Listeria monocytogenes Internalin B Activates Junctional Endocytosis to Accelerate Intestinal Invasion

Listeria monocytogenes (Lm) uses InlA to invade the tips of the intestinal villi, a location at which cell extrusion generates a transient defect in epithelial polarity that exposes the receptor for InlA, E-cadherin, on the cell surface. As the dying cell is removed from the epithelium, the surrounding cells reorganize to form a multicellular junction (MCJ) that Lm exploits to find its basolateral receptor and invade. By examining individual infected villi using 3D-confocal imaging, we uncovered a novel role for the second major invasin, InlB, during invasion of the intestine. We infected mice intragastrically with isogenic strains of Lm that express or lack InlB and that have a modified InlA capable of binding murine E-cadherin and found that Lm lacking InlB invade the same number of villi but have decreased numbers of bacteria within each infected villus tip. We studied the mechanism of InlB action at the MCJs of polarized MDCK monolayers and find that InlB does not act as an adhesin, but instead accelerates bacterial internalization after attachment. InlB locally activates its receptor, c-Met, and increases endocytosis of junctional components, including E-cadherin. We show that MCJs are naturally more endocytic than other sites of the apical membrane, that endocytosis and Lm invasion of MCJs depends on functional dynamin, and that c-Met activation by soluble InlB or hepatocyte growth factor (HGF) increases MCJ endocytosis. Also, in vivo, InlB applied through the intestinal lumen increases endocytosis at the villus tips. Our findings demonstrate a two-step mechanism of synergy between Lm's invasins: InlA provides the specificity of Lm adhesion to MCJs at the villus tips and InlB locally activates c-Met to accelerate junctional endocytosis and bacterial invasion of the intestine

Listeria pathogenesis and molecular virulence determinants

The gram-positive bacterium Listeria monocytogenes is the causative agent of listeriosis, a highly fatal opportunistic foodborne infection. Pregnant women, neonates, the elderly, and debilitated or immunocompromised patients in general are predominantly affected, although the disease can also develop in normal individuals. Clinical manifestations of invasive listeriosis are usually severe and include abortion, sepsis, and meningoencephalitis. Listeriosis can also manifest as a febrile gastroenteritis syndrome. In addition to humans, L. monocytogenes affects many vertebrate species, including birds. Listeria ivanovii, a second pathogenic species of the genus, is specific for ruminants. Our current view of the pathophysiology of listeriosis derives largely from studies with the mouse infection model. Pathogenic listeriae enter the host primarily through the intestine. The liver is thought to be their first target organ after intestinal translocation. In the liver, listeriae actively multiply until the infection is controlled by a cell-mediated immune response. This initial, subclinical step of listeriosis is thought to be common due to the frequent presence of pathogenic L. monocytogenes in food. In normal indivuals, the continual exposure to listerial antigens probably contributes to the maintenance of anti-Listeria memory T cells. However, in debilitated and immunocompromised patients, the unrestricted proliferation of listeriae in the liver may result in prolonged low-level bacteremia, leading to invasion of the preferred secondary target organs (the brain and the gravid uterus) and to overt clinical disease. L. monocytogenes and L. ivanovii are facultative intracellular parasites able to survive in macrophages and to invade a variety of normally nonphagocytic cells, such as epithelial cells, hepatocytes, and endothelial cells. In all these cell types, pathogenic listeriae go through an intracellular life cycle involving early escape from the phagocytic vacuole, rapid intracytoplasmic multiplication, bacterially induced actin-based motility, and direct spread to neighboring cells, in which they reinitiate the cycle. In this way, listeriae disseminate in host tissues sheltered from the humoral arm of the immune system. Over the last 15 years, a number of virulence factors involved in key steps of this intracellular life cycle have been identified. This review describes in detail the molecular determinants of Listeria virulence and their mechanism of action and summarizes the current knowledge on the pathophysiology of listeriosis and the cell biology and host cell responses to Listeria infection. This article provides an updated perspective of the development of our understanding of Listeria pathogenesis from the first molecular genetic analyses of virulence mechanisms reported in 1985 until the start of the genomic era of Listeria research

Low molecular weight rat fibroblast tropomyosin 5 (TM-5): cDNA cloning, actin-binding, localization, and coiled-coil interactions

