Figure 5

Semi-quantitative RT-PCR assessment of TvBspA expression in T. vaginalis bound to ECM proteins. Upon contact to the ECM proteins T. vaginalis cells strongly bind to the substrate and the majority of cells cannot be washed off after 60 min of incubation. RT-PCR for nine selected TvBspA-like genes and control actin (no major change) and alpha-actinin (up-regulated) genes was performed on T. vaginalis ECM proteins bound cells after 60 min of incubation (BC) and control free swiming trophozoites incubated in parallel (T). The shown results are representative of 6 to 15 independent PCR and at least five independent binding experiments/RNA extractions. TvBspA gene-specific primers were designed to produce amplicons of different sizes and avoid cross-amplification between TvBspA genes, in particular between the closely related TvBspA625 (TVAG_073760) and TvBspA805 (TVAG_154640) indicated by a star. A control PCR was also performed on genomic DNA (gDNA) confirming that the designed primers are efficient in generating the specific amplicons (size are indicated). Actin amplicons are equivalent for cDNA preparations from both trophozoite and ECM bound cells indicating similar total cDNA load whereas the cDNA specific for alpha-actinin increases upon T. vaginalis binding to substrate as described [97]. Five TvBspA genes show clear increase in the amount of amplicons for the ECM bound cells suggesting transcription up-regulation or higher stability of their mRNA in this condition. For TvBspA605 no amplicon could be detected suggesting that it is not transcribed in either tested conditions. For TvBspA805 the amount of cDNA was doubled to allow the detection of the shown signal indicating that the mRNA encoding this protein is not as abundant as for the paralogue TvBspA625.