Cellular cholesterol licenses Legionella pneumophila intracellular replication in macrophages

Host membranes are inherently critical for niche homeostasis of vacuolar pathogens. Thus, intracellular bacteria frequently encode the capacity to regulate host lipogenesis as well as to modulate the lipid composition of host membranes. One membrane component that is often subverted by vacuolar bacteria is cholesterol – an abundant lipid that mammalian cells produce de novo at the endoplasmic reticulum (ER) or acquire exogenously from serum-derived lipoprotein carriers. Legionella pneumophila is an accidental human bacterial pathogen that infects and replicates within alveolar macrophages causing a severe atypical pneumonia known as Legionnaires’ disease. From within a unique ER-derived vacuole L. pneumophila promotes host lipogenesis and experimental evidence indicates that cholesterol production might be one facet of this response. Here we investigated the link between cellular cholesterol and L. pneumophila intracellular replication and discovered that disruption of cholesterol biosynthesis or cholesterol trafficking lowered bacterial replication in infected cells. These growth defects were rescued by addition of exogenous cholesterol. Conversely, bacterial growth within cholesterol-leaden macrophages was enhanced. Importantly, the growth benefit of cholesterol was observed strictly in cellular infections and L. pneumophila growth kinetics in axenic cultures did not change in the presence of cholesterol. Microscopy analyses indicate that cholesterol regulates a step in L. pneumophila intracellular lifecycle that occurs after bacteria begin to replicate within an established intracellular niche. Collectively, we provide experimental evidence that cellular cholesterol promotes L. pneumophila replication within a membrane bound organelle in infected macrophages

Novel Role for the AnxA1-Fpr2/ALX Signaling Axis as a Key Regulator of Platelet Function to Promote Resolution of Inflammation

Background: Ischemia reperfusion injury (I/RI) is a common complication of cardiovascular diseases. Resolution of detrimental I/RI-generated prothrombotic and proinflammatory responses is essential to restore homeostasis. Platelets play a crucial part in the integration of thrombosis and inflammation. Their role as participants in the resolution of thromboinflammation is underappreciated; therefore we used pharmacological and genetic approaches, coupled with murine and clinical samples, to uncover key concepts underlying this role. Methods: Middle cerebral artery occlusion with reperfusion was performed in wild-type or annexin A1 (AnxA1) knockout (AnxA1-/-) mice. Fluorescence intravital microscopy was used to visualize cellular trafficking and to monitor light/dye-induced thrombosis. The mice were treated with vehicle, AnxA1 (3.3 mg/kg), WRW4 (1.8 mg/kg), or all 3, and the effect of AnxA1 was determined in vivo and in vitro. Results: Intravital microscopy revealed heightened platelet adherence and aggregate formation post I/RI, which were further exacerbated in AnxA1-/- mice. AnxA1 administration regulated platelet function directly (eg, via reducing thromboxane B2 and modulating phosphatidylserine expression) to promote cerebral protection post-I/RI and act as an effective preventative strategy for stroke by reducing platelet activation, aggregate formation, and cerebral thrombosis, a prerequisite for ischemic stroke. To translate these findings into a clinical setting, we show that AnxA1 plasma levels are reduced in human and murine stroke and that AnxA1 is able to act on human platelets, suppressing classic thrombin-induced inside-out signaling events (eg, Akt activation, intracellular calcium release, and Ras-associated protein 1 [Rap1] expression) to decrease IIbβ3 activation without altering its surface expression. AnxA1 also selectively modifies cell surface determinants (eg, phosphatidylserine) to promote platelet phagocytosis by neutrophils, thereby driving active resolution. (n=5-13 mice/group or 7-10 humans/group.) Conclusions: AnxA1 affords protection by altering the platelet phenotype in cerebral I/RI from propathogenic to regulatory and reducing the propensity for platelets to aggregate and cause thrombosis by affecting integrin (IIbβ3) activation, a previously unknown phenomenon. Thus, our data reveal a novel multifaceted role for AnxA1 to act both as a therapeutic and a prophylactic drug via its ability to promote endogenous proresolving, antithromboinflammatory circuits in cerebral I/RI. Collectively, these results further advance our knowledge and understanding in the field of platelet and resolution biology.Fil: Senchenkova, Elena Y.. State University of Louisiana; Estados UnidosFil: Ansari, Junaid. State University of Louisiana; Estados UnidosFil: Becker, Felix. University Hospital Muenster; AlemaniaFil: Vital, Shantel A.. State University of Louisiana; Estados UnidosFil: Al-Yafeai, Zaki. State University of Louisiana; Estados UnidosFil: Sparkenbaugh, Erica M.. University North Carolina Chapel Hill; Estados UnidosFil: Pawlinski, Rafal. University North Carolina Chapel Hill; Estados UnidosFil: Stokes, Karen Y.. State University of Louisiana; Estados UnidosFil: Carroll, Jennifer L.. State University of Louisiana; Estados UnidosFil: Dragoi, Ana-Maria. State University of Louisiana; Estados UnidosFil: Qin, Cheng Xue. Baker Heart And Diabetes Institute; AustraliaFil: Ritchie, Rebecca H.. Baker Heart And Diabetes Institute; AustraliaFil: Sun, Hai. University Hospital Muenster; AlemaniaFil: Cuellar-Saenz, Hugo H.. State University of Louisiana; Estados UnidosFil: Rubinstein Guichon, Mara Roxana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Biomédicas; Argentina. Columbia University; Estados UnidosFil: Han, Yiping W.. Columbia University; Estados UnidosFil: Orr, A. Wayne. University Hospital Muenster; AlemaniaFil: Perretti, Mauro. Queen Mary University Of London; Reino UnidoFil: Granger, D. Neil. State University of Louisiana; Estados UnidosFil: Gavins, Felicity N.E.. State University of Louisiana; Estados Unido

