<i>Salmonella</i> exploits the host endolysosomal tethering factor HOPS complex to promote its intravacuolar replication

<div><p><i>Salmonella enterica</i> serovar <i>typhimurium</i> extensively remodels the host late endocytic compartments to establish its vacuolar niche within the host cells conducive for its replication, also known as the <i>Salmonella</i>-containing vacuole (SCV). By maintaining a prolonged interaction with late endosomes and lysosomes of the host cells in the form of interconnected network of tubules (<i>Salmonella</i>-induced filaments or SIFs), <i>Salmonella</i> gains access to both membrane and fluid-phase cargo from these compartments. This is essential for maintaining SCV membrane integrity and for bacterial intravacuolar nutrition. Here, we have identified the multisubunit lysosomal tethering factor—HOPS (<u>HO</u>motypic fusion and <u>P</u>rotein <u>S</u>orting) complex as a crucial host factor facilitating delivery of late endosomal and lysosomal content to SCVs, providing membrane for SIF formation, and nutrients for intravacuolar bacterial replication. Accordingly, depletion of HOPS subunits significantly reduced the bacterial load in non-phagocytic and phagocytic cells as well as in a mouse model of <i>Salmonella</i> infection. We found that <i>Salmonella</i> effector SifA in complex with its binding partner; SKIP, interacts with HOPS subunit Vps39 and mediates recruitment of this tethering factor to SCV compartments. The lysosomal small GTPase Arl8b that binds to, and promotes membrane localization of Vps41 (and other HOPS subunits) was also required for HOPS recruitment to SCVs and SIFs. Our findings suggest that <i>Salmonella</i> recruits the host late endosomal and lysosomal membrane fusion machinery to its vacuolar niche for access to host membrane and nutrients, ensuring its intracellular survival and replication.</p></div

Descriptions of the strains of S. typhimurium and the plasmids used in this study.

<p>Descriptions of the strains of S. typhimurium and the plasmids used in this study.</p

Recruitment of HOPS subunit Vps41 to SCVs requires SifA-SKIP interaction.

<p><b>a and b)</b> Representative confocal micrographs of control siRNA- or SKIP siRNA-treated HeLa cells infected with <i>Salmonella</i>, followed by co-transtection with plasmids expressing GFP-Vps41 (green) and untagged-Arl8b. Cells were fixed at 10 hr p.i., and immunostained with antibodies to <i>Salmonella</i> (blue) and LAMP1 (red). Arrowheads in the insets depict localization of Vps41 around SCV membranes (marked by LAMP1). <b>c and d)</b> Representative confocal micrographs of HeLa cells infected with <i>Salmonella</i> strains <i>sifA/</i>pSifA (WT)-2HA (<b>c</b>) or <i>sifA/</i>pSifA (L130D)-2HA (<b>d</b>), followed by co-transfection with plasmids expressing GFP-Vps41 (green) and untagged-Arl8b. Cells were fixed 10 hr p.i., and immunostained with antibodies to <i>Salmonella</i> (blue) and LAMP1 (red). Arrowheads in the insets depict localization of Vps41 around SCV membranes (marked by LAMP1). <b>e and f)</b> Representative confocal micrographs of HeLa cells treated with SKIP siRNA, infected with DsRed-expressing <i>Salmonella</i>, followed by transfection with plasmids expressing HA-Vps41, untagged-Arl8b and vector (<b>e</b>) or siRNA-resistant FLAG-SKIP (rescue construct) (<b>f</b>). Cells were fixed 10 hr p.i., and immunostained with antibodies to HA tag (green) and LAMP1 (blue). Different panels represent a higher magnification of the boxed areas, indicating Vps41 localization around SCV membranes (marked by arrowheads). Bars: (main) 10 μm; (insets) 5 μm. <b>g)</b> Quantification of HA-Vps41-postive SCVs at 10 hr p.i. in indicated siRNA treated HeLa cells. Data represent mean ± S.D. of ~50 SCVs scored in each experiment for three independent experiments (n.s., not significant; ***, P < 0.001; ****, P < 0.0001; Student’s <i>t</i> test). <b>h)</b> Quantification of HA-Vps41-positive SCVs at 10 hr p.i. in HeLa cells infected with indicated <i>Salmonella</i> strains. Data represent mean ± S.D. of ~100 SCVs scored in each experiment for three independent experiments (****, P < 0.0001; Student’s <i>t</i> test).</p

Depletion of HOPS subunits does not alter Rab7 recruitment to SCV.

<p><b>a-f)</b> Representative confocal images of control-, Vps41-, or Vps39-siRNA treated HeLa cells, and subsequently transfected with GFP-Rab7 and infected with DsRed-expressing <i>Salmonella</i> (red). At different times p.i., cells were fixed and stained for LAMP1 (blue). Insets depict higher magnification of the boxed areas showing localization of Rab7 and LAMP1 around the SCVs. Bars: (main) 10 μm; (insets) 5 μm. <b>g)</b> Quantification of Rab7-positive SCVs in control-, Vps41- or Vps39-siRNA treated HeLa cells. Data represent mean ± S.D. over three independent experiments at the indicated time points where ~100 SCVs were counted in each experiment. <b>h and i)</b> Quantification of GFP-Rab7 intensity around the SCVs in control-, Vps41- or Vps39-siRNA treated cells over three independent experiments at the indicated time points p.i. where intensity profile of ~50 SCVs were quantified in each experiment. Data represent mean ± S.E.M. <b>j-m)</b> Representative immunogold EM images of control siRNA (<b>j</b> and <b>k</b>)- or Vps41 siRNA (<b>l</b> and <b>m</b>)-treated HeLa cells infected with <i>Salmonella</i> for 2 hr and transfected with HA-tagged Rab7 and fixed at 10 hr p.i. Cells were processed for immunogold labeling with anti-LAMP1 (10 nm) and anti-HA (15 nm) antibodies. Arrowheads indicate localization of Rab7 (red) and LAMP1 (black) around the SCVs. Bar: 500 nm.</p

Depletion of HOPS subunits delays but does not block SCV maturation.

