Publications by authors named "Paulo L Ho"

53 Publications

Molecular alterations in the extracellular matrix in the brains of newborns with congenital Zika syndrome.

Sci Signal 2020 06 9;13(635). Epub 2020 Jun 9.

Department of Clinical and Toxicological Analyses, School of Pharmaceutical Sciences, University of São Paulo, São Paulo, Brazil.

Zika virus (ZIKV) infection during pregnancy can cause a set of severe abnormalities in the fetus known as congenital Zika syndrome (CZS). Experiments with animal models and in vitro systems have substantially contributed to our understanding of the pathophysiology of ZIKV infection. Here, to investigate the molecular basis of CZS in humans, we used a systems biology approach to integrate transcriptomic, proteomic, and genomic data from the postmortem brains of neonates with CZS. We observed that collagens were greatly reduced in expression in CZS brains at both the RNA and protein levels and that neonates with CZS had several single-nucleotide polymorphisms in collagen-encoding genes that are associated with osteogenesis imperfecta and arthrogryposis. These findings were validated by immunohistochemistry and comparative analysis of collagen abundance in ZIKV-infected and uninfected samples. In addition, we showed a ZIKV-dependent increase in the expression of cell adhesion factors that are essential for neurite outgrowth and axon guidance, findings that are consistent with the neuronal migration defects observed in CZS. Together, these findings provide insights into the underlying molecular alterations in the ZIKV-infected brain and reveal host genes associated with CZS susceptibility.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1126/scisignal.aay6736DOI Listing
June 2020

New strategies for Leptospira vaccine development based on LPS removal.

PLoS One 2020 27;15(3):e0230460. Epub 2020 Mar 27.

Laboratory of Bacteriology, Butantan Institute, São Paulo, Brazil.

Pathogenic spirochetes from genus Leptospira are etiologic agents of leptospirosis. Cellular vaccines against Leptospira infection often elicit mainly response against the LPS antigen of the serovars present in the formulation. There is no suitable protein candidate capable of replacing whole-cell vaccines, thus requiring new approaches on vaccine development to improve leptospirosis prevention. Our goal was to develop a whole-cell vaccine sorovar-independent based on LPS removal and conservation of protein antigens exposure, to evaluate the protective capacity of monovalent or bivalent vaccines against homologous and heterologous virulent Leptospira in hamster. Leptospire were subjected to heat inactivation, or to LPS extraction with butanol and in some cases further inactivation with formaldehyde. Hamsters were immunized and challenged with homologous or heterologous virulent serovars, blood and organs were collected from the survivors for bacterial quantification, chemokine evaluation, and analysis of sera antibody reactivity and cross-reactivity by Western blot. Immunization with either heated or low LPS vaccines with serovar Copenhageni or Canicola resulted in 100% protection of the animals challenged with homologous virulent bacteria. Notably, different from the whole-cell vaccine, the low LPS vaccines produced with serovar Canicola provided only partial protection in heterologous challenge with the virulent Copenhageni serovar. Immunization with bivalent formulation results in 100% protection of immunized animals challenged with virulent serovar Canicola. All vaccines produced were able to eliminate bacteria from the kidney of challenged animals. All the vaccines raised antibodies capable to recognize antigens of serovars not present in the vaccine formulation. Transcripts of IFNγ, CXCL16, CCL5, CXCL10, CXCR6, and CCR5, increased in all immunized animals. Conclusion: Our results showed that bivalent vaccines with reduced LPS may be an interesting strategy for protection against heterologous virulent serovars. Besides the desirable multivalent protection, the low LPS vaccines are specially promising due to the expected lower reatogenicity.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0230460PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7100938PMC
June 2020

Evaluation of inactivated Bordetella pertussis as a delivery system for the immunization of mice with Pneumococcal Surface Antigen A.

PLoS One 2020 16;15(1):e0228055. Epub 2020 Jan 16.

Laboratório de Bacteriologia, Instituto Butantan, São Paulo, SP, Brazil.

Pneumococcal Surface Protein A (PspA) has been successfully tested as vaccine candidate against Streptococcus pneumoniae infections. Vaccines able to induce PspA-specific antibodies and Th1 cytokines usually provide protection in mice. We have shown that the whole cell pertussis vaccine (wP) or components from acellular pertussis vaccines, such as Pertussis Toxin or Filamentous Hemagglutinin (FHA), are good adjuvants to PspA, suggesting that combined pertussis-PspA vaccines would be interesting strategies against the two infections. Here, we evaluated the potential of wP as a delivery vector to PspA. Bordetella pertussis strains producing a PspA from clade 4 (PspA4Pro) fused to the N-terminal region of FHA (Fha44) were constructed and inactivated with formaldehyde for the production of wPPspA4Pro. Subcutaneous immunization of mice with wPPspA4Pro induced low levels of anti-PspA4 IgG, even after 3 doses, and did not protect against a lethal pneumococcal challenge. Prime-boost strategies using wPPspA4Pro and PspA4Pro showed that there was no advantage in using the wPPspA4Pro vaccine. Immunization of mice with purified PspA4Pro induced higher levels of antibodies and protection against pneumococcal infection than the prime-boost strategies. Finally, purified Fha44:PspA4Pro induced high levels of anti-PspA4Pro IgG, but no protection, suggesting that the antibodies induced by the fusion protein were not directed to protective epitopes.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0228055PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6964896PMC
May 2020

Expression, Polyubiquitination, and Therapeutic Potential of Recombinant E6E7 from HPV16 Antigens Fused to Ubiquitin.

Mol Biotechnol 2017 Jan;59(1):46-56

Laboratório de Biotecnologia Molecular I, Centro de Biotecnologia, Instituto Butantan, Av. Vital Brasil 1500, São Paulo, SP, Brazil.

Ubiquitin-proteasome system plays an essential role in the immune response due to its involvement in the antigen generation and presentation to CD8 T cells. Hereby, ubiquitin fused to antigens has been explored as an immunotherapeutic strategy that requires the activation of cytotoxic T lymphocytes. Here we propose to apply this ubiquitin fusion approach to a recombinant vaccine against human papillomavirus 16-infected cells. E6E7 multi-epitope antigen was fused genetically at its N- or C-terminal end to ubiquitin and expressed in Escherichia coli as inclusion bodies. The antigens were solubilized using urea and purified by nickel affinity chromatography in denatured condition. Fusion of ubiquitin to E6E7 resulted in marked polyubiquitination in vitro mainly when fused to the E6E7 N-terminal. When tested in a therapeutic scenario, the fusion of ubiquitin to E6E7 reinforced the anti-tumor protection and increased the E6/E7-specific cellular immune responses. Present results encourage the investigation of the adjuvant potential of the ubiquitin fusion to recombinant vaccines requiring CD8 T cells.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s12033-016-9990-6DOI Listing
January 2017

Proteomic identification of gender molecular markers in Bothrops jararaca venom.

