthcare providers in diagnosing symptomatic GAS pharyngitis, as well as could potentially contribute to the development of a GAS vaccine. Currently, data on GAS carriage prevalence and M-type distribution in African countries are largely scant. We therefore undertook to perform a systematic review to determine the prevalence and type distribution of asymptomatic streptococcus carriage in children aged 5 -15years, residing in African countries. Methods: We conducted a comprehensive literature search among a number of databases, using an African search filter to identify GAS prevalence studies that report on children between the ages of 5 - 15 years who reside in African countries. Electronic searches were complemented by a hand search performed on reference lists of potentially included studies. The search was not limited by year of publication and language. Two evaluators independently reviewed, rated, and abstracted data from each article. Prevalence estimates were pooled in a meta-analysis and stratified according to region and study design using Stata®. Specifically, we applied the random effects metaprop routine to aggregate prevalence estimates and account for between study variability in calculating the overall pooled estimates and 95% CI for GAS carriage prevalence.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第20566号 / 医博第4251号 / 新制||医||1022(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 竹内 理, 教授 小柳 義夫, 教授 秋山 芳展 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

No / Streptococcus agalactiae is a major neonatal pathogen that is able to colonise various host environments and is associated with both gastrointestinal and vaginal maternal carriage. Maternal vaginal carriage represents the major source for transmission of S. agalactiae to the foetus/neonate and thus is a significant risk factor for neonatal disease. In order to understand factors influencing maternal carriage we have investigated growth and long term survival of S. agalactiae under conditions of low pH and nutrient stress in vitro. Surprisingly, given that vaginal pH is normally <4.5, S. agalactiae was found to survive poorly at low pH and failed to grow at pH 4.3. However, biofilm growth, although also reduced at low pH, was shown to enhance survival of S. agalactiae. Proteomic analysis identified 26 proteins that were more abundant under nutrient stress conditions (extended stationary phase), including a RelE family protein, a universal stress protein family member and four proteins that belong to the Gls24 (PF03780) stress protein family. Cumulatively, these data indicate that novel mechanisms are likely to operate that allow S. agalactiae survival at low pH and under nutrient stress during maternal vaginal colonisation and/or that the bacteria may access a more favourable microenvironment at the vaginal mucosa. As current in vitro models for S. agalactiae growth appear unsatisfactory, novel methods need to be developed to study streptococcal colonisation under physiologically-relevant conditions.

No / Metal ion acquisition and homeostasis are essential for bacterial survival, growth and physiology. A family of metal ion, ABC-type import systems have been identified in Gram-positive bacteria, in which the solute-binding proteins are predicted to be membrane-anchored lipoproteins. The prediction that the MtsA protein of Streptococcus agalactiae A909 is a lipoprotein was confirmed. The expression of MtsA was co-ordinately regulated by the presence of both manganese and ferrous ions suggesting that MtsA may be involved in the uptake of both these ions. MtsA was shown to be expressed at levels of ferrous ions known to be present in amniotic fluid, a growth medium for S. agalactiae during neonatal infection.

No / Streptococcus agalactiae is a significant pathogen causing invasive disease in neonates and thus an understanding of the molecular basis of the pathogenicity of this organism is of importance. N-terminal lipidation is a major mechanism by which bacteria can tether proteins to membranes. Lipidation is directed by the presence of a cysteine-containing 'lipobox' within specific signal peptides and this feature has greatly facilitated the bioinformatic identification of putative lipoproteins. We have designed previously a taxon-specific pattern (G+LPP) for the identification of Gram-positive bacterial lipoproteins, based on the signal peptides of experimentally verified lipoproteins (Sutcliffe I.C. and Harrington D.J. Microbiology 148: 2065-2077). Patterns searches with this pattern and other bioinformatic methods have been used to identify putative lipoproteins in the recently published genomes of S. agalactiae strains 2603/V and NEM316. A core of 39 common putative lipoproteins was identified, along with 5 putative lipoproteins unique to strain 2603/V and 2 putative lipoproteins unique to strain NEM316. Thus putative lipoproteins represent ca. 2% of the S. agalactiae proteome. As in other Gram-positive bacteria, the largest functional category of S. agalactiae lipoproteins is that predicted to comprise of substrate binding proteins of ABC transport systems. Other roles include lipoproteins that appear to participate in adhesion (including the previously characterised Lmb protein), protein export and folding, enzymes and several species-specific proteins of unknown function. These data suggest lipoproteins may have significant roles that influence the virulence of this important pathogen.

No / Streptococcus equi is the aetiological agent of strangles, one of the most prevalent diseases of the horse. The animal suffering and economic burden associated with this disease necessitate effective treatment. Current antibiotic therapy is often ineffective and thus recent attention has focused on vaccine development. A systematic understanding of S. equi virulence, leading to the identification of targets to which protective immunity can be directed, is a prerequisite of the development of such a vaccine. Here, the virulence factors of S. equi are reviewed.

Heme is vital to a variety of cellular functions in bacteria ranging from energy generation to iron reserve. Group A streptococcus (GAS) is a prevalent bacterial pathogen that is responsible for an array of human diseases ranging from simple, self-limiting, mucosal and skin infections to invasive and systemic manifestations. GAS needs iron for growth and can satisfy this nutritional requirement by scavenging the metal from heme. The pathogen produces powerful hemolysins that facilitate heme release during infection. Heme is captured and relayed through the GAS cell wall and cytoplasmic membrane by dedicated receptors and transporters. To-date, the fate of the acquired heme is unknown in Streptococci. Although heme is nutritionally beneficial for GAS growth, its pro-oxidant and lipophilic nature makes it a liability with damaging effects on cellular components. The conundrum associated with heme use is particularly pertinent to GAS pathophysiology since invasive GAS infections involve massive hemolysis and the generation of unescorted heme in excess. In this dissertation, I aimed to describe the mechanisms that GAS uses for heme catabolism while managing its toxicity. I conducted a biochemical characterization of a new enzyme, HupZ in GAS that degrades heme in vitro. Similar to the heme oxygenase-1 (HO-1), HupZ activity leads to the formation of iron, CO, and a biliverdin-like product. I also investigated the impact of heme on GAS physiology and identified key mediators in the repair and detoxification process. This study demonstrated that heme exposure leads to a general stress response that involves the activation of antioxidant defense pathways to restore redox balance. Further, I studied a 3-gene cluster, pefRCD (porphyrin-regulated efflux RCD), which was activated by environmental heme, and provided support to my hypothesis that the pefRCD gene encodes a heme-sensing regulator (PefR) and heme efflux system (PefCD). I showed that the pef system protects GAS cells from heme-induced damage to the membrane and DNA by preventing cellular accumulation of heme. In conclusion, this dissertation addresses key knowledge gaps in GAS physiology and provides new insights into heme metabolism of GAS.