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D A Portnoy

Publications and source records attributed to D A Portnoy.

At least 19 recordsLinked to original sources
PubMed1998

Interaction of human Arp2/3 complex and the Listeria monocytogenes ActA protein in actin filament nucleation.

Actin filament assembly at the cell surface of the pathogenic bacterium Listeria monocytogenes requires the bacterial ActA surface protein and the host cell Arp2/3 complex. Purified Arp2/3 complex accelerated the nucleation of actin polymerization in vitro, but pure ActA had no effect. However, when combined, the Arp2/3 complex and ActA synergistically stimulated the nucleation of actin filaments. This mechanism of activating the host Arp2/3 complex at the L. monocytogenes surface may be similar to the strategy used by cells to control Arp2/3 complex activity and hence the spatial and temporal distribution of actin polymerization.

Actin-Related Protein 2↗
PubMed1998

Pathogenicity and immunogenicity of a Listeria monocytogenes strain that requires D-alanine for growth.

Listeria monocytogenes is an intracellular bacterial pathogen that elicits a strong cellular immune response following infection and therefore has potential use as a vaccine vector. However, while infections by L. monocytogenes are fairly rare and can readily be controlled by a number of antibiotics, the organism can nevertheless cause meningitis and death, particularly in immunocompromised or pregnant patients. We therefore have endeavored to isolate a highly attenuated strain of this organism for use as a vaccine vector. D-Alanine is required for the synthesis of the mucopeptide component of the cell walls of virtually all bacteria and is found almost exclusively in the microbial world. We have found in L. monocytogenes two genes that control the synthesis of this compound, an alanine racemase gene (dal) and a D-amino acid aminotransferase gene (dat). By inactivating both genes, we produced an organism that could be grown in the laboratory when supplemented with D-alanine but was unable to grow outside the laboratory, particularly in the cytoplasm of eukaryotic host cells, the natural habitat of this organism during infection. In mice, the double-mutant strain was completely attenuated. Nevertheless, it showed the ability, particularly under conditions of transient suppression of the mutant phenotype, to induce cytotoxic T-lymphocyte responses and to generate protective immunity against lethal challenge by wild-type L. monocytogenes equivalent to that induced by the wild-type organism.

Alanine↗
PubMed1997

Proteolytic pathways of activation and degradation of a bacterial phospholipase C during intracellular infection by Listeria monocytogenes.

Listeria monocytogenes is a facultative intracellular bacterial pathogen that spreads cell to cell without exposure to the extracellular environment. Bacterial cell-to-cell spread is mediated in part by two secreted bacterial phospholipases C (PLC), a broad spectrum PLC (PC-PLC) and a phosphatidylinositolspecific PLC (PI-PLC). PI-PLC is secreted in an active state, whereas PC-PLC is secreted as an inactive proenzyme (proPC-PLC) whose activation is mediated in vitro by an L. monocytogenes metalloprotease (Mpl). Analysis of PI-PLC, PC-PLC, and Mpl single and double mutants revealed that Mpl also plays a role in the spread of an infection, but suggested that proPC-PLC has an Mpl-independent activation pathway. Using biochemical and microscopic approaches, we describe three intracellular proteolytic pathways regulating PCPLC activity. Initially, proPC-PLC secreted in the cytosol of infected cells was rapidly degraded in a proteasome-dependent manner. Later during infection, PCPLC colocalized with bacteria in lysosome-associated membrane protein 1-positive vacuoles. Activation of proPC-PLC in vacuoles was mediated by Mpl and an Mpl-independent pathway, the latter being sensitive to inhibitors of cysteine proteases. Lastly, proPC-PLC activation by either pathway was sensitive to bafilomycin A1, a specific inhibitor of vacuolar ATPase, suggesting that activation was dependent on acidification of the vacuolar compartment. These results are consistent with a model in which proPC-PLC activation is compartment specific and controlled by a combination of bacterial and host factors.

