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1.  Toll-Like Receptor 6 Plays an Important Role in Host Innate Resistance to Brucella abortus Infection in Mice 
Infection and Immunity  2013;81(5):1654-1662.
Brucella abortus is recognized by several Toll-like receptor (TLR)-associated pathways triggering proinflammatory responses that affect both the nature and intensity of the immune response. Previously, we demonstrated that B. abortus-mediated dendritic cell (DC) maturation and control of infection are dependent on the adaptor molecule MyD88. However, the involvement of all TLRs in response to B. abortus infection is not completely understood. Therefore, we decided to evaluate the requirement for TLR6 in host resistance to B. abortus. Here, we demonstrated that TLR6 is an important component for triggering an innate immune response against B. abortus. An in vitro luciferase assay indicated that TLR6 cooperates with TLR2 to sense Brucella and further activates NF-κB signaling. However, in vivo analysis showed that TLR6, not TLR2, is required for the efficient control of B. abortus infection. Additionally, B. abortus-infected dendritic cells require TLR6 to induce tumor necrosis factor alpha (TNF-α) and interleukin-12 (IL-12). Furthermore, our findings demonstrated that the mitogen-activated protein kinase (MAPK) signaling pathway is impaired in TLR2, TLR6, and TLR2/6 knockout (KO) DCs when infected with B. abortus, which may account for the lower proinflammatory cytokine production observed in TLR6 KO mouse dendritic cells. In summary, the results presented here indicate that TLR6 is required to trigger innate immune responses against B. abortus in vivo and is required for the full activation of DCs to induce robust proinflammatory cytokine production.
PMCID: PMC3647997  PMID: 23460520
2.  Schistosomes Enhance Plasminogen Activation: The Role of Tegumental Enolase 
PLoS Pathogens  2015;11(12):e1005335.
Schistosoma mansoni is a blood fluke parasite that causes schistosomiasis, a debilitating disease of global public health importance. These relatively large parasites are able to survive prolonged periods in the human vasculature without inducing stable blood clots around them. We show here that the intravascular life stages (schistosomula and adult males and females) can all promote significant plasminogen (PLMG) activation in the presence of tissue plasminogen activator (tPA). This results in the generation of the potent fibrinolytic agent plasmin which could degrade blood clots forming around the worms in vivo. We demonstrate that S. mansoni enolase (SmEno) is a host-interactive tegumental enzyme that, in recombinant form, can bind PLMG and promote its activation. Like classical members of the enolase protein family, SmEno can catalyze the interconversion of 2-phospho-D-glycerate (2-PGA) and phosphoenolpyruvate (PEP). The enzyme has maximal activity at pH 7.5, requires Mg2+ for optimal activity and can be inhibited by NaF but not mefloquin. Suppressing expression of the SmEno gene significantly diminishes enolase mRNA levels, protein levels and surface enzyme activity but, surprisingly, does not affect the ability of the worms to promote PLMG activation. Thus, while SmEno can enhance PLMG activation, our analysis suggests that it is not the only contributor to the parasite’s ability to perform this function. We show that the worms possess several other PLMG-binding proteins in addition to SmEno and these may have a greater importance in schistosome-driven PLMG activation.
Author Summary
Schistosomiasis affects more than 200 million people worldwide and causes up to 280,000 deaths per year. In terms of global mortality and morbidity, this disease is the most important human helminth infection. Schistosoma mansoni parasites can live for years within human blood vessels and seem to be refractory to intravascular thrombus formation. We hypothesize that the parasites are able to promote fibrinolysis in order to avoid firm clot formation around them. In this work, we characterize S. mansoni enolase (SmEno), an enzyme that is found both inside and at the surface of the intravascular worms, and we evaluate its involvement in plasminogen (PLMG) activation. We demonstrate that SmEno is highly expressed in schistosomula, the larval intravascular stages of S. mansoni, as well as adult worms and eggs. We confirm the localization of SmEno within the parasites and at the host/parasite interface. We demonstrate that tegumental SmEno is enzymatically functional and it is able to bind to and enhance the activation of human PLMG. Live parasites whose SmEno gene has been suppressed using RNAi display lower surface enolase activity, but their ability to activate PLMG is unchanged compared to control parasites. Thus, our data show that SmEno can contribute to PLMG activation, although it is not the only schistosome molecule responsible for this activity.
PMCID: PMC4676649  PMID: 26658895
3.  Vaccination with Enzymatically Cleaved GPI-Anchored Proteins from Schistosoma mansoni Induces Protection against Challenge Infection 
The flatworm Schistosoma mansoni is a blood fluke parasite that causes schistosomiasis, a debilitating disease that occurs throughout the developing world. Current schistosomiasis control strategies are mainly based on chemotherapy, but many researchers believe that the best long-term strategy to control schistosomiasis is through immunization with an antischistosomiasis vaccine combined with drug treatment. In the search for potential vaccine candidates, numerous tegument antigens have been assessed. As the major interface between parasite and mammalian host, the tegument plays crucial roles in the establishment and further course of schistosomiasis. Herein, we evaluated the potential of a GPI fraction, containing representative molecules located on the outer surface of adult worms, as vaccine candidate. Immunization of mice with GPI-anchored proteins induced a mixed Th1/Th2 type of immune response with production of IFN-γ and TNF-α, and low levels of IL-5 into the supernatant of splenocyte cultures. The protection engendered by this vaccination protocol was confirmed by 42% reduction in worm burden, 45% reduction in eggs per gram of hepatic tissue, 29% reduction in the number of granulomas per area, and 53% reduction in the granuloma fibrosis. Taken together, the data herein support the potential of surface-exposed GPI-anchored antigens from the S. mansoni tegument as vaccine candidate.
