Molecular modeling and in-silico engineering of Cardamom mosaic virus coat protein for the presentation of immunogenic epitopes of Leptospira LipL32

Expression of Cardamom mosaic virus (CdMV) coat protein (CP) in E. coli forms virus-like particles. In this study, the structure of CdMV CP was predicted and used as a platform to display epitopes of the most abundant surface-associated protein, LipL32 of Leptospira at C, N, and both the termini of CdMV CP. In silico, we have mapped sequential and conformational B-cell epitopes from the crystal structure of LipL32 of Leptospira interrogans serovar Copenhageni str. Fiocruz L1-130 using IEDB Elipro, ABCpred, BCPRED, and VaxiJen servers. Our results show that the epitopes displayed at the N-terminus of CdMV CP are promising vaccine candidates as compared to those displayed at the C-terminus or at both the termini. LipL32 epitopes, EP2, EP3, EP4, and EP6 are found to be promising B-cell epitopes for vaccine development. Based on the type of amino acids, length, surface accessibility, and docking energy with CdMV CP model, the order of antigenicity of the LipL32 epitopes was found to be EP4 > EP3 > EP2 > EP6.     

Enzymatic properties of a new peptide deformylase from pathogenic bacterium Leptospira interrogans

Membrane-associated prostaglandin E synthase (mPGE synthase) was previously purified to apparent homogeneity from the microsomal fraction of bovine heart (Watanabe, K., et al., Biochim. Biophys. Acta 1439, 406--414, 1999). The N-terminal 22-amino acid sequence of the purified enzyme was identical to that of the 88th to 109th amino acids deduced from the monkey (AB046026) or human (AK024100) cDNA that encodes a hypothetical protein with unknown function. The primary structure has the consensus region of glutaredoxin and of thioredoxin. We constructed an expression plasmid, using the vector (pTrc-HisA) and the monkey cDNA for the 290-amino-acid polypeptide. The recombinant protein with a M(r) of 33 kDa exhibited PGE synthase activity and was purified to apparent homogeneity by nickel-chelating column chromatography. The V(max) and K(m) values for PGH(2) of the purified recombinant mPGE synthase were about 3.3 mumol/min center dot mg of protein and 28 muM, respectively. The recombinant enzyme was activated by various SH-reducing reagents, i.e., dithiothreitol, glutathione (GSH), and beta-mercaptoethanol, in order of decreasing effectiveness. Moreover, the mRNA distribution was high in the heart and brain, but the mRNA was not expressed in the seminal vesicles. These results indicate that the recombinant mPGE synthase is identical to the enzyme purified from the microsomal fraction of bovine heart, and is a novel type of mPGE synthase based on the primary structure, a broad specificity of thiol requirement, and tissue distribution.     

Calcium Binds to LipL32, a Lipoprotein from Pathogenic Leptospira, and Modulates Fibronectin Binding

Tubulointerstitial nephritis is a cardinal renal manifestation of leptospirosis. LipL32, a major lipoprotein and a virulence factor, locates on the outer membrane of the pathogen Leptospira. It evades immune response by recognizing and adhering to extracellular matrix components of the host cell. The crystal structure of Ca²⁺-bound LipL32 was determined at 2.3 Å resolution. LipL32 has a novel polyD sequence of seven aspartates that forms a continuous acidic surface patch for Ca²⁺ binding. A significant conformational change was observed for the Ca²⁺-bound form of LipL32. Calcium binding to LipL32 was determined by isothermal titration calorimetry. The binding of fibronectin to LipL32 was observed by Stains-all CD and enzyme-linked immunosorbent assay experiments. The interaction between LipL32 and fibronectin might be associated with Ca²⁺ binding. Based on the crystal structure of Ca²⁺-bound LipL32 and the Stains-all results, fibronectin probably binds near the polyD region on LipL32. Ca²⁺ binding to LipL32 might be important for Leptospira to interact with the extracellular matrix of the host cell.     

