Birth-and-death evolution of the internalin multigene family in Listeria

The birth-and-death model of multigene family evolution describes patterns of gene origination, diversification and loss within multigene families. Since it was first developed in the 1990s, the model has been found to characterize a large number of eukaryotic multigene families. In this paper, we report for the first time a bacterial multigene family that undergoes birth-and-death evolution. By analyzing the evolutionary relationships among internalins, a relatively large and diverse family of genes associated with key virulence functions in Listeria, we demonstrate the importance of birth-and-death evolution in the diversification of this important bacterial pathogen. We also detected two instances of lateral gene transfer within the internalins, but the estimated frequency would have been much higher had it not been analyzed within the context of birth-and-death evolutionary dynamics and a phenomenon that we term "paralog-sorting", which involves the unequal transmittal of gene duplicates during or subsequent to the speciation process. As such, in addition to providing the first demonstration of birth-and-death evolution within a bacterial multigene family, our results indicate that the extent of lateral transfer in bacterial multigene families should be re-examined in the light of birth-and-death evolution.     

Entry of L. monocytogenes into cells is mediated by internalin, a repeat protein reminiscent of surface antigens from gram-positive cocci

We report the identification of a previously unknown gene, inlA, which is necessary for the gram-positive intracellular pathogen Listeria monocytogenes to invade cultured epithelial cells. The inlA region was localized by transposon mutagenesis, cloned, and sequenced. inlA was introduced into Listeria innocua and shown to confer on this normally noninvasive species the ability to enter cells. Sequencing of inlA predicts an 80 kd protein, internalin. Two-thirds of internalin is made up of two regions of repeats, region A and region B, and the C-terminus of the molecule is similar to that of surface proteins from gram-positive cocci. Internalin has a high content of threonine and serine residues, and the repeat motif of region A has regularly spaced leucine residues. As evidenced by Southern blot analysis, inlA is part of a gene family. One of them is the gene situated directly downstream of inlA, called inlB, which also encodes a leucine-rich repeat protein.     

Internalins from the human pathogen Listeria monocytogenes combine three distinct folds into a contiguous internalin domain

The pathology of type II diabetes includes deposition of amyloid in the extra cellular space surrounding the beta-cells of the endocrine pancreas. The principle component of these deposits is an insoluble fibrillar form of a normally soluble 37 residue peptide hormone, islet amyloid polypeptide. Multiple sequence analysis and peptide synthesis have identified a core set of residues (20 to 29) as intrinsically amyloidogenic. As the fibrillogenesis of the 20-29 peptide often requires conditions that deviate considerably from physiological, residues 20 to 29 may be necessary, but not sufficient, for amyloidosis. We aim to determine the structural role of residues outside this core in the context of in vitro fibrillogenesis of the wild-type peptide at physiological pH and ionic strength. Specifically, we make use of an intrinsic fluorescent probe, tyrosine 37 (Y37), to explore the role of the C terminus in fibrillogenesis. Our protocol permits steady state measurement of the lag phase and fiber conformational states of the protein under identical conditions. These are compared to a non-amyloidogenic variant of islet amyloid polypeptide from rat and N-acetyl-tyrosinamide as models of the unfolded state under matched conditions. Spectral, quenching and anisotropic properties of Y37 in the fiber state indicate that the C terminus is packed in a well-defined environment with near frozen rigidity. The presence of a fluorescence resonance energy transfer pathway shows Y37 is near F15 and F23. The lag-phase conformation, while considerably less ordered than the fiber, is more ordered than unfolded models. Differences in anisotropy between the lag and fiber state were used to monitor fibrillogenesis in real time. Parallel assessment of fiber formation using the histological dye, ThT, indicate that ordering at the C terminus of islet amyloid polypeptide is coincident with, and thus indicative of, fiber formation.     

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.     

Structure of internalin,a major invasion protein of Listeria monocytogenes,in complex with its human receptor E-cadherin

Listeria monocytogenes, a food-borne bacterial pathogen, enters mammalian cells by inducing its own phagocytosis. The listerial protein internalin (InlA) mediates bacterial adhesion and invasion of epithelial cells in the human intestine through specific interaction with its host cell receptor E-cadherin. We present the crystal structures of the functional domain of InlA alone and in a complex with the extracellular, N-terminal domain of human E-cadherin (hEC1). The leucine rich repeat (LRR) domain of InlA surrounds and specifically recognizes hEC1. Individual interactions were probed by mutagenesis and analytical ultracentrifugation. These include Pro16 of hEC1, a major determinant for human susceptibility to L. monocytogenes infection that is essential for intermolecular recognition. Our studies reveal the structural basis for host tro-pism of this bacterium and the molecular deception L. monocytogenes employs to exploit the E-cadherin system.     

