The leptospiral antigen Lp49 is a two-domain protein with putative protein binding function

Pathogenic Leptospira is the etiological agent of leptospirosis, a life-threatening disease that affects populations worldwide. Currently available vaccines have limited effectiveness and therapeutic interventions are complicated by the difficulty in making an early diagnosis of leptospirosis. The genome of Leptospira interrogans was recently sequenced and comparative genomic analysis contributed to the identification of surface antigens, potential candidates for development of new vaccines and serodiagnosis. Lp49 is a membrane-associated protein recognized by antibodies present in sera from early and convalescent phases of leptospirosis patients. Its crystal structure was determined by single-wavelength anomalous diffraction using selenomethionine-labelled crystals and refined at 2.0 Å resolution. Lp49 is composed of two domains and belongs to the all-beta-proteins class. The N-terminal domain folds in an immunoglobulin-like beta-sandwich structure, whereas the C-terminal domain presents a seven-bladed beta-propeller fold. Structural analysis of Lp49 indicates putative protein–protein binding sites, suggesting a role in Leptospira–host interaction. This is the first crystal structure of a leptospiral antigen described to date.     

Cloning and nucleotide sequence of a cDNA encoding a major fucoxanthin-, chlorophylla/c-containing protein from the chrysophyteIsochrysis galbana: implications for evolution of thecab gene family

We investigated the primary structure of a cDNA encoding a light-harvesting protein from the marine chrysophyteIsochrysis galbana. Antibodies raised against the major fucoxanthin, chlorophylla/c-binding light-harvesting protein (FCP) ofI. galbana were used to select a cDNA clone encoding one of the FCP apoproteins. The nucleic acid and deduced amino acid sequences reveal conserved regions within the first and third transmembrane spans with Chla/b-binding proteins and with FCPs of another chromophyte. However, the amino acid identity betweenI. galbana FCP and othercab genes of FCPs is only ca. 30%. Phylogenetic analyses demonstrated that the FCP genes of both diatoms and chrysophytes sequenced to date are more closely related tocab genes encoding LHC I, CP 29, and CP 24 of higher plants than tocab genes encoding LHC II of chlorophytes. We propose that LHC I, CP 24 and CP 29 and FCP might have originated from a common ancestral chl binding protein and that the major LHC II of Chla/b-containing organisms arose after the divergence between the chromophytes and the chlorophytes.     

Analysis of differential proteomes in pathogenic and non‐pathogenic Leptospira: Potential pathogenic and virulence factors

Leptospirosis is a bacterial zoonotic disease caused by spirochetes in the genus Leptospira. To date, factors determining the pathogenicity and virulence of leptospires remain unclear. We performed a gel‐based proteomic analysis to evaluate differential leptospiral proteomes in the pathogenic L. interrogans (serovars Australis, Bratislava, Autumnalis, and Icterohaemorrhagiae) and the non‐pathogenic L. biflexa (serovar Patoc). Quantitative proteome analysis and MS protein identification revealed 42 forms of 33 unique proteins whose levels were significantly greater in the pathogenic serovars compared with the non‐pathogenic serovar. Among the four pathogenic serovars, the more virulent serovar Icterohaemorrhagiae (which is most commonly associated with severe leptospirosis in patients) had significantly greater levels of 14 forms of 12 unique proteins, when compared with the other three pathogenic serovars. Some of these identified proteins may serve as the pathogenic and/or virulence factors of leptospirosis.     

Leptospira antibodies in wild boars (<Emphasis Type="Italic">Sus scrofa</Emphasis>) in Slovenia

Serum samples collected from 437 shot wild boars (Sus scrofa) were tested for the presence of antibodies against Leptospira interrogans sensu lato in wild boar in Slovenia. Assessment of leptospira-specific antibodies was performed by microscopic agglutination test. Antibodies against at least one of the pathogenic serovars were detected in 200 (45.8%) sera. From 200 positive samples, 100 samples (50%) had positive titre against a single serovar, while 100 (50%) samples had positive titres against two or more serovars. The most frequently detected antibodies were those against serovar Tarassovi. This investigation confirmed the presence of different pathogenic serovars in wild boar across Slovenia. It can be concluded that wild boars are natural reservoirs of at least some of the leptospiral serovars that represent a potential source of leptospirosis for other wild and domestic animals, as well as for humans.     