Crystal structure of SAM-dependent O-methyltransferase from pathogenic bacterium Leptospira interrogans

The S-adenosylmethionine (SAM)-dependent O-methyltransferase from Leptospira interrogans (LiOMT) expressed by gene LA0415 belongs to the Methyltransf_3 family (Pfam PF01596). In this family all of the five bacterial homologues with known function are reported as SAM-dependent O-methylstransferases involved in antibiotic production. The crystal structure of LiOMT in complex with S-adenosylhomocysteine reported here is the first bacterial protein structure in this family. The LiOMT structure shows a conserved SAM-binding region and a probable metal-dependent catalytic site. The molecules of LiOMT generate homodimers by N-terminal swapping, which assists the pre-organization of the substrate-binding site. Based on the sequence and structural analysis, it is implied by the catalytic and substrate-binding site that the substrate of LiOMT is a phenolic derivative, which probably has a large ring-shaped moiety.     

The use of halloysite clay and carboxyl-functionalised multi-walled carbon nanotubes for recombinant LipL32 antigen delivery enhanced the IgG response

We studied the feasibility of using halloysite clay nanotubes (HNTs) and carboxyl-functionalised multi-walled carbon nanotubes (COOH-MWCNTs) as antigen carriers to improve immune responses against a recombinant LipL32 protein (rLipL32). Immunisation using the HNTs or COOH-MWCNTs significantly increased the rLipL32-specific IgG antibody titres (p < 0.05) of Golden Syrian hamsters. None of the vaccines tested conferred protection against a challenge using a virulent Leptospira interrogans strain. These results demonstrated that nanotubes can be used as antigen carriers for delivery in hosts and the induction of a humoral immune response against purified leptospiral antigens used in subunit vaccine preparations.     

Leptospira interrogans requires a functional heme oxygenase to scavenge iron from hemoglobin

The transposon TnSC189 was used to construct a mutant in the putative heme oxygenase gene hemO (LB186) of Leptospira interrogans. Unlike its parent strain, the mutant grew poorly in medium in which hemoglobin was the sole iron source. The putative heme oxygenase was over expressed in a His-tagged form, purified and was demonstrated to degrade heme in vitro. Unexpectedly, it was also found that the L. interrogans growth rate was significantly increased when medium was supplemented with hemoglobin, but only if ferrous iron sources were absent. This result was mirrored in the expression of some iron-related genes and suggests the presence of regulatory mechanisms detecting Fe²⁺ and hemoglobin. This is the first demonstration of a functional heme oxygenase from a spirochete.     

Purification and proteomic analysis of outer membrane vesicles from a clinical isolate of Leptospira interrogans serovar Copenhageni

The severe pulmonary form of leptospirosis (SPFL) is an especially serious and rapid disease process characterized by alveolar hemorrhage and acute respiratory failure. The outer membrane of Leptospira facilitates direct interactions with the environs and likely contains important constituents involved during infection, transmission, survival, and adaptation to environmental conditions, including putative vaccinogen and diagnostic candidates. Outer membrane vesicles (OMVs) were purified by incubation in low-pH citrate buffer, treatment in a French press, and centrifugation over a continuous sucrose gradient. OMVs characterized by two-dimensional gel electrophoresis (2-DE) contained the previously described outer membrane proteins OmpL1, Qlp42, LipL32, LipL41, LipL36 and Loa22. In addition, unknown, hypothetical and putative outer membrane proteins were identified. High-performance liquid chromatography (HPLC) coupled with mass spectrometry and fraction collection (LC-MS+) measured the intact mass profile of the major outer membrane protein, LipL32, and the putative lipoprotein Qlp42. In contrast to a predicted molecularmass of 27,653.5 Da for LipL32 after cleavage of its signal peptide, intact mass proteomics measured the mass as ranging from 28,468 to 28,583 Da, consistent with lipidation of LipL32. In contrast to a predicted molecular mass of 39.8 kDa for Qlp42, the actual mass was measured as 24,811 and 26,461 Da consistent with a 30 kDa doublet observed on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels and processing of the N-terminus of the mature protein. These studies indicate that purified OMVs are highly compatible with proteomics technologies including 2-DE and intact mass proteomics using LC-MS+ that facilitates definition of actual molecular masses of intact outer membrane proteins, and heterogeneity associated with them.     

