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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. Fil: de Moreno, Maria Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina Fil: del Carmen, Silvina Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina Fil: Zurita Turk, Meritxell. Universidade Federal de Minas Gerais; Brasil Fil: Santos Rocha, Clarissa. Universidade Federal de Minas Gerais; Brasil Fil: van de Guchte, Maarten. Institut National de la Recherche Agronomique; Francia Fil: Azevedo, Vasco. Universidade Federal de Minas Gerais; Brasil Fil: Miyoshi, Anderson. Universidade Federal de Minas Gerais; Brasil Fil: Leblanc, Jean Guy Joseph. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina

p. 1-5 Submitted by Edileide Reis (leyde-landy@hotmail.com) on 2014-03-13T17:06:10Z No. of bitstreams: 1 Roberto Meyer.pdf: 740113 bytes, checksum: f881405072d7590d7eee75b5cc556ce8 (MD5) Approved for entry into archive by Patricia Barroso (pbarroso@ufba.br) on 2014-03-13T18:25:25Z (GMT) No. of bitstreams: 1 Roberto Meyer.pdf: 740113 bytes, checksum: f881405072d7590d7eee75b5cc556ce8 (MD5) Made available in DSpace on 2014-03-13T18:25:25Z (GMT). No. of bitstreams: 1 Roberto Meyer.pdf: 740113 bytes, checksum: f881405072d7590d7eee75b5cc556ce8 (MD5) Previous issue date: 2011 Background 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. Results We assessed the seroprevalence of infection with C. pseudotuberculosis in 805 sheep from 23 sheep farms that supply slaughterhouses in the state of Minas Gerais; we also analyzed management practices that could be associated with CLA occurrence, used on these and nearby farms that also supplied animals to the slaughterhouse (n = 60). The serum samples for assaying CLA infection were taken at the slaughterhouse. Frequency of infection with C. pseudotuberculosis was estimated at 43.7%, and farm frequency was estimated at 100%. Management practices were analyzed through a questionnaire. All farmers (60/60) had extensive/semi-extensive rearing system; 70.0% (42/60) identified sheep individually; 11.7% (7/60) had periodical technical assistance; 41.7% (25/60) disinfected the facilities; 86.7% (52/60) used barbed wire fences and did not implement adequate CLA control measures; only 11.7% (7/60) of breeders reported vaccination against C. pseudotuberculosis; 13.3% (8/60) took note of animals with clinical signs of CLA; 1.7% (1/60) opened and sanitized abscesses, and isolated the infected animals; 10.0% (6/60) knew the zoonotic potential of this disease and 1.7% (1/60) of the farmers culled animals in case of recurrence of abscesses. Conclusions It can be concluded that C. pseudotuberculosis infection is widely spread in sheep flocks in Minas Gerais state in Brazil and that there is a lack of good management measures and vaccination, allowing transmission of this infectious agent throughout the production network.

Background 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. Results Sequencing and assembly of the C. pseudotuberculosis FRC41 genome yielded a circular chromosome with a size of 2,337,913 bp and a mean G+C content of 52.2%. Specific gene sets associated with iron and zinc homeostasis were detected among the 2,110 predicted protein-coding regions and integrated into a gene-regulatory network that is linked with both the central metabolism and the oxidative stress response of FRC41. Two gene clusters encode proteins involved in the sortase-mediated polymerization of adhesive pili that can probably mediate the adherence to host tissue to facilitate additional ligand-receptor interactions and the delivery of virulence factors. The prominent virulence factors phospholipase D (Pld) and corynebacterial protease CP40 are encoded in the genome of this human isolate. The genome annotation revealed additional serine proteases, neuraminidase H, nitric oxide reductase, an invasion-associated protein, and acyl-CoA carboxylase subunits involved in mycolic acid biosynthesis as potential virulence factors. The cAMP-sensing transcription regulator GlxR plays a key role in controlling the expression of several genes contributing to virulence. Conclusion The functional data deduced from the genome sequencing and the extended knowledge of virulence factors indicate that the human isolate C. pseudotuberculosis FRC41 is equipped with a distinct gene set promoting its survival under unfavorable environmental conditions encountered in the mammalian host.

During the last decades, the majority of Brazilian Corynebacterium diphtheriae isolates were shown to be capable to metabolize sucrose, sometimes leading to erroneous identification as a non-diphtheric Corynebacterium species. The sequencing of the polymorphic region of the RNA polymerase beta subunit-encoding gene (rpoB) is an important taxonomic tool for identification of corynebacteria. The present study aimed to investigate the rpoB gene polymorphic features of sucrose-fermenting and non sucrose-fermenting strains. The results showed that sucrose-fermenting strains presented rpoB gene polymorphic regions with more than 98% similarity with the sequences deposited in the gene bank corresponding to non sucrose-fermenting strains. Data indicate that sucrose-fermenting isolates may act as a variant of C. diphtheriae biotype mitis. In addition we alert that sucrose-fermenting strains should not be discarded as contaminants mainly in countries where the possibility of isolation of this variant is higher.

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.

Previously, we isolated two strains of spontaneous oxidative (SpOx2 and SpOx3) stress mutants of Lactococcus lactis subsp cremoris. Herein, we compared these mutants to a parental wild-type strain (J60011) and a commercial starter in experimental fermented milk production. Total solid contents of milk and fermentation temperature both affected the acidification profile of the spontaneous oxidative stress-resistant L. lactis mutants during fermented milk production. Fermentation times to pH 4.7 ranged from 6.40 h (J60011) to 9.36 h (SpOx2); V(max) values were inversely proportional to fermentation time. Bacterial counts increased to above 8.50 log(10) cfu/mL. The counts of viable SpOx3 mutants were higher than those of the parental wild strain in all treatments. All fermented milk products showed post-fermentation acidification after 24 h of storage at 4 degrees C; they remained stable after one week of storage.

We conducted a seroepidemiological survey to determine the prevalence of caseous lymphadenitis (CLA) in goat herds in Minas Gerais state, Brazil. Serum samples were collected from goats (n = 676) from 108 rural properties in 2001, covering most of the sub-regions of this ca. 586,500 square kilometer state. Antibodies against Corynebacterium pseudotuberculosis secreted proteins were detected by an indirect enzyme-linked immunosorbent assay (ELISA). Most of the animals (78.9%) tested positive for CLA; 98% of flocks presented at least one seropositive animal. Goats managed under an extensive production system had a significantly higher seroprevalence of CLA than those in intensive and semi-intensive operations. The age distribution of the animals in the flocks affected the prevalence of this disease; however, goat breed did not. We found seropositivity against C. pseudotuberculosis to be highly prevalent in these Brazilian goat herds; consequently, appropriate management practices for the control of CLA should be implemented.

