FIG 3.

EsxE and EsxF are required for CpnT secretion. (A) Secretion analysis of WT+ CpnT, _AxxxECpnT, and ΔesxEF-CpnT strains. Whole-cell lysate (WCL) and supernatant preparations were immunoblotted for CpnT detection (anti-HA antibody); immunodetection of GroEL was used as a lysis control, whereas PE_PGRS was used as a control for secretion. Surface proteins were extracted from intact cells with the detergent Genapol X-080 (Genapol supernatant); nonextracted proteins remained in the Genapol pellet fraction. GroEL was used as a cytosolic control, whereas PE_PGRS was used as a control for protein extraction. (B) Subcellular fractions of WT+ CpnT and WT+ ΔesxEF-CpnT strains were obtained using differential centrifugation steps. Whole-cell lysate (WCL), soluble proteins (S), and pellet fraction (P, insoluble proteins) were collected. Detection of GroEL (cytosolic protein) and EccB₅ (membrane-associated protein) with specific antibodies was used as a control for the supernatant and pellet fraction, respectively.