Influence of N-Glycosylation on the Morphogenesis and Growth of Paracoccidioides brasiliensis and on the Biological Activities of Yeast Proteins

The fungus Paracoccidioides brasiliensis is a human pathogen that causes paracoccidioidomycosis, the most prevalent systemic mycosis in Latin America. The cell wall of P. brasiliensis is a network of glycoproteins and polysaccharides, such as chitin, that perform several functions. N-linked glycans are involved in glycoprotein folding, intracellular transport, secretion, and protection from proteolytic degradation. Here, we report the effects of tunicamycin (TM)-mediated inhibition of N-linked glycosylation on P. brasiliensis yeast cells. The underglycosylated yeasts were smaller than their fully glycosylated counterparts and exhibited a drastic reduction of cell budding, reflecting impairment of growth and morphogenesis by TM treatment. The intracellular distribution in TM-treated yeasts of the P. brasiliensis glycoprotein paracoccin was investigated using highly specific antibodies. Paracoccin was observed to accumulate at intracellular locations, far from the yeast wall. Paracoccin derived from TM-treated yeasts retained the ability to bind to laminin despite their underglycosylation. As paracoccin has N-acetyl-β-d-glucosaminidase (NAGase) activity and induces the production of TNF-α and nitric oxide (NO) by macrophages, we compared these properties between glycosylated and underglycosylated yeast proteins. Paracoccin demonstrated lower NAGase activity when underglycosylated, although no difference was detected between the pH and temperature optimums of the two forms. Murine macrophages stimulated with underglycosylated yeast proteins produced significantly lower levels of TNF-α and NO. Taken together, the impaired growth and morphogenesis of tunicamycin-treated yeasts and the decreased biological activities of underglycosylated fungal components suggest that N-glycans play important roles in P. brasiliensis yeast biology

Search for the Xb and other hidden-beauty states in the π+π−ϒ(1S) channel at ATLAS

This Letter presents a search for a hidden-beauty counterpart of the X(3872) in the mass ranges of 10.05–10.31 GeV and 10.40–11.00 GeV, in the channel Xb→π+π−ϒ(1S)(→μ+μ−), using 16.2 fb−1 of pp   collision data collected by the ATLAS detector at the LHC. No evidence for new narrow states is found, and upper limits are set on the product of the Xb cross section and branching fraction, relative to those of the ϒ(2S), at the 95% confidence level using the CLS approach. These limits range from 0.8% to 4.0%, depending on mass. For masses above 10.1 GeV, the expected upper limits from this analysis are the most restrictive to date. Searches for production of the ϒ(13DJ), , and states also reveal no significant signals

The complete genome sequence of Chromobacterium violaceum reveals remarkable and exploitable bacterial adaptability

Chromobacterium violaceum is one of millions of species of free-living microorganisms that populate the soil and water in the extant areas of tropical biodiversity around the world. Its complete genome sequence reveals (i) extensive alternative pathways for energy generation, (ii) ≈500 ORFs for transport-related proteins, (iii) complex and extensive systems for stress adaptation and motility, and (iv) wide-spread utilization of quorum sensing for control of inducible systems, all of which underpin the versatility and adaptability of the organism. The genome also contains extensive but incomplete arrays of ORFs coding for proteins associated with mammalian pathogenicity, possibly involved in the occasional but often fatal cases of human C. violaceum infection. There is, in addition, a series of previously unknown but important enzymes and secondary metabolites including paraquat-inducible proteins, drug and heavy-metal-resistance proteins, multiple chitinases, and proteins for the detoxification of xenobiotics that may have biotechnological applications

Cloning, expression and purification of a glycosylated form of the DNA-binding protein Dps from Salmonella enterica Typhimurium

Dps, found in many eubacterial and archaebacterial species, appears to protect cells from oxidative stress and/or nutrient-limited environment. Dps has been shown to accumulate during the stationary phase, to bind to DNA non-specifically, and to form a crystalline structure that compacts and protects the chromosome. Our previous results have indicated that Dps is glycosylated at least for a certain period of the bacterial cell physiology and this glycosylation is thought to be orchestrated by some factors not yet understood, explaining our difficulties in standardizing the Dps purification process. In the present work, the open reading frame of the dps gene, together with all the upstream regulatory elements, were cloned into a PCR cloning vector. As a result, the expression of dps was also controlled by the plasmid system introduced in the bacterial cell. The gene was then over-expressed regardless of the growth phase of the culture and a glycosylated fraction was purified to homogeneity by lectin-immobilized chromatography assay. Unlike the high level expression of Dps in Salmonella cells, less than 1% of the recombinant protein was purified by affinity chromatography using jacalin column. Sequencing and mass spectrometry data confirmed the identity of the dps gene and the protein, respectively. In spite of the low level of purification of the jacalin-binding Dps, this work shall aid further investigations into the mechanism of Dps glycosylation. (C) 2008 Elsevier Inc. All rights reserved

