Stephen Vadia

The Pore-Forming Toxin Listeriolysin O Mediates a Novel Entry Pathway of L. monocytogenes into Human Hepatocytes

Intracellular pathogens have evolved diverse strategies to invade and survive within host cells. Among the most studied facultative intracellular pathogens, Listeria monocytogenes is known to express two invasins-InlA and InlB-that induce bacterial internalization into nonphagocytic cells. The pore-forming toxin listeriolysin O (LLO) facilitates bacterial escape from the internalization vesicle into the cytoplasm, where bacteria divide and undergo cell-to-cell spreading via actin-based motility. In the present study we demonstrate that in addition to InlA and InlB, LLO is required for efficient internalization of L. monocytogenes into human hepatocytes (HepG2). Surprisingly, LLO is an invasion factor sufficient to induce the internalization of noninvasive Listeria innocua or polystyrene beads into host cells in a dose-dependent fashion and at the concentrations produced by L. monocytogenes. To elucidate the mechanisms underlying LLO-induced bacterial entry, we constructed novel LLO derivatives locked at different stages of the toxin assembly on host membranes. We found that LLO-induced bacterial or bead entry only occurs upon LLO pore formation. Scanning electron and fluorescence microscopy studies show that LLO-coated beads stimulate the formation of membrane extensions that ingest the beads into an early endosomal compartment. This LLO-induced internalization pathway is dynamin-and F-actin-dependent, and clathrin-independent. Interestingly, further linking pore formation to bacteria/bead uptake, LLO induces F-actin polymerization in a tyrosine kinase-and pore-dependent fashion. In conclusion, we demonstrate for the first time that a bacterial pathogen perforates the host cell plasma membrane as a strategy to activate the endocytic machinery and gain entry into the host cell.

Human trophoblasts confer resistance to viruses implicated in perinatal infection

OBJECTIVE Primary human trophoblasts were previously shown to be resistant to viral infection, and able to confer this resistance to nontrophoblast cells. Can trophoblasts protect nontrophoblastic cells from infection by viruses or other intracellular pathogens that are implicated in perinatal infection? STUDY DESIGN Isolated primary term human trophoblasts were cultured for 48-72 hours. Diverse nonplacental human cell lines (U2OS, human foreskin fibroblast, TZM-bl, MeWo, and Caco-2) were preexposed to either trophoblast conditioned medium, nonconditioned medium, or miR-517-3p for 24 hours. Cells were infected with several viral and nonviral pathogens known to be associated with perinatal infections. Cellular infection was defined and quantified by plaque assays, luciferase assays, microscopy, and/or colonization assays. Differences in infection were assessed by Student t test or analysis of variance with Bonferroni correction. RESULTS Infection by rubella and other togaviruses, human immunodeficiency virus-1, and varicella zoster was attenuated in cells preexposed to trophoblast-conditioned medium (P < .05), and a partial effect by the chromosome 19 microRNA miR-517-3p on specific pathogens. The conditioned medium had no effect on infection by Toxoplasma gondii or Listeria monocytogenes. CONCLUSION Our findings indicate that medium conditioned by primary human trophoblasts attenuates viral infection in nontrophoblastic cells. Our data point to a trophoblast-specific antiviral effect that may be exploited therapeutically.

Growth rate and cell size: a re-examination of the growth law

Research into the mechanisms regulating bacterial cell size has its origins in a single paper published over 50 years ago. In it Schaechter and colleagues made the observation that the chemical composition and size of a bacterial cell is a function of growth rate, independent of the medium used to achieve that growth rate, a finding that is colloquially referred to as the growth law. Recent findings hint at unforeseen complexity in the growth law, and suggest that nutrients rather than growth rate are the primary arbiter of size. The emerging picture suggests that size is a complex, multifactorial phenomenon mediated through the varied impacts of central carbon metabolism on cell cycle progression and biosynthetic capacity.

