Fig. 2: Pat1 facilitates escape from single and double membrane compartments following invasion.
From: A patatin-like phospholipase mediates Rickettsia parkeri escape from host membranes
a TEM images of WT and pat1::Tn mutant bacteria in HMECs at 1 hpi. “R” indicates R. parkeri and arrowheads point to membrane surrounding the bacteria. Scale bar 1 µm. b quantification of (a), percentage of single and double membrane-bound or cytosolic bacteria (n = 3 independent experiments, >50 bacteria quantified), MOI 5. Data are mean ± SEM (n = 3 independent experiments); ****p < 0.0001 (single membranes) ***p = 0.0002 (cytosol) *p = 0.0115 (double membranes) relative to WT (unpaired t-test (two-tailed)). c Images of cytosolic bacteria (magenta) in HMECs infected with WT, pat1::Tn, or pat1::Tn pat1 + bacteria (green in merge) at 1.5 hpi. Scale bar 5 µm. d Quantification of (c), percentage of vacuolar bacteria at 1.5 and 4 hpi in HMECs, MOI 5 (n = 2 independent experiments; WT n = 32 fields (1.5 hpi), 33 fields (4 hpi), pat1::Tn n = 33 fields (1.5 hpi, 4 hpi), pat1::Tn pat1+ n = 33 fields (1.5 hpi), 32 fields (4 hpi). e Quantification of vacuolar bacteria at 1.5 hpi in BMDMs, MOI 5 (n = 3 independent experiments; WT n = 65 fields, pat1::Tn n = 77 fields). Data in (d, e) represent individual fields imaged representing n > 1500 bacteria for each strain and timepoint; bars are mean ± SD. Data in (d) ****p < 0.0001 relative to WT (one-way ANOVA with Dunnett’s post hoc test. Data in (e) ****p < 0.0001 relative to WT (unpaired t-test (two-tailed)). f Images of early (30 mpi) actin tails in HMECs, (magenta, F-actin; green, R. parkeri), asterisk indicates bacterium with actin tail. Scale Bar 3 µm. g Quantification of (f), percentage of bacteria with actin tails at 30 mpi and 1 hpi (>500 bacteria counted per time point), MOI 5. Data are mean ± SEM (n = 3 independent experiments); *p = 0.01738 (0.5 hpi), p = 0.02461 (1 hpi) relative to WT (unpaired t-test (two-tailed)). h Percentage of bacteria with actin in untreated cells or cells that have undergone hypotonic shock treatment to lyse vacuoles (images not shown; >1000 bacteria counted per strain/condition), MOI 5. Data are mean ± SEM (n = 3 independent experiments); *p = 0.0325 relative to untreated (paired t-test (two-tailed)). Source data are provided as a Source Data file.