J Proteomics 2016 Apr 2;139:26-37. Epub 2016 Mar 2.

Laboratório Especial de Toxinologia Aplicada, Center of Toxins, Immune-Response and Cell Signaling, Instituto Butantan, São Paulo, SP, Brazil. Electronic address:

Unlabelled: Variation in the snake venom proteome is a well-documented phenomenon; however, sex-based variation in the venom proteome/peptidome is poorly understood. Bothrops jararaca shows significant sexual size dimorphism and here we report a comparative proteomic/peptidomic analysis of venoms from male and female specimens and correlate it with the evaluation of important venom features. We demonstrate that adult male and female venoms have distinct profiles of proteolytic activity upon fibrinogen and gelatin. These differences were clearly reflected in their different profiles of SDS-PAGE, two-dimensional electrophoresis and glycosylated proteins. Identification of differential protein bands and spots between male or female venoms revealed gender-specific molecular markers. However, the proteome comparison by in-solution trypsin digestion and label-free quantification analysis showed that the overall profiles of male and female venoms are similar at the polypeptide chain level but show striking variation regarding their attached carbohydrate moieties. The analysis of the peptidomes of male and female venoms revealed different contents of peptides, while the bradykinin potentiating peptides (BPPs) showed rather similar profiles. Furthermore we confirmed the ubiquitous presence of four BPPs that lack the C-terminal Q-I-P-P sequence only in the female venom as gender molecular markers. As a result of these studies we demonstrate that the sexual size dimorphism is associated with differences in the venom proteome/peptidome in B. jararaca species. Moreover, gender-based variations contributed by different glycosylation levels in toxins impact venom complexity.

Biological Significance: Bothrops jararaca is primarily a nocturnal and generalist snake species, however, it exhibits a notable ontogenetic shift in diet and in venom proteome upon neonate to adult transition. As is common in the Bothrops genus, B. jararaca shows significant sexual dimorphism in snout-vent length and weight, with females being larger than males. This sexual size dimorphism suggests the tendency for female specimens to feed on larger prey, and for male specimens to go on a diet similar to that of juveniles. Variation in the snake venom proteome is a ubiquitous phenomenon occurring at all taxonomic levels. At the intraspecific variation level, the individual contribution to the venom proteome is important but effects contributed by age and feeding habits may also affect the proteome phenotype. Whether sex-based factors play a role in venom variation of a species that shows sexual size dimorphism is poorly known. The use of proteomic strategies supported by transcriptomic data allows a more comprehensive assessment of venom proteomes uncovering components that are gender-specific.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jprot.2016.02.030DOI Listing
April 2016

Analysis of LexA binding sites and transcriptomics in response to genotoxic stress in Leptospira interrogans.

Nucleic Acids Res 2016 Feb 13;44(3):1179-91. Epub 2016 Jan 13.

Centro de Ciências Naturais e Humanas, Universidade Federal do ABC, Santo André, São Paulo 09210580, Brazil

We determined the effects of DNA damage caused by ultraviolet radiation on gene expression in Leptospira interrogans using DNA microarrays. These data were integrated with DNA binding in vivo of LexA1, a regulator of the DNA damage response, assessed by chromatin immunoprecipitation and massively parallel DNA sequencing (ChIP-seq). In response to DNA damage, Leptospira induced expression of genes involved in DNA metabolism, in mobile genetic elements and defective prophages. The DNA repair genes involved in removal of photo-damage (e.g. nucleotide excision repair uvrABC, recombinases recBCD and resolvases ruvABC) were not induced. Genes involved in various metabolic pathways were down regulated, including genes involved in cell growth, RNA metabolism and the tricarboxylic acid cycle. From ChIP-seq data, we observed 24 LexA1 binding sites located throughout chromosome 1 and one binding site in chromosome 2. Expression of many, but not all, genes near those sites was increased following DNA damage. Binding sites were found as far as 550 bp upstream from the start codon, or 1 kb into the coding sequence. Our findings indicate that there is a shift in gene expression following DNA damage that represses genes involved in cell growth and virulence, and induces genes involved in mutagenesis and recombination.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/nar/gkv1536DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4756842PMC
February 2016

Evaluation of a vaccine formulation against Streptococcus pneumoniae based on choline-binding proteins.

Clin Vaccine Immunol 2015 Feb 17;22(2):213-20. Epub 2014 Dec 17.

Centro de Biotecnologia, Instituto Butantan, São Paulo, SP, Brazil Bioindustrial Division, Instituto Butantan, São Paulo, SP, Brazil.

Streptococcus pneumoniae has proteins that are attached to its surface by binding to phosphorylcholine of teichoic and lipoteichoic acids. These proteins are known as choline-binding proteins (CBPs). CBPs are an interesting alternative for the development of a cost-effective vaccine, and PspA (pneumococcal surface protein A) is believed to be the most important protective component among the different CBPs. We sought to use CBPs eluted from pneumococci as an experimental vaccine. Since PspA shows variability between isolates, we constructed strains producing different PspAs. We used the nonencapsulated Rx1 strain, which produces PspA from clade 2 (PspA2), to generate a pspA-knockout strain (Rx1 ΔpspA) and strains expressing PspA from clade 1 (Rx1 pspA1) and clade 4 (Rx1 pspA4). We grew Rx1, Rx1 ΔpspA, Rx1 pspA1, and Rx1 pspA4 in Todd-Hewitt medium containing 0.5% yeast extract and washed cells in 2% choline chloride (CC). SDS-PAGE analysis of the proteins recovered by a CC wash showed few bands, and the CBPs PspA and PspC (pneumococcal surface protein C) were identified by mass spectrometry analysis. Subcutaneous immunization of mice with these full-length native proteins without adjuvant led to significantly higher rates of survival than immunization with diluent after an intranasal lethal challenge with two pneumococcal strains and also after a colonization challenge with one strain. Importantly, immunization with recombinant PspA4 (rPspA4) without adjuvant did not elicit significant protection.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/CVI.00692-14DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4308862PMC
February 2015

Pertussis toxin improves immune responses to a combined pneumococcal antigen and leads to enhanced protection against Streptococcus pneumoniae.

