Immune evasion by a staphylococcal inhibitor of myeloperoxidase

Abstract

Staphylococcus aureus is highly adapted to its host and has evolved many strategies to resist opsonization and phagocytosis. Even after uptake by neutrophils, S. aureus shows resistance to killing, which suggests the presence of phagosomal immune evasion molecules. With the aid of secretome phage display, we identified a highly conserved protein that specifically binds and inhibits human myeloperoxidase (MPO), a major player in the oxidative defense of neutrophils. We have named this protein "staphylococcal peroxidase inhibitor" (SPIN). To gain insight into inhibition of MPO by SPIN, we solved the cocrystal structure of SPIN bound to a recombinant form of human MPO at 2.4-Å resolution. This structure reveals that SPIN acts as a molecular plug that prevents H 2 O 2 substrate access to the MPO active site. In subsequent experiments, we observed that SPIN expression increases inside the neutrophil phagosome, where MPO is located, compared with outside the neutrophil. Moreover, bacteria with a deleted gene encoding SPIN showed decreased survival compared with WT bacteria after phagocytosis by neutrophils. Taken together, our results demonstrate that S. aureus secretes a unique proteinaceous MPO inhibitor to enhance survival by interfering with MPO-mediated killing.

A structurally dynamic N-terminal region drives function of the staphylococcal peroxidase inhibitor (SPIN)

“…In summary, while SPIN in the bound state displays a β-hairpin structure at its Nterminus, our studies presented here strongly suggest that this region is dynamic and has a random coil character prior to MPO binding. (17), as judged by the EBI-PISA server (22). Although the SPIN/rMPO interface is contiguous on the SPIN surface, it is useful for purposes of analysis to consider it comprised of two distinct binding sites (17).…”

“…Since MPO is the most abundant component of azurophilic granules (4,16), it represented an promising initial candidate for this approach. We succeded in identifying a novel protein of 8.4 kDa that binds and inhibits human MPO and named it SPIN, for Staphylococcal Peroxidase INhibitor (17). Gene expression studies revealed that spn transcription is upregulated following phagocytosis of S. aureus cells by neutrophils, suggesting that the SPIN protein operates primarily within the phagosomal compartment (17).…”

“…We succeded in identifying a novel protein of 8.4 kDa that binds and inhibits human MPO and named it SPIN, for Staphylococcal Peroxidase INhibitor (17). Gene expression studies revealed that spn transcription is upregulated following phagocytosis of S. aureus cells by neutrophils, suggesting that the SPIN protein operates primarily within the phagosomal compartment (17). Through subsequent studies, we found that SPIN protects S. aureus from MPOmediated killing and thereby established SPIN as a novel immune evasion protein of S. aureus (17).…”

“…To circumvent this limitation, we solved a 2.4 Å resolution crystal structure of SPIN bound to a recombinant form of human MPO (i.e. rMPO) (17). This structure suggested that SPIN acts by occluding the exchange of substrate, product, and bulk solvent with the reactive heme that lies the within the MPO active site.…”

“…S1), inspection of the crystal structure suggested that this helical bundle might not be responsible per se for inhibiting MPO. Instead, an approximately 10-residue extension at the SPIN N-terminus assumes a β-hairpin structure which almost completely occupies the MPO active site channel (17). Further examination of the SPIN structure suggested that this N-terminal β-hairpin might not be intrinsically stable in the absence of its MPO ligand, however, as it lacks features such as disulfide bonds, etc., that would constrain these two short β-strands into their MPO-bound conformation.…”