Animals↗
PubMed1997

Influenza-specific immunity induced by recombinant Listeria monocytogenes vaccines.

In this study, we evaluate two Listeria monocytogenes strains that express influenza nucleoprotein (NP) sequences for their ability to protect against challenge with influenza-virus. The construction of one strain, which expresses only the Kd restricted NP epitope (NP 147-155), is described in this study; the other strain, which expresses the full NP sequence in the form of a fusion protein, has been described previously. The ability of the two strains to present the Kd restricted NP epitope in vitro and induce NP-specific CTL in vivo is also described. Mice immunized by the intravenous route with either strain cleared a subsequent (3 weeks post-immunization) influenza virus infection more rapidly as indicated by reduced virus titers in the lungs 5 days after challenge. Efficacy of both recombinant L. monocytogenes strains as vaccines in this system was equivalent and equal to that of recombinant vaccinia expressing NP.

Animals↗
PubMed1997

How the Listeria monocytogenes ActA protein converts actin polymerization into a motile force.

The ActA protein is an essential determinant of pathogenicity that is responsible for the actin-based motility of Listeria monocytogenes in mammalian cells and cell-free extracts. ActA appears to control at least four functions that collectively lead to actin-based motility: (1) initiation of actin polymerization, (2) polarization of ActA function, (3) transformation of actin polymerization into a motile force and (4) acceleration of movement mediated by the host protein profilin.

Actins↗
PubMed1996

Delivery of macromolecules into cytosol using liposomes containing hemolysin from Listeria monocytogenes.

The cytosolic space of cells is an important but relatively inaccessible target for the delivery of therapeutic macromolecules. Here we describe the efficient delivery of macromolecules into the cytosolic space of macrophages from liposomes that contain listeriolysin O (LLO), the hemolytic protein of Listeria monocytogenes that normally mediates bacterial passage from phagosomes into cytosol. LLO was purified and encapsulated inside pH-sensitive liposomes, along with other molecules to be delivered. When internalized by bone marrow-derived macrophages, these liposomes rapidly released encapsulated fluorescent dye, first into endosomes and then into the cytosol, without measurably harming the cells. Furthermore, these liposomes efficiently delivered encapsulated ovalbumin to the cytosolic pathway of antigen processing and presentation, as measured by the major histocompatibility complex (MHC) class I-restricted presentation of peptides derived from ovalbumin. Delivery was significantly better than that obtained by other currently available liposome formulations. LLO-containing liposomes should therefore provide an efficient vehicle for delivery of antigens or therapeutic molecules in vivo.

Animals↗
PubMed1996

Conversion of an extracellular cytolysin into a phagosome-specific lysin which supports the growth of an intracellular pathogen.

Listeria monocytogenes is a facultative intracellular pathogen which secretes a pore-forming cytolysin, listeriolysin O (LLO), necessary for intracellular growth. Clostridium perfringens is an extracellular pathogen which secretes a related cytolysin, perfringolysin O (PFO). When PFO is secreted by intracellular L. monocytogenes, it is toxic to the infected host cell. PFO-mediated toxicity renders the infected host cell permeable to gentamicin and leads to the death of the intracellular bacteria. In this study, we selected for L. monocytogenes mutants in which PFO supported the intracellular growth of L. monocytogenes. Six independent mutants were isolated, each containing a single amino acid change within the PFO protein. Three classes of PFO mutations were identified, all capable of mediating lysis of the vacuole but without a toxic effect upon the infected host cell. The first class had a severe defect in haemolytic activity. The second class had a change in the pH optimum of PFO. The third class had nearly wild-type levels of haemolytic activity, but had a decrease in protein half-life in the host-cell cytosol. Acquisition of single amino acid changes in PFO were sufficient to convert an extracellular cytolysin into a vacuole-specific lysin which mediated growth of L. monocytogenes in cultured cells.

Bacterial Proteins↗
PubMed1996

The tandem repeat domain in the Listeria monocytogenes ActA protein controls the rate of actin-based motility, the percentage of moving bacteria, and the localization of vasodilator-stimulated phosphoprotein and profilin.

