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Find video protocols related to scientific articles indexed in Pubmed.
Protective Effects of Lactococci Strains Delivering Either IL-10 Protein or cDNA in a TNBS-induced Chronic Colitis Model.
J. Clin. Gastroenterol.
PUBLISHED: 10-08-2014
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Oral treatment with Lactococcus lactis strains secreting the anti-inflammatory cytokine interleukin (IL)-10 has previously shown success as a therapy for inflammatory bowel diseases (IBD).
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Milk Fermented with a 15-Lipoxygenase-1-Producing Lactococcus Lactis Alleviates Symptoms of colitis in a Murine Model.
Curr Pharm Biotechnol
PUBLISHED: 09-26-2014
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Inflammatory bowel disease (IBD), such as Crohn's disease and ulcerative colitis, are characterized by extensive inflammation due to dysregulation of the innate and adaptive immune system whose exact etiology is not yet completely understood. Currently there is no cure for IBD, thus the search for new molecules capable of controlling IBD and their delivery to the site of inflammation are the goal of many researchers. The aim of this work was to evaluate the anti-inflammatory effect of the administration of milks fermented by a Lactococcus (L.) lactis strain producing 15-lipoxygenase-1 (15-LOX-1) using a trinitrobenzenesulfonic acid-induced IBD mouse model. The results obtained demonstrated that 15-LOX-1 producing L. lactis was effective in the prevention of the intestinal damage associated to inflammatory bowel disease in a murine model. The work also confirmed previous studies showing that fermented milk is an effective form of administration of recombinant lactic acid bacteria expressing beneficial molecules.
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Recombinant Lactococcus lactis fails to secrete bovine chymosine.
Bioengineered
PUBLISHED: 09-03-2014
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Bovine chymosin is an important milk-clotting agent used in the manufacturing of cheeses. Currently, the production of recombinant proteins by genetically modified organisms is widespread, leading to greatly reduced costs. Lactococcus (L.) lactis, the model lactic acid bacterium, was considered a good candidate for heterologous chymosin production for the following reasons: (1) it is considered to be a GRAS (generally regarded as safe) microorganism, (2) only one protease is present on its surface, (3) it can secrete proteins of different sizes, and (4) it allows for the direct production of protein in fermented food products. Thus, three genetically modified L. lactis strains were constructed to produce and target the three different forms of bovine chymosin, prochymosin B, chymosin A and chymosin B to the extracellular medium. Although all three proteins were stably produced in L. lactis, none of the forms were detected in the extracellular medium or showed clotting activity in milk. Our hypothesis is that this secretion deficiency and lack of clotting activity can be explained by the recombinant protein being attached to the cell envelope. Thus, the development of other strategies is necessary to achieve both production and targeting of chymosin in L. lactis, which could facilitate the downstream processing and recovery of this industrially important protein.
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Safety and protective effectiveness of two strains of Lactobacillus with probiotic features in an experimental model of salmonellosis.
Int J Environ Res Public Health
PUBLISHED: 08-26-2014
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Two strains of Lactobacillus, previously isolated from bovine faeces and tested in vitro for properties desired in probiotics, were evaluated for their in vivo effectiveness in protecting against experimental salmonellosis. L. salivarius L38 and L. acidophilus L36 previously demonstrated the ability to successfully colonize the gastrointestinal tract of germ-free mice and stimulate the immune system associated with the intestinal mucosa. L38- or L36-feeding showed no detrimental effect on the general health indicators and did not induce changes in normal architecture of liver and small intestine, indicating that the use of these strains is apparently safe. In control animals fed L38 strain, several cytokines had augmented mRNA levels that can be associated with a homeostatic state of intestinal mucosa, while L36 had less diverse regulation. IgA production and secretion in the intestinal lumen induced by infection was abrogated by pretreating with both lactobacilli. In addition, liver and small intestine histological scores and, translocation of Salmonella cells to liver and spleen, indicated that these strains did not confer protection against the infection. So, the IL-12:IL-18àIFN-g axis, essential for an effective immune response against Salmonella, was not favored with L38 or L36 strains. However, increased expression of IL-10 in different portions of the gastrointestinal tract of L38-fed animals is indicative of anti-inflammatory effect to be explored furthermore.
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Lactococcus lactis carrying the pValac DNA expression vector coding for IL-10 reduces inflammation in a murine model of experimental colitis.
BMC Biotechnol.
PUBLISHED: 08-09-2014
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Inflammatory bowel diseases (IBD) are intestinal disorders characterized by inflammation in the gastrointestinal tract. Interleukin-10 is one of the most important anti-inflammatory cytokines involved in the intestinal immune system and because of its role in downregulating inflammatory cascades, its potential for IBD therapy is under study. We previously presented the development of an invasive strain of Lactococcus lactis (L. lactis) producing Fibronectin Binding Protein A (FnBPA) which was capable of delivering, directly to host cells, a eukaryotic DNA expression vector coding for IL-10 of Mus musculus (pValac:il-10) and diminish inflammation in a trinitrobenzene sulfonic acid (TNBS)-induced mouse model of intestinal inflammation. As a new therapeutic strategy against IBD, the aim of this work was to evaluate the therapeutic effect of two L. lactis strains (the same invasive strain evaluated previously and the wild-type strain) carrying the therapeutic pValac:il-10 plasmid in the prevention of inflammation in a dextran sodium sulphate (DSS)-induced mouse model.
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Anti-inflammatory effects of Lactococcus lactis NCDO 2118 during the remission period of chemically induced colitis.
Gut Pathog
PUBLISHED: 07-29-2014
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Many probiotic bacteria have been described as promising tools for the treatment and prevention of inflammatory bowel diseases (IBDs). Most of these bacteria are lactic acid bacteria, which are part of the healthy human microbiota. However, little is known about the effects of transient bacteria present in normal diets, including Lactococcus lactis.
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Correlation between fibronectin binding protein A expression level at the surface of recombinant lactococcus lactis and plasmid transfer in vitro and in vivo.
BMC Microbiol.
PUBLISHED: 07-08-2014
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BackgroundFibronectin Binding Protein A (FnBPA) is an invasin from Staphylococcus aureus that allows this pathogen to internalize into eukaryote cells. It was previously demonstrated that recombinant Lactococcus lactis expressing FnBPA were invasive and able to transfer a plasmid to eukaryotic cells in vitro and in vivo. In this study, the invasivity of recombinant strains of Lactococcus lactis that express FnBPA under the control of its constitutive promoter or driven by the strong nisin inducible expression system (NICE) were studied.ResultsIt was demonstrated that the nisA promoter allows an increase of FnBPA expression on the surface of Lactococcus lactis surface, as shown by flow cytometry, which subsequently enhanced internalization and plasmid transfer properties in vitro in Caco2 cells and Bone Marrow Dendritic Cells. In vivo, the use of nisA promoter increase the plasmid transfer in cells of both the small and large intestine of mice.ConclusionFnBPA expression at the surface of recombinant L. lactis is positively correlated to internalization and DNA transfer properties. The recombinant strains of L. lactis that expresses FnBPA under the control of the nisin inducible expression system could thus be considered as an improved tool in the field of DNA transfer.
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Fine-tuned characterization of Staphylococcus aureus Newbould 305, a strain associated with mild and chronic mastitis in bovines.
Vet. Res.
PUBLISHED: 06-25-2014
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S. aureus is a major aetiological agent of ruminant mastitis worldwide. The chronic nature of S. aureus mastitis makes it difficult to cure and prone to resurgence. In order to identify the bacterial factors involved in this chronicity, Newbould 305 (N305), a strain that can reproducibly induce mild and chronic mastitis in an experimental setting, was characterized in depth. We employed genomic and proteomic techniques combined with phenotype characterization, in order to comprehensively analyse N305. The results were compared with data obtained on S. aureus RF122, a strain representative of the major clone involved in severe bovine mastitis worldwide. Five mobile genetic elements were identified in the N305 genome as carrying virulence factors which correlated with phenotypic features such as cytotoxicity, mammary epithelial cell invasion or host-adaptation. In particular, the presence and characteristics of surface exposed proteins correlated well with the greater adhesion and internalization capacities of N305 in bovine mammary epithelial cells. N305 also displayed less diversity of toxin genes but secreted larger quantities of these toxins, associated with a higher cytotoxicity potential. Our data are consistent with the invasiveness and host-adaptation features which contribute to the chronicity of S. aureus mastitis. Mobile genetic elements, exoproteins and surface exposed proteins constitute good targets for further research to explore the underlying mechanisms related to mastitis chronicity.
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?(ECF) factors of gram-positive bacteria: a focus on Bacillus subtilis and the CMNR group.