Background 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. Results After transfection with pValac:gfp, we observed GFP expression in PK15 cells. L. lactis inlA+ were able to invade Caco-2 cells and delivered a functional expression cassette (pCMV:gfp) into epithelial cells. Conclusion We showed the potential of an invasive L. lactis harbouring pValac to DNA delivery and subsequent triggering DNA expression by epithelial cells. Further work will be to examine whether these strains are able to deliver DNA in intestinal cells in vivo.

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.

Lactic acid bacteria (LAB) represent a heterogeneous group of microorganisms that are naturally present in many foods. Some selected strains are frequently added as probiotics in order to confer specific benefits to consumers. Many studies have shown that LAB possess therapeutic properties since they are able to prevent the development of some diseases, as shown mostly on animal models for cancer, infections and gastrointestinal disorders such as intestinal inflammation. They have been shown to regulate mucosal immune responses by modulating the production and liberation of regulatory agents such as cytokines by the host. Some of these cytokines, such as the anti-inflammatory interleukin-10 (IL-10), modulate the inflammatory immune response, thus immunomodulation is a mechanism by which LAB can prevent certain inflammatory bowel diseases (IBD). Since oxidative stress participates to the inflammatory processes and to the appearance of damages in pathologies of the gastrointestinal tract of humans such as IBD, LAB could also prevent inflammation by eliminating reactive oxygen species (ROS) through the activity of antioxidant enzymes. Engineering LAB to produce either antioxidant enzymes (such as catalases and superoxide dismutases) or anti-inflammatory cytokines (such as IL-10) is a strategy currently exploited by several groups. These novel strains have successfully been used to prevent inflammatory bowel diseases in animal models and could be evaluated in human clinical trials. Here, we present an overview of the current knowledge of the mechanisms by which LAB can be used to prevent undesired intestinal inflammatory responses. Fil: Leblanc, Jean Guy Joseph. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina Fil: de Moreno, Maria Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina Fil: Perdigon, Gabriela del Valle. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina. Universidad Nacional de Tucumán. Facultad de Bioquímica, Química y Farmacia; Argentina Fil: Miyoshi, Anderson. Universidade Federal de Minas Gerais; Brasil Fil: Rochat, Tatiana. Institut National de la Recherche Agronomique; Francia Fil: Bermudez Humaran, Luis. Institut National de la Recherche Agronomique; Francia Fil: Langella, Philippe. Institut National de la Recherche Agronomique; Francia Fil: Sesma, Fernando Juan Manuel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina Fil: Azevedo, Vasco. Universidade Federal de Minas Gerais; Brasil

The protection against Schistosoma mansoni infection was evaluated in SWISS mice orally vaccinated with an attenuated strain of Salmonella carrying a Sm14-based DNA vaccine. Although this formulation was not able to afford a reduction in the worm burden, a non-antigen-specific decrease in schistosome-induced granulomatous reaction was verified in livers of mice that received Salmonella.

Reactive oxygen species, such as hydrogen peroxide (H2O 2), are involved in various aspects of tumour development. Decreasing their levels can therefore be a promising approach for colon cancer prevention. The objective of this study was to evaluate the effect of catalase-producing Lactococcus lactis on the prevention of an experimental murine 1,2-dimethylhydrazine (DMH)-induced colon cancer. DMH-treated BALB/c mice received either a catalase-producing L. lactis strain or the isogenic non-catalase-producing strain as a control, whereas other untreated mice did not receive bacterial supplementation. Catalase activity and H2O 2 levels in intestinal fluids and blood samples were measured, and changes in the histology of the large intestines during tumour progression were evaluated. The catalase-producing L. lactis strain used in this study was able to slightly increase catalase activities in DMH-treated mice (1.19 ± 0.08 U ml-1) and reduce H2O2 levels (3.4 ± 1.1 μM) compared to (i) animals that received the non-catalase-producing strain (1.00 ± 0.09 U ml-1, 9.0 ± 0.8 μM), and (ii) those that did not receive bacterial supplementation (1.06 ± 0.07 U ml-1, 10.0 ± 1.1 μM). Using the histopathological grading scale of chemically induced colorectal cancer, animals that received the catalase-producing L. lactis had a significantly lesser extent of colonic damage and inflammation (2.0 ± 0.4) compared to animals that received the non-catalase-producing L. lactis (4.0 ± 0.3) or those that did not receive bacterial supplementation (4.7 ± 0.5). The catalase-producing L. lactis strain used in this study was able to prevent tumour appearance in an experimental DMH-induced colon cancer model. Fil: de Moreno, Maria Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina Fil: Leblanc, Jean Guy Joseph. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina Fil: Perdigon, Gabriela del Valle. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina. Universidad Nacional de Tucumán. Facultad de Bioquímica, Química y Farmacia; Argentina Fil: Miyoshi, Anderson. Universidade Federal de Minas Gerais; Brasil Fil: Langella, Philippe. Institut National de la Recherche Agronomique; Francia Fil: Azevedo, Vaso. Universidade Federal de Minas Gerais; Brasil Fil: Sesma, Fernando Juan Manuel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina

The frequency and severity of human infections associated with Corynebacterium ulcerans appear to be increasing in different countries. Here, we describe the first C. ulcerans strain producing a diphtheria-like toxin isolated from an elderly woman with a fatal pulmonary infection and a history of leg skin ulcers in the Rio de Janeiro metropolitan area.

Brucella species are important zoonotic pathogens affecting a wide variety of mammals. Therefore, the identification of new Brucella virulence factors is of great interest in understanding bacterial pathogenesis and immune evasion. In this study, we have identified Brucella abortus vacB gene that presents 2343 nucleotides and 781 amino acids and it shows 39% identity with Shigella flexneri vacB gene that encodes an exoribonuclease RNase R involved in bacterial virulence. Further, we have inactivated Brucella vacB by gene replacement strategy generating a deletion mutant strain. In order to test the role of Brucella vacB in pathogenesis, BALB/c and interferon regulatory factor-1 (IRF-1) knockout (KO) mice received Brucella vacB mutant, the virulent parental strain 2308 or the vaccine strain RB51 and the bacterial CFU numbers in spleens and mous survival were monitored. Our results demonstrated that the B. abortus ΔvacB mutant and the wild type strain 2308 showed similar CFU numbers in BALB/c mice. Additionally, IRF-1 KO mice that received either the vacB mutant or S2308 strain died in 12-14 days postinfection; in contrast, all animals that received the RB51 vaccine strain survived for 30 days postinoculation. In summary, this study reports that the vacB gene in B. abortus has no impact on bacterial pathogenesis. © 2007 Elsevier Masson SAS. All rights reserved.

