Molecular cloning, nucleotide sequence, and characterization of lppB, encoding an antigenic 40-kilodalton lipoprotein of Haemophilus somnus.

Haemophilus somnus is a facultative intracellular pathogen which causes a wide range of diseases in cattle. To identify putative virulence determinants, a genomic library of H. somnus in Escherichia coli was screened for Congo red binding, a property associated with virulence in pathogenic bacteria, and subsequently with bovine hyperimmune sera raised against H. somnus HS25. A Congo red-binding clone carrying a 1.8-kb DNA insert was found to encode a strongly seroreactive LppB protein with an apparent molecular weight of 40,000. The nucleotide sequence of the entire DNA insert was determined. Two open reading frames coding for polypeptides with calculated molecular weights of 21,893 and 30,721 were identified. The larger open reading frame encoded LppB, while the smaller reading frame encoded a nonseroreactive protein with a relative molecular mass of approximately 18 kDa. The 16 amino-terminal amino acids of the deduced LppB polypeptide showed strong sequence homology to the signal peptide of secreted bacterial proteins, and the sequence Leu-Ala-Ala-Cys at the putative cleavage site corresponds to the consensus cleavage sequence of bacterial lipoproteins. Synthesis of the mature LppB lipoprotein in H. somnus was inhibited by globomycin, a specific inhibitor of signal peptidase II. LppB was localized to the outer membrane of H. somnus.     

Isolation, sequencing and expression of Bartonella henselae omp43 and predicted membrane topology of the deduced protein

The infection of and interaction of human endothelial cells with Bartonella henselae is one of the most interesting aspects of Bartonella -associated disease. The gene encoding the 43 kDa B. henselae outer membrane protein (Omp43) that binds endothelial cells was cloned and sequenced. Sequence analysis revealed an open reading frame of 1206 nucleotides coding for a protein of 402 amino acids. Analysis of the deduced amino acid sequence shows 38% identity over the entire sequence to the Brucella spp. In addition to this Omp2b porin also shows a signal sequence and peptidase cleavage site. Cleavage of the signal peptide results in a mature 380 amino acid polypeptide with a predicted molecular weight of 42 kDa. Omp43 was expressed in Escherichia coli as a fusion protein. Purified recombinant Omp43 at concentrations of 11 and 2.75 microg/ml bound to intact human umbilical vein endothelial cells. Membrane topology analysis predicts that Omp43 exists as a 16 stranded beta barrel protein, similar to that predicted for the Omp2b Brucella abortus porin. Characterization and expression of the gene encoding Omp43 should provide a tool for further investigation of the role of adherence to endothelial cells in the pathogenesis of B. henselae.     

Cloning and sequencing of the mumps virus fusion protein gene

The fusion protein (F) gene of mumps virus was cloned from a cDNA library constructed from infected cell mRNA. The F-specific plasmids were identified by hybridization to a degenerate oligonucleotide probe whose sequence was deduced from the N-terminal amino acid sequence of the F₂ protein. The complete nucleotide sequence of the F gene was determined. The gene is 1786 nucleotides long and encodes one long open reading frame of 538 amino acids. The F protein has a 19-amino acid signal peptide cleaved between Cys and Val residues. The cleavage site for activation of the F₀ protein into themature F_1,2 is ArgArgHisLysArg. A stretch of 30 hydrophobic amino acids near the C-terminus of the protein is followed by serveral charged amino acids and appears to serve as the anchoring domain for the protein in the lipid bilayer. The F gene of mumps virus is highly related to the F gene of the paramyxovirus SV-5.     

Sequence and analysis of the capsid protein of Nudaurelia capensis omega virus, an insect virus with T = 4 icosahedral symmetry.

