Molecular Characterization of Nonhemolytic and Nonpigmented Group B Streptococci Responsible for Human Invasive Infections

Group B Streptococcus (GBS) is a common commensal bacterium in adults, but is also the leading cause of invasive bacterial infections in neonates in developed countries. The β-hemolysin/cytolysin (β-h/c), which is always associated with the production of an orange-to-red pigment, is a major virulence factor that is also used for GBS diagnosis. A collection of 1,776 independent clinical GBS strains isolated in France between 2006 and 2013 was evaluated on specific medium for β-h/c activity and pigment production. The genomic sequences of nonhemolytic and nonpigmented (NH/NP) strains were analyzed to identify the molecular basis of this phenotype. Gene deletions or complementations were carried out to confirm the genotype-phenotype association. Sixty-three GBS strains (3.5%) were NH/NP, and 47 of these (74.6%) originated from invasive infections, including bacteremia and meningitis, in neonates or adults. The mutations are localized predominantly in the cyl operon, encoding the β-h/c pigment biosynthetic pathway and, in the abx1 gene, encoding a CovSR regulator partner. In conclusion, although usually associated with GBS virulence, β-h/c pigment production is not absolutely required to cause human invasive infections. Caution should therefore be taken in the use of hemolysis and pigmentation as criteria for GBS diagnosis in routine clinical laboratory settings.     

载脂蛋白B研究进展

载脂蛋白B(apolipoprotein B,ApoB)在脂类代谢、胆固醇的运输中起关键性的作用,其在心血管疾病、家族性β脂蛋白血症、肥胖和胆囊疾病的研究中尤为重要。本文对ApoB独特的基因结构、表达模式及其多态性与疾病关系的研究进展进行了综述。     

Molecular Control of the Hyaluronan Biosynthesis

Hyaluronan (HA) is the only unsulfated glycosaminoglycan (GAG) composed of repeating units of D-glucuronic acid and N-acetylglucosamine. The amount and the molecular weight of HA are important factors that regulate the physiology and pathology in several mammalian tissues. In fact hydrated HA makes ECM an ideal environment in which cells can move and proliferate. HA interacting with several receptors at the cellular level plays a critical role in signal transduction responses. The control of the HA synthesis is therefore a critical aspect in ECM and cells biology, but so far the information about this question is scanty. The synthesis of HA is due to several enzymes activities which not only involves its synthetic enzymes on the membranes of the cells (HA synthases 1, 2, 3, isoforms) but also the cytoplasmatic enzymes producing the UDP-sugar precursors. The UDP-sugars availability in cytoplasm is a critical point for the GAG synthesis and it seems to affect particularly the HA production. Eventually, the activity control of the enzymes involved in HA metabolism is obtained throughout both enzyme amount and their postsynthetic covalent modification, as phosphorylation. In fact, it was recently reported that HA synthase 3 may be phosphorylated after specific stimuli, and an increasing body of evidence supports the idea that the synthetic pathway of HA may be carefully regulated in all steps.     

Molecular forms of neurotoxins in proteolytic Clostridium botulinum type B cultures.

A modified purification method was used to isolate the neurotoxin of proteolytic Clostridium botulinum type B strain Lamanna. The preparation was found to be a mixture of two protein forms. They were of molecular weight 152,000 and could not be separated by ion-exchange chromatography or electrophoresis in polyacrylamide gel. One was a single polypeptide chain, and the other was a dichain molecule (nicked toxin) held together by an interchain disulfide bond(s). Trypsinization increased the toxicity of the toxin preparation and converted the single-chain molecules into dichain forms that were indistinguishable from the endogenously generated nicked toxin. A protease of the type B culture, with substrate specificity similar to that of trypsin, did not change detectably the molecular form of unnicked type E toxin, although toxicity was increased. Higher toxicity was obtained when unnicked type E was trypsinized; the resulting preparation contained only nicked toxin molecules.     

The Apolipoprotein B/Apolipoprotein A 1 ratio in relation to metabolic syndrome and its components in a sample of the Tunisian population

Objective

This study was undertaken to investigate the relationship between the Apolipoprotein B/Apolipoprotein A 1 (ApoB/ApoA 1) ratio and various characteristics of the metabolic syndrome (MetS) in a sample of the Tunisian population.

Methods

The study included 330 adults aged 35–74 (172 patients with MetS and 158 controls). Waist circumference (WC), blood pressure (BP), HDL-cholesterol (HDL-C), triglycerides (TG), glucose, insulin, and apolipoprotein concentrations were measured. Homeostasis model assessment (HOMA) was used to assess insulin resistance (IR). MetS was defined by NCEP-ATPIII report.

