Fig 5. PI(4,5)P₂ interacts with RalF_Rt and mediates R. typhi infection.

(A) RalF_RtCTD co-localizes with PI(4,5)P₂. HeLa cells transfected with pEYFP-C1 empty vector, GFP-C1-PLCδ-PH (a PI(4,5)P2 biosensor), EYFP–RalF_RtCTD, or EYFP–RalF_RbCTD were treated with 5 μM ionomycin alone, with Ca²⁺, or with Ca²⁺ and EGTA. Nuclei were stained with DAPI (blue). (Scale bar: 10 μm). (B) PI(4,5)P₂ is recruited during R. typhi infection. HeLa cells transfected with GFP-C1-PLCδ-PH (green) were infected with R. typhi (MOI ~100:1) for indicated times. R. typhi was detected with rat anti-R. typhi serum and Alexa Fluor 594 anti-rat antibody (red). Nuclei were stained with DAPI (blue). Boxed regions are enlarged to show detail (inset). (Scale bar: 1 μm). (C) RalF localizes to PI(4,5)P₂-enriched regions of the plasma membrane during R. typhi infection. HeLa cells transfected with GFP-C1-PLCδ-PH (green) were infected with R. typhi (MOI ~100:1) for indicated times. RalF_Rt was detected with rabbit anti-RalF_Rt and Alexa Fluor 594 anti-rabbit antibodies (red). Nuclei were stained with DAPI (blue). Boxed regions are enlarged to show detail (inset). (Scale bar: 1 μm). (D) Ionomycin and Ca²⁺ treatment decreases R. typhi infection. HeLa cells treated with 5 μM ionomycin and Ca²⁺ or no treatment were infected with R. typhi (MOI ~100:1) for 2 hrs. R. typhi was detected with rat anti-R. typhi serum and Alexa Fluor 488 anti-rat antibody. Cell membrane was stained with Alexa Fluor 594 wheat germ agglutinin. The number of infected host cells was counted, with percent infection of three independent experiments (100 host cells counted for each) plotted. Error bars represent mean ± SD (Student’s two-sided t-test).