Virulence
PUBLISHED: 06-12-2014
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The survival of bacteria to different environmental conditions depends on the activation of adaptive mechanisms, which are intricately driven through gene regulation. Because transcriptional initiation is considered to be the major step in the control of bacterial genes, we discuss the characteristics and roles of the sigma factors, addressing (1) their structural, functional and phylogenetic classification; (2) how their activity is regulated; and (3) the promoters recognized by these factors. Finally, we focus on a specific group of alternative sigma factors, the so-called ?(ECF) factors, in Bacillus subtilis and some of the main species that comprise the CMNR group, providing information on the roles they play in the microorganisms' physiology and indicating some of the genes whose transcription they regulate.
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Characterization of the Opp peptide transporter of Corynebacterium pseudotuberculosis and its role in virulence and pathogenicity.
Biomed Res Int
PUBLISHED: 03-07-2014
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Despite the economic importance of caseous lymphadenitis (CLA), a chronic disease caused by Corynebacterium pseudotuberculosis, few genes related to the virulence of its etiologic agent have been characterized. The oligopeptide permease (Opp) transporters are located in the plasma membrane and have functions generally related to the uptake of peptides from the extracellular environment. These peptide transporters, in addition to having an important role in cell nutrition, also participate in the regulation of various processes involving intercellular signaling, including the control of the expression of virulence genes in pathogenic bacteria. To study the role of Opp in C. pseudotuberculosis, an OppD deficient strain was constructed via simple crossover with a nonreplicative plasmid carrying part of the oppD gene sequence. As occurred to the wild-type, the ?oppD strain showed impaired growth when exposed to the toxic glutathione peptide (GSH), indicating two possible scenarios: (i) that this component can be internalized by the bacterium through an Opp-independent pathway or (ii) that there is toxicity while the peptide is extracellular. Additionally, the ?oppD mutant presented a reduced ability to adhere to and infect macrophages compared to the wild-type, although both strains exhibit the same potential to colonize spleens and cause injury and death to infected mice.
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An iron-acquisition-deficient mutant of Corynebacterium pseudotuberculosis efficiently protects mice against challenge.
Vet. Res.
PUBLISHED: 02-25-2014
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Caseous lymphadenitis (CLA) is a chronic disease that affects sheep and goats worldwide, and its etiological agent is Corynebacterium pseudotuberculosis. Despite the economic losses caused by CLA, there is little information about the molecular mechanisms of bacterial pathogenesis, and current immune prophylaxis against infection has been unable to reduce the incidence of CLA in goats. Recently, 21 different mutant strains of C. pseudotuberculosis were identified by random mutagenesis. In this study, these previously generated mutants were used in mice vaccination trials to develop new immunogens against CLA. Based on this analysis, CZ171053, an iron-acquisition-deficient mutant strain, was selected. After challenge with a virulent strain, 80% of the animals that were immunized with the CZ171053 strain survived. Furthermore, this vaccination elicited both humoral and cellular responses. Intracellular survival of the bacterium was determined using murine J774 cells; in this assay, the CZ171053 had reduced intracellular viability. Because iron acquisition in intracellular bacteria is considered one of their most important virulence factors during infection, these results demonstrate the immunogenic potential of this mutant against CLA.
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Oral combined therapy with probiotics and alloantigen induces B cell-dependent long-lasting specific tolerance.
J. Immunol.
PUBLISHED: 01-22-2014
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Allogeneic hematopietic stem cell transplantation (aHSCT) is widely used for the treatment of hematologic malignancies. Although aHSCT provides a good response against the malignant cells (graft-versus-leukemia [GVL]), it also leads to the development of graft-versus-host disease (GVHD), a severe disease with high mortality and morbidity rates. Therapy for GVHD is commonly based on nonspecific immunosupression of the transplanted recipient, resulting in the concomitant inhibition of the GVL effect. In this study, we propose an alternative approach to specifically suppress GVHD while sparing the GVL, based on oral treatment of transplant donors with recipient Ags, associated with the intake of probiotic Lactococcus lactis as tolerogenic adjuvant (combined therapy). We show that treatment of C57BL/6 donor mice with combined therapy before the transplant protects the recipients F1 (C57BL/6 × BAL/c) mice from clinical and pathological manifestations of disease, resulting in 100% survival rate. Importantly, the animals keep the immunological competence maintaining the GVL response as well as the response to third-party Ags. The protection is specific, long lasting and dependent on donor IL-10-sufficient B cells activity, which induces regulatory T cells in the host. These data suggest that combined therapy is a promising strategy for prevention of GVHD with preservation of GVL, opening new possibilities to treat human patients subjected to transplantation.
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Value of a newly sequenced bacterial genome.
World J Biol Chem
PUBLISHED: 01-14-2014
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Next-generation sequencing (NGS) technologies have made high-throughput sequencing available to medium- and small-size laboratories, culminating in a tidal wave of genomic information. The quantity of sequenced bacterial genomes has not only brought excitement to the field of genomics but also heightened expectations that NGS would boost antibacterial discovery and vaccine development. Although many possible drug and vaccine targets have been discovered, the success rate of genome-based analysis has remained below expectations. Furthermore, NGS has had consequences for genome quality, resulting in an exponential increase in draft (partial data) genome deposits in public databases. If no further interests are expressed for a particular bacterial genome, it is more likely that the sequencing of its genome will be limited to a draft stage, and the painstaking tasks of completing the sequencing of its genome and annotation will not be undertaken. It is important to know what is lost when we settle for a draft genome and to determine the "scientific value" of a newly sequenced genome. This review addresses the expected impact of newly sequenced genomes on antibacterial discovery and vaccinology. Also, it discusses the factors that could be leading to the increase in the number of draft deposits and the consequent loss of relevant biological information.
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Serological proteome analysis of Corynebacterium pseudotuberculosis isolated from different hosts reveals novel candidates for prophylactics to control caseous lymphadenitis.
Vet. Microbiol.
PUBLISHED: 01-11-2014
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Caseous lymphadenitis (CLA) is a highly prevalent disease in goats and sheep worldwide, which is caused by Corynebacterium pseudotuberculosis. Although several prophylactic methods against CLA have been proposed previously, the identification of new C. pseudotuberculosis proteins that are really produced during the infectious process is still needed to improve efficiency and accuracy in vaccines and diagnostics. In this study, we used optimized conditions for serological proteome analysis (SERPA) in order to identify new immune-reactive proteins in C. pseudotuberculosis culture supernatants of two strains, 1002 and C231, isolated from goats and sheep, respectively. Using a sheep and goat serum pool, 13 novel immune-reactive exoproteins common to the two strains were identified. Four of these proteins present known functions and were already described as immune-reactive proteins in other microorganisms, whereas the other nine are of unknown function and show low similarity with proteins from other bacterial species. These data reveal promising targets for immunoprophylactic methods against CLA.
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Differential transcriptional profile of Corynebacterium pseudotuberculosis in response to abiotic stresses.
BMC Genomics
PUBLISHED: 01-09-2014
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The completion of whole-genome sequencing for Corynebacterium pseudotuberculosis strain 1002 has contributed to major advances in research aimed at understanding the biology of this microorganism. This bacterium causes significant loss to goat and sheep farmers because it is the causal agent of the infectious disease caseous lymphadenitis, which may lead to outcomes ranging from skin injury to animal death. In the current study, we simulated the conditions experienced by the bacteria during host infection. By sequencing transcripts using the SOLiDTM 3 Plus platform, we identified new targets expected to potentiate the survival and replication of the pathogen in adverse environments. These results may also identify possible candidates useful for the development of vaccines, diagnostic kits or therapies aimed at the reduction of losses in agribusiness.
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Evaluation of ERIC-PCR as genotyping method for Corynebacterium pseudotuberculosis isolates.
PLoS ONE
PUBLISHED: 01-01-2014
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The aim of this study was to evaluate the Enterobacterial Repetitive Intergenic Consensus (ERIC-PCR) as a tool for molecular typing of C. pseudotuberculosis isolates from eight different hosts in twelve countries. Ninety-nine C. pseudotuberculosis field strains, one type strain (ATCC 19410T) and one vaccine strain (1002) were fingerprinted using the ERIC-1R and ERIC-2 primers, and the ERIC-1R+ERIC-2 primer pair. Twenty-nine different genotypes were generated by ERIC 1-PCR, 28 by ERIC 2-PCR and 35 by ERIC 1+2-PCR. The discriminatory index calculated for ERIC 1, ERIC 2, and ERIC 1+2-PCR was 0.89, 0.86, and 0.92, respectively. Epidemiological concordance was established for all ERIC-PCR assays. ERIC 1+2-PCR was defined as the best method based on suitability of the amplification patterns and discriminatory index. Minimal spanning tree for ERIC 1+2-PCR revealed three major clonal complexes and clustering around nitrate-positive (biovar Equi) and nitrate-negative (biovar Ovis) strains. Therefore, ERIC 1+2-PCR proved to be the best technique evaluated in this study for genotyping C. pseudotuberculosis strains, due to its usefulness for molecular epidemiology investigations.
