Characterisation of the spatio-temporal localisation of a pan-Mucorales specific antigen during germination and immunohistochemistry.

ABSTRACT

BACKGROUND: Mucormycosis is an aggressive, invasive fungal infection caused by moulds in the order Mucorales. Early diagnosis is key to improving patient prognosis, yet relies on insensitive culture or non-specific histopathology. A pan-Mucorales specific monoclonal antibody (mAb), TG11, was recently developed. Here, we investigate the spatio-temporal localisation of the antigen and specificity of the mAb for immunohistochemistry. METHODS: We use immunofluorescence (IF) microscopy to assess antigen localisation in eleven Mucorales species of clinical importance and live imaging of Rhizopus arrhizus germination. Immunogold transmission electron microscopy (immunoTEM) reveals the sub-cellular location of mAb TG11 binding. Finally, we perform immunohistochemistry of R. arrhizus in an ex vivo murine lung infection model alongside lung infection by Aspergillus fumigatus. RESULTS: IF revealed TG11 antigen production at the emerging hyphal tip and along the length of growing hyphae in all Mucorales except Sakasenea. Timelapse imaging revealed early antigen exposure during spore germination and along the growing hypha. ImmunoTEM confirmed mAb TG11 binding to the hyphal cell wall only. The TG11 mAb specifically stained Mucorales but not Aspergillus hyphae in infected murine lung tissue. CONCLUSIONS: TG11 detects early hyphal growth and has valuable potential for diagnosing mucormycosis by enhancing discriminatory detection of Mucorales in tissue.