Results

The ApoB/ApoA 1 ratio was significantly higher in patients with MetS versus normal control subjects (p < 0.001). Mean values of ApoB/ApoA 1 ratio increased significantly as the numbers of MetS components increased in men (p < 0.001) and women (p < 0.001). ApoB/ApoA 1 ratio showed statistically significant associations with WC, HDL-C, TG, systolic and diastolic BP, and HOMA-IR. After adjusting for age and gender, the high ApoB/ApoA 1 ratio was significantly associated with the presence of MetS (odds ratio [OR] = 6.10), IR (OR = 1.88), and with each of the MetS components, including: high WC (OR = 2.43), High TG (OR = 6.14), and low HDL-C (OR = 6.92).

Conclusions

Our findings suggest that the ApoB/ApoA 1 ratio is strongly associated with MetS and its components, as well as with IR.     

Molecular Characterization of EmABP,an Apolipoprotein A-I Binding Protein Secreted by the Echinococcus multilocularis Metacestode

Cestodes are unable to synthesize de novo most of their own membrane lipids, including cholesterol, and have to take them up from the host during an infection. The underlying molecular mechanisms are so far unknown. Here we report the identification and characterization of a novel gene, Emabp, which is expressed by larval stages and adults of the fox tapeworm Echinococcus multilocularis. The encoded protein, EmABP, displays significant homologies to apolipoprotein A-I binding protein (AI-BP) of mammalian origin and to metazoan YjeF_N domain proteins. Like mammalian AI-BP, EmABP carries an export-directing signal sequence which is absent in predicted AI-BP orthologs from the related flatworms Schistosoma japonicum and Schmidtea mediterranea. Using a specific antibody and immunoprecipitation techniques, we demonstrate that EmABP is secreted into the extraparasitic environment and into the hydatid fluid of in vitro-cultivated metacestode vesicles. Furthermore, we show that apolipoprotein A-I (apoA-I), a major constituent of cholesterol-transporting high-density lipoproteins, is present in hydatid fluid. By pulldown experiments, we demonstrate that recombinantly expressed, purified EmABP interacts with purified human apoA-I and is able to precipitate apoA-I from human serum. On the basis of these features and the suggested function of AI-BP in cholesterol transport in higher eukaryotes, we propose a role for EmABP in cholesterol and lipid uptake mechanisms of larval E. multilocularis.The metacestode larval stage of the fox tapeworm Echinococcus multilocularis is the causative agent of alveolar echinococcosis (AE) in humans, one of the most serious and life-threatening parasitoses of the Northern Hemisphere (16). The E. multilocularis life cycle involves an adult stage which dwells in the intestines of definitive hosts, such as foxes or dogs, and produces infective eggs that contain the parasite''s oncosphere larva. Upon oral ingestion of the eggs by intermediate hosts (rodents and, occasionally, humans), the oncosphere is activated, hatches, and penetrates the intestinal barrier. Within the liver of the intermediate host, the oncosphere undergoes a metamorphosis toward the bladder-like metacestode stage which grows infiltratively, like a malignant tumor, into the surrounding host tissue. At a later stage of the infection, numerous protoscoleces are formed from the parasite''s germinal tissue, which are passed onto the definitive host when it takes the prey (6-8, 16, 49). Human E. multilocularis infections are relatively rare but pose serious problems to surgical and/or chemotherapeutic treatment (28). A very similar life cycle is displayed by the closely related dog tapeworm Echinococcus granulosus, the causative agent of cystic echinococcosis (CE), with several modifications concerning the spectrum of host species (domestic animals), metacestode morphology (unilocular versus multilocular), and organ tropism (the lung, kidney, and brain in addition to the liver) (6, 16).Although E. multilocularis and E. granulosus contain complex mixtures of lipids, including cholesterol, in their membranes, they are unable to synthesize most of these molecules de novo and share this trait with other cestodes (35). As a consequence, they have to take up host-derived lipids during an infection. Particularly in the case of cholesterol, Frayha (19, 20) already demonstrated that this compound cannot be synthesized by both E. multilocularis and E. granulosus and that at least E. granulosus incorporates radioactively labeled, host-derived cholesterol during experimental infection of mice. Although several Echinococcus proteins with fatty acid and hydrophobic ligand binding properties have been reported (12, 25), none of these displayed cholesterol binding activities nor has, as yet, any cestode molecule been identified that interacts with components of the host''s cholesterol transport machinery.Mammalian cells acquire exogenous cholesterol mainly from low-density lipoprotein (LDL) particles via the LDL receptor pathway. During this process, the LDL receptor specifically interacts with the major protein component of LDL particles, apolipoprotein B-100 (apoB-100), resulting in the formation of clathrin-coated vesicles which are processed via the classical endocytic pathway. Upon fusion of the vesicles with lysosomes, the