Clin Vaccine Immunol 2014 Jul 7;21(7):972-81. Epub 2014 May 7.

Centro de Biotecnologia, Instituto Butantan, São Paulo, Brazil

Pneumococcal surface protein A (PspA) is a candidate antigen for the composition of protein-based vaccines against Streptococcus pneumoniae. While searching for efficient adjuvants for PspA-based vaccines, our group has described the potential of combining PspA with the whole-cell pertussis vaccine (wP). When given to mice through the nasal route, a formulation composed of PspA from clade 5 (PspA5) and wP (PspA5-wP) induced high levels of antibodies and protection against challenges with different pneumococcal strains. PspA5-wP also induced the secretion of interleukin 17 (IL-17) by splenocytes and the infiltration of leukocytes in the lungs after challenge. Here, we show that protection against a pneumococcal invasive challenge was completely abrogated in μMT(-/-) mice, which are deficient in the maturation of B cells, illustrating the importance of antibodies in the survival elicited by the PspA5-wP vaccine. Moreover, passive immunization showed that IgG purified from the sera of mice immunized with PspA5-wP conferred significant protection to naive mice, whereas the respective F(ab')2 did not. Additionally, in vivo depletion of complement abolished protection against the pneumococcal challenge. The combination of PspA5 with wild-type or mutant Bordetella pertussis strains or with purified components showed that the pertussis toxin (PT)-containing formulations induced the highest levels of antibodies and protection. This suggests that the adjuvant activity of wP in the PspA5 model is mediated at least in part by PT. The sera from mice immunized with such formulations displayed high IgG binding and induction of complement deposition on the pneumococcal surface in vitro, which is consistent with the in vivo results.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/CVI.00134-14DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4097443PMC
July 2014

Mapping of epitopes recognized by antibodies induced by immunization of mice with PspA and PspC.

Clin Vaccine Immunol 2014 Jul 7;21(7):940-8. Epub 2014 May 7.

Centro de Biotecnologia, Instituto Butantan, São Paulo, Brazil

Pneumococcal surface protein A (PspA) and pneumococcal surface protein C (PspC) are important candidates for an alternative vaccine against pneumococcal infections. Since these antigens show variability, the use of variants that do not afford broad protection may lead to the selection of vaccine escape bacteria. Epitopes capable of inducing antibodies with broad cross-reactivities should thus be the preferred antigens. In this work, experiments using peptide arrays show that most linear epitopes recognized by antibodies induced in mice against different PspAs were located at the initial 44 amino acids of the mature protein and that antibodies against these linear epitopes did not confer protection against a lethal challenge. Conversely, linear epitopes recognized by antibodies to PspC included the consensus sequences involved in the interaction with human factor H and secretory immunoglobulin A (sIgA). Since linear epitopes of PspA were not protective, larger overlapping fragments containing 100 amino acids of PspA of strain Rx1 were constructed (fragments 1 to 7, numbered from the N terminus) to permit the mapping of antibodies with conformational epitopes not represented in the peptide arrays. Antibodies from mice immunized with fragments 1, 2, 4, and 5 were capable of binding onto the surface of pneumococci and mediating protection against a lethal challenge. The fact that immunization of mice with 100-amino-acid fragments located at the more conserved N-terminal region of PspA (fragments 1 and 2) induced protection against a pneumococcal challenge indicates that the induction of antibodies against conformational epitopes present at this region may be important in strategies for inducing broad protection against pneumococci.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/CVI.00239-14DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4097434PMC
July 2014

Leptospira interrogans serovar copenhageni harbors two lexA genes involved in SOS response.

PLoS One 2013 3;8(10):e76419. Epub 2013 Oct 3.

Centro de Biotecnologia, Instituto Butantan, São Paulo, Brazil ; Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, São Paulo, Brazil.

Bacteria activate a regulatory network in response to the challenges imposed by DNA damage to genetic material, known as the SOS response. This system is regulated by the RecA recombinase and by the transcriptional repressor lexA. Leptospira interrogans is a pathogen capable of surviving in the environment for weeks, being exposed to a great variety of stress agents and yet retaining its ability to infect the host. This study aims to investigate the behavior of L. interrogans serovar Copenhageni after the stress induced by DNA damage. We show that L. interrogans serovar Copenhageni genome contains two genes encoding putative LexA proteins (lexA1 and lexA2) one of them being potentially acquired by lateral gene transfer. Both genes are induced after DNA damage, but the steady state levels of both LexA proteins drop, probably due to auto-proteolytic activity triggered in this condition. In addition, seven other genes were up-regulated following UV-C irradiation, recA, recN, dinP, and four genes encoding hypothetical proteins. This set of genes is potentially regulated by LexA1, as it showed binding to their promoter regions. All these regions contain degenerated sequences in relation to the previously described SOS box, TTTGN 5CAAA. On the other hand, LexA2 was able to bind to the palindrome TTGTAN10TACAA, found in its own promoter region, but not in the others. Therefore, the L. interrogans serovar Copenhageni SOS regulon may be even more complex, as a result of LexA1 and LexA2 binding to divergent motifs. New possibilities for DNA damage response in Leptospira are expected, with potential influence in other biological responses such as virulence.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0076419PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3789691PMC
July 2014

Pneumococcal Surface Protein A does not affect the immune responses to a combined diphtheria tetanus and pertussis vaccine in mice.

Vaccine 2013 May 26;31(20):2465-70. Epub 2013 Mar 26.

Centro de Biotecnologia, Instituto Butantan, São Paulo, SP, Brazil.