PMCID: PMC3426240  PMID: 22927873
4.  The role of innate immune signals in immunity to Brucella abortus 
Innate immunity serves as the first line of defense against infectious agents such as intracellular bacteria. The innate immune platform includes Toll-like receptors (TLRs), retinoid acid-inducible gene-I-like receptors and other cytosolic nucleic acid sensors, nucleotide-binding and oligomerization domain-like receptors, adaptors, kinases and other signaling molecules that are required to elicit effective responses against different pathogens. Our research group has been using the Gram-negative bacteria Brucella abortus as a model of pathogen. We have demonstrated that B. abortus triggers MAPK and NF-κB signaling pathways in macrophages in a MyD88 and IRAK-4-dependent manner. Furthermore, we claimed that so far TLR9 is the most important single TLR during Brucella infection. The identification of host receptors that recognize pathogen-derived nucleic acids has revealed an essential role for nucleic acid sensing in the triggering of immunity to intracellular pathogens. Besides TLRs, herein we describe recent advances in NOD1, NOD2, and type I IFN receptors in innate immune pathways during B. abortus infection.
PMCID: PMC3480720  PMID: 23112959
Brucella abortus; innate immunity; type I interferon; TLR signaling; NLR
5.  Host Susceptibility to Brucella abortus Infection Is More Pronounced in IFN-γ knockout than IL-12/β2-Microglobulin Double-Deficient Mice 
Brucella abortus is a facultative intracellular bacterial pathogen that causes abortion in domestic animals and undulant fever in humans. IFN-γ, IL-12, and CD8+ T lymphocytes are important components of host immune responses against B. abortus. Herein, IFN-γ and IL-12/β2-microglobulin (β2-m) knockout mice were used to determine whether CD8+ T cells and IL-12-dependent IFN-γ deficiency would be more critical to control B. abortus infection compared to the lack of endogenous IFN-γ. At 1 week after infection, IFN-γ KO and IL-12/β2-m KO mice showed increased numbers of bacterial load in spleens; however, at 3 weeks postinfection (p.i.), only IFN-γ KO succumbed to Brucella. All IFN-γ KO had died at 16 days p.i. whereas death within the IL-12/β2-m KO group was delayed and occurred at 32 days until 47 days postinfection. Susceptibility of IL-12/β2-m KO animals to Brucella was associated to undetectable levels of IFN-γ in mouse splenocytes and inability of these cells to lyse Brucella-infected macrophages. However, the lack of endogenous IFN-γ was found to be more important to control brucellosis than CD8+ T cells and IL-12-dependent IFN-γ deficiencies.
PMCID: PMC3238360  PMID: 22194770
6.  Nucleotide-Binding Oligomerization Domain-1 and -2 Play No Role in Controlling Brucella abortus Infection in Mice 
Nucleotide-binding oligomerization domain proteins (NODs) are modular cytoplasmic proteins implicated in the recognition of peptidoglycan-derived molecules. Further, several in vivo studies have demonstrated a role for Nod1 and Nod2 in host defense against bacterial pathogens. Here, we demonstrated that macrophages from NOD1-, NOD2-, and Rip2-deficient mice produced lower levels of TNF-α following infection with live Brucella abortus compared to wild-type mice. Similar reduction on cytokine synthesis was not observed for IL-12 and IL-6. However, NOD1, NOD2, and Rip2 knockout mice were no more susceptible to infection with virulent B. abortus than wild-type mice. Additionally, spleen cells from NOD1-, NOD2-, and Rip2-deficient mice showed unaltered production of IFN-γ compared to C57BL/6 mice. Taken together, this study demonstrates that NOD1, NOD2 and Rip2 are dispensable for the control of B. abortus during in vivo infection.
PMCID: PMC3235452  PMID: 22203860
7.  Computational Vaccinology: An Important Strategy to Discover New Potential S. mansoni Vaccine Candidates 
The flatworm Schistosoma mansoni is a blood fluke parasite that causes schistosomiasis, a debilitating disease that occurs throughout the developing world. Current schistosomiasis control strategies are mainly based on chemotherapy, but many researchers believe that the best long-term strategy to control schistosomiasis is through immunization with an antischistosomiasis vaccine combined with drug treatment. Several papers on Schistosoma mansoni vaccine and drug development have been published in the past few years, representing an important field of study. The advent of technologies that allow large-scale studies of genes and proteins had a remarkable impact on the screening of new and potential vaccine candidates in schistosomiasis. In this postgenomic scenario, bioinformatic technologies have emerged as important tools to mine transcriptomic, genomic, and proteomic databases. These new perspectives are leading to a new round of rational vaccine development. Herein, we discuss different strategies to identify potential S. mansoni vaccine candidates using computational vaccinology.
PMCID: PMC3196198  PMID: 22013383
8.  Human T Cell and Antibody-Mediated Responses to the Mycobacterium tuberculosis Recombinant 85A, 85B, and ESAT-6 Antigens 
Tuberculosis remains a major health problem throughout the world causing large number of deaths. Effective disease control and eradication programs require the identification of major antigens recognized by the protective responses against M. tuberculosis. In this study, we have investigated humoral and cellular immune responses to M. tuberculosis-specific Ag85A, Ag85B, and ESAT-6 antigens in Brazilian patients with pulmonary (P, n = 13) or extrapulmonary (EP, n = 12) tuberculosis, patients undergoing chemotherapy (PT, n = 23), and noninfected healthy individuals (NI, n = 7). Compared to NI, we observed increased levels of IgG1 responses to Ag85B and ESAT-6 in P and PT groups. Regarding cellular immunity, Ag85A and ESAT-6 were able to discriminate P, PT, and EP patients from healthy individuals by IFN-γ production and P and PT groups from EP individuals by production of TNF-α. In summary, these findings demonstrate the ability of Ag85A, Ag85B, and ESAT-6 to differentiate TB patients from controls by IgG1, IFN-γ and TNF-α production.