Background Brucella abortus is a facultative intracellular pathogen that mainly infects cattle and humans. Current vaccines rely on live attenuated strains of B. abortus, which can revert to their pathogenic status and thus are not totally safe for use in humans. Therefore, the development of mucosal live vaccines using the food-grade lactic acid bacterium, Lactococcus lactis, as an antigen delivery vector, is an attractive alternative and a safer vaccination strategy against B. abortus. Here, we report the construction of L. lactis strains genetically modified to produce B. abortus GroEL heat-shock protein, a candidate antigen, in two cellular locations, intracellular or secreted. Results Only the secreted form of GroEL was stably produced in L. lactis, suggesting a detrimental effect of GroEL protein when intracellularly produced in this bacterium. Only trace amounts of mature GroEL were detected in the supernatant fraction of induced lactococcal cultures, and the GroEL precursor remained stacked in the cell fraction. Attempts to raise the secretion yields were made, but even when GroEL was fused to a synthetic propeptide, secretion of this antigen was not improved. Conclusion We found that L. lactis is able to produce, and to secrete, a stable form of GroEL into the extracellular medium. Despite the low secretion efficiency of GroEL, which suggest that this antigen interacts with the cell envelope of L. lactis, secretion seems to be the best way to achieve both production and protein yields, regardless of cellular location. The L. lactis strain secreting GroEL has potential for in vivo immunization.

International audience; Corynebacterium pseudotuberculosis is the etiological agent of caseous lymphadenitis (CLA), a common disease in small ruminant populations throughout the world. Once established, this disease is difficult to eradicate because drug therapy is not effective and because the clinical detection of infected animals is of limited efficiency. We reviewed the microbiological, biochemical and taxonomic features of C. pseudotuberculosis, general aspects of infection, the main virulence determinants and currently available commercial vaccines. We also examined the current molecular strategies for the study of virulence in C. pseudotuberculosis, including the latest research on the identification of novel virulence factors and genes, which will help us to better understand the biology of this microorganism. This knowledge may also contribute to the development of improved CLA vaccines, including subunit and DNA-based types, as well as to improve the diagnosis, treatment and control of this disease.

Lactococcus lactis, a lactic acid bacterium widely used for food fermentations, is often exposed to damaging stress conditions. In particular, oxidative stress leads to DNA, protein and membrane damages that can be lethal. AsL. lactishas no catalase, the impact of production of theBacillus subtilishaem catalase KatE on its oxidative stress resistance was tested. This cytoplasmic catalase was engineered for extracellular expression inL. lactiswith an optimization strategy based on fusion to the nisin-inducible promoter and a lactococcal signal peptide (SPUsp45). The production of KatE byL. lactisconferred an 800-fold increase in survival after 1 h exposure to 4 mM hydrogen peroxide, and a 160-fold greater survival in long-term (3 days) survival of aerated cultures in acydAmutant, which is unable to respire. The presence of KatE protected DNA from oxidative damage and limited its degradation after long-term aeration in acydA/recAmutant, defective in DNA repair.L. lactisis thus able to produce active catalase that can provide efficient antioxidant activity.

We report the oral vaccination of SWISS mice with an Aro attenuated Salmonella enterica var. Typhimurium vaccine strain expressing the 14-kDa Schistosoma mansoni antigen, Sm14. Bacterial adjuvants, including (i) Lactococcus lactis expressing interleukin-12 (IL-12) and (ii) Lactobacillus delbrueckii UFV-H2b20, were also employed in oral immunization assays. Detection assays to specific IgG and IgA anti-Sm14 antibodies were performed to evaluate humoral immune responses in vaccinated mice. An increase in specific IgG titers was observed; however, no IgA production was detected. The protection levels against schistosomiasis (34.9-49.5%) obtained with all experimental formulations in this work were very similar to values reported by previous studies, which used purified recombinant Sm14 for parenteral vaccination of mice. There was a slight reduction in hepatic granulomas of mice vaccinated with Salmonella. Oogram studies showed diminished numbers of S. mansoni eggs in the intestinal wall of vaccinated mice, but individual female worm fecundity did not seem to be affected by our immunization protocol.

Since the Haemophilus influenzae genome sequence was completed in 1995, 172 other prokaryotic genomes have been completely sequenced, while 508 projects are underway. Besides pathogens, organisms important in several other fields, such as biotechnology and bioremediation, have also been sequenced. Institutions choose the organisms they wish to sequence according to the importance that these species represent to them, the availability of the microbes, and based on the similarity of a species of interest with others that have been sequenced previously. Improvements in sequencing techniques and in associated methodologies have been achieved; however, scientists need to continue working on the development of this field. In Brazil, a multicentered, centrally coordinated and research-focused network was adopted and successfully used for the sequencing of several important organisms. We analyzed the current status of microbial genomes, the trends for criteria used to choose new sequencing projects, the future of microbial sequencing, and the Brazilian genome network.

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 live vehicles for the production and delivery of heterologous proteins of vaccinal, medical or technological interest has therefore been extensively investigated. Lactococcus lactis, a LAB species, is a potential candidate for the production of biologically useful proteins. Several delivery systems have been developed to target heterologous proteins to a specific cell location (i.e., cytoplasm, cell wall or extracellular medium). A promising application of L. lactis is its use as an antigen delivery vehicle, for the development of live mucosal vaccines. The expression of heterologous proteins and antigens as well as the various delivery systems developed in L. lactis, and its use as an oral vaccine carrier are discussed.

International audience; The Brucella abortus ribosomal protein L7/L12 is an immunodominant antigen and an interesting candidate for the development of oral live vaccines against brucellosis. Here, a recombinant Lactococcus lactis strain producing L7/L12 under the control of nisin inducible promoter was orally administered to BALB/c mice. Significant levels of anti-L7/L12 specific IgA detected in feces revealed an induced local humoral immune response. However, serum analysis did not reveal any anti-L7/L12 antibodies suggesting the absence of a systemic response. Nevertheless, the vaccinated mice showed a partial protective immunity against B. abortus virulent strain (S2308) challenged by intraperitoneal inoculation.

Here, we report the whole-genome sequences of two ovine-pathogenic Corynebacterium pseudotuberculosis isolates: strain 3/99-5, which represents the first C. pseudotuberculosis genome originating from the United Kingdom, and 42/02-A, the second from Australia. These genome sequences will contribute to the objective of determining the global pan-genome of this bacterium.

The E7 protein of human papillomavirus type 16 was produced in Lactococcus lactis. Secretion allowed higher production yields than cytoplasmic production. In stationary phase, amounts of cytoplasmic E7 were reduced, while amounts of secreted E7 increased, suggesting a phase-dependent intracellular proteolysis. Fusion of E7 to the staphylococcal nuclease, a stable protein, resulted in a highly stable cytoplasmic protein. This work provides new candidates for development of viral screening systems and for oral vaccine against cervical cancer.