The Shigella flexneri type 3 secretion system is required for tyrosine kinase-dependent protrusion resolution, and vacuole escape during bacterial dissemination.

Shigella flexneri is a human pathogen that triggers its own entry into intestinal cells and escapes primary vacuoles to gain access to the cytosolic compartment. As cytosolic and motile bacteria encounter the cell cortex, they spread from cell to cell through formation of membrane protrusions that resolve into secondary vacuoles in adjacent cells. Here, we examined the roles of the Type 3 Secretion System (T3SS) in S. flexneri dissemination in HT-29 intestinal cells infected with the serotype 2a strain 2457T. We generated a 2457T strain defective in the expression of MxiG, a central component of the T3SS needle apparatus. As expected, the ΔmxiG strain was severely affected in its ability to invade HT-29 cells, and expression of mxiG under the control of an arabinose inducible expression system (ΔmxiG/pmxiG) restored full infectivity. In this experimental system, removal of the inducer after the invasion steps (ΔmxiG/pmxiG (Ara withdrawal)) led to normal actin-based motility in the cytosol of HT-29 cells. However, the time spent in protrusions until vacuole formation was significantly increased. Moreover, the number of formed protrusions that failed to resolve into vacuoles was also increased. Accordingly, the ΔmxiG/pmxiG (Ara withdrawal) strain failed to trigger tyrosine phosphorylation in membrane protrusions, a signaling event that is required for the resolution of protrusions into vacuoles. Finally, the ΔmxiG/pmxiG (Ara withdrawal) strain failed to escape from the formed secondary vacuoles, as previously reported in non-intestinal cells. Thus, the T3SS system displays multiple roles in S. flexneri dissemination in intestinal cells, including the tyrosine kinase signaling-dependent resolution of membrane protrusions into secondary vacuoles, and the escape from the formed secondary vacuoles

MTOR-Driven Metabolic Reprogramming Regulates Legionella pneumophila Intracellular Niche Homeostasis.

