Final Report Summary - CELLCOM-GBS (Control of Streptococcus agalactiae virulence genes via peptide-based cell to cell communication)
Streptococcus agalactiae (Group B streptococci or GBS) is a pathogen bacteria for humans (especially newborns and immunocompromised adults as well as for animals (cows, fishs). In humans, it is a commensal bacterium of the lower gastrointestinal tract and vaginal genital mucosa of approximately 30% of healthy women. However, this bacterium is the most common cause of life-threatening bacterial infection in human newborns. No vaccines are available to protect the population at risk. The effective way of preventing GBS disease is intrapartum antibiotic prophylaxis. However, this approach results in increasing emergence of antibiotic-resistant isolates as observed for erythromycin and clindamycin resistances. In a previous study, we described a new cell-to-cell communication system in streptococci that is formed by a transcriptional regulator belonging to the Rgg family and a short hydrophobic peptide (SHP) that acts as a signaling molecule for Streptococcus thermophilus. One copy of this shp/rgg locus is present in the genome of nearly all streptococci including GBS. The main goal of CELLCOM-GBS project was to elucidate the role of this new cell-cell communication system in the virulence of GBS.
During this project, we have validated the functionality of this cell-cell communication mechanism in GBS made of a transcriptional regulator RovS, belonging to the Rgg family, and a small hydrophobic peptide, SHP. By constructing different isogenic mutants, we have also identified different additional partners of the mechanism: the Eep membrane peptidase implicated in the peptide maturation; the PptAB oligopeptide transporter involved in peptide secretion, a step that has been identified for the first time in Streptococci; and the Opp1A-F oligopeptide transporter, essential for mature peptide re-importing into the cell (Figure 1).
An in-depth search of rgg and shp genes in all GBS genomes deposited in GenBank has revealed that rovS, and shp genes are highly conserved at the DNA level in the 300 sequenced genomes of GBS available in the databases, meaning that they belong to the core genome of GBS and suggesting that whatever the origin of S. agalactiae isolates (human, bovine, fish, etc.), this cell-cell communication system has an important relevance in the biology of this bacterium.
Furthermore, our results are the first to indicate the SHP/RovS system is involved in the virulence of GBS. Indeed, SHP/RovS is necessary for GBS successful persistence in host liver and spleenand influences S. agalactiae’s ability to adhere to and invade HepG-2 hepatic cells. Hence, the SHP/RovS cell-to-cell communication system appears to be an essential mechanism that regulates pathogenicity in S. agalactiae and represents an attractive target for the development of new therapeutic strategies.
We have also identified a new target gene directly regulated by this system, gbs1556, which encodes a secreted protein with a protease/glutaminase domain but with unknown function. The expression of gbs1556 target is positively controlled by both SHP and RovS; this gene is located downstream of shp gene and both are co-transcribed in operon. The function of Gbs1556 protein has been studied as well its implication in GBS’s pathogenesis using different eukaryotic cell-lines and finally an in vivo murine model (unpublished results). In the other hand, we have re-examined the regulation of a previously proposed target gene, fbsA (Samen et al., 2006), in the context of the SHP-associated RovS system. We have confirmed that both, SHP and RovS are needed for the correct expression of this gene. Based on these results, we hypothesize that RovS is a global regulator of GBS and that shp and gbs1556 are direct targets of the SHP/RovS system, whereas the fbsA gene is probably an indirect target. The indirect control activity of SHP/RovS system remains unclear yet. Finally, in order to in-depth in the regulatory network controlled by SHP/RovS mechanism in GBS, we have characterized the regulon controlled by this system using a label free proteomic approach, identifying several putative genes which expression is positively or negatively modulated (unpublished results). The confirmation of these targets genes by a genetic approach is ongoing.
During this project, we have validated the functionality of this cell-cell communication mechanism in GBS made of a transcriptional regulator RovS, belonging to the Rgg family, and a small hydrophobic peptide, SHP. By constructing different isogenic mutants, we have also identified different additional partners of the mechanism: the Eep membrane peptidase implicated in the peptide maturation; the PptAB oligopeptide transporter involved in peptide secretion, a step that has been identified for the first time in Streptococci; and the Opp1A-F oligopeptide transporter, essential for mature peptide re-importing into the cell (Figure 1).
An in-depth search of rgg and shp genes in all GBS genomes deposited in GenBank has revealed that rovS, and shp genes are highly conserved at the DNA level in the 300 sequenced genomes of GBS available in the databases, meaning that they belong to the core genome of GBS and suggesting that whatever the origin of S. agalactiae isolates (human, bovine, fish, etc.), this cell-cell communication system has an important relevance in the biology of this bacterium.
Furthermore, our results are the first to indicate the SHP/RovS system is involved in the virulence of GBS. Indeed, SHP/RovS is necessary for GBS successful persistence in host liver and spleenand influences S. agalactiae’s ability to adhere to and invade HepG-2 hepatic cells. Hence, the SHP/RovS cell-to-cell communication system appears to be an essential mechanism that regulates pathogenicity in S. agalactiae and represents an attractive target for the development of new therapeutic strategies.
We have also identified a new target gene directly regulated by this system, gbs1556, which encodes a secreted protein with a protease/glutaminase domain but with unknown function. The expression of gbs1556 target is positively controlled by both SHP and RovS; this gene is located downstream of shp gene and both are co-transcribed in operon. The function of Gbs1556 protein has been studied as well its implication in GBS’s pathogenesis using different eukaryotic cell-lines and finally an in vivo murine model (unpublished results). In the other hand, we have re-examined the regulation of a previously proposed target gene, fbsA (Samen et al., 2006), in the context of the SHP-associated RovS system. We have confirmed that both, SHP and RovS are needed for the correct expression of this gene. Based on these results, we hypothesize that RovS is a global regulator of GBS and that shp and gbs1556 are direct targets of the SHP/RovS system, whereas the fbsA gene is probably an indirect target. The indirect control activity of SHP/RovS system remains unclear yet. Finally, in order to in-depth in the regulatory network controlled by SHP/RovS mechanism in GBS, we have characterized the regulon controlled by this system using a label free proteomic approach, identifying several putative genes which expression is positively or negatively modulated (unpublished results). The confirmation of these targets genes by a genetic approach is ongoing.