Journal: Science Advances

Article Title: Bacterial pathogens hijack host cell peroxisomes for replication vacuole expansion and integrity

doi: 10.1126/sciadv.adr8005

Figure Lengend Snippet: ( A ) RAW264.7 macrophage knockout (KO) cell lines depleted of PEX19 or PEX5 based on Western analysis of whole-cell lysates. ( B ) L. pneumophila growth is impaired in peroxisome-deficient cells. Cells in (A) were infected with the indicated L. pneumophila strains. Bacterial growth was quantified on the basis of recovered cfus from host cell lysates at 24 hours and normalized to the WT bacteria in WT host cells by the number of intracellular bacteria at 1 hpi. Data are the mean ± SD of three to four biological replicates consisting of three technical replications each. An asterisk indicates a Student’s t test P < 0.05 compared to WT bacteria in WT host cells.

Article Snippet: Equal amounts of protein were examined by Western analysis, probing with the target specific antibody, rabbit α-PEX5 (1:500) (Proteintech), rabbit α-GNPAT (1:1000) (Proteintech), or mouse α-AGPS (1:500) (Santa Cruz) overnight at 4°C, followed by HRP-conjugated α-rabbit IgG (1:5000) (Sigma-Aldrich) or HRP-conjugated α-mouse IgG (1:5000) (Sigma-Aldrich), as appropriate.

Techniques: Knock-Out, Western Blot, Infection, Bacteria

Journal: Science Advances

Article Title: Bacterial pathogens hijack host cell peroxisomes for replication vacuole expansion and integrity

doi: 10.1126/sciadv.adr8005

Figure Lengend Snippet: ( A ) LCVs in macrophages lacking functional peroxisomes exhibit compact architecture. Wild-type (WT) immortalized murine macrophages (RAW264.7 cells) lacking either PEX19 or PEX5 or cells lacking PEX5 that were exogenously supplemented with palmitate (PA) were infected with L. pneumophila for 12 hours, fixed, and visualized by fluorescence microscopy. ( B ) LCVs in (A) were scored as spread (the majority of bacteria aligned end to end or at various angles) or compact (the majority of bacteria bundled together with their sidewalls juxtaposed to one another, with no bacteria extending beyond the boundary of a spherical shape) based on the organization of the bacteria in three-dimensional space . ( C ) Peroxisome defects lead to increased numbers of ruptured LCVs. Infected cells as in (A) were stained for L. pneumophila ( Lp ) and Galectin-3 (Gal3). ( D ) LCVs in (C) were scored for colocalization with Gal3. ( E ) Disruption of peroxisome lipid metabolic pathways limits LCV expansion and causes LCV destabilization. WT human monocyte–derived macrophages (THP-1 cells) lacking PEX5, GNPAT, or AGPS (Fig. 5B) were infected with L. pneumophila for 12 hours, fixed, and visualized by fluorescence microscopy. ( F ) LCVs in (E) were scored as spread or compact, as described in (B). ( G ) Gal3 colocalization with LCVs in (E) was quantified. [(B), (C), (F), (G)] Data are the mean ± SD of three biological replicates consisting of three technical replications each, scoring 50 to 100 vacuoles per technical replicate. An asterisk indicates a Student’s t test P < 0.05 compared to WT, unless otherwise indicated. ns, not significant.

Article Snippet: Equal amounts of protein were examined by Western analysis, probing with the target specific antibody, rabbit α-PEX5 (1:500) (Proteintech), rabbit α-GNPAT (1:1000) (Proteintech), or mouse α-AGPS (1:500) (Santa Cruz) overnight at 4°C, followed by HRP-conjugated α-rabbit IgG (1:5000) (Sigma-Aldrich) or HRP-conjugated α-mouse IgG (1:5000) (Sigma-Aldrich), as appropriate.

Techniques: Functional Assay, Infection, Fluorescence, Microscopy, Bacteria, Staining, Disruption, Derivative Assay

Journal: Science Advances

Article Title: Bacterial pathogens hijack host cell peroxisomes for replication vacuole expansion and integrity

doi: 10.1126/sciadv.adr8005

Figure Lengend Snippet: ( A ) S . Typhimurium growth is reduced in peroxisome-deficient cells. Wild-type (WT), PEX19-, and PEX5-deficient murine macrophages (RAW264.7 cells) were infected with WT S . Typhimurium ( St ) or an avirulent phoP - mutant strain. Bacterial growth was measured on the basis of recovered cfus from host cell lysates at 21 hpi, normalized to the WT strain in WT host cells by the number of intracellular bacteria at 2 hpi. Data are the mean ± SD of three biological replicates consisting of three technical replications each. An asterisk indicates a Student’s t test P < 0.05 compared to WT bacteria in WT host cells. ( B ) Defects in peroxisome function lead to more compact S . Typhimurium vacuoles. WT RAW264.7 cells or cells lacking functional peroxisomes ( Pex5 −/− ) were infected for 20 hours with S . Typhimurium constitutively expressing mCherry fluorescent protein, fixed, stained and visualized by fluorescence microscopy (left panel). The area of the vacuoles, based on the contour of the bacteria, and the number of bacteria within the vacuole, based on fluorescence intensity, were measured and the concentration of bacteria per vacuole (area/bacteria) was determined (right panels). Data are the combined measurements of three biological replicates, scoring 100 vacuoles per replicate. An asterisk indicates a two-tailed, nonparametric unpaired t test with Mann-Whitney correction P < 0.0001 relative to WT cells. ( C ) Peroxisome dysfunction leads to the destabilization of Salmonella -containing vacuoles (SCVs). Cells in (B) were stained for Galectin-3 (Gal3) (left panel) and the percentage of SCVs colocalizing with Galectin-3 were quantified (right panel). Data are the mean ± SD of three biological replicates consisting of three technical replications each, scoring 100 vacuoles per technical replicate. An asterisk indicates a Student’s t test P < 0.05 compared to WT cells.

Article Snippet: Equal amounts of protein were examined by Western analysis, probing with the target specific antibody, rabbit α-PEX5 (1:500) (Proteintech), rabbit α-GNPAT (1:1000) (Proteintech), or mouse α-AGPS (1:500) (Santa Cruz) overnight at 4°C, followed by HRP-conjugated α-rabbit IgG (1:5000) (Sigma-Aldrich) or HRP-conjugated α-mouse IgG (1:5000) (Sigma-Aldrich), as appropriate.

Techniques: Infection, Mutagenesis, Bacteria, Functional Assay, Expressing, Staining, Fluorescence, Microscopy, Concentration Assay, Two Tailed Test, MANN-WHITNEY