Vacuolar bacterial pathogens are sheltered within unique membrane-bound organelles that expand over time to support bacterial replication. These compartments sequester bacterial molecules away from host cytosolic immunosurveillance pathways that induce antimicrobial responses. The mechanisms by which the human pulmonary pathogen Legionella pneumophila maintains niche homeostasis are poorly understood. We uncovered that the Legionella-containing vacuole (LCV) required a sustained supply of host lipids during expansion. Lipids shortage resulted in LCV rupture and initiation of a host cell death response, whereas excess of host lipids increased LCVs size and housing capacity. We found that lipids uptake from serum and de novo lipogenesis are distinct redundant supply mechanisms for membrane biogenesis in Legionella-infected macrophages. During infection, the metabolic checkpoint kinase Mechanistic Target of Rapamycin (MTOR) controlled lipogenesis through the Serum Response Element Binding Protein 1 and 2 (SREBP1/2) transcription factors. In Legionella-infected macrophages a host-driven response that required the Toll-like receptors (TLRs) adaptor protein Myeloid differentiation primary response gene 88 (Myd88) dampened MTOR signaling which in turn destabilized LCVs under serum starvation. Inactivation of the host MTOR-suppression pathway revealed that L. pneumophila sustained MTOR signaling throughout its intracellular infection cycle by a process that required the upstream regulator Phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) and one or more Dot/Icm effector proteins. Legionella-sustained MTOR signaling facilitated LCV expansion and inhibition of the PI3K-MTOR-SREPB1/2 axis through pharmacological or genetic interference or by activation of the host MTOR-suppression response destabilized expanding LCVs, which in turn triggered cell death of infected macrophages. Our work identified a host metabolic requirement for LCV homeostasis and demonstrated that L. pneumophila has evolved to manipulate MTOR-dependent lipogenesis for optimal intracellular replication

Quantification of <i>S. flexneri</i> dissemination in HT-29 cells.

<p>(A,B) Infection of HT-29 cells with CFP-expressing <i>S. flexneri</i>. (A) Representative images showing the size of infection foci 8 h post infection comparing wild-type 2457T to the Δ<i>mxiG</i> strain complemented with p<i>mxiG</i> with 1.0% arabinose removed after the initial 30 minutes of infection (Δ<i>mxiG/pmxiG</i> (Ara withdrawal)), and the Δ<i>mxiG</i> mutant complemented with p<i>mxiG</i> with 1.0% arabinose throughout infection (Δ<i>mxiG/pmxiG</i> 1.0% Ara). Green, <i>Shigella</i>; red, DNA. Scale bar, 200 µm. (B) Computer-assisted image analysis was used to quantify the size of the infection foci and the average focus size was determined. Individual points represent individual foci, red bars indicate the mean +/−SD. Statistical analysis; ****, p<0.0001, unpaired t-test.</p

Quantification of <i>S. flexneri</i> invasion of HT-29 cell monolayer.

<p>(A) Representative images (XY and XZ planes) of HT-29 cells expressing plasma membrane-targeted YFP at 4 h post infection with the wild type strain 2457T, the isogenic Δ<i>mxiG</i> strain and the complemented Δ<i>mxiG/pmxiG</i>. Bacteria (red) were stained with an anti-<i>Shigella</i> antibody. Arrows indicate the presence or absence of host cell membrane surrounding the bacteria. Scale bar, 2.5 µm. (B) Percent invasion of HT-29 cell monolayers with wild-type 2457T strain set to 100%. Values represent the mean +/−SD of three independent experiments. Statistical analysis; ** p = 0.0095, **** p<0.0001, unpaired t test.</p

Quantification of intracellular motility.

<p>(A and B) Infection of HT-29 cells with CFP-expressing wild type and Δ<i>mxiG/pmxiG</i> (Ara withdrawal) strains. (A) Graph showing the percent of bacteria with actin tail in the cytoplasm of HT-29 cells 2 h post infection. Points represent the percent of bacteria with actin tail per infected cell. Red bars indicate the mean +/- SD of three independent experiments. Statistical analysis: p = 0.73. (B) Graph showing the velocity of wild type and Δ<i>mxiG/pmxiG</i> (Ara withdrawal) bacteria in the cytoplasm of HT-29 cells 2 h post infection. Points represent the average velocity of individual bacteria. Red bars indicate the mean +/−SD of three independent experiments. Statistical analysis: p = 0.39, unpaired t test.</p

Dynamics of wild-type 2457T dissemination in HT-29 cells.