entire LDL particle is disassembled by enzymatic hydrolysis, releasing cholesterol and lipids for cellular metabolism (9, 36, 41). The majority of LDL receptors expressed in mammals are on the surfaces of liver cells, although a certain level of LDL receptor expression also occurs in the peripheral tissue (9). The LDL/LDL receptor lipid transport system appears to be evolutionarily conserved, since apoB-100-like cholesterol binding proteins (vitellogenins) have already been identified in yolk of invertebrates, such as Caenorhabditis elegans and Drosophila melanogaster (29, 34, 45). Furthermore, surface receptors of the LDL receptor family have been reported to be expressed by invertebrates (34).In addition to exogenous uptake of cholesterol, nearly all mammalian cells are able to also synthesize cholesterol de novo. In cells of peripheral tissues, excess cholesterol needs to be removed and transported to the liver for reutilization and excretion. The underlying mechanism of “reverse cholesterol transport” is mediated by high-density lipoprotein (HDL) particles, the major component of which is apolipoprotein A-I (apoA-I) (38). Lipid-free apoA-I is secreted predominantly by the liver and intestine and acquires phospholipids and cholesterol via cellular efflux from peripheral tissue cells and macrophages, giving rise to nascent HDL. Once mature, HDL particles are transported to the liver, adrenal glands, and steroidogenic tissue where they are recognized by the HDL receptor, scavenger receptor type B class I, upon which the process of “selective lipid uptake” by the target cell is induced, which fundamentally differs from receptor-mediated endocytosis (9, 36, 38, 39). During “selective lipid uptake,” cholesterol and phospholipids are effectively transferred to target cells, releasing extracellular, lipid-depleted HDL particles which can reenter circulation. Although LDL particles are the major carriers of cholesterol in human blood, sera from rodents and ungulates typically contain much higher levels of HDL components than LDL components (10). Another difference concerns the extracellular transfer of cholesteryl esters from HDL particles to other lipoproteins (e.g., LDL) for further transport, which can be observed only in humans and not in rodents (9). Although as in the case of LDL receptors, the scavenger receptor type B family appears to be evolutionarily conserved and occurs also in invertebrates (15), soluble apolipoproteins, such as apoA-I or apoE, are probably deuterostome specific and may have first appeared around 450 million years ago in an Ordovician vertebrate (27).As yet, only two parasitic flatworm proteins which can interact with the cholesterol and lipid transport machinery of mammalian hosts have been reported and both derive from trematodes, an LDL receptor-like very low-density lipoprotein binding protein from Schistosoma japonicum (17) and a CD36-like class B scavenger receptor from Schistosoma mansoni (15) which interacts with modified host LDL at the tegumental surface. As a first step toward the characterization of cestode molecules that are involved in cholesterol uptake during infections of the intermediate host, we herein report the identification and characterization of a secreted E. multilocularis protein, EmABP, which interacts with human apoA-I, the major component of HDL particles for reverse cholesterol transport. The role of EmABP in Echinococcus cholesterol uptake mechanisms is discussed in the background of what is known on the function of the homologous apolipoprotein A-I binding protein (AI-BP) (26, 42, 44) in humans.     

Molecular Modelling of HLA-DQ Suggests a Mechanism of Resistance in Type 1 Diabetes

Genetic resistance and susceptibility to insulin-dependent diabetes mellitus (IDDM) have been associated with the HLA class II region on chromosome 6. In many races, the DQB1*0602 allele has been observed at a decreased frequency in IDDM, suggesting a protective role. A DNA sequence analysis of five patients, previously typed as having allele DQB1*0602, revealed sequence variation: one is DQB1*0603 and four possess unique sequences related to DQB1*0602 (one patient) or DQB1*0603 (three patients). Samples from four unaffected controls possessed normal DQB1*0602 sequences, and all patients and controls have normal DQA1 sequences. Each of the four unique patient sequences yields predicted amino acid sequences differing from the more common DQB1 alleles by variation at codons 9, 38 (silent), 59, and/or 62. Molecular modelling of the predicted protein sequence of these permissive variants reveals an HLA-DQ structure from diabetic patients that differs in the surface contour of the peptide-binding groove from normal DQB1*0602 sequence. In all the models of permissive molecules, the surface area corresponding to the HLA-DR pockets 6, 7 and 9 are modified. These pockets accommodate side chains of the bound peptide; thus modification of this region could alter peptide specificity. This 'pocket change' suggests that the normal allele could confer dominant protection against the development of IDDM by affecting peptide and/or T cell receptor (TCR) binding. This could regulate the deletion or suppression of T cell clones inappropriately recognizing the beta cells of the pancreas.     

