Molecular analysis of an enigmatic Streptococcus pneumoniae virulence factor: The raffinose-family oligosaccharide utilization system

Abstract

Streptococcus pneumoniae is an opportunistic respiratory pathogen that can spread to other body sites, including the ears, brain, and blood. The ability of this bacterium to break down, import, and metabolize a wide range of glycans is key to its virulence. Intriguingly, S. pneumoniae can utilize several plant oligosaccharides for growth in vitro, including raffinose-family oligosaccharides (RFOs, which are α-(1→6)-galactosyl extensions of sucrose). An RFO utilization locus has been identified in the pneumococcal genome; however, none of the proteins encoded by this locus have been biochemically characterized. The enigmatic ability of S. pneumoniae to utilize RFOs has recently received attention because mutations in two of the RFO locus genes have been linked to the tissue tropism of clinical pneumococcal isolates. Here, we use functional studies combined with X-ray crystallography to show that although the pneumococcal RFO locus encodes for all the machinery required for uptake and degradation of RFOs, the individual pathway components are biochemically inefficient. We also demonstrate that the initiating enzyme in this pathway, the α-galactosidase Aga (a family 36 glycoside hydrolase), can cleave α-(1→3)-linked galactose units from a linear blood group antigen. We propose that the pneumococcal RFO pathway is an evolutionary relic that is not utilized in this streptococcal species and, as such, is under no selection pressure to maintain binding affinity and/or catalytic efficiency. We speculate that the apparent contribution of RFO utilization to pneumococcal tissue tropism may, in fact, be due to the essential role the ATPase RafK plays in the transport of other carbohydrates.

Streptococcus pneumoniae Strains Isolated From a Single Pediatric Patient Display Distinct Phenotypes

“…A sucrose phosphorylase, GtfA, cleaves sucrose into glucose and fructose, both of which can enter further metabolic pathways and be utilized by the cell. ( Hobbs et al., 2019 ). Created with .…”

Co-cultures of Propionibacterium freudenreichii and Bacillus amyloliquefaciens cooperatively upgrade sunflower seed milk to high levels of vitamin B12 and multiple co-benefits

“…Between 30 and 48 h, however, the sucrose level remained constant, while raffinose started to be consumed. The metabolization of this trisaccharide involves enzymatic cleavage into sucrose and galactose [ 42 ]. The constant sucrose level therefore resulted from a superposition of sucrose release from the cleavage of raffinose and (the probably) on-going sucrose consumption by NCC 156.…”

“…Like raffinose, the tetrasaccharide stachyose belongs to the raffinose oligosaccharide family. It yields raffinose plus galactose during initial cleavage [ 42 ]. Regarding propionate, certain Bacillus strains can use it as a carbon source but B. amyloliquefaciens cannot [ 44 ].…”

Structural characterization of two solute-binding proteins for N,N′-diacetylchitobiose/N,N′,N′′-triacetylchitotoriose of the gram-positive bacterium, Paenibacillus sp. str. FPU-7

“…for the 396 Cα atoms = 2.5 Å and an a.a. sequence identity = 19%), xylooligosaccharide-binding protein BxlE (PDB: 3VXB) from S. thermoviolaceus OPC-520 ( Tomoo et al, 2017 ) with a Z-score of 38.3 (r.m.s.d. = 4.3 Å (399 atoms) and a.a. sequence identity = 24%), and raffinose binding protein RafE (PDB: 6PRE) of Streptococcus pneumoniae ( Hobbs et al, 2019 ) with a Z-score of 38.0 (r.m.s.d. = 2.6 Å (386 atoms) and a.a. sequence identity = 20%).…”