Thesis
The interactions of pathogenic methicillin resistant and methicillin sensitive Staphylococcus aureus strains with host cell autophagic machinery
- Creator
- Rights statement
- Awarding institution
- University of Strathclyde
- Date of award
- 2012
- Thesis identifier
- T13493
- Qualification Level
- Qualification Name
- Department, School or Faculty
- Abstract
- A specific form of autophagy (known as xenophagy) is involved in the protection of cells from intracellular pathogens. Recent research has shown that Staphylococcus aureus is able to subvert autophagy and create a protective niche within autophagosomes, where it is able to survive and divide. We studied 3 strains of S. aureus: an epidemic methicillin-resistant strain (EMRSA-15), a pathogenic methicillin-sensitive strain (ATCC 29213) and an agr-mutant MRSA strain (NRS 144). We first wished to observe autophagy activation induced by these three S. aureus strains within a well characterised cell type (Human embryonic kidney 293 cells expressing green florescent protein labelled microtubule associated protein light chain 3 (GFP-LC3)). As LC3 is only attached to autophagic membranes, this gave a good indication of autophagy activation. In these cells, EMRSA-15 activated autophagy at a much higher rate than the other two strains. A small proportion of all three strains were observed to co-localise within GFP-LC3 positive membranes. This indicated that S. aureus both activated and interacted with autophagy and so we wished to investigate the importance of autophagy proteins. EMRSA-15 effectively activated and interacted with autophagy in parental MEFs but when autophagy related gene 5 (Atg5) (an essential for the activation of autophagy) was knocked-out in MEFs, no activation was every observed (as expected). We also observed that ULK (unc-51-like kinase) 1 and 2 are important for S. aureus induced activation of autophagy and that both ULK-1 and Atg9 interact with membranes surrounding invading S. aureus, but this interaction was transient. Next, we wished to investigate the mechanism for bacterial targeting (i.e. whether adaptor molecules were important). We observed that S. aureus are likely ubiquitinated once internalised and specifically targeted toward autophagosomes via the attachment of ubiquitin-binding adaptor molecules (as both p62 and NBR-1 (neighbour of BRCA1 gene 1) co-localised with EMRSA-15). Due to the low frequency of S.aureus-autophagosome interaction, we wished to investigate the effect further activation of autophagy had upon the formation of autophagosomes. The formation of large autophagosomes was not induced by the further activation of autophagy in infected cells, but small autophagosomes were, suggesting the presence of a specific bacterial factor which specifically activates enlarged xenophagy-associated structures, but not general autophagy. Finally, we wished to observe the intracellular growth of EMRSA-15 and ATCC 29213 and the importance of autophagy for this growth. We observed that ATCC 29213 was able to grow effectively within HEK 293 cells. However, within MEFs, bacteria required activation of autophagy (either through amino acid starvation or rapamycin treatment) in order to efficiently divide intracellularly. This intracellular division is dependent on autophagy and it is likely that the presence of a functioning agr-pathogenicity island within S. aureus is important for intracellular survival and growth. This work demonstrates that host cell autophagy is activated following S. aureus infection and degree of activation is dependent on bacterial pathogenicity. Internalised S. aureus are enclosed by enlarged autophagic membranes and part of this targeting may be mediated by ubiquitin-binding autophagy adaptor proteins. Host cell autophagy can be combating bacterial infection via xenophagy but in some contexts autophagy can also support S. aureus intracellular replication highlighting multiple types of possible bacteria-host cell interactions. This work outlines a number of areas for further extensive study.
- Resource Type
- DOI
- Date Created
- 2012
- Former identifier
- 991657
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File | 2021-07-02 | University of Strathclyde |