Characterization of the cheY genes from Leptospira interrogans and their effects on the behavior of Escherichia coli

The motility and chemotaxis system are critical for the virulence of pathogenic leptospire, which enable them to penetrate host tissue barriers during infection. The completed genome sequence of a representative virulent serovar type strain (Lai) of Leptospira interrogans serogroups Icterohaemorrhagiae (L. interrogans strain Lai) suggested that there were multiple copies of putative chemotaxis homologues located at its large chromosome. In order to verify the function of these proteins, the putative cheY genes were cloned into pQE31 vector and then expressed, respectively, in wild-type Escherichia coli strain RP437 and cheY defective strain RP5232. The results showed that all the five cheYs could restore the swarming of RP5232 strain to some extend. Overexpression of CheYs in RP437 showed inhibited swarming of RP437. To investigate the mechanism of chemotaxis signaling in L. interrogans strain Lai, certain aspartates (Asp-53, Asp-61, Asp-70, Asp-62, and Asp-66 for L. interrogans strain Lai CheY1, CheY2, CheY3, CheY4, and CheY5, respectively) were mutated. Expression of these mutated cheYs manifested neither restoration of the swarming ability of RP5232 nor inhibition on swarming ability of RP437. Multiple amino acid sequence alignment predicted ternary structures and the result of mutation experiment suggested that these conserved aspartate residues of L. interrogans were analogous to that in E. coli CheY in function and structure. So, L. interrogans and E. coli may have similar mechanisms of activation of the chemotaxis phosphorelay pathway, but there are differences in their control by signal terminator.     

Crystal structure of homoserine O-acetyltransferase from Leptospira interrogans

Homoserine O-acetyltransferase (HTA, EC 2.3.1.31) initiates methionine biosynthesis pathway by catalyzing the transfer of acetyl group from acetyl-CoA to homoserine. This study reports the crystal structure of HTA from Leptospira interrogans determined at 2.2 Å resolution using selenomethionyl single-wavelength anomalous diffraction method. HTA is modular and consists of two structurally distinct domains—a core α/β domain containing the catalytic site and a helical bundle called the lid domain. Overall, the structure fold belongs to α/β hydrolase superfamily with the characteristic ‘catalytic triad’ residues in the active site. Detailed structure analysis showed that the catalytic histidine and serine are both present in two conformations, which may be involved in the catalytic mechanism for acetyl transfer.     

Identification of three extra-chromosomal replicons in Leptospira pathogenic strain and development of new shuttle vectors

Background

The genome of pathogenic Leptospira interrogans contains two chromosomes. Plasmids and prophages are known to play specific roles in gene transfer in bacteria and can potentially serve as efficient genetic tools in these organisms. Although plasmids and prophage remnants have recently been reported in Leptospira species, their characteristics and potential applications in leptospiral genetic transformation systems have not been fully evaluated.

Results

Three extrachromosomal replicons designated lcp1 (65,732 bp), lcp2 (56,757 bp), and lcp3 (54,986 bp) in the L. interrogans serovar Linhai strain 56609 were identified through whole genome sequencing. All three replicons were stable outside of the bacterial chromosomes. Phage particles were observed in the culture supernatant of 56609 after mitomycin C induction, and lcp3, which contained phage-related genes, was considered to be an inducible prophage. L. interrogans–Escherichia coli shuttle vectors, constructed with the predicted replication elements of single rep or rep combined with parAB loci from the three plasmids were shown to successfully transform into both saprophytic and pathogenic Leptospira species, suggesting an essential function for rep genes in supporting auto-replication of the plasmids. Additionally, a wide distribution of homologs of the three rep genes was identified in L. interrogans isolates, and correlation tests showed that the transformability of the shuttle vectors in L. interrogans isolates depended, to certain extent, on genetic compatibility between the rep sequences of both plasmid and host.