The Pneumococcal Surface Protein A (PspA) is a promising candidate for the composition of a protein vaccine against Streptococcus pneumoniae. We have previously shown that the whole cell Bordetella pertussis vaccine (wP) is a good adjuvant to PspA, inducing protective responses against pneumococcal infection in mice. In Brazil, wP is administered to children, formulated with diphtheria and tetanus toxoids (DTPw) and aluminum hydroxide (alum) as adjuvant. A single subcutaneous dose of PspA5-DTPlow (a formulation containing PspA from clade 5 and a new generation DTPw, containing low levels of B. pertussis LPS and Alum) induced high levels of systemic anti-PspA5 antibodies in mice and conferred protection against respiratory lethal challenges with two different pneumococcal strains. Here we evaluate the mucosal immune responses against PspA5 as well as the immune responses against the DTP antigens in mice vaccinated with PspA5-DTPlow. Subcutaneous immunization of mice with PspA5-DTPlow induced high levels of anti-PspA5 IgG in the airways but no IgA. In addition, no differences in the influx of cells to the respiratory mucosa, after the challenge, were observed in vaccinated mice, when compared with control mice. The levels of circulating anti-pertussis, -tetanus and -diphtheria antibodies were equivalent in mice vaccinated with DTPlow or PspA5-DTPlow. Antibodies induced by DTPlow or PspA5-DTPlow showed similar ability to neutralize the cytotoxic effects of the diphtheria toxin on Vero cells. Furthermore, combination with PspA5 did not affect protection against B. pertussis and tetanus toxin challenges in mice. Our results support the proposal for a combined PspA-DTP vaccine.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.vaccine.2013.03.026DOI Listing
May 2013

Characterization of the antibody response elicited by immunization with pneumococcal surface protein A (PspA) as recombinant protein or DNA vaccine and analysis of protection against an intranasal lethal challenge with Streptococcus pneumoniae.

Microb Pathog 2012 Nov-Dec;53(5-6):243-9. Epub 2012 Sep 7.

Centro de Biotecnologia, Instituto Butantan, Av Vital Brasil, 1500, 05503-900, São Paulo, SP, Brazil.

Pneumococcal surface protein A (PspA) is an important candidate for a vaccine against pneumococcal infections. DNA vaccines expressing PspA were shown to protect mice against intraperitoneal and colonization challenge models in mice. We now show that a DNA vaccine expressing PspA from clade 4 (pSec-pspA4Pro) is also able to elicit protection against an intranasal lethal challenge model at levels similar to the recombinant protein PspA4Pro adjuvanted with alum. PspA4Pro + alum induced an IgG response characterized by a high IgG1/IgG2a ratio, leading to a lack of binding of anti-PspA IgG2a antibodies to intact pneumococci in vitro, which is in contrast to the response elicited by pSec-pspA4Pro. Epitopes recognized by the sera were mapped and antibodies induced by immunization with PspA4Pro + alum showed positive reaction with several synthetic peptides, mostly located in the first half of the protein. On the other hand, antibodies induced by the DNA vaccine showed reactivity with only two peptides. Though both strategies were protective against the intranasal lethal challenge model, the elicited humoral responses differ significantly, with the detection of important differences in the Fc (IgG1/IgG2a ratios) and Fab (recognized epitopes) regions of the induced antibodies.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.micpath.2012.08.007DOI Listing
March 2013

Controlled inflammatory responses in the lungs are associated with protection elicited by a pneumococcal surface protein A-based vaccine against a lethal respiratory challenge with Streptococcus pneumoniae in mice.

Clin Vaccine Immunol 2012 Sep 3;19(9):1382-92. Epub 2012 Jul 3.

Centro de Biotecnologia, Instituto Butantan, São Paulo, Brazil.

Streptococcus pneumoniae is a pathogen of great importance worldwide. We have previously described the efficacy of a nasal vaccine composed of the pneumococcal surface protein A and the whole-cell pertussis vaccine as an adjuvant against a pneumococcal invasive challenge in mice. Spread of bacteria to the bloodstream was probably prevented by the high levels of systemic antibodies induced by the vaccine, but bacteria were only cleared from the lungs 3 weeks later, indicating that local immune responses may contribute to survival. Here we show that a strict control of inflammatory responses in lungs of vaccinated mice occurs even in the presence of high numbers of pneumococci. This response was characterized by a sharp peak of neutrophils and lymphocytes with a simultaneous decrease in macrophages in the respiratory mucosa at 12 h postchallenge. Secretion of interleukin-6 (IL-6) and gamma interferon (IFN-γ) was reduced at 24 h postchallenge, and the induction of tumor necrosis factor alpha (TNF-α) secretion, observed in the first hours postchallenge, was completely abolished at 24 h. Before challenge and at 12 h postchallenge, vaccinated mice displayed higher numbers of CD4(+) T, CD8(+) T, and B lymphocytes in the lungs. However, protection still occurs in the absence of each of these cells during the challenge, indicating that other effectors may be related to the prevention of lung injuries in this model. High levels of mucosal anti-PspA antibodies were maintained in vaccinated mice during the challenge, suggesting an important role in protection.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/CVI.00171-12DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3428385PMC
September 2012

Cross-reactivity of antipneumococcal surface protein C (PspC) antibodies with different strains and evaluation of inhibition of human complement factor H and secretory IgA binding via PspC.

Clin Vaccine Immunol 2012 Apr 15;19(4):499-507. Epub 2012 Feb 15.

Centro de Biotecnologia, Instituto Butantan, São Paulo, Brazil.

Pneumococcal surface protein C (PspC) is an important candidate for a cost-effective vaccine with broad coverage against pneumococcal diseases. Previous studies have shown that Streptococcus pneumoniae is able to bind to both human factor H (FH), an inhibitor of complement alternative pathway, and human secretory IgA (sIgA) via PspC. PspC was classified into 11 groups based on variations of the gene. In this work, we used three PspC fragments from different groups (PspC3, PspC5, and PspC8) to immunize mice for the production of antibodies. Immunization with PspC3 induced antibodies that recognized the majority of the clinical isolates as analyzed by Western blotting of whole-cell extracts and flow cytometry of intact bacteria, while anti-PspC5 antibodies showed cross-reactivity with the paralogue pneumococcal surface protein A (PspA), and anti-PspC8 antibodies reacted only with the PspC8-expressing strain. Most of the isolates tested showed strong binding to FH and weaker interaction with sIgA. Preincubation with anti-PspC3 and anti-PspC5 IgG led to some inhibition of binding of FH, and preincubation with anti-PspC3 partially inhibited sIgA binding in Western blotting. The analysis of intact bacteria through flow cytometry showed only a small decrease in FH binding after incubation of strain D39 with anti-PspC3 IgG, and one clinical isolate showed inhibition of sIgA binding by anti-PspC3 IgG. We conclude that although anti-PspC3 antibodies were able to recognize PspC variants from the majority of the strains tested, partial inhibition of FH and sIgA binding through anti-PspC3 antibodies in vitro could be observed for only a restricted number of isolates.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/CVI.05706-11DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3318277PMC
April 2012

Venomics profiling of Thamnodynastes strigatus unveils matrix metalloproteinases and other novel proteins recruited to the toxin arsenal of rear-fanged snakes.

