The bulk of protein secretion in bacteria occurs through the general secretion (Sec) pathway. Proteins interact via their N-terminal leader peptides with the membrane-associated Sec system where they undergo processing, secretion and folding within the cell wall environment. Some Gram-positive bacteria and mycobacteria have been shown to possess a second Sec pathway known as the accessory Sec system. This exports proteins with specific N-terminalleader peptides and assists in the secretion of other proteins that lack N-terminal leaders. The core component of this system is SecA'2, a functional homologue of SecA in the canonical system. Other core components include SecY2 (homologue of SecY) and accessory Sec proteins Asp l-Asp5. The objective of this study was to characterise the composition and activities of the accessory Sec systems in commensal Streptococcus gordonii and pathogenic Streptococcus pneumoniae.

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Clostridiom kluyveri, growing anaerobically in a medium containing ethanol, acetate and bicarbonate as sole carbon sources, rapidly incorporated radioactivity from 14C-bicarbonate into a wide variety of cell constituents. The first two compounds to become appreciably labelled were alanine and aspartate : after 6 seconds' incubation, alanine contained 84% of the total incorporated radioactivity, but this percentage subsequently decreased, while that in aspartate and in most other compounds increased. This suggested that alanine was derived through the carboxylation of a non-radioactive 2-carbon precursor, any intermediates in its formation being of only transitory existence, while other evidence suggested that a second carboxylation was involved in the formation of aspartate. The formation of alaninein cell-free extracts of C.kluyveri required the presence of acetyl-CoA, bicarbonate, hydrogen, and certain cofactors, and evidence was obtained for the participation of three enzymes; hydrogenase, catalysing the reduction of ferredoxin with hydrogen; pyruvate synthase, catalysing the reductive carboxylation of acetyl-CoA to pyruvate, and requiring reduced ferredoxin, thiamine pyrophosphate, and a divalent cation for its activity; and alanine transaminase. Evidence was also obtained that the formation of aspartate required two enzymes: a typical biotin- and ATP-dependent pyruvate carboxylase, catalysing the formation of oxaloacetate from pyruvate; and aspartate trans- aminase. These various enzymic activities were separated by standard fractionation procedures, but were mostly unstable, and little overall purification was achieved. The decarboxylation of oxaloacetate by C.kluyveri extracts was stimulated by ATP, suggesting the participation of PEP carb-oxykinase. The formation of phosphoenolpyruvate in crude extracts was obscured by the presence of pyruvate kinase, which catalysed its hydrolysis. The sources of energy and reducing power for biosynthetic reactions in C.kluyveri were discussed. A complete tricarboxylic acid cycle could not be demonstrated in cell-free extracts, although certain of the enzymes were present, and evidence was obtained for their participation in the synthesis of glutamate.

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Cronobacter sakazakii is in the Cronobacter genus (previously known as Enterobacter sakazakii), which consists in total of seven species. C. sakazakii strains in the clonal complex 4 (CC4), including sequence type 4 (ST4), have been strongly associated with neonatal meningitis. In recent years, research on this organism has made substantial progress using improved identification and molecular methods including multilocus sequence typing. A number of virulence traits have been proposed but have not been studied to date with respect to detailed aspects of the virulence potential and host response. Therefore this project compared 34 isolates of C. sakazakii made up of clonal complex 4 (CC4; 21 isolates) and non-clonal complex 4 (13 isolates) strains for their virulence potential, and investigated whether CC4 strains have the ability to overcome the host barriers more than the other sequence types. The attachment and invasion of mammalian intestinal and brain cells by these strains were evaluated using colorectal adenocarcinoma epithelial cells (Caco-2), human brain microvascular endothelial cells (HBMEC), and rat brain capillary endothelial (rBCEC4) cell lines. Furthermore, the ability of the organism to translocate through different cell lines, including Caco-2 and HBMEC, was assessed. The project also studied the survival of C. sakazakii strains in human macrophages (U937) and human microglial cell lines, and the response of these cells in eliminating the infection as a part of the immune response. In addition, it examined the host response to C. sakazakii infection. C. sakazakii strains were motile except for three strains 1223, 1224, and 680. Moreover, the majority of the strains were able to produce iron siderophores except for strains 6 and 520. Additionally, a group of C. sakazakii strains were able to withstand serum-mediated killing, whereas strains 6 and 680 were sensitive. The previous traits are important for bacterial growth and survival inside the host. C. sakazakii strains showed the ability to adhere and invade the Caco-2, HBMEC, and rBCEC4 cell lines, especially CC4 strains (Caco-2 0.29%, HBMEC 0.13, rBCEC4 0.02%) that displayed the highest invasion levels compared to non-CC4 strains (Caco-2 0.16%, HBMEC 0.1, rBCEC4 0.016%), supporting the clinical evidence that it can overcome the intestinal and brain barriers. Furthermore, C. sakazakii strains, including CC4 strains, were able to translocate through the intact monolayers of the Caco-2 and HBMEC cell lines, and CC4 strains (HBMEC translocation 4.92%) were higher in translocation compared to non-CC4 strains (HBMEC translocation 1.67%). The translocation through Caco-2 and HBMEC is a crucial sign of their invasiveness. The test isolates were able to survive and multiply inside macrophages and microglia. This process is advantageous for the bacterium to survive within the host and evade the immune system. The test strains, including CC4 strains, triggered the HBMEC cell line to produce iNOS that could lead to elevated levels of NO production leading to cell line permeability. Additionally, the organism was able to induce apoptosis in HBMEC and microglial cells and two markers were detected, caspase-3 and annexin V. Inducing apoptosis in the blood brain barrier cells and microglia is a major threat to the central nervous system (CNS). A number of cytokines were produced by HBMEC and microglial cell lines as a result of C. sakazakii exposure. These cytokines included the pro-inflammatory IL-1β, TNF-α, IL-6, and IL-8 in addition to GM-CSF and the anti-inflammatory IL-10 and IL-4. These might contribute to increased blood brain barrier permeability and host damage.

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