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Characterisation of lipoproteins in staphylococcus aureus

Elgallali, A Characterisation of lipoproteins in staphylococcus aureus , PhD thesis, University of Salford.

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The Gram-positive bacterium Staphylococcus aureus is an extremely successful opportunistic bacterium capable of causing a wide range of hospital-acquired and community-acquired infections, and is becoming increasingly virulent and resistant to antibiotics. In order to investigate this pathogen, various methods have been used to analyse the pathogenic behaviour including genomics, transcriptomics and proteomics. S. aureus expresses approximately 55-70 lipoproteins with only about half with known functions. Little is known about the biochemical functions of many individual lipoproteins and their proteomics has not been investigated in detail. Lipoproteins have a broad ranging functionality and perform various roles in bacterial activity and attract a particular interest to investigate their virulence and survival influences in the course of host infection. The initial part of this study was to find out whether the lipoproteins of S. aureus have similar genetic characteristics among all strains. PCR and Quantitative Real-Time PCR experiments were performed to analyse the genetic and the expression levels for some lipoprotein genes. The majority of PCR results showed high similarity in lipoprotein genetic structure among the examined strains. Phylogenetic trees from concatenated lipoprotein genes alignment were generated to represent the lipoprotein genes distribution of S. aureus strains. To identify and characterise proteomic of S. aureus lipoproteins a comprehensive quantitative proteome profiling of S. aureus lipoproteins using gel-free /in-solution trypsin digestion system followed by LC-MS/MS quantification identified 38 lipoproteins that represent two-thirds of the S. aureus MRSA252 lipoprotein. In addition, S. aureus-mediated infections with live C. elegans were performed on solid assays to investigate the host-pathogen relationships. S. aureus MRSA252 exhibited a high level of nematocidal activity with average time for half of the worms to die of ~ 2 d and infected C. elegans showed visible signs of illness. To evaluate lipoprotein transcripts expression level and microbe/host-specific pathogenic factors RNA of both S. aureus and C. elegans were characterised after isolation from the infected C. elegans and subjected to RNA Sequencing, the large-scale data has provided useful information on pathogen and host activities during infection. RNA sequencing analysis showed different types of regulations and interactions of lipoprotein transcripts during host exposures to indicate 3 transcripts significantly were up-regulated and 11 down-regulated. RNA sequencing analysis showed that 62 lipoprotein transcripts were expressed during C. elegans infection model. Proteomic analysis using the application of gel-free proteomic technique identified 38 lipoproteins that were expressed in the non-infection condition representing approx. two-thirds of the S. aureus MRSA252 lipoproteins. The results suggest that some lipoproteins were involved in pathogenesis of C. elegans but their function were not clear. More research is needed for explore the roles of lipoproteins in pathogenesis and the interactions of S. aureus with the host immune responses.

Item Type: Thesis (PhD)
Schools: Schools > School of Environment and Life Sciences
Funders: University of Salford
Depositing User: A Elgallali
Date Deposited: 02 Sep 2016 08:13
Last Modified: 02 Sep 2016 08:13

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