J Proteome Res 2012 Feb 20;11(2):1152-62. Epub 2012 Jan 20.

Centro de Biotecnologia, Instituto Butantan, Av. Vital Brazil, 1500, São Paulo, SP, 05503-900, Brazil.

Rear-fanged and aglyphous snakes are usually considered not dangerous to humans because of their limited capacity of injecting venom. Therefore, only a few studies have been dedicated to characterizing the venom of the largest parcel of snake fauna. Here, we investigated the venom proteome of the rear-fanged snake Thamnodynastes strigatus , in combination with a transcriptomic evaluation of the venom gland. About 60% of all transcripts code for putative venom components. A striking finding is that the most abundant type of transcript (∼47%) and also the major protein type in the venom correspond to a new kind of matrix metalloproteinase (MMP) that is unrelated to the classical snake venom metalloproteinases found in all snake families. These enzymes were recently suggested as possible venom components, and we show here that they are proteolytically active and probably recruited to venom from a MMP-9 ancestor. Other unusual proteins were suggested to be venom components: a protein related to lactadherin and an EGF repeat-containing transcript. Despite these unusual molecules, seven toxin classes commonly found in typical venomous snakes are also present in the venom. These results support the evidence that the arsenals of these snakes are very diverse and harbor new types of biologically important molecules.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/pr200876cDOI Listing
February 2012

Bothrops jararaca venom proteome rearrangement upon neonate to adult transition.

Proteomics 2011 Nov 16;11(21):4218-28. Epub 2011 Sep 16.

Laboratório Especial de Toxinologia Aplicada-CAT/cepid, Instituto Butantan, Brazil.

The pharmacological activities displayed by Bothrops jararaca venom undergo a significant ontogenetic shift. Similarly, the diet of this species changes from ectothermic prey in early life to endothermic prey in adulthood. In this study we used large and representative newborn and adult venom samples consisting of pools from 694 and 110 specimens, respectively, and demonstrate a significant ontogenetic shift in the venom proteome complexity of B. jararaca. 2-DE coupled to MS protein identification showed a clear rearrangement of the toxin arsenal both in terms of the total proteome, as of the glycoproteome. N-glycosylation seems to play a key role in venom protein variability between newborn and adult specimens. Upon the snake development, the subproteome of metalloproteinases undergoes a shift from a P-III-rich to a P-I-rich profile while the serine proteinase profile does not vary significantly. We also used isobaric tag labeling (iTRAQ) of venom tryptic peptides for the first time to examine the quantitative changes in the venom toxins of B. jararaca upon neonate to adult transition. The iTRAQ analysis showed changes in various toxin classes, especially the proteinases. Our study expands the in-depth understanding of venom complexity variation particularly with regard to toxin families that have been associated with envenomation pathogenesis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/pmic.201100287DOI Listing
November 2011

Snake venomics and venom gland transcriptomic analysis of Brazilian coral snakes, Micrurus altirostris and M. corallinus.

J Proteomics 2011 Aug 12;74(9):1795-809. Epub 2011 Apr 12.

Instituto de Bioquímica Médica, Programa de Biologia Estrutural and Instituto Nacional de Biologia Estrutural e Bioimagem, Universidade Federal do Rio de Janeiro, Brazil.

The venom proteomes of Micrurus altirostris and M. corallinus were analyzed by combining snake venomics and venom gland transcriptomic surveys. In both coral snake species, 3FTx and PLA(2) were the most abundant and diversified toxin families. 33 different 3FTxs and 13 PLA(2) proteins, accounting respectively for 79.5% and 13.7% of the total proteins, were identified in the venom of M. altirostris. The venom of M. corallinus comprised 10 3FTx (81.7% of the venom proteome) and 4 (11.9%) PLA(2) molecules. Transcriptomic data provided the full-length amino acid sequences of 18 (M. altirostris) and 10 (M. corallinus) 3FTxs, and 3 (M. altirostris) and 1 (M. corallinus) novel PLA(2) sequences. In addition, venom from each species contained single members of minor toxin families: 3 common (PIII-SVMP, C-type lectin-like, L-amino acid oxidase) and 4 species-specific (CRISP, Kunitz-type inhibitor, lysosomal acid lipase in M. altirostris; serine proteinase in M. corallinus) toxin classes. The finding of a lipase (LIPA) in the venom proteome and in the venom gland transcriptome of M. altirostris supports the view of a recruitment event predating the divergence of Elapidae and Viperidae more than 60 Mya. The toxin profile of both M. altirostris and M. corallinus venoms points to 3FTxs and PLA(2) molecules as the major players of the envenoming process. In M. altirostris venom, all major, and most minor, 3FTxs display highest similarity to type I α-neurotoxins, suggesting that these postsynaptically acting toxins may play the predominant role in the neurotoxic effect leading to peripheral paralysis, respiratory arrest, and death. M. corallinus venom posesses both, type I α-neurotoxins and a high-abundance (26% of the venom proteome) protein of subfamily XIX of 3FTxs, exhibiting similarity to bucandin from Malayan krait, Bungarus candidus, venom, which enhances acetylcholine release presynaptically. This finding may explain the presynaptic neurotoxicity of M. corallinus venom and the lack of this effect in M. altirostris venom. The anti-Micrurus (corallinus and frontalis) antivenom produced by Instituto Butantan quantitatively immunodepleted the minor toxins from M. altirostris and M. corallinus venoms but showed impaired crossreactivity towards their major 3FTx and PLA(2) molecules. The structural diversity of 3FTxs among Micrurus sp. may underlay the impaired cross-immunoreactivity of the Butantan antivenom towards M. altirostris and M. corallinus toxins, hampering the possibility to raise an antivenom against a simple venom mixture exhibiting paraspecific neutralization of other Micrurus venoms.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jprot.2011.04.003DOI Listing
August 2011

Evaluation of the use of selective PCR amplification of LPS biosynthesis genes for molecular typing of leptospira at the serovar level.

