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  • Staphylococcus pseudintermedius 5'-nucleotidase suppresses canine phagocytic activity.

Staphylococcus pseudintermedius 5'-nucleotidase suppresses canine phagocytic activity.

Veterinary microbiology (2020-07-02)
Mohamed A Abouelkhair, Linda A Frank, David A Bemis, Richard J Giannone, Stephen A Kania
ABSTRACT

Staphylococcus pseudintermedius is a major opportunistic bacterial pathogen and the leading cause of pyoderma in dogs. In canines it is also often associated with infections of the urinary system and wounds and occasionally infects people. Widespread antimicrobial resistance has made the development of alternative treatments a high priority. The development of a staphylococcal vaccine, however, has proven challenging. Identification of virulence factors that inhibit phagocytosis and avoid innate immunity may play a significant role in preventing or treating infection with S. pseudintermedius. In this study, we identified a putative 5'-nucleotidase provisionally named SpAdsA, a S. pseudintermedius cell- wall protein encoded by SpAdsA. SpAdsA shares approximately 52% identity with the orthologous protein of Staphylococcus aureus and 14.8% identity with that of Streptococcus suis type2. It catalyzes the dephosphorylation of adenosine triphosphate and attenuation of this enzyme with critical amino acid substitutions nearly eliminated its hydrolytic activity. Exogenous adenosine inhibited phagocytosis of S. pseudintermedius by canine neutrophils and monocytes. Conversely, the addition of SpAdsA inhibitor or A2A adenosine receptor antagonist impaired the capacity of S. pseudintermedius to escape from killing by phagocytic cells. The neutralizing ability of canine antibody produced against SpAdsA-M was determined. Taken together, these results suggest that SpAdsA likely plays an important role in S. pseudintermedius virulence and that attenuated SpAdsA may be a good candidate for inclusion in a vaccine against S. pseudintermedius.

MATERIALS
Product Number
Brand
Product Description

Millipore
Protease Inhibitor Cocktail Set III, EDTA-Free, Protease inhibitor cocktail III, EDTA-free for inhibiting aspartic, cysteine, and serine proteases as well as aminopeptidases in mammalian cells and tissues.
Supelco
Malachite Green chloride, analytical standard
Sigma-Aldrich
Adenosine 5′-triphosphate disodium salt hydrate, 99%
Sigma-Aldrich
Ampicillin, Ready Made Solution, 100 mg/mL, 0.2 μm filtered
Sigma-Aldrich
Red Blood Cell Lysing Buffer Hybri-Max, liquid, sterile-filtered, suitable for hybridoma
Sigma-Aldrich
Malachite Green Phosphate Assay Kit, sufficient for 2500 colorimetric tests