The Pore-Forming Toxin Listeriolysin O Is Degraded by Neutrophil Metalloproteinase-8 and Fails To Mediate Listeria monocytogenes Intracellular Survival in Neutrophils

The pore-forming toxin listeriolysin O (LLO) is a major virulence factor secreted by the facultative intracellular pathogen Listeria monocytogenes. This toxin facilitates L. monocytogenes intracellular survival in macrophages and diverse nonphagocytic cells by disrupting the internalization vesicle, releasing the bacterium into its replicative niche, the cytosol. Neutrophils are innate immune cells that play an important role in the control of infections, yet it was unknown if LLO could confer a survival advantage to L. monocytogenes in neutrophils. We report that LLO can enhance the phagocytic efficiency of human neutrophils and is unable to protect L. monocytogenes from intracellular killing. To explain the absence of L. monocytogenes survival in neutrophils, we hypothesized that neutrophil degranulation leads to the release of LLO-neutralizing molecules in the forming phagosome. In support of this, L. monocytogenes is a potent inducer of neutrophil degranulation, since its virulence factors, such as LLO, facilitate granule exocytosis. Within the first few minutes of interaction with L. monocytogenes, granules can fuse with the plasma membrane at the bacterial interaction site before closure of the phagosome. Furthermore, granule products directly degrade LLO, irreversibly inhibiting its activity. The matrix metalloproteinase-8, stored in secondary granules, was identified as an endoprotease that degrades LLO, and blocking neutrophil proteases increased L. monocytogenes intracellular survival. In conclusion, we propose that LLO degradation by matrix metalloproteinase-8 during phagocytosis protects neutrophil membranes from perforation and contributes to maintaining L. monocytogenes in a bactericidal phagosome from which it cannot escape.

Fluxes of Ca2+ and K+ Are Required for the Listeriolysin O-Dependent Internalization Pathway of Listeria monocytogenes

ABSTRACT Listeria monocytogenes is responsible for the life-threatening food-borne disease listeriosis. This disease mainly affects elderly and immunocompromised individuals, causing bacteremia and meningoencephalitis. In pregnant women, L. monocytogenes infection leads to abortion and severe infection of the fetus or newborn. The L. monocytogenes intracellular life cycle is critical for pathogenesis. Previous studies have established that the major virulence factor of L. monocytogenes, the pore-forming toxin listeriolysin O (LLO), is sufficient to induce L. monocytogenes internalization into human epithelial cell lines. This internalization pathway strictly requires the formation of LLO pores in the plasma membrane and can be stimulated by the heterologous pore-forming toxin pneumolysin, suggesting that LLO acts nonspecifically by forming transmembrane pores. The present work tested the hypothesis that Ca2+ and K+ fluxes subsequent to perforation by LLO control L. monocytogenes internalization. We report that L. monocytogenes perforates the host cell plasma membrane in an LLO-dependent fashion at the early stage of invasion. In response to perforation, host cells undergo Ca2+-dependent but K+-independent resealing of their plasma membrane. In contrast to the plasma membrane resealing process, LLO-induced L. monocytogenes internalization requires both Ca2+ and K+ fluxes. Further linking ion fluxes to bacterial internalization, treating cells with a combination of Ca2+ and K+ ionophores but not with individual ionophores is sufficient to induce efficient internalization of large cargoes, such as 1-μm polystyrene beads and bacteria. We propose that LLO-induced L. monocytogenes internalization requires a Ca2+- and K+-dependent internalization pathway that is mechanistically distinct from the process of plasma membrane resealing.

CXCR3 expression defines a novel subset of innate CD8+ T cells that enhance immunity against bacterial infection and cancer upon stimulation with IL-15

Innate CD8+ T cells are a heterogeneous population with developmental pathways distinct from conventional CD8+ T cells. However, their biology, classification, and functions remain incompletely understood. We recently demonstrated the existence of a novel population of chemokine (C‐X‐C motif) receptor 3 (CXCR3)‐positive innate CD8+ T cells. Here, we investigated the functional properties of this subset and identified effector molecules and pathways which mediate their function. Adoptive transfer of IL‐15 activated CXCR3+ innate CD8+ T cells conferred increased protection against Listeria monocytogenes infection in susceptible IFN‐γ–/– mice compared with similarly activated CXCR3– subset. This was associated with enhanced proliferation and IFN‐γ production in CXCR3+ cells. Further, CXCR3+ innate cells showed enhanced cytotoxicity against a tumor cell line in vitro. In depth analysis of the CXCR3+ subset showed increased gene expression of Ccl5, Klrc1, CtsW, GP49a, IL‐2Rβ, Atp5e, and Ly6c but reduced IFN‐γR2 and Art2b. Ingenuity pathway analysis revealed an up‐regulation of genes associated with T‐cell activation, proliferation, cytotoxicity, and translational initiation in CXCR3+ populations. Our results demonstrate that CXCR3 expression in innate CD8+ T cells defines a subset with enhanced cytotoxic potential and protective antibacterial immune functions. Immunotherapeutic approaches against infectious disease and cancer could utilize CXCR3+ innate CD8+ T‐cell populations as novel clinical intervention strategies.—Oghumu, S., Terrazas, C. A., Varikuti, S., Kimble, J., Vadia, S., Yu, L., Seveau, S., Satoskar, A. R., CXCR3 expression defines a novel subset of innate CD8+ T cells that enhance immunity against bacterial infection and cancer upon stimulation with IL‐15. FASEB J. 29, 1019–1028 (2015). www.fasebj.org