The ActA protein is responsible for the actin-based movement of Listeria monocytogenes in the cytosol of eukaryotic cells. Analysis of mutants in which we varied the number of proline-rich repeats (PRR; consensus sequence DFPPPPTDEEL) revealed a linear relationship between the number of PRRs and the rate of movement, with each repeat contributing approximately 2-3 microns/min. Mutants lacking all functional PRRs (generated by deletion or point mutation) moved at rates 30% of wild-type. Indirect immunofluorescence indicated that the PRRs were directly responsible for binding of vasodilator-stimulated phosphoprotein (VASP) and for the localization of profilin at the bacterial surface. The long repeats, which are interdigitated between the PRRs, increased the frequency with which actin-based motility occurred by a mechanism independent of the PRRs, VASP, and profilin. Lastly, a mutant which expressed low levels of ActA exhibited a phenotype indicative of a threshold; there was a very low percentage of moving bacteria, but when movement did occur, it was at wild-type rates. These results indicate that the ActA protein directs at least three separable events: (1) initiation of actin polymerization that is independent of the repeat region; (2) initiation of movement dependent on the long repeats and the amount of ActA; and (3) movement rate dependent on the PRRs.

Actins↗
PubMed1995

Asymmetric distribution of the Listeria monocytogenes ActA protein is required and sufficient to direct actin-based motility.

Listeria monocytogenes is a Gram-positive facultative intracytoplasmic bacterial pathogen that exhibits rapid actin-based motility in eukaryotic cells and in cell-free cytoplasmic extracts. The protein product of the actA gene is required for bacterial movement and is normally expressed in a polarized fashion on the bacterial surface. Here we demonstrate that the ActA protein is sufficient to direct motility in the absence of other L. monocytogenes gene products, and that polarized localization of the protein is required for efficient unidirectional movement. We have engineered a fusion protein combining ActA with the C-terminal domain of the LytA protein of Streptococcus pneumoniae, which mediates high-affinity binding to DEAE-cellulose and to choline moieties present in the S. pneumoniae cell wall. DEAE-cellulose fragments or S. pneumoniae coated uniformly with the ActA/LytA fusion protein nucleate actin filament growth in cytoplasmic extracts, but do not move efficiently. However, when ActA/LytA-coated S. pneumoniae is grown to polarize the distribution of the fusion protein, the bacteria exhibit unidirectional actin-based movement similar to the normal movement of L. monocytogenes.

Actins↗
PubMed1995

The two distinct phospholipases C of Listeria monocytogenes have overlapping roles in escape from a vacuole and cell-to-cell spread.

Listeria monocytogenes secretes two distinct phospholipases C, a phosphatidylinositol-specific phospholipase C (PI-PLC) and a broad-range phospholipase C (PC-PLC). In this study, single in-frame deletion mutants with mutations in each PLC and a double mutant lacking both PLCs were characterized with regard to virulence in mice, escape from a primary vacuole, and cell-to-cell spread in cell culture. The mutant lacking PI-PLC, previously shown to be twofold less virulent than the wild type in mice, had a minor defect in escape from a primary vacuole but was not notably affected in cell-to-cell spread. The mutant lacking PC-PLC was 20-fold less virulent in mice and was defective in cell-to-cell spread but had no measurable defect in escape from a primary vacuole. The mutant lacking both PLCs was 500-fold less virulent in mice and was severely diminished in its ability to escape from the primary vacuole and to spread cell to cell. Cellular levels of diacylglycerol and ceramide, products of PLC activity, accumulated beginning 3 to 4 h after infection of cells with wild-type bacteria. The bacterial PLCs were partially responsible for this activity, since cells infected with the mutant lacking both PLCs had a reduced increase in diacylglycerol and no increase in ceramide. Elevation of diacylglycerol in the absence of bacterial PLCs indicated that host cell phospholipase(s) was activated during infection. The results of this study were consistent with the two bacterial PLCs having overlapping functions throughout the course of intracellular infection. Furthermore, the PC-PLC, and possibly PI-PLC, appeared to be enzymatically active intracellularly.

