TcpC inhibits neutrophil extracellular trap formation by enhancing ubiquitination mediated degradation of peptidylarginine deiminase 4

TcpC is a multifunctional virulence factor of uropathogenic E. coli (UPEC). Neutrophil extracellular trap formation (NETosis) is a crucial anti-infection mechanism of neutrophils. Here we show the influence of TcpC on NETosis and related mechanisms. We show NETosis in the context of a pyelonephritis mouse model induced by TcpC-secreting wild-type E. coli CFT073 (CFT073wt) and LPS-induced in vitro NETosis with CFT073wt or recombinant TcpC (rTcpC)-treated neutrophils are inhibited. rTcpC enters neutrophils through caveolin-mediated endocytosis and inhibits LPS-induced production of ROS, proinflammatory cytokines and protein but not mRNA levels of peptidylarginine deiminase 4 (PAD4). rTcpC treatment enhances PAD4 ubiquitination and accumulation in proteasomes. Moreover, in vitro ubiquitination kit analyses show that TcpC is a PAD4-targetd E3 ubiquitin-ligase. These data suggest that TcpC inhibits NETosis primarily by serving as an E3 ligase that promotes degradation of PAD4. Our findings provide a novel mechanism underlying TcpC-mediated innate immune evasion.

The fixed tissues were incubated with rabbit anti-Ly-6G-IgG, rabbit anti-CitH3-IgG, and rabbit anti-MPO-IgG (Abcam, UK, 1:200 dilution) overnight at 4C and stained with different fluorescent secondary antibody (Alexa fluor 546 nm goat anti-rabbit IgG, Alexa fluor 488nm goat anti-rabbit IgG and Alexa fluor 647nm goat anti-rabbit IgG, 1:500 dilution) in dark respectively. Purity of the human neutrophils was evaluated by FACS using antibodies against Ly-6G/Ly-6C (1:500 dilution, Ly-6G/Ly-6C monoclonal antibody, PE, Thermo, USA) and CD11b (1:500 dilution, CD11b monoclonal antibody, FITC, Thermo, USA). Confocal microscopy to examine NETosis:rabbit anti-MPO antibodies (Abcam, UK, 1:200 diluted in 0.1% Tween 20 in PBS) overnight at 4C. Next, neutrophils were gently washed three times with PBS and incubated in the dark with goat anti-rabbit IgG antibody (Alex Flour 647 nm, 1:500 diluted in the PBS) for 2 h at room temperature.neutrophils were incubated with rabbit anti-CitH3 antibodies Note that full information on the approval of the study protocol must also be provided in the manuscript.

Human research participants
Policy information about studies involving human research participants Population characteristics

Recruitment
Ethics oversight Note that full information on the approval of the study protocol must also be provided in the manuscript.

ChIP-seq Data deposition
Confirm that both raw and final processed data have been deposited in a public database such as GEO.
Confirm that you have deposited or provided access to graph files (e.g. BED files) for the called peaks.

Data access links
May remain private before publication.