The role of CpsABCD in Streptococcus agalactiae capsule biosynthesis

Streptococcus agalactiae or group B Streptococcus (GBS) is a Gram-positive bacterium asymptomatically colonizing 15-35% of women in the gastrointestinal and urogenital tracts. During delivery, neonates born to mothers who carry GBS can be infected themselves and develop severe diseases such as sepsis, pneumonia and meningitis. Pre-partum screenings and prophylactic treatment with antibiotics have reduced the incidence of neonatal GBS disease to 0.04% in USA. But still, in the western world, S. agalactiae represents the major cause of bacterial meningitis in newborns and half of the infected suffer long-term neurodevelopmental defects. Moreover, GBS has also emerged as a pathogen in other patient populations such as the elderly, pregnant women, diabetics and individuals who are immunocompromised. Vaccines based on the capsule polysaccharide (CPS) of this pathogen are currently under development. The CPS is the main virulence factor of GBS, preventing complement deposition and opsonophagocytosis. The production of a CPS is ubiquitous in bacteria, and the Wzy pathway constitutes one of the prototypical mechanisms to produce these structures. This pathway has been characterized in detail in S. pneumoniae. Briefly, the repeating units of sugars composing the CPS are synthesized inside the cell by a group of glycosyltransferases. The repeating units are then flipped outside the membrane and incorporated into the growing polysaccharide chain by a polymerase. Lastly, the polysaccharide is attached to the cell wall peptidoglycan to create the CPS layer surrounding the bacterium. All the enzymes involved in this process are encoded in a single operon. The aim of this work is to investigate the role of the CpsABCD proteins encoded in the cps operon of GBS. These proteins are highly conserved in all GBS serotypes, as well as in some other related bacteria, but they are not involved in the synthesis of the basic repeating units of sugars. CpsA is reported to be a transcriptional regulator and/or an enzyme attaching the CPS to the cell wall. CpsBCD homologous proteins in S. pneumoniae constitute a putative phosphoregulatory system, but their role in GBS capsule biosynthesis is unclear. To investigate the role of these proteins we developed twelve knockout and functional GBS mutant strains and we examined them for CPS quantity, size, and attachment to the cell surface, as well as CpsD phosphorylation. Moreover, we used a bacterial two hybrid assay to investigate interdependencies between these proteins. We observed that in GBS CpsB, C and D constitute a phosphoregulatory system where the CpsD autokinase phosphorylates its C-terminal tyrosines in a CpsC-dependent manner. These Tyr residues are also the target of the cognate CpsB phosphatase. Analysis of cps operon transcription by qRT-PCR on the mutant strains suggested that CpsABCD are not involved in transcriptional regulation of this operon. Furthermore, all the mutant strains retained the capability to produce a CPS, confirming that these proteins are not involved in the synthesis of polysaccharides, however, differences in CPS length and attachment to the cell wall were observed. In particular, we observed that the CpsC extracellular domain appeared necessary for the production of high molecular weight polysaccharides and that the LytR domain of CpsA is required for the attachment of the CPS to the bacterial cell surface. Protein-protein interactions between CpsD and CpsC and between CpsA and CpsC were observed. These results allowed us to propose tentative roles for the proteins and their interdependencies. We propose a model where these proteins are fine-tuning the steps terminating the CPS biosynthesis, i.e. the balance between polymerization and attachment to the cell wall. In said model, CpsA competes with the CPS polymerase and attaches the CPS to the cell wall. This interplay depends on the cyclic phosphorylation of the CpsCD complex which modulates the activity of CpsA balancing the two competing activities. Ultimately, to investigate how differences in CPS length, amount and localization impact on S. agalactiae ability to interact with cells, an in vitro adhesion-invasion assay, using lung epithelial cells have been tested. Our results showed that strains with CPS length different from the wild type were defective in associations to cells. Moreover, strains lacking the capsule or producing very little CPS were more efficient in invading cells irrespective of the CPS length.