Interactions between epsilon, the proofreading subunit of DNA polymerase III, and proteins involved in the SOS response of Escherichia coli

Summary Epsilon, a fidelity subunit of Escherichia coli DNA Polymerase III, is encoded by dnaQ ⁺. dnaQ49 is a recessive allele that confers temperature-sensitive and saltsuppressible phenotypes for both replication fidelity and viability. SOS mutagenesis in E. coli is regulated by LexA and requires activated RecA (RecA^*) and the products of the umuDC operon. dnaQ49 strains with various recA, lexA and umuDC alleles were constructed to determine if activities induced as part of the SOS response influence epsilon activity. We found: (1) both UmuDC and RecA^* independently enhance the dnaQ49 mutator phenotype, and (2) expression of RecA^* activity in the absence of UmuDC function increases the temperature sensitivity for viability of dnaQ49. These results support the hypothesis that RecA and one or both of the UmuDC proteins interact with the replication complex during SOS mutagenesis.     

Differential reactivity of filarial antigens with human sera from bancroftian filariasis endemic zone

The reacting pattern of circulating filarial antigen fraction-2 fromWuchereria bancrofti and soluble antigen from adultBrugia malayi with bancroftian filarial sera were analysed by immunoblotting technique and enzyme linked immunosorbent assay. Microfilaraemic sera reacted specifically with proteins of molecular weight 200, 120, 97, 56, 54, 43, 26 and 17 kDa of circulating Filarial antigen fraction-2 and 44, 40, 38, 31, 22 and 18 kDa ofBrugia malayi adult soluble antigen. Clinical filarial sera identified protein molecules of 56, 54 and 42 kDa of circulating filarial antigen fraction-2 and 19, 16 and 14 kDa ofBrugia malayi adult soluble antigen. Some components of both the antigen preparation were also identified by endemic normal serai.e.proteins 120, 97, 62, 43 and 33 kDa of circulating filarial antigen fraction-2 and 170, 120, 43, 31 and 12 kDa ofBrugia malayi adult soluble antigen. One of the sodium dodecyl sulphate-polyacrylamide gel electropherosis fractions of circulating filarial antigen fraction-2 (CFA₂-8) andBrugia malayi adult soluble antigen fraction-6 when used in enzyme linked immunosorbent assay could differentiate microfilaraemic sera from endemic normal and clinical filarial sera. The other antigen fractions (CFA₂-2, 6 and 7 andBmA-2) showed a high geometric mean titre of filarial immunoglobulin G antibodies in endemic normal sera when compared to microfilaraemia and clinical filarial sera. These proteins need to be further studied to assess their involvement in protecting from filarial infection in an endemic area.     

Whole-genome analysis of Leptospira interrogans to identify potential vaccine candidates against leptospirosis

Leptospirosis is an important global human and veterinary health problem. Humans can be infected by exposure to chronically infected animals and their environment. An important focus of the current leptospiral research is the identification of outer membrane proteins (OMPs). Due to their location, leptospiral OMPs are likely to be relevant in host-pathogen interactions, hence their potential ability to stimulate heterologous immunity. The existing whole-genome sequence of Leptospira interrogans serovar Copenhageni offers a unique opportunity to search for cell surface proteins. Predicted genes encoding potential surface proteins were amplified from genomic DNA by PCR methodology and cloned into an Escherichia coli expression system. The partially purified recombinant proteins were probed by Western blotting with sera from human patients diagnosed with leptospirosis. Sixteen proteins, out of a hundred tested, were recognized by antibodies present in human sera. Four of these proteins were conserved among eight serovars of L. interrogans and absent in the non-pathogenic Leptospira biflexa. These proteins might be useful for the diagnosis of the disease as well as potential vaccine candidates.     

