The role of S.aureus cell wall structure during host:pathogen interaction

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S. aureus is a major, antibiotic resistant, human pathogen. Targeting bacterial cell wall, peptidoglycan (PG) biosynthesis via antibiotics such as the beta-lactams has been a crucial part in our fight against S. aureus. PG is also recognized by the host, and is involved in shock, arthritis etc. Despite this importance we do not know the structure of the PG during infection, hampered by technological difficulties in obtaining enough material. The interdisciplinary project, in infectious disease will span the length scales from single molecules to whole animals, combining host:pathogen interaction, molecular imaging and biochemistry to define the first PG structure during infection and the role of specific in vivo produced structures in disease. Scientific Objectives:1. Production and analysis of peptidoglycan structure in vitroThe structure of S. aureus PG will be determined using bacteria grown in vitro in rich media and under conditions that mimic in vivo conditions (whole human blood). RP-HPLC separation of muropeptides, coupled with MS and NMR will define the structure and modification of S. aureus PG (Newcastle and Sheffield). Glycan strand length will be measured using a protocol developed in Sheffield. Atomic force microscopy will determine PG architecture. Analysis of the sensitivity of the assays will determine the number of bacteria required (important for the in vivo analysis). 2. Analysis of the role of modification of peptidoglycan during host:pathogen interactionThe student will be trained in in vivo analysis of S. aureus infection, using our well-established mouse models. This requires a Home Office personal licence. The student will then use the mouse sepsis model, a primary bacterial output of the model being abscesses in organs. All the abscess-associated bacteria have grown in vivo as each is founded by an individual bacterium. Abscesses can contain up to 109 cfu, which preliminary data suggests is enough PG for analysis. A protocol has already been established for harvesting bacteria from abscesses. PG will be purified and its structure and architecture compared to in vitro derived material. 3. Analysis of the role of PG modifications in host:pathogen interaction.The importance of in vivo associated cell wall features will be tested by the use of specific mutants (an ordered library of S. aureus mutants in genes such as PG hydrolases, O-acetyltransferase etc.is available). It is known that modifications such as O-acetylation have an important role in host:pathogen interaction. PG structure has multiple functions that will be tested including resistance to host attack via enzymes and the effect of shed material on the immune system (cytokine analysis etc). 4. Role of antibiotic resistance and treatment on PG in vivo structureMethicillin Resistant S. aureus (MRSA) is a major healthcare problem. The target of methicillin and other beta-lactams is PG biosynthesis. The in vivo structure of MRSA PG and how antibiotics effect this is unknown. If we are to identify new treatment regimes it is important to determine how existing resistance modalities prevent effective control.