Identification of genes implicated in the virulence of streptococcus agalactiae
Group B streptococcus is an important cause of maternal and neonatal morbidity and mortality in many part of the world. The invention is a method of identification of novel targets for inhibitors preventing septicemic dissemination of Streptococcus agalactiae , a model of Gram positive bacteria, in order to treat bacterial infections using these virulence determinant.
1 . A method for the insertion mutagenesis of GBS comprising the use of vector pTCV-Tase.
2 . The insertion mutant of genes gbs 0052 (SEQ ID N° 1), gbs 0100 (SEQ ID N° 2), gbs 0307 (SEQ ID N° 3), gbs 0582 (SEQ ID N° 4), gbs 0653 (SEQ ID N° 5), gbs 0683 (SEQ ID N° 6), gbs 1787 (SEQ ID N° 7), gbs 2100 (SEQ ID N° 8).
3 . In vitro Screening Method of the mutants library comprising using colistine as a mimic of innate immunity components that are the antibacterial cationic peptides and using novobiocine to detect mutant with defect in outer membrane permeability.
4 . A methods combining the in vitro screening results with in vivo effect of mutations, to identify target proteins having en essential function for in vivo virulence.
5 . Proteins sequences of the targets in GBS identified claim 1 as useful to find drugs preventing bacterial dissemination in the host.
6 . The proteins of claim 1 which are in GBS and homologous sequences in gram positive bacteria with at least 22% identify on the full length seq and 25% identity in a 100 continuous amino acid sequence.
7 . The proteins according to claim 6 , wherein the homologous proteins are present in Streptococcus Pneumoniae (SPN), to find drugs for treating Gram positive infections.
8 . A biochemical assay for screening inhibitors for GBS 0307
characterized in that they are based either on luminescent ATP or fluorescent ADP detection.
9 . The biochemical assay of claim 8 , comprising:
adding a substrate mixture comprising GBS, myelin basic protein and ATP to an assay buffer preincubated with DMSO or an analog or inhibitor dissolved in DMSO or analog,
incubating at room temperature,
adding a revelation mixture, and
measuring luminescence.
10 . The biochemical assay of claim 6 , comprising:
adding a substrate mixture comprising GBS, myelin basic protein; ATP, pyruvate and NaDH,
measuring fluorescence intensity of NaDH (λ ex =360 nm, λ em =520 nm),
deriving the inhibition % from fitted initial velocities.