The
antibiotic-tolerant biofilms present in tuberculous
granulomas add an additional layer of complexity when treating mycobacterial
infections, including
tuberculosis (TB). For a more efficient treatment of TB, the biofilm forms of mycobacteria warrant specific attention. Here, we used Mycobacterium marinum (Mmr) as a biofilm-forming model to identify the abundant
proteins covering the biofilm surface. We used biotinylation/
streptavidin-based proteomics on the
proteins exposed at the Mmr biofilm matrices in vitro to identify 448
proteins and ex vivo proteomics to detect 91 Mmr
proteins from the mycobacterial
granulomas isolated from adult zebrafish. In vitro and ex vivo proteomics data are available via ProteomeXchange with identifiers PXD033425 and PXD039416, respectively. Data comparisons pinpointed the
molecular chaperone GroEL2 as the most abundant Mmr
protein within the in vitro and ex vivo
proteomes, while its paralog, GroEL1, with a known role in biofilm formation, was detected with slightly lower intensity values. To validate the surface exposure of these targets, we created in-house synthetic
nanobodies (sybodies) against the two chaperones and identified sybodies that bind the mycobacterial biofilms in vitro and those present in ex vivo
granulomas. Taken together, the present study reports a proof-of-concept showing that surface proteomics in vitro and ex vivo proteomics combined is a valuable strategy to identify surface-exposed
proteins on the mycobacterial biofilm. Biofilm surface-binding
nanobodies could be eventually used as homing agents to deliver biofilm-targeting treatments to the sites of persistent biofilm
infection. IMPORTANCE With the currently available
antibiotics, the treatment of TB takes months. The slow response to treatment is caused by
antibiotic tolerance, which is especially common among bacteria that form biofilms. Such biofilms are composed of bacterial cells surrounded by the extracellular matrix. Both the matrix and the dormant lifestyle of the bacterial cells are thought to hinder the efficacy of
antibiotics. To be able to develop faster-acting treatments against TB, the biofilm forms of mycobacteria deserve specific attention. In this work, we characterize the
protein composition of Mmr biofilms in bacterial cultures and in mycobacteria extracted from infected adult zebrafish. We identify abundant surface-exposed targets and develop the first sybodies that bind to mycobacterial biofilms. As
nanobodies can be linked to other therapeutic compounds, in the future, they can provide means to target
therapies to biofilms.