Mycobacterium tuberculosis (MTB) causes both acute and
chronic infections in humans characterized by tolerance to antibiotics and
reactivation to cause secondary tuberculosis. These characteristics have led to
renewed interested in the in vitro pellicle, or biofilm mode of
growth, where bacteria grow to produce a thick aggregate at the air-liquid
interface and exhibit increased phenotypic resistance to antibiotics. We
infected guinea pigs with the virulent H37Rv strain of MTB for 60 days at which
point we collected blood. To identify antigenic proteins, membrane protein
extracts of MTB H37Ra pellicles and shaken cultures grown for 3, 5, or 7 weeks
were probed with the infected animals’ sera after the proteins were
separated by two-dimensional gel electrophoresis (2DGE). Antigenic proteins were
then identified using MALDI-TOF/TOF mass spectrometry peptide mass
fingerprinting. Antigenic pellicle proteins were compared across the three
timepoints to identify those that were produced consistently during pellicle
growth. They were also compared to those membrane proteins identified from
harvested shaken cultures to determine pellicle-specific versus
universally-expressed proteins. Using this technique we identified 44 distinct
antigenic proteins, nine of which were pellicle-specific. The sequence of
antigenic pellicle-specific proteins was checked for sequence conservation
across 15 sequenced MTB clinical isolates, three other members of the MTB
complex, as well as Mycobacterium avium and
Mycobacterium smegmatis. The antigenic pellicle-specific
protein Rv0097 was found to have very high sequence conservation within the MTB
complex but not with related mycobacteria while FabG4 was highly conserved in
all mycobacteria analyzed. These conserved pellicle-specific proteins represent
targets for the development of future diagnostic tests and vaccines.