A Metachromatic Dye-Atpase Method for The Simultaneous Identification of Skeletal Muscle Fiber Types I, IIA, IIB and IIC

The histochemical demonstration of quantitative differences in myofibrillar ATPase activity at the selective pH optima of the various types of human skeletal muscle fibers is the most widely used technique for their differentiation. The basis of the reaction is the deposition of insoluble salts of inorganic phosphate cleaved from ATP by myofibrillar ATPase(s) followed by substitution of the phosphates with less soluble chromogenic salts. Doriguzzi and associates reported using metachromatic dyes to demonstrate quantitative differences in phosphate deposition among different fiber types. Following routine ATPase histochemistry and staining with either azure A or toluidine blue, fibers with low ATPase activity (and low phosphate content) were stained metachromatically while fibers with high ATPase activity (and high phosphate content) were orthochromatic with the intensity of color proportional to the content of insoluble phosphate. The metachromasia was readily lost after immoderate washing in aqueous solutions or routine dehydration in ethanol, with consequent diminished fiber type distinction. A critical modification of this technique is reported in which incubation of frozen sections of human skeletal muscle in ATP-containing medium is carried out at room temperature (22-24 C), rather than the usual 37 C., followed by a revised washing and dehydration protocol. With these modifications, the four human skeletal muscle fiber types (types I, HA, IIB, and IIC) can be identified rapidly and reliably in single sections, obviating the need for examination of serial sections. The tinctorial differentiation allows fiber type identification even in black and white photographs.     

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-β-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.     

A metachromatic dye-ATPase method for the simultaneous identification of skeletal muscle fiber types I, IIA, IIB and IIC.

The histochemical demonstration of quantitative differences in myofibrillar ATPase activity at the selective pH optima of the various types of human skeletal muscle fibers is the most widely used technique for their differentiation. The basis of the reaction is the deposition of insoluble salts of inorganic phosphate cleaved from ATP by myofibrillar ATPase(s) followed by substitution of the phosphates with less soluble chromogenic salts. Doriguzzi and associates reported using metachromatic dyes to demonstrate quantitative differences in phosphate deposition among different fiber types. Following routine ATPase histochemistry and staining with either azure A or toluidine blue, fibers with low ATPase activity (and low phosphate content) were stained metachromatically while fibers with high ATPase activity (and high phosphate content) were orthochromatic with the intensity of color proportional to the content of insoluble phosphate. The metachromasia was readily lost after immoderate washing in aqueous solutions or routine dehydration in ethanol, with consequent diminished fiber type distinction. A critical modification of this technique is reported in which incubation of frozen sections of human skeletal muscle in ATP-containing medium is carried out at room temperature (22-24 C), rather than the usual 37 C, followed by a revised washing and dehydration protocol. With these modifications, the four human skeletal muscle fiber types (types I, IIA, IIB, and IIC) can be identified rapidly and reliably in single sections, obviating the need for examination of serial sections. The tinctorial differentiation allows fiber type identification even in black and white photographs.     

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.     

Entry of Listeria monocytogenes into hepatocytes requires expression of InIB, a surface protein of the internalin multigene family

The intracellular bacterium Listeria monocytogenes can invade several types of normally non-phagocytic cells. Entry into cultured epithelial cells requires the expression of inIA, the first gene of an operon, comprising two genes: inIA, which encodes internalin, an 800-amino-acid protein, and inIB, which encodes a 630-amino-acid protein. Several genes homologous to inIA are detected in the genome of L. monocytogenes; InIB is one of them. We have assessed the role of inIB In invasiveness of L. monocytogenes by constructing isogenic chromosomal deletion mutants in the inIAB locus. Our findings indicate that: i) inIB is required for entry of L. monocytogenes into hepatocytes, but not into intestinal epithelial cells; ii) inIB encodes a surface protein; iii) internalin plays a role for entry into some hepatocyte cell lines. These results provide the first insight into the cell tropism displayed by L. monocytogenes.     

