Citrus Sudden Death Is Transmitted by Graft-Inoculation and Natural Transmission Is Prevented by Individual Insect-Proof Cages

Citrus sudden death (CSD) transmission was studied by graft-inoculation and under natural conditions. Young sweet orange trees on Rangpur rootstock were used as indicator plants. They were examined regularly for one or two characteristic markers of CSD: (i) presence of a yellow-stained layer of thickened bark on the Rangpur rootstock, and (ii) infection with the CSD-associated marafivirus. Based on these two markers, transmission of CSD was obtained, not only when budwood for graft-inoculation was taken from symptomatic, sweet orange trees on Rangpur, but also when the budwood sources were asymptomatic sweet orange trees on Cleopatra mandarin, indicating that the latter trees are symptomless carriers of the CSD agent. For natural transmission, 80 young indicator plants were planted within a citrus plot severely affected by CSD. Individual insect-proof cages were built around 40 indicator plants, and the other 40 indicator plants remained uncaged. Only two of the 40 caged indicator plants were affected by CSD, whereas 17 uncaged indicator plants showed CSD symptoms and were infected with the marafivirus. An additional 12 uncaged indicator plants became severely affected with citrus variegated chlorosis and were removed. These results strongly suggest that under natural conditions, CSD is transmitted by an aerial vector, such as an insect, and that the cages protected the trees against infection by the vector.

Mycovirus in Pseudocercospora griseola, the causal agent of angular leaf spot in common bean

Pseudocercospora griseola (Sacc.) Crous &. Braun is a widespread fungal phytopathogen that is responsible for angular leaf spot in the common bean (Phaseolus vulgaris L.). A number of fungal phytopathogens have been shown to harbour mycoviruses, and this possibility was investigated in populations of Pseudocercospora griseola. The total nucleic acid extracts of 61 fungal isolates were subjected to agarose gel electrophoresis. Small fragments (800-4800 bp) could be identified in 42 of the samples. The presence of dsRNA in isolate Ig838 was confirmed by treatment of total nucleic acid with DNase, RNase A, and nuclease S I. Transmission electron microscopy revealed the presence of viral-like particles 40 nm in diameter in the mycelia of 2 fungal isolates, namely 29-3 and Ig838. The transmission of dsRNA by means of conidia was 100% for isolate 29-3, but there was loss of 1-6 fragments of dsRNA in monosporic colonies of isolate Ig848. Cycloheximide treatment failed to inhibit the mycovirus in isolate 29-3, but proved efficient in the elimination of the 2.2, 2.0, 1.8, 1.2 and 1.0 kb fragments in 2 colonies of isolate Ig848. The occurrence of a mycovirus in Pseudocercospora griseola was demonstrated for the first time in the present study.

Functional characterization of the Aspergillus fumigatus PHO80 homologue

Phosphate is an ion that is essential for fungal growth. The systems for inorganic phosphate (Pi) acquisition in eukaryotic cells (PHO) have been characterized as a low-affinity (that assures a supply of Pi at normal or high external Pi concentrations) and a high-affinity (activated in response to Pi starvation). Here, as an initial step to understand the PHO pathway in Aspergillus fumigatus, we characterized the PH080 homologue, PhoB(PHO80). We show that the Delta phoB(PHO80) mutant has a polar growth defect (i.e., a delayed germ tube emergence) and, by phenotypic and phosphate uptake analyses, establish a link between PhoB(PHO80), calcineurin and calcium metabolism. Microarray hybridizations carried out with RNA obtained from wild-type and Delta phoB(PHO80) mutant cells identify Afu4g03610 (phoD(PHO84)), Afu7g06350 (phoE(PHO89)), Afu4g06020 (phoC(PHO81)), and Afu2g09040 (vacuolar transporter Vtc4) as more expressed both in the Delta phoB(PHO80) mutant background and under phosphate-limiting conditions of 0.1 mM P-i. Epifluorescence microscopy revealed accumulation of poly-phosphate in Delta phoB(PHO80) vacuoles, which was independent of extracellular phosphate concentration. Surprisingly, a phoD(PHO84) deletion mutant is indistinguishable phenotypically from the corresponding wild-type strain. mRNA analyses suggest that protein kinase A absence supports the expression of PHO genes in A. fumigatus. Furthermore, Delta phoB(PHO80) and Delta phoD(PHO84) mutant are fully virulent in a murine low dose model for invasive aspergillosis. (C) 2008 Elsevier Inc. All rights reserved.

