Impact of reducing and oxidizing agents on the infectivity of Qβ phage and the overall structure of its capsid

Abstract

Qβ phages infect Escherichia coli in the human gut by recognizing F-pili as receptors. Infection therefore occurs under reducing conditions induced by physiological agents (e.g. glutathione) or the intestinal bacterial flora. After excretion in the environment, phage particles are exposed to oxidizing conditions and sometimes disinfection. If inactivation does not occur, the phage may infect new hosts in the human gut through the oral route. During such a life cycle, we demonstrated that, outside the human gut, cysteines of the major protein capsid of Qβ phage form disulfide bonds. Disinfection with NaClO does not allow overoxidation to occur. Such oxidation induces inactivation rather by irreversible damage to the minor proteins. In the presence of glutathione, most disulfide bonds are reduced, which slightly increases the capacity of the phage to infect E. coli in vitro Such reduction is reversible and barely alters infectivity of the phage. Reduction of all disulfide bonds by dithiothreitol leads to complete capsid destabilization. These data provide new insights into how the phages are impacted by oxidizing-reducing conditions outside their host cell and raises the possibility of the intervention of the redox during life cycle of the phage.

Free Chlorine and Peroxynitrite Alter the Capsid Structure of Human Norovirus GII.4 and Its Capacity to Bind Histo-Blood Group Antigens

“…TEM was used to observe 10 μl of untreated/oxidation-treated GII.4 VLPs following negative staining with phosphotungstic acid, as previously described (Loison et al, 2016). The number of GII.4 VLPs was estimated for each condition using eight representative pictures per field at a magnification of X 15,000.…”

“…SDS-PAGE was used to study both the free chlorine/ONOO − treated and the untreated GII.4 VLPs. They were denatured as previously described, with minor adjustments (Loison et al, 2016). 15 μl of GII.4 VLPs or GII.4 HuNoVs were denatured using 5 μl of 5X Laemmli buffer [2.5 M Tris pH 6.8, 25% (w/v) SDS, 2.5% (w/v) bromophenol blue, 1 M dithiothreitol (DTT), 70% (v/v) glycerol, and distilled water to give a final volume of 10 ml].…”

“…Protein migration at 130 V was applied in a Mini-PROTEAN 3 cell set up (Biorad) in a tris-glycine buffer. Gel staining was performed using Oriole fluorescent stain (Biorad) for 60 min and then observed using a Gel Doc system (Loison et al, 2016). The intensity of the bands was measured using ImageJ software (Schindelin et al, 2012).…”

The effect of proteolytic enzymes and pH on GII.4 norovirus, during both interactions and non-interaction with Histo-Blood Group Antigens

“…VLPs was studied as previously described 62 , with slight modifications. Tubes were filled with 5 µL of 150 mM Tris-HCl pH 8.0, 5 μL of VLPs, with or without acid pH or proteolytic enzyme treatments, and 13 µL of distilled water.…”

“…The tubes were prepared on ice and a control prepared under the same conditions was added to each experiment. Equilibration was performed and fluorescence intensity recorded as previously described 62 . This allowed the Tm values that corresponded to the temperatures at which viral hydrophobic domains became accessible to the hydrophobic probe to be determined.…”