Recombinant human P450 forms 1A1, 1A2, and 1B1 catalyze the bioactivation of heterocyclic amine mutagens in Escherichia coli lacZ strains

Three recombinant human P450 enzymes, forms 1A1, 1A2, and 1B1, were coexpressed with NADPH-cytochrome P450 reductase in an E. coli lacZ strain suitable for detection of the mutagenicity of heterocyclic and aromatic amines. The resulting strains expressed the recombinant P450 holoenzymes at high levels. MeIQ (2-amino-3,4-dimethylimidazo[4,5-f]quinoline) was activated effectively by P450 1A2, weakly by P450 1A1, and not detectably by P450 1B1. MeIQx (2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline) and Trp-P-2 (3-amino-1-methyl-5H-pyrido[4,3-b]indole) were activated by all three enzymes, with form 1A2 the most effective. These strains facilitate analysis of the substrate specificity of human P450 forms that participate in the metabolic activation of carcinogens.     

Mechanism of the Adherence of Streptococcus mutans to Smooth Surfaces III. Purification and Properties of the Enzyme Complex Responsible for Adherence

Enzymes which possess the ability to cause the adherence of Streptococcus mutans cells to a smooth glass surface were purified 1,100 times by chromatography on agarose gel followed by hydroxylapatite gel. During the purification procedures, the enzymes from strain HS6 (group a) were examined for the synthesis of water-soluble and water-insoluble polysaccharide and the ability to produce adherence. The enzyme preparations producing adherence of the S. mutans cells in the presence of sucrose possessed a molecular size of about 400,000 to 2,000,000 and were composed of approximately equivalent amounts of dextran and levan sucrases and 5 to 30% polysaccharide. The most highly purified preparation contained a negligible amount of contaminating protein as judged by polyacrylamide gel electrophoresis, immunoelectrophoresis, and gel diffusion. In these three tests, the location of the enzyme responsible for the synthesis of insoluble polymer was detected by embedding or covering the enzyme-containing gel with a layer of sucrose-containing agarose gel and observing the formation of insoluble polymer. During purification the ability of all fractions to produce adherence was parallel with the enzyme activity responsible for the synthesis of insoluble polysaccharide from sucrose. About two-thirds of the sucrase enzyme complex in the S. mutans culture fluid synthesized water-soluble polymer. This complex, obtained by filtration through agarose gel, was smaller in molecular size, lower in sugar content, and did not produce adherence, in contrast to the enzyme complex which possessed adherence activity. The inhibition of the enzyme complex synthesizing soluble polymer required more anti-synthetase serum than that required to inhibit the synthesis of water-insoluble polymer. It is not known whether the lack of adherence activity in this enzyme was due to its smaller size and lower sugar content or the absence of unknown factors which are essential for its activity. The carbohydrate in these enzyme preparations, composed of glucose, may represent a primer molecule and/or a remnant of the polymer synthesized by the enzyme. The enzyme activity was not inhibited by anti-dextran globulin.     

Monoclonal antibodies for fumonisins B1, B2 and B3

There has been only one report of fumonisin antibodies produced from a fumonisin-bovine serum albumin (BSA) immunogen in the past. Other, more exoteric, carriers, such as cholera toxin and keyhole limpet hemocyanin, have also been used. The resulting antibodies have a wide range of sensitivity and selectivity for the most common fumonisins B₁, B₂ and B₃. We report on fumonisin monoclonal antibodies produced in typical fashion from an immunogen consisting of FB₁ conjugated to BSA through its terminal amino group. These antibodies have excellent cross-reactivity with the three main fumonisins: FB₁, 100%, FB₂, 94%, and FB₃, 72%, adequate sensitivity with an IC₅₀ of 430 ng/mL for FB₁, and show excellent correlation between ELISA and total fumonisins as determined by HPLC, with a slope of 0.985 and an r² of 0.987.     