Cloning, expression, and homology modeling of GroEL protein from Leptospira interrogans serovar autumnalis strain N2

Leptospirosis is an infectious bacterial disease caused by Leptospira species. In this study, we cloned and sequenced the gene encoding the immunodominant protein GroEL from L. interrogans serovar Autumnalis strain N2, which was isolated from the urine of a patient during an outbreak of leptospirosis in Chennai, India. This groEL gene encodes a protein of 60 kDa with a high degree of homology (99% similarity) to those of other leptospiral serovars. Recombinant GroEL was overexpressed in Escherichia coli. Immunoblot analysis indicated that the sera from confirmed leptospirosis patients showed strong reactivity with the recombinant GroEL while no reactivity was observed with the sera from seronegative control patient. In addition, the 3D structure of GroEL was constructed using chaperonin complex cpn60 from Thermus thermophilus as template and validated. The results indicated a Z-score of ?8.35, which is in good agreement with the expected value for a protein. The superposition of the Cα traces of cpn60 structure and predicted structure of leptospiral GroEL indicates good agreement of secondary structure elements with an RMSD value of 1.5 Å. Further study is necessary to evaluate GroEL for serological diagnosis of leptospirosis and for its potential as a vaccine component.     

The biology and immunogenicity of a 34-kDa antigen of Stachybotrys chartarum sensu lato

We are interested in isolating and identifying antigenic fungal proteins from species that grow on damp building materials. Fungal spores contain a modest number of proteins present in high concentrations and mycelia can contain large numbers of such proteins. Their distribution in mycelium when grown on laboratory media often does not reflect their occurrence in nature. Stachybotrys chartarum occurs on wet cellulose based building materials including paper-faced gypsum board and wood. To find potentially characteristic antigenic proteins from this fungus, we screened mycelium and spores from geographically representative strains using sera from a large number of patients who had been identified as having antibodies to fungi that grow in the built environment. Proteins were sought that were common across strains and for which antibodies were present in many sera. The use of human sera as the source of antibodies permitted the conclusion that any proteins of interest were produced in nature. The target proteins were shown to be present in spores/mycelium produced on natural substrata including straw and paper. This report concerns the isolation and partial characterization of a 34-kDa exoantigen. It is a glycoprotein that appears to comprise two subunits with molecular weights of 34 and 21 kDa which are not bound together through disulfide bonds. Both proteins have pIs in the acidic range. The sequences indicate that the protein is related to an extracellular DNase from Nectria haematococca. The partial sequences of the larger subunit are similar to that of a hypothetical protein from Gibberella zeae.     

Sequence analysis of a group of low molecular-weight plasmids carrying multiple IS903 elements flanking a kanamycin resistance aph gene in Salmonella enterica serovars

A group of low molecular-weight ColE1-like plasmids carrying the aph sequence type aph(ii) from three different Salmonella serovars were sequenced. These plasmids carry two or more copies of IS903 elements, with up to 21bp sequence differences to one another, two of which flank the aph gene. This group of plasmids did not appear to carry any known mobilization genes and instead carry three open reading frames encoding hypothetical proteins of unknown function possibly organized in an operon. The plasmid replication region (RNA I/II--rom) of this plasmid group showed extensive homology to that of pKPN2 plasmid of Klebsiella pneumoniae and pCol-let plasmid of Escherichia coli. Three of the four plasmids had identical sequences, and the fourth had an extra copy of IS903 with target duplication, suggesting a recent divergence in the different Salmonella serovars from a common ancestor.     

Analysis of storage proteins in normal and aborted seeds from embryo-lethal mutants of Arabidopsis thaliana

The major storage proteins isolated from wild-type seeds of Arabidopsis thaliana (L.) Heynh., strain Columbia, were studied by sucrose gradient centrifugation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Both the hypocotyl and cotyledons of mature embryos contained abundant 12 S (cruciferin) and 2 S (arabin) proteins that appeared similar in size and subunit composition to the cruciferin (12 S) and napin (1.7 S) seed-storage proteins of Brassica napus. The 12 S protein from Arabidopsis was resolved by SDS-PAGE into two groups of subunits with approximate relative molecular weights of 22–23 kDa (kilodalton) and 30–34 kDa. These polypeptides accumulated late in embryo development, disappeared early in germination, and were not detected in other vegetative or reproductive tissues. Accumulation of the 12 S proteins in aborted seeds from nine embryo-lethal mutants with different patterns of abnormal development was studied to determine the extent of cellular differentiation in arrested embryos from each mutant line. Abundant 12 S proteins were found in arrested embryos from two mutants with late lethal phases, but not in seven other mutants with lethal phases ranging from the globular to the cotyledon stages of embryo development. These results indicate that the accumulation of seed-storage proteins in wild-type embryos of Arabidopsis is closely tied to morphogenetic changes that occur during embryo development. Embryo-lethal mutants may therefore be useful in future studies on the developmental regulation of storage-protein synthesis.Abbreviations kDa kilodalton - M_r relative molecular weight - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulfate     

