Interactions of Listeria monocytogenes with mammalian cells during entry and actin-based movement: bacterial factors, cellular ligands and signaling.

Although <50 kb of its 3.3 megabase genome is known, Listeria monocytogenes has received much attention and an impressive amount of data has contributed in raising this bacterium among the best understood intracellular pathogens. The mechanisms that Listeria uses to enter cells, escape from the phagocytic vacuole and spread from one cell to another using an actin-based motility process have been analysed in detail. Several bacterial proteins contributing to these events have been identified, including the invasion proteins internalin A (InlA) and B (InlB), the secreted pore-forming toxin listeriolysin O (LLO) which promotes the escape from the phagocytic vacuole, and the surface protein ActA which is required for actin polymerization and bacterial movement. While LLO and ActA are critical for the infectious process and are not redundant with other listerial proteins, the precise role of InlA and InlB in vivo remains unclear. How InlA, InlB, LLO or ActA interact with the mammalian cells is beginning to be deciphered. The picture that emerges is that this bacterium uses general strategies also used by other invasive bacteria but has evolved a panel of specific tools and tricks to exploit mammalian cell functions. Their study may lead to a better understanding of important questions in cell biology such as ligand receptor signalling and dynamics of actin polymerization in mammalian cells.     

Interactions between Listeria monocytogenes and host mammalian cells

Bacterial pathogens have developed a variety of strategies to induce their own internalization into mammalian cells which are normally nonphagocytic. The Gram-positive bacterium Listeria monocytogenes enters into many cultured cell types using two bacterial surface proteins, InlA (internalin) and InlB. In both cases, entry takes place after engagement of a receptor and induction of a series of signaling events.     

Entry of the bacterial pathogen Listeria monocytogenes into mammalian cells

The bacterial pathogen Listeria monocytogenes causes food-borne illnesses leading to meningitis or abortion. Listeria provokes its internalization ('entry') into mammalian cells that are normally non-phagocytic, such as intestinal epithelial cells and hepatocytes. Entry provides access to a nutrient-rich cytosol and allows translocation across anatomical barriers. Here I discuss the two major internalization pathways used by Listeria. These pathways are initiated by binding of the bacterial surface proteins InlA or InlB to their respective host receptors, E-cadherin or Met. InlA mediates traversal of the intestinal barrier, whereas InlB promotes infection of the liver. At the cellular level, both InlA- and InlB-dependent entry require host signalling that promotes cytoskeletal rearrangements and pathogen engulfment. However, many of the specific signalling proteins in the two entry routes differ. InlA-mediated uptake uses components of adherens junctions that are coupled to F-actin and myosin, whereas InlB-dependent entry involves cytosolic adaptors that bridge Met to regulators of F-actin, including phosphoinositide 3-kinase and activators of the Arp2/3 complex. Unexpectedly, entry directed by InlB also involves endocytic components. Future work on InlA and InlB will lead to a better understanding of virulence, and may also provide novel insights into the normal biological functions of E-cadherin and Met.     

Invasion of mammalian cells by Listeria monocytogenes: functional mimicry to subvert cellular functions

The bacterium Listeria monocytogenes has the unusual capacity to enter and to multiply in nonphagocytic cells. Bacterially induced phagocytosis is triggered mainly by the two surface proteins internalin (also called InlA) and InlB, which interact with host cell receptors and either mimic or act in place of the normal cellular ligands. Internalin interacts specifically with human E-cadherin, whereas InlB activates the tyrosine kinase receptor Met and also interacts with the ubiquitous receptor gC1qR and proteoglycans. Signals induced by crosstalk between the bacterium and the host cell allow internalization, which is a prelude to intracellular multiplication, actin-based movement and spread of the bacterium from cell to cell. Manipulating the bacterial invasion proteins offers us an unprecedented tool with which to understand the complex phenomenon of phagocytosis.     

Interactions between somatic cells and germ cells throughout mammalian oogenesis

Oocytes and their companion somatic cells maintain a close association throughout oogenesis and this association is essential for normal oocyte and follicular development. This review summarizes current concepts of the role of the somatic cells in the regulation of mammalian oocyte growth, the maintenance of meiotic arrest, the induction of oocyte maturation, and the acquisition of full embryonic developmental competence during oocyte maturation in vitro. Gap junctions appear to mediate these regulatory processes. The regulatory interaction of oocytes and somatic cells, however, is not unidirectional; the oocyte participates in the proliferation, development, and function of the follicular somatic cells. The oocyte secretes factors that enable the cumulus cells to synthesize hyaluronic acid and undergo cumulus expansion in response to hormonal stimulation. In addition, the oocyte produces factors that promote the proliferation of granulosa cells. These interactions in vitro do not appear to require the mediation of gap junctions. The oocyte also promotes the differentiation of granulosa cells into functional cumulus cells, but this function of the oocyte appears to require the continued presence and close association of the oocyte and granulosa cells. Therefore, oocytes and follicular somatic cells are interdependent for development and function.     

