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Asp²² drives the protonation state of the Staphylococcus epidermidis glucose/H⁺ symporter.

ABSTRACT: The Staphylococcus epidermidis glucose/H⁺ symporter (GlcP_Se) is a membrane transporter highly specific for glucose and a homolog of the human glucose transporters (GLUT, SLC2 family). Most GLUTs and their bacterial counterparts differ in the transport mechanism, adopting uniport and sugar/H⁺ symport, respectively. Unlike other bacterial GLUT homologs (for example, XylE), GlcP_Se has a loose H⁺/sugar coupling. Asp²² is part of the proton-binding site of GlcP_Se and crucial for the glucose/H⁺ co-transport mechanism. To determine how pH variations affect the proton site and the transporter, we performed surface-enhanced IR absorption spectroscopy on the immobilized GlcP_Se We found that Asp²² has a pK_a of 8.5 ± 0.1, a value consistent with that determined previously for glucose transport, confirming the central role of this residue for the transport mechanism of GlcP_Se A neutral replacement of the negatively charged Asp²² led to positive charge displacements over the entire pH range, suggesting that the polarity change of the WT reflects the protonation state of Asp²² We expected that the substitution of the residue Ile¹⁰⁵ for a serine, located within hydrogen-bonding distance to Asp²², would change the microenvironment, but the pK_a of Asp²² corresponded to that of the WT. A167E mutation, selected in analogy to the XylE, introduced an additional protonatable site and perturbed the protonation state of Asp²², with the latter now exhibiting a pK_a of 6.4. These studies confirm that Asp²² is the proton-binding residue in GlcP_Se and show that charged residues in its vicinity affect the pK_a of glucose/H⁺ symport.

SUBMITTER: Seica AFS

PROVIDER: S-EPMC7650245 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:The species- and clone-specific susceptibility of Staphylococcus cells for bacteriophages is governed by the structures and glycosylation patterns of wall teichoic acid (WTA) glycopolymers. The glycosylation-dependent phage-WTA interactions in the opportunistic pathogen Staphylococcus epidermidis and in other coagulase-negative staphylococci (CoNS) have remained unknown. We report a new S. epidermidis WTA glycosyltransferase TagE whose deletion confers resistance to siphoviruses such as ΦE72 but enables binding of otherwise unbound podoviruses. S. epidermidis glycerolphosphate WTA was found to be modified with glucose in a tagE-dependent manner. TagE is encoded together with the enzymes PgcA and GtaB providing uridine diphosphate-activated glucose. ΦE72 transduced several other CoNS species encoding TagE homologs, suggesting that WTA glycosylation via TagE is a frequent trait among CoNS that permits interspecies horizontal gene transfer. Our study unravels a crucial mechanism of phage-Staphylococcus interaction and horizontal gene transfer, and it will help in the design of anti-staphylococcal phage therapies.IMPORTANCEPhages are highly specific for certain bacterial hosts, and some can transduce DNA even across species boundaries. How phages recognize cognate host cells remains incompletely understood. Phages infecting members of the genus Staphylococcus bind to wall teichoic acid (WTA) glycopolymers with highly variable structures and glycosylation patterns. How WTA is glycosylated in the opportunistic pathogen Staphylococcus epidermidis and in other coagulase-negative staphylococci (CoNS) species has remained unknown. We describe that S. epidermidis glycosylates its WTA backbone with glucose, and we identify a cluster of three genes responsible for glucose activation and transfer to WTA. Their inactivation strongly alters phage susceptibility patterns, yielding resistance to siphoviruses but susceptibility to podoviruses. Many different CoNS species with related glycosylation genes can exchange DNA via siphovirus ΦE72, suggesting that glucose-modified WTA is crucial for interspecies horizontal gene transfer. Our finding will help to develop antibacterial phage therapies and unravel routes of genetic exchange.

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Asp²² drives the protonation state of the Staphylococcus epidermidis glucose/H⁺ symporter.

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Asp²² drives the protonation state of the Staphylococcus epidermidis glucose/H⁺ symporter.