Abstract
OBJECTIVES: To optimize an enzyme-linked immunosorbent assay (ELISA) for measuring the HspX protein (α-crystallin) levels and then evaluate its correlation with the accumulation of lipid bodies in Mycobacterium bovis (M. bovis) during hypoxia and exposure to nitric oxide. METHODS: This study was conducted at Prince Sultan Military Medical City, Riyadh, Saudi Arabia between 2016 and 2017. We first optimized ELISA conditions for the detection of HspX. The optimization protocol focused on minimizing concentrations of the capture antibody, detection antibody, and conjugated secondary antibody, and determining the minimum detection limit of the antigen, HspX. Bacteria were grown either in shaking culture or in stationary flasks mimicking hypoxic environments. A standard Bradford assay was used to determine the total protein and HspX was detected using the optimized ELISA protocol. The effect of hypoxic environment and nitric oxide on the levels of HspX and lipid bodies, detected by staining with Nile red, was also evaluated. RESULTS: An optimized ELISA protocol was established for the detection of HspX from M. bovis. Exposure to nitric oxide and hypoxic conditions led to an increase in the levels of HspX protein. The increase in HspX associated with nitric oxide treatment and hypoxic conditions correlated with higher levels of lipid bodies mainly found in pathogenic mycobacteria. CONCLUSIONS: The optimized ELISA protocol in this study can detect HspX protein levels in M. bovis growing in normal and hypoxic environments. Importantly, hypoxia led to enhanced expression of HspX protein, which correlated with the enhanced production of lipid bodies. Lipid body production is a survival strategy of pathogenic mycobacteria.