PMCID: PMC3023041  PMID: 21253450
9.  Schistosome Syntenin Partially Protects Vaccinated Mice against Schistosoma mansoni Infection 
Schistosomiasis is a neglected tropical disease caused by several species of trematode of the genus Schistosoma. The disease affects more than 200 million people in the world and causes up to 280,000 deaths per year, besides having high morbidity due to chronic illness that damages internal organs. Current schistosomiasis control strategies are mainly based on chemotherapy, but many researchers believe that the best long-term strategy to control disease is a combination of drug treatment and immunization with an anti-schistosome vaccine. Among the most promising molecules as vaccine candidates are the proteins present in the tegument and digestive tract of the parasite.
Methodology/Principal Findings
In this study, we describe for the first time Schistosoma mansoni syntenin (SmSynt) and we evaluate its potential as a recombinant vaccine. We demonstrate by real-time PCR that syntenin is mainly expressed in intravascular life stages (schistosomula and adult worms) of the parasite life cycle and, by confocal microscopy, we localize it in digestive epithelia in adult worms and schistosomula. Administration of siRNAs targeting SmSynt leads to the knock-down of syntenin gene and protein levels, but this has no demonstrable impact on parasite morphology or viability, suggesting that high SmSynt gene expression is not essential for the parasites in vitro. Mice immunization with rSmSynt, formulated with Freund's adjuvant, induces a Th1-type response, as suggested by the production of IFN-γ and TNF-α by rSmSynt-stimulated cultured splenocytes. The protective effect conferred by vaccination with rSmSynt was demonstrated by 30–37% reduction of worm burden, 38–43% reduction in the number, and 35–37% reduction in the area, of liver granulomas.
Our report is the first characterization of syntenin in Schistosoma mansoni and our data suggest that this protein is a potential candidate for the development of a multi-antigen vaccine to control schistosomiasis.
Author Summary
Schistosomiasis affects more than 200 million people worldwide and causes up to 280,000 deaths per year. In terms of global mortality and morbidity, this disease is the most important human helminth infection. Current control strategies are based on chemotherapy, but recurrent re-infection of people living in endemic areas makes many researchers, and also the World Health Organization, search for an effective vaccine to provide protection against schistosomiasis. Substantial efforts have been committed to the characterization of new antigens for an anti-schistosome vaccine and, in order to find new targets for vaccine and/or drug development, we searched transcriptomics and proteomics of Schistosoma mansoni and identified the protein syntenin (SmSynt) for analysis. In this study, we characterize SmSynt and evaluate its potential as a vaccine candidate to protect mice against S. mansoni infection. We demonstrate that SmSynt is expressed in schistosomula and adult worms, the intravascular stages of S. mansoni and it is located in the intestinal tract of the worms, an important host/parasite interface. Furthermore, vaccination of mice with rSmSynt confers partial protection against S. mansoni challenge infection and ameliorates parasite-induced liver pathology. Our data suggest that SmSynt is a potential candidate in the development of a vaccine against schistosomiasis.
PMCID: PMC4140676  PMID: 25144756
10.  Key Role of Toll-Like Receptor 2 in the Inflammatory Response and Major Histocompatibility Complex Class II Downregulation in Brucella abortus-Infected Alveolar Macrophages 
Infection and Immunity  2014;82(2):626-639.
Alveolar macrophages (AM) seem to constitute the main cellular target of inhaled brucellae. Here, we show that Brucella abortus invades and replicates in murine AM without inducing cytotoxicity. B. abortus infection induced a statistically significant increase of tumor necrosis factor alpha (TNF-α), CXCL1 or keratinocyte chemoattractant (KC), interleukin-1β (IL-1β), IL-6, and IL-12 in AM from C57BL/6 mice and BALB/c mice, but these responses were generally weaker and/or delayed compared to those elicited in peritoneal macrophages. Studies using knockout mice for TLR2, TLR4, and TLR9 revealed that TNF-α and KC responses were mediated by TLR2 recognition. Brucella infection reduced in a multiplicity of infection-dependent manner the expression of major histocompatibility complex class II (MHC-II) molecules induced by gamma interferon (IFN-γ) in AM. The same phenomenon was induced by incubation with heat-killed B. abortus (HKBA) or the lipidated form of the 19-kDa outer membrane protein of Brucella (L-Omp19), and it was shown to be mediated by TLR2 recognition. In contrast, no significant downregulation of MHC-II was induced by either unlipidated Omp19 or Brucella LPS. In a functional assay, treatment of AM with either L-Omp19 or HKBA reduced the MHC-II-restricted presentation of OVA peptides to specific T cells. One week after intratracheal infection, viable B. abortus was detected in AM from both wild-type and TLR2 KO mice, but CFU counts were higher in the latter. These results suggest that B. abortus survives in AM after inhalatory infection in spite of a certain degree of immune control exerted by the TLR2-mediated inflammatory response. Both the modest nature of the latter and the modulation of MHC-II expression by the bacterium may contribute to such survival.
PMCID: PMC3911380  PMID: 24478078
11.  Brucella Cyclic β-1,2-Glucan Plays a Critical Role in the Induction of Splenomegaly in Mice 
PLoS ONE  2014;9(7):e101279.
