No description available.

No description available.

No description available.

Sequence typing is a rapidly evolving field and offers improved analysis into the genetic background and lineages of organisms compared to serological or DNA banding pattern based analysis. However, the resolution of molecular typing schemes varies between organisms and often loci used in sequence typing lack discriminatory power and give limited information into the evolution of the organism. This is particularly true of group B streptococci (GBS), where the same sequence types appear worldwide, which is unlikely for such a pathogen. This project aimed to develop a two component pathogen profiling approach which accurately reflected the phylogeny of GBS isolates, using elements of the core genome and elements of the variable genome. To address this a bioinformatic approach which selected loci for sequence typing based on predicting genes which evolve in the same manner as the average for the core genomes was adapted from a previously applied study for designing genus level sequence typing schemes. This informed the selection of candidate loci which were then experimentally verified, using a collection of 135 GBS clinical isolates. It was demonstrated that it was possible to obtain greater resolution and accuracy using only three unique genes that are intelligently selected, rather than using seven known housekeeping genes that are selected at random. Sources of hyper variability within the genome, in particular the presence of mononucleotide repeats (MNR) were investigated in non-coding DNA. It was postulated that these regions of DNA are more prone to mutation due to the lack of selective pressures, the presence of MNR repeats make these regions more unstable during replication and that in core genes these regions may be involved in genomic regulation by slipped strand mispairing. Results did confirm that non-coding DNA containing MNR repeats were more variable than DNA without them but these did not match the discriminatory power of MlST typing or the new three gene typing scheme. However, it was observed that one MNR tract was an insertion site for one of two insertion sequences and that typing using the presence/absence of these insertion sequences further enhanced discriminatory

Murein and its turnover products are recognised by components of the innate immune system leading to an inflammatory response and resulting in the killing of bacteria. To counter the clearance by the innate immune system pathogens like Streptococcus pneumoniae have enzymes to modify their cell wall so that they are no longer recognised. The most common modifications are the N-deacetylation and O-acetylation of the glycan strands in the murein. Deacetylated murein was shown to be a poor substrate for lysozyme which is an important factor of the innate immune system capable of lysing sensitive bacteria that have unmodified murein. The single pneumococcal protein responsible for the deacetylation of murein is the murein N- acetylglucosamine deacetylase A (PgdA). Thus PgdA is a possible target for antibacterial therapy. An economical and ease-to-use assay is required to screen for inhibitors of PgdA. Within this work, such an assay has been established using recombinant PgdA with chromogenic substrates. For the first time the activity of purified PgdA with a natural substrate, murein from S. pneumoniae, has been demonstrated. Another interesting candidate as a possible target for inhibitors is the putative murein hydrolase PcsB. However, there are no biochemical data available yet, and in silico comparison and deletion mutants gave only vague hints for a hypothetical function as murein hydrolase. In this work a recombinant, soluble version of PcsB was purified. It showed no significant enzymatic activity against pneumococcal cell wall or murein.

The Gram-positive bacterium Streptococcus pneumoniae is a major human pathogen causing pneumoniae, blood-stream infections and meningitis. Many of the interactions of this bacterium in humans are mediated by components of the pneumococcal cell wall. The cell wall has a complex structure and is made of a thick peptidoglycan with a covalently linked wall teichoic acid (WTA), cell surface proteins and capsular polysaccharides. In addition, there are surface-exposed, lipid-linked components of the cell envelope such as lipoteichoic acid (LTA) and lipoproteins. The repeating units of WTA and LTA have an identical chemical structure, and both polymers are substituted with phosphorylcholine (PCho) residues.