We have determined the nucleotide sequence of the RNA2 segment of the Nudaurelia capensis omega virus genome. It was found to consist of 2448 nucleotides and contained one long open reading frame (ORF) encoding the 644 residue capsid protein. The deduced amino acid sequence of this protein reveals a positively charged amino terminus, a characteristic exhibited by several other viral capsid proteins, that is thought to be important for interactions between the capsid and the genomic RNA. There are 366 and 150 bases of untranslated sequence on the 5' and 3' ends, respectively. The ORF encoding the capsid protein initiates at the second AUG from the 5' end. The 5' proximal AUG specifies a short ORF (30 codons) which terminates 1 base before the initiation codon for the coat protein. Our analysis also revealed the presence of a second, previously unidentified polypeptide associated with Nudaurelia capensis omega virus particles. The amino terminal sequence of this protein corresponds to a portion of the long ORF beginning at codon 571. The lack of an initiation codon near this sequence indicates that the small polypeptide is most likely produced as a carboxy terminal cleavage product from a 70-kDa capsid protein precursor, yielding the previously identified 62-kDa protein and the 8-kDa protein that we have observed. The putative cleavage site would be at an Asn/Phe pair, somewhat resembling known cleavage sites (Asn/Ala) in the T = 3 Nodaviridae. In addition, we have found that there is also a second polypeptide similar in size to that from Nudaurelia capensis omega virus associated with particles of Nudaurelia capensis beta virus, the type member of Tetraviridae.     

The nucleotide sequence of the gene encoding the F protein of canine distemper virus: a comparison of the deduced amino acid sequence with other paramyxoviruses

The nucleotide sequence of the gene encoding the fusion protein of canine distemper virus was determined from cDNA clones derived from virus genome RNA and poly(A)+ RNA extracted from infected cells. The mRNA encoding the F protein is about 2300 nucleotides in length including the 3' poly(A) tail. There is a large open reading frame from nucleotides 86 to 2071 which begins at the first AUG codon in the F mRNA. This reading frame encodes a protein of 662 amino acid residues with a calculated mol. wt. of 73001. The first major hydrophobic domain in the amino acid sequence of the deduced protein (residues 104 to 130) may represent all or part of a signal sequence for cleavage of the N terminal part of the F2 protein. There are four potential N glycosylation sites in the F protein located within the F2 part of the molecule or the putative signal sequence, and one in the F1 portion. A second hydrophobic region corresponds to the proteolytic cleavage site which generates the F2 and F1 subunits. This stretches from residue 225 to 262 and the N terminal part of the F1 protein shows sequence conservation with the other paramyxoviruses. A third major hydrophobic domain near the C terminus of the F protein probably represents the membrane anchor for the F protein (residues 602 to 630). The F1 proteins of six paramyxoviruses are compared and shown to have substantial conservation of those residues important in the maintenance of tertiary structure of this protein.     

Molecular cloning, nucleotide sequence, and occurrence of a 16.5-kilodalton outer membrane protein of Brucella abortus with similarity to pal lipoproteins.

Recombinant lambda gt11 phages were selected by screening a genomic library of Brucella abortus DNA with monoclonal antibodies specific for a 16.5-kDa Brucella outer membrane protein (Omp16). The corresponding gene, named pal, was subcloned on a 0.7-kb AluI fragment. Immunoblotting confirmed the expression of a recombinant Omp16 in the transformants. DNA sequence analysis revealed an open reading frame of 168 codons. The deduced amino acid sequence agrees with an internal peptide sequence of native Omp16 and contains a potential lipoprotein signal peptide cleavage site, giving rise to a predicted mature protein of 144 amino acids. The predicted sequence of Omp16 also shows a remarkable degree of similarity to the sequences of three peptidoglycan-associated bacterial lipoproteins. In immunoblotting with a monoclonal antibody specific for Omp16, we demonstrated that Omp16 was expressed in the 34 Brucella strains tested, representing all six species and known biovars.     

The hbpA gene of Haemophilus influenzae type b encodes a heme-binding lipoprotein conserved among heme-dependent Haemophilus species.