Brucella, the etiological agent of animal and human brucellosis, is a bacterium with the capacity to modulate the inflammatory response. Cyclic β-1,2-glucan (CβG) is a virulence factor key for the pathogenesis of Brucella as it is involved in the intracellular life cycle of the bacteria. Using comparative studies with different CβG mutants of Brucella, cgs (CβG synthase), cgt (CβG transporter) and cgm (CβG modifier), we have identified different roles for this polysaccharide in Brucella. While anionic CβG is required for bacterial growth in low osmolarity conditions, the sole requirement for a successful Brucella interaction with mammalian host is its transport to periplasmic space. Our results uncover a new role for CβG in promoting splenomegaly in mice. We showed that CβG-dependent spleen inflammation is the consequence of massive cell recruitment (monocytes, dendritics cells and neutrophils) due to the induction of pro-inflammatory cytokines such as IL-12 and TNF-α and also that the reduced splenomegaly response observed with the cgs mutant is not the consequence of changes in expression levels of the characterized Brucella PAMPs LPS, flagellin or OMP16/19. Complementation of cgs mutant with purified CβG increased significantly spleen inflammation response suggesting a direct role for this polysaccharide.
PMCID: PMC4077732  PMID: 24983999
12.  Sm10.3, a Member of the Micro-Exon Gene 4 (MEG-4) Family, Induces Erythrocyte Agglutination In Vitro and Partially Protects Vaccinated Mice against Schistosoma mansoni Infection 
The parasitic flatworm Schistosoma mansoni is a blood fluke that causes schistosomiasis. Current schistosomiasis control strategies are mainly based on chemotherapy, but many researchers believe that the best long-term strategy to control disease is a combination of drug treatment and immunization with an anti-schistosome vaccine. Numerous antigens that are expressed at the interface between the parasite and the mammalian host have been assessed. Among the most promising molecules are the proteins present in the tegument and digestive tract of the parasite.
Methodology/Principal Findings
In this study, we evaluated the potential of Sm10.3, a member of the micro-exon gene 4 (MEG-4) family, for use as part of a recombinant vaccine. We confirmed by real-time PCR that Sm10.3 was expressed at all stages of the parasite life cycle. The localization of Sm10.3 on the surface and lumen of the esophageal and intestinal tract in adult worms and lung-stage schistosomula was confirmed by confocal microscopy. We also show preliminary evidence that rSm10.3 induces erythrocyte agglutination in vitro. Immunization of mice with rSm10.3 induced a mixed Th1/Th2-type response, as IFN-γ, TNF-α, and low levels of IL-5 were detected in the supernatant of cultured splenocytes. The protective effect conferred by vaccination with rSm10.3 was demonstrated by 25.5–32% reduction in the worm burden, 32.9–43.6% reduction in the number of eggs per gram of hepatic tissue, a 23.8% reduction in the number of granulomas, an 11.8% reduction in the area of the granulomas and a 39.8% reduction in granuloma fibrosis.
Our data suggest that Sm10.3 is a potential candidate for use in developing a multi-antigen vaccine to control schistosomiasis and provide the first evidence for a possible role for Sm10.3 in the blood feeding process.
Author Summary
Schistosomiasis mainly occurs in developing countries and is the most important human helminth infection in terms of global mortality. This parasitic disease affects more than 200 million people worldwide and causes more than 250,000 deaths per year. Current schistosomiasis control strategies are mainly based on chemotherapy, but many researchers believe that the best long-term strategy for controlling schistosomiasis is a combination of drug treatment and immunization with an anti-schistosome vaccine. Consequently, significant effort has been dedicated to developing and characterizing an anti-schistosome vaccine. Over the last five years, considerable data have been generated regarding the genomics, transcriptomics and proteomics of Schistosoma mansoni. In the present study, we characterize the Sm10.3 protein and evaluate its potential to protect against S. mansoni infection in a murine model. We demonstrate that Sm10.3 is primarily expressed during the stages of the parasite life cycle that involve infection and disease development in the human host. Sm10.3 is located on the surface of the digestive epithelia of adult female worms, an important host/parasite interface. Moreover, the vaccination of mice with rSm10.3 confers partial protection against S. mansoni. Taken together, our data suggest that Sm10.3 may be a useful component of a multi-antigen vaccine against schistosomiasis.
PMCID: PMC3961193  PMID: 24651069
13.  Lack of Endogenous IL-10 Enhances Production of Proinflammatory Cytokines and Leads to Brucella abortus Clearance in Mice 
PLoS ONE  2013;8(9):e74729.
IL-10 is a cytokine that regulates the balance between pathogen clearance and immunopathology. Brucella abortus is an intracellular bacterium that causes chronic disease in humans and domestic animals. Here we evaluated the contribution of IL-10 in host immune response and pathology during B. abortus infection. To assess the role of IL-10 in vivo, IL-10 knockout (KO) or 129 Sv/Ev (wild-type) mice were infected with B. abortus and the number of viable bacteria from the spleen was determined at 1, 2, 3, 6 and 14-weeks postinfection. IL-10 KO mice showed reduced bacterial loads in the spleen when compared to wild-type mice during all time points studied. Additionally, at 14-weeks postinfection IL-10 KO mice had totally cleared the infection. This clearance was preceded by an enhanced IFN-γ, TNF-α and IL-17 responses in both the serum and the spleen of IL-10 KO mice. Additionally, dendritic cells from infected IL-10 KO mice produced elevated levels of IL-12 and TNF-α compared to wild-type animals. Histopathology analysis was performed and both KO and wild-type mice developed multifocal granulomas and necrosis in the liver. However, at six-weeks postinfection reduced numbers of granulomas was detected in IL-10 KO mice compared to wild-type animals. This reduced liver pathology at later stage of infection was accompanied by increased numbers of CD4+CD25+foxp3+ T cells and expression of TGF-β in IL-10 KO splenocytes. Taken together, our findings demonstrate that IL-10 modulates the proinflammatory immune response to B. abortus infection and the lack of IL-10 increases resistance to Brucella infection.