The use of Lactococcus lactis (the most extensively characterized lactic acid bacterium) as a delivery organism for heterologous proteins is, in some cases, limited by low production levels and poor-quality products due to surface proteolysis. In this study, we combined in one L. lactis strain use of the nisin-inducible promoter P nisA and inactivation of the extracellular housekeeping protease HtrA. The ability of the mutant strain, designated htrA -NZ9000, to produce high levels of stable proteins was confirmed by using the staphylococcal nuclease (Nuc) and the following four heterologous proteins fused or not fused to Nuc that were initially unstable in wild-type L. lactis strains: (i) Staphylococcus hyicus lipase, (ii) the bovine rotavirus antigen nonstructural protein 4, (iii) human papillomavirus antigen E7, and (iv) Brucella abortus antigen L7/L12. In all cases, protein degradation was significantly lower in strain htrA -NZ9000, demonstrating the usefulness of this strain for stable heterologous protein production.

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.

Background. 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 (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 non-specific immunosupression of the transplanted recipient, resulting in the concomitant inhibition of the GVL effect. Material and Methods. Here we propose an alternative approach to specifically suppress GVHD, while sparing the GVL, based on oral treatment of transplant donors with recipient antigens, associated with the intake of probiotic Lactococcus lactis as tolerogenic adjuvant (combined therapy). Results. We show that treatment of C57Bl/6 donor mice with combined therapy before the transplant protects the recipients F1 (C57Bl/6XBAL/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 antigens. The protection is specific, long lasting and dependent on donor IL-10 sufficient B cells activity which induces regulatory T cells in the host. Conclusion. 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 transplantation. Note: This abstract was not presented at the meeting. Citation Format: Ana Carolina T. Mercadante, Suelen Perobelli, Ana Paula G. Alves, Triciana Gonçalves-Silva, Wallace Mello, Ana C Gomes-Santos, Anderson Miyoshi, Ana Maria C. Faria, Adriana Bonomo. Oral combined therapy with probiotics and alloantigen induces B cell dependent long lasting specific tolerance. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2590. doi:10.1158/1538-7445.AM2014-2590

Corynebacterium pseudotuberculosis (Cp) is a pathogenic bacterium that causes caseous lymphadenitis (CLA), ulcerative lymphangitis, mastitis, and edematous to a broad spectrum of hosts, including ruminants, thereby threatening economic and dairy industries worldwide. Currently there is no effective drug or vaccine available against Cp. To identify new targets, we adopted a novel integrative strategy, which began with the prediction of the modelome (tridimensional protein structures for the proteome of an organism, generated through comparative modeling) for 15 previously sequenced C. pseudotuberculosis strains. This pan-modelomics approach identified a set of 331 conserved proteins having 95-100% intra-species sequence similarity. Next, we combined subtractive proteomics and modelomics to reveal a set of 10 Cp proteins, which may be essential for the bacteria. Of these, 4 proteins (tcsR, mtrA, nrdI, and ispH) were essential and non-host homologs (considering man, horse, cow and sheep as hosts) and satisfied all criteria of being putative targets. Additionally, we subjected these 4 proteins to virtual screening of a drug-like compound library. In all cases, molecules predicted to form favorable interactions and which showed high complementarity to the target were found among the top ranking compounds. The remaining 6 essential proteins (adk, gapA, glyA, fumC, gnd, and aspA) have homologs in the host proteomes. Their active site cavities were compared to the respective cavities in host proteins. We propose that some of these proteins can be selectively targeted using structure-based drug design approaches (SBDD). Our results facilitate the selection of C. pseudotuberculosis putative proteins for developing broad-spectrum novel drugs and vaccines. A few of the targets identified here have been validated in other microorganisms, suggesting that our modelome strategy is effective and can also be applicable to other pathogens.

Background 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. Results Under the 3 simulated conditions (acid, osmotic and thermal shock stresses), 474 differentially expressed genes exhibiting at least a 2-fold change in expression levels were identified. Important genes to the infection process were induced, such as those involved in virulence, defence against oxidative stress, adhesion and regulation, and many genes encoded hypothetical proteins, indicating that further investigation of the bacterium is necessary. The data will contribute to a better understanding of the biology of C. pseudotuberculosis and to studies investigating strategies to control the disease. Conclusions Despite the veterinary importance of C. pseudotuberculosis, the bacterium is poorly characterised; therefore, effective treatments for caseous lymphadenitis have been difficult to establish. Through the use of RNAseq, these results provide a better biological understanding of this bacterium, shed light on the most likely survival mechanisms used by this microorganism in adverse environments and identify candidates that may help reduce or even eradicate the problems caused by this disease.

DNA-based immunization has initiated a new era of vaccine research. One of the main goals of gene vaccine development is the control of the levels of expression in vivo for efficient immunization. Modifying the vector to modulate expression or immunogenicity is of critical importance for the improvement of DNA vaccines. The most frequently used vectors for genetic immunization are plasmids. In this article, we review some of the main elements relevant to their design such as strong promoter/enhancer region, introns, genes encoding antigens of interest from the pathogen (how to choose and modify them), polyadenylation termination sequence, origin of replication for plasmid production in Escherichia coli, antibiotic resistance gene as selectable marker, convenient cloning sites, and the presence of immunostimulatory sequences (ISS) that can be added to the plasmid to enhance adjuvanticity and to activate the immune system. In this review, the specific modifications that can increase overall expression as well as the potential of DNA-based vaccination are also discussed.

Corynebacterium pseudotuberculosis causa uma infeccao cronica, denominada Linfadenite caseosa em caprinos e ovinos causando grandes perdas economicas. A caracterizacao das vias e genes de Reparo presentes neste parasito pode contribuir para o conhecimento genetico deste, revelando alvos potenciais para intervencao terapeutica. A hidrolase MutT, componente do Sistema GO de reparo, hidrolisa 8-oxodGTP, que pode gerar mutacoes, impedindo sua incorporacao no DNA. Foram realizadas analises in silico, mostrando que a proteina MutT tem um dominio conservado Nudix hidrolase. O gene foi amplificado por PCR e clonado no vetor pProEX HTb. Celulas competentes de E. coli BH600 mutT mutantes foram transformadas com o plasmideo. A expressao da proteina esta sendo padronizada. Posteriormente, serao feitos testes de complementacao funcional apos tratamento com agentes oxidantes a fim de revelar seu papel no sistema GO de C. pseudotuberculosis.