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Local and systemic immune mechanisms underlying the anti-colitis effects of the dairy bacterium Lactobacillus delbrueckii.
PLoS ONE
PUBLISHED: 01-01-2014
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Several probiotic bacteria have been proposed for treatment or prevention of inflammatory bowel diseases (IBD), showing a protective effect in animal models of experimental colitis and for some of them also in human clinical trials. While most of these probiotic bacteria are isolated from the digestive tract, we recently reported that a Lactobacillus strain isolated from cheese, L. delbrueckii subsp. lactis CNRZ327 (Lb CNRZ327), also possesses anti-inflammatory effects in vitro and in vivo, demonstrating that common dairy bacteria may be useful in the treatment or prevention of IBD. Here, we studied the mechanisms underlying the protective effects of Lb CNRZ327 in vivo, in a mouse dextran sodium sulfate (DSS) colitis model. During colitis, Lb CNRZ327 modulated the production of TGF-?, IL-6, and IL-12 in colonic tissue and of TGF-? and IL-6 in the spleen, and caused an expansion of CD4+Foxp3+ regulatory T cells in the cecal lymph nodes. Moreover, a strong tendency to CD4+Foxp3+ expansion was also observed in the spleen. The results of this study for the first time show that orally administered dairy lactobacilli can not only modulate mucosal but also systemic immune responses and constitute an effective treatment of IBD.
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Immune response elicited by DNA vaccination using Lactococcus lactis is modified by the production of surface exposed pathogenic protein.
PLoS ONE
PUBLISHED: 01-01-2014
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In this study, we compared immune responses elicited by DNA immunization using Lactococcus lactis or L. lactis expressing the Staphylococcus aureus invasin Fibronectin Binding Protein A (FnBPA) at its surface. Both strains carried pValac:BLG, a plasmid containing the cDNA of Beta-Lactoglobulin (BLG), and were designated LL-BLG and LL-FnBPA+ BLG respectively. A TH2 immune response characterized by the secretion of IL-4 and IL-5 in medium of BLG reactivated splenocytes was detected after either oral or intranasal administration of LL-FnBPA+ BLG. In contrast, intranasal administration of LL-BLG elicited a TH1 immune response. After BLG sensitization, mice previously intranasally administered with LL-BLG showed a significantly lower concentration of BLG-specific IgE than the mice non-administered. Altenatively administration of LL-FnBPA+ BLG didn't modify the BLG-specific IgE concentration obtained after sensitization, thus confirming the TH2 orientation of the immune response. To determine if the TH2-skewed immune response obtained with LL-FnBpA+ BLG was FnBPA-specific or not, mice received another L. lactis strain producing a mutated form of the Listeria monocytogenes invasin Internalin A intranasally, allowing thus the binding to murine E-cadherin, and containing pValac:BLG (LL-mInlA+ BLG). As with LL-FnBPA+ BLG, LL-mInlA+ BLG was not able to elicit a TH1 immune response. Furthermore, we observed that these difference were not due to the peptidoglycan composition of the cell wall as LL-FnBPA+ BLG, LL-mInlA+ BLG and LL-BLG strains shared a similar composition. DNA vaccination using LL-BLG elicited a pro-inflammatory TH1 immune response while using LL-FnBPA+ BLG or LL-mInlA+ BLG elicited an anti-inflammatory TH2 immune response.
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Complete genome sequence of Streptococcus agalactiae strain SA20-06, a fish pathogen associated to meningoencephalitis outbreaks.
Stand Genomic Sci
PUBLISHED: 06-15-2013
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Streptococcus agalactiae (Lancefield group B; GBS) is the causative agent of meningoencephalitis in fish, mastitis in cows, and neonatal sepsis in humans. Meningoencephalitis is a major health problem for tilapia farming and is responsible for high economic losses worldwide. Despite its importance, the genomic characteristics and the main molecular mechanisms involved in virulence of S. agalactiae isolated from fish are still poorly understood. Here, we present the genomic features of the 1,820,886 bp long complete genome sequence of S. agalactiae SA20-06 isolated from a meningoencephalitis outbreak in Nile tilapia (Oreochromis niloticus) from Brazil, and its annotation, consisting of 1,710 protein-coding genes (excluding pseudogenes), 7 rRNA operons, 79 tRNA genes and 62 pseudogenes.
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Identification of 11 new exoproteins in Corynebacterium pseudotuberculosis by comparative analysis of the exoproteome.
Microb. Pathog.
PUBLISHED: 04-23-2013
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This study involves the comparison between the exoproteomes of two different strains of Corynebacterium pseudotuberculosis, the etiologic agent of caseous lymphadenitis in small ruminants. In a previous study, based on a gel-free system (TPP-LC/MS(E)), 70 exoproteins for the strain 1002 and 67 for the strain C231, totaling 93 different extracellular proteins for C. pseudotuberculosis, were identified. In the present work, we have used 2D gel electrophoresis to resolve the extracellular proteins of both strains, which were then digested with trypsin, analyzed by MALDI-TOF/TOF and identified with the software MASCOT(®). A total of 45 extracellular proteins of C. pseudotuberculosis were identified by this approach. The comparative analysis between the strains 1002 and C231 identified 13 and 3 strain-specific proteins, respectively, 11 of which are novel. These newly identified proteins may play an important role in the physiology and virulence of C. pseudotuberculosis.
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Identification of a vaccine against schistosomiasis using bioinformatics and molecular modeling tools.
Infect. Genet. Evol.
PUBLISHED: 03-29-2013
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Schistosomiasis is a serious public health problem in Brazil and worldwide. Although the drugs used to treatment schistosomiasis are effective, the disease continues to expand in all endemic countries due to constant reinfection, poor sanitation, and the lack of effective programs for disease control. However, advances generated through genome projects have provided important information that has improved the understanding of the biology of this parasite. These advances, associated with the advent of bioinformatic analysis, are becoming an important tool in reverse vaccinology. Through database access to the DNA and protein sequences of Schistosoma mansoni and the use of bioinformatics programs, fourteen epitopes were identified. Five epitopes were obtained from proteins whose immunogenic potential had already been assessed in other studies (KP), and nine whose immunogenic potential is unknown (UP). To improve stimulation of the host immune system, the selected epitopes were modeled with a sugar moiety. After this addition, all of the epitopes showed structures similar to those observed in the native proteins, but only eleven of the peptides presented thermodynamically stable structures. Prediction analysis and molecular modeling showed that the glycopeptides presented here are important targets in the search for a vaccine against schistosomiasis. Additionally, they suggest that these molecules may be used in immunological assays to evaluate the level of protection, the effect on pathology reduction and the profile of cytokines and antibodies induced by them.
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Oral immunization with Lactococcus lactis secreting attenuated recombinant staphylococcal enterotoxin B induces a protective immune response in a murine model.
Microb. Cell Fact.
PUBLISHED: 03-22-2013
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Staphylococcus aureus is unrestrictedly found in humans and in animal species that maintain thermal homeostasis. Inadequate cleaning of processing equipment or inappropriate handling can contaminate processed food and cause severe food poisoning. Staphylococcal enterotoxin B (SEB), a potent superantigenic exotoxin, is produced by 50% of clinical isolates of S. aureus and is associated with massive food poisoning and with the induction of toxic shock syndrome.
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Mucosal targeting of therapeutic molecules using genetically modified lactic acid bacteria: an update.
FEMS Microbiol. Lett.
PUBLISHED: 03-10-2013
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Lactic acid bacteria (LAB) represent a heterogeneous group of microorganisms naturally present in many foods and those have proved to be effective mucosal delivery vectors. Moreover, some specific strains of LAB exert beneficial properties (known as probiotic effect) on both human and animal health. Although probiotic effects are strain-specific traits, it is theoretically possible, using genetic engineering techniques, to design strains that can exert a variety of beneficial properties. During the two past decades, a large variety of therapeutic molecules has been successfully expressed in LAB, and although this field has been largely reviewed in recent years, approximately 20 new publications appear each year. Thus, the aim of this minireview is not to extensively assess the entire literature but to update progress made within the last 2 years regarding the use of the model LAB Lactococcus lactis and certain species of lactobacilli as live recombinant vectors for the development of new safe mucosal vaccines.
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Differential Exoproteome analysis of two corynebacterium pseudotuberculosis biovar ovis strains isolated from goat (1002) and sheep (C231).
Curr. Microbiol.