Host cell perforation by listeriolysin O (LLO) activates a Ca2+-dependent cPKC/Rac1/Arp2/3 signaling pathway that promotes Listeria monocytogenes internalization independently of membrane resealing

Pathogen-induced host plasma membrane damage is a recently recognized mechanism used by pathogens to promote their entry into host cells. We identified key transducers activated upon host cell perforation by the pore-forming toxin LLO to promote Listeria entry. This pathway is distinct from the pathway that reseals the toxin-perforated cell.

Relative Roles of Listeriolysin O, InlA, and InlB in Listeria monocytogenes Uptake by Host Cells

Listeria monocytogenes is a facultative intracellular pathogen that infects a wide variety of cells, causing the life-threatening disease listeriosis. L. monocytogenes virulence factors include two surface invasins, InlA and InlB, known to promote bacterial uptake by host cells, and the secreted pore-forming toxin listeriolysin O (LLO), which disrupts the phagosome to allow bacterial proliferation in the cytosol. ABSTRACT Listeria monocytogenes is a facultative intracellular pathogen that infects a wide variety of cells, causing the life-threatening disease listeriosis. L. monocytogenes virulence factors include two surface invasins, InlA and InlB, known to promote bacterial uptake by host cells, and the secreted pore-forming toxin listeriolysin O (LLO), which disrupts the phagosome to allow bacterial proliferation in the cytosol. In addition, plasma membrane perforation by LLO has been shown to facilitate L. monocytogenes internalization into epithelial cells. In this work, we tested the host cell range and importance of LLO-mediated L. monocytogenes internalization relative to the canonical invasins, InlA and InlB. We measured the efficiencies of L. monocytogenes association with and internalization into several human cell types (hepatocytes, cytotrophoblasts, and endothelial cells) using wild-type bacteria and isogenic single, double, and triple deletion mutants for the genes encoding InlA, InlB and LLO. No role for InlB was detected in any tested cells unless the InlB expression level was substantially enhanced, which was achieved by introducing a mutation (prfA*) in the gene encoding the transcription factor PrfA. In contrast, InlA and LLO were the most critical invasion factors, although they act in a different manner and in a cell-type-dependent fashion. As expected, InlA facilitates both bacterial attachment and internalization in cells that express its receptor, E-cadherin. LLO promotes L. monocytogenes internalization into hepatocytes, but not into cytotrophoblasts and endothelial cells. Finally, LLO and InlA cooperate to increase the efficiency of host cell invasion by L. monocytogenes.

Bacterial Size: Can’t Escape the Long Arm of the Law

Environmental perturbations can lead to changes in bacterial cell size that are not predicted by current models. A recent study presents a model that accurately predicts cell size under a variety of environmental conditions, from just a few measurable variables.

Flux through lipid synthesis dictates bacterial cell size.

Nutrients—and by extension biosynthetic capacity—positively impact cell size in organisms throughout the tree of life. In bacteria, cell size is reduced three-fold in response to nutrient starvation or accumulation of the alarmone ppGpp, a global inhibitor of biosynthesis. However, whether biosynthetic capacity as a whole determines cell size or if particular anabolic pathways are more important than others remains an open question. Utilizing a top-down approach, here we identify flux through lipid synthesis as the primary biosynthetic determinant of Escherichia coli cell size. Altering flux through lipid synthesis recapitulated the impact of altering nutrients on cell size and morphology, while defects in other biosynthetic pathways either did not impact size or altered size in a lipid-dependent manner. Together our findings support a model in which lipid availability dictates cell envelope capacity and ppGpp functions as a linchpin linking surface area expansion with cytoplasmic volume to maintain cellular integrity.