smegmatis SMR5. The type strain M. fortuitum DSM 46621 exhibited porin amounts close to those of M. smegmatis, whereas the other two strains showed clearly decreased porin amounts (Figure 5B). Notably, M. fortuitum 10851/03
exhibited the lowest amount of porin very close to the background, which was represented by the control M. bovis BCG. Figure 5 Detection of PorMs in M. fortuitum and M. smegmatis . 2D-Electrophoresis, Western Blot, ELISA and qRT-PCR experiments proved PorMs to be expressed in the analysed strains. selleck chemicals Section A shows 2D-Electrophoresis of protein isolation from the strain M. fortuitum 10860/03 using the detergent nOPOE. The arrow indicates the porin spot proven by Western Blot analysis (see Additional file 2). Section B and C show comparative PF-04929113 ic50 analysis of porin expression among RGM. Expression of porin was detected by means of ELISA (B) and qRT-PCR (C). Each value represents the mean (± SD) of at least three independent experiments. B: Quantification of porin by means of ELISA in cell extracts of different mycobacteria using the polyclonal antibody pAk MspA#813. C: RT-Real-time-PCR quantification of porin mRNA in various RGM using specific primers and probes for mspA or porM, respectively. Comparative expression analysis was also performed by means of quantitative reverse transcription polymerase chain reaction
(qRT-PCR) using sequence-specific primers and probes (Table 1). The values were compared to porin expression in M. smegmatis. Because of the high degree of sequence conservation of the two paralogs porM1 and porM2, a qRT-PCR approach was established using primers and a dually labelled probe that hybridised to a region where both genes are identical (porM1 amplicon: nucleotide 132–232 and porM2 amplicon nucleotide 144–244, see also Table 1). This PCR approach enabled the quantification of the
overall expression of the paralogs. As was already proven by the ELISA results, the highest porin mRNA expression was measured in M. smegmatis. second It showed transcription rates about twice as high as the highest level among the M. fortuitum strains, which was detected in the type strain M. fortuitum DSM 46621. M. fortuitum 10851/03 exhibited the lowest transcription rate among all M. fortuitum strains (Figure 5C). The quantification of porM mRNA as well as the protein isolated from the various strains demonstrated consistence of transcriptional and translational levels and underlined the differential porin expression among the analysed M. fortuitum strains. MspA was shown to be accessible on the cell surface of M. smegmatis by using pAK MspA#813 . Since the expression analysis showed a differing amount of porin in M. fortuitum strains, M. fortuitum DSM 46621 and M. fortuitum 10860/03 (the strains with the highest porin expressions) were employed for detection of porins at the surface of intact mycobacteria. Porins were accessible at the surface of intact cells of M. fortuitum and were detected by the NVP-LDE225 chemical structure porin-specific antibody.