A membrane-associated lipoprotein of Haemophilus influenzae type b has previously been shown to bind heme in vitro and to promote binding of this compound by Escherichia coli recombinants expressing this protein. The H. influenzae type b heme-binding protein A (HbpA) was found to be highly conserved with respect to both antigenicity and apparent molecular weight among heme-requiring Haemophilus species pathogenic for humans. To further the characterization of the structure and function of HbpA, the complete nucleotide sequence of its gene, hbpA, was determined. Analysis of the nucleotide sequence revealed a single large open reading frame of 1,638 bp encoding a protein of 546 amino acid residues, with a molecular weight of 60,695. The sequence of the amino-terminal end of this protein contained a potential site for lipid acylation and for cleavage by signal peptidase II, consistent with earlier biochemical evidence which indicated that HbpA is a lipoprotein. A search of GenBank for proteins with amino acid sequence similarity to HbpA revealed that the periplasmic dipeptide transport protein of E. coli, DppA, has 53% sequence identity to HbpA.     

Cloning and characterisation of malE in Burkholderia pseudomallei

No recombinant protein is available for serodiagnosis or skin test in the diagnosis of melioidosis. This report describes the cloning of the malE gene, which encodes an immunogenic protein of Burkholderia pseudomallei. Bi-directional DNA sequencing of malE revealed that the gene contained a single open reading frame encoding 416 amino acid residues with a predicted molecular mass of 44.4 kDa. BLAST analysis showed that the putative protein encoded by malE is homologous to the maltose-binding protein (MBP) of other bacteria. It has 48% and 63% amino acid identity and similarity with the MBP of Brucella abortus, and malE complementation assay showed that it partially complemented the function of the MBP of Escherichia coli. Several highly conserved regions among the MBP of B. pseudomallei, Br. abortus, Salmonella enterica serotype Typhimurium, E. coli and Enterobacter aerogenes were observed. These regions represent signatures A, B, C, D and F identified in the MBP of E. coli. Further sequence analysis revealed that the first 24 amino acid residues of the MBP of B. pseudomallei probably represent the N-terminal signal peptide of the protein. Similar to the signal peptide of the MBP of E. coli, Ent. aerogenes and S. Typhimurium, the MBP of B. pseudomallei contains two basic residues in the first eight amino acids, followed by a hydrophobic core, with the last three amino acids in the signal peptide being Ala-Gln-Ala, conforming to the consensus sequence Ala-X-Ala at positions -3 to -1 relative to the site of proteolytic cleavage for recognition by signal peptidase I. Further studies on serodiagnosis of melioidosis with recombinant MBP should be performed.     

Genetic and functional analysis of Haemophilus somnus high molecular weight-immunoglobulin binding proteins

Haemophilus somnus immunoglobulin binding proteins (IgBPs) are virulence associated but only one (p76) has been genetically defined. We determined the nucleotide sequence of the 5'-flanking region of the p76 gene. This region had been identified as the coding region for a series of high molecular weight (HMW)-IgBPs. Analysis of the nucleotide sequence indicated the gene (immunoglobulin binding protein A, ibpA) encoding the HMW and p76 IgBPs comprised a single open reading frame of 12,285 base pairs (bp). The ibpA gene is flanked by an upstream ORF of 1758bp, designated ibpB. The predicted amino acid sequences of these two genes demonstrate similarity to virulence exoproteins and their transporter proteins that comprise a two-partner secretion pathway in various Gram-negative bacteria. Motifs associated with binding to mammalian cells were also identified within the sequence. Competitive inhibition studies implicated a putative heparin-binding domain in adherence to bovine endothelial cells. Expression plasmids for glutathione S-transferase (GST)-fused recombinant fragments covered amino acid residues 972-3201. IgG2 Fc binding studies identified fragment 972-1515 (GST-IbpA3) as an Fc binding peptide. This peptide and GST-IbpA5 (aa 2071-2730) reacted strongly with convalescent phase serum. In a small preliminary study, calves immunized with the purified GST-IbpA3 peptide were protected against an intrabronchial H. somnus challenge.     