Isolation of Beauveria bassiana and Metarhizium anisopliae var. anisopliae from Boophilus microplus tick (Canestrini, 1887), in Rio de Janeiro State,Brazil

The objective of this work were to isolate and identify strains of entomopathogenic fungi from ingurgitated female Boophilus microplus ticks, collected from the soil in the municipality of Paracambi, Rio de Janeiro State, Brazil. The ingurgitated females were inoculated in the selective medium oat dodine agar (oda), where 49 colonies of Beauveria bassiana (71%) and 20 of Metarhizium anisopliae var. anisopliae (29%) were isolated. These isolated strains characterize for the first time in Brazil the natural occurrence of these species of fungi in this tick, and will be used to conduct bioassays to evaluate the pathogenicity and virulence of these strains for ticks of the genus Boophilus microplus. This revised version was published online in June 2006 with corrections to the Cover Date.     

Analysis of the <Emphasis Type="Italic">MAT1-2-1</Emphasis> gene of <Emphasis Type="Italic">Colletotrichum lindemuthianum</Emphasis>

A single MAT1-2-1 gene was identified from a mating pair of the filamentous ascomycete Colletotrichum lindemuthianum. The MAT1-2-1 genes from both mating partners carried an open reading frame (ORF) of 870 bp encoding a putative protein of 290 amino acids that includes the highly conserved high mobility group (HMG) domain typical of the fungal MAT1-2-1 genes. Three introns were confirmed within the C. lindemuthianum ORF, two of which were found to be conserved relative to a previously reported MAT1-2-1 gene from C. gloeosporioides. The amino acid sequence of the HMG domain from C. lindemuthianum MAT1-2-1 was also compared with those from other ascomycetes. These results suggest that although the MAT1-2-1 genes are highly conserved among ascomycetes, the mechanism which defines mating partners in the genus Colletotrichum is distinct to the idiomorph system described for other members of this phylum.     

Constitutive expression of two endochitinases from root nodules ofElaeagnus umbellata confers resistance on transgenicArabidopsis plants against the fungal pathogenBotrytis cinerea

Plant chitinases have been known as pathogenesis-related (PR) proteins, but recent studies suggest that they play functional roles during normal plant growth and development. We previously isolated two cDNA clones encoding endochitinases,EuNOD-CHT1 and -CHT2, from the root nodules ofElaeagnus umbellata. These genes show differential expression patterns, with theEuNOD-CHT1 gene being active in the root nodules and meristems, whileEuNOD-CHT2 is preferentially expressed in the infected cells of those nodules. To elucidate the functional roles of these two endochitinases, we have now constitutively expressed each gene in a heterologous plant system,Arabidopsis thaliana. Stable inheritance and expression of the transgenes were confirmed by genomic Southern hybridization and RT-PCR. Our transgenic plants did not differ morphologically from the wild types. However, constitutive expression ofEuNOD-CHT1 and -CHT2 inArabidopsis resulted in increased resistance against a fungal pathogen,Botrytis cinerea, but not against a bacterial agent,Pseudomonas syringae pv. Tomato DC3000. Expression levels were enhanced by both wounding and jasmonic acid treatments (forEuNOD-CHT1), or by jasmonic acid only (forEuNOD-CHT2). These data suggest thatEuNOD-CHT1 and -CHT2 primarily play defensive roles during root nodule development inE. umbellata.     

Acidic and basic class III chitinase mRNA accumulation in response to TMV infection of tobacco

Complementary DNA clones encoding acidic and basic isoforms of the class III chitinase were isolated from Nicotiana tabacum. The clones share ca. 65% identity, are equally homologous to the class III chitinases from cucumber and Arabidopsis, and are members of small gene families in tobacco. An acidic class III chitinase was purified from the intercellular fluid of tobacco leaves infected with tobacco mosaic virus (TMV). Partial amino acid sequencing of the protein confirmed that it was encoded by one of the cDNA clones. The mRNAs of the class III chitinases are coordinately expressed in response to TMV infection, both in infected and uninfected tissue. The acidic and basic class III chitinases constitute previously undescribed pathogenesis-related proteins in tobacco.     

