DiMeN Doctoral Training Partnership: The intracellular environment of human neutrophils permits the formation of a persister phenotype in S. aureus

Neutrophils are circulating white blood cells and a powerful innate immune defence against bacterial infection. Despite this, the important human pathogen, Staphylococcus aureus, is able to resist the potent anti-microbial functions of neutrophils, which can lead to potentially life-threatening infections including pneumonia and septicaemia. S. aureus infections are notoriously difficult to treat due to multi-drug resistance, in particular methicillin resistant S. aureus (MRSA), and there is a great need to develop novel therapeutics to promote immunity against this pathogen. To achieve this, we must first understand this very complex host-pathogen relationship.

Neutrophils avidly phagocytose (internalise into a phagosomal compartment) S. aureus and yet this pathogen is able to survive within the inhospitable intracellular environment. Because neutrophils are motile cells, carrying viable S. aureus cargo can lead to the rapid dissemination of infection to other sites in the body. We hypothesise that exposure to endogenous microbicidal molecules such as proteases and oxidative molecules within the neutrophil intraphagosomal space, induces a ‘persister’ phenotype of S. aureus. Persister cells are slow growing, metabolically depressed and resistant to antibiotic treatment. This phenotype is well described for S. aureus, for example, following oxidative stress. Although macrophages can induce a persister phenotype in a number of bacteria, it is not known whether S. aureus survival within neutrophils is due to persister formation and if so, how. Understanding this will ultimately allow therapeutic manipulation to prevent persister formation within neutrophils, thereby improving immune responses to this difficult to treat pathogen.

This project aims to understand the mechanisms by which S. aureus remain viable within neutrophils. To do this we will:

Aim 1: Interrogate the presence and phenotype of S. aureus persister cells within human neutrophils.
1. Infect primary circulating neutrophils isolated from the blood of healthy subjects with the clinical MRSA isolate, JE2. Bacterial viability assays will confirm the presence of viable intracellular bacteria as previously shown by our group and others (Gresham et al Journal of Immunology, 2000).
2. Identify active and persister phenotypes and correlate phenotypes with bacterial growth rate and other functional assays.
3. Determine the ability of persister cells to establish infection in vivo.

Aim 2. Study the genetic mechanisms of persister formation using unbiased and candidate approaches.
1. An unbiased strategy will utilise a genome-wide S. aureus mutant library and a more focussed approach will use mutants known to have roles in persister formation. Mutants will be screened in neutrophil infection experiments in order to identify mutants that fail to enter into a persister phenotype.

This multi-disciplinary and innovative project straddles two extremely successful Faculties here at the University of Sheffield, namely the Faculty of Medicine, Dentistry and Health and the Faculty of Science. The supervisors, Dr Lynne Prince and Dr Rebecca Corrigan have proven track records in understanding human neutrophil biology and host-pathogen interactions (Prince) and the bacterial mechanisms that underpin S. aureus persister formation (Corrigan). The student will join a collegiate and productive research environment and will benefit from excellent training both in specific research skills and also wider personal development opportunities.

http://www.sheffield.ac.uk/iicd/profiles/prince

http://www.sheffield.ac.uk/mbb/staff/rebeccacorrigan

http://www.floreyinstitute.com

Key references:
Anwar S et al, The rise and rise of Staphylococcus aureus: laughing in the face of granulocytes. Clin Exp Immunol (2009) 157(2), 216-224.
Gresham et al, Survival of Staphylococcus aureus Inside Neutrophils
Contributes to Infection, The Journal of Immunology (2000) 164: 3713–3722.
Corrigan et al, ppGpp negatively impacts ribosome assembly affecting growth and antimicrobial tolerance in Gram-positive bacteria, PNAS, (2016)  vol. 113 no. 12 E1710-E1719.