Vibrio deploys type 2 secreted lipase to esterify cholesterol with host fatty acids and mediate cell egress
- Department of Molecular Biology, University of Texas Southwestern Medical Center, United States
- Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, United States
- Department of Microbiology, University of Texas Southwestern Medical Center, United States
- Department of Physiology, University of Texas Southwestern Medical Center, United States
- Department of Internal Medicine, University of Texas Southwestern Medical Center, United States
- Center for Human Nutrition, University of Texas Southwestern Medical Center, United States
- Faculty of Agriculture and Biology, Warsaw University of Life Sciences, Poland
- Department of Molecular Genetics, University of Texas Southwestern Medical Center, United States
Figures
Figure 1 with 2 supplements
VPA0226 mediates bacterial egress from the host cell.
(A) HeLa cells were infected with CAB2 or CAB2Δvpa0226 for 1, 3, 5, and 7 hr PGT. Host cell lysates were serially diluted and plated onto MMM agar plates for intracellular bacterial colony counting …
Figure 1—figure supplement 1
VPA0226 is GCAT lipase that contributes to bacterial cell egress but does not contribute to V. parahaemolyticus’ escape from its containing vacuole.
(A) Sequence alignment between the Salmonella T3SS2 effector SseJ and the V. parahaemolyticus lipase VPA0226. Conserved active sites are boxed in red. (B) Sequence alignment between V. …
Figure 1—figure supplement 2
Extended time course for gentamicin protection assay.
(A) HeLa cells were infected with CAB2, CAB2Δvpa0226, CAB2Δvpa0226+WT, or CAB2Δvpa0226+S/A for 2 hr, treated with 100 μg/mL gentamicin for 1 hr followed by treatment with 10 μg/mL gentamicin for the …
Figure 2 with 1 supplement
VPA0226 is a T2SS secreted protein.
(A) Expression (pellet) and secretion (supernatant) of VPA0226 from CAB2, CAB3, CAB4 and CAB2Δvpa0226 detected by immunoblotting with anti-VPA0226 antibody. LC: loading control from total bacterial …
Figure 2—figure supplement 1
Signal P-5 prediction of VPA0226.
VPA0226 Prediction: Signal peptide (Sec/SPI), (------) denotes the length of the signal peptide). Cleavage site between positions 20 and 21 (-----).
Figure 3 with 2 supplements
VPA0226 localizes to and fragments mitochondria in the host cell.
(A) Confocal micrographs of HeLa cells transiently transfected with empty vector, VPA0226- or VPA0226-H/A-sfGFPN1 (green) for 14 hr. Mitochondria were stained with anti-COXIV antibody (red) and DNA …
Figure 3—figure supplement 1
VPA0226 exclusively localizes to the mitochondria.
Confocal micrographs of HeLa cells transiently transfected with VPA0226-sfGFPN1 (green) for 24 hr. Early endosomes, late endosomes/lysosomes, and endoplasmic reticulum were stained with (A) …
Figure 3—figure supplement 2
Bacterially invaded cells display apoptotic signatures in a VPA0226-dependent manner.
(A) Confocal micrographs of HeLa cells infected with GFP-expressing (green) CAB2, CAB2Δvpa0226, CAB2Δvpa0226+WT, or CAB2Δvpa0226+S/A tested for release of Cytochrome C (Cyto C) from the mitochondria …
Figure 4 with 2 supplements
VPA0226 changes lipid profile in cells.
Quantification of polyunsaturated and saturated fatty acyl cholesteryl esters extracted from transfected HeLa cells (empty vector (EV), wildtype VPA0226-sfGFPN1 and VPA0226-H/A-sfGFPN1). Numbers are …
Figure 4—figure supplement 1
VPA0226 displays phospholipase 2 (PLA 2) activity.
(A) Increasing amounts of purified VPA0226WT or catalytically inactive S/A version were incubated with the fluorogenic PLA2-specific substrate BODIPY PC-A2 (EnzCheck Phospholipase A2 Assay kit, …
Figure 4—figure supplement 2
CAB2Δvpa0226+H/A mutant displays an egression defect similar to CAB2Δvpa0226 and CAB2Δvpa0226+S/A.
HeLa cells were infected with either CAB2, CAB2Δvpa0226, CAB2Δvpa0226+WT, CAB2Δvpa0226+S/A or CAB2Δvpa0226+H/A for 7 hr PGT. Host cell lysates were serially diluted and plated onto MMM agar plates …
Figure 5 with 1 supplement
VPA0226 modifies cholesterol content in the plasma membrane, upregulates cholesterol synthesis gene expression in cells and permeabilizes the host cell plasma membrane.
(A) qPCR analysis for relative expression of HMGCR, HMGCS and DHCR24, signature genes involved in cholesterol synthesis (SREBP2) pathway in HeLa cells transfected with empty vector (EV), wildtype …
Figure 5—figure supplement 1
Subcellular fractionation and sytox staining of HeLa cells infected with GFP-expressing (green) CAB2.
(A) Western blot analysis for subcellular fractionation to isolate plasma membrane fraction from HeLa cells. (B) Schematic representation of subcellular fractionation through differential …