Counterimmunoelectrophoresis and opportunistic fungal infections

Sera from 35 apparently normal humans, 37 compromised human patients, 30 hedgehogs and 30 sheep, were examined for precipitating antibodies to four opportunistic fungi — Absidia corymbifera, Aspergillus fumigatus, Candida albicans and Rhizopus arrhizus — using Counterimmunoelectrophoresis (CIE).Precipitins to A. fumigatus were almost exclusively confined to specimens obtained from the compromised human group (51% of those examined) while Candida precipitating antibodies were detected in the sera of both normal (26%) and compromised (49%) humans and in 10% of the hedgehog specimens. Serum precipitins against the two phycomycetes included in the investigations were rare.Because of the complexity of most fungal antigen extracts, it appears essential that sera be tested against a number of different antigen concentrations if CIE is to be used with confidence in fungal serology.     

Identification and nucleotide sequence of the Acinetobacter calcoaceticus encoded trpE gene

Summary The trpE gene from Acinetobacter calcoaceticus encoding the anthranilate synthase component I was cloned, identified by deletion analysis and sequenced. It encodes a predicted polypeptide of 497 amino acids with a calculated molecular weight of 55323. Its primary structure shows 49% identical amino acids with the enzyme from Clostridium thermocellum, 45% with that of Thermus thermophilus and only 35% with that of Escherichia coli. The codon usage of the trpE genes encoding the most homologous enzymes differs greatly indicating selection for amino acid maintainance. The homologies are clustered in the C-terminal 200 amino acids of the sequences indicating that this part is important for enzymic activity.     

Leptospiral glycolipoprotein as a candidate antigen for serodiagnosis of human leptospirosis

Aims:  Development of a simple, specific, rapid and inexpensive Dot-ELISA test for early diagnosis of human leptospirosis.
Methods and Results:  Serum samples from 90 patients diagnosed with leptospirosis were analysed by Dot-ELISA test incorporating Glycolipoprotein (GLP) antigen from serovars Copenhageni and Patoc. Results were compared with those obtained with microscopic agglutination test, currently, the gold standard reference serological method. Serum samples from healthy blood bank donors and patients diagnosed with diseases other than leptospirosis were used as negative controls. The specificities of both GLP-based assays were 97·1% and 100% with serum samples from patients with other diseases and with serum samples from healthy control group, respectively. With serum samples from patients with acute leptospirosis, sensitivity was 76·6% with Dot-ELISA Copenhageni and 90·0% with Dot-ELISA Patoc. With serum samples from patients in convalescence, sensitivity was 100% with both GLP-based assays.
Conclusions:  This Dot-ELISA provides a candidate antigen for serodiagnosis of leptospirosis during all phases of illness and could be a good alternative method for the early diagnosis of leptospirosis.
Significance and Impact of the Study:  The Dot-ELISA test is simple, specific, rapid and inexpensive. It is suitable for identifying a large number of samples and, hence, reducing the death rate of patients with leptospirosis.     

巴西橡胶树HbWRKY1基因的克隆及转化烟草的初步研究

利用RACE结合RT-PCR技术,从巴西橡胶树(Hevea brasiliensis)总RNA中扩增得到长度为1234 bp的WRKY基因cDNA全长编码序列。通过氨基酸同源性比对,该序列推导的氨基酸序列与蓖麻、白杨的WRKY同源性分别为79%和73%,表明分离的cDNA序列为橡胶树WRKY基因,命名为HbWRKY1。通过构建pCAMBIA1304-HbWRKY1植物表达载体,经农杆菌GV3101介导,将HbWRKY1基因导入烟草(Nicotiana tabacum)中,对所获得的潮霉素抗性烟草株系进行PCR鉴定。结果表明,HbWRKY1基因已整合到65株转基因植株中。干旱胁迫试验表明,HbWRKY1的过量表达可以明显提高转基因烟草对干旱胁迫的耐受能力。这说明WRKY基因与橡胶树抗旱能力之间存在一定的关系。