A Linfadenite caseosa e uma doenca causada por Corynebacterium pseudotuberculosis que afeta ovinos e caprinos. O Reparo de DNA e vital para qualquer organismo, pois evita possiveis efeitos deleterios. O objetivo deste estudo foi caracterizar o gene uvrA de C.psudotuberculosis para futuros ensaios de complementacao funcional. Analises in silico foram feitas para verificar os parâmetros fisicos e quimicos, busca por dominios conservados e modelagem da estrutura 3D. As analises mostram que a proteina tem um peso molecular de cerca de 104,3 kDa e um dominio conservado de exonuclease. A amplificacao do gene uvrA foi realizada utilizando um kit de PCR Long Range e o inserto foi clonado no vetor pCR 2.1 TOPO. Linhagens de E. coli DH5a foram eletroporadas com o plasmideo recombinante e depois estes foram digeridos com enzimas de restricao BamH1 e Xho1 para subsequente clonagem num vetor de expressao pProEX HTB. As construcoes serao confirmadas por sequenciamento.

Corynebacterium pseudotuberculosis is a bacterium which causes diseases such as caseous lymphadenitis in small ruminants, resulting in large-scale economic losses for agribusiness worldwide. Consequently, this bacterium including its transcriptional profile analysis has been the focus of various studies. Identification of the transcripts that appear under conditions that simulate the environment encountered by this bacterial species in the host is of great importance in discovering new targets for the production of more efficient vaccines. We sequenced the cDNA of Corynebacterium pseudotuberculosis strain 1002, using the SOLiD V3 system, under the following conditions: osmotic stress (2 M), acidity (pH), heat shock (50 °C) and control condition (N). To identify the transcripts shared among the stimulons and integrate this information with the results from BLAST and BLAST2GO, we developed the software CoreStImulon (CSI) which allows the user to individually distinguish the genes in terms of their participation in biological processes, their function and cellular location. In the biosynthetic processes, eleven genes represented in the core stimulon and twenty genes in the control were observed. This validates the hypothesis that the organisms strategy for surviving in a hostile environment is through growth reduction. The oxidation reduction process, response to stress process, and cell adhesion are controlled by genes that contribute to bacterial cell maintenance under stress conditions; these could be involved in their pathogenicity. The methodology for identification of transcripts obtained by ab initio assembly and shared among the stimulons permitted candidates selection for vaccine studies. CSI is available at https://sourceforge.net/projects/corestimulon/.

Background 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. Results A total of 489 LAB isolates were obtained from four distilleries in 2007 and 2008. The abundance of LAB in the fermentation tanks varied between 6.0 × 105 and 8.9 × 108 CFUs/mL. Crude sugar cane juice contained 7.4 × 107 to 6.0 × 108 LAB CFUs. Most of the LAB isolates belonged to the genus Lactobacillus according to rRNA operon enzyme restriction profiles. A variety of Lactobacillus species occurred throughout the bioethanol process, but the most frequently found species towards the end of the harvest season were L. fermentum and L. vini. The different rep-PCR patterns indicate the co-occurrence of distinct populations of the species L. fermentum and L. vini, suggesting a great intraspecific diversity. Representative isolates of both species had the ability to grow in medium containing up to 10% ethanol, suggesting selection of ethanol tolerant bacteria throughout the process. Conclusions This study served as a first survey of the LAB diversity in the bioethanol process in Brazil. The abundance and diversity of LAB suggest that they have a significant impact in the bioethanol process.

Since the first successful attempt at sequencing the Corynebacterium pseudotuberculosis genome, large amounts of genomic, transcriptomic and proteomic data have been generated. C. pseudotuberculosis is an interesting bacterium due to its great zoonotic potential and because it causes considerable economic losses worldwide. Furthermore, different strains of C. pseudotuberculosis are capable of causing various diseases in different hosts. Currently, we seek information about the phylogenetic relationships between different strains of C. pseudotuberculosis isolates from different hosts across the world and to employ these data to develop tools to diagnose and eradicate the diseases these strains cause. In this review, we present the latest findings on C. pseudotuberculosis that have been obtained with the most advanced techniques for sequencing and genomic organization. We also discuss the development of in silico tools for processing these data to prompt a better understanding of this pathogen.

International audience; Background: Inflammatory bowel diseases are characterized by chronic intestinal inflammation that leads to severe destruction of the intestinal mucosa. Therefore, the understanding of their aetiology as well as the development of new medicines is an important step for the treatment of such diseases. Consequently, the development of Lactococcus lactis strains capable of delivering a eukaryotic expression vector encoding the interleukin 4 (IL-4) of Mus musculus would represent a new strategy for the elaboration of a more effective alternative therapy against Crohn's disease. Results: The murine IL-4 ORF was cloned into the eukaryotic expression vector pValac(sic)dts. The resulting plasmid-pValac(sic)dts(sic)IL-4-was transfected into CHO cells so that its functionality could be evaluated in vitro. With fluorescent confocal microscopy, flow cytometry and ELISA, it was observed that pValac(sic)dts(sic)IL-4-transfected cells produced-IL-4, while non-transfected cells and cells transfected with the empty vector did not. Then, pValac(sic)dts(sic)IL-4 was inserted into L. lactis MG1363 FnBPA(+) in order to evaluate the therapeutic potential of the recombinant strain against TNBS-induced colitis. Intragastric administration of L. lactis MG1363 FnBPA(+) (pValac(sic)dts(sic)IL-4) was able to decrease the severity of colitis, with animals showing decreased levels of IL-12, IL-6 and MPO activity; and increased levels of IL-4 and IL-10. Finally, LP-isolated cells from mice administered TNBS were immunophenotyped so that the main IL-4 and IL-10 producers were identified. Mice administered the recombinant strain presented significantly higher percentages of F4/80(+)MHCII(+)Ly6C-IL-4(+), F4/80(+)MHCII(+)Ly6C-IL-10(+), F4/80(+)MHCII(+)Ly6C-CD206(+)CD124(+)IL-10(+) and CD4(+)Foxp3(+)IL10(+) cells compared to the other groups. Conclusions: This study shows that L. lactis MG1363 FnBPA(+) (pValac(sic)dts(sic)IL-4) is a good candidate to maintain the anti-inflammatory and proinflammatory balance in the gastrointestinal tract, increasing the levels of IL-10-secreting regulatory cells and, thus, demonstrating the effectiveness of this novel DNA delivery-based strategy.