PUBLISHED: 02-19-2013
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Corynebacterium pseudotuberculosis is the etiologic agent of caseous lymphadenitis a chronic infectious disease affecting small ruminants. The 2D-DIGE technique was used to compare the exoproteomes of two C. pseudotuberculosis biovar ovis strains isolated from goat (strain 1002) and sheep (strain C231). Seventeen proteins differentially produced were identified here. Nine proteins appeared over-produced in the exoproteome of 1002 goat strain and 8 in that of C231 sheep strain. These proteins were related to various biological functions, such as the cell envelope, respiratory metabolism and proteolysis. This proteomic analysis revealed strain-specific exoproteins although each of the corresponding genes was found in both strain genomes. Such differential expression pattern may reflect inter-strain differences in adaptation to a specific host, in pathogenicity and or in antigenicity of this pathogenic bacterium.
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Exoproteome and secretome derived broad spectrum novel drug and vaccine candidates in Vibrio cholerae targeted by Piper betel derived compounds.
PLoS ONE
PUBLISHED: 01-30-2013
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Vibrio cholerae is the causal organism of the cholera epidemic, which is mostly prevalent in developing and underdeveloped countries. However, incidences of cholera in developed countries are also alarming. Because of the emergence of new drug-resistant strains, even though several generic drugs and vaccines have been developed over time, Vibrio infections remain a global health problem that appeals for the development of novel drugs and vaccines against the pathogen. Here, applying comparative proteomic and reverse vaccinology approaches to the exoproteome and secretome of the pathogen, we have identified three candidate targets (ompU, uppP and yajC) for most of the pathogenic Vibrio strains. Two targets (uppP and yajC) are novel to Vibrio, and two targets (uppP and ompU) can be used to develop both drugs and vaccines (dual targets) against broad spectrum Vibrio serotypes. Using our novel computational approach, we have identified three peptide vaccine candidates that have high potential to induce both B- and T-cell-mediated immune responses from our identified two dual targets. These two targets were modeled and subjected to virtual screening against natural compounds derived from Piper betel. Seven compounds were identified first time from Piper betel to be highly effective to render the function of these targets to identify them as emerging potential drugs against Vibrio. Our preliminary validation suggests that these identified peptide vaccines and betel compounds are highly effective against Vibrio cholerae. Currently we are exhaustively validating these targets, candidate peptide vaccines, and betel derived lead compounds against a number of Vibrio species.
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Implications of the human microbiome in inflammatory bowel diseases.
FEMS Microbiol. Lett.
PUBLISHED: 01-25-2013
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The study of the human microbiome or community of microorganisms and collection of genomes found in the human body is one of the fastest growing research areas because many diseases are reported to be associated with microbiome imbalance or dysbiosis. With the improvement in novel sequencing techniques, researchers are now generating millions of sequences of different sites from the human body and evaluating specific differences in microbial communities. The importance of microbiome constituency is so relevant that several consortia like the Human Microbiome project (HMP) and Metagenomics of the Human Intestinal Tract (MetaHIT) project are focusing mainly on the human microbiome. The aim of this review is to highlight points of research in this field, mainly focusing on particular factors that modulate the microbiome and important insights into its potential impact on our health and well-being.
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Ion Torrent-based transcriptional assessment of a Corynebacterium pseudotuberculosis equi strain reveals denaturing high-performance liquid chromatography a promising rRNA depletion method.
Microb Biotechnol
PUBLISHED: 01-15-2013
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Corynebacterium pseudotuberculosis equi is a Gram-positive pathogenic bacterium which affects a variety of hosts. Besides the great economic losses it causes to horse-breeders, this organism is also known to be an important infectious agent to cattle and buffaloes. As an outcome of the efforts in characterizing the molecular basis of its virulence, several complete genome sequences were made available in recent years, enabling the large-scale assessment of genes throughout distinct isolates. Meanwhile, the RNA-seq stood out as the technology of choice for comprehensive transcriptome studies, which may bring valuable information regarding active genomic regions, despite of the still impeditive associated costs. In an attempt to increase the use of generated reads per instrument run, by effectively eliminating unwanted rRNAs from total RNA samples without relying on any commercially available kits, we applied denaturing high-performance liquid chromatography (DHPLC) as an alternative method to assess the transcriptional profile of C. pseudotuberculosis. We have found that the DHPLC depletion method, allied to Ion Torrent sequencing, allows mapping of transcripts in a comprehensive way and identifying novel transcripts when a de novo approach is used. These data encourage us to use DHPLC in future transcriptional evaluations in C. pseudotuberculosis.
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The pan-genome of the animal pathogen Corynebacterium pseudotuberculosis reveals differences in genome plasticity between the biovar ovis and equi strains.
PLoS ONE
PUBLISHED: 01-14-2013
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Corynebacterium pseudotuberculosis is a facultative intracellular pathogen and the causative agent of several infectious and contagious chronic diseases, including caseous lymphadenitis, ulcerative lymphangitis, mastitis, and edematous skin disease, in a broad spectrum of hosts. In addition, Corynebacterium pseudotuberculosis infections pose a rising worldwide economic problem in ruminants. The complete genome sequences of 15 C. pseudotuberculosis strains isolated from different hosts and countries were comparatively analyzed using a pan-genomic strategy. Phylogenomic, pan-genomic, core genomic, and singleton analyses revealed close relationships among pathogenic corynebacteria, the clonal-like behavior of C. pseudotuberculosis and slow increases in the sizes of pan-genomes. According to extrapolations based on the pan-genomes, core genomes and singletons, the C. pseudotuberculosis biovar ovis shows a more clonal-like behavior than the C. pseudotuberculosis biovar equi. Most of the variable genes of the biovar ovis strains were acquired in a block through horizontal gene transfer and are highly conserved, whereas the biovar equi strains contain great variability, both intra- and inter-biovar, in the 16 detected pathogenicity islands (PAIs). With respect to the gene content of the PAIs, the most interesting finding is the high similarity of the pilus genes in the biovar ovis strains compared with the great variability of these genes in the biovar equi strains. Concluding, the polymerization of complete pilus structures in biovar ovis could be responsible for a remarkable ability of these strains to spread throughout host tissues and penetrate cells to live intracellularly, in contrast with the biovar equi, which rarely attacks visceral organs. Intracellularly, the biovar ovis strains are expected to have less contact with other organisms than the biovar equi strains, thereby explaining the significant clonal-like behavior of the biovar ovis strains.
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Conserved host-pathogen PPIs. Globally conserved inter-species bacterial PPIs based conserved host-pathogen interactome derived novel target in C. pseudotuberculosis, C. diphtheriae, M. tuberculosis, C. ulcerans, Y. pestis, and E. coli targeted by Piper b
Integr Biol (Camb)
PUBLISHED: 01-05-2013
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Although attempts have been made to unveil protein-protein and host-pathogen interactions based on molecular insights of important biological events and pathogenesis in various organisms, these efforts have not yet been reported in Corynebacterium pseudotuberculosis (Cp), the causative agent of Caseous Lymphadenitis (CLA). In this study, we used computational approaches to develop common conserved intra-species protein-protein interaction (PPI) networks first time for four Cp strains (Cp FRC41, Cp 316, Cp 3/99-5, and Cp P54B96) followed by development of a common conserved inter-species bacterial PPI using conserved proteins in multiple pathogens (Y. pestis, M. tuberculosis, C. diphtheriae, C. ulcerans, E. coli, and all four Cp strains) and E. Coli based experimentally validated PPI data. Furthermore, the interacting proteins in the common conserved inter-species bacterial PPI were used to generate a conserved host-pathogen interaction (HP-PPI) network considering human, goat, sheep, bovine, and horse as hosts. The HP-PPI network was validated, and acetate kinase (Ack) was identified as a novel broad spectrum target. Ceftiofur, penicillin, and two natural compounds derived from Piper betel were predicted to inhibit Ack activity. One of these Piper betel compounds found to inhibit E. coli O157:H7 growth similar to penicillin. The target specificity of these betel compounds, their effects on other studied pathogens, and other in silico results are currently being validated and the results are promising.
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Staphylococcus aureus-induced G2/M phase transition delay in host epithelial cells increases bacterial infective efficiency.
PLoS ONE
PUBLISHED: 01-01-2013
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Staphylococcus aureus is a highly versatile, opportunistic pathogen and the etiological agent of a wide range of infections in humans and warm-blooded animals. The epithelial surface is its principal site of colonization and infection. In this work, we investigated the cytopathic effect of S. aureus strains from human and animal origins and their ability to affect the host cell cycle in human HeLa and bovine MAC-T epithelial cell lines. S. aureus invasion slowed down cell proliferation and induced a cytopathic effect, resulting in the enlargement of host cells. A dramatic decrease in the number of mitotic cells was observed in the infected cultures. Flow cytometry analysis revealed an S. aureus-induced delay in the G2/M phase transition in synchronous HeLa cells. This delay required the presence of live S. aureus since the addition of the heat-killed bacteria did not alter the cell cycle. The results of Western blot experiments showed that the G2/M transition delay was associated with the accumulation of inactive cyclin-dependent kinase Cdk1, a key inducer of mitosis entry, and with the accumulation of unphosphorylated histone H3, which was correlated with a reduction of the mitotic cell number. Analysis of S. aureus proliferation in asynchronous, G1- and G2-phase-enriched HeLa cells showed that the G2 phase was preferential for bacterial infective efficiency, suggesting that the G2 phase delay may be used by S. aureus for propagation within the host. Taken together, our results divulge the potential of S. aureus in the subversion of key cellular processes such as cell cycle progression, and shed light on the biological significance of S. aureus-induced host cell cycle alteration.
