Enzymatic properties of Staphylococcus aureus adenosine synthase (AdsA)

<p>Abstract</p> <p>Background</p> <p><it>Staphylococcus aureus </it>is a human pathogen that produces extracellular adenosine to evade clearance by the host immune system, an activity attributed to the 5'-nucleotidase activity of adenosine synthase (AdsA). In mammals, conversion of adenosine triphosphate to adenosine is catalyzed in a two-step process: ecto-nucleoside triphosphate diphosphohydrolases (ecto-NTDPases) hydrolyze ATP and ADP to AMP, whereas 5'-nucleotidases hydrolyze AMP to adenosine. NTPDases harbor apyrase conserved regions (ACRs) that are critical for activity.</p> <p>Results</p> <p>NTPDase ACR motifs are absent in AdsA, yet we report here that recombinant AdsA hydrolyzes ADP and ATP in addition to AMP. Competition assays suggest that hydrolysis occurs following binding of all three substrates at a unique site. Alanine substitution of two amino acids, aspartic acid 127 and histidine 196 within the 5'-nucleotidase signature sequence, leads to reduced AMP or ADP hydrolysis but does not affect the binding of these substrates.</p> <p>Conclusion</p> <p>Collectively, these results provide insight into the unique ability of AdsA to produce adenosine through the consecutive hydrolysis of ATP, ADP and AMP, thereby endowing <it>S. aureus </it>with the ability to modulate host immune responses.</p

Staphylococcus aureus synthesizes adenosine to escape host immune responses

Staphylococcus aureus infects hospitalized or healthy individuals and represents the most frequent cause of bacteremia, treatment of which is complicated by the emergence of methicillin-resistant S. aureus. We examined the ability of S. aureus to escape phagocytic clearance in blood and identified adenosine synthase A (AdsA), a cell wall–anchored enzyme that converts adenosine monophosphate to adenosine, as a critical virulence factor. Staphylococcal synthesis of adenosine in blood, escape from phagocytic clearance, and subsequent formation of organ abscesses were all dependent on adsA and could be rescued by an exogenous supply of adenosine. An AdsA homologue was identified in the anthrax pathogen, and adenosine synthesis also enabled escape of Bacillus anthracis from phagocytic clearance. Collectively, these results suggest that staphylococci and other bacterial pathogens exploit the immunomodulatory attributes of adenosine to escape host immune responses

Assembly and intracellular trafficking of human leukocyte antigens B (HLA-B): Differences and functional consequences.

Recognition of MHC class I/antigenic peptide complexes by CD8+ cytotoxic T cells (CTL) is central to the immune response to virally infected and cancerous cells. The assembly of MHC class I molecules with peptides within the endoplasmic reticulum (ER) is assisted by many proteins, which include the generic ER chaperones (calnexin and calreticulin), transporter associated with antigen presentation (TAP), tapasin, ERp57, and protein disulfide isomerase (PDI). This thesis was focused on (1) understanding the molecular basis of differences in assembly characteristics of polymorphic MHC class I variants; (2) elucidating the role of the MHC dedicated chaperone tapasin; and (3) examining in vitro interactions between calnexin and MHC class I molecules. The particular MHC class I alleles that are expressed in an individual can profoundly influence infectious disease outcome. For example, HLA-B*4402 and HLA-B*4405, which differ only at position 116 of their sequences, have differential outcomes in the context various viral infections. HLA-B*3501 and HLA-B*3503 also differ only at position 116, and are associated with differences in AIDS progression rates. We compared the rates at which HLA-B*44 and HLA-B*35 molecules were being assembled within a CD+4 T cell line, in addition to examining how efficiently these class I molecules were being loaded with peptides. The studies showed that a reciprocal relationship exists between inherent efficiencies of peptide loading and intracellular trafficking rates as well as associations with assembly factors in the ER, which could help explain differences in infectious disease susceptibility and resolution. Tapasin's precise role in class I assembly is widely debated. By examining the effect tapasin had on intracellular assembly and cell surface expression of different HLA-B molecules in tapasin deficient and sufficient cells, we concluded that a primary function of tapasin was to promote class I assembly with peptides. Tapasin could be promoting assembly by recruiting other assembly factors to class I molecules. In independent analyses, calnexin and calreticulin could enhance class I assembly under certain conditions in vitro. These results allow us to propose a model for tapasin function in which tapasin may be recruiting other ER assembly factors, thus localizing the class I molecule in an environment that is more favorable for peptide loading.Ph.D.Health and Environmental SciencesImmunologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/126577/2/3253420.pd