<p><b>a-f)</b> Representative confocal micrographs of control siRNA-, Vps41 siRNA- or Vps39 siRNA-treated HeLa cells infected with DsRed-expressing <i>Salmonella</i> (red). At different time points p.i., cells were fixed and stained for early endosomes marker, EEA1 (green), and LAMP1 (blue). Insets depict higher magnification of the boxed areas showing localization of different markers on the SCVs. Intensity line scan profile of EEA1/LAMP1 across the width of a single SCV (indicated by an arrow in the boxed region) is shown below the individual image. Bars: (main) 10 μm; (insets) 5 μm. <b>g and h)</b> Quantification of percentage of infected cells displaying EEA1/LAMP1-accumulation around SCVs at the indicated time point p.i. Data represent mean ± S.D. for ~50 SCVs from three independent experiments (n.s., not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; Student’s <i>t</i> test).</p

Silencing of Vps41 abrogates docking of late endosomes and lysosomes at SCVs and impairs nutrient acquisition by auxotrophic strain of <i>Salmonella</i>.

<p><b>a-d)</b> Representative TEM images of control (<b>a</b> and <b>b</b>) and Vps41 (<b>c</b> and <b>d</b>) siRNA treated HeLa cells infected with <i>Salmonella</i> for 10 hr. Higher magnification of multiple SCVs (marked by yellow arrowheads) interacting with late endosomes (containing MVBs) and lysosomes (containing lamellar membrane sheets) in control siRNA treated cells are shown in the panels on the right. In Vps41 siRNA treated cells, white arrowheads depict the MVBs containing late endosomal compartments. Bar: 500 nm. <b>e</b>) Intracellular replication of wild-type (WT) <i>Salmonella</i> and a mutant strain auxotrophic for proline (<i>proC</i>) was determined at indicated times p.i. in control siRNA- or Vps41 siRNA-transfected HeLa cells. The fold change in intracellular proliferation was calculated as the ratio of CFU at 16 hr p.i./CFU at 2 hr p.i. To complement the intracellular proliferation of <i>proC Salmonella</i> strain, cell culture medium was supplemented with proline (0.8 mM). Shown are the means ± S.D. from three independent experiments (n.s., not significant; ***, P < 0.001; ****, P < 0.0001; Student’s <i>t</i> test).</p

Deletion of <i>Salmonella</i> effector SifA abrogates Vps41 recruitment to SCV membranes.

<p><b>a-f)</b> HeLa cells were infected with either DsRed expressing-wild-type (WT) strain of <i>Salmonella</i> (NCTC 12023 in (<b>a</b>) and SL1344 in (<b>c</b>)) or <i>sifA sseJ</i>, <i>sseF</i>, <i>sseG</i>, and <i>pipB2</i> strains, followed by transfection with HA tagged-Vps41. Cells were fixed at 10 hr p.i., and co-stained with anti-HA (green) and anti-LAMP1 (blue) antibodies. Different panels represent a higher magnification of the boxed areas. Bars: (main) 10 μm; (insets) 5 μm. <b>g and h)</b> Quantification of Vps41-positive SCVs in HeLa cells infected with different <i>Salmonella</i> strains (as labeled) and fixed at 10 hr p.i. Data represent mean ± S.D. from three independent experiments where ~100 SCVs were counted in each experiment (n.s., not significant; ****, P < 0.0001; Student’s <i>t</i> test).</p

SifA-dependent lysosome clustering requires both SKIP and Vps39.

<p><b>a-b)</b> Representative confocal micrographs of HeLa cells co-transfected with Myc-SifA and GFP-LAMP1 plasmids. The cells were fixed and co-stained using anti-Myc (blue) and anti-Vps18 (<b>a</b>, red) or anti-Vps41 (<b>b</b>, red) antibodies. Different panels represent a higher magnification of the boxed areas and HOPS subunits enrichment on clustered lysosomes is indicated by the arrowheads. <b>c-e</b>) HeLa cells treated with indicated siRNA were transfected with Myc-SifA expressing plasmid. The cells were fixed and co-stained using anti-Myc (green) and anti-LAMP1 (red) antibodies. Insets show higher magnification of the boxed areas clustered lysosomes induced by SifA expression, which is dependent upon Vps39 and SKIP expression. Bars: (main) 10 μm; (insets) 5 μm. <b>f)</b> Average size of LAMP1-positive compartments calculated in cells treated with indicated siRNA and transfected with Myc-SifA plasmid. Data represent mean ± S.D. of ~25–30 transfected per experiment over three independent experiments (**, P < 0.01; Student’s <i>t</i> test). <b>g)</b> Schematic depicting the molecular machinery required for SCV fusion with late endosomes and lysosomes. Multisubunit tethering factor-HOPS complex is a target for <i>Salmonella</i> effector SifA, which associates with its known binding partner-SKIP to recruit HOPS complex to SCV membranes, thereby enabling SCV fusion with Arl8b-positive lysosomes.</p