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.     

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.     

The 46-kDa mannose 6-phosphate receptor contains multiple binding sites for clathrin adaptors

The two known mannose 6-phosphate receptors (MPR46 and MPR300) both mediate the transport of Man-6-P-containing lysosomal proteins to lysosomes. However, the MPRs cannot be detected in lysosomes, instead they recycle between the plasma membrane and endosomes and between endosomes and the trans-Golgi network. Both, endocytosis from the plasma membrane and budding of transport vesicles from the trans-Golgi network involves the interaction of the receptor with the clathrin-coated vesicles-associated protein complexes AP1 and AP2. We have analyzed this interaction between the Golgi-restricted AP1 complex and the plasma membrane-restricted AP2 complex with the MPR46 tail in vitro by using a biosensor. AP1 and AP2 both bind to and dissociate from the MPR46 tail with similar kinetics. Using synthetic peptides corresponding to different MPR receptor tail regions in inhibition and binding studies, a common high affinity binding site for AP1 and AP2 and two separate high affinity binding sites for AP1 and AP2, respectively, were identified.     

The role of actin binding proteins in epithelial morphogenesis: models based upon Listeria movement

We summarize recent findings on the organization of the protein actin in eucaryotic cells. In particular we focus on how actin can be used to generate a vectorial force that is required for cell movement. These forces arise from protein molecules that recruit actin to the plasma membrane in such a manner that actin filaments extend outward from the cell body. This type of actin dependent force generation has been described in a nucleation-release model, which is one of several models currently being tested to explain actin dependent cell movement. Data in support of this model has arisen unexpectedly from studies of an intracellular bacteria, Listeria monocytogenes. This bacteria uses actin to propel itself during infection of eucaryotic cells. By studying Listeria movement, the roles of several eucaryotic actin interacting proteins have been identified. One of these is zyxin, a human protein that shares important structural and possibly functional properties with ActA, an actin dependent force generating protein of Listeria. We intend to test the function of these and other actin interacting proteins in a simplified system that should facilitate precise measurement of their properties of force generation in vitro.     

Efficient induction of cytotoxic CD8+ T cells against exogenous proteins: establishment and characterization of a T cell line specific for the membrane protein ActA of Listeria monocytogenes

The property of listeriolysin (LLO) to introduce soluble passenger proteins into the cytosol of antigen-presenting cells allows the induction of CD8+ cytotoxic T cells against such antigens. To overcome the potential problem of presentation of the immunodominant epitope LL091-99 by H-2Kd, a variant LLO92A was established in which Tyr 92 was replaced by Ala. Immunization of BALB/c mice with purified LLO92A failed to stimulate cytotoxic T cells specific for either the epitope LLO91-99 or for any other LLO-derived peptide. Injection of mixtures of purified LLO92A and soluble nucleoprotein (NP) of influenza virus into mice resulted in a strong cytotoxic T cell response exclusively directed against NP. The LLO92A variant was successfully used to generate, propagate and characterize a CD8 T cell line specific for the membrane-bound virulence factor ActA of Listeria monocytogenes. Interestingly, wildtype ActA bound to the surface of live L. monocytogenes was not presented by MHC class I molecules to the CD8+ T cell line.     

Gelsolin, a protein that caps the barbed ends and severs actin filaments, enhances the actin-based motility of Listeria monocytogenes in host cells