Conclusions

Three extrachromosomal replicons co-exist in L. interrogans, one of which we consider to be an inducible prophage. The vectors constructed with the rep genes of the three replicons successfully transformed into saprophytic and pathogenic Leptospira species alike, but this was partly dependent on genetic compatibility between the rep sequences of both plasmid and host.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1321-y) contains supplementary material, which is available to authorized users.     

Functional characterization of Helicobacter pylori TlyA: Pore-forming hemolytic activity and cytotoxic property of the protein

Helicobacter pylori is a human specific gastric pathogen. H. pylori pathogenesis process involves a number of well-studied virulence factors that include the ‘vacuolating cytotoxin’ and the ‘cytotoxin associated gene A’. Analysis of the H. pylori genome, however, indicates presence of additional virulence factors that are yet to be characterized in molecular detail. For example, H. pylori genome harbors a gene that has potential to encode a protein with sequence similarity to those of the TlyA-like proteins of several pathogenic bacteria. Earlier studies have indicated potential association of this H. pylori tlyA gene in the virulence mechanism of the organism. Despite such notions, however, the TlyA-like protein of H. pylori has not been studied previously in molecular detail. In particular, purified form of H. pylori TlyA has never been studied before toward exploring its functional properties. Here, we report characterization of the H. pylori TlyA protein purified from the recombinant over-expression system in Escherichia coli. Purified form of the recombinant TlyA exhibits prominent hemolytic activity against human erythrocytes, presumably via formation of pores of specific diameter in the cell membrane. Purified TlyA also triggers prominent cytotoxic responses in human gastric adenocarcinoma cells. Altogether, our study establishes H. pylori TlyA as a potential virulence factor of the organism.     

Calcium binding to leptospira outer membrane antigen LipL32 is not necessary for its interaction with plasma fibronectin, collagen type IV, and plasminogen

LipL32 is the most abundant outer membrane protein from pathogenic Leptospira and has been shown to bind extracellular matrix (ECM) proteins as well as Ca(2+). Recent crystal structures have been obtained for the protein in the apo- and Ca(2+)-bound forms. In this work, we produced three LipL32 mutants (D163-168A, Q67A, and S247A) and evaluated their ability to interact with Ca(2+) and with ECM glycoproteins and human plasminogen. The D163-168A mutant modifies aspartate residues involved in Ca(2+) binding, whereas the other two modify residues in a cavity on the other side of the protein structure. Loss of calcium binding in the D163-D168A mutant was confirmed using intrinsic tryptophan fluorescence, circular dichroism, and thermal denaturation whereas the Q67A and S247A mutants presented the same Ca(2+) affinity as the wild-type protein. We then evaluated if Ca(2+) binding to LipL32 would be crucial for its interaction with collagen type IV and plasma proteins fibronectin and plasminogen. Surprisingly, the wild-type protein and all three mutants, including the D163-168A variant, bound to these ECM proteins with very similar affinities, both in the presence and absence of Ca(2+) ions. In conclusion, calcium binding to LipL32 may be important to stabilize the protein, but is not necessary to mediate interaction with host extracellular matrix proteins.     