Sequence and analysis of the capsid protein of Nudaurelia capensis ω virus, an insect virus with T = 4 icosahedral symmetry

We have determined the nucleotide sequence of the RNA2 segment of the Nudaurelia capensis ω virus genome. It was found to consist of 2448 nucleotides and contained one long open reading frame (ORF) encoding the 644 residue capsid protein. The deduced amino acid sequence of this protein reveals a positively charged amino terminus, a characteristic exhibited by several other viral capsid proteins, that is thought to be important for interactions between the capsid and the genomic RNA. There are 366 and 150 bases of untranslated sequence on the 5′ and 3′ ends, respectively. The ORF encoding the capsid protein initiates at the second AUG from the Fend. The 5′ proximal AUG specifies a short ORF (30 codons) which terminates 1 base before the initiation codon for the coat protein. Our analysis also revealed the presence of a second, previously unidentified polypeptide associated with Nudaurelia capensis ω virus particles. The amino terminal sequence of this protein corresponds to a portion of the long ORF beginning at codon 571. The lack of an initiation codon near this sequence indicates that the small polypeptide is most likely produced as a carboxy terminal cleavage product from a 70-kDa capsid protein precursor, yielding the previously identified 62-kDa protein and the 8-kDa protein that we have observed. The putative cleavage site would be at an Asn/Phe pair, somewhat resembling known cleavage sites (Asn/Ala) in the T = 3 Nodaviridae. In addition, we have found that there is also a second polypeptide similar in size to that from Nudaurelia capensis ω virus associated with particles of Nudaurelia capensis β virus, the type member of Tetraviridae.     

Cloning, characterization, and expression of a novel secretory lipase-like gene from Clonorchis sinensis

A secretory lipase-like gene was isolated from total cDNA of adult Clonorchis sinensis. The gene has an open reading frame of 1,218 bp long and encodes for a protein of 406 amino acids including a putative signal peptide of 20 amino acids. The deduced amino acid sequence including signal peptide has 42–45% identity with lipase of other species and two typical enzymic active sites that contain consensus sequence (Gly-X-Ser-X-Gly) of lipase. The cDNA encoding this protein was subcloned into pET-28a (+) expression vector and expressed in Escherichia coli. The expressed fusion protein has a molecular mass of about 45 kDa. Prediction of signal peptide and Western blot analysis indicated that the secretory lipase-like protein is an excretory–secretory product of C. sinensis. Immunostaining revealed that the secretory lipase-like protein was localized in the tegument of the adult worm and metacercaria. These results provide basis for further studies on the nutrition taking and invasion of C. sinensis mediated by the secretory lipase-like protein.     

Nucleotide sequence of the gene encoding infectious laryngotracheitis virus glycoprotein B.

The nucleotide sequence of the infectious laryngotracheitis virus (ILTV) gene encoding the 205K complex glycoprotein (gp205) was determined. The gene is contained within a 3-kb EcoRI restriction fragment mapping at approximately map coordinates 0.23 to 0.25 in the UL region of the ILTV genome and is transcribed from right to left. Nucleotide sequence analysis of the DNA fragment identified a single, long open reading frame capable of encoding 873 amino acids. The predicted precursor polypeptide derived from this open reading frame would have a calculated Mr of 98,895 Da and contains nine potential glycosylation sites. Hydropathic analysis indicates the presence of an amino terminal hydrophobic sequence and hydrophobic carboxyl terminal domain which may function as a signal peptide and a membrane anchor sequence, respectively. Comparison of the predicted ILTV gp205 protein sequence with those of other herpesviruses revealed a significant sequence similarity with gB-like glycoproteins. Extensive homology was observed throughout the molecule except for the amino and carboxyl termini. The high homology in predicted primary and secondary structures is consistent with the essential role of the gB family of proteins for viral infectivity and pathogenesis.     

F17-like fimbriae from an invasive Escherichia coli strain producing cytotoxic necrotizing factor type 2 toxin.