Curr Microbiol 2011 Feb 19;62(2):518-24. Epub 2010 Aug 19.

Centro de Biotecnologia, Instituto Butantan, 05503-900, São Paulo, SP, Brazil.

Leptospirosis is an important epidemic zoonosis worldwide. Currently, there are more than 250 Leptospira pathogenic serovars known that can potentially infect humans. Conventional classification of leptospires with the serovar as the basic taxon, based on serological recognition of lipopolysaccharide (LPS) composition does not correlate well with species determination, based on general genomic features. Here, we investigate the selective amplification of polymorphic regions from the LPS biosynthesis loci (rfb) as a potential tool for serovar typing of Leptospira interrogans species. Eight pairs of primers were designed to target six ORFs from the rfb operon with varying levels of sequence polymorphism. They were tested both separately and multiplexed. Half of these primer pairs produced serovar-specific amplicons, allowing the identification of some specific serovars and also groups of serovars. It was shown that the serovar classification of Leptospira can be accessed by selective amplification of rfb operons in some cases, which may permit a parallel between the serological and the genomic classifications of Leptospira. As a conclusion, the selective amplification of rfb generated promising and already useful results, but it appears necessary to characterize a larger variety of Leptospira genomes or rfb operons to fully develop this method.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s00284-010-9738-7DOI Listing
February 2011

Increased immunogenicity to LipL32 of Leptospira interrogans when expressed as a fusion protein with the cholera toxin B subunit.

Curr Microbiol 2011 Feb 19;62(2):526-31. Epub 2010 Aug 19.

Instituto de Biotecnología y Biología Molecular, Centro Científico Tecnológico CONICET La Plata, La Plata, Argentina.

Leptospirosis is one of the most widespread zoonosis in the world. The development of a recombinant leptospira vaccine remains a challenge. In this study, we cloned the Leptospira interrogans open reading frame (ORF) coding the external membrane protein LipL32, an immunodominant antigen found in all pathogenic leptospira, downstream of the highly immunogenic cholera toxin B subunit (CTB) ORF. Expression and assembly of the CTB-LipL32 fusion protein into oligomeric structures of pentameric size were observed in soluble fractions by Western blot analysis. The CTB-LipL32 protein demonstrated strong affinity for monosialotetrahexosylgaglioside (GM1-ganglioside) in an enzyme-linked immunosorbent assay (ELISA), suggesting that the antigenic sites for binding and proper folding of the pentameric CTB structure were conserved. Furthermore, antisera against LipL32 also recognized the CTB-LipL32 fusion protein, suggesting that LipL32 also conserved its antigenic sites, a fact confirmed by an ELISA assay showing soluble CTB-LipL32 recognition by sera from convalescent patients. In addition, soluble CTB-LipL32 generated higher specific titers in mice immunized without external adjuvant than co-administration of CTB with LipL32. The data presented here provide support for CTB-LipL32 as a promising antigen for use in the control and study of leptospirosis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s00284-010-9739-6DOI Listing
February 2011

Combination of pneumococcal surface protein A (PspA) with whole cell pertussis vaccine increases protection against pneumococcal challenge in mice.

PLoS One 2010 May 27;5(5):e10863. Epub 2010 May 27.

Centro de Biotecnologia, Instituto Butantan, São Paulo, São Paulo, Brazil.

Streptococcus pneumoniae is the leading cause of respiratory acute infections around the world. In Latin America, approximately 20,000 children under 5 years of age die of pneumococcal diseases annually. Pneumococcal surface protein A (PspA) is among the best-characterized pneumococcal antigens that confer protection in animal models of pneumococcal infections and, as such, is a good alternative for the currently available conjugated vaccines. Efficient immune responses directed to PspA in animal models have already been described. Nevertheless, few low cost adjuvants for a subunit pneumococcal vaccine have been proposed to date. Here, we have tested the adjuvant properties of the whole cell Bordetella pertussis vaccine (wP) that is currently part of the DTP (diphtheria-tetanus-pertussis) vaccine administrated to children in several countries, as an adjuvant to PspA. Nasal immunization of BALB/c mice with a combination of PspA5 and wP or wP(low)--a new generation vaccine that contains low levels of B. pertussis LPS--conferred protection against a respiratory lethal challenge with S. pneumoniae. Both PspA5-wP and PspA5-wP(low) vaccines induced high levels of systemic and mucosal antibodies against PspA5, with similar profile, indicating no essential requirement for B. pertussis LPS in the adjuvant properties of wP. Accordingly, nasal immunization of C3H/HeJ mice with PspA5-wP conferred protection against the pneumococcal challenge, thus ruling out a role for TLR4 responses in the adjuvant activity and the protection mechanisms triggered by the vaccines. The high levels of anti-PspA5 antibodies correlated with increased cross-reactivity against PspAs from different clades and also reflected in cross-protection. In addition, passive immunization experiments indicated that antibodies played an important role in protection in this model. Finally, subcutaneous immunization with a combination of PspA5 with DTP(low) protected mice against challenge with two different pneumococcal strains, opening the possibility for the development of a combined infant vaccine composed of DTP and PspA.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0010863PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2877721PMC
May 2010

Protection against nasal colonization with Streptococcus pneumoniae by parenteral immunization with a DNA vaccine encoding PspA (Pneumococcal surface protein A).

Microb Pathog 2010 Jun 4;48(6):205-13. Epub 2010 Mar 4.

Centro de Biotecnologia, Instituto Butantan, Av Vital Brasil, 1500, 05503-900, São Paulo, SP, Brazil.