Biosynthesis of a novel prostaglandin, delta 17-PGE1, in the ram

The prostaglandin, delta 17-PGE1, was purified from extracts of ram seminal fluid by reversed phase high performance liquid chromatography (HPLC) and identified after conversion to delta 17-PGB1 by UV-analysis and by capillary column gas chromatography-mass spectrometry (GC-MS). It was also formed by incubation of a homogenate of ram vesicular glands. The amount of delta 17-PGE1 in the seminal fluid and in the homogenate averaged 12% and 25% of the amount of PGE2, respectively. The results show that cis-8,11,14,17-eicosatetraenoic acid can be metabolized to prostaglandins in vivo in the ram.     

Molecular determinants of disease in coxsackievirus B1 murine infection

To understand better how different genomic regions may confer pathogenicity for the coxsackievirus B (CVB), two intratypic CVB1 variants, and a number of recombinant viruses were studied. Sequencing analysis showed 23 nucleotide changes between the parental non-pathogenic CVB1N and the pathogenic CVB1Nm. Mutations present in CVB1Nm were more conserved than those in CVB1N when compared to other CVB sequences. Inoculation in C3H/HeJ mice showed that the P1 region is critical for pathogenicity in murine pancreas and heart. The molecular determinants of disease for these organs partially overlap. Several P1 region amino acid differences appear to be located in the decay-accelerating factor (DAF) footprint CVBs. CVB1N and CVB1Nm interacted with human CAR, but only CVB1N seemed to interact with human DAF, as determined using soluble receptors in a plaque-reduction assay. However, the murine homolog Daf-1 did not interact with any virus assessed by hemagglutination. The results of this study suggest that an unknown receptor interaction with the virus play an important role in the pathogenicity of CVB1Nm. Further in vivo studies may clarify this issue.     

Factor B of the Alternative Complement Pathway on Human Lymphocytes

A factor on human lymphocytes has been identified as factor B of the alternative pathway. Lymphocytes can replace factor B in the fluid phase formation of C3 convertase with cobra venom factor (CVF). This lymphocyte activity is inhibited by specific anti-human factor B, and it is shown by Burkitt lymphoma cell lines cultured in the absence of any factor B source. After the reaction with CVF all the C3-converting activity is found in the cell supernatant, and the same cells can undergo several successive cycles of 'activation' by CVF. Factor B is distinct from the C3b receptor, and its presence could not be detected antigenically on the lymphocyte membrane. It may be secreted by the cells, but the reaction was not affected by sodium azide or cytochalasin B. No detectable factor B activity was found in the culture medium of cells grown in the absence of CVF.     

Apolipoprotein B detected in the plasma of a patient with homozygous hypobetalipoproteinaemia: implications for aetiology.

A hypobetalipoproteinaemic kindred is described in which the proband manifested the clinical and biochemical features of the homozygous state. Unlike the apparent complete absence of apolipoprotein B in the plasma of the five cases of homozygous hypobetalipoproteinaemia reported so far, we were able to demonstrate minute quantities of this protein (approximately 0.025% of normal) in the plasma of the proband. This finding suggests that the disorder may not result from a structural gene defect but may rather reflect a failure of secretion.     

Apolipoprotein A-I,apolipoprotein B,high-sensitivity C-reactive protein and severity of coronary artery disease in tunisian population

BackgroundThe relationship between Apolipoprotein A-I, Apolipoprotein B, C-reactive protein and the severity coronary artery disease in Tunisian CAD patients has not been examined. We investigated the association between serum ApoA-I, ApoB, hs-CRP and the severity of coronary artery disease.MethodsThis study was carried out on 180 patients who underwent angiography and 129 healthy controls. ApoA-I and ApoB as well as the serum total cholesterol, HDL, triglyceride, LDL and hs-CRP levels were measured. The ApoB/ApoA-I ratio was calculated.ResultsWe showed a decreased level of ApoA-I and an increased level of ApoB, ApoB/ApoA-I ratio and hs-CRP in CAD patients compared to the control group (P<.001). In addition, we showed a significant increase of ApoB, ApoB/ApoA-I ratio and hs-CRP in CAD patients presenting 0 to 3 vessels stenosis (P<.001). Multivariate analysis showed that ApoB (P<.001), and hs-CRP (P<.001) were independent predictors of the severity of CAD.ConclusionIn this study, ApoB and hs-CRP levels were markedly associated with the severity of CAD in Tunisian patients. We suggested that synergistic effects between dyslipidemia and inflammation led to increase the risk of the severity of CAD.