The F17b fimbriae encoded by the transmissible virulence plasmid Vir, also coding for cytotoxic necrotizing factor type 2, were characterized. A 5.7-kb region of Vir mediates in vitro N-acetylglucosamine-sensitive adhesion to calf intestinal villi. Sequence analysis revealed that this region codes for a structural subunit and an adhesin closely related to the F17-A and F17-G proteins encoded by the F17 fimbrial gene cluster. The F17b-A gene presents an open reading frame of 540 bp encoding a polypeptide of 180 amino acids with a putative signal peptide of 21 residues. The mature protein shows an identity of 74% with the F17-A structural subunit. This 20-kDa protein is recognized by antiserum directed against F17 fimbriae. The F17b-G gene shows an open reading frame of 1,029 bp encoding a polypeptide of 343 amino acids with a putative signal peptide of 22 residues. The F17b-G polypeptide exhibits 95% identity with the F17-G adhesin. The functional homology of the gene products was further confirmed by demonstrating that mutants in the F17-A gene can be complemented by the F17b-A gene and vice versa. These results prove that fimbriae belonging to the F17 family are also found on pathogenic Escherichia coli strains other than enterotoxigenic isolates producing heat-labile or heat-stable enterotoxin.     

Mapping and sequence of the gene encoding protein p37, A major structural protein of African swine fever virus

The gene encoding protein p37, one of the major structural proteins of African swine fever (ASF) virus has been mapped and sequenced. Protein p37 was obtained from purified virions and the first 27 amino acids from its NH₂-terminal end were identified by automatic Edman degradation. To map the gene encoding protein p37, a mixture of 20-mer deoxyoligonucleotides based upon a part of this amino acid sequence was hybridized to cloned ASF virus restriction fragments. This allowed localization of the gene in fragmentKpnIF/HindIII G₁ of the African swine fever virus genome. An analysis of the DNA sequence from this region revealed an open reading frame encoding 418 amino acids. In this sequence, the 27 NH₂-terminal amino acids determined by sequence analysis of protein p37 are preceded by a stretch of 132 amino acids residues, indicating that protein p37 is synthesized as a polypeptide of higher molecular weight and then posttranslationally processed by cleavage of a Gly-Ala bond. This processing event accounts for the antigenic relationship of protein p37 to a virus-induced, nonstructural protein with a relative molecular weight of 60 kD.     

Molecular cloning, expression, and DNA sequence analysis of the gene that encodes the 16-kilodalton outer membrane lipoprotein of Serpulina hyodysenteriae.

The gene (smpA) that encodes the 16-kDa outer membrane lipoprotein of Serpulina hyodysenteriae was cloned in Escherichia coli, and its primary structure was determined by nucleotide sequencing. The putative open reading frame encodes a prolipoprotein of 16.8 kDa which in its fully acylated and cleaved form is 15.1 kDa. Analysis of the N-terminal amino acid sequence derived from the DNA sequence revealed the presence of a signal sequence and a putative acylation and signal peptidase II cleavage site (Phe-Ala-Val-Ser-Cys). In E. coli, processing of the prolipoprotein was less efficient than that observed in S. hyodysenteriae, and globomycin, an inhibitor of signal peptidase II, inhibited cleavage of the lipoprotein expressed in E. coli but did not inhibit cleavage in S. hyodysenteriae.     

The leptospiral major outer membrane protein LipL32 is a lipoprotein expressed during mammalian infection