Caissarolysin I (Bcs I), a new hemolytic toxin from the Brazilian sea anemone Bunodosoma caissarum: Purification and biological characterization

Two cationic proteins, C1 and C3, were purified to homogeneity from the hemolytic fraction of the venom of Bunodosoma caissarum sea anemone. The purification processes employed gel filtration followed by ion exchange chromatography, being the purity and molecular mass confirmed by SDS-PAGE and mass spectrometry. Protein C1 represented the second major peak of the hemolytic fraction and was previously believed to be a cytolysin belonging to a new class of hemolysins. The C1 protein has a molecular mass of 15495 Da and was assayed for hemolysis, PLA₂ activity and acute toxicity in crabs and mice, showing no activity in these assays. It has an amino terminal with no similarity to all known hemolysins and, therefore, should not be considered a toxin, being its function completely unknown. The protein C3 (19757 Da), that also lacks PLA₂ activity, was recognized by antiserum against Eqt II and presented high hemolytic activity to human erythrocytes (ED₅₀ of 0.270 μg/ml), being named Caissarolysin I (Bcs I). Its activity was inhibited by pre-incubation with sphingomyelin (SM) and also when in presence of erythrocytes pre-treated with the SMase P2, a phospholipase D from the brown spider Loxosceles intermedia, indicating that SM is the main target of Bcs I. Caissarolysin I is the first hemolysin purified from a sea anemone belonging to the genus Bunodosoma and belongs to the Actinoporin family of sea anemone hemolysins.     

First report of an anti-tumor, anti-fungal, anti-yeast and anti-bacterial hemolysin from Albizia lebbeck seeds

A monomeric 5.5-kDa protein with hemolytic activity toward rabbit erythrocytes was isolated from seeds of Albizia lebbeck by using a protocol that involved ion-exchange chromatography on Q-Sepharose and SP-Sepharose, hydrophobic interaction chromatography on Phenyl-Sepharose, and gel filtration on Superdex 75. It was unadsorbed on both Q-Sepharose and SP-Sepharose, but adsorbed on Phenyl-Sepharose. Its hemolytic activity was fully preserved in the pH range 0-14 and in the temperature range 0-100 °C, and unaffected in the presence of a variety of metal ions and carbohydrates. The hemolysin reduced viability of murine splenocytes and inhibited proliferation of MCF-7 breast cancer cells and HepG2 hepatoma cells with an IC₅₀ of 0.21, 0.97, and 1.37 μM, respectively. It impeded mycelial growth in the fungi Rhizoctonia solani with an IC₅₀ of 39 μM but there was no effect on a variety of other filamentous fungi, including Fusarium oxysporum, Helminthosporium maydis, Valsa mali and Mycosphaerella arachidicola. Lebbeckalysin inhibited growth of Escherichia coli with an IC₅₀ of 0.52 μM.     

Sequence analysis and characterization of sulfonamide resistance plasmid pRF-1 from Salmonella enterica serovar Choleraesuis

The nucleotide sequence of a small plasmid, designated pRF-1, isolated from Salmonella enterica serovar Choleraesuis, was determined. We identified seven open reading frames (ORFs) encoded by 6066 nucleotides with a total G + C content of 53.6%. Analysis of the complete nucleotide sequence revealed a replicon of pRF-1 to have high similarity to the p15A origin of replication, with a possible cer-like region. ORF1, which is composed of 816 nucleotides, shows a high degree of similarity to dihydropteroate synthetase encoded by the sulII gene from plasmids in several enteropathogenic bacteria, which functions as the sulfonamide resistance determinant. In fact, Salmonella and Escherichia coli strains carrying pRF-1 were found to show strong resistance to sulfathiazole, suggesting that orf1 is a functional gene. Four of seven ORFs were found to encode putative proteins of unknown function.     