Pneumococcal surface protein A (PspA) is an important candidate for a cost-effective vaccine with broad coverage against Streptococcus pneumoniae. We have previously shown that intramuscular immunization with PspA as a DNA vaccine induces an immune response characterized by the induction of a balanced IgG1/IgG2a antibody response in BALB/c mice, which was able to efficiently mediate complement deposition onto intact bacteria and to induce protection against an intraperitoneal challenge. We now confirm the results in C57BL/6 mice and further show that the response induced by the DNA vaccine expressing PspA is able to mediate protection against colonization of the nasopharyngeal mucosa even though immunization was given parenterally. Moreover, a positive correlation was observed between IgG1 and the numbers of CFU recovered, whereas an inverse correlation was observed between nasal CFU levels and IgG2a. A positive correlation was also found for IgG1/IgG2a antibody ratios with CFU recovered from the nasopharynx. Therefore, reduction of nasal colonization was strongly associated with increased levels of serum IgG2a complement fixing antibody and low levels of IgG1 antibody which has much less complement fixing activity. Passive transfer of serum from animals immunized with the DNA vaccine expressing PspA was also able to reduce the fraction of mice with high density of colonization of the nasopharynx. Secretion of IFN-gamma, but not IL-17, was observed in splenocytes from mice immunized with the DNA vaccine.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.micpath.2010.02.009DOI Listing
June 2010

Analysis of the ontogenetic variation in the venom proteome/peptidome of Bothrops jararaca reveals different strategies to deal with prey.

J Proteome Res 2010 May;9(5):2278-91

Laboratório Especial de Toxinologia Aplicada-CAT/cepid, Instituto Butantan, Brazil.

Previous studies have demonstrated that the pharmacological activities displayed by Bothrops jararaca venom undergo a significant ontogenetic shift. Variation in the venom proteome is a well-documented phenomenon; however, variation in the venom peptidome is poorly understood. We report a comparative proteomic and peptidomic analysis of venoms from newborn and adult specimens of B. jararaca and correlate it with the evaluation of important venom features. We demonstrate that newborn and adult venoms have similar hemorrhagic activities, while the adult venom has a slightly higher lethal activity in mice; however, the newborn venom is extremely more potent to kill chicks. The coagulant activity of newborn venom upon human plasma is 10 times higher than that of adult venom. These differences were clearly reflected in their different profiles of SDS-PAGE, gelatin zimography, immunostaining using specific antibodies, glycosylation pattern, and concanavalin A-binding proteins. Furthermore, we report for the first time the analysis of the peptide fraction of newborn and adult venoms by MALDI-TOF mass spectrometry and LC-MS/MS, which revealed different contents of peptides, while the bradykinin potentiating peptides (BPPs) showed rather similar profiles and were detected in the venoms showing their canonical sequences and also novel sequences corresponding to BPPs processed from their precursor protein at sites so far not described. As a result of these studies, we demonstrated that the ontogenetic shift in diet, from ectothermic prey in early life to endothermic prey in adulthood, and in animal size are associated with changes in the venom proteome in B. jararaca species.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/pr901027rDOI Listing
May 2010

Immunization of mice with single PspA fragments induces antibodies capable of mediating complement deposition on different pneumococcal strains and cross-protection.

Clin Vaccine Immunol 2010 Mar 20;17(3):439-46. Epub 2010 Jan 20.

Centro de Biotecnologia, Instituto Butantan, São Paulo, SP, Brazil.

PspA is an important candidate for a vaccine with serotype-independent immunity against pneumococcal infections. Based on sequence relatedness, PspA has been classified into three families comprising six clades. We have previously addressed the cross-reactivity of antibodies against PspA fragments containing the N-terminal and proline-rich regions of PspA from clades 1 to 5 (PspA1, PspA2, PspA3, PspA4, and PspA5) by Western blot analysis and reported that anti-PspA4 and anti-PspA5 were able to recognize pneumococci expressing PspA proteins from all of the clades analyzed. We have now analyzed the functional capacity of these antibodies to bind and to mediate complement deposition on intact bacteria in vitro. Our results show that both PspA4 and PspA5 elicit antibodies that are able to bind and to mediate complement deposition efficiently on pneumococcal strains bearing PspA proteins from clades 1 to 5. Moreover, mice immunized with PspA4 and PspA5 were protected against an intranasal lethal challenge with strains expressing PspA proteins from the two major families. PspA4 and PspA5 are thus able to induce antibodies with a high degree of cross-reactivity in vitro, which is reflected in cross-protection of mice. We have also analyzed the contribution of the nonproline (NonPro) block within the conserved proline-rich region to the reactivity of anti-PspA antibodies, and the results indicate that N-terminal alpha-helical region, the blocks of proline repeats, and the NonPro region can influence the degree of cross-reactivity of antibodies to PspA.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/CVI.00430-09DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2837969PMC
March 2010

The action of topical basic fibroblast growth factor in facial nerve regeneration.

Otol Neurotol 2010 Apr;31(3):498-505

Department of Otorhinolaryngology-Head and Neck Surgery, Federal University of São Paulo, Rua dos Otonis 700, São Paulo, Brazil.

Objective: To analyze the influence of the topical use of basic fibroblast growth factor (bFGF) in the regeneration of the facial nerve in rats.

Study Design: Experimental study.

Materials And Methods: Twenty-eight Wistar adult male rats underwent complete section of the facial nerve trunk, followed by end-to-end anastomosis with epineural sutures. An osmotic minipump equipped with a delivery catheter was implanted subcutaneously near the neural anastomosis. During the subsequent 14 days, 14 animals received a solution containing 25 microg/ml of bFGF, 250 UI/ml of sodium heparin, and 1,000 microg/ml of human albumin diluted in Ringer lactate, and 14 animals received a control solution of the same components without bFGF. To evaluate facial nerve regeneration, the number of myelinated fibers evident on histologic sections was counted on the 14th (7 experimental and 8 control animals) and the 28th days (7 experimental and 6 control animals) after surgery, and the facial movements of vibrissae and the blink reflex were evaluated on alternate days until the 28th day.

Results: On histologic evaluation, the number of myelinated fibers was similar between groups on the 14th day and greater in the group that received bFGF on the 28th day. Behavioral evaluation showed that the animals of the bFGF group presented better functional results between the 6th and 16th days for the blink test and the 14th to the 16th days for vibrissae movements.

Conclusion: This study showed that the regeneration of the facial nerve occurred earlier and resulted in significantly more myelinated nerve fibers in the animals that received topical bFGF.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1097/MAO.0b013e3181c0e7e9DOI Listing
April 2010

Evaluation of the expression and protective potential of Leptospiral sphingomyelinases.

Curr Microbiol 2010 Feb 14;60(2):134-42. Epub 2009 Oct 14.

Centro de Biotecnologia, Instituto Butantan, São Paulo, SP, Brazil.