The roles played by Aspergillus nidulans apoptosis-inducing factor (AIF)-like mitochondrial oxidoreductase (AifA) and NADH-ubiquinone oxidoreductases (NdeA-B and NdiA) in farnesol resistance

Farnesol (FOH) is a nonsterol isoprenold produced by dephosphorylanon of farnesyl pyrophosphate a catabolite of the cholesterol biosynthetic pathway These isoprenoids inhibit proliferation and induce apoptosis Here we show that Aspergillus nidulans MA encoding the apoptosis-Inducing factor (AIF)-like mitochondrial oxidoreductase plays a role in the function of the mitochondrial Complex I Additionally we demonstrated that ndeA B and ndiA encode external and internal alternative NADH dehydrogenases respectively that have a function in FOH resistance When exposed to FOH the Delta aifA and Delta ndeA strains have increased ROS production while Delta ndeB Delta ndeA Delta ndeB and Andul mutant strains showed the same ROS accumulation than in the absence of FOH We observed several compensatory mechanisms affecting the differential survival of these mutants to FOH (C) 2010 Elsevier Inc All rights reserved

The Paracoccidioides brasiliensis gp70 antigen is encoded by a putative member of the flavoproteins monooxygenase family

Glycoprotein gp70 is an important intracellular antigen from Paracoccidioides brasillensis that elicits both humoral and cellular immune responses. Herein, the PbGP70 gene cloning from isolate Pb18 using internal peptide sequence information is reported. The deduced protein sequence bears two N-glycosylation sites, antigenic sites and two mouse T-cell epitopes. Anti-recombinant gp70 (rPbgp70) polyclonal antibodies reacted with a 70-kDa component in total cell extract of A brasiliensis, while MAbC5F11 and paracoccidioiclomycosis patients` sera recognized rPbgp70. Confocal microscopy with anti-rPbgp70 and MAbC5F11 showed intense staining and cytoplasmatic co-localization. The protein sequence belongs to the flavoprotein monooxygenase family which groups important anti-oxidative bioactive compounds. We found increased PbGP70 transcript accumulation under oxidative stress induced by H(2)O(2), during fungal growth and in macrophage phagocyted/bound yeasts. Therefore, gp70 might play a dual role in P. brasiliensis by both eliciting immune cellular and humoral responses in the host and protecting the fungus from oxidative stress generated by phagocytic cells. (c) 2009 Elsevier Inc. All rights reserved.

Cdc42p controls yeast-cell shape and virulence of Paracoccidioides brasiliensis

Paracoccidioides brasiliensis is characterized by a multiple budding phenotype and a polymorphic cell growth, leading to the formation of cells with extreme variations in shape and size. Since Cdc42 is a pivotal molecule in establishing and maintaining polarized growth for diverse cell types, as well as during pathogenesis of certain fungi, we evaluated its role during cell growth and virulence of the yeast-form of P. brasiliensis. We used antisense technology to knock-down PbCDC42`s expression in P. brasiliensis yeast cells, promoting a decrease in cell size and more homogenous cell growth, altering the typical polymorphism of wild-type cells. Reduced expression levels also lead to increased phagocytosis and decreased virulence in a mouse model of infection. We provide genetic evidences underlying Pbcdc42p as an important protein during host-pathogen interaction and the relevance of the polymorphic nature and cell size in the pathogenesis of P. brasiliensis. (C) 2009 Elsevier Inc. All rights reserved.

Phenotypic analysis of genes whose mRNA accumulation is dependent on calcineurin in Aspergillus fumigatus

Calcineurin plays an important role in the control of cell morphology and virulence in fungi. Calcineurin is a serine/threonine-specific protein phosphatase heterodimer consisting of a catalytic subunit A and a regulatory subunit B. A mutant of Aspergillus fumigatus lacking the calcineurin A (calA) catalytic subunit exhibited defective hyphal morphology related to apical extension and branching growth, which resulted in drastically decreased filamentation. Here, we investigated which pathways are influenced by A. fumigatus calcineurin during proliferation by comparatively determining the transcriptional profile of A. fumigatus wild type and Delta calA mutant strains. Our results showed that the mitochondrial copy number is reduced in the Delta calA mutant strain, and the mutant has increased alternative oxidase (aoxA) mRNA accumulation and activity. Furthermore, we identified four genes that encode transcription factors that have increased mRNA expression in the Delta calA mutant. Deletion mutants for these transcription factors had reduced susceptibility to itraconazole, caspofungin, and sodium dodecyl sulfate (SDS). (C) 2009 Elsevier Inc. All rights reserved.