PMCID: PMC3775771  PMID: 24069337
14.  Unlipidated Outer Membrane Protein Omp16 (U-Omp16) from Brucella spp. as Nasal Adjuvant Induces a Th1 Immune Response and Modulates the Th2 Allergic Response to Cow’s Milk Proteins 
PLoS ONE  2013;8(7):e69438.
The discovery of novel mucosal adjuvants will help to develop new formulations to control infectious and allergic diseases. In this work we demonstrate that U-Omp16 from Brucella spp. delivered by the nasal route (i.n.) induced an inflammatory immune response in bronchoalveolar lavage (BAL) and lung tissues. Nasal co-administration of U-Omp16 with the model antigen (Ag) ovalbumin (OVA) increased the amount of Ag in lung tissues and induced OVA-specific systemic IgG and T helper (Th) 1 immune responses. The usefulness of U-Omp16 was also assessed in a mouse model of food allergy. U-Omp16 i.n. administration during sensitization ameliorated the hypersensitivity responses of sensitized mice upon oral exposure to Cow’s Milk Protein (CMP), decreased clinical signs, reduced anti-CMP IgE serum antibodies and modulated the Th2 response in favor of Th1 immunity. Thus, U-Omp16 could be used as a broad Th1 mucosal adjuvant for different Ag formulations.
PMCID: PMC3703917  PMID: 23861971
15.  A Role for Sigma Factor σE in Corynebacterium pseudotuberculosis Resistance to Nitric Oxide/Peroxide Stress 
Pathogenic intracellular bacteria can respond to antimicrobial mechanisms of the host cell through transient activation of stress-responsive genes by alternative sigma (σ) factors of the RNA polymerase. We evaluated the contribution of the extracytoplasmic function sigma factor σE for Corynebacterium pseudotuberculosis resistance to stress conditions resembling those found intracellularly during infection. A sigE-null mutant strain (ΔsigE) of this bacterium was more susceptible in vitro to acidic pH, cell surface stressors, and biologically relevant concentrations of nitric oxide (NO). The same mutant strain was unable to persist in C57BL/6 mice but remained infective in mice lacking inducible nitric oxide synthase (iNOS), confirming the significance of σE for resistance to nitric oxide/peroxide stress in vivo. High-throughput proteomic analysis identified NO-responsive extracellular proteins of C. pseudotuberculosis and demonstrated the participation of σE in composition of this bacterium’s exoproteome.
PMCID: PMC3322355  PMID: 22514549
Corynebacterium pseudotuberculosis; sigma factor; nitric oxide; inducible nitric oxide synthase
16.  MyD88 and STING Signaling Pathways Are Required for IRF3-Mediated IFN-β Induction in Response to Brucella abortus Infection 
PLoS ONE  2011;6(8):e23135.
Type I interferons (IFNs) are cytokines that orchestrate diverse immune responses to viral and bacterial infections. Although typically considered to be most important molecules in response to viruses, type I IFNs are also induced by most, if not all, bacterial pathogens. In this study, we addressed the role of type I IFN signaling during Brucella abortus infection, a facultative intracellular bacterial pathogen that causes abortion in domestic animals and undulant fever in humans. Herein, we have shown that B. abortus induced IFN-β in macrophages and splenocytes. Further, IFN-β induction by Brucella was mediated by IRF3 signaling pathway and activates IFN-stimulated genes via STAT1 phosphorylation. In addition, IFN-β expression induced by Brucella is independent of TLRs and TRIF signaling but MyD88-dependent, a pathway not yet described for Gram-negative bacteria. Furthermore, we have identified Brucella DNA as the major bacterial component to induce IFN-β and our study revealed that this molecule operates through a mechanism dependent on RNA polymerase III to be sensed probably by an unknown receptor via the adaptor molecule STING. Finally, we have demonstrated that IFN-αβR KO mice are more resistant to infection suggesting that type I IFN signaling is detrimental to host control of Brucella. This resistance phenotype is accompanied by increased IFN-γ and NO production by IFN-αβR KO spleen cells and reduced apoptosis.
PMCID: PMC3149075  PMID: 21829705
17.  Involvement of an Alternative Oxidase in Oxidative Stress and Mycelium-to-Yeast Differentiation in Paracoccidioides brasiliensis ▿ † 
Eukaryotic Cell  2011;10(2):237-248.
Paracoccidioides brasiliensis is a thermodimorphic human pathogenic fungus that causes paracoccidioidomycosis (PCM), which is the most prevalent systemic mycosis in Latin America. Differentiation from the mycelial to the yeast form (M-to-Y) is an essential step for the establishment of PCM. We evaluated the involvement of mitochondria and intracellular oxidative stress in M-to-Y differentiation. M-to-Y transition was delayed by the inhibition of mitochondrial complexes III and IV or alternative oxidase (AOX) and was blocked by the association of AOX with complex III or IV inhibitors. The expression of P. brasiliensis aox (Pbaox) was developmentally regulated through M-to-Y differentiation, wherein the highest levels were achieved in the first 24 h and during the yeast exponential growth phase; Pbaox was upregulated by oxidative stress. Pbaox was cloned, and its heterologous expression conferred cyanide-resistant respiration in Saccharomyces cerevisiae and Escherichia coli and reduced oxidative stress in S. cerevisiae cells. These results reinforce the role of PbAOX in intracellular redox balancing and demonstrate its involvement, as well as that of other components of the mitochondrial respiratory chain complexes, in the early stages of the M-to-Y differentiation of P. brasiliensis.