We report the cloning of the gene encoding the 32-kDa lipoprotein, designated LipL32, the most prominent protein in the leptospiral protein profile. We obtained the N-terminal amino acid sequence of a staphylococcal V8 proteolytic-digest fragment to design an oligonucleotide probe. A Lambda-Zap II library containing EcoRI fragments of Leptospira kirschneri DNA was screened, and a 5.0-kb DNA fragment which contained the entire structural lipL32 gene was identified. Several lines of evidence indicate that LipL32 is lipid modified in a manner similar to that of other procaryotic lipoproteins. The deduced amino acid sequence of LipL32 would encode a 272-amino-acid polypeptide with a 19-amino-acid signal peptide, followed by a lipoprotein signal peptidase cleavage site. LipL32 is intrinsically labeled during incubation of L. kirschneri in media containing [(3)H]palmitate. The linkage of palmitate and the amino-terminal cysteine of LipL32 is acid labile. LipL32 is completely solubilized by Triton X-114 extraction of L. kirschneri; phase separation results in partitioning of LipL32 exclusively into the hydrophobic, detergent phase, indicating that it is a component of the leptospiral outer membrane. CaCl(2) (20 mM) must be present during phase separation for recovery of LipL32. LipL32 is expressed not only during cultivation but also during mammalian infection. Immunohistochemistry demonstrated intense LipL32 reactivity with L. kirschneri infecting proximal tubules of hamster kidneys. LipL32 is also a prominent immunogen during human leptospirosis. The sequence and expression of LipL32 is highly conserved among pathogenic Leptospira species. These findings indicate that LipL32 may be important in the pathogenesis, diagnosis, and prevention of leptospirosis.     

Cloning and Sequence Analysis of a Non-Structural Gene of an Aquareovirus

The nucleotide and deduced amino acid sequence of genome segment 11 encoding a nonstructural protein of an aquareovirus strain SBR have been determined. Nucleotide sequence analysis showed that the genome segment 11 of SBR virus is 780 nucleotides long and contains a major open reading frame that codes for a polypeptide of 236 amino acids with a predicted molecular weight of 25,504 Da. The second reading frame of genome segment 11 was 480 nucleotides long and codes for a polypeptide of 145 with a predicted molecular weight of 15,715 Da. The genome segment 11 contains 24 nontranslated nucleotides at the 5′-end and 48 nontranslated nucleotides at the 3′-end. This gene codes for two nonstructural polypeptides NS29 and NS15. Comparison of the deduced amino acid sequence of this gene with the published sequences of other members of the family Reoviridae indicated no sequence relatedness. This revised version was published online in July 2006 with corrections to the Cover Date.     

Identification of an immunogenic 18-kDa protein of Helicobacter pylori by alkaline phosphatase gene fusions

The use of alkaline phosphatase fusion methodology to identify Helicobacter pylori exported proteins enabled the identification of an open reading frame (ORF) encoding a highly immunogenic, previously uncharacterised exported protein. The predicted aminoacid sequence displays a typical N-terminal signal peptide and contains regions of C-terminal hydrophobicity consistent with a membrane-associated protein. Southern blot analysis revealed that the gene encoding the protein was absent in several Helicobacter spp. and a combination of PCR and sequence analysis of the amplified gene showed that it is highly conserved amongst isolates of H. pylori. To obtain pure recombinant protein, the gene encoding the protein was cloned and expressed as a beta-galactosidase (beta-gal) fusion in Escherichia coli and the protein was purified by affinity chromatography and proteolytic cleavage of the beta-gal portion. The purified protein, which has an apparent mol. wt of 18 kDa, was recognised by antibody present in 71% of sera from patients infected with H. pylori, but in only 16% of sera from patients with unrelated or no gastrointestinal disease, by Western blot assays. These results indicate that the 18-kDa protein from H. pylori is immunogenic and is expressed in vivo.     

Characterization of a 34-Kilodalton Protein of Mycobacterium leprae That Is Isologous to the Immunodominant 34-Kilodalton Antigen of Mycobacterium paratuberculosis

During DNA sequence analysis of cosmid L373 from the Mycobacterium leprae genome, an open reading frame of 1.4 kb encoding a protein with some homology to the immunodominant 34-kDa protein of Mycobacterium paratuberculosis, but lacking significant serological activity, was detected. The DNA sequence predicted a signal peptide with a modified lipoprotein consensus sequence, but the protein proved to be devoid of lipid attachment.