Functional characterization of the Aspergillus nidulans methionine sulfoxide reductases (msrA and msrB)

Proteins are subject to modification by reactive oxygen species (ROS), and oxidation of specific amino acid residues can impair their biological function, leading to an alteration in cellular homeostasis. Sulfur-containing amino acids as methionine are the most vulnerable to oxidation by ROS, resulting in the formation of methionine sulfoxide [Met(O)] residues. This modification can be repaired by methionine sulfoxide reductases (Msr). Two distinct classes of these enzymes, MsrA and MsrB, which selectively reduce the two methionine sulfoxide epimers, methionine-S-sulfoxide and methionine-R-sulfoxide, respectively, are found in virtually all organisms. Here. we describe the homologs of methionine sulfoxide reductases, msrA and msrB, in the filamentous fungus Aspergillus nidulans. Both single and double inactivation mutants were viable, but more sensitive to oxidative stress agents as hydrogen peroxide, paraquat, and ultraviolet light. These strains also accumulated more carbonylated proteins when exposed to hydrogen peroxide indicating that MsrA and MsrB are active players in the protection of the cellular proteins from oxidative stress damage. (C) 2009 Elsevier Inc. All rights reserved.

The 2008 update of the Aspergillus nidulans genome annotation: A community effort

The identification and annotation of protein-coding genes is one of the primary goals of whole-genome sequencing projects, and the accuracy of predicting the primary protein products of gene expression is vital to the interpretation of the available data and the design of downstream functional applications. Nevertheless, the comprehensive annotation of eukaryotic genomes remains a considerable challenge. Many genomes submitted to public databases, including those of major model organisms, contain significant numbers of wrong and incomplete gene predictions. We present a community-based reannotation of the Aspergillus nidulans genome with the primary goal of increasing the number and quality of protein functional assignments through the careful review of experts in the field of fungal biology. (C) 2009 Elsevier Inc. All rights reserved.

Transcription regulation of the PbGP43 gene by nitrogen in the human pathogen Paracoccidioides brasiliensis

We show indirect evidences for the possible involvement of NIT-2-like binding motifs in transcription modulation of the PbGP43 gene, which codes for an important antigen from the human fungal pathogen Paracoccidioides brasiliensis. This investigation was motivated by the finding of 23 NIT2-like sites within the proximal -2047 nucleotides of the PbGP43 5` intergenic region from the Pb339 isolate. They compose four clusters, two of them identical. We found four NIT2-containing probes that were positive in electrophoretic mobility shift assays and further analyzed them. PbGP43 could be modulated by nitrogen primary sources in Pb339, Pb3 and Pb18 isolates, as observed by reverse transcription (RT) real time-PCR. Gene reporter assays conducted in Aspergillus nidulans suggested that the minimal fragment responsible for nitrogen modulation lies within -480 bp of the PbGP43 gene. This is the first report on PbGP43 transcription modulation in response to nitrogen primary sources, which might help understand its regulation during infection. (C) 2008 Elsevier Inc. All rights reserved.

Heterozygosity for the S180L Variant of MAL/TIRAP, a Gene Expressing an Adaptor Protein in the Toll-Like Receptor Pathway, Is Associated with Lower Risk of Developing Chronic Chagas Cardiomyopathy

Background. Chagas disease is caused by the protozoan parasite Trypanosoma cruzi. Among T. cruzi-infected individuals, only a subgroup develops severe chronic Chagas cardiomyopathy (CCC); the majority remain asymptomatic. T. cruzi displays numerous ligands for the Toll-like receptors (TLRs), which are an important component of innate immunity that lead to the transcription of proinflammatory cytokines by nuclear factor-kappa B. Because proinflammatory cytokines play an important role in CCC, we hypothesized that single-nucleotide polymorphisms (SNPs) in the genes that encode proteins in the TLR pathway could explain differential susceptibility to CCC among T. cruzi-infected individuals. Methods. For 169 patients with CCC and 76 T. cruzi-infected, asymptomatic individuals, we analyzed SNPs by use of polymerase chain reaction-restriction fragment length polymorphism analysis for the genes TLR1, TLR2, TLR4, TLR5, TLR9, and MAL/TIRAP, which encodes an adaptor protein. Results. Heterozygous carriers of the MAL/TIRAP variant S180L were more prevalent in the asymptomatic group (24 [32%] of 76 subjects) than in the CCC group (21 [12%] of 169) (chi(2) = 12.6; P = .0004 [adjusted P (P(c)) = .0084]; odds ratio [OR], 0.31 [95% confidence interval {CI}, 0.16-0.60]). Subgroup analysis showed a stronger association when asymptomatic patients were compared with patients who had severe CCC (i.e., patients with left-ventricular ejection fraction <= 40%) (chi(2) = 11.3; P = .0008 [P(c) = .017]; OR, 0.22 [95% CI, 0.09-0.56]) than when asymptomatic patients were compared with patients who had mild CCC (i.e., patients with left-ventricular ejection fraction >40%) (chi(2) = 7.7; P = .005 [P(c) = .11]; OR, 0.33 [95% CI, 0.15-0.73]). Conclusion. T. cruzi-infected individuals who are heterozygous for the MAL/TIRAP S180L variant that leads to a decrease in signal transduction upon ligation of TLR2 or TLR4 to their respective ligand may have a lower risk of developing CCC.

