Possible Association of GroES and Antigen 85 Proteins with Heat Resistance ofMycobacterium paratuberculosis

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Abstract
Conflicting reports on the heat resistance ofMycobacterium paratuberculosisprompted an examination of the effect of culture medium on this property of the organism.M. paratuberculosiswas cultured in three types of media (fatty acid-containing medium 7H9-OADC (oleic acid-albumin-dextrose-catalase supplement) and glycerol-containing media WR-GD and 7H9-GD [glycerol-dextrose supplement]) at pH 6.0.M. paratuberculosisgrown under these three culture conditions was then tested for heat resistance in distilled water at 65°C. Soluble proteins and mycolic acids ofM. paratuberculosiswere evaluated by two-dimensional electrophoresis (2-DE) and thin-layer chromatography (TLC), respectively. The type of culture medium used significantly affected the heat resistance ofM. paratuberculosis. The decimal reduction times at 65°C (D65°Cvalues; times required to reduce the concentration of bacteria by a factor of 10 at 65°C) forM. paratuberculosisstrains grown in 7H9-OADC were significantly higher than those for the organisms grown in WR-GD medium (P< 0.01). When the glycerol-dextrose supplement of WR was substituted for the fatty acid supplement (OADC) in 7H9 medium (resulting in 7H9-GD), theD65°Cvalue was significantly lower than that for the organism grown in 7H9-OADC medium (P =0.022) but higher than that when it was cultured in WR-GD medium (P =0.005). Proteomic analysis by 2-DE of soluble proteins extracted fromM. paratuberculosisgrown without heat stress in the three media (7H9-OADC, 7H9-GD, and WR-GD) revealed that seven proteins were more highly expressed in 7H9-OADC medium than in the other two media. When the seven proteins were subjected to matrix-assisted laser desorption ionization-mass spectrometric analysis, four of the seven protein spots were unidentifiable. The other three proteins were identified as GroES heat shock protein, alpha antigen, and antigen 85 complex B (Ag85B; fibronectin-binding protein). These proteins may be associated with the heat resistance ofM. paratuberculosis. Alpha antigen and Ag85B are both trehalose mycolyltransferases involved in mycobacterial cell wall assembly. TLC revealed that 7H9-OADC medium supported production of more trehalose dimycolates and cell wall-bound mycolic acids than did WR-GD medium. The present study shows that in vitro culture conditions significantly affect heat resistance, cell wall synthesis, and protein expression ofM. paratuberculosisand emphasize the importance of culture conditions on in vitro and ex vivo studies to estimate heat resistance.