Lung cancer accounts for the highest number of cancer-related deaths worldwide. Early diagnosis significantly increases the disease-free survival rate and a large amount of effort has been expended in screening trials and the development of early molecular diagnostics. However, a gold standard diagnostic strategy is not yet available. Here, based on miRNA expression profile in lung cancer and using a novel in silico reverse-transcriptomics approach, followed by analysis of the interactome; we have identified potential transcription factor (TF) markers that would facilitate diagnosis of subtype specific lung cancer. A subset of seven TF markers has been used in a microarray screen and was then validated by blood-based qPCR using stage-II and IV non-small cell lung carcinomas (NSCLC). Our results suggest that overexpression of HMGA1, E2F6, IRF1, and TFDP1 and downregulation or no expression of SUV39H1, RBL1, and HNRPD in blood is suitable for diagnosis of lung adenocarcinoma and squamous cell carcinoma sub-types of NSCLC. Here, E2F6 was, for the first time, found to be upregulated in NSCLC blood samples. The miRNA-TF-miRNA interaction based molecular mechanisms of these seven markers in NSCLC revealed that HMGA1 and TFDP1 play vital roles in lung cancer tumorigenesis. The strategy developed in this work is applicable to any other cancer or disease and can assist in the identification of potential biomarkers.

Lactococcus lactis, the model lactic acid bacterium, is a good candidate for heterologous protein production in both foodstuffs and the digestive tract. We attempted to produce Streptomyces tendae antifungal protein 1 (Afp1) in L. lactis with the objective of constructing a strain able to limit fungal growth. Since Afp1 activity requires disulfide bond (DSB) formation and since intracellular redox conditions are reportedly unfavorable for DSB formation in prokaryotes, Afp1 was produced as a secreted form. An inducible expression-secretion system was used to drive Afp1 secretion by L. lactis; Afp1 was fused or not with LEISSTCDA, a synthetic propeptide (LEISS) that has been described to be a secretion enhancer. Production of Afp1 alone was not achieved, but production of LEISS-Afp1 was confirmed by Western blot and immunodetection with anti-Afp1 antibodies. This protein (molecular mass: 9.8 kDa) is the smallest non-bacteriocin heterologous protein ever reported to be secreted in L. lactis via the Sec-dependent pathway. However, no anti-fungal activity was detected, even in concentrated samples of induced supernatant. This could be due to a too low secretion yield of Afp1 in L. lactis, to the absence of DSB formation, or to an improper DSB formation involving the additional cysteine residue included in LEISS propeptide. This raises questions about size limits, conformation problems, and protein secretion yields in L. lactis.

Inflammatory bowel diseases (IBD) describe a group of complex intestinal disorders characterized by inflammation in the gastrointestinal tract. Current treatments for IBD include the use of anti-inflammatory drugs; furthermore, recombinant lactic acid bacteria have been used as a therapeutic vehicle for anti-inflammatory agents in IBD models. Interleukin-10 (IL-10) is one of the most important anti-inflammatory cytokines; however, its oral administration is limited because it is quickly degraded in the gastrointestinal tract and systemic treatments have led to undesirable side effects. In this study, an engineered invasive strain of Lactococcus (L.) lactis producing Fibronectin Binding Protein A (FnBPA+), from Staphylococcus aureus capable of delivering, directly inside eukaryotic cells, an eukaryotic DNA expression vector containing the ORF coding for IL-10 of Mus musculus (pValac:il-10) was developed and its functionality was evaluated using in vitro and in vivo assays. Functionality of the plasmid and the invasive strain was demonstrated by transfection and invasiveness assays using cell cultures and in vivo in mice by fluorescence microscopy. TNBS inoculated mice that received this novel strain showed lower damage scores in their large intestines (at both macroscopic and microscopic levels), lower microbial translocation to liver, and increased anti-inflammatory/pro-inflammatory cytokine ratios compared to mice that received L. lactis FnBPA+ without the pValac:il-10 plasmid. The effectiveness was demonstrated of this novel DNA delivery therapeutic strategy in the prevention of inflammation using a murine model of colitis. Fil: del Carmen, Silvina Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Tucumán. Centro de Referencia para Lactobacilos (i); Argentina; Fil: Zurita-Turk, Meritxell. Universidade Federal Do Minas Gerais; Brasil; Fil: Alvarenga Lima, Fernanda. Universidade Federal Do Minas Gerais; Brasil; Fil: Coelho Dos Santos, Janete. Fundaçăo Ezequiel Dias. Laboratório de Inovaçăo Biotecnológica; Brasil; Fil: Leclercq, Sophie Yvette. Fundaçăo Ezequiel Dias. Laboratório de Inovaçăo Biotecnológica; Brasil; Fil: Chatel, Jean-Marc. Institut National de la Recherche Agronomique; Francia; Fil: Azevedo, Vasco. Universidade Federal Do Minas Gerais; Brasil; Fil: de Moreno, Maria Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Tucumán. Centro de Referencia para Lactobacilos (i); Argentina; Fil: Miyoshi, Anderson. Universidade Federal Do Minas Gerais; Brasil; Fil: Leblanc, Jean Guy Joseph. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Tucumán. Centro de Referencia para Lactobacilos (i); Argentina; Universidad Nacional de Tucuman. Facultad de Medicina. Departamento de Investigacion. Catedra de Metodologia de la Invest.cientifica; Argentina

Background 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. Results The complete genome of five C. pseudotuberculosis strains (1002, C231, I19, FRC41 and PAT10) were submitted to pan-genomics analysis, yielding 306, 59 and 12 gene sets, respectively, representing the core, dispensable and unique in silico predicted exported pan-genomes. These sets bear 150 genes classified as secreted (SEC) and 227 as potentially surface exposed (PSE). Our findings suggest that the main C. pseudotuberculosis in vitro exoproteome could be greater, appended by a fraction of the 35 proteins formerly predicted as making part of the variant in vitro exoproteome. These genomes were manually curated for correct methionine initiation and redeposited with a total of 1885 homogenized genes. Conclusions The in silico prediction of exported proteins has allowed to define a list of putative vaccine candidate genes present in all five complete C. pseudotuberculosis genomes. Moreover, it has also been possible to define the in silico predicted dispensable and unique C. pseudotuberculosis exported proteins. These results provide in silico evidence to further guide experiments in the areas of vaccines, diagnosis and drugs. The work here presented is the first whole C. pseudotuberculosis in silico predicted pan-exoproteome completed till today.