Health in Brazil 4 Chronic non-communicable diseases in Brazil: burden and current challenges

Non-communicable diseases (NCDs) have become a major health priority in Brazil-72% of all deaths were attributable to NCDs in 2007. They are also the main source of disease burden, with neuropsychiatric disorders being the single largest contributor. Morbidity and mortality due to NCDs are greatest in the poor population. Although the crude NCD mortality increased 5% between 1996 and 2007, age-standardised mortality declined by 20%. Declines were primarily for cardiovascular and chronic respiratory diseases, in association with the successful implementation of health policies that lead to decreases in smoking and the expansion of access to primary health care. Of note, however, the prevalence of diabetes and hypertension is rising in parallel with that of excess weight; these increases are associated with unfavourable changes of diet and physical activity. Brazil has implemented major policies for the prevention of NCDs, and its age-adjusted NCD mortality is falling by 1.8% per year. However, the unfavourable trends for most major risk factors pose an enormous challenge and call for additional and timely action and policies, especially those of a legislative and regulatory nature and those providing cost-effective chronic care for individuals affected by NCDs.

Extracellular matrix components and regulators in the airway smooth muscle in asthma

There is an intimate relationship between the extracellular matrix (ECM) and smooth muscle cells within the airways. Few studies have comprehensively assessed the composition of different ECM components and its regulators within the airway smooth muscle (ASM) in asthma. With the aid of image analysis, the fractional areas of total collagen and elastic fibres were quantified within the ASM of 35 subjects with fatal asthma (FA) and compared with 10 nonfatal asthma (NFA) patients and 22 nonasthmatic control cases. Expression of collagen I and III, fibronectin, versican, matrix metalloproteinase (MMP)-1, -2, -9 and -12 and tissue inhibitor of metalloproteinase-1 and -2 was quantified within the ASM in 22 FA and 10 control cases. In the large airways of FA cases, the fractional area of elastic fibres within the ASM was increased compared with NFA and controls. Similarly, fibronectin, MMP-9 and MMP-12 were increased within the ASM in large airways of FA cases compared with controls. Elastic fibres were increased in small airways in FA only in comparison with NFA cases. There is altered extracellular matrix composition and a degradative environment within the airway smooth muscle in fatal asthma patients, which may have important consequences for the mechanical and synthetic functions of airway smooth muscle.

Airway smooth muscle thickness in asthma is related to severity but not duration of asthma

Asthma is characterised by an increased airway smooth muscle (ASM) area (ASMarea) within the airway wall. The present study examined the relationship of factors including severity and duration of asthma to ASMarea. The perimeter of the basement membrane (PBM) and ASMarea were measured on transverse sections of large and small airways from post mortem cases of fatal (n=107) and nonfatal asthma (n=37) and from control subjects (n=69). The thickness of ASM (ASMarea/PBM) was compared between asthma groups using multivariate linear regression. When all airways were considered together, ASMarea/PBM (in millimetres) was increased in nonfatal (median 0.04; interquartile range 0.013-0.051; p=0.034) and fatal cases of asthma (0.048; 0.025-0.078; p<0.001) compared with controls (0.036; 0.024-0.042). Compared with cases of nonfatal asthma, ASMarea/PBM was greater in cases of fatal asthma in large (p<0.001) and medium (p<0.001), but not small, airways. ASMarea/PBM was not related to duration of asthma, age of onset of asthma, sex or smoking. No effect due to study centre, other than that due to sampling strategy, was found. The thickness of the ASM layer is increased in asthma and is related to the severity of asthma but not its duration.

Giant multilocular thymic cyst in an HIV-infected adolescent

A girl with vertically acquired HIV infection presented with a 6-month history of dyspnea and chest pain. Computed tomography of the thorax showed a heterogenous mass measuring 13 x 9 x 17 cm located in the anterior mediastinum. Complete surgical resection was accomplished with no complications. The final diagnosis was multilocular thymic cyst, a distinct pathologic entity that is morphologically distinguishable and unrelated to congenital thymic cyst. (C) 2011 Elsevier Inc. All rights reserved.