Leptospirosis is a zoonotic disease of global distribution, which affects both animals and humans. Pathogenic leptospires, the bacteria that cause this disease, require iron for their growth, and these spirochetes probably use their hemolysins, such as the sphingomyelinases, as a way to obtain this important nutrient from host red blood cells during infection. We expressed and purified the leptospiral sphingomyelinases Sph1, Sph2, Sph4, and SphH in a heterologous system. However, the recombinant proteins were not able to lyse sheep erythrocytes, despite having regular secondary structures. Transcripts for all sphingomyelinases tested were detected by RT-PCR analyses, but only Sph2 and SphH native proteins could be detected in Western blot assays using Leptospira whole extracts as well as in renal tubules of infected hamsters. Moreover, antibodies present in the serum of a human patient with laboratory-confirmed leptospirosis recognized Sph2, indicating that this sphingomyelinase is expressed and exposed to the immune system during infection in humans. However, in an animal challenge model, none of the sphingomyelinases tested conferred protection against leptospirosis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s00284-009-9519-3DOI Listing
February 2010

Comparison of the pulmonary response against lethal and non-lethal intranasal challenges with two different pneumococcal strains.

Microb Pathog 2009 Sep 23;47(3):157-63. Epub 2009 May 23.

Centro de Biotecnologia, Instituto Butantan, São Paulo, SP, Brazil.

The differences between the immune response elicited during a self-limiting and a life-threatening lung infection with Streptococcus pneumoniae was analyzed in a mouse model of intranasal challenge using two different pneumococcal strains. M10, a serotype 11A strain, induced an early response within the first 12h after the challenge, which was characterized by the early local secretion of TNF-alpha and IL-6, followed by a sharp and rapid neutrophil influx. Bacterial loads in the lungs already started to fall at 12h after the challenge and no pneumococci could be recovered after 36h, at the time point when the animals started to show improvement in disease symptoms. ATCC6303, a serotype 3 strain, on the other hand, showed only a late increase in local TNF-alpha and IL-6 levels, when bacterial growth already seems to be out of control. Although cell influx was also observed, neutrophil rise was not as marked as with M10 (type 11A). Pneumococcal loads increased constantly and bacteria started to be recovered from the blood at 30h after the challenge. After this time point, animals showed worsening of symptoms and became lethargic. The resolution of the acute infection could be thus correlated with the early induction of proinflammatory cytokines, which could be due to the presence of a thinner polysaccharide capsule in M10 (type 11A), rendering bacterial components capable of activating the innate immune response more accessible.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.micpath.2009.05.005DOI Listing
September 2009

Leptospiral TlyC is an extracellular matrix-binding protein and does not present hemolysin activity.

FEBS Lett 2009 Apr 27;583(8):1381-5. Epub 2009 Mar 27.

Centro de Biotecnologia, Instituto Butantan, 05503-900 São Paulo - SP, Brazil.

The role of TlyA, TlyB and TlyC proteins in the biology of Leptospira is still uncertain. Although these proteins have been considered as putative hemolysins, we demonstrate that leptospiral recombinant TlyB and TlyC do not possess hemolytic activity. However, further experiments showed that TlyC is a surface-exposed protein that seems to bind to laminin, collagen IV and fibronectin. The expression of both proteins was detected both in vitro and in vivo. Our findings suggest that TlyB and TlyC are not directly involved in hemolysis, and that TlyC may contribute to Leptospira binding to extracellular matrix (ECM) during host infection.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.febslet.2009.03.050DOI Listing
April 2009

Transcriptomic basis for an antiserum against Micrurus corallinus (coral snake) venom.

BMC Genomics 2009 Mar 16;10:112. Epub 2009 Mar 16.

Centro de Biotecnologia, Instituto Butantan, São Paulo, SP, Brazil.

Background: Micrurus corallinus (coral snake) is a tropical forest snake belonging to the family Elapidae. Its venom shows a high neurotoxicity associated with pre- and post-synaptic toxins, causing diaphragm paralysis, which may result in death. In spite of a relatively small incidence of accidents, serum therapy is crucial for those bitten. However, the adequate production of antiserum is hampered by the difficulty in obtaining sufficient amounts of venom from a small snake with demanding breeding conditions. In order to elucidate the molecular basis of this venom and to uncover possible immunogens for an antiserum, we generated expressed sequences tags (ESTs) from its venom glands and analyzed the transcriptomic profile. In addition, their immunogenicity was tested using DNA immunization.

Results: A total of 1438 ESTs were generated and grouped into 611 clusters. Toxin transcripts represented 46% of the total ESTs. The two main toxin classes consisted of three-finger toxins (3FTx) (24%) and phospholipases A(2) (PLA(2)s) (15%). However, 8 other classes of toxins were present, including C-type lectins, natriuretic peptide precursors and even high-molecular mass components such as metalloproteases and L-amino acid oxidases. Each class included an assortment of isoforms, some showing evidence of alternative splicing and domain deletions. Five antigenic candidates were selected (four 3FTx and one PLA(2)) and used for a preliminary study of DNA immunization. The immunological response showed that the sera from the immunized animals were able to recognize the recombinant antigens.

Conclusion: Besides an improvement in our knowledge of the composition of coral snake venoms, which are very poorly known when compared to Old World elapids, the expression profile suggests abundant and diversified components that may be used in future antiserum formulation. As recombinant production of venom antigens frequently fails due to complex disulfide arrangements, DNA immunization may be a viable alternative. In fact, the selected candidates provided an initial evidence of the feasibility of this approach, which is less costly and not dependent on the availability of the venom.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/1471-2164-10-112DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2662881PMC
March 2009

Crystallization and preliminary X-ray analysis of LipL32 from Leptospira interrogans serovar Copenhageni.

Acta Crystallogr Sect F Struct Biol Cryst Commun 2009 Mar 26;65(Pt 3):307-9. Epub 2009 Feb 26.

Centro de Biotecnologia, Instituto Butantan, São Paulo-SP, Brazil.

LipL32 is a major surface protein that is expressed during infection by pathogenic Leptospira. Here, the crystallization of recombinant LipL32(21-272), which corresponds to the mature LipL32 protein minus its N-terminal lipid-anchored cysteine residue, is described. Selenomethionine-labelled LipL32(21-272) crystals diffracted to 2.25 A resolution at a synchrotron source. The space group was P3(1)21 or P3(2)21 and the unit-cell parameters were a = b = 126.7, c = 96.0 A.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1107/S1744309109005533DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2650462PMC
March 2009