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A singular value decomposition approach for improved taxonomic classification of biological sequences.
BMC Genomics
PUBLISHED: 12-22-2011
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Singular value decomposition (SVD) is a powerful technique for information retrieval; it helps uncover relationships between elements that are not prima facie related. SVD was initially developed to reduce the time needed for information retrieval and analysis of very large data sets in the complex internet environment. Since information retrieval from large-scale genome and proteome data sets has a similar level of complexity, SVD-based methods could also facilitate data analysis in this research area.
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Complete genome sequence of Corynebacterium pseudotuberculosis strain CIP 52.97, isolated from a horse in Kenya.
J. Bacteriol.
PUBLISHED: 11-30-2011
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In this work, we report the whole-genome sequence of Corynebacterium pseudotuberculosis bv. equi strain CIP 52.97 (Collection Institut Pasteur), isolated in 1952 from a case of ulcerative lymphangitis in a Kenyan horse, which has evidently caused significant losses to agribusiness. Therefore, obtaining this genome will allow the detection of important targets for postgenomic studies, with the aim of minimizing problems caused by this microorganism.
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High sero-prevalence of caseous lymphadenitis identified in slaughterhouse samples as a consequence of deficiencies in sheep farm management in the state of Minas Gerais, Brazil.
BMC Vet. Res.
PUBLISHED: 11-08-2011
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Caseous lymphadenitis (CLA), caused by Corynebacterium pseudotuberculosis, is one of the most important diseases of sheep and goats, causing considerable economic losses for herd owners.
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Whole-genome sequence of Corynebacterium pseudotuberculosis PAT10 strain isolated from sheep in Patagonia, Argentina.
J. Bacteriol.
PUBLISHED: 11-01-2011
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In this work, we report the complete genome sequence of a Corynebacterium pseudotuberculosis PAT10 isolate, collected from a lung abscess in an Argentine sheep in Patagonia, whose pathogen also required an investigation of its pathogenesis. Thus, the analysis of the genome sequence offers a means to better understanding of the molecular and genetic basis of virulence of this bacterium.
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Anti-inflammatory properties of dairy lactobacilli.
Inflamm. Bowel Dis.
PUBLISHED: 06-02-2011
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The intestinal microbiota plays an important role in human health through the modulation of innate immune responses. While selected commensal bacteria are marketed in specific probiotic products to control these responses, relatively little is known about the immune modulation potential of dairy bacteria that have principally been selected for their fermentation properties. The modulation of innate immune responses may reduce chronic inflammation in inflammatory bowel diseases like ulcerative colitis.
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Comparative analysis of two complete Corynebacterium ulcerans genomes and detection of candidate virulence factors.
BMC Genomics
PUBLISHED: 06-01-2011
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Corynebacterium ulcerans has been detected as a commensal in domestic and wild animals that may serve as reservoirs for zoonotic infections. During the last decade, the frequency and severity of human infections associated with C. ulcerans appear to be increasing in various countries. As the knowledge of genes contributing to the virulence of this bacterium was very limited, the complete genome sequences of two C. ulcerans strains detected in the metropolitan area of Rio de Janeiro were determined and characterized by comparative genomics: C. ulcerans 809 was initially isolated from an elderly woman with fatal pulmonary infection and C. ulcerans BR-AD22 was recovered from a nasal sample of an asymptomatic dog.
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A novel comparative genomics analysis for common drug and vaccine targets in Corynebacterium pseudotuberculosis and other CMN group of human pathogens.
Chem Biol Drug Des
PUBLISHED: 05-25-2011
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Caseous lymphadenitis is a chronic goat and sheep disease caused by Corynebacterium pseudotuberculosis (Cp) that accounts for a huge economic loss worldwide. Proper vaccination or medication is not available because of the lack of understanding of molecular biology of the pathogen. In a recent approach, four Cp (CpFrc41, Cp1002, CpC231, and CpI-19) genomes were sequenced to elucidate the molecular pathology of the bacteria. In this study, using these four genome sequences along with other eight genomes (total 12 genomes) and a novel subtractive genomics approach (first time ever applied to a veterinary pathogen), we identified potential conserved common drug and vaccine targets of these four Cp strains along with other Corybacterium, Mycobacterium and Nocardia (CMN) group of human pathogens (Corynebacterium diphtheriae and Mycobacterium tuberculosis) considering goat, sheep, bovine, horse, and human as the most affected hosts. The minimal genome of Cp1002 was found to consist of 724 genes, and 20 conserved common targets (to all Cp strains as well as CMN group of pathogens) from various metabolic pathways (13 from host-pathogen common and seven from pathogens unique pathways) are potential targets irrespective of all hosts considered. ubiA from host-pathogen common pathway and an ABC-like transporter from unique pathways may serve dual (drug and vaccine) targets. Two Corynebacterium-specific (mscL and resB) and one broad-spectrum (rpmB) novel targets were also identified. Strain-specific targets are also discussed. Six important targets were subjected to virtual screening, and one compound was found to be potent enough to render two targets (cdc and nrdL). We are currently validating all identified targets and lead compounds.
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Molecular characterization of Corynebacterium pseudotuberculosis isolates using ERIC-PCR.
Vet. Microbiol.
PUBLISHED: 05-23-2011
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Caseous lymphadenitis is an infectious sheep and goats disease caused by Corynebacterium pseudotuberculosis and characterized by abscesses in superficial and visceral lymph nodes. C. pseudotuberculosis strains isolated from these hosts have been shown to be very difficult to type by the existing methods. The aim of this study is evaluating the potential of the Enterobacterial Repetitive Intergenic Consensus (ERIC-PCR) as a tool for molecular typing of C. pseudotuberculosis strains isolated in sheep. One hundred and twenty seven isolates of C. pseudotuberculosis were isolated from lesions suspected to have had caseous lymphadenitis collected from sheep at the slaughterhouse. Animals were from 24 flocks in 13 municipalities of the Minas Gerais State, Brazil. Species identification of the isolates was performed by routine biochemical tests and mPCR. Fingerprint was performed by RAPD using ERIC-1R, ERIC-2 and ERIC-1R+ERIC-2 primers. Seventeen different genotypes were generated by ERIC 1-PCR, 21 genotypes by ERIC 2-PCR and 21 genotypes by ERIC 1+2-PCR. Hunter-Gaston Discrimination Index (HGDI) found for ERIC 1, ERIC 2, ERIC 1+2 PCR were 0.69, 0.87, and 0.84, respectively. For most herds evaluated observed at most three different genotypes among isolates from animals of these property, in all ERIC-PCR assays. However a few flocks observed between four and nine genotypes per flock. The W Kendall value found for correlation among the three techniques of ERIC-PCR was 0.91 (P<5.0 x 10(-6)). The results show that ERIC-PCR has good discriminatory power and advantages over other DNA-based typing methods, making it a useful tool to discriminate C. pseudotuberculosis isolates.
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The phage display technique: advantages and recent patents.
Recent Pat DNA Gene Seq
PUBLISHED: 05-09-2011
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Phage display technology has advanced considerably since its creation, and the number of research projects using this technique is constantly increasing, generating numerous antibody and antigen libraries. These libraries, besides expediting library screening, improving selection methods and allowing evaluation of novel applications, have great potential for the development of new vaccines, drugs and diagnosis tests. Consequently, patent registries for the protection of these sequences are essential.
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Evaluation of the anti-inflammatory effect of milk fermented by a strain of IL-10-producing Lactococcus lactis using a murine model of Crohns disease.
J. Mol. Microbiol. Biotechnol.
PUBLISHED: 05-03-2011
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Interleukin-10 (IL-10) is the most important anti-inflammatory cytokine at intestinal level, and its absence is involved in inflammatory bowel diseases. However, oral treatment with IL-10 is difficult because of its low survival in the gastrointestinal tract and systemic treatments lead to undesirable side effects. The aim of this paper was to evaluate the anti-inflammatory effect of the administration of milks fermented by Lactococcus lactis strains that produce IL-10 under the control of the xylose-inducible expression system using a trinitrobenzenesulfonic acid-induced colitis murine model. Mice that received milks fermented by L. lactis strains producing IL-10 in the cytoplasm (Cyt strain) or secreted to the product (Sec strain) showed lower damage scores in their large intestines, decreased IFN-? levels in their intestinal fluids and lower microbial translocation to liver, compared to mice receiving milk fermented by the wild-type strain or those not receiving any treatment. The results obtained in this study show that the employment of fermented milks as a new form of administration of IL-10-producing L. lactisis effective in the prevention of inflammatory bowel disease in a murine model.