The genus Peptostreptococcus comprises a heterogeneous collection of species that are gram-positive cocci, non-spore forming and obligately anaerobic. They represent a significant component of the normal human flora but remain poorly studied. They are asaccharolytic or weakly saccharolytic and therefore not easily delineated by conventional biochemical tests. No virulence mechanisms have been reported to date, yet peptostreptococci are nearly always present in human anaerobic infections, and growing evidence indicate that they are likely to play a key role in disease development. In order to study the role of these organisms in disease, it is necessary to reliably identify isolates and gain insight into their metabolic activities. Consequently, the aim of this study was two fold: undertake a 'polyphasic' approach to the taxonomic structure of the genus, and to study aspects of their metabolism with particular reference to the genes that regulate haem biosynthesis. It was shown early in this study that glutamate may have a central role in metabolism and the enzymic properties of glutamate dehydrogenase was used as a preliminary method to study the intra-generic structure of the genus. Several species possessed the enzyme and the electrophoretic mobilities correlated with some. The glutamate dehydrogenase gene was sequenced in 21 strains of peptostreptococci and phylogenetically closely related Clostridium species (sp.) and carried out in parallel with 16S rDNA sequencing. Comparison of the phylogenetic trees constructed using gdh sequences and 16S rDNA sequence of Peptostreptococcus showed similarities. P. anaerobius, the type species of the genus clustered with some species of Clostridium in both trees indicating that P. anaerobius is not only distantly related to other peptostreptococci but is more closely related to clostridia. Consequently, during the course of this work, it was proposed that the genus Peptostreptococcus be restricted to only P. anaerobius and other species were designated incertae sedis. Further extensive analysis on 44 strains of peptostreptococci and several related Clostridium sp. were undertaken using gas chromatography of their long chain fatty acid methyl esters (FAME), while MALDI-TOF (cell surface-associated molecules) and SELDI-TOF (intracellular proteins) mass spectrometry were also explored in an attempt to find new characters to delineate species. MALDI-TOF-MS was successfully used to discriminate between rough and smooth morphotypes of Peptostreptococcus micros strains, but these analyses remain inconclusive at the species level. However, there was sufficient data generated to begin the reclassification of the remaining taxa. Consequently, the following new genera were created to accommodate these; Finegoldia magna comb. nov. (Prevot 1933; Holdeman and Moore 1972) for Peptostreptococcus magnus\ Micromonas micros comb. nov. (Prevot 1933; Smith 1957) for Peptostreptococcus micros', Schleiferella asaccharolytica comb. nov. (Distaso 1912; Ezaki, Yamamoto, Ninomiya, Suzuki and Yabuuchi 1983) for Peptostreptococcus asaccharolyticus; Schleiferella indolica comb. nov. (Christiansen 1934; Ezaki, Yamamoto, Ninomiya, Suzuki and Yabuuchi 1983) for Peptostreptococcus indolicus; Schleiferella lacrimalis comb. nov. (Li, Hashimoto, Adnan, Miura, Yamamoto and Ezaki 1992) for Peptostreptococcus lacrimalis and Schleiferella harei comb. nov. (Murdoch, Collins, Willems, Hardie, Young and Magee 1997) for Peptostreptococcus harei. The second phase of this study focussed on investigating the diversity of the tetrapyrrole biosynthetic pathway among Peptostreptococcus. This pathway leads to the synthesis of haem, cobalamin (vitamin 612) and other metal ion chelated tetrapyrrole molecules. Haem is an iron chelated tetrapyrrole derivative that is essential for several physiological functions and is one of the prerequisites for the pathogenicity of many bacteria. Intracellular levels of haem are known to regulate bacterial biological properties and their ability to initiate infection. Molecular characterisation of important haem regulatory genes including hemA, hemB, hemH, cysG, cbiP and btuR were investigated. Gene fragments of hemB, cbiF and cbiP were amplified, cloned and sequenced. The presence of the hemB, cbiF and cbiP which encodes for aminolevulinic acid dehydratase (ALAD), precorrin-4 C-ll methyltransferase and cobyric-acid synthase respectively indicated the presence of a branched pathway leading to cobalamin biosynthesis. In-vitro transcriptional expression of hemB and cbiF were investigated by reverse-transcriptase (RT)-PCR in parallel with enzymatic assays for ALAD activity. The results of this study provided unambiguous evidence for the presence of a functional tetrapyrrole biosynthetic pathway. This is in contrast to many anaerobic taxa such as Porphyrinmonas gingivalis where only vestiges of a tetrapyrrole biosynthetic pathway occur so that the organism has an absolute dependence on in vitro haem. Peptostreptococci are therefore able to biosynthesise haem from basic precursors and this may confer significant advantages for this species in deep-seated infections where it thrives, and where the basic tetrapyrrole precursors are available.

No description available.