Helicobacter pylori: bacterial factors and interaction with the epithelial cells.

Helicobacter pylori has been recognized as a major cause of most of the diseases of the stomach. These diseases are preceded by lesions of gastritis induced by H. pylori. This long-standing infection gives us a very good model of interaction between a bacterium and its host. We will review the direct and indirect effects of H. pylori.     

Interactions of MCP1 with components of the replication machinery in mammalian cells

Eukaryotic DNA replication starts with the assembly of a pre-replication complex (pre-RC) at replication origins. We have previously demonstrated that Metaphase Chromosome Protein 1 (MCP1) is involved in the early events of DNA replication. Here we show that MCP1 associates with proteins that are required for the establishment of the pre-replication complex. Reciprocal immunoprecipitation analysis showed that MCP1 interacted with Cdc6, ORC2, ORC4, MCM2, MCM3 and MCM7, with Cdc45 and PCNA. Immunofluorescence studies demonstrated the co-localization of MCP1 with some of those proteins. Moreover, biochemical studies utilizing chromatin-immunoprecipitation (ChIP) revealed that MCP1 preferentially binds replication initiation sites in human cells. Interestingly, although members of the pre-RC are known to interact with some hallmarks of heterochromatin, our co-immunoprecipitation and immunofluorescence analyses showed that MCP1 did not interact and did not co-localize with heterochromatic proteins including HP1β and MetH3K9. These observations suggest that MCP1 is associated with replication factors required for the initiation of DNA replication and binds to the initiation sites in loci that replicate early in S-phase. In addition, immunological assays revealed the association of MCP1 forms with histone H1 variants and mass spectrometry analysis confirmed that MCP1 peptides share common sequences with H1.2 and H1.5 subtypes.     

Dendritic cells are early cellular targets of Listeria monocytogenes after intestinal delivery and are involved in bacterial spread in the host

We studied the sequence of cellular events leading to the dissemination of Listeria monocytogenes from the gut to draining mesenteric lymph nodes (MLNs) by confocal microscopy of immunostained tissue sections from a rat ligated ileal loop system. OX-62-positive cells beneath the epithelial lining of Peyer's patches (PPs) were the first Listeria targets identified after intestinal inoculation. These cells had other features typical of dendritic cells (DCs): they were large, pleiomorphic and major histocompatibility complex class II^hi. Listeria were detected by microscopy in draining MLNs as early as 6 h after inoculation. Some 80–90% of bacteria were located in the deep paracortical regions, and 100% of the bacteria were present in OX-62-positive cells. Most infected cells contained more than five bacteria each, suggesting that they had arrived already loaded with bacteria. At later stages, the bacteria in these areas were mostly present in ED1-positive mononuclear phagocytes. These cells were also infected by an actA mutant defective in cell-to-cell spreading. This suggests that Listeria are transported by DCs from PPs to the deep paracortical regions of draining MLNs and are then transmitted to other cell populations by mechanisms independent of ActA. Another pathway of dissemination to MLNs was identified, probably involving free Listeria and leading to the infection of ED3-positive mononuclear phagocytes in the subcapsular sinus and adjacent paracortical areas. This study provides evidence that DCs are major cellular targets of L. monocytogenes in PPs and that DCs may be involved in the early dissemination of this pathogen. DCs were not sites of active bacterial replication, making these cells ideal vectors of infection.     

Interactions between folding factors and bacterial outer membrane proteins

The outer membrane is the first line of contact between Gram-negative bacteria and their external environment. Embedded in the outer membrane are integral outer membrane proteins (OMPs) that perform a diverse range of tasks. OMPs are synthesized in the cytoplasm and are translocated across the inner membrane and probably diffuse through the periplasm before they are inserted into the outer membrane in a folded and biologically active form. Passage through the periplasm presents a number of challenges, due to the hydrophobic nature of the OMPs and the choice of membranes into which they can insert. Recently, a number of periplasmic proteins and one OMP have been shown to play a role in OMP biogenesis. In this review, we describe what is known about these folding factors and how they function in a biological context. In particular, we focus on how they interact with the OMPs at the molecular level and present a comprehensive overview of data relating to a possible effect on OMP folding yield and kinetics. Furthermore, we discuss the role of lipo-chaperones, i.e. lipopolysaccharide and phospholipids, in OMP folding. Important advances have clearly been made in the field, but much work remains to be done, particularly in terms of describing the biophysical basis for the chaperone-OMP interactions which so intricately regulate OMP biogenesis.     