A tandem repeat of a fragment of Listeria monocytogenes internalin B protein induces cell survival and proliferation

Hepatocyte growth factor (HGF) is critical for tissue homeostasis and repair in many organs including the lung, heart, kidney, liver, nervous system, and skin. HGF is a heterodimeric protein containing 20 disulfide bonds distributed among an amino-terminal hairpin, four kringle domains, and a serine protease-like domain. Due to its complex structure, recombinant production of HGF in prokaryotes requires denaturation and refolding, processes that are impractical for large-scale manufacture. Thus, pharmaceutical quantities of HGF are not available despite its potential applications. A fragment of the Listeria monocytogenes internalin B protein from amino acids 36-321 (InlB??????) was demonstrated to bind to and partially activate the HGF receptor Met. InlB?????? has a stable β-sheet structure and is easily produced in its native conformation by Escherichia coli. We cloned InlB?????? (1×InlB??????) and engineered a head-to-tail repeat of InlB?????? with a linker peptide (2×InlB??????); 1×InlB?????? and 2×InlB?????? were purified from E. coli. Both 1× and 2×InlB?????? activated the Met tyrosine kinase. We subsequently compared signal transduction of the two proteins in primary lung endothelial cells. 2×InlB?????? activated ERK1/2, STAT3, and phosphatidylinositol 3-kinase/Akt pathways, whereas 1×InlB?????? activated only STAT3 and ERK1/2. The 2×InlB?????? promoted improved motility compared with 1×InlB?????? and additionally stimulated proliferation equivalent to full-length HGF. Both the 1× and 2×InlB?????? prevented apoptosis by the profibrotic peptide angiotensin II in cell culture and ex vivo lung slice cultures. The ease of large-scale production and capacity of 2×InlB?????? to mimic HGF make it a potential candidate as a pharmaceutical agent for tissue repair.     

单核增生李斯特菌Internalin A的克隆表达与抗体制备

目的克隆表达单核增生李斯特菌Internalin A（InlA）,并以之为抗原制备检测单核增生李斯特菌的抗体。方法通过PCR技术从单核细胞增生李斯特菌4b中扩增出inlA基因,克隆筛选和测序鉴定后,最终构建该基因的原核表达质粒pGEX-4T-InlA,谷胱甘肽树脂亲和层析纯化表达产物后,免疫小鼠分别制备相应的多抗和单抗。结果在大肠埃希菌中成功表达了InlA,并对其进行了纯化,融合表达产物分子量约为110 kD;免疫小鼠获得的抗血清效价达到1∶1600;得到了3株抗InlA的单克隆抗体杂交瘤细胞株,腹水单抗效价为1∶1×10^5-1∶3×10^5。2种抗体与其他病原菌均无交叉反应。结论通过表达单核增生李斯特菌的特异性蛋白制备的抗体,能有效地消除交叉反应,提高检测的特异性。     

Mechanism of polarization of Listeria monocytogenes surface protein ActA

The polar distribution of the ActA protein on the surface of the Gram-positive intracellular bacterial pathogen, Listeria monocytogenes, is required for bacterial actin-based motility and successful infection. ActA spans both the bacterial membrane and the peptidoglycan cell wall. We have directly examined the de novo ActA polarization process in vitro by using an ActA-RFP (red fluorescent protein) fusion. After induction of expression, ActA initially appeared at distinct sites along the sides of bacteria and was then redistributed over the entire cylindrical cell body through helical cell wall growth. The accumulation of ActA at the bacterial poles displayed slower kinetics, occurring over several bacterial generations. ActA accumulated more efficiently at younger, less inert poles, and proper polarization required an optimal balance between protein secretion and bacterial growth rates. Within infected host cells, younger generations of L. monocytogenes initiated motility more quickly than older ones, consistent with our in vitro observations of de novo ActA polarization. We propose a model in which the polarization of ActA, and possibly other Gram-positive cell wall-associated proteins, may be a direct consequence of the differential cell wall growth rates along the bacterium and dependent on the relative rates of protein secretion, protein degradation and bacterial growth.     

