The structure of NAD+ consuming protein Acinetobacter baumannii TIR domain shows unique kinetics and conformations.

Toll-like and interleukin-1/18 receptor/resistance (TIR) domain-containing proteins function as important signaling and immune regulatory molecules. TIR domain-containing proteins identified in eukaryotic and prokaryotic species also exhibit NAD+ hydrolase activity in select bacteria, plants, and mammalian cells. We report the crystal structure of the Acinetobacter baumannii TIR domain protein (AbTir-TIR) with confirmed NAD+ hydrolysis and map the conformational effects of its interaction with NAD+ using hydrogen-deuterium exchange-mass spectrometry. NAD+ results in mild decreases in deuterium uptake at the dimeric interface. In addition, AbTir-TIR exhibits EX1 kinetics indicative of large cooperative conformational changes, which are slowed down upon substrate binding. Additionally, we have developed label-free imaging using the minimally invasive spectroscopic method 2-photon excitation with fluorescence lifetime imaging, which shows differences in bacteria expressing native and mutant NAD+ hydrolase-inactivated AbTir-TIRE208A protein. Our observations are consistent with substrate-induced conformational changes reported in other TIR model systems with NAD+ hydrolase activity. These studies provide further insight into bacterial TIR protein mechanisms and their varying roles in biology.

Surface Contamination of Reusable Respirators and Face Shields During Care of Critically Ill COVID-19 Patients.

BACKGROUND: With the emergence of SARS-CoV-2, healthcare workers (HCW) have relied on reusable personal protective equipment (PPE), including respirators and face shields (FSs). The effectiveness of decontamination procedures outside experimental settings is unclear. We examined the prevalence of surface contamination on reusable PPE used by HCWs at a hospital incorporating daily centralized decontamination and post-use wiping by sampling for common pathogens. METHOD: Samples were collected from HCWs' CleanSpace Halo respirator face masks (FMs) and FSs at the start of shift, immediately after use, and after cleaning with disinfecting wipes. Samples were analyzed for pathogens using the Applied Biosystems™ TaqPath™ COVID-19 Combo Kit and ThermoFisher TaqMan Array Card. Patient charts were reviewed for clinical correlation. FINDINGS: Of the 89 samples, 51 from FMs and 38 from FSs, none tested positive for SARS-CoV-2, despite 58 being obtained from PPE used in the care of patients with COVID-19, many with recent aerosol-generating procedures. Four samples tested positive (4.5%) for Staphylococcus aureus, two each from FMs and FSs. FMs that tested positive were not worn concurrently with FSs that tested positive. The FM and FS samples testing positive were worn in the care of patients without diagnosed S. aureus infection. No FMs tested positive following wipe-based disinfection, but both positive FS samples were found after disinfection wiping. CONCLUSION/APPLICATION TO PRACTICE: Contamination of reusable PPE appears uncommon, especially with SARS-CoV-2, when regular decontamination programs are in place. The rare presence of S. aureus highlights the importance of doffing procedures and hand hygiene by HCW to prevent surface contamination.