PMCID: PMC3067407  PMID: 21183691
18.  An Oral Vaccine Based on U-Omp19 Induces Protection against B. abortus Mucosal Challenge by Inducing an Adaptive IL-17 Immune Response in Mice 
PLoS ONE  2011;6(1):e16203.
As Brucella infections occur mainly through mucosal surfaces, the development of mucosal administered vaccines could be radical for the control of brucellosis. In this work we evaluated the potential of Brucella abortus 19 kDa outer membrane protein (U-Omp19) as an edible subunit vaccine against brucellosis. We investigated the protective immune response elicited against oral B. abortus infection after vaccination of mice with leaves from transgenic plants expressing U-Omp19; or with plant-made or E. coli-made purified U-Omp19. All tested U-Omp19 formulations induced protection against Brucella when orally administered without the need of adjuvants. U-Omp19 also induced protection against a systemic challenge when parenterally administered. This built-in adjuvant ability of U-Omp19 was independent of TLR4 and could be explained at least in part by its capability to activate dendritic cells in vivo. While unadjuvanted U-Omp19 intraperitoneally administered induced a specific Th1 response, following U-Omp19 oral delivery a mixed specific Th1-Th17 response was induced. Depletion of CD4+ T cells in mice orally vaccinated with U-Omp19 resulted in a loss of the elicited protection, indicating that this cell type mediates immune protection. The role of IL-17 against Brucella infection has never been explored. In this study, we determined that if IL-17A was neutralized in vivo during the challenge period, the mucosal U-Omp19 vaccine did not confer mucosal protection. On the contrary, IL-17A neutralization during the infection did not influence at all the subsistence and growth of this bacterium in PBS-immunized mice. All together, our results indicate that an oral unadjuvanted vaccine based on U-Omp19 induces protection against a mucosal challenge with Brucella abortus by inducing an adaptive IL-17 immune response. They also indicate different and important new aspects i) IL-17 does not contribute to reduce the bacterial burden in non vaccinated mice and ii) IL-17 plays a central role in vaccine mediated anti-Brucella mucosal immunity.
PMCID: PMC3021544  PMID: 21264260
19.  The Brucella abortus Phosphoglycerate Kinase Mutant Is Highly Attenuated and Induces Protection Superior to That of Vaccine Strain 19 in Immunocompromised and Immunocompetent Mice ▿  
Infection and Immunity  2010;78(5):2283-2291.
Brucella abortus is a facultative intracellular bacterial pathogen that causes abortion in domestic animals and undulant fever in humans. The mechanism of virulence of Brucella spp. is not yet fully understood. Therefore, it is crucial to identify new molecules that can function as virulence factors to better understand the host-pathogen interplay. Herein, we identified the gene encoding the phosphoglycerate kinase (PGK) of B. abortus strain 2308. To test the role of PGK in Brucella pathogenesis, a pgk deletion mutant was constructed. Replacement of the wild-type pgk by recombination was demonstrated by Southern and Western blot analyses. The B. abortus Δpgk mutant strain exhibited extreme attenuation in bone marrow-derived macrophages and in vivo in BALB/c, C57BL/6, 129/Sv, and interferon regulatory factor-1 knockout (IRF-1 KO) mice. Additionally, at 24 h postinfection the Δpgk mutant was not found within the same endoplasmic reticulum-derived compartment as the wild-type bacteria, but, instead, over 60% of Brucella-containing vacuoles (BCVs) retained the late endosomal/lysosomal marker LAMP1. Furthermore, the B. abortus Δpgk deletion mutant was used as a live vaccine. Challenge experiments revealed that the Δpgk mutant strain induced protective immunity in 129/Sv or IRF-1 KO mice that was superior to the protection conferred by commercial strain 19 or RB51. Finally, the results shown here demonstrated that Brucella PGK is critical for full bacterial virulence and that a Δpgk mutant may serve as a potential vaccine candidate in future studies.
PMCID: PMC2863508  PMID: 20194591
20.  Schistosoma mansoni Antigens Modulate Experimental Allergic Asthma in a Murine Model: a Major Role for CD4+ CD25+ Foxp3+ T Cells Independent of Interleukin-10▿  
Infection and Immunity  2008;77(1):98-107.
In areas where schistosomiasis is endemic, a negative correlation is observed between atopy and helminth infection, associated with a low prevalence of asthma. We investigated whether Schistosoma mansoni infection or injection of parasite eggs can modulate airway allergic inflammation in mice, examining the mechanisms of such regulation. We infected BALB/c mice with 30 S. mansoni cercariae or intraperitoneally injected 2,500 schistosome eggs, and experimental asthma was induced by ovalbumin (OVA). The number of eosinophils in bronchoalveolar lavage fluid was higher in the asthmatic group than in asthmatic mice infected with S. mansoni or treated with parasite eggs. Reduced Th2 cytokine production, characterized by lower levels of interleukin-4 (IL-4), IL-5, and immunoglobulin E, was observed in both S. mansoni-treated groups compared to the asthmatic group. There was a reduction in the number of inflammatory cells in lungs of S. mansoni-infected and egg-treated mice, demonstrating that both S. mansoni infection and the egg treatment modulated the lung inflammatory response to OVA. Only allergic animals that were treated with parasite eggs had increased numbers of CD4+ CD25+ Foxp3+ T cells and increased levels of IL-10 and decreased production of CCL2, CCL3, and CCL5 in the lungs compared to the asthmatic group. Neutralization of IL-10 receptor or depletion of CD25+ T cells in vivo confirmed the critical role of CD4+ CD25+ Foxp3+ regulatory T cells in experimental asthma modulation independent of IL-10.
