Abstract
Staphylococcus aureus gene locus sdr is encoding proteins responsible for adhesion to bone tissue and therefore for the invasiveness of this bacteria. The aim of the study was to determine possible mechanism of activation of this gene and its role in bone infections.
S. aureus strain 838/05 was isolated from the bone adjacent to infected joint prostheses. The strain contains all sdrC,D, and E genes. It was grown in liquid TSB medium (Bio Merieux) with gentle aeration or on Columbia agar plates containing 5% sheep blood (Bio Merieux). The presence of putative promoters and transcriptional organization of the sdr region was detected using BPROM and FGENESB algorithms (www.softberry.com) based on region 611262bp-623152bp (GeneBank number CP000730.1) of the Staphylococcus aureus subsp. aureus USA300_TCH1516 complete genome sequence. In order to determine interaction of S. aureus with human blood, 50 ml of bacteria grown in TSB medium to ML growth phase were collected and washed twice with sterile PBS to remove the medium and re-suspended in 50ml of sterile PBS. 10 ml of cells were mixed with ∼100ml of mixed fresh human blood collected from healthy volunteers. Immediately after mixing blood with bacteria, 30 ml of the sample (Time 0) were mixed with two volumes of RNA Protect reagent (Qiagen), centrifuged to sediment cells, and frozen at −800C. Additional samples were collected after 30 and 90 minutes of incubation of bacteria with blood. Samples collected after 30 and 90 minutes were treated the same as samples collected at the beginning of the experiment. RNA was isolated, and cDNA was generated. The expression level of the sdr genes was calibrated to the gyrA level and then normalized to the expression level at time 0 using the ΔΔCT method.
Results and conclusions. The study confirms separation of the Sdr region into three transcriptional units and suggests its dissimilar functions, based on differential reaction of the sdrD transcript to environmental conditions and blood.
SdrE has been previously proposed to play role in bone infections; our results indicate that sdrD can play a role in the interactions between the pathogen and human immune system.