Glucose and nutrient concentrations affect the expression of a 104-kilodalton Listeria adhesion protein in Listeria monocytogenes

Growth media and environmental conditions influence the expression of adhesion and invasion proteins in Listeria monocytogenes. Here, the expression of the 104-kDa Listeria adhesion protein (LAP) was studied in nutrient-rich media (Trypticase soy broth [TSB] and brain heart infusion [BHI]), minimal medium (Luria-Bertani [LB]), or nutrient-deficient medium (peptone water [PW]) by immunoblotting, enzyme-linked immunosorbent assay (ELISA), and immunoelectron microscopy. Also, the effect of incorporating different concentrations of glucose on LAP expression was studied. Immunoblotting showed that LAP expression was at least twofold higher in LB medium than in TSB or BHI, while PW supported very poor cell growth and LAP expression. ELISA and immunoblotting results showed that higher concentrations of glucose (>1.6 g/liter) lowered the culture pH and suppressed LAP expression by more than 75%; however, the addition of K(2)HPO(4) reduced this effect. L. monocytogenes cells grown in LB media with lower concentrations of glucose showed higher adhesion to Caco-2 cells (3,716 and 4,186 cpm of attached bacteria for 0 and 0.2 g of glucose/liter, respectively), while L. monocytogenes cells grown in LB with higher glucose concentrations exhibited lower adhesion (2,126 and 2,221 cpm for 1.6 and 3.2 g of glucose/liter, respectively). A LAP-negative L. monocytogenes strain (A572) showed low adhesion profiles regardless of the amount of glucose added. Transmission electron microscopy revealed that LAP is localized mainly in the cytoplasm, with only a few molecules located on the cell surface. Growth in LB with high glucose (3.2 g/liter) showed the presence of only a few molecules in the cells, corroborating the results observed with ELISA or immunoblotting. In summary, nutrient-rich media and high concentrations of glucose suppressed LAP expression, which possibly is due to the changes in the pH of the media during growth from the accumulation of sugar fermentation by-products.     

Cloning,expression, and localization of a new member of a Paracentrotus lividus cell surface multigene family

We have isolated and characterized a cDNA clone corresponding to a new member of bep (butanol, extracted, proteins) Paracentrotus lividus multigene family coding for cell surface proteins. The cDNA, called bep3, encodes a 370 amino acid protein and shares the same structural organization in the coding region with other members of the same gene family already characterized. Expression of this clone studied by Northern blot and by whole mount hybridization shows that the bep3 messenger is transcribed during oogenesis and utilized till the gastrula stage, whereas at the prism stage, unlike other members of the same gene family, new synthesis of messenger occurs. By whole mount hybridization spatial distribution of bep3 messenger in egg and embryos is established. This messenger appears located in the animal half of the unfertilized egg and moves to the cortical zone after fertilization; it is not present in the structures derived by the vegetal part of the embryo, such as the micromeres of the 16-cell stage, the primary mesenchyme cells of the blastula, and the primary intestine of the gastrula. At the prism stage instead, hybridization of bep3 messenger is restricted to the part of the embryo that will give origin to the oral region as successively confirmed by hybridization at the pluteus stage. The result of whole mount hybridization was confirmed by Northern blot hybridization of separated meso-macromere and micromere RNAs. A Southern blot experiment demonstrates that bep3 is codified by a single copy gene. Conservation of the bep multigene family in several Mediterranean and Japanese sea urchin species has also been analyzed. © 1996 Wiley-Liss, Inc.     

Cell-surface anchoring of Listeria adhesion protein on L. monocytogenes is fastened by internalin B for pathogenesis

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Genetic organization of ascB-dapE internalin cluster serves as a potential marker for Listeria monocytogenes sublineages IIA, IIB, and IIC

Listeria monocytogenes is an important foodborne pathogen that comprises four genetic lineages: I, II, III, and IV. Of these, lineage II is frequently recovered from foods and environments and responsible for the increasing incidence of human listeriosis. In this study, the phylogenetic structure of lineage II was determined through sequencing analysis of the ascB-dapE internalin cluster. Fifteen sequence types proposed by multilocus sequence typing based on nine housekeeping genes were grouped into three distinct sublineages, IIA, IIB, and IIC. Organization of the ascBdapE internalin cluster could serve as a molecular marker for these sublineages, with inlGHE, inlGC2DE, and inlC2DE for IIA, IIB, and IIC, respectively. These sublineages displayed specific genetic and phenotypic characteristics. IIA and IIC showed a higher frequency of recombination (rho/theta). However, recombination events had greater effect (r/m) on IIB, leading to its high nucleotide diversity. Moreover, IIA and IIB harbored a wider range of internalin and stress-response genes, and possessed higher nisin tolerance, whereas IIC contained the largest portion of low-virulent strains owing to premature stop codons in inlA. The results of this study indicate that IIA, IIB, and IIC might occupy different ecological niches, and IIB might have a better adaptation to a broad range of environmental niches.     

