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.
Highlights
Streptococcus pneumoniae is an opportunistic respiratory pathogen that can spread to other body sites, including the ears, brain, and blood
We use functional studies combined with X-ray crystallography to show that the pneumococcal raffinose-family oligosaccharides (RFOs) locus encodes for all the machinery required for uptake and degradation of RFOs, the individual pathway components are biochemically inefficient
Given the ability of RafE to accommodate glycans of different lengths and monosaccharide composition, we explored the molecular basis of its ligand binding by X-ray crystallography
Summary
Ing to GH family 13; an ATP-binding cassette (ABC) transporter, consisting of the solute-binding protein (SBP) RafE [26] and the putative permeases RafF and RafG; RafR and RafS, two putative transcriptional regulators/repressors; and RafX, a putative protein of unknown function (Fig. 1). Minhas et al [28] compared the genomes of serotype- and sequence type–matched isolates from blood and ear infections and identified SNPs in rafK or rafR among the ear isolates. The ability of these isolates to grow on raffinose was significantly reduced compared with their paired blood isolates, and reversal of the rafR mutation restored full raffinose utilization competency. Expression of genes from each of the transcriptional units within the RFO locus (aga, rafG, and rafK) was significantly lower in the ear isolates compared with the blood isolates. We speculate that the reported link between the RFO locus and virulence may, be due to the promiscuous role RafK plays in the import of several human-derived carbohydrates
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