Streptococcus pneumoniae, also known as the pneumococcus, is an important human pathogen, with high burdens of disease and mortality worldwide. There are over 90 serotypes of this pathogen, demonstrating the vast amounts of diversity present. Currently, there are two pneumococcal vaccines, both targeting the polysaccharide capsule. However, one vaccine is ineffective in the paediatric population, whilst the other only targets a minority of disease-causing serotypes, and has increased disease caused by serotypes not present in the vaccine. One solution is a new pneumococcal vaccine targeting a protein virulence factor possessed by all pneumococci, which would afford cross-serotype protection. As a result, it is important to assess the diversity of pneumococcal virulence factors in order to determine their potential as vaccine candidates, as excess diversity present may prevent full serotype-independent protection of a vaccine. Furthermore, diversity studies offer important insight on pneumococcal biology, epidemiology and pathogenesis. The diversity in the toxin pneumolysin (Ply) was greater than previously thought, with 14 protein alleles discovered. However, diversity remained significantly lower than surface-exposed virulence factors, indicating this toxin may be more suitable as a vaccine candidate. Furthermore, all 14 alleles were recognised by polyclonal antibodies, showing the potential cross-serotype protection of a vaccine targeting this toxin. A novel non-haemolytic Ply allele was associated with clones recently expanding in the pneumococcal population, as well as serotypes associated with outbreaks of pneumococcal disease. The non-haemolytic toxin may therefore play a role in driving clonal expansion in certain genetic backgrounds, or be involved in establishing outbreaks of pneumococcal disease. The diversity in the neuraminidase A (NanA) enzyme was significantly higher than in Ply, with many point mutations, mosaic blocks and insertions regions present in 18 divergent alleles. This level of diversity should not be prohibitive to the use of this protein as a vaccine candidate, as polyclonal antibodies recognised 4 NanA alleles of significant diversity, indicating the possibility of cross-serotype protection. The role of NanA in pathogenesis of pneumococcal haemolytic uraemic syndrome (p-HUS) was investigated, although there was no correlation between p-HUS and NanA allele or activity. The novel discovery that pneumococcal NanA was inhibited by viral neuraminidase inhibitors of influenza allowed insight into the synergistic relationship between these two deadly pathogens, and showed for the first time that treatment with these drugs acts on both the primary and secondary pathogen. One of these inhibitors, Oseltamivir, was found to have potential in treating secondary pneumococcal pneumonia, which may help decrease the significant burden of this disease, as well as reducing the over-reliance on antibiotics for treating pneumococcal diseases. Homologues of Ply and NanA were identified and characterised in the related species Streptococcus mitis and Streptococcus pseudopneumoniae, giving insight into the evolutionary relationships between these species. Furthermore, the presence of these homologues in related species gives rise to the possibility of pneumococci acquiring altered genes through horizontal gene transfer. The selective pressure of a vaccine targeting these proteins may give evolutionary advantage to these pneumococci, resulting in evasion of a vaccine. Microarray studies have been used to assess pneumococcal diversity at a genome-wide level. Gene expression studies identified candidate genes which may play a role in p-HUS pathogenesis. Further studies into this area will improve the diagnosis and treatment of this disease. Furthermore, a large number of established pneumococcal virulence factors, many of which are vaccine candidates, were found to have homologues in closely related commensal species. These results must be taken into consideration for future protein-based pneumococcal vaccine design.

Streptococcus pyogenes (group A streptococcus, GAS) is a major human Gram-positive pathogen that causes infections that normally occur in the respiratory tract, the skin, the wound, the lung, the bloodstream and/or muscle tissues and result in millions of deaths every year. To cause such infections, S. pyogenes produces a wide range of virulence factors. The destruction of connective tissue and the hyaluronic acid therein plays an important role in pathogenesis. S. pyogenes was propagated in hyaluronic acid rich growth media in an attempt to create a simple biological system that could reflect some elements of the pathogenesis. The growth of bacteria was analyzed in the hyaluronic acid rich media and control media and a proteomic approach was applied to identify those proteins that were differentially expressed by the streptococcal pathogens growing in the different media. The techniques of two dimensional gel electrophoresis and static nanospray mass spectrometry were optimized and proteome maps for S. pyogenes grown in both media were constructed. The differentially expressed proteins by S. pyogenes were identified and analyzed using bioinformatics. Our results showed that several recognized virulence factors of S. pyogenes were upregulated in hyaluronic acid rich media, including the Ml protein, a collagen-like surface protein and the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase, which has been shown to play important roles in streptococcal pathogenesis. Interestingly, two hypothetical proteins of unknown function were also up-regulated and detailed bioinformatics analysis showed that at least one of these hypothetical proteins is likely to be involved in GAS pathogenesis. It was therefore concluded that this simple biological system provided a valuable tool for the identification of potential streptococcal pathogens virulence factors.