Fig. 1: Pat1 is a PLA2 enzyme important for infection of cells and in mice.
From: A patatin-like phospholipase mediates Rickettsia parkeri escape from host membranes
a Graph of fluorescence emissions intensity of fluorogenic PLA2 substrate Red/Green BODIPY PC-A2 at 535 nm after a 2 h incubation with the indicated protein. Bar represents mean  from n = 2 independent experiments, each with three technical replicates. b Genomic loci of pat1 (top) and pat1 insertion site for complementation (bottom). Triangle represents transposon insertion sites, and nucleotide numbers indicate the position in the R. parkeri genome. Genes upstream and downstream are included to show intergenic regions. c Western blot of purified R. parkeri strains, WT, pat1::Tn, and complemented strain (pat1::Tn pat1+), probed with anti-Pat1 antibody; anti-RickA was used as a loading control. Pat1 has a predicted size of 55kD (asterisk, beneath a higher molecular weight background band that is observed in all samples). The image represents results from three independent experiments. The numbers on left are molecular weight in kD. d Images of plaques stained with neutral red at 6 dpi (WT and pat1::Tn pat1+) and 8 dpi (pat1::Tn). Scale bar 10 mm. Represents results from two independent experiments. e Plaque areas in Vero cells infected with WT (7 dpi), pat1::Tn (8 dpi), and complemented strain (7 dpi) (n = 2 independent experiments; WT n = 263 plaques, pat1::Tn n = 109 plaques, pat1::Tn pat1+ n = 141 plaques). Data are mean ± SD; ****p < 0.0001 relative to WT (one-way ANOVA with Dunnett’s post hoc test). f Bacterial abundance of WT, pat1::Tn, and complemented strain in (f) HMECs (n = 3 independent experiments), MOI 0.01 and (g) BMDMs (n = 3 independent experiments), MOI 0.5. Data in (f, g) are mean ± SEM; p-values were significantly different in (g) at 72 hpi (WT vs pat1::Tn p = 0.0154, WT vs pat1::Tn pat1 + p = 0.6871, pat1::Tn vs pat1::Tn pat1::Tn p = 0.3479) (two-way ANOVA with Tukey’s post hoc test). h Survival of Ifnar1−/−;Ifngr1−/− mice infected intravenously (i.v.) with 5 × 106 WT or pat1::Tn mutant (n = 10 mice for WT, n = 7 mice for pat1::Tn, n = 2 independent experiments). Data were analyzed using a log-rank (Mantel-Cox) test, ***p = 0.0001. i Temperature changes over time in i.v. infection of Ifnar1−/−;Ifngr1−/− mice with 5 × 106 WT or pat1::Tn mutant bacteria; graphs represent data from individual mice. j Weight change over time expressed as percent change from initial weight in i.v. infection of Ifnar1−/−;Ifngr1−/− mice with 5 × 106 WT or pat1::Tn mutant bacteria. Data are mean ± SEM and were analyzed using a two-way ANOVA from 0 to 7 dpi, *p = 0.0308. Source data are provided as a Source Data file. .