Cloning and characterization of the chromosomal replication origin region of Amycolatopsis mediterranei U32

The chromosomal replication origins (oriC) of gram positive, acid-fast actinomycetes have been investigated in streptomycetes and mycobacteria. A 1339 bp DNA fragment of the putative oriC region from the rifamycin SV producer Amycolatopsis mediterranei U32 was cloned by PCR amplification employing primers designed based on the conserved flanking genes of dnaA and dnaN. The 884 bp sequence of the intergenic region between dnaA and dnaN genes consists of 19 DnaA-boxes and two 13-mer AT-rich sequences, which is similar to the oriC structure of Streptomyces lividans. A mini-chromosome constructed by cloning the putative U32 oriC DNA fragment into an Escherichia coli plasmid was able to replicate autonomously, but was unstable, in A. mediterranei U32 with an estimated copy number of two per cell. Although efficient replication of the mini-chromosome in U32 requires the complete set of DnaA-boxes and AT-rich regions, only one of the AT-rich sequences together with part of the DnaA-boxes is sufficient, suggesting the presence of combinatorial alternatives for a functional oriC region of A. mediterranei U32. Phylogenetic analysis based on definite oriC sequences among eubacteria reflects well the relationship between these species.     

Genotypes of pathogenic Leptospira spp isolated from rodents in Argentina

Leptospirosis is the most widespread zoonosis in the world and significant efforts have been made to determine and classify pathogenic Leptospira strains. This zoonosis is maintained in nature through chronic renal infections of carrier animals, with rodents and other small mammals serving as the most important reservoirs. Additionally, domestic animals, such as livestock and dogs, are significant sources of human infection. In this study, a multiple-locus variable-number tandem repeat analysis (MLVA) was applied to genotype 22 pathogenic Leptospira strains isolated from urban and periurban rodent populations from different regions of Argentina. Three MLVA profiles were identified in strains belonging to the species Leptospira interrogans (serovars Icterohaemorrhagiae and Canicola); one profile was observed in serovar Icterohaemorrhagiae and two MLVA profiles were observed in isolates of serovars Canicola and Portlandvere. All strains belonging to Leptospira borgpetersenii serovar Castellonis exhibited the same MLVA profile. Four different genotypes were isolated from urban populations of rodents, including both mice and rats and two different genotypes were isolated from periurban populations.     

Two-step purification and in vitro characterization of a hemolysin from the venom of jellyfish Cyanea nozakii Kishinouye

Hemolysin is one of the most hazardous components in the venom of Cyanea nozakii Kishinouye. Here we describe the purification and in vitro characterization of the hemolysin, which we named CnPH. The CnPH was isolated by anion-exchange and size-exclusion chromatography from the nematocyst venom. Two protein bands with molecular masses of 20 kDa, 60 kDa respectively were shown in the reducing SDS-PAGE analysis of the CnPH. And Approximately 5 μg/mL of the CnPH resulted in 50% hemolysis of the erythrocyte suspension. The hemolytic activity of the CnPH was both temperature and pH dependent. Moreover, it was significantly inhibited in the presence of divalent metal cations, including Cu²⁺, Mg²⁺, Mn²⁺, Zn²⁺ and Ca²⁺, but enhanced in the presence of EDTA. However, how CnPH performs its hemolytic activity is not yet clear, therefore the mechanism of the hemolytic activity of the CnPH is under research.     

Expression of peptidylarginine deiminase from Porphyromonas gingivalis in Escherichia coli: Enzyme purification and characterization

Porphyromonas gingivalis peptidylarginine deiminase (PAD) catalyzes the deimination of peptidylarginine residues of various peptides to produce peptidylcitrulline and ammonia. P. gingivalis is associated with adult-onset periodontitis and cardiovascular disease, and its proliferation depends on secretion of PAD. We have expressed two recombinant forms of the P. gingivalis PAD in Escherichia coli, a truncated form with a 43-amino acid N-terminal deletion and the full-length form of PAD as predicted from the DNA sequence. Both forms contain a poly-His tag and Xpress epitope at the N-terminus to aid in detection and purification. The activities and stabilities of these two forms have been evaluated. PAD is cold sensitive; it aggregates within 30 min at 4 °C, and optimal storage conditions are at 25 °C in the presence of a reducing agent. PAD is not a metalloenzyme and does not need a cofactor for catalysis or stability. Multiple l-arginine analogs, various arginine-containing peptides, and free l-arginine were used to evaluate substrate specificity and determine kinetic parameters.