The development of a 'labeless' immunosensor for the detection of Listeria monocytogenes cell surface protein, Internalin B

Electrochemical impedance spectroscopy (EIS) is a widely used technique for probing bioaffinity interactions at the surfaces of electrically conducting polymers. EIS methods can be employed to investigate 'labeless' detection of analytes via impedimetric transduction. This paper describes the development of a direct immunosensor for the detection of a cell-surface protein on Listeria monocytogenes, an extremely important food-borne pathogen. L. monocytogenes are facultative anaerobic, non-sporing, Gram-positive, motile rods that employ the surface bound protein, Internalin B (InlB), to promote invasion into host cells. A recombinant form of InlB was previously cloned and expressed in Escherichia coli and a panel of antibodies and antibody fragments directed against the protein were also produced. Here, we describe how a portion of the recombinant InlB protein, the F3 fragment, and an anti-InlB polyclonal antibody, were used to develop a platform for the labeless immunosensing of InlB. Sensors were fabricated by electropolymerisation of planar screen-printed carbon electrodes with polyaniline (PANI), to produce a conductive substrate. Polyclonal anti-InlB antibody was subsequently incorporated onto the PANI layer using a biotin-avidin system for site-specific immobilisation. The sensors were then probed with varying concentrations of InlB antigen and the impedimetric response at each concentration was recorded. An anti-IgG antibody was immobilised at the electrode surface, as a control and subsequently exposed to the same concentrations of InlB. Impedimetric data for the control sensors were also recorded. Upon exposure to a range of concentrations of antigen, complex plane impedance analyses were used to relate the differing redox states of the polymer layer, to the possible charge transfer at the surface, with respect to the related mechanisms between the antibody and the polymer. These effects were subsequently monitored to assess the impedance of the polymer thereby determining the amount of bound antigen at the sensor surface. Calibration profiles for both sample (InlB) and control (IgG) sensors were constructed. A limit of detection of 4.1 pg/ml was achieved for Internalin B.     

Auto, a surface associated autolysin of Listeria monocytogenes required for entry into eukaryotic cells and virulence

Listeria monocytogenes is an opportunistic food-borne human and animal pathogen. Several surface proteins expressed by this intracellular pathogen are critical for the infectious process. By in silico analysis we compared the surface protein repertories of L. monocytogenes and of the non-pathogenic species Listeria innocua and identified a gene encoding a surface protein of L. monocytogenes absent in L. innocua. This gene that we named aut encodes a protein (Auto) of 572 amino acids containing a signal sequence, a N-terminal autolysin domain and a C-terminal cell wall-anchoring domain made up of four GW modules. We show here that the aut gene is expressed independently of the virulence gene regulator PrfA and encodes a surface protein with an autolytic activity. We provide evidence that Auto is required for entry of L. monocytogenes into cultured non-phagocytic eukaryotic cells. The low invasiveness of an aut deletion mutant correlates with its reduced virulence following intravenous inoculation of mice and oral infection of guinea pigs. During infection, the autolytic activity of Auto may also be critical. Auto appears thus as a novel type of L. monocytogenes virulence factor.     

Listeria monocytogenes, a Food-Borne Pathogen

[This corrects the article on p. 485 in vol. 55.].     

Purification of a delayed hypersensitivity-inducing protein from Listeria monocytogenes

Abstract Cell-envelope fragments were prepared from Listeria monocytogenes L242, serotype 4b. Delayed hypersensitivity (DH)-inducing proteins were extracted with deoxycholate and separated into two fractions by filtration through a Sephacryl S-200 column equilibrated with deoxycholate buffer. The second peak eluting from the Sephacryl column was fractionated using ion exchange chromatography on a DEAE Sepharose CL-6B column in the presence of 6 M urea. A purified 20 400-Da protein which induced DH against L. monocytogenes was obtained by isocratic elution. Three other DH-inducing fractions containing several protein bands were eluted by a gradient of potassium thiocyanate (KSCN) in urea buffer. Our results indicate that denaturing conditions can be employed for the fractionation and purification of DH inducing proteins from L. monocytogenes . In addition, it is suggested that the procedure described might also be useful for the purification of other antigens involved in cellular immune reactions.