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Lactococcus lactis as a live vector: heterologous protein production and DNA delivery systems.
Protein Expr. Purif.
PUBLISHED: 04-12-2011
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Lactic acid bacteria (LAB), widely used in the food industry, are present in the intestine of most animals, including humans. The potential use of these bacteria as mucosal delivery vehicles for vaccinal, medical or technological use has been extensively investigated. Lactococcus lactis, a LAB species, is a potential candidate for the production of biologically useful proteins and for plasmid DNA delivery to eukaryotic cells. Several delivery systems have been developed to target heterologous proteins to a specific cell location (i.e., cytoplasm, cell wall or extracellular medium) and more recently to efficiently transfer DNA to eukaryotic cells. A promising application of L. lactis is its use for the development of live mucosal vaccines. Here, we have reviewed the expression of heterologous protein and the various delivery systems developed for L. lactis, as well as its use as an oral vaccine carrier.
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Corynebacterium pseudotuberculosis secreted antigen-induced specific gamma-interferon production by peripheral blood leukocytes: potential diagnostic marker for caseous lymphadenitis in sheep and goats.
J. Vet. Diagn. Invest.
PUBLISHED: 03-15-2011
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In the current study, the applicability of the quantification of gamma interferon (IFN-?) levels for the detection of animals infected with Corynebacterium pseudotuberculosis and for determining caseous lymphadenitis (CLA) clinical status was evaluated. Peripheral blood leukocytes were collected from CLA nonendemic areas animals, from CLA seropositive animals without clinical signs of the disease, and from seropositive animals presenting CLA clinical signs. The leukocytes were stimulated with C. pseudotuberculosis-secreted antigens that were concentrated by the three-phase partitioning technique. An ovine IFN-? enzyme-linked immunosorbent assay was used to quantify IFN-? production. Goats and sheep with CLA had higher IFN-? levels than uninfected seronegative animals. Leukocytes from sheep with CLA chronic abscesses produced higher IFN-? levels when compared with seropositive sheep without CLA clinical signs, but this difference was not significant in goats. The sensitivity of the assay was 55.8% and 56%, whereas the specificity was 100% and 93%, for goats and sheep, respectively. In conclusion, IFN-? is a potential marker for the determination of CLA infection status in small ruminants; however, further research is needed to improve assay sensitivity.
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Evidence for reductive genome evolution and lateral acquisition of virulence functions in two Corynebacterium pseudotuberculosis strains.
PLoS ONE
PUBLISHED: 03-11-2011
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Corynebacterium pseudotuberculosis, a gram-positive, facultative intracellular pathogen, is the etiologic agent of the disease known as caseous lymphadenitis (CL). CL mainly affects small ruminants, such as goats and sheep; it also causes infections in humans, though rarely. This species is distributed worldwide, but it has the most serious economic impact in Oceania, Africa and South America. Although C. pseudotuberculosis causes major health and productivity problems for livestock, little is known about the molecular basis of its pathogenicity.
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Importance of IL-10 modulation by probiotic microorganisms in gastrointestinal inflammatory diseases.
ISRN Gastroenterol
PUBLISHED: 02-08-2011
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Lactic acid bacteria (LAB) represent a heterogeneous group of microorganisms that are naturally present in many foods and possess a wide range of therapeutic properties. The aim of this paper is to present an overview of the current expanding knowledge of one of the mechanisms by which LAB and other probiotic microorganisms participate in the prevention and treatment of gastrointestinal inflammatory disease through their immune-modulating properties. A special emphasis will be placed on the critical role of the anti-inflammatory cytokine IL-10, and a brief overview of the uses of genetically engineered LAB that produce this important immune response mediator will also be discussed. Thus, this paper will demonstrate the critical role that IL-10 plays in gastrointestinal inflammatory diseases and how probiotics could be used in their treatment.
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Molecular characterization of the Corynebacterium pseudotuberculosis hsp60-hsp10 operon, and evaluation of the immune response and protective efficacy induced by hsp60 DNA vaccination in mice.
BMC Res Notes
PUBLISHED: 01-27-2011
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Heat shock proteins (HSPs) are important candidates for the development of vaccines because they are usually able to promote both humoral and cellular immune responses in mammals. We identified and characterized the hsp60-hsp10 bicistronic operon of the animal pathogen Corynebacterium pseudotuberculosis, a Gram-positive bacterium of the class Actinobacteria, which causes caseous lymphadenitis (CLA) in small ruminants.
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Homology modeling, molecular dynamics and QM/MM study of the regulatory protein PhoP from Corynebacterium pseudotuberculosis.
J Mol Model
PUBLISHED: 01-20-2011
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Corynebacterium pseudotuberculosis is a facultatively intracellular Gram-positive bacterium that causes caseous lymphadenitis, principally in sheep and goats, though sometimes in other species of animals, leading to considerable economic losses. This pathogen has a TCS known as PhoPR, which consists of a sensory histidine kinase protein (PhoR) and an intracellular response regulator protein (PhoP). This system is involved in the regulation of proteins present in various processes, including virulence. The regulation is activated by PhoP protein phosphorylation, an event that requires a magnesium (Mg(2+)) ion. Here we describe the 3D structure of the regulatory response protein (PhoP) of C. pseudotuberculosis through molecular modeling by homology. The model generated provides the first structural information on a full-length member of the OmpR/PhoP subfamily. Classical molecular dynamics was used to investigate the stability of the model. In addition, we used quantum mechanical/molecular mechanical techniques to perform (internal, potential) energy optimizations to determine the interaction energy between the Mg(2+) ion and the structure of the PhoP protein. Analysis of the interaction energy residue by residue shows that Asp-16 and Asp-59 play an important role in the protein-Mg(2+) ion interactions. These results may be useful for the future development of a new vaccine against tuberculosis based on genetic attenuation via a point mutation that results in the polar residue Asp-16 and/or Asp-59 being replaced with a nonpolar residue in the DNA-binding domain of PhoP of C. pseudotuberculosis.
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A combined approach for comparative exoproteome analysis of Corynebacterium pseudotuberculosis.
BMC Microbiol.
PUBLISHED: 01-17-2011
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Bacterial exported proteins represent key components of the host-pathogen interplay. Hence, we sought to implement a combined approach for characterizing the entire exoproteome of the pathogenic bacterium Corynebacterium pseudotuberculosis, the etiological agent of caseous lymphadenitis (CLA) in sheep and goats.
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Complete genome sequence of Corynebacterium pseudotuberculosis I19, a strain isolated from a cow in Israel with bovine mastitis.
J. Bacteriol.
PUBLISHED: 10-29-2010
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This work reports the completion and annotation of the genome sequence of Corynebacterium pseudotuberculosis I19, isolated from an Israeli dairy cow with severe clinical mastitis. To present the whole-genome sequence, a de novo assembly approach using 33 million short (25-bp) mate-paired SOLiD reads only was applied. Furthermore, the automatic, functional, and manual annotations were attained with the use of several algorithms in a multistep process.
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The complete genome sequence of Corynebacterium pseudotuberculosis FRC41 isolated from a 12-year-old girl with necrotizing lymphadenitis reveals insights into gene-regulatory networks contributing to virulence.
BMC Genomics
PUBLISHED: 09-08-2010
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Corynebacterium pseudotuberculosis is generally regarded as an important animal pathogen that rarely infects humans. Clinical strains are occasionally recovered from human cases of lymphadenitis, such as C. pseudotuberculosis FRC41 that was isolated from the inguinal lymph node of a 12-year-old girl with necrotizing lymphadenitis. To detect potential virulence factors and corresponding gene-regulatory networks in this human isolate, the genome sequence of C. pseudotuberculosis FCR41 was determined by pyrosequencing and functionally annotated.
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Diversity of lactic acid bacteria of the bioethanol process.
BMC Microbiol.
PUBLISHED: 07-31-2010
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Bacteria may compete with yeast for nutrients during bioethanol production process, potentially causing economic losses. This is the first study aiming at the quantification and identification of Lactic Acid Bacteria (LAB) present in the bioethanol industrial processes in different distilleries of Brazil.
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Use of superoxide dismutase and catalase producing lactic acid bacteria in TNBS induced Crohns disease in mice.