<p>(A–C) Time-lapse microscopy of plasma membrane-targeted YFP-expressing HT-29 cells infected with CFP-expressing wild-type strain 2757T. Yellow, plasma membrane; Cyan, <i>Shigella</i>. (A,B) Representative images showing the progression of a single bacterium over time. For each panel, the top image shows a low magnification image of infected cells and the bottom images show an enlargement of the tracked bacterium (merged bacterium and membrane channels, left; membrane channel only, right). (A) Successful progression of a bacterium (white arrows, top panel) from the primary cell cytoplasm (0′, Primary Cell), into a membrane protrusion (15′, Protrusion) that resolve into a secondary vacuole (50′, Vacuole) from which the bacterium escape and gains access to the cytoplasm of the adjacent cell (75′, Free bacteria). (B) Unsuccessful progression of a bacterium (white arrows, top panel) from the primary cell cytoplasm (0′, Primary Cell) into a membrane protrusion (10′, Protrusion) that retracted towards the primary infected cell (20′, Protrusion) and returned the bacterium to the cytosol of the primary infected cell (25′, Primary Cell). (C) Tracking analysis of 60 bacteria, which formed protrusions in 20 independent foci. All bacteria were tracked for at least 180 minutes and the progression of the dissemination process was depicted using the color key shown at the bottom of panel C. Primary cell, dark blue; Protrusion, light blue; Vacuole, yellow; Free bacteria in adjacent cell, red. Scale bars, 5 µm.</p

Dynamics of Δ<i>mxiG</i> dissemination in HT-29 cells.

<p>(A–C) Time-lapse microscopy of plasma membrane-targeted YFP-expressing HT-29 cells infected with the CFP-expressing Δ<i>mxiG/pmxiG</i> (Ara withdrawal) strain. Yellow, plasma membrane; Cyan, <i>Shigella</i>. (A,B) Representative images showing the progression of a single bacterium over time. For each panel, the top image shows a low magnification image of infected cells and the bottom images show an enlargement of the tracked bacterium (merged bacterium and membrane channels, left; membrane channel only, right). (A) Unsuccessful progression of a bacterium (white arrows, top panel) from the primary cell cytoplasm (0′, Primary Cell) into a membrane protrusion (10′, Protrusion) that resolved into a vacuole (30′, Vacuole) from which the pathogen did not escape (180′, vacuole). Note that the trapped bacterium divided into at least 5 bacteria. (B) Unsuccessful progression of a bacterium (white arrows, top panel) from the primary cell cytoplasm (0′, Primary Cell) into a membrane protrusion (15′, Protrusion) that retracted towards the primary infected cell (65′, Protrusion) and returned the pathogen to the cytosol of the primary infected cell (70′, Primary Cell). (C) Tracking analysis of 60 bacteria, which formed protrusions in 25 independent foci. All bacteria were tracked for at least 180 minutes and the progression of the dissemination process was depicted using the color key shown at the bottom of panel C. Primary cell, dark blue; Protrusion, light blue; Vacuole, yellow; Free bacteria in adjacent cell, red. Scale bars, 5 µm.</p

The T3SS is required for activation of tyrosine kinase signaling in protrusions.

<p>(A) Representative images of HT-29 cells expressing a YFP-membrane marker (yellow), infected with the CFP-expressing wild-type strain 2457T (cyan) and stained for phospho-tyrosine residues (red). (B) Representative images of HT-29 cells expressing membrane-targeted YFP (yellow), infected with the CFP-expressing Δ<i>mxiG/pmxiG</i> (Ara withdrawal) strain (cyan) and stained for phospho-tyrosine residues (red). Scale bar 5 µm. (C) Graph showing the percent of phospho-tyrosine positive and phospho-tyrosine negative protrusions for the wild-type and the Δ<i>mxiG/pmxiG</i> (Ara withdrawal) strains. Values indicate the mean +/SD of three independent experiments. Statistical analysis; p-Tyr positive p<0.0001, unpaired t test.</p