Animals↗
PubMed1995

The broad-range phospholipase C and a metalloprotease mediate listeriolysin O-independent escape of Listeria monocytogenes from a primary vacuole in human epithelial cells.

Intracellular growth of Listeria monocytogenes begins after lysis of the primary vacuole formed upon bacterial entry into a host cell. Listeriolysin O (LLO), a pore-forming hemolysin encoded by hly, is essential for vacuolar lysis in most cell types. However, in human epithelial cells, LLO- mutants are capable of growth, suggesting that gene products other than LLO are capable of mediating escape from a vacuole. In this study, we investigated the role of other bacterial gene products in lysis of the primary vacuole in the human epithelial cell line Henle 407. Double internal in-frame deletion mutants were constructed by introducing a mutated hly allele into strains harboring deletions in either of the phospholipase C (PLC)-encoding genes or a metalloprotease-encoding gene. Bacterial escape from the primary vacuole, intracellular growth, and cell-to-cell spread were evaluated in Henle 407 cells. The results indicated that, in the absence of LLO, the broad-range PLC and the metalloprotease were both required for lysis of the primary vacuole in Henle 407 cells. Although phosphatidylinositol-specific PLC was not required, the efficiency of escape was reduced in an LLO phosphatidylinositol-specific PLC double mutant. These observations suggest that the relative importance of LLO, the phospholipases, and the metalloprotease may vary in different cell types or in cells from different species. In addition, these studies provide insight into the mechanisms of action of virulence determinants involved in the lysis of vacuolar membranes.

Bacterial Toxins↗
PubMed1994

Delivery of a viral antigen to the class I processing and presentation pathway by Listeria monocytogenes.

Listeria monocytogenes is a facultative intracellular pathogen that grows in the cytoplasm of infected host cells. We examined the capacity of L. monocytogenes to introduce influenza nucleoprotein (NP) into the class I pathway of antigen presentation both in vitro and in vivo. Recombinant L. monocytogenes secreting a fusion of listeriolysin O and NP (LLO-NP) targeted infected cells for lysis by NP-specific class I-restricted cytotoxic T cells. Antigen presentation occurred in the context of three different class I haplotypes in vitro. A hemolysin-negative L. monocytogenes strain expressing LLO-NP was able to present in a class II-restricted manner. However, it failed to target infected cells for lysis by CD8+ T cells, indicating that hemolysin-dependent bacterial escape from the vacuole is necessary for class I presentation in vitro. Immunization of mice with a recombinant L. monocytogenes strain that stably expressed and secreted LLO-NP induced NP-specific CD8+ cytotoxic T lymphocytes. These studies have implications for the use of L. monocytogenes to deliver potentially any antigen to the class I pathway in vivo.

Animals↗
PubMed1994

Involvement of profilin in the actin-based motility of L. monocytogenes in cells and in cell-free extracts.

Within hours of Listeria monocytogenes infection, host cell actin filaments form a dense cloud around the intracytoplasmic bacteria and then rearrange to form a polarized comet tail that is associated with moving bacteria. We have devised a cell-free extract system capable of faithfully reconstituting L. monocytogenes motility, and we have used this system to demonstrate that profilin, a host actin monomer-binding protein, is necessary for bacterial actin-based motility. We find that extracts from which profilin has been depleted do not support comet tail formation or bacterial motility. In extracts and host cells, profilin is localized to the back half of the surface of motile L. monocytogenes, the site of actin filament assembly in the tail. This association is not observed with L. monocytogenes mutants that do not express the ActA protein, a bacterial gene product necessary for motility and virulence. Profilin also fails to bind L. monocytogenes grown outside of host cytoplasm, suggesting that at least one other host cell factor is required for this association.

Actins↗