Cloning, expression and purification of a glycosylated form of the DNA-binding protein Dps from Salmonella enterica Typhimurium

Dps, found in many eubacterial and archaebacterial species, appears to protect cells from oxidative stress and/or nutrient-limited environment. Dps has been shown to accumulate during the stationary phase, to bind to DNA non-specifically, and to form a crystalline structure that compacts and protects the chromosome. Our previous results have indicated that Dps is glycosylated at least for a certain period of the bacterial cell physiology and this glycosylation is thought to be orchestrated by some factors not yet understood, explaining our difficulties in standardizing the Dps purification process. In the present work, the open reading frame of the dps gene, together with all the upstream regulatory elements, were cloned into a PCR cloning vector. As a result, the expression of dps was also controlled by the plasmid system introduced in the bacterial cell. The gene was then over-expressed regardless of the growth phase of the culture and a glycosylated fraction was purified to homogeneity by lectin-immobilized chromatography assay. Unlike the high level expression of Dps in Salmonella cells, less than 1% of the recombinant protein was purified by affinity chromatography using jacalin column. Sequencing and mass spectrometry data confirmed the identity of the dps gene and the protein, respectively. In spite of the low level of purification of the jacalin-binding Dps, this work shall aid further investigations into the mechanism of Dps glycosylation2197202

Recombinant paracoccin reproduces the biological properties of the native protein and induces protective Th1 immunity against Paracoccidioides brasiliensis infection.

BACKGROUND: Paracoccin is a dual-function protein of the yeast Paracoccidioides brasiliensis that has lectin properties and N-acetylglucosaminidase activities. Proteomic analysis of a paracoccin preparation from P. brasiliensis revealed that the sequence matched that of the hypothetical protein encoded by PADG-3347 of isolate Pb-18, with a polypeptide sequence similar to the family 18 endochitinases. These endochitinases are multi-functional proteins, with distinct lectin and enzymatic domains. METHODOLOGY/PRINCIPAL FINDINGS: The multi-exon assembly and the largest exon of the predicted ORF (PADG-3347), was cloned and expressed in Escherichia coli cells, and the features of the recombinant proteins were compared to those of the native paracoccin. The multi-exon protein was also used for protection assays in a mouse model of paracoccidioidomycosis. CONCLUSIONS/SIGNIFICANCE: Our results showed that the recombinant protein reproduced the biological properties described for the native protein-including binding to laminin in a manner that is dependent on carbohydrate recognition-showed N-acetylglucosaminidase activity, and stimulated murine peritoneal macrophages to produce high levels of TNF-α and nitric oxide. Considering the immunomodulatory potential of glycan-binding proteins, we also investigated whether prophylactic administration of recombinant paracoccin affected the course of experimental paracoccidioidomycosis in mice. In comparison to animals injected with vehicle (controls), mice treated with recombinant paracoccin displayed lower pulmonary fungal burdens and reduced pulmonary granulomas. These protective effects were associated with augmented pulmonary levels of IL-12 and IFN-γ. We also observed that injection of paracoccin three days before challenge was the most efficient administration protocol, as the induced Th1 immunity was balanced by high levels of pulmonary IL-10, which may prevent the tissue damage caused by exacerbated inflammation. The results indicated that paracoccin is the protein encoded by PADG-3347, and we propose that this gene and homologous proteins in other P. brasiliensis strains be called paracoccin. We also concluded that recombinant paracoccin confers resistance to murine P. brasiliensis infection by exerting immunomodulatory effects

Drug Resistance In Chromobacterium Violaceum.