PMCID: PMC2612239  PMID: 18824533
21.  Schistosoma mansoni Tegument Protein Sm29 Is Able to Induce a Th1-Type of Immune Response and Protection against Parasite Infection 
Schistosomiasis continues to be a significant public health problem. This disease affects 200 million people worldwide and almost 800 million people are at risk of acquiring the infection. Although vaccine development against this disease has experienced more failures than successes, encouraging results have recently been obtained using membrane-spanning protein antigens from the tegument of Schistosoma mansoni. Our group recently identified Sm29, another antigen that is present at the adult worm tegument surface. In this study, we investigated murine cellular immune responses to recombinant (r) Sm29 and tested this protein as a vaccine candidate.
Methods and Findings
We first show that Sm29 is located on the surface of adult worms and lung-stage schistosomula through confocal microscopy. Next, immunization of mice with rSm29 engendered 51%, 60% and 50% reduction in adult worm burdens, in intestinal eggs and in liver granuloma counts, respectively (p<0.05). Protective immunity in mice was associated with high titers of specific anti-Sm29 IgG1 and IgG2a and elevated production of IFN-γ, TNF-α and IL-12, a typical Th1 response. Gene expression analysis of worms recovered from rSm29 vaccinated mice relative to worms from control mice revealed a significant (q<0.01) down-regulation of 495 genes and up-regulation of only 22 genes. Among down-regulated genes, many of them encode surface antigens and proteins associated with immune signals, suggesting that under immune attack schistosomes reduce the expression of critical surface proteins.
This study demonstrates that Sm29 surface protein is a new vaccine candidate against schistosomiasis and suggests that Sm29 vaccination associated with other protective critical surface antigens is the next logical strategy for improving protection.
Author Summary
Schistosomiasis is the most important human helminth infection in terms of morbidity and mortality. Although the efforts to develop a vaccine against this disease have experienced failures, a new generation of surface antigens revealed by proteomic studies changed this scenario. Our group has characterized the protein Sm29 described previously as one of the most exposed and expressed antigens in the outer tegument of Schistosoma mansoni. Studies in patients living in endemic areas for schistosomiasis revealed high levels of IgG1 and IgG3 anti-Sm29 in resistant individuals. In this study, confocal microscope analysis showed Sm29 present in the surface of lung-stage schistosoluma and adult worms. Recombinant Sm29, when used as vaccine candidate, induced high levels of protection in mice. This protection was associated with a typical Th1 immune response and reduction of worm burden, liver granulomas and in intestinal eggs. Further, microarray analysis of worms recovered from vaccinated mice showed significant down-regulation of several genes encoding previously characterized vaccine candidates and/or molecules exposed on the surface, suggesting an immune evasion strategy of schistosomes under immune attack. These results demonstrated that Sm29 as one of the important antigens with potential to compose a vaccine against schistosomiasis.
PMCID: PMC2553283  PMID: 18827884
22.  Polymyxin B as inhibitor of LPS contamination of Schistosoma mansoni recombinant proteins in human cytokine analysis 
Recombinant proteins expressed in Escherichia coli vectors are generally contaminated with endotoxin. In this study, we evaluated the ability of Polymyxin B to neutralize the effect of LPS present as contaminant on Schistosoma mansoni recombinant proteins produced in E. coli in inducing TNF-α and IL-10. Peripheral blood mononuclear cells from individuals chronically infected with S. mansoni were stimulated in vitro with recombinant Sm22.6, Sm14 and P24 antigens (10 μg/mL) in the presence of Polymyxin B (10 μg/mL).
The levels of cytokines were measured using ELISA. There was greater than 90 % reduction (p < 0.05) in the levels of TNF-α and IL-10 when Polymyxin B was added to the cultures stimulated with LPS. In cultures stimulated with S. mansoni recombinant proteins in the presence of Polymyxin B, a reduction in the levels of TNF-α and IL-10 was also observed. However, the percentage of reduction was lower when compared to the cultures stimulated with LPS, probably because these proteins are able to induce the production of these cytokines by themselves.
This study showed that Polymyxin B was able to neutralize the effect of endotoxin, as contaminant in S. mansoni recombinant antigens produced in E. coli, in inducing TNF-α and IL-10 production.
PMCID: PMC1766364  PMID: 17201926
23.  DNA Vaccine Using Mycobacterium bovis Ag85B Antigen Induces Partial Protection against Experimental Infection in BALB/c Mice 
Clinical and Vaccine Immunology  2006;13(8):930-935.
Bovine tuberculosis is a major cause of economic loss in countries where it is endemic, and in some countries, it may be a significant zoonotic disease problem. Therefore, new strategies for vaccine development are required, and among them, genetic immunization has potential value. The main goal of this study was to test the Mycobacterium bovis Ag85B gene as a DNA vaccine following challenge with an M. bovis virulent strain (ATCC 19274). Groups of BALB/c mice (n = 10) were immunized four times intramuscularly with the pCI-Ag85B construct or the pCI vector alone as the control. High titers of total immunoglobulin G (IgG), IgG1, and IgG2a anti-Ag85B were measured in pCI-Ag85B immunized mice when compared to the pCI control group. Regarding cellular immunity, significant levels of gamma interferon (IFN-γ) (1,100 ± 157 pg/ml) and tumor necrosis factor alpha (650 ± 42 pg/ml) but not interleukin-4 were detected in splenocyte culture supernatants of pCI-Ag85B-vaccinated mice following stimulation with recombinant Ag85B. Further, the main source of IFN-γ is CD8+ T cells, as demonstrated by intracellular cytokine staining. As far as protection, a significant reduction in bacterial load in spleens (P < 0.05) was detected in pCI-Ag85B-immunized mice compared to the pCI vector control group. The results obtained here suggest that use of the Ag85B DNA vaccine is a promising strategy to control M. bovis infection due to its ability to induce a Th1 type of immune response. However, protective efficacy needs to be improved, since partial protection was achieved in spleens but not in lungs of vaccinated mice.