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。     

The alpha-kinases TRPM6 and TRPM7, but not eEF-2 kinase, phosphorylate the assembly domain of myosin IIA, IIB and IIC

TRPM6 and TRPM7 encode channel-kinases. While these channels share electrophysiological properties and cellular functions, TRPM6 and TRPM7 are non-redundant genes raising the possibility that the kinases have distinct substrates. Here, we demonstrate that TRPM6 and TRPM7 phosphorylate the assembly domain of myosin IIA, IIB and IIC on identical residues. Whereas phosphorylation of myosin IIA is restricted to the coiled-coil domain, TRPM6 and TRPM7 also phosphorylate the non-helical tails of myosin IIB and IIC. TRPM7 does not phosphorylate eukaryotic elongation factor-2 (eEF-2) and myosin II is a poor substrate for eEF-2 kinase. In conclusion, TRPM6 and TRPM7 share exogenous substrates among themselves but not with functionally distant alpha-kinases. STRUCTURED SUMMARY:     

Monoclonal antibody specific for Listeria monocytogenes, Listeria innocua, and Listeria welshimeri

Eight hundred fifty-nine murine hybridomas were produced from eight fusions, and 27 were characterized for secretion of antibodies reactive to Listeria monocytogenes. One monoclonal antibody (MAb), P5C9, reacted with all test strains of L. monocytogenes (31 of 31), L. innocua (3 of 3), and L. welshimeri (1 of 1) but not with any strains of the other four Listeria species or with any of 22 gram-positive or 11 gram-negative species of bacteria when tested in microtiter and dot blot enzyme immunoassays. Of the other 26 antibodies, 20 reacted with either L. monocytogenes Scott A or V7 and with some or all of the other six Listeria species but also cross-reacted with some or all of the non-Listeria bacteria tested. MAb P5C9 is of the immunoglobulin G1 murine subclass. In Western blot (immunoblot) analyses, this MAb reacted with a single antigen with a molecular weight of 18,500, and it is shared in common with all three reactive species, L. monocytogenes, L. innocua, and L. welshimeri. This antigen was extracted with detergent and appeared to be cell bound.     

Surface proteins and the pathogenic potential of Listeria monocytogenes

On the basis of the recently determined genome sequence of Listeria monocytogenes, we performed a global analysis of the surface-protein-encoding genes. Only proteins displaying a signal peptide were taken into account. Forty-one genes encoding LPXTG proteins, including the previously known internalin gene family, were detected. Several genes encoding proteins that, like InlB and Ami, possess GW modules that attach them to lipoteichoic acids were also identified. Additionally, the completed genome sequence revealed genes encoding proteins potentially anchored in the cell membrane by a hydrophobic tail as well as genes encoding P60-like proteins and lipoproteins. We describe these families and discuss their putative implications for host-pathogen interactions.     

Genetic Diversity of Listeria monocytogenes Strains from a High-Prevalence Dairy Farm

Listeria monocytogenes is a significant food-borne human and veterinary pathogen. Contaminated silage commonly leads to disease in livestock, but the pervasive nature of the bacterium can make it difficult to identify the source of infection. An investigation of bovine listeriosis that occurred on a Pacific Northwest dairy farm (“farm A”) revealed that the clinical strain was closely related to fecal strains from asymptomatic cows, and that farm environment was heavily contaminated with a diversity of L. monocytogenes strains. In addition, the farm A clinical strain was closely related to clinical and environmental strains obtained 1 year prior from a second Northwest dairy farm (“farm B”). To investigate the source(s) of contamination on farm A, environmental samples were collected from farm A at two time points. Pulsed-field gel electrophoresis characterization of 538 isolates obtained from that farm identified 57 different AscI pulsovars. Fecal isolates obtained from individual cows were the most genetically diverse, with up to 94% of fecal samples containing more than one pulsovar. The maximum numbers of pulsovars and serotypes isolated from a fecal sample of one cow were 6 and 4, respectively. Serotype 1/2a was isolated most frequently at both time points. Microarray genotyping of bovine listeriosis, fecal, and silage strains from both farms identified four probes that differentiated listeriosis strains from environmental strains; however, no probe was common to both bovine listeriosis strains.     