J. Biotechnol.
PUBLISHED: 06-25-2010
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Reactive oxygen species are involved in various aspects of intestinal inflammation and tumor development. Decreasing their levels using antioxidant enzymes, such as catalase (CAT) or superoxide dismutase (SOD) could therefore be useful in the prevention of certain diseases. Lactic acid bacteria (LAB) are ideal candidates to deliver these enzymes in the gut. In this study, the anti-inflammatory effects of CAT or SOD producing LAB were evaluated using a trinitrobenzenesulfonic acid (TNBS) induced Crohns disease murine model. Engineered Lactobacillus casei BL23 strains producing either CAT or SOD, or the native strain were given to mice before and after intrarectal administration of TNBS. Animal survival, live weight, intestinal morphology and histology, enzymatic activities, microbial translocation to the liver and cytokines released in the intestinal fluid were evaluated. The mice that received CAT or SOD-producing LAB showed a faster recovery of initial weight loss, increased enzymatic activities in the gut and lesser extent of intestinal inflammation compared to animals that received the wild-type strain or those that did not receive bacterial supplementation. Our findings suggest that genetically engineered LAB that produce antioxidant enzymes could be used to prevent or decrease the severity of certain intestinal pathologies.
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Corynebacterium ulcerans isolated from an asymptomatic dog kept in an animal shelter in the metropolitan area of Rio de Janeiro, Brazil.
Vector Borne Zoonotic Dis.
PUBLISHED: 01-07-2010
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Corynebacterium ulcerans was isolated from nares of one asymptomatic dog kept in an animal shelter in the metropolitan area of Rio de Janeiro, Brazil. The RNA polymerase beta subunit-encoding gene was sequenced to confirm the species identity. C. ulcerans strains producing phospholipase D, but not diphtheria toxin, are able to cause severe disease in humans, such as pneumonia and granulomatous nodules in pulmonary tissues. The infection rate varies really widely by region, probably because of the variations in the reported infection rates. Dogs with unapparent C. ulcerans infections may be considered as potentially capable of infecting other animals and humans, including pet owners. Medical and veterinary staff should be aware that asymptomatic animals can carry C. ulcerans and cooperate in eliminating infections and monitoring animals also in the developing countries.
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Lactococcus lactis expressing either Staphylococcus aureus fibronectin-binding protein A or Listeria monocytogenes internalin A can efficiently internalize and deliver DNA in human epithelial cells.
Appl. Environ. Microbiol.
PUBLISHED: 05-29-2009
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Lactococci are noninvasive bacteria frequently used as protein delivery vectors and, more recently, as in vitro and in vivo DNA delivery vehicles. We previously showed that a functional eukaryotic enhanced green fluorescent protein (eGFP) expression plasmid vector was delivered in epithelial cells by Lactococcus lactis producing Listeria monocytogenes internalin A (L. lactis InlA(+)), but this strategy is limited in vivo to transgenic mice and guinea pigs. In this study, we compare the internalization ability of L. lactis InlA(+) and L. lactis producing either the fibronectin-binding protein A of Staphylococcus aureus (L. lactis FnBPA(+)) or its fibronectin binding domains C and D (L. lactis CD(+)). L. lactis FnBPA(+) and L. lactis InlA(+) showed comparable internalization rates in Caco-2 cells, while the internalization rate observed with L. lactis CD(+) was lower. As visualized by conventional and confocal fluorescence microscopy, large clusters of L. lactis FnBPA(+), L. lactis CD(+), and L. lactis InlA(+) were present in the cytoplasm of Caco-2 cells after internalization. Moreover, the internalization rates of Lactobacillus acidophilus NCFM and of an NCFM mutant strain with the gene coding for the fibronectin-binding protein (fbpA) inactivated were also evaluated in Caco-2 cells. Similar low internalization rates were observed for both wild-type L. acidophilus NCFM and the fbpA mutant, suggesting that commensal fibronectin binding proteins have a role in adhesion but not in invasion. L. lactis FnBPA(+), L. lactis CD(+), and L. lactis InlA(+) were then used to deliver a eukaryotic eGFP expression plasmid in Caco-2 cells: flow cytometry analysis showed that the highest percentage of green fluorescent Caco-2 cells was observed after coculture with either L. lactis FnBPA(+) or L. lactis InlA(+). Analysis of the in vivo efficiency of these invasive recombinant strains is currently in progress to validate their potential as DNA vaccine delivery vehicles.
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Survey of genome organization and gene content of Corynebacterium pseudotuberculosis.
Microbiol. Res.
PUBLISHED: 05-27-2009
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Corynebacterium pseudotuberculosis is an intracellular pathogen that causes Caseous lymphadenitis (CLA) disease in sheep and goats. The widespread occurrence and the economic importance of this pathogen have prompted investigation of its pathogenesis. We used a genomic library of C. pseudotuberculosis to generate 1440 genomic survey sequences (GSSs); these were analyzed in silico with bioinformatics tools, using public databases for comparative analyses. We employed non-redundant unique sequences as a query for BLAST searches against the genome, the translated genome and the proteome of four other Corynebacterium species that have been completely sequenced. We were able to characterize approximately 8% of the genome of C. pseudotuberculosis, including previously undescribed functional group genes, based on the COG database; the GSSs classification into categories gave 13% information storage and processing, 14% cellular processes and 23% metabolism. We found a close relation between C. pseudotuberculosis and C. diphtheriae conserved-gene synteny in Corynebacteria species.
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A new plasmid vector for DNA delivery using lactococci.
Genet Vaccines Ther
PUBLISHED: 02-10-2009
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The use of food-grade lactococci as bacterial carriers to DNA delivery into epithelial cells is a new strategy to develop live oral DNA vaccine. Our goal was to develop a new plasmid, named pValac, for antigen delivery for use in lactococci. The pValac plasmid was constructed by the fusion of: i) a eukaryotic region, allowing the cloning of an antigen of interest under the control of the pCMV eukaryotic promoter to be expressed by a host cell and ii) a prokaryotic region allowing replication and selection of bacteria. In order to evaluate pValac functionality, the gfp ORF was cloned into pValac (pValac:gfp) and was analysed by transfection in PK15 cells. The applicability of pValac was demonstrated by invasiveness assays of Lactococcus lactis inlA+ strains harbouring pValac:gfp into Caco-2 cells.
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Antigens of Corynebacterium pseudotuberculosis and prospects for vaccine development.
Expert Rev Vaccines
PUBLISHED: 02-07-2009
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Corynebacterium pseudotuberculosis continues to cause considerable economic losses in ovine and caprine herds worldwide, causing caseous lymphadenitis. Nevertheless, the immunology of this disease is relatively unknown. Novel antigens may provide vaccines that are more effective and improve diagnostic methods for better control of this disease. The available commercial vaccines are not able to fully protect susceptible animals, cannot be used in all host species and are not licensed for use in many countries. Recent studies on the genomics of C. pseudotuberculosis and on its molecular determinants of virulence should bring us new alternatives for more effective vaccine formulations.
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Complete genome sequence of Corynebacterium pseudotuberculosis biovar ovis strain P54B96 isolated from antelope in South Africa obtained by rapid next generation sequencing technology.
Stand Genomic Sci
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The Actinobacteria, Corynebacterium pseudotuberculosis strain P54B96, a nonmotile, non-sporulating and a mesophile bacterium, was isolated from liver, lung and mediastinal lymph node lesions in an antelope from South Africa. This strain is interesting in the sense that it has been found together with non-tuberculous mycobacteria (NTMs) which could nevertheless play a role in the lesion formation. In this work, we describe a set of features of C. pseudotuberculosis P54B96, together with the details of the complete genome sequence and annotation. The genome comprises of 2.34 Mbp long, single circular genome with 2,084 protein-coding genes, 12 rRNA, 49 tRNA and 62 pseudogenes and a G+C content of 52.19%. The analysis of the genome sequence provides means to better understanding the molecular and genetic basis of virulence of this bacterium, enabling a detailed investigation of its pathogenesis.
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In vitro and in vivo characterization of DNA delivery using recombinant Lactococcus lactis expressing a mutated form of L. monocytogenes Internalin A.
BMC Microbiol.
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The use of food-grade Lactic Acid Bacteria (LAB) as DNA delivery vehicles represents an attractive strategy to deliver DNA vaccines at the mucosal surfaces as they are generally regarded as safe (GRAS). We previously showed that either native Lactococcus lactis (LL) or recombinant invasive LL expressing Fibronectin Binding Protein A of Staphylococcus aureus (LL-FnBPA+) or Internalin A of Listeria monocytogenes (LL-InlA+), were able to deliver and trigger DNA expression by epithelial cells, either in vitro or in vivo. InlA does not bind to its receptor, the murine E-cadherin, thus limiting the use of LL-InlA+ in in vivo murine models. Moreover, FnBPA binds to its receptors, integrins, via fibronectin introducing another limiting factor. In order to avoid the limitations of LL-InlA+ and LL-FnBPA+, a new L. lactis strain was engineered to produce a previously described mutated form of InlA (LL-mInlA+) allowing the binding of mInlA on murine E-cadherin.
