Uncovering the molecular mechanisms behind mycetoma

Abstract

Mycetoma is a chronic, painless, inflammatory condition, caused by either invading fungi or bacteria. It is one of twenty neglected tropical diseases formally recognised by the World Health Organisation. Following inoculation of the causative organisms into the subcutaneous tissue of a host, they organise into structures called grains. These in turn initiate the formation of granulomas and the development of a large, tumour-like mass. This lesion growth is reported to be painless by the majority of patients. Additionally, through unknown mechanisms, the pathogens appear to be able to persist in the host and evade their immune response. This thesis focuses on actinomycetoma, which is exclusively caused by bacteria of the phylum Actinobacteria. Such bacteria are a major source of specialised metabolites, such as antibiotics, antitumour compounds and immunosuppressants. The central hypothesis of the thesis is that bacterial pathogens produce one or more specialised metabolites that mediate painless lesion development and pathogen persistence. A key aim was therefore to isolate and characterise any such compounds, which may also have therapeutic potential. A new host-pathogen interaction assay was developed and applied to study virulence mechanisms of the actinomycetoma pathogen Streptomyces sudanensis. RNAseq, cytokine ELISAs, an NF-κB activity assay and microscopy were deployed to observe how murine macrophages and S. sudanensis interact. A unique immune profile was observed to be induced within the macrophages, characterised by a mixture of pro- and anti-inflammatory features. Multiple potential virulence factors were also identified within S. sudanensis. Additionally, human tissue culture cells were shown to undergo pyroptosis when interacting with the pathogen. Two related compounds that appear to be responsible for this activity were isolated from the bacteria and identified as 2,5-diketopiperazines. This work provides novel insights into how mycetoma pathogens interact with the immune system and of the molecular mechanisms underlying this disease.