Dynamics ofMycobacterium tuberculosisAg85B revealed by sensitive ELISA

Abstract

31Secretion of specific proteins contributes to pathogenesis and immune responses in tuberculosis and 32 other bacterial infections, yet the kinetics of protein secretion and fate of secreted proteins in vivo are 33 poorly understood. We generated new monoclonal antibodies that recognize the M. tuberculosis 34 secreted protein, Ag85B, and used them to establish and characterize a sensitive ELISA to quantitate 35 Ag85B in samples generated in vitro and in vivo. We found that nutritional or culture conditions had 36 little impact on secretion of Ag85B, and that there is considerable variation in Ag85B secretion by 37 distinct strains in the M. tuberculosis complex: compared with the commonly-used H37Rv strain 38 (Lineage 4), M. africanum (Lineage 6) secretes less, and two strains from Lineage 2 secrete more 39Ag85B. We also used the ELISA to determine that the rate of secretion of Ag85B is 10-to 100-fold 40 lower than that of proteins secreted by gram-negative and gram-positive bacteria, respectively. ELISA 41 quantitation of Ag85B in lung homogenates of M. tuberculosis H37Rv-infected mice revealed that 42 although Ag85B accumulates in the lungs as the bacterial population expands, the amount of Ag85B 43 per bacterium decreases nearly 10,000-fold at later stages of infection, coincident with development of 44 T cell responses and arrest of bacterial population growth. These results indicate that bacterial protein 45 secretion in vivo is dynamic and regulated, and quantitation of secreted bacterial proteins can 46 contribute to understanding pathogenesis and immunity in tuberculosis and other infections. 47Bacterial protein secretion contributes to host-pathogen interactions, yet the process and 49 consequences of bacterial protein secretion during infection are poorly understood. We developed a 50 sensitive ELISA to quantitate a protein (termed Ag85B) secreted by M. tuberculosis and used it to find 51 that Ag85B secretion occurs with slower kinetics than for proteins secreted by gram positive and gram 52 negative bacteria, and that accumulation of Ag85B in the lungs is markedly regulated as a function of 53 the bacterial population density. Our results demonstrate that quantitation of bacterial proteins during 54 infection can reveal novel insights into host-pathogen interactions. 55 56