Chromobacterium violaceum is a free-living bacterium commonly found in aquatic habitats of tropical and subtropical regions of the world. This bacterium is able to produce a large variety of products of biotechnological and pharmacological use. Although C. violaceum is considered to be non-pathogenic, some cases of severe infections in humans and other animals have been reported. Genomic data on the type strain ATCC 12472(T) has provided a comprehensive basis for detailed studies of pathogenicity, virulence and drug resistance genes. A large number of open reading frames associated with various mechanisms of drug resistance were found, comprising a remarkable feature of this organism. Amongst these, beta-lactam (penicillin and cephalosporin) and multidrug resistance genes (drug efflux pumps) were the most numerous. In addition, genes associated with bacitracin, bicyclomycin, chloramphenicol, kasugamycin, and methylenomycin were also found. It is postulated that these genes contribute to the ability of C. violaceum to compete with other bacteria in the environment, and also may help to explain the common drug resistance phenotypes observed in infections caused by this bacterium.3134-4

Oral immunization with attenuated Salmonella vaccine expressing Escherichia coli O157:H7 intimin gamma triggers both systemic and mucosal humoral immunity in mice

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Human infections with EHEC such as O157:H7 have been a great concern for worldwide food-industry surveillance. This pathogen is commonly associated with bloody diarrhea that can evolve to the life-threatening hemolytic uremic syndrome. Animals are the natural reservoir where this pathogen remains asymptomatically, in steps of ingestion and colonization of the bowel. The bacterium is shed in the feces, contaminating the surroundings, including water and food that are directed for human consumption. A major player in this colonization process is intimin, an outer membrane adhesion molecule encoded by the E. coli attachment and effacement (eae) gene that has been shown to be essential for intimate bacterial attachment to eukaryotic host cells. In an attempt to reduce the colonization of animal reservoirs with EHEC O157:H7, we designed a vaccine model to induce an immune response against intimin gamma. The model is based on its recombinant expression in attenuated Salmonella, used as a suitable vaccine vector because of its recognized ability to deliver recombinant antigens and to elicit all forms of immunity: mucosal, systemic, and humoral responses. To test this model, mice were orally immunized with a S. enterica serovar Typhimurium strain carrying the pYA3137eaeA vector, and challenged with E. coli O157:H7. Here we show that immunization induced the production of high levels of specific IgG and IgA antibodies and promoted reduction in the fecal shedding of EHEC after challenge. The live recombinant vaccine reported herein may contribute to the efforts of reducing animal intestinal mucosa colonization.568513522Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq

Nasal vaccination with attenuated Salmonella expressing VapA: TLR2 activation is not essential for protection against R. equi infection

AbstractVirulent strains of Rhodococcus equi have a large plasmid of 80–90kb, which encodes several virulence-associated proteins (Vap), including VapA, a lipoprotein highly associated with disease. We have previously demonstrated that oral immunisation with attenuated Salmonella enterica Typhimurium strain expressing the antigen VapA (STM VapA+) induces specific and long-term humoral and cellular immunity against R. equi. It was shown that VapA activates Toll-like receptor 2 (TLR2) on macrophages by establishing an interaction that ultimately favours immunity against R. equi infection. The purpose of this study was to evaluate the immune response triggered by nasal immunisation with STM VapA+ and to determine whether TLR2 supports the vaccine effect. We developed an optimised protocol for a single nasal immunisation that conferred protection against R. equi infection in mice, which was manifested by efficient R. equi clearance in challenged animals. Nasal vaccination with STM VapA+ has also induced protection in Tlr2−/− mice and mice with non-functional TLR4. Moreover, spleen cells of vaccinated mice augmented T-bet expression, as well as the production of IL-12, IFN-γ, nitric oxide and hydrogen peroxide. Notably, the population of CD4+ T cells with memory phenotype significantly increased in the spleens of vaccinated mice challenged 1 or 5 months after immunisation. In these animals, the spleen bacterial burden was also reduced. When similar experimental procedures were performed in TLR2 knockout mice, an increase in CD4+ T cells with memory phenotype was not observed. Consequently, we conclude that nasal vaccination with attenuated Salmonella expressing the R. equi virulence factor VapA confers long-lasting protection against experimental rhodoccocosis and that TLR2 engagement was not crucial to induce this protection but may be required for a long-term immune response

Anti-rPCN_full antibody reactivity on the yeast cell surface.

<p>Fluorescence labeling with anti-rPCN_full (<b>Panel A–D</b>) was evenly distributed over the yeast cell surface, with more intense labeling in some budding regions. Panel D is the merge of panels B and C.</p