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Genome sequence of Corynebacterium pseudotuberculosis biovar equi strain 258 and prediction of antigenic targets to improve biotechnological vaccine production.
J. Biotechnol.
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Corynebacterium pseudotuberculosis is the causative agent of several veterinary diseases in a broad range of economically important hosts, which can vary from caseous lymphadenitis in sheep and goats (biovar ovis) to ulcerative lymphangitis in cattle and horses (biovar equi). Existing vaccines against C. pseudotuberculosis are mainly intended for small ruminants and, even in these hosts, they still present remarkable limitations. In this study, we present the complete genome sequence of C. pseudotuberculosis biovar equi strain 258, isolated from a horse with ulcerative lymphangitis. The genome has a total size of 2,314,404 bp and contains 2088 predicted protein-coding regions. Using in silico analysis, eleven pathogenicity islands were detected in the genome sequence of C. pseudotuberculosis 258. The application of a reverse vaccinology strategy identified 49 putative antigenic proteins, which can be used as candidate vaccine targets in future works.
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Complete genome sequence of Corynebacterium pseudotuberculosis Cp31, isolated from an Egyptian buffalo.
J. Bacteriol.
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Corynebacterium pseudotuberculosis is of major veterinary importance because it affects many animal species, causing economically significant livestock diseases and losses. Therefore, the genomic sequencing of various lines of this organism, isolated from different hosts, will aid in the development of diagnostic methods and new prevention and treatment strategies and improve our knowledge of the biology of this microorganism. In this study, we present the genome of C. pseudotuberculosis Cp31, isolated from a buffalo in Egypt.
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Genome sequence of the Corynebacterium pseudotuberculosis Cp316 strain, isolated from the abscess of a Californian horse.
J. Bacteriol.
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The bacterium Corynebacterium pseudotuberculosis is of major veterinary importance because it affects livestock, particularly sheep, goats, and horses, in several countries, including Australia, Brazil, the United States, and Canada, resulting in significant economic losses. In the present study, we describe the complete genome of the Corynebacterium pseudotuberculosis Cp316 strain, biovar equi, isolated from the abscess of a North American horse.
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The Corynebacterium pseudotuberculosis in silico predicted pan-exoproteome.
BMC Genomics
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Pan-genomic studies aim, for instance, at defining the core, dispensable and unique genes within a species. A pan-genomics study for vaccine design tries to assess the best candidates for a vaccine against a specific pathogen. In this context, rather than studying genes predicted to be exported in a single genome, with pan-genomics it is possible to study genes present in different strains within the same species, such as virulence factors. The target organism of this pan-genomic work here presented is Corynebacterium pseudotuberculosis, the etiologic agent of caseous lymphadenitis (CLA) in goat and sheep, which causes significant economic losses in those herds around the world. Currently, only a few antigens against CLA are known as being the basis of commercial and still ineffective vaccines. In this regard, the here presented work analyses, in silico, five C. pseudotuberculosis genomes and gathers data to predict common exported proteins in all five genomes. These candidates were also compared to two recent C. pseudotuberculosis in vitro exoproteome results.
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Genome sequence of Staphylococcus aureus Newbould 305, a strain associated with mild bovine mastitis.
J. Bacteriol.
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Staphylococcus aureus is a major etiological agent of mastitis in ruminants. We report here the genome sequence of bovine strain Newbould 305, isolated in the 1950s in a case of bovine mastitis and now used as a model strain able to reproducibly induce chronic mastitis in cows.
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Production of Fibronectin Binding Protein A at the surface of Lactococcus lactis increases plasmid transfer in vitro and in vivo.
PLoS ONE
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Lactococci are noninvasive lactic acid bacteria frequently used as protein delivery vectors and, more recently, as DNA delivery vehicles. We previously showed that Lactococcus lactis (LL) expressing the Fibronectin-Binding Protein A of Staphylococcus aureus (LL-FnBPA+) showed higher internalization rates in vitro in Caco-2 cells than the native (wt) lactococci and were able to deliver a eukaryotic Green Fluorescent Protein (GFP) expression plasmid in 1% of human Caco-2 cells. Here, using the bovine beta-lactoglobulin (BLG), one of the major cows milk allergen, and GFP we characterized the potential of LL-FnBPA+ as an in vivo DNA vaccine delivery vehicle. We first showed that the invasive strain LL-FnBPA+ carrying the plasmid pValac:BLG (LL-FnBPA+ BLG) was more invasive than LL-BLG and showed the same invasivity as LL-FnBPA+. Then we demonstrated that the Caco-2 cells, co-incubated with LL-FnBPA+ BLG produced up to 30 times more BLG than the Caco-2 cells co-incubated with the non invasive LL-BLG. Using two different gene reporters, BLG and GFP, and two different methods of detection, EIA and fluorescence microscopy, we showed in vivo that: i) in order to be effective, LL-FnBPA+ required a pre-coating with Fetal Calf Serum before oral administration; ii) plasmid transfer occurred in enterocytes without regard to the strains used (invasive or not); iii) the use of LL-FnBPA+ increased the number of mice producing BLG, but not the level of BLG produced. We thus confirmed the good potential of invasive recombinant lactic acid bacteria as DNA delivery vector in vivo.
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Whole-genome sequence of Corynebacterium pseudotuberculosis strain Cp162, isolated from camel.
J. Bacteriol.
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Corynebacterium pseudotuberculosis is a pathogen of great veterinary and economic importance, since it affects livestock, mainly sheep and goats, worldwide, together with reports of its presence in camels in several Arabic, Asiatic, and East and West African countries, as well as Australia. In this article, we report the genome sequence of Corynebacterium pseudotuberculosis strain Cp162, collected from the external neck abscess of a camel in the United Kingdom.
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Hsp65-producing Lactococcus lactis prevents experimental autoimmune encephalomyelitis in mice by inducing CD4+LAP+ regulatory T cells.
J. Autoimmun.
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Heat shock proteins (Hsps) participate in the cellular response to stress and they are hiperexpressed in inflammatory conditions. They are also known to play a major role in immune modulation, controlling, for instance, autoimmune responses. In this study, we showed that oral administration of a recombinant Lactococcus lactis strain that produces and releases LPS-free Hsp65 prevented the development of experimental autoimmune encephalomyelitis (EAE) in C57BL/6 mice. This was confirmed by the reduced inflammatory cell infiltrate and absence of injury signs in the spinal cord. The effect was associated with reduced IL-17 and increased IL-10 production in mesenteric lymph node and spleen cell cultures. Hsp65-producing-L. lactis-fed mice had a remarkable increase in the number of natural and inducible CD4+Foxp3+ regulatory T (Treg) cells and CD4+LAP+ (Latency-associated peptide) Tregs - which express the membrane-bound TGF-? - in spleen, inguinal and mesenteric lymph nodes as well as in spinal cord. Moreover, many Tregs co-expressed Foxp3 and LAP. In vivo depletion of LAP+ cells abrogated the effect of Hsp65-producing L. lactis in EAE prevention and worsened disease in medium-fed mice. Thus, Hsp65-L.lactis seems to boost this critical regulatory circuit involved in controlling EAE development in mice.
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Campylobacter fetus subspecies: comparative genomics and prediction of potential virulence targets.
Gene
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The genus Campylobacter contains pathogens causing a wide range of diseases, targeting both humans and animals. Among them, the Campylobacter fetus subspecies fetus and venerealis deserve special attention, as they are the etiological agents of human bacterial gastroenteritis and bovine genital campylobacteriosis, respectively. We compare the whole genomes of both subspecies to get insights into genomic architecture, phylogenetic relationships, genome conservation and core virulence factors. Pan-genomic approach was applied to identify the core- and pan-genome for both C. fetus subspecies and members of the genus. The C. fetus subspecies conserved (76%) proteome were then analyzed for their subcellular localization and protein functions in biological processes. Furthermore, with pathogenomic strategies, unique candidate regions in the genomes and several potential core-virulence factors were identified. The potential candidate factors identified for attenuation and/or subunit vaccine development against C. fetus subspecies contain: nucleoside diphosphate kinase (Ndk), type IV secretion systems (T4SS), outer membrane proteins (OMP), substrate binding proteins CjaA and CjaC, surface array proteins, sap gene, and cytolethal distending toxin (CDT). Significantly, many of those genes were found in genomic regions with signals of horizontal gene transfer and, therefore, predicted as putative pathogenicity islands. We found CRISPR loci and dam genes in an island specific for C. fetus subsp. fetus, and T4SS and sap genes in an island specific for C. fetus subsp. venerealis. The genomic variations and potential core and unique virulence factors characterized in this study would lead to better insight into the species virulence and to more efficient use of the candidates for antibiotic, drug and vaccine development.
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JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.

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