Many probiotic bacteria have been described as a promising tool in the therapy of Inflammatory Bowel Diseases (IBD). Most of them belong to the Lactic Acid Bacteria (LAB) group, which makes part of our healthy microbiota. However, little is known about the effects of transiting dairy bacteria that make part of our diet, including Lactococcus lactis. In the present study, we analyzed the immune modulation effects of three L. lactis strains in vitro using intestinal epithelial cells and the effectiveness of a selected strain in a murine model of colitis. Only one strain, L. lactis NCDO 2118, was able to reduce the IL1-β-induced IL-8 secretion in Caco-2 cells, suggesting a potential anti-inflammatory effect of this strain. In vivo, L. lactis NCDO 2118 was administered for 4 days to C57BL/6 mice during a remission period between a first and second course of colitis induced by dextran sulfate sodium. This resulted in a milder form of recurrent colitis than observed in mice administered medium during this same period. This protective effect was not attributable to changes in secretory IgA, however NCDO 2118 administration was associated with early increase in IL-6 production and by the maintenance of IL-10 in colonic tissue. Mice fed with L. lactis NCDO 2118 increased the number of regulatory CD4+ T cells bearing surface TGF-β in the form of latency-associated protein in mesenteric lymph node and spleen. The results of this study allowed us to identify a new probiotic strain that may be used in the treatment of IBD as well as some of the mechanism involved in its anti-inflammatory effect. Fil: Diniz Luerce, Tessália. Universidade Federal de Minas Gerais. Instituto de Ciências Biológicas. Departamento de Biologia Geral; Brasil Fil: Gomes Santos, Ana Cristina. Universidade Federal de Minas Gerais. Instituto de Ciências Biológicas. Departamento de Bioquímica e Imunologia; Brasil Fil: Santos Rochat, Clarissa. Universidade Federal de Minas Gerais. Instituto de Ciências Biológicas. Departamento de Biologia Geral; Brasil Fil: Garcias Moreira, Thais. Universidade Federal de Minas Gerais. Faculdade de Farmácia. Departamento de Ciência de Alimentos.; Brasil Fil: Nogueira Cruz, Déborah. Universidade Federal de Minas Gerais. Instituto de Ciências Biológicas. Departamento de Bioquímica e Imunologia; Brasil Fil: Lemos, Luísa. Universidade Federal de Minas Gerais. Instituto de Ciências Biológicas. Departamento de Bioquímica e Imunologia; Brasil Fil: Sousa, Adna Luciana. Universidade Federal de Minas Gerais. Instituto de Ciências Biológicas. Departamento de Bioquímica e Imunologia; Brasil Fil: Bastos Pereira, Vanessa. Universidade Federal de Minas Gerais. Instituto de Ciências Biológicas. Departamento de Biologia Geral; Brasil Fil: de Azevedo, Marcela. Universidade Federal de Minas Gerais. Instituto de Ciências Biológicas. Departamento de Biologia Geral; Brasil Fil: Moraes, Kátia. Universidade Federal de Minas Gerais. Instituto de Ciências Biológicas. Departamento de Biologia Geral; Brasil Fil: Carmona Cara, Denise. Universidade Federal de Minas Gerais. Instituto de Ciências Biológicas. Departamento de Morfologia; Brasil Fil: Leblanc, Jean Guy Joseph. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Tucumán. Centro de Referencia para Lactobacilos (i); Argentina Fil: Azevedo, Vasco. Universidade Federal de Minas Gerais. Instituto de Ciências Biológicas. Departamento de Biologia Geral; Brasil Fil: Caetano Faria, Ana Maria. Universidade Federal de Minas Gerais. Instituto de Ciências Biológicas. Departamento de Bioquímica e Imunologia; Brasil Fil: Miyoshi, Anderson. Universidade Federal de Minas Gerais. Instituto de Ciências Biológicas. Departamento de Biologia Geral; Brasil

International audience; 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.

CNPq - Conselho Nacional de Desenvolvimento Científico e Tecnológico FAPEMIG - Fundação de Amparo à Pesquisa do Estado de Minas Gerais CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior As doenças inflamatórias intestinais (IBDs) compreendem um conjunto de distúrbios que afetam o trato gastrointestinal. As IBDs abrangem, essencialmente, duas enfermidades: a doença de Crohn (CD) e a colite ulcerativa (UC), que são caracterizadas por uma inflamação crônica e recorrente da mucosa. Além disso, uma das principais complicações associadas à CD é o desenvolvimento de fibrose, resultante do acúmulo excessivo de colágeno nos tecidos intestinais. O tratamento, geralmente, é realizado com aminossalicilatos, corticosteroides, imunomoduladores, terapias biológicas, antibióticos e cirurgia, contudo, está atrelado à vários efeitos adversos. Em vista desse problema, há necessidade de se desenvolver tratamentos mais eficientes aos pacientes com CD. Assim, as proteínas do choque térmico (HSPs) podem ser uma alternativa, haja vista que são consideradas antígenos importantes na regulação das células T efetoras. Logo, este trabalho teve como objetivo avaliar o potencial imunomodulatório da linhagem invasiva e produtora de Hsp65 [L. lactis NCDO2118 FnBPA+ (pXYCYT:Hsp65)] em modelos murinos de colite aguda e crônica, como uma estratégia terapêutica alternativa contra a CD experimental. Para tal, o plasmídeo pXYCYT:Hsp65 foi transformado na linhagem L. lactis NCDO2118 FnBPA+, resultando na linhagem L. lactis NCDO2118 FnBPA+ (pXYCYT:Hsp65). Em seguida, a funcionalidade dessa linhagem foi avaliada, in vitro, para a produção de Hsp65 por Western blotting e para invasão em células Caco-2. Os resultados demonstraram que a linhagem foi capaz de produzir Hsp65 e invadir eficientemente células eucarióticas. Posteriormente, in vivo, a colite experimental foi induzida em camundongos BALB/c pelo ácido trinitrobenzeno sulfônico (TNBS) e o tratamento realizado oralmente com L. lactis NCDO2118 FnBPA+ (pXYCYT:Hsp65) por gavagem intragástrica. A administração oral dessa linhagem recombinante foi capaz de atenuar a severidade da colite aguda, por meio, principalmente, da redução dos níveis de IL-12 e IL-17 e aumento dos níveis de IL-10 e de sIgA. Em seguida, essa linhagem também foi capaz de melhorar a colite crônica (inflamação e fibrose), através da regulação das citocinas pró-fibróticas IL-13 e TGF-β e da citocina regulatória IL-10. Finalmente, este trabalho constitui uma abordagem inovadora e promissora para o tratamento alternativo da CD experimental, utilizando a imunidade de mucosas e a imunomodulação para restaurar a homeostase intestinal dessa IBD. Inflammatory bowel diseases (IBDs) are a set of disorders that affect the gastrointestinal tract. IBDs essentially comprise two diseases, Crohn’s disease (CD) and ulcerative colitis (UC), which are characterized by chronic and recurrent inflammation of the mucosa. In addition, one of the main complications of CD is the development of fibrosis, which results from excessive accumulation of collagen in the intestine layers. The treatment is usually performed with anti-inflammatory drugs, immunosuppressants, antibiotics, biological drugs and surgery, but these treatments are linked to several adverse effects. In view of this problem, there is a need to develop more effective treatments for patients with CD. Thus, heat shock proteins (HSPs) might serve as an alternative treatment because these antigens play important roles in the regulation of effector T cells. Therefore, the present study aimed to evaluate the immunomodulatory potential of the invasive and Hsp65-producing strain [L. lactis NCDO2118 FnBPA+ (pXYCYT:Hsp65)] in murine models of acute and chronic colitis as an alternative therapeutic strategy against experimental CD. For this, the pXYCYT:Hsp65 plasmid was transformed into the L. lactis NCDO2118 FnBPA+ strain, resulting in the L. lactis NCDO2118 FnBPA+ (pXYCYT:Hsp65) strain. Then, the functionality of the strain was evaluated in vitro for Hsp65 production by Western blotting and for invasion into Caco-2 cells. The results demonstrated that the strain was able to produce Hsp65 and efficiently invade eukaryotic cells. Subsequently, in vivo, experimental colitis was induced by trinitrobenzene sulfonic acid (TNBS) in BALB/c mice, and the mice were treated orally with L. lactis NCDO2118 FnBPA+ (pXYCYT:Hsp65) via intragastric gavage. Oral administration of the recombinant strain was able to attenuated the severity of acute colitis by mainly reducing IL-12 and IL-17 levels and increasing IL-10 and sIgA levels. Next, this strain was also able ameliorated chronic colitis (inflammation and fibrosis) through regulation of the pro-fibrotic cytokines IL-13 and TGF-β and the regulatory cytokine IL-10. Finally, this work describes an innovative and promising approach for alternative treatment of experimental CD using mucosal immunity and immunomodulation to restore the intestinal homeostasis of this IBD.