The actin-based motility of Listeria monocytogenes requires the addition of actin monomers to the barbed or plus ends of actin filaments. Immunofluorescence micrographs have demonstrated that gelsolin, a protein that both caps barbed ends and severs actin filaments, is concentrated directly behind motile bacteria at the junction between the actin filament rocket tail and the bacterium. In contrast, CapG, a protein that strictly caps actin filaments, fails to localize near intracellular Listeria. To explore the effect of increasing concentrations of gelsolin on bacterial motility, NIH 3T3 fibroblasts stably transfected with gelsolin cDNA were infected with Listeria. The C5 cell line containing 2.25 times control levels of gelsolin supported significantly higher velocities of bacterial movement than did control fibroblasts (mean +/- standard error of the mean, 0.09 +/- 0.003 micro(m)/s [n = 176] versus 0.05 +/- 0.003 micro(m)/s [n = 65]). The rate of disassembly of the Listeria-induced actin filament rocket tail was found to be independent of gelsolin content. Therefore, if increases in gelsolin content result in increases in Listeria-induced rocket tail assembly rates, a positive correlation between gelsolin content and tail length would be expected. BODIPY-phalloidin staining of four different stably transfected NIH 3T3 fibroblast cell lines confirmed this expectation (r = 0.92). Rocket tails were significantly longer in cells with a high gelsolin content. Microinjection of gelsolin 1/2 (consisting of the amino-terminal half of native gelsolin) also increased bacterial velocity by more than 2.2 times. Microinjection of CapG had no effect on bacterial movement. Cultured skin fibroblasts derived from gelsolin-null mice were capable of supporting intracellular Listeria motility at velocities comparable to those supported by wild-type skin fibroblasts. These experiments demonstrated that the surface of Listeria contains a polymerization zone that can block the barbed-end-capping activity of both gelsolin and CapG. The ability of Listeria to uncap actin filaments combined with the severing activity of gelsolin can accelerate actin-based motility. However, gelsolin is not absolutely required for the actin-based intracellular movement of Listeria because its function can be replaced by other actin regulatory proteins in gelsolin-null cells, demonstrating the functional redundancy of the actin system.     

The amino-terminal part of ActA is critical for the actin-based motility of Listeria monocytogenes; the central proline-rich region acts as a stimulator

The intracellular bacterial pathogen Listeria monocytogenes moves inside the host-cell cytoplasm propelled by continuous actin assembly at one pole of the bacterium. This process requires expression of the bacterial surface protein ActA. Recently, in order to identify the regions of ActA which are required for actin assembly, we and others have expressed different domains of ActA by transfection in eukaryotic cells. As this type of approach cannot address the role of ActA in the actin-driven bacterial propulsion, we have now generated several L. monocytogenes strains expressing different domains of ActA and analysed the ability of the different domains to trigger actin assembly and bacterial movement in both infected cells and cytoplasmic extracts. We show here that the amino-terminal part is critical for F-actin assembly and movement. The internal proline-rich repeats and the carboxy-terminal domains are not essential. However, in vitro motility assays have demonstrated that mutants lacking the proline-rich repeats domain of ActA moved two times slower (6+/-2 micrometers min(-1)) than the wild type (13 +/-3 micrometers min(-1)). In addition, phosphatase treatment of protein extracts of cells infected with the L. monocytogenes strains expressing the ActA variants suggested that phosphorylation may not be essential for ActA activity.     

Caldesmon-actin-tropomyosin contains two types of binding sites for myosin S1

Caldesmon inhibits the activation of myosin ATPase activity by actin-tropomyosin. Caldesmon also inhibits the binding of myosin to actin. There is disagreement as to the degree to which competitive displacement of myosin subfragment binding to actin is responsible for the inhibition of ATPase activity. We have examined the possibility that one or more molecules of S1 may bind to actin-tropomyosin-caldesmon without having the normal actin activation of ATPase activity. The effect of caldesmon on the binding and ATPase activity of S1 was measured at several initial levels of saturation of S1 to determine if a fraction of the bound S1 was resistant to displacement by caldesmon. In the case of both unmodified S1 and rhoPDM-modified S1, most, but not all, of the S1 was displaced by caldesmon. The results are consistent with a single molecule of S1 binding with low affinity for each seven actin monomers that are fully saturated with caldesmon and tropomyosin. This single S1 is not necessarily bound directly to actin but may be attached to the NH2-terminal region of caldesmon.     

Developmental regulation of the Drosophila Tropomyosin I (TmI) gene is controlled by a muscle activator enhancer region that contains multiple cis-elements and binding sites for multiple proteins

We have analyzed in Paramecium cells the occurrence and intracellular distribution of the high capacity/low affinity calcium-binding proteins, calsequestrin (CS) and calreticulin (CR) using antibodies against CS from rat skeletal muscle and against CR from rat liver, respectively. As revealed by Western blots, a CS-like protein isolated by affinity chromatography from Paramecium cells comigrated with CS isolated from rat skeletal muscle. The immunoreactivity of this 53 kDa protein band was blocked when the antibodies had been preadsorbed with purified rat CS. A band of identical molecular size was shown to bind 45Ca in overlays. By immunofluorescence and immunogold labeling this CS-like protein was localized selectively to the extended subplasmalemmal calcium stores, the "alveolar sacs", which cover almost the entire cell surface. Concomitantly the 53 kDa 45Ca-binding band became increasingly intense in overlays as we increasingly enriched alveolar sacs. Antibodies against rat CR react with a 61 kDa band but do not cross-react with CS-like protein in Paramecium. These antibodies selectively stained intracellular reticular structures, identified bona fide as endoplasmic reticulum.     