Sensing, signaling, and responding to DNA damage: organization of the checkpoint pathways in mammalian cells

The DNA damage and replication checkpoints are signaling mechanisms that regulate and coordinate cellular responses to genotoxic conditions. Unlike typical signal transduction mechanisms that respond to one or a few stimuli, checkpoints can be activated by a broad spectrum of extrinsically or intrinsically derived DNA damage or replication interference. Recent investigations have shed light on how the damage and replication checkpoints are able to respond to such diverse stimuli. The activation of checkpoints not only attenuates cell cycle progression but also facilitates DNA repair and recovery of faltered replication forks, thereby preventing DNA lesions from being converted to inheritable mutations. Recently, more checkpoint targets from the cell cycle and DNA replication apparatus have been identified, revealing the increasing complexity of the checkpoint control of the cell cycle. In this article, we discuss current models of the DNA damage and replication checkpoints and highlight recent advances in the field.     

Genetic toxicity of the benzene metabolite trans, trans-muconaldehyde in mammalian and bacterial cells

Previous studies in our laboratory identified trans,trans-muconaldehyde (MUC), a six-carbon diene dialdehyde, as a microsomal metabolite of benzene. This ring-opened metabolite of benzene was also shown to be hematotoxic in mice in a manner similar to benzene. To further explore the role of MUC in relation to benzene toxicity, a number of test systems were utilized to determine its genotoxic potential. In B6C3F1 mice, MUC induced a highly significant increase in sister-chromatid exchange (SCE), the lowest effective dose being 3 mg/kg, but failed to induce any micronuclei (MN). In Chinese hamster ovary (CHO) cells, MUC at concentrations up to 0.8 micrograms/ml was negative in the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) assay. Dose-related increases in the percentage of cells with MN were observed in CHO cells treated with 0.4-0.8 micrograms/ml MUC. MUC did not-cause unscheduled DNA synthesis in rat primary hepatocytes. Treatment of Salmonella typhimurium TA97 with MUC induced a low level of mutations at concentrations ranging from 10 to 70 micrograms/ml with or without S9 activation. MUC was inactive in strains TA1535, TA100, TA1538 and TA98. In CHO cells and rat primary hepatocytes, MUC was cytotoxic at 0.4 and 4.0 micrograms/ml, respectively. Concentrations of 100 micrograms/plate MUC were toxic for bacterial cells. The present findings indicate that MUC is nonmutagenic or minimally mutagenic in bacterial and mammalian in vitro systems. In mammalian cells, MUC is highly cytotoxic and genotoxic.     

The amino acid composition of mammalian and bacterial cells

Summary High performance liquid chromatography was used to analyze the amino acid composition of cells. A total of 17 amino acids was analyzed. This method was used to compare the amino acid compositions of the following combinations: primary culture and established cells, normal and transformed cells, mammalian and bacterial cells, andEscherichia coli andStaphylococcus aureus. The amino acid compositions of mammalian cells were similar, but the amino acid compositions ofEscherichia coli andStaphylococcus aureus differed not only from mammalian cells, but also from each other. It was concluded that amino acid composition is almost independent of cell establishment and cell transformation, and that the amino acid compositions of mammalian and bacterial cells differ. Thus, it is likely that changes in amino acid composition due to cell transformation or species differences between mammalian cells are negligible compared with the differences between mammalian and bacterial cells, which are more distantly related.     

Interactions of mammalian cells with lipid dispersions containing novel metabolizable cationic amphiphiles

Several novel cationic amphiphiles, based on a hydrophobic cholesteryl or dioleoylglyceryl moiety, have been prepared whose hydrophobic and cationic portions are linked by ester bonds to facilitate efficient degradation in animal cells. Dispersions combining such cationic species with phosphatidylethanolamine (PE), certain structural analogues of PE or diacylglycerol can mediate efficient transfer of both nonexchangeable lipid probes and the DNA plasmid pSV2cat into cultured mammalian (CV-1 and 3T3) cells. The abilities of different types of cationic lipid dispersions to mediate transfection of mammalian cells with pSV2cat could not be directly correlated with their abilities to coalesce with other membranes, as assessed by their ability to intermix lipids efficiently with large unilamellar phosphatidylcholine/phosphatidylserine vesicles in the presence or absence of DNA. The cytotoxicities toward CV-1 cells of dispersions combining PE with most of the degradable cationic amphiphiles studied here compare favorably with those reported previously for similar dispersions containing other types of cationic amphiphiles. Fluorescent analogues of two of the diacylglycerol-based cationic amphiphiles examined in this study are shown to be readily degraded after incorporation into CV-1 cells from PE/cationic lipid dispersions.