Previous studies have shown that three distinct genes encode six isoforms of tropomyosin (TM) in rat fibroblasts: the alpha gene encodes TM-2, TM-3, TM-5a, and TM-5b, the beta gene encodes TM-1, and the TM-4 gene encodes TM-4. Here we report the characterization of a cDNA clone encoding the most recent rat fibroblast TM to be identified, herein referred to as TM-5, that is the product of a fourth gene that is homologous to the human hTMnm gene, herein referred to as the rat slow-twitch alpha TM gene. The cDNA clone is approximately 1.7 kb and encodes a protein of 248 amino acids. Using two-dimensional gel electrophoresis, the TM-5 protein was found to co-migrate with fibroblast TM-5a and 5b. Comparison of the amino acid sequences of TM-5 to other fibroblast isoforms encoded by the alpha, beta, and TM-4 genes revealed a high degree of homology, although there were regions of divergence among the different isoforms. The gene encoding TM-5 is expressed in all tissues examined including skeletal muscle, stomach, heart, liver, kidney, uterus, spleen, brain, and diaphragm. However, Northern blot and RNase protection analyses revealed the presence of different mRNAs in fibroblasts, striated muscle (skeletal and diaphragm), and brain, which are expressed via alternative RNA splicing and the use of alternative promoters. The TM-5 protein was expressed in a bacterial system and tested for its ability to bind actin in vitro and in vivo. The apparent TM association constant (Ka) was taken as the free concentration at half saturation and was found to be 3 microM for TM-5 compared to 2 microM for TM-5b at an F-actin concentration of 42 microM. When fluorescently-labeled TM-5 was microinjected into living rat fibroblasts, it localized to the stress fibers and ruffles of the leading lamella. The coiled-coil interactions of TM-5 with other low and high molecular weight TM isoforms were studied. TM-5 and TM-4 were capable of dimerizing with each other as well as with other low molecular weight isoforms (TM-5a and TM-5b), but not with the HMW isoforms (TM-1, TM-2, and TM-3). In addition, TM-5a and TM-5b were unable to heterodimerize with each other. The implications of these results in understanding the role of TM diversity in cytoskeletal dynamics are discussed

Distinct localizations of tropomyosin isoforms in LLC-PK1 epithelial cells suggests specialized function at cell-cell adhesions

At least eight nonmuscle, nonbrain tropomyosin isoforms have been described. We used antibodies, microinjection, and transfection to characterize their expression and localization in LLC-PK1 kidney epithelial cells and compared them with other cells. Similar to primary enterocytes, LLC-PK1 cells exhibited predominantly TM-1 and TM-3 of the high-molecular-weight (HMW) isoforms; TM-5 and TM-5b of the low-molecular-weight (LMW) isoforms. Neither TM-4 nor TM-5a was detectable in the LLC-PKI cells. Immunofluorescence studies revealed that HMW isoforms were localized only on stress fibers, not adhesion belts, whereas the adhesion belts were stained by LMW isoform antibodies. When exogenous proteins are introduced either by transfection or microinjection, the HMW isoforms do not incorporate into the adhesion belt, whereas the LMW isoforms can incorporate into the stress fibers, thus indicating there are different mechanisms at work for the selective localization. Temporal changes in the microfilament system of the LLC-PK1 cells were studied during differentiation in culture as defined by spectrin expression and F-actin architecture. Western blot analysis indicated that TM-5b is only expressed in the LLC-PK1 cells after a certain degree of maturation in culture, which suggests isoform switching after the cell-cell contacts are developed. Collectively these results demonstrate that epithelial cells express a complex pattern of TM isoforms, which exhibit differential localizations within the cells and different patterns of expression depending on their origin and stage of differentiation. The implication of differential localization of TM isoforms on their specific functions is discussed

Tropomyosin isoform 5b is expressed in human erythrocytes: implications of tropomodulin-TM5 or tropomodulin-TM5b complexes in the protofilament and hexagonal organization of membrane skeletons

The human erythrocyte membrane skeleton consists of hexagonal lattices with junctional complexes containing F-actin protofilaments of approximately 33-37 nm in length. We hypothesize that complexes formed by tropomodulin, a globular capping protein at the pointed end of actin filaments, and tropomyosin (TM), a rod-like molecule of approximately 33-35 nm, may contribute to the formation of protofilaments, We have previously cloned the human tropomodulin complementary DNA and identified human TM isoform 5 (hTM5), a product of the gamma-TM gene, as one of the major TM isoforms in erythrocytes, We now identify TM5b, a product of the alpha-TM gene, to-be the second major TM isoform, TM5a, the alternatively spliced isoform of the (U-TM gene, which differs by 1 exon and has a weaker actin-binding affinity, however, is not present. TM4, encoded by the delta-TM gene, is not present either. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis, hTM5 comigrated with the slower TM major species in erythrocyte membranes, and hTM5b comigrated with the faster TM major species. TM5b, like TM5, binds strongly to tropomodulin, more so than other TM isoforms, The 2 major TM isoforms, therefore, share several common features: They have 248 residues, are approximately 33-35 nm long, and have high affinities toward F-actin and tropomodulin. These common features may be the key to the mechanism by which protofilaments are formed. Tropomodulin-TM5 or tropomodulin-TM5b complexes may stabilize F-actin in segments of approximately 33-37 nm during erythroid terminal differentiation and may, therefore, function as a molecular ruler. TM5 and TM5b further define the hexagonal geometry of the skeletal network and allow actin-regulatory functions of TMs to be modulated by tropomoduli