Determination of SUMO1 and ATP affinity for the SUMO E1by quantitative FRET technology.

SUMOylation plays important roles in many key physiological and pathological processes. The SUMOylation cascade involves a heterodimer of activating enzyme, E1 (Aos1/Uba2); a conjugating enzyme, E2 (Ubc9); and many ligase enzymes, E3. Focusing on the activation step of the SUMOylation process, we examined the interaction of E1 with its substrates. Previous studies reported the Km of E1 enzymes in ubiquitin and other ubiquitin-like pathways, but the Km of the SUMO paralogs (SUMO2 and SUMO3) is unknown. Here, by using quantitative FRET to measure the SUMO E1 enzyme kinetics of SUMO1, 2, and 3 and ATP under steady state conditions, we found that the enzyme kinetics from the quantitative FRET method are comparable to those from conventional radioactive assays. Additionally, the kinetic constants, Km , of SUMO2 (3.418 ± 0.9131 µM) and SUMO3 (2.764 ± 0.75 µM) [FW1] are approximately four to five times higher than that of SUMO1 Km (0.7458 ± 0.1105 µM). These results demonstrate the advantages of FRET technology for determining Km , including the ability to monitor reaction progress in real-time with high-throughput and high-sensitivity in an environmentally friendly manner. The processes discussed here extend the utility of quantitative FRET in characterizing protein-protein interactions and enzyme kinetics.

School-located influenza vaccination decreases laboratory-confirmed influenza and improves school attendance.

BACKGROUND: School-located influenza vaccination (SLV) programs can efficiently immunize large numbers of school-aged children. We evaluated the impact of SLV on laboratory-confirmed influenza and absenteeism. METHODS: Active surveillance for influenza-like illness (ILI) was conducted on 4455 children in 4 SLV intervention and 4 control elementary schools (grades K-6) matched for sociodemographic characteristics during the 2010-2011 influenza season in Los Angeles County, California. Combined nose/throat swabs were collected from febrile children with ILI at presentation to the school nurse or during absenteeism. RESULTS: In SLV schools, 26.9%-46.6% of enrolled students received at least 1 dose of either inactivated or live attenuated influenza vaccine compared with 0.8%-4.3% in control schools. Polymerase chain reaction for respiratory viruses (PCR) was performed on 1021 specimens obtained from 898 children. Specimens were positive for influenza in 217 (21.3%), including 2009 H1N1 (30.9%), H3 (9.2%), and B (59.9%). Children attending SLV schools, regardless of vaccination status, were 30.8% (95% confidence interval, 10.1%-46.8%) less likely to acquire influenza compared with children at control schools. Unvaccinated children were indirectly protected in the school with nearly 50% vaccination coverage compared with control schools (influenza rate, 27.1 vs 60.0 per 1000 children; P = .023). Unvaccinated children missed more school days than vaccinated children (4.3 vs 2.8 days per 100 school days; P < .001). CONCLUSIONS: Vaccination of at least a quarter of the school population resulted in decreased influenza rates and improved school attendance. Herd immunity for unvaccinated children may occur in schools with vaccination coverage approaching 50%.

Anaplasma phagocytophilum dihydrolipoamide dehydrogenase 1 affects host-derived immunopathology during microbial colonization.

Anaplasma phagocytophilum is a tick-borne rickettsial pathogen that provokes an acute inflammatory response during mammalian infection. The illness caused by A. phagocytophilum, human granulocytic anaplasmosis, occurs irrespective of pathogen load and results instead from host-derived immunopathology. Thus, characterizing A. phagocytophilum genes that affect the inflammatory process is critical for understanding disease etiology. By using an A. phagocytophilum Himar1 transposon mutant library, we showed that a single transposon insertion into the A. phagocytophilum dihydrolipoamide dehydrogenase 1 gene (lpda1 [APH_0065]) affects inflammation during infection. A. phagocytophilum lacking lpda1 revealed enlargement of the spleen, increased splenic extramedullary hematopoiesis, and altered clinicopathological abnormalities during mammalian colonization. Furthermore, LPDA1-derived immunopathology was independent of neutrophil infection and correlated with enhanced reactive oxygen species from NADPH oxidase and nuclear factor (NF)-κB signaling in macrophages. Taken together, these findings suggest the presence of different signaling pathways in neutrophils and macrophages during A. phagocytophilum invasion and highlight the importance of LPDA1 as an immunopathological molecule.