Elucidating the gene encoding the R3 surface protein in Streptococcus agalactiae
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Introduction: S. agalactiae, Lancefield’s group B Streptococcus (GBS), occurs as part of the normal microbial flora of the gastrointestinal and genital tract and can cause critical invasive disease in neonates, including meningitis, pneumonia and septicaemia, as well as systemic infections in pregnant women and non-pregnant adults. R3 is a surface protein which has not been studied much because it has been considered to be of low prevalence in GBS strains in Western countries and the gene encoding R3 is still unknown. In a study of serosubtype protein markers found in GBS strains from Zimbabwe, R3 was common, and 97% of strains which expressed R3, contained the sar5 gene which has previously been reported to encode another surface protein, R5. The aim of this study was to elucidate the gene encoding R3 by analysing whether the sar5 gene encodes R3. Materials and methods: A collection of 142 GBS strains previously tested for R3 expression, was tested for sar5 by PCR to investigate the association between R3 protein expression and presence of the sar5 gene in these strains. The nucleotide sequences of the sar5 sequences of the two R3 negative GBS strains were aligned with the sar5 sequences of three R3 positive and sar5 positive GBS strains. The strains and nucleotide sequences were available from the strain collection at the national reference laboratory for GBS, Department of Medical Microbiology, St Olavs Hospital, Trondheim, Norway. In addition, to experimentally verify that the sar5 gene encodes the R3 protein, the sar5 gene was cloned behind an inducible promoter on a plasmid expression vector, using Gibson Assembly Cloning. The cloned plasmid was then transformed into a sar5 and R3 negative Escherichia coli strain for subsequent expression and R3 detection by Western blot. To ensure that the cloned sar5 gene was properly expressed, a FLAG-tag sequence was inserted either at the 5’-end or at the 3’-end of the gene. Thus, the expressed protein could be detected using α-FLAG antibodies in western blotting, in addition to the R3 antibody. Results: There was correspondence between R3 expression and presence of the sar5 gene in 139 (97.9%) of the 142 GBS strains tested. Two R3 negative strains were sar5 positive by PCR, and one strain previously reported as R3 positive, was sar5 negative by PCR. The sar5 sequences of the two R3 negative GBS strains were aligned with the sar5 sequences of three R3 positive and sar5 positive GBS strains. Both R3 negative GBS strains had multiple deletions near the 3’-end of the sar5 gene, creating a frameshift and a stop codon, leading to a truncated protein. This is most likely the reason why the R3 protein was not expressed in these two strains, even though the strains were sar5 PCR positive. In addition, when data were re-examined at the reference laboratory for the R3 positive sar5 negative strain, it appears that there might have been a switch of strains some time in the past. To experimentally verify that the sar5 gene encodes the R3 protein, the sar5 gene was cloned behind an inducible promoter on a plasmid expression vector. The cloned plasmid was then transformed into a sar5 and R3 negative bacterial strain. After induction of protein expression, Western blotting was then used to detect the protein product of the sar5 gene. In the Western blot, the R3 antibody was bound to a ladder like forming protein in the size range of between 30 kDa and 140 kDa, similar to previously shown for R3. Conclusion: In the PCR-based study there was close correspondence between R3 expression and presence of the sar5 gene in the majority of strains tested, and the lack of correspondence could be explained for the few remaining strains. By cloning the sar5 gene into an R3 and sar5 negative strain, it was possible to verify that the sar5 gene encodes the R3 protein. Therefore, the main hypothesis of this thesis has been proven, that the R3 protein is encoded by the sar5 gene.