Genetic diversity of Listeria monocytogenes strains from a high-prevalence dairy farm

Listeria monocytogenes is a significant food-borne human and veterinary pathogen. Contaminated silage commonly leads to disease in livestock, but the pervasive nature of the bacterium can make it difficult to identify the source of infection. An investigation of bovine listeriosis that occurred on a Pacific Northwest dairy farm ("farm A") revealed that the clinical strain was closely related to fecal strains from asymptomatic cows, and that farm environment was heavily contaminated with a diversity of L. monocytogenes strains. In addition, the farm A clinical strain was closely related to clinical and environmental strains obtained 1 year prior from a second Northwest dairy farm ("farm B"). To investigate the source(s) of contamination on farm A, environmental samples were collected from farm A at two time points. Pulsed-field gel electrophoresis characterization of 538 isolates obtained from that farm identified 57 different AscI pulsovars. Fecal isolates obtained from individual cows were the most genetically diverse, with up to 94% of fecal samples containing more than one pulsovar. The maximum numbers of pulsovars and serotypes isolated from a fecal sample of one cow were 6 and 4, respectively. Serotype 1/2a was isolated most frequently at both time points. Microarray genotyping of bovine listeriosis, fecal, and silage strains from both farms identified four probes that differentiated listeriosis strains from environmental strains; however, no probe was common to both bovine listeriosis strains.     

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

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Production, characterisation and potential application of a novel monoclonal antibody for rapid identification of virulent Listeria monocytogenes

A panel of hybridomas was produced using intact Listeria monocytogenes serotype 1/2a cells as the immunogen. An IgG2a monoclonal antibody (mAb) 'mAb2B3' was isolated that reacted with L. monocytogenes but not with a representative panel of related Listeria spp. and non-Listeria spp. Binding activity was greatest against L. monocytogenes serotype 1/2a and was significantly enhanced when cells were prepared in Listeria enrichment broth (LEB). The reactive epitope was deduced, by immunoblot analysis, to be a surface localised protein of approximately 80 kilodaltons (kDa), putatively assumed to be internalin A (InlA). Recombinant InlA protein was subsequently expressed in Escherischia coli. When crude E. coli cell lysates were subjected to immunoblot analysis, it was demonstrated that the mAb bound specifically to the heterologously expressed recombinant InlA protein, thus confirming the specificity of the mAb. The mAb was further evaluated in a series of enzyme-linked-immunosorbent assay (ELISA)-based formats and in a surface plasmon resonance (SPR)-based biosensor platform. Both configurations were capable of differential identification of virulent L. monocytogenes at concentrations greater than or equal to 1x10(7) cells/ml. Notwithstanding the apparent insensitivity, the results indicate that InlA could be exploited as a marker for highly specific confirmatory identification of pathogenic L. monocytogenes following primary enrichment of suspect food samples, using the anti-InlA antibody 'mAb2B3', described herein.     

Genetic diversity and molecular typing of Listeria monocytogenes in China

ABSTRACT: BACKGROUND: Listeria monocytogenes can cause invasive diseases in humans and farm animals and is frequently isolated from dairy products and poultry. Listeriosis is uncommon in China but L. monocytogenes has been isolated from foods and food processing environments in China. However little is known of genetic diversity of Chinese L. monocytogenes isolates and their relationships with global isolates. RESULTS: Two hundred and twelve isolates of L. monocytogenes from food sources from 12 provinces/cities in China were analysed by serotyping, Pulsed Field Gel Electrophoresis (PFGE) and Multi-locus Sequence Typing (MLST). The predominant serotypes are 1/2a, 1/2b and 1/2c accounting for 90.1% of the isolations. PFGE divided the isolates into 61 pulse types (PTs). Twenty nine PTs were represented by more than one isolates with PT GX6A16.0004 containing the most number of isolates. MLST differentiated the isolates into 36 STs, among which 15 were novel. The most common 3 STs were ST9 (29.1%), ST8 (10.7%) and ST87 (9.2%), accounting for 49.0% of the isolates. CONCLUSIONS: STs prevalent in other parts of the world are also prevalent in China including 7 STs (ST1-ST3, ST5, ST6, ST8, ST9) which caused maternal fetal infections or outbreaks, suggesting that these STs potentially can also cause severe human infections or outbreaks in China. Surveillance of these STs will provide important information for prevention of listeriosis. This study also enhances our understanding of genetic diversity of L. monocytogenes in China.     

