Bacteriosomes in axenic plants: endophytes as stable endosymbionts

Observations of cells of axenic peach palm (Bactris gasipaes) microplants by light microscopy revealed movements of small particles within the cells. The phenomenon was characterized initially as Brownian movement, but electron microscopy revealed the presence of an intracellular bacterial community in these plants. Microscopy observations revealed the particular shapes of bacterial cells colonizing inner tissues of analyzed plants. Applying a molecular characterization by polymerase chain reaction and denaturing gradient gel electrophoresis, it was revealed the existence of bacterial rRNA within the plants. Sequencing of the rRNA identified three different phylogenetic groups; two bands had a high degree of similarity to sequences from Moraxella sp. and Brevibacillus sp., and a third sequence was similar to a non-cultivated cyanobacterium. The presence of those endosymbionts, called bacteriosomes, in axenic peach palm microplants raises the question of whether these stable endosymbionts were acquired in the process of evolution and how could they benefit the process of plants micropropagation.

Characterization of fungal soil communities by F-RISA and arbuscular mycorrhizal fungi from Araucaria angustifolia forest soils after replanting and wildfire disturbances

The Fungal Ribosomal Intergenic Spacer Analysis (F-RISA) was used to characterize soil fungal communities from three ecosystems of Araucaria angustifolia from Brazil: a native forest and two replanted forest ecosystems, one of them with a past history of wildfire. The arbuscular mycorrhizal fungi (AMF) infection was evaluated in Araucaria roots of 18-month-old axenic plants previously inoculated with soils collected from those areas in a greenhouse experiment. The principal component analysis of F-RISA profiles showed different soil fungal community between the three studied areas. Sixty three percent of F-RISA fragments amplified in the soil and the substrate samples presented lengths between 500 and 700 bp. The number of Operational Taxonomic Units (OTUs) was 34 for soil and 38 for substrate, however, more fragments were detected in soil (214) than in substrate (163). An in silico F-RISA analysis to compare our data with ITS1-5.8S-ITS2 sequences from NCBI database showed the presence of Ascomycota, Basidiomycota and Glomeromycota among the soil and substrate fungal communities. AMF infection was higher in plants inoculated with soil from the native forest and the replanted forest with wildfire, both presenting similar chemical characteristics but with different disturbance levels. These results indicate that soil chemical composition may influence the soil fungal community structures rather than the anthropogenic or fire disturbances.

Endophytic population of Pantoea agglomerans in citrus plants and development of a cloning vector for endophytes

Harmless bacteria inhabiting inner plant tissues are termed endophytes. Population fluctuations in the endophytic bacterium Pantoea agglomerans associated with two species of field cultured citrus plants were monitored over a two-year period. The results demonstrated that populations of P. agglomerans fluctuated in Citrus reticulata but not C. sinensis. A cryptic plasmid pPA3.0 (2.9 kb) was identified in 35 out of 44 endophytic isolates of P. agglomerans and was subsequently sequenced. The origins of replication were identified and nine out of 18 open reading frames (ORFs) revealed homology with described proteins. Notably, two ORFs were related to cellular transport systems and plasmid maintenance. Plasmid pPA3.0 was cloned and the gfp gene inserted to generate the pPAGFP vector. The vector was introduced into P. agglomerans isolates and revealed stability was dependent on the isolate genotype, ninety-percent stability values were reached after 60 hours of bacterial cultivation in most evaluated isolates. In order to definitively establish P. agglomerans as an endophyte, the non-transformed bacterium was reintroduced into in vitro cultivated seedlings and the density of inner tissue colonization in inoculated plants was estimated by bacterium re-isolation, while the tissue niches preferred by the bacterium were investigated by scanning electronic microscopy (SEM). Cells from P. agglomerans (strain ARB18) at similar densities were re-isolated from roots, stems and leaves and colonization of parenchyma and xylem tissues were observed. Data suggested that P. agglomerans is a ubiquitous citrus endophyte harboring cryptic plasmids. These characteristics suggest the potential to use the bacterium as a vehicle to introduce new genes in host plants via endophytic bacterial transformation.

Restoration of Pattern Recognition Receptor Costimulation to Treat Chromoblastomycosis, a Chronic Fungal Infection of the Skin

Chromoblastomycosis is a chronic skin infection caused by the fungus Fonsecaea pedrosoi. Exploring the reasons underlying the chronic nature of F. pedrosoi infection in a murine model of chromoblastomycosis, we find that chronicity develops due to a lack of pattern recognition receptor (PRR) costimulation. F. pedrosoi was recognized primarily by C-type lectin receptors (CLRs), but not by Toll-like receptors (TLRs), which resulted in the defective induction of proinflammatory cytokines. Inflammatory responses to F. pedrosoi could be reinstated by TLR costimulation, but also required the CLR Mincle and signaling via the Syk/CARD9 pathway. Importantly, exogenously administering TLR ligands helped clear F. pedrosoi infection in vivo. These results demonstrate how a failure in innate recognition can result in chronic infection, highlight the importance of coordinated PRR signaling, and provide proof of the principle that exogenously applied PRR agonists can be used therapeutically.

Leukotrienes Are Potent Adjuvant during Fungal Infection: Effects on Memory T Cells

Leukotrienes (LTs) are potent lipid mediators involved in the control of host defense. LTB(4) induces leukocyte accumulation, enhances phagocytosis and bacterial clearance, and increases NO synthesis. LTB(4) is also important in early effector T cell recruitment that is mediated by LTB(4) receptor 1, the high-affinity receptor for LTB(4). The aims of this study were to evaluate whether LTs are involved in the secondary immune response to vaccination in a murine model of Histoplasma capsulatum infection. Our results demonstrate that protection of wild-type mice immunized with cell-free Ags from H. capsulatum against histoplasmosis was associated with increased LTB(4) and IFN-gamma production as well as recruitment of memory T cells into the lungs. In contrast, cell-free Ag-immunized mice lacking 5-lipoxygenase(-/-), a critical enzyme involved in LT synthesis, displayed a marked decrease on recruitment of memory T cells to the lungs associated with increased synthesis of TGF-beta as well as IL-10. Strikingly, these effects were associated with increased mortality to 5-lipoxygenase(-/-)-infected mice. These data establish an important immunomodulatory role of LTs, in both the primary and secondary immune responses to histoplasmosis. The Journal of Immunology, 2008,181: 8544-8551.

Fungal tyrosine betaine, a novel secondary metabolite from conidia of entomopathogenic Metarhizium spp. fungi

Fungi, including the entomopathogenic deuteromycete Metarhizium anisopliae, produce a wide diversity of secondary metabolites that either can be secreted or stored in