The gene cluster inlC2DE of Listeria monocytogenes contains additional new internalin genes and is important for virulence in mice

In this work we identified and characterized a gene cluster containing three internalin genes of Listeria monocytogenes EGD. These genes, termed inlG, inlH and inlE, encode proteins of 490, 548 and 499 amino acids, respectively, which belong to the family of large, cell wall-bound internalins. The inlGHE gene cluster is flanked by two listerial house-keeping genes encoding proteins homologous to the 6-phospho-beta-glucosidase and the succinyl-diaminopimelate desuccinylase of E. coli. A similar internalin gene cluster, inlC2DE, localised to the same position on the L. monocytogenes EGD chromosome was recently described in a different isolate (Dramsi S, Dehoux P, Lebrun M, Goossens PL, Cossart P (1997) Infect Immun 65: 1615-1625). Sequence comparison of the two inl gene clusters indicates that inlG is a new internalin gene, while inlH was generated by a site-specific recombination, leading to an in-frame deletion which removed the 3'-terminal end of inlC2 and the 5'-terminal part of inlD. The third gene of the inlGHE cluster, inlE, is almost identical to the previously reported inlE gene. Our data show that the inlGHE gene cluster is probably transcribed from a major PrfA-independent promoter located upstream of inlG. PCR analysis revealed the presence of the newly identified inl genes inlG and inlH in most L. monocytogenes isolates tested. A mutant which has lost inlG, inlH and inlE by an in-frame deletion exhibited, after oral infection of mice, a significant loss in virulence and shows drastically reduced numbers of viable bacteria in both liver and spleen when compared to the wild-type strain.     

Synthetic peptides derived from the Listeria monocytogenes p60 protein as antigens for the generation of polyclonal antibodies specific for secreted cell-free L. monocytogenes p60 proteins

All species of the genus Listeria secrete a major extracellular protein called p60. A comparison of the deduced amino acid sequences of all listerial p60 proteins previously indicated there were only a few regions which were unique to the pathogenic, food-borne species Listeria monocytogenes. Two of these p60 regions were chosen for the development of antibodies specific for the facultative intracellular species L. monocytogenes. Initially, these regions were characterized via epitope mapping, and this led to the development of two different synthetic peptides. Rabbits immunized with these synthetic peptides generated polyclonal antibodies that were then used in Western blot (immunoblot) analyses. Antiserum against peptide A (PepA) recognized the p60 protein in the supernatants collected from most L. monocytogenes serotypes except for several strains belonging to serotypes 4a and 4c. No p60-related protein was detected in the supernatants from other Listeria species with this anti-PepA antiserum. Antibodies raised against peptide D (PepD) reacted with p60 from all L. monocytogenes serotypes, including all 4a and 4c strains that were tested, and also showed no cross-reactivity with supernatant proteins from other Listeria species. Both antisera also detected p60 in supernatants of a large number of environmental isolates of L. monocytogenes. Besides Western blot analyses, these antisera to PepA and PepD reacted with secreted p60 in an enzyme-linked immunosorbent assay, indicating recognition of the native antigen in addition to the denatured form. These data suggest that synthetic peptides derived from the variable region of the L. monocytogenes p60 protein may be useful for the development of an immunological diagnostic assay.     

Identification of the binding site on S100B protein for the actin capping protein CapZ

The calcium-binding protein S100B binds to several potential target proteins, but there is no detailed information showing the location of the binding site for any target protein on S100B. We have made backbone assignments of the calcium-bound form of S100B and used chemical-shift changes in spectra of 15N-labeled protein to locate the site that binds a peptide corresponding to residues 265-276 from CapZ alpha, the actin capping protein. The largest chemical-shift changes are observed for resonances arising from residues around the C terminus of the C-terminal helix of S100B and residues Val-8 to Asp-12 of the N-terminal helix. These residues are close to but not identical to residues that have been identified by mutational analysis to be important in other S100 protein-protein interactions. They make up a patch across the S100B dimer interface and include some residues that are quite buried in the structure of calcium-free S100B. We believe we may have identified a binding site that could be common to many S100 protein-protein interactions.     

