Abstract
Secretion of specific proteins contributes to pathogenesis and immune responses in tuberculosis and other bacterial infections, yet the kinetics of protein secretion and fate of secreted proteins in vivo are poorly understood. We generated new monoclonal antibodies that recognize the Mycobacteriumtuberculosis secreted protein Ag85B and used them to establish and characterize a sensitive enzyme-linked immunosorbent assay (ELISA) to quantitate Ag85B in samples generated in vitro and in vivo We found that nutritional or culture conditions had little impact on the secretion of Ag85B and that there is considerable variation in Ag85B secretion by distinct strains in the M. tuberculosis complex: compared with the commonly used H37Rv strain (lineage 4), Mycobacteriumafricanum (lineage 6) secretes less Ag85B, and two strains from lineage 2 secrete more Ag85B. We also used the ELISA to determine that the rate of secretion of Ag85B is 10- to 100-fold lower than that of proteins secreted by Gram-negative and Gram-positive bacteria, respectively. ELISA quantitation of Ag85B in lung homogenates of M. tuberculosis H37Rv-infected mice revealed that although Ag85B accumulates in the lungs as the bacterial population expands, the amount of Ag85B per bacterium decreases nearly 10,000-fold at later stages of infection, coincident with the development of T cell responses and arrest of bacterial population growth. These results indicate that bacterial protein secretion in vivo is dynamic and regulated, and quantitation of secreted bacterial proteins can contribute to the understanding of pathogenesis and immunity in tuberculosis and other infections.IMPORTANCE Bacterial protein secretion contributes to host-pathogen interactions, yet the process and consequences of bacterial protein secretion during infection are poorly understood. We developed a sensitive ELISA to quantitate a protein (termed Ag85B) secreted by M. tuberculosis and used it to find that Ag85B secretion occurs with slower kinetics than for proteins secreted by Gram-positive and Gram-negative bacteria and that accumulation of Ag85B in the lungs is markedly regulated as a function of the bacterial population density. Our results demonstrate that quantitation of bacterial proteins during infection can reveal novel insights into host-pathogen interactions.
Highlights
Secretion of specific proteins contributes to pathogenesis and immune responses in tuberculosis and other bacterial infections, yet the kinetics of protein secretion and fate of secreted proteins in vivo are poorly understood
We developed a sensitive enzymelinked immunosorbent assay (ELISA) to quantitate a protein secreted by M. tuberculosis and used it to find that Ag85B secretion occurs with slower kinetics than for proteins secreted by Gram-positive and Gram-negative bacteria and that accumulation of Ag85B in the lungs is markedly regulated as a function of the bacterial population density
These results indicate that monoclonal antibodies (mAbs) 710, 711, and 712 preferentially recognize Ag85B, each of the mAbs binds Ag85A and Ab85C when these antigens are present at high concentrations
Summary
Secretion of specific proteins contributes to pathogenesis and immune responses in tuberculosis and other bacterial infections, yet the kinetics of protein secretion and fate of secreted proteins in vivo are poorly understood. ELISA quantitation of Ag85B in lung homogenates of M. tuberculosis H37Rv-infected mice revealed that Ag85B accumulates in the lungs as the bacterial population expands, the amount of Ag85B per bacterium decreases nearly 10,000-fold at later stages of infection, coincident with the development of T cell responses and arrest of bacterial population growth These results indicate that bacterial protein secretion in vivo is dynamic and regulated, and quantitation of secreted bacterial proteins can contribute to the understanding of pathogenesis and immunity in tuberculosis and other infections. Transferases, in which they catalyze transesterification reactions to synthesize trehalose monomycolate (TMM), trehalose dimycolate (TDM), and mycolated arabinogalactan [10, 11] Because of these enzymatic activities and their importance in constructing the mycobacterial envelope, Ag85A, Ag85B, and Ag85C have been considered potential drug targets for treatment of tuberculosis (TB) [10]. Consistent with the results of bacterial RNA quantitation, CD4 T cells specific for Ag85B are activated in the lungs between 2 and 3 weeks after infection of mice, but their activation markedly decreases concurrent with decreased bacterial expression of the fbpB gene [12, 15]
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