Cloning of a gene encoding a major secreted polypeptide of Listeria monocytogenes and its potential use as a species-specific probe

A gene, designated msp, that encodes a major secreted polypeptide with a molecular mass of approximately 60 kilodaltons (kDa) was cloned from Listeria monocytogenes 10403. DNA hybridization analysis indicated that the msp gene was highly conserved among 15 independent L. monocytogenes isolates and that each of 5 isolates tested secreted a 60-kDa polypeptide that was immunologically related to the msp gene product. DNA sequences related to msp were not detected in any other Listeria species or in strains of Bacillus cereus, Bacillus thuringiensis, Streptococcus pyogenes, or Streptococcus pneumoniae when standard stringent DNA hybridization conditions were used. Under nonstringent conditions, related sequences were detected in Listeria ivanovii, Listeria seeligeri, and Listeria innocua, and immunoblot analysis indicated that these strains secreted polypeptides of about 60 kDa that were immunologically related to the msp gene product. The possibility of using the msp gene as a probe for the detection of L. monocytogenes and the potential functions of the msp gene product are discussed.     

Genetic characterization of Listeria monocytogenes food isolates and pathogenic potential within serovars 1/2a and 1/2b

A total of 39 Listeria monocytogenes strains isolated from raw milk, smoked meat, chicken carcass and reference strains, belonging to serovars 1/2a, 4a, 1/2b, 3b and 4b, were analysed by RAPD and by polymorphisms of the virulent genes inlAB and iap. Ten isolates, belonging to serovars 1/2a and 1/2b and, collected from raw milk and smoked meat, were further tested for pathogenicity by IP injection into mice. The clustering of the 39 L. monocytogenes strains in 3 groups at 0.45 similarity level, based on molecular typing, was observed. Distribution of serovars in these clusters was in agreement with the proposed three Listeria monocytogenes lineages. Within serovar 1/2b, the 50% lethal dose (LD50) ranged from 8.4 x 10(4) to 1.7 x 10(6) cfu.ml(-1). One of the serovar 1/2b strains, isolated from smoked meat, exhibited the lowest virulence potential evaluated by LD50 and by mean time to death (MTD) and, from this point of view, was completely different from the other strains. Our results suggest the existence of heterogeneity in virulence levels within serovars 1/2a and 1/2b. However, when comparing the isolates based on genotyping, virulence indicators and food origin, no relation could be assessed.     

Cloning of a gene encoding a major secreted polypeptide of Listeria monocytogenes and its potential use as a species-specific probe.

A gene, designated msp, that encodes a major secreted polypeptide with a molecular mass of approximately 60 kilodaltons (kDa) was cloned from Listeria monocytogenes 10403. DNA hybridization analysis indicated that the msp gene was highly conserved among 15 independent L. monocytogenes isolates and that each of 5 isolates tested secreted a 60-kDa polypeptide that was immunologically related to the msp gene product. DNA sequences related to msp were not detected in any other Listeria species or in strains of Bacillus cereus, Bacillus thuringiensis, Streptococcus pyogenes, or Streptococcus pneumoniae when standard stringent DNA hybridization conditions were used. Under nonstringent conditions, related sequences were detected in Listeria ivanovii, Listeria seeligeri, and Listeria innocua, and immunoblot analysis indicated that these strains secreted polypeptides of about 60 kDa that were immunologically related to the msp gene product. The possibility of using the msp gene as a probe for the detection of L. monocytogenes and the potential functions of the msp gene product are discussed.