PMCID: PMC1539111  PMID: 16893994
24.  Swine and Poultry Pathogens: the Complete Genome Sequences of Two Strains of Mycoplasma hyopneumoniae and a Strain of Mycoplasma synoviae†  
Vasconcelos, Ana Tereza R. | Ferreira, Henrique B. | Bizarro, Cristiano V. | Bonatto, Sandro L. | Carvalho, Marcos O. | Pinto, Paulo M. | Almeida, Darcy F. | Almeida, Luiz G. P. | Almeida, Rosana | Alves-Filho, Leonardo | Assunção, Enedina N. | Azevedo, Vasco A. C. | Bogo, Maurício R. | Brigido, Marcelo M. | Brocchi, Marcelo | Burity, Helio A. | Camargo, Anamaria A. | Camargo, Sandro S. | Carepo, Marta S. | Carraro, Dirce M. | de Mattos Cascardo, Júlio C. | Castro, Luiza A. | Cavalcanti, Gisele | Chemale, Gustavo | Collevatti, Rosane G. | Cunha, Cristina W. | Dallagiovanna, Bruno | Dambrós, Bibiana P. | Dellagostin, Odir A. | Falcão, Clarissa | Fantinatti-Garboggini, Fabiana | Felipe, Maria S. S. | Fiorentin, Laurimar | Franco, Gloria R. | Freitas, Nara S. A. | Frías, Diego | Grangeiro, Thalles B. | Grisard, Edmundo C. | Guimarães, Claudia T. | Hungria, Mariangela | Jardim, Sílvia N. | Krieger, Marco A. | Laurino, Jomar P. | Lima, Lucymara F. A. | Lopes, Maryellen I. | Loreto, Élgion L. S. | Madeira, Humberto M. F. | Manfio, Gilson P. | Maranhão, Andrea Q. | Martinkovics, Christyanne T. | Medeiros, Sílvia R. B. | Moreira, Miguel A. M. | Neiva, Márcia | Ramalho-Neto, Cicero E. | Nicolás, Marisa F. | Oliveira, Sergio C. | Paixão, Roger F. C. | Pedrosa, Fábio O. | Pena, Sérgio D. J. | Pereira, Maristela | Pereira-Ferrari, Lilian | Piffer, Itamar | Pinto, Luciano S. | Potrich, Deise P. | Salim, Anna C. M. | Santos, Fabrício R. | Schmitt, Renata | Schneider, Maria P. C. | Schrank, Augusto | Schrank, Irene S. | Schuck, Adriana F. | Seuanez, Hector N. | Silva, Denise W. | Silva, Rosane | Silva, Sérgio C. | Soares, Célia M. A. | Souza, Kelly R. L. | Souza, Rangel C. | Staats, Charley C. | Steffens, Maria B. R. | Teixeira, Santuza M. R. | Urmenyi, Turan P. | Vainstein, Marilene H. | Zuccherato, Luciana W. | Simpson, Andrew J. G. | Zaha, Arnaldo
Journal of Bacteriology  2005;187(16):5568-5577.
This work reports the results of analyses of three complete mycoplasma genomes, a pathogenic (7448) and a nonpathogenic (J) strain of the swine pathogen Mycoplasma hyopneumoniae and a strain of the avian pathogen Mycoplasma synoviae; the genome sizes of the three strains were 920,079 bp, 897,405 bp, and 799,476 bp, respectively. These genomes were compared with other sequenced mycoplasma genomes reported in the literature to examine several aspects of mycoplasma evolution. Strain-specific regions, including integrative and conjugal elements, and genome rearrangements and alterations in adhesin sequences were observed in the M. hyopneumoniae strains, and all of these were potentially related to pathogenicity. Genomic comparisons revealed that reduction in genome size implied loss of redundant metabolic pathways, with maintenance of alternative routes in different species. Horizontal gene transfer was consistently observed between M. synoviae and Mycoplasma gallisepticum. Our analyses indicated a likely transfer event of hemagglutinin-coding DNA sequences from M. gallisepticum to M. synoviae.
PMCID: PMC1196056  PMID: 16077101
25.  Protein secretion in Lactococcus lactis : an efficient way to increase the overall heterologous protein production 
Lactococcus lactis, the model lactic acid bacterium (LAB), is a food grade and well-characterized Gram positive bacterium. It is a good candidate for heterologous protein delivery in foodstuff or in the digestive tract. L. lactis can also be used as a protein producer in fermentor. Many heterologous proteins have already been produced in L. lactis but only few reports allow comparing production yields for a given protein either produced intracellularly or secreted in the medium. Here, we review several works evaluating the influence of the localization on the production yields of several heterologous proteins produced in L. lactis. The questions of size limits, conformation, and proteolysis are addressed and discussed with regard to protein yields. These data show that i) secretion is preferable to cytoplasmic production; ii) secretion enhancement (by signal peptide and propeptide optimization) results in increased production yield; iii) protein conformation rather than protein size can impair secretion and thus alter production yields; and iv) fusion of a stable protein can stabilize labile proteins. The role of intracellular proteolysis on heterologous cytoplasmic proteins and precursors is discussed. The new challenges now are the development of food grade systems and the identification and optimization of host factors affecting heterologous protein production not only in L. lactis, but also in other LAB species.
PMCID: PMC545053  PMID: 15631634

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