Expression studies
During the experiments of MG-63 infection, RNA was extracted and the expression of some regulatory, adhesion and toxin genes was evaluated after 3h and 24h of intracellular persistence, in comparison with the non-internalized bacteria (basal condition). All experiments were the average of three biological experiments and are reported in Figure 3 and Table 4, subdivided with respect to their function.
In ATCC12598, sig B was significantly up-regulated after 24h p.i,sar A and sar S were up-regulated after 3h p.i. In particular, sig B gene mRNA expression showed a strong statistically significant increase at 24h p.i. (+2.92±0.27-fold change (fd); p≤ 0.001) vs the basal condition, and comparing 24hvs 3h p.i. (p≤ 0.01). sar A gene mRNA expression showed an increase at 3h vs basal condition (+4.4±0.98 fd, p≤ 0.05) and a statistically significant decrease at 24h vs basal condition (+0.79±0.36 fd, p≤ 0.05). sar S gene mRNA expression showed an increase at 3h vs basal condition (+7.45±0.65 fd, p≤ 0.01) and a statistically significant decrease at 24h vs basal condition (+1.83±0.45 fd, p≤ 0.01).
The rot gene was significantly down-regulated after 24h p.i. only in the ST228 strain. rot gene mRNA expression of the ST228 strain showed a statistically significant decrease at 24h vs basal condition (+0.23±0.016 fd, p≤ 0.05) and vs 3h (p≤ 0.05).
For the other genes involved in regulation, no other statistical differences were observed in any condition tested. Expression of the MSCRAMM genes were reported in Figure 4 and Table 4. All the MSCRAMM genes were differentially modulated in our sample.
In the ATCC12598 strain, fnb A gene mRNA expression showed a statistically significant increase at 3h vs basal condition (+4255.6±1052.8 fd, p≤ 0.05) and a statistically significant decrease at 24h p.i. vs 3h (+86.9± 28.03 fd, p≤ 0.05). sdr E gene mRNA expression showed a statistically significant increase at 3h vsbasal condition (+10.63±2.48 fd, p≤ 0.05) and a statistically significant decrease at 24h p.i. vs 3h (+1.55± 0.1 fd, p≤ 0.05).
bbp gene mRNA expression showed a statistically significant increase (+2318.32± 801.5 fd, p≤ 0.05) in the ST239 strain at 3h p.i.vs basal condition, and at 24h vs basal condition (+2897.22±156.12 fd, p≤ 0.05). For the fnb B gene and the other strains, no other statistical differences were observed in each condition considered. Expression of toxin genes and the sugar phosphate antiporter gene are reported in Figure 5 and Table 4.
psm A gene mRNA expression of the ATCC12598 strain showed a statistically significant increase (+6.75± 3.75 fd, p≤ 0.05) at 3h p.i.vs 24h. In the ST228 strain, it was statistically significantly up-regulated (+32.84±11.5 fd, p≤ 0.05) at 3h p.i vs basal condition.
hla gene mRNA expression showed a significant increase in the ATCC12598 strain (+1308.64± 323.75 fd, p≤ 0.05) at 3h p.i. vsbasal condition and a statistically significant decrease comparing 24hvs 3h p.i. (17.58±6.4 fd; p≤ 0.05). In the ST239 strain, it showed a statistically significant increase at 3h and 24h when comparing 3h vs basal condition (4922.01±840.23 fd, p≤ 0.01 and 26698.18±130.85 fd, p≤ 0.001), and also when comparing 3h vs 24h (p≤ 0.001). hld gene mRNA expression showed a significant decrease in ATCC12598 at 24h vs basal condition (0.04±0.04 fd; p≤ 0.05). For the other strains, in each condition considered, no statistically significant differences were observed. The mRNA expression of the uhp T gene showed a statistically significative difference only in the ATCC12598 strain, in particular an up-regulation when comparing 3h vs basal condition (16.85±1.5 fd; p≤ 0.05) and a down-regulation when comparing 24h vs 3h (5.32±2.27 fd; p≤ 0.05).