The Listeria monocytogenes iap gene as an indicator gene for the study of PrfA-dependent regulation

In this study the effects of hypoxanthine (HX) on meiotic maturation were compared using oocytes from mice possessing a hypoxanthine phosphoribosyltransferase null mutation (HPRT-) and from the corresponding HPRT-competent background strain (HPRT+). Oocyte-cumulus cell complexes and cumulus cell-enclosed oocytes (oocytes cultured while enclosed by cumulus cells) from HPRT+, but not HPRT-, mice took up HX and contained significant levels of HPRT activity. In addition, FSH increased, and HX suppressed, the de novo synthesis of purines in HPRT+ complexes, whereas de novo synthesis was elevated in HPRT complexes and was unaffected by FSH or HX. After 3 h of HX treatment, lower frequencies of germinal vesicle breakdown (GVB) were observed in cumulus cell-enclosed than in denuded HPRT+ oocytes; however, identical frequencies of maturation were observed in denuded and cumulus cell-enclosed HPRT oocytes. This demonstrates a direct inhibitory action of HX on the oocyte that does not depend on salvage, plus an additional action of the cumulus cells that requires HPRT activity. Nevertheless, cumulus cells from HPRT- mice are capable of exerting an additional inhibitory action of dibutyryl cAMP (dbcAMP) on the oocyte. A kinetics analysis of FSH action on HX-arrested cumulus cell-enclosed HPRT+ and HPRT- oocytes revealed, first, that the inhibitory effect of the cumulus cells is transient and, second, that HPRT activity is not required for FSH induction of GVB in HX-arrested oocytes. When dbcAMP- or HX-arrested oocytes were treated with FSH, GVB was blocked to the same extent in HPRT- oocytes with the purine de novo synthesis inhibitor, azaserine, but this drug was less effective in HX-treated HPRT+ oocytes. These results confirm the importance of the de novo pathway in hormone-induced maturation and also support a role for purine salvage as an alternative source of nucleotide in this process.     

Importance of specific amino acids in protein binding sites for heparin and heparan sulfate

The present study tests whether lesions small enough to allow the rapid reestablishment of a normally aligned tract glial framework would provide a permissive environment for the regeneration of cut adult CNS axons. We made penetrating microlesions which cut a narrow beam of axons in the adult rat cingulum, but caused minimal damage to the tract glial framework and no cavitation. The proximal tips of cut axons were identified by enhanced immunoreactivity for low affinity neurotrophin receptor, p75. From 1 day they became expanded into large growth-cone-like structures. At later times some axons turned back and extended in the reverse direction. Up to 14 days (after which time p75 could no longer be used as a marker), no axons advanced beyond the line of the lesion. From 1 to 2 days, OX42 immunostaining and electron microscopy showed that the lesion site was densely infiltrated by macrophages, which disappeared by 3 to 4 days. This was followed by a local hypertrophy of the OX42 immunoreactive resident tract microglial cells and an increase in both GFAP and vimentin immunoreactivity of the tract astrocytes. These responses were greatly reduced by 8 days, when the longitudinal alignment of glial processes across the lesion site was similar to that of an undamaged tract. The large growth-cone-like structures formed at the ends of the cut axons resemble those of developing axons exposed to chemorepulsive factors. This suggests that cellular elements in adult tract lesions may also exert chemorepulsive influences blocking regeneration of axons even in an apparently "open" tract framework.     

Design superiority of palindromic DNA sites for site-specific recognition of proteins: tests using protein stitchery

Using protein stitchery with appropriate attachment of cysteines linking to either C or N termini of the basic region of the v-Jun leucine zipper gene-regulatory protein, we constructed three dimers--pCC, pCN, and pNN. All three bind specifically to the appropriately rearranged DNA recognition sites for v-Jun: ATGAcgTCAT, ATGAcgATGA, and TCATcgTCAT, respectively (Kd, approximately 4 nM at 4 degrees C). Results of DNase I footprinting provide strong support for bent recognition helices in leucine zipper protein-DNA complexes. Comparison of the results for pCC and pNN with those for pCN shows the design superiority of palindromic sequences for protein recognition.