Proteomic analysis of Mycobacterial Biofilm Matrix and Development of Biofilm-binding Synthetic Nanobodies

Abstract Tissue-penetrating nanobodies as drug-delivery vehicles have shown efficacy in treating hard-to-reach and -treat diseases. The present study reports nanobodies with application potential in the treatment of tuberculosis (TB), especially the antibiotic tolerant biofilms that form in TB granulomas. Using Mycobacterium marinum (Mmr) as a model pathogen, we identified the most abundant matrix proteins in cultured biofilms and granuloma biofilms from infected zebrafish, and used them as targets to produce synthetic nanobodies (sybodies). Surface-exposed proteins on Mmr biofilms were identified using a combination of in vitro cell surface biotinylation proteomics and ex vivo proteomics on granulomas extracted from Mmr-infected adult zebrafish. We identified a total of 3080 and 41 mycobacterial biofilm matrix-associated proteins by in vitro and ex vivo proteomics, respectively. The molecular chaperones GroEL1 and GroEL2 were identified in both datasets, and fulfilled the criteria of ideal nanobody targets. We created sybodies against GroEL1 and GroEL2, and showed that they bind the intact in vitro and ex vivo Mmr-biofilms. Taken together, the present study reports a proof-of-concept showing that biotinylation proteomics of biofilms complemented with ex vivo proteomics of granuloma biofilms is a valuable strategy to uncover optimal nanobody targets within the biofilm matrix. Nanobody-based strategies have great potential to provide new and much-needed solutions for more effective treatment of tuberculosis, as well as other chronic biofilm infections with increased antibiotic tolerance and high risk for emerging antibiotic resistance.