Identification and characterisation of novel surface proteins of the urinary tract pathogen Staphylococcus saprophyticus

Nathan King (2011). Identification and characterisation of novel surface proteins of the urinary tract pathogen Staphylococcus saprophyticus PhD Thesis, School of Chemistry & Molecular Biosciences, The University of Queensland.

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Author Nathan King
Thesis Title Identification and characterisation of novel surface proteins of the urinary tract pathogen Staphylococcus saprophyticus
School, Centre or Institute School of Chemistry & Molecular Biosciences
Institution The University of Queensland
Publication date 2011-07
Thesis type PhD Thesis
Supervisor Prof. Mark Schembri
Dr Scott Beatson
Total pages 203
Total colour pages 13
Total black and white pages 190
Subjects 03 Chemical Sciences
Abstract/Summary Urinary tract infections (UTIs) are one of the most common human bacterial infections, placing major burdens on affected individuals and health care systems. Staphylococcus saprophyticus is the leading Gram-positive aetiological agent of UTI, contributing to up to 20 % of all cases. Young sexually active women are particularly at risk of infection with this organism and 10 % of infected women suffer recurrent UTI episodes. Despite the established reputation of S. saprophyticus as an important human pathogen, relatively little is known about the virulence factors and mechanisms involved in S. saprophyticus-mediated UTI. The genome sequence of S. saprophyticus type strain ATCC 15305, released in 2005, provided critical insights into the pathogenicity of S. saprophyticus UTI. A notable outcome from this work was the identification and partial characterisation of a novel putative virulence factor: the cell wallanchored adhesive protein UafA. We are in the final stages of genome sequence annotation of a second clinical isolate of S. saprophyticus. The original genome assembly of this strain, MS1146, contained a 2.5 Mb circular chromosome and two endogenous plasmids – pSSAP1 and pSSAP2. In this study, a number of sequence gaps were closed, allowing the finalisation of the genome sequence. These gaps included six highly similar rRNA operons which were PCR amplified and sequenced individually. This revealed differences between the rRNA regions of strains MS1146 and ATCC 15305 which were supported by Southern blotting. Cloning and random transposon mutagenesis was employed to sequence across the extensive repeats of the MS1146 uafA gene. Annotation of the pSSAP1 plasmid identified a novel gene encoding a putative cell wall-anchored protein. This gene, termed uro-adherence factor B (uafB), contains a long highly repetitive region that similarly necessitated the use of cloning and transposon mutagenesis to allow uafB sequence completion. UafB is a high-molecular-mass glycosylated serine-rich repeat protein that is expressed on the surface of S. saprophyticus MS1146. UafB also functions as a major cell surface hydrophobicity factor. To characterise the role of UafB, an isogenic uafB mutant was constructed in S. saprophyticus MS1146 by interruption with a group II intron. The uafB mutant had a significantly reduced ability to bind to fibronectin and fibrinogen. Furthermore, a recombinant protein containing the putative binding domain of UafB bound specifically to fibronectin and fibrinogen. UafB was not involved in adhesion in a mouse UTI model, however a striking UafB2 mediated adhesion phenotype to human uroepithelial cells was observed. Genes homologous to uafB were identified in other staphylococci which, like uafB, appear to be located on transposable elements. Annotation of the pSSAP2 plasmid revealed the presence of a gene for a previously undescribed 73.5 kDa cell wall-anchored protein, that has since been termed fatty acid resistance protein of S. saprophyticus (Frs). The frs gene has a high prevalence of 87 % in S. saprophyticus clinical isolates collected from Australia, Germany and the USA. Frs was demonstrated to be expressed at the cell surface, but extensive experiments have not shown a role in adhesion for this protein. Frs shares moderate sequence identity to SasF, a surface protein of Staphylococcus aureus recently shown to be an important mediator of resistance to linoleic acid - an antibacterial free fatty acid found on skin especially active against skin-colonising staphylococci. Using a heterologous complementation approach in a S. aureus sasF null genetic background, Frs was demonstrated to be associated with resistance to linoleic acid. Every staphylococcal genome sequenced to date encodes frs and sasF homologues, suggesting that Frs and SasF are members of an emerging protein family widely disseminated throughout the staphylococci. During this project a third endogenous plasmid (pSSAP3) of S. saprophyticus MS1146 was discovered, embedded in the misassembled pSSAP1 sequence. Amongst a number of genes encoding hypothetical proteins, this plasmid possesses heavy metal resistance determinants as well as a locus of putative metabolism-associated genes of unknown uropathogenic significance. Despite the lack of any established virulence factors, we found strong evidence that plasmid pSSAP3 is conjugative. Also incorporated into this study is virulence-associated gene prevalence data for a collection of 76 clinical S. saprophyticus isolates, as well as selected phenotypic data for the same strain set. Further characterisation of the UafA adhesin was investigated and preliminary findings are discussed, including the possible glycosylation of UafA and size variability of UafA proteins of different S. saprophyticus strains. Overall, this thesis has contributed to our understanding of the pathogenicity of S. saprophyticus and has identified several new avenues for further research.
Keyword Staphylococcus saprophyticus
genome sequence
surface protein
virulence factor
Additional Notes Colour pages: 24, 54, 58, 60, 78, 81, 108, 109, 112, 133, 188, 189, 202.

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Created: Tue, 18 Oct 2011, 15:16:01 EST by Mr Nathan King on behalf of Library - Information Access Service