Aeromonas hydrophila utilizes TLR4 topology for synchronous activation of MyD88 and TRIF to orchestrate anti-inflammatory responses in zebrafish.

Toll-like receptor 4 (TLR4) plays a critical role in host immunity against Gram-negative bacteria. It transduces signals through two distinct TIR-domain-containing adaptors, MyD88 and TRIF, which function at the plasma membrane and endosomes, respectively. Using zebrafish Aeromonas hydrophila infection model, we demonstrate that synchronization of MyD88 and TRIF dependent pathways is critical for determining the fate of infection. Zebrafish were infected with A. hydrophila, and bacterial recovery studies suggested its effective persistence inside the host. Histopathological assessment elucidates that A. hydrophila did not provoke inflammatory responses in the spleen. Immunofluorescence revealed the presence of TLR4-bound A. hydrophila on the plasma membrane at 3 h post-infection (p.i.), and inside endosomes 1 day p.i. Quantitative PCR studies suggest that TLR4 activates the downstream pathway of MyD88-IRAK4 axis at early stages followed by a shift to TRIF-TRAF6 axis at late stages of infection coupled with fold increase in NFκB. Our results implicated the involvement of p110δ isoform of PI(3)Kinase in this transition. Coupled to this, we noted that the TLR4-TRIF-NFκB axis prompted burgeoned secretion of anti-inflammatory cytokines. We observed that A. hydrophila inhibits endosome maturation and escapes to cytoplasm. Significant downregulation of cytosolic-NLR receptors further suggested that A. hydrophila represses pro-inflammatory responses in cytosol aiding its persistence. Our findings suggest a novel role of 'TLR4 topology' in A. hydrophila-induced pathogenesis. We propose that A. hydrophila manipulates translocation of TLR4 and migrates to endosome, where it triggers TRIF-dependent anti-inflammatory responses, interferes with endosomal maturation and escapes to cytosol. Inside the cytosol, A. hydrophila avoids detection by suppressing NLRs, facilitating its survival and ensuing pathogenesis.