CNPq - Conselho Nacional de Desenvolvimento Científico e Tecnológico CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior Immunization is a crucial strategy to prevent the dissemination of pathogens without aggravating resistance. The society is facing an unprecedented hazard due to the exacerbate use of antimicrobials that accelerated the overall antimicrobial resistant problematic that utterly leads to an increase in the cost within the health system. Furthermore, the continuous outbreaks of previously controlled pathogens can be linked with a low immunization coverage due to conventional vaccines drawbacks and socioeconomic difficulties. The use of cutting-edge technology may resolute many of those problems in a customized and cost-effective way. Among the bacterial pathogens that present a high morbidity and mortality rates, we highlight two crucial pathogens: Corynebacterium diphtheriae and Clostridioides difficile, the causative agents of diphtheria and C. difficile infections (CDI) (e.g. diarrhea to pseudomembrane colitis). Diphtheria was considered the main cause of child mortality but the development of the diphtheria vaccine, which is based in an inactivated form of the diphtheria toxin, significantly decrease the number of cases. However, this disease continuous to be a problem, especially considering the emergence of variants of non-toxigenic C. diphtheriae and multidrug resistant strains. Likewise, the occurrence of C. difficile multidrug resistance strains became an issue since it became more challenging to treat the disease. CDI recommended treatment is based on specific antibiotics that possible, in a near future, may became obsolete due to the high capacity of the bacterium to adapt to the surrounding environment (variable genome due to the presence of mobile elements). Therefore, the design of innovative and more inclusive vaccines may circumvent the hitches and ameliorate/resolve the spread of those life-threatening pathogens. Bearing this, we applied an immunoinformatics strategy (in silico approach) to design two multi-epitope vaccines (one for each pathogen) encompassing toxigenic and non-toxigenic strains of C. diphtheriae and C. difficile. The results predict that both vaccines can induce cellular and humoral response due to the presence of predicted MHC-I, MHC-II and B cell epitopes. Furthermore, the vaccines and the complex (vaccine + Toll-like receptors) were classified as stable, non-allergenic and antigenic. The in silico approach considerable reduced the cost and time that would be spend designing the vaccines and selecting the vaccine (reverse vaccinology) and drug targets (subtractive genomics); nonetheless, considering that the analyses were performed only in silico, experimental validations are required to confirm the results. Imunização é uma estratégia crucial para prevenir a disseminação de patógenos sem agravar a resistência. A sociedade está enfrentando uma ameaça sem precedentes devido ao uso exacerbado de antimicrobianos, que são responsáveis por acelerar a problemática da resistência que provavelmente nos conduzirá a um aumento do custo com saúde pública. Ademais, o crescente número de epidemias de patógenos anteriormente controlados está diretamente associado com uma baixa abrangência de programas de imunização, desvantagens das vacinas convencionais e dificuldades socioeconômicas. O uso de tecnologias inovadoras pode resultar na resolução de muitos desses problemas de forma customizada e com um custo efetivo reduzido. Dentre os patógenos bacterianos que apresentam uma alta taxa de morbidade e mortalidade, nós destacamos dois cruciais: Corynebacterium diphtheriae e Clostridioides difficile, os agentes causadores da difteria e C. difficile infecções (CDI) (e.g. diarreia até colite pseudomembranosa). A difteria era considerada a principal causadora da mortalidade infantil, mas com o advento da vacina diftérica (i.e. vacina fundamentada na toxina diftérica inativada), o número de casos da doença reduziu significativamente. Entretanto, esta moléstia continua a ser um problema, especialmente considerando variantes não toxigênicos de C. diphtheriae e a emergência de linhagens multidroga resistentes. Tal-qualmente, a conjuntura do surgimento de linhagens C. difficile multidroga resistentes se tornou um transtorno, visto que afeta o tratamento recomendado das CDI, posto que são baseados em antibióticos específicos. Considerando isto, possivelmente, o tratamento via antibióticos se tornará obsoleto devido à alta capacidade de adaptação desta bactéria ao meio ambiente (genoma variável em razão da presença de elementos móveis). Isto posto, a concepção de vacinas inovadoras e mais inclusivas pode sanar os inconvenientes e melhor/resolver a disseminação destes patógenos Portanto, nós utilizamos a estratégia de imunoinformática (abordagem in silico) para conceber duas vacinas multiepitopo (uma para cada patógeno) considerando as linhagens toxigênicas e não toxigênicas de C. diphtheriae e C. difficile. Os resultados preditos para cada vacina demonstraram que ambas induzem resposta imune celular e humoral devido a presença da epitopos MHC-I, MHC-II e células B. Ademais, as vacinas e os complexos (vacina + receptores Toll-like) foram classificados como estáveis, não alergênicos e antigênicos. A abordagem in silico reduz consideravelmente o custo e o tempo gasto com o design e das vacinas e com a seleção dos alvos vacinais (vacinologia reversa) e drogas (genômica subtrativa), entretanto, considerando que as análises foram realizadas apenas in silico, a validação experimental é imprescindível para confirmar os resultados.