Staphylococcus aureus subversion of phagosomal maturation in macrophages

Staphylococcus aureus is a common bacteria that can cause infections ranging from skin infections to blood poisoning. These can complicate hospital care leading to infections that prolong hospital stay. Many people carry S. aureus on their skin, leaving a large number of patients at risk of infection.

A key immune cell that protects against bacteria is the macrophage, which exerts their effect by eating and subsequently killing bacteria. Macrophages are not good at killing all the S. aureus they have eaten. S. aureus persist inside macrophages and can evade antibiotics and spread to other sites around the body. Normally a macrophage contains bacteria in a cell membrane-bound compartment, called a 'phagosome', which must fuse with other cell compartments that deliver factors that help kill the bacteria. These other compartments are also membrane-bound to stop the harmful factors they contain, such as acid, from leaking into other parts of the cell. Phagosomes containing S. aureus don't fuse with these other compartments effectively and thus don't mature to kill S. aureus as they should.

I will investigate how S. aureus stop phagosomes from maturing appropriately. I will use a large panel of S. aureus strains, each of which lack one particular bacterial factor (gene) and a microscopy-based approach that uses labelled bacteria as well as a chemical detection system that uses a marker of phagosomal maturation. These will allow me to tell when the phagosomes that contain bacteria have matured. I will identify which bacterial factors stop the phagosome maturing and confirm if these bacterial factors prevent S. aureus killing in macrophages. I will also test if strains of S. aureus from patients with certain infections are more adept at modifying phagosomal maturation.

Normally as the phagosome matures and interacts with other cellular compartments it gains markers on its surface from the cell membranes of these other compartments that fuse with it. I will determine which of these markers the S. aureus containing phagosomes lack. I will start by looking at candidate factors involved in controlling the fusion of the phagosome with a particular compartment that my results to date suggest may be defective. I will also manipulate macrophages by modifying a whole range of genes that regulate the interaction of different membrane bound compartments to take a broader overview of factors that might regulate the maturation of the phagosome containing S. aureus. I will use this panel of altered macrophages and challenge them with bacteria that lack the bacterial factor I have found that stops the phagosome maturing. This bacterial strain should allow the phagosome to mature normally but I will determine if removal of any of the regulators of interaction between cell compartments produce the same defect of phagosome maturation that I observe with unaltered S. aureus. I will again perform this experiment with my microscopy-based and chemical detection system analyses, allowing me to check a large number of these regulators of interactions between different cellular compartments. I will confirm that removal of any of the regulators of phagosomal maturation that copy the phagosomal defect seen with S. aureus, also prevent bacterial killing.

Finally, I will test if these factors I have identified as regulating phagosomal maturation alter clearance of bacteria by macrophages using fish to model an infection. This will confirm that the pathway I identify is important in regulating bacterial clearance when macrophages are working with other cells in the body to control infection. Overall this will provide valuable information on how a major cause of human disease manipulates the immune system and will suggest new approaches that can be used to correct the defect and prevent bacterial persistence.

Staphylococcus aureus is a major human pathogen with several adaptations to aid immune evasion. Macrophages that phagocytose S. aureus struggle to kill internalised bacteria. I have shown S. aureus inhibits phagosomal maturation. I hypothesise that S. aureus gene expression impairs critical steps in phagosomal maturation and inhibits phagosome fusion with lysosomes, leading to bacterial persistence.

My aims are to:
1) Identify microbial determinants of impaired phagosomal maturation;
2) Characterise the host responses critical for phagosomal maturation and establish the steps inhibited by S. aureus;
3) Confirm that phagosomal maturation plays a critical role in S. aureus killing in vivo.

I will establish high-content automated microscopy screens of phagosomal maturation in differentiated THP-1 cells. These screens will involve challenging macrophages with S. aureus labelled with pHrodo, to measure bacteria in a phagosome of pH 6 or less and an automated fluorometric assay of activation of the lysosomal protease cathepsin D as markers of phagosomal maturation. These screens will be used with the Nebraska transposon mutant library of a USA300 S. aureus strain and with a targeted siRNA library of host factors regulating membrane trafficking to identify factors influencing the maturation of S. aureus containing phagosomes. A candidate approach using a panel of antibodies against phagosomal markers of lysosomal tethering and fusion will also be studied to characterise the features of S. aureus containing phagosomes. Key findings will be confirmed with clinical strains of S. aureus, in primary human macrophages using CRISPR gene-editing and in a zebrafish model of S. aureus infection, to establish the role of regulators of phagosomal maturation in bacterial killing.

This will define the S. aureus induced phagosomal maturation block and it's role in intracellular bacterial persistence in vivo.

Staphylococcal disease is a major health problem and findings relating to its persistence and host subversion will have wide-reaching consequences. Improved understanding of these processes will have implications for antimicrobial use and the development of antimicrobial resistance with profound cross-sector implications.

Academic sector.
Identification of immune evasion mechanisms involving subversion of phagosomal maturation will be of interest, not just to those studying Staphylococcus aureus but also more broadly to those studying other infections that persist in the intracellular environment. These include pathogens of global impact such as Mycobacterium tuberculosis. Understanding processes that ultimately contribute to effective antimicrobial killing, release of microbial factors to stimulate inflammatory responses and antigen processing can be applied by those studying a range of infections and immunological processes. This includes those developing novel approaches to vaccines. Staphylococcal disease and the general implications of my research will also be of importance to those studying animal health. Models and methods developed by my research will be shared with academic colleagues and can inform other projects.

NHS.
Staphylococcal disease is a global problem, with high associated mortality and morbidity. It adds significantly to health-care costs. Understanding the factors that contribute to persistence will lead to new approaches to combat this pathogen therapeutically and can help limit the use of ineffective courses of prolonged antimicrobials that foster antimicrobial resistance. Improving management of this infection would decrease health-care associated costs. The clinical implications will extend beyond the UK and be applicable in health settings around the world. Increasing public consciousness of antimicrobial resistance will also highlight the importance of alternative approaches to the use of antimicrobials and relieve inappropriate pressure on antimicrobial prescribing in the NHS.

Industry.
Factors I identify that regulate phagosomal maturation may be amenable to drug targeting. The high-throughput screen I develop would be of interest to industry colleagues screening compound libraries to target this response. Microbial determinants of subversion may be targeted through biological therapy and my host laboratory has research links with MedImmune, who are targeting S. aureus virulence factors with monoclonal antibodies. Given the limited pipeline of new antimicrobials with which to combat antimicrobial resistance, it is likely that findings generated from my project could have impact in the pharmaceutical industry.

Policy Makers.
The identification of new pathways of microbial immune evasion recognition, and the development of new therapeutic strategies, would need to be reflected in national and international guidelines. If the work outlined in this application, and its subsequent therapeutic development, results in new approaches, I will engage with policy makers where appropriate, using contacts through my professional body the British Infection Association.

Society.
The complications of Staphylococcal disease come with a cost to the individual and to society. Improved management of this common disease has the potential to lessen loss of time from work, disability and the complications of healthcare for a range of medical conditions. The potential development of alternative strategies to combat bacterial infection will ease the burden on antimicrobial agents and could help reduce resistance. Even if my research does not directly identify alternative therapies, further understanding of pathogenesis and new models of infection will high-light the importance of alternative strategies to prevent antimicrobial resistance and focus debate on the threats provided by antimicrobial resistance in general.