Mosaic Structure and Molecular Evolution of the Leukotoxin Operon (lktCABD) in Mannheimia (Pasteurella) haemolytica, Mannheimia glucosida, and Pasteurella trehalosi

The mosaic structure and molecular evolution of the leukotoxin operon (lktCABD) was investigated by nucleotide sequence comparison of the lktC, lktB, and lktD genes in 23 Mannheimia (Pasteurella) haemolytica, 6 Mannheimia glucosida, and 4 Pasteurella trehalosi strains. Sequence variation in the lktA gene has been described previously (R. L. Davies et al., J. Bacteriol. 183:1394-1404, 2001). The leukotoxin operon of M. haemolytica has a complex mosaic structure and has been derived by extensive inter- and intraspecies horizontal DNA transfer and intragenic recombination events. However, the pattern of recombination varies throughout the operon and among the different evolutionary lineages of M. haemolytica. The lktA and lktB genes have the most complex mosaic structures with segments derived from up to four different sources, including M. glucosida and P. trehalosi. In contrast, the lktD gene is highly conserved in M. haemolytica. The lktC, lktA, and lktB genes of strains representing the major ovine lineages contain recombinant segments derived from bovine or bovine-like serotype A2 strains. These findings support the previous conclusion that host switching of bovine A2 strains from cattle to sheep has played a major role in the evolution of the leukotoxin operon in ovine strains of M. haemolytica. Homologous segments of donor and recipient alleles are identical, or nearly identical, indicating that the recombinational exchanges occurred relatively recent in evolutionary terms. The 5' and 3' ends of the operon are highly conserved in M. haemolytica, which suggests that multiple horizontal exchanges of the complete operon have occurred by a common mechanism such as transduction. Although the lktA and lktB genes both have complex mosaic structures and high nucleotide substitution rates, the amino acid diversity of LktB is significantly lower than that of LktA due to a higher degree of evolutionary constraint against amino acid replacement. The recombinational exchanges within the leukotoxin operon have had greatest effect on LktA and probably provide an adaptive advantage against the host antibody response by generating novel antigenic variation at surface-exposed sites.     

Deletion analysis resolves cell-binding and lytic domains of the Pasteurella leukotoxin

A series of internal deletions in the lktA gene of Pasteurella haemolytica has been constructed. All of the deletions eliminated the lytic activity of the leukotoxin towards the bovine lymphoma cell line, BL-3. Deletions removing segments of the amino-proximal hydrophobic region, which is thought to constitute an essential membrane-spanning domain, were found to agglutinate BL-3 cells. Agglutination was similar to lysis by the wild-type toxin in that it was dependent upon the presence of calcium and required expression of the lktC gene. The agglutinating deletion proteins protected BL-3 cells from lysis by the wild-type toxin in a competitive fashion. This suggests that these mutants bind to a surface feature of the leukocyte which interacts with the native leukotoxin. These findings demonstrate that the cell-binding and lytic domains of the leukotoxin are separable.     

Sequence diversity and molecular evolution of the leukotoxin (lktA) gene in bovine and ovine strains of Mannheimia (Pasteurella) haemolytica

The molecular evolution of the leukotoxin structural gene (lktA) of Mannheimia (Pasteurella) haemolytica was investigated by nucleotide sequence comparison of lktA in 31 bovine and ovine strains representing the various evolutionary lineages and serotypes of the species. Eight major allelic variants (1.4 to 15.7% nucleotide divergence) were identified; these have mosaic structures of varying degrees of complexity reflecting a history of horizontal gene transfer and extensive intragenic recombination. The presence of identical alleles in strains of different genetic backgrounds suggests that assortative (entire gene) recombination has also contributed to strain diversification in M. haemolytica. Five allelic variants occur only in ovine strains and consist of recombinant segments derived from as many as four different sources. Four of these alleles consist of DNA (52.8 to 96.7%) derived from the lktA gene of the two related species Mannheimia glucosida and Pasteurella trehalosi, and four contain recombinant segments derived from an allele that is associated exclusively with bovine or bovine-like serotype A2 strains. The two major lineages of ovine serotype A2 strains possess lktA alleles that have very different evolutionary histories and encode divergent leukotoxins (5.3% amino acid divergence), but both contain segments derived from the bovine allele. Homologous segments of donor and recipient alleles are identical or nearly identical, indicating that the recombination events are relatively recent and probably postdate the domestication of cattle and sheep. Our findings suggest that host switching of bovine strains from cattle to sheep, together with inter- and intraspecies recombinational exchanges, has played an important role in generating leukotoxin diversity in ovine strains. In contrast, there is limited allelic diversity of lktA in bovine strains, suggesting that transmission of strains from sheep to cattle has been less important in leukotoxin evolution.     

Proteomic analysis using an unfinished bacterial genome: the effects of subminimum inhibitory concentrations of antibiotics on Mannheimia haemolytica virulence factor expression

Here we identify, using nonelectrophoretic proteomics, effects of subminimum inhibitory concentrations (subMIC) of two antibiotic preparations, chlortetracycline (CTC), and chlortetracycline-sulfamethazine (CTC + SMZ), on protein expression in the bovine respiratory pathogen Mannheimia haemolytica. The M. haemolytica genome is currently in draft form, and annotation is incomplete. Relying on the principle of gene sequence conservation across species, we used annotated genomes from closely related species to identify, confirm, and functionally annotate 495 M. haemolytica proteins. To conduct quantitative comparative proteomics, we developed a protein quantitation method based on the cross correlation function of the SEQUEST algorithm. When M. haemolytica was cultivated in the presence of 1/4 MIC of CTC and CTC + SMZ, expression of proteins involved in energy production, nucleotide metabolism, translation, and the bacterial stress response (chaperones) were affected. The most notable subMIC effect was a significant decrease in the expression of leukotoxin A, which is an important M. haemolytica virulence factor. Reduction in leukotoxin expression could be one of the molecular mechanisms responsible for the efficacy of these antibiotics against bovine respiratory disease.     

Multi-sample acoustic biosensing microsystem for protein interaction analysis

The current work describes a novel setup for multi-sample biomolecular analysis. It is based on the assembly of a dual acoustic device chip with a four-channel microfluidic module, forming an array of eight available domains for experiments. Initially, multiple detection was demonstrated via the specific interaction of neutravidin with four different biotinylated proteins, namely protein G, protein A, bovine serum albumin, and immunoglobulin G; results revealed a reproducibility between the microchannel domains better than 90%. Real-time analysis of the binding interactions was used to calculate the affinity and kinetic constants of the four biotinylated molecules binding to surface-immobilized neutravidin; this was the first time that this information was derived using a biosensing device and four biotinylated molecules. Interestingly, all calculated kinetic and affinity constants resemble those typical of antibody-antigen interactions, although the investigated specific binding was of avidin-biotin nature. Finally, under device pre-functionalization conditions, it was possible to probe eight interactions all together, exploiting the full capacity of the microsystem and reducing significantly the analysis time, contrary to the use of the standard acoustic device configuration. The outcome of this full-scale validation opens the way for the integrated acoustic platform to be implemented in even higher throughput detection for future diagnostic/biomedical applications, as well as in fundamental research studies regarding biomolecular interaction investigation and characterization.     

The leukotoxin of Pasteurella haemolytica binds to β2 integrins on bovine leukocytes

The putative receptor proteins of Pasteurella haemolytica leukotoxin were isolated from bovine polymorphonuclear neutrophil lysate by affinity chromatography on a leukotoxin-specific monoclonal antibody column to which the leukotoxin was pre-bound. SDS-PAGE of the purified proteins showed four bands at 180 kDa, 170 kDa, 150 kDa and 95 kDa, in addition to the expected 102-kDa leukotoxin band and a series of bands with molecular masses lower than 102 kDa representing the disintegrated leukotoxin. N-terminal amino acid sequencing of the 170-kDa band showed homology with human and murine CD11b. The purified proteins reacted specifically with monoclonal antibodies specific for CD11a, CD11b, CD11c (the alpha chains of beta(2) integrins), and CD18 (the beta chain of beta(2) integrins). Pre-incubation of polymorphonuclear neutrophils with a monoclonal antibody specific for CD18 reduced the cytotoxicity of the leukotoxin to the cells. These results indicate that the leukotoxin binds to the beta(2) integrins on bovine leukocytes, very likely via CD18.     

Characterization of the caprine (Capra hircus) beta-2 integrin CD18-encoding cDNA and identification of mutations potentially responsible for the ruminant-specific virulence of Mannheimia haemolytica

The leukocyte integrins play a critical role in a great number of cellular adhesive interactions during the immune response. We describe here the isolation and characterization of the caprine beta(2) (CD18) sub-unit, common to the leukocyte beta(2)-integrin family. The deduced 770-amino-acid sequence reveals a transmembrane protein with 80, 81, 83, 96 and 99% identity with its canine, murine, human, bovine and ovine homologues respectively. Analysis of CD18 sequences emphasizes the functional importance of the beta(2) sub-unit I-like domain, and included metal ion-dependent adhesion site-like motif and confirms that of the cytoplasmic tail. Moreover, comparisons of ruminant versus non-ruminant CD18 sequences allowed the identification of 16 potential mutation sites that could be held responsible for the unique virulence of Mannheimia haemolytica for ruminants. Mannheimiosis is known to be the major respiratory disease among ruminants, whereas it is not pathogenic for other mammals, an observation that has been attributed to a specific interaction between M. haemolytica leukotoxin and ruminants' CD18. Therefore, the data provided here offer the possibility to explore new avenues in studies based on the caprine model and provide key information for future studies aimed at elucidating the molecular mechanisms underlying the ruminant-specific virulence of M. haemolytica.     

Analysis of the Actinobacillus actinomycetemcomitans leukotoxin gene. Delineation of unique features and comparison to homologous toxins

Actinobacillus actinomycetemcomitans leukotoxin has been implicated as a virulence factor in human infections. To initiate delineation of leukotoxin structure/function relationships, molecular cloning of the leukotoxin gene was carried out. When an A. actinomycetemcomitans genomic DNA library in lambda EMBL3 was screened using a 1.3-kilobase pair restriction fragment containing a portion of the leukotoxin gene, 13 positive recombinants were identified. One recombinant, designated lambda OP8, containing a 16-kilobase pair insert was selected for detailed study. Lysates from lambda OP8, but not control lysates, exhibited leukotoxic activity with target cell specificity identical to the native toxin. Western blots identified the recombinant-produced toxin as a 125-kDa protein doublet identical in mobility to the native toxin. Restriction enzyme and extensive DNA analyses demonstrated that the leukotoxin gene showed strong homology to two other toxins produced by Escherichia coli and Pasteurella haemolytica. As in the other two species, the A. actinomycetemcomitans toxin is contained in a cluster of four genes in which the A gene encodes the toxin and the products of the B, C, and D genes are involved in posttranslational modification of the toxin and its membrane insertion and secretion. The target cell specificity of the A. actinomycetemcomitans toxin differs from the other two toxins and is restricted to human and some non-human primate cells of the monomyelocytic lineage. The A. actinomycetemcomitans leukotoxin is not secreted but remains associated with the bacterial membrane, possibly through a hydrophobic domain at the carboxyl terminus which distinguishes it from the E. coli and P. haemolytica toxins.     

Mannheimia haemolytica leukotoxin-induced increase in leukotriene B4 production by bovine neutrophils is mediated by a sustained and excessive increase in intracellular calcium concentration

Isolated bovine neutrophils were used to study the relationship between the duration and magnitude of the Mannheimia haemolytica leukotoxin-induced increase in intracellular calcium concentration and leukotriene B4 synthesis. In contrast to recombinant human C5a, which caused a transient, small increase in intracellular calcium concentration and no effects on leukotriene B4 synthesis, exposure of neutrophils to leukotoxin resulted in a rapid, sustained, large increase in intracellular calcium concentration, followed by leukotriene B4 synthesis. This leukotoxin-induced response was similar to those produced by the calcium ionophore, A23187, and phorbol myristate acetate, which also caused significant leukotriene B4 production. Manipulation of the duration and magnitude of leukotoxin- and A23187-induced intracellular calcium concentration increase confirmed that a high and sustained intracellular calcium concentration was necessary to stimulate production of leukotriene B4, which is believed to play an important role in the pathogenesis of pulmonary M. haemolytica infection.     

Effect of pH, temperature and nutrient limitations on growth and leukotoxin production by Mannheimia haemolytica in batch and continuous culture

AIMS: Quantification of the effects of pH, temperature and nutrient limitations on the growth and leukotoxin (LKT) production parameters of Mannheimia haemolytica in batch and chemostat culture. METHODS AND RESULTS: Mannheimia haemolytica strains OVI-1 and PH12296 were grown aerobically in two semi-defined media. In amino acid-limited cultures, the LKT concentration and yield in terms of biomass (Y(LKT/x)) were up to eightfold greater than in carbon-limited cultures. Supplementing amino acid-limited chemostat cultures with cysteine, glutamine, ferric iron and manganese further enhanced the Y(LKT/x) values up to threefold. Supplementation of an amino acid-limited batch culture of M. haemolytica strain OVI-1 with these nutrients resulted in an LKT concentration of 1.77 g l(-1) that was 45-fold greater than that obtained in RPMI 1640 medium. Aerobiosis enhanced LKT production. High acetic acid concentrations were produced under carbon-sufficient conditions. The highest maximum specific growth rates were recorded in the range of pH 6.8 to 7.8 and 37 to 40 degrees C. CONCLUSIONS: An amino acid-limited culture medium greatly improved LKT production in aerobic batch culture, which could be further enhanced by supplementation with cysteine, glutamine, ferric iron and manganese. SIGNIFICANCE AND IMPACT OF THE STUDY: It was demonstrated that LKT production by M. haemolytica could be dramatically increased through manipulation of the culture medium composition, which could benefit the production of LKT-based vaccines against bovine shipping fever pneumonia.     

Identification and expression of the Actinobacillus actinomycetemcomitans leukotoxin gene

The leukotoxin produced by the oral bacterium Actinobacillus actinomycetemcomitans has been implicated in the pathogenesis of juvenile periodontitis. In order to elucidate the structure of the leukotoxin, molecular cloning of the leukotoxin gene was carried out. A DNA library of A. actinomycetemcomitans, strain JP2, was constructed by partial digestion of genomic DNA with Sau3AI and ligation of 0.5 to 5.0 kilobase pair fragments into the Bam HI site of the plasmid vector pENN-vrf. After transformation into E. coli RR1 (lambda cI857), the clones were screened for the production of A. actinomycetemcomitans leukotoxin with polyclonal antibody. Six immunoreactive clones were identified. The clones expressed proteins which ranged from 21-80 kilodaltons, and the clone designated pII-2, producing the largest protein was selected for further study. Antibodies eluted from immobilized pII-2 protein also recognized the native A. actinomycetemcomitans leukotoxin molecule indicating that both molecules shared at least one epitope. DNA sequence analysis demonstrated that there are regions of significant amino acid sequence homology between the cloned A. actinomycetemcomitans leukotoxin and two other cytolysins, Escherichia coli alpha-hemolysin and Pasteurella haemolytica leukotoxin, suggesting that a family of cytolysins may exist which share a common mechanism of killing but vary in their target cell specificity.     

Different respiratory syncytial virus and Quillaja saponin formulations induce murine peritoneal cells to express different proinflammatory cytokine profiles

The recognition of a pathogen or a vaccine antigen formulation by cells in the innate immune system leads to production of proinflammatory cytokines, which will determine the ensuing acquired immune response quantitatively and qualitatively. Tumour necrosis factor (TNF)-alpha, interleukin (IL)-1 and IL-6 are the first set of cytokines produced upon such an encounter, which have roles both in protective immunity and immunopathogenesis evident with respiratory syncytial virus (RSV). RSV antigens in different physical adjuvant-vaccine formulations were analysed for their capacity to provoke cultured murine peritoneal cells to produce these three proinflammatory cytokines. RSV immunostimulating complex (ISCOM), i.e. both antigen and adjuvant are incorporated in the same particle, induced high levels of IL-1alpha being of the same magnitude or higher than those of live RSV and lipopolysaccharide (LPS). Live virus and LPS induced higher levels of IL-6 and TNF-alpha than ISCOM and so did non-adjuvanted UV-inactivated RSV but only at high doses. ISCOM-Matrix, i.e. ISCOM without antigens, admixed as a separate entity to inactivated RSV, downregulated or blocked the cytokine response to the inactivated RSV in contrast to ISCOM. Kinetic studies showed that ISCOM induced cytokine production first detected at hours 1, 2, 4 for TNF-alpha, IL-6 and IL-1alpha respectively, which was earlier than for the other antigen formulations containing corresponding doses of antigen and/or Quillaja adjuvant. Peak values for production of TNF-alpha and IL-6 were at 8 h and for IL-1alpha at 72 h following stimulation with ISCOM. The delayed appearance of IL-1alpha may reflect the cell-bound nature of this cytokine.     

成人腹泻轮状病毒的提纯及其高价免疫血清的制备

本文采用交叉免疫电泳技术,首先提纯了成人腹泻轮状病毒(Adult DiarrhoeaRotavirus简称:ADRV),并经电镜确认,用作免疫抗原,首次制备了高价兔与豚鼠抗ADRV血清和小鼠抗ADRV腹水,其特异性经交叉免疫电泳和对流免疫电泳鉴定,其效价经酶联免疫吸附试验(ELISA)检测均在1:2,000以上。     

Quantum dot-doped silica nanoparticles as probes for targeting of T-lymphocytes

To enhance diagnostic or therapeutic efficacy, novel nanomaterials must be engineered to function in biologically relevant environments, be visible by conventional fluorescent microscopy, and have multivalent loading capacity for easy detection or effective drug delivery. Here we report the fabrication of silica nanoparticles doped with quantum dots and superficially functionalized with amino and phosphonate groups. The amino groups were acylated with a water-soluble biotin-labeling reagent. The biotinylated nanoparticles were subsequently decorated with neutravidin by exploiting the strong affinity between neutravidin and biotin. The resultant neutravidin-decorated fluorescent silica nanoparticles stably dispersed under physiological conditions, were visible by conventional optical and confocal fluorescent microscopy, and could be further functionalized with macromolecules, nucleic acids, and polymers. We also coated the surface of the nanoparticles with biotinylated mouse anti-human CD3 (alphaCD3). The resultant fluorescent nanoassembly was taken up by Jurkat T cells through receptor-mediated endocytosis and was partially released to lysosomes. Thus, quantum dot-doped silica nanoparticles decorated with neutravidin represent a potentially excellent scaffold for constructing specific intracellular nanoprobes and transporters.     

Quantification of intact 146S foot-and-mouth disease antigen for vaccine production by a double antibody sandwich ELISA using monoclonal antibodies

A double antibody sandwich (DAS) enzyme-linked immunosorbent assay (ELISA) was developed to quantify 146S antigen of foot-and-mouth disease virus (FMDV) strain A10 Holland grown in suspension cultures of surviving bovine tongue epithelium. When virus harvests were incubated with trypsin--which affects VP1, the most immunogenic structural protein of FMDV--the concentration of 146S antigen as determined by ELISA was reduced by greater than 90%. Therefore, the test detected essentially only those virus particles with intact VP1. When the test was compared with the sucrose density gradient method, concentrations of 146S antigen correlated well (r = 0.87). The rate of variation in both tests was the same. In contrast to the sucrose density gradient method, the DAS-ELISA can simultaneously quantify 146S antigen in many samples, and also indicates when VP1 of 146S particles has disintegrated by the action of proteases.     

Kinetics of growth and leukotoxin production by <Emphasis Type="Italic">Mannheimia haemolytica</Emphasis> in continuous culture

The growth and product formation kinetics of the bovine pathogen Mannheimia (Pasteurella) haemolytica strain OVI-1 in continuous culture were investigated. The leukotoxin (LKT) concentration and yield on biomass could substantially be enhanced by supplementation of a carbon-limited medium with an amino acid mixture or a mixture of cysteine and glutamine. Acetic acid was a major product, increasing to 1.66 g l(-1) in carbon-limited chemostat culture at intermediate dilution rates and accounting for more than 80% of the glucose carbon, whereas in amino acid-limited cultures high acetic acid concentrations were produced at low dilution rates, suggesting a carbon-overflow metabolism. The maintenance coefficients of carbon-limited and carbon-sufficient cultures were 0.07 and 0.88 mmol glucose g(-1) h(-1), respectively. LKT production was partially growth-associated and the LKT concentration was maximised to 0.15 g l(-1) and acetic acid production minimised by using a carbon-limited medium and a low dilution rate.     

牛奶过敏原β-乳球蛋白的重组制备及磁微粒化学发光检测法的建立

运用原核系统表达牛奶β-乳球蛋白(Bos d5)蛋白,建立一种纳米磁微粒化学发光方法用于检测牛奶组分过敏原β-乳球蛋白特异性Ig E抗体的含量。通过优化合成牛奶β-乳球蛋白基因,与质粒重组导入Rosetta原核表达菌株中表达目标蛋白,纯化的重组蛋白采用生物素标记后,在化学发光平台上进行过敏原项目检测。获得纯度﹥85%的22.5 kD重组牛β-乳球蛋白,将生物素化的重组蛋白用于牛奶β-乳球蛋白纳米磁微粒检测试剂盒,检测临床110例血清样本,和Phadia进行方法学比对阳性符合率为88.9%,阴性符合率97.3%,总符合率94.5%,P<0.001,χ^2=84.238,Kappa=0.874,其与Phadia参照试剂盒同样具有良好的检测能力。原核表达系统中获得的重组牛奶过敏组分β-乳球蛋白具有较好的免疫活性,开发的免疫诊断试剂盒具备良好的性能,可用于临床辅助诊断。     

Generic Method for Attaching Biomolecules via Avidin-Biotin Complexes Immobilized on Films of Regenerated and Nanofibrillar Cellulose

We investigated the adsorption and chemical conjugation of avidin and its deglycosylated form, neutravidin, on films of regenerated and nanofibrillar cellulose. The dynamics and extent of biomolecular attachment were monitored in situ by quartz crystal microbalance microgravimetry and ex situ via surface analyses with atomic force microscopy and X-ray photoelectron spectroscopy. The installation of carboxyl groups on cellulose after modification with carboxymethylated cellulose (CMC) or TEMPO-oxidation significantly increases physisorption of avidins, which can be then covalently conjugated by using 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride/N-hydroxysuccinimide (EDS/NHS) coupling chemistries. The developed cellulose-avidin biointerfaces are able to scavenge biotinylated molecules from solution as demonstrated by successful surface complexation of biotinylated bovine serum albumin (Biotin-BSA) and antihuman immunoglobulin G (Biotin-anti-hIgG). Finally, we show that cellulose substrates carrying immobilized anti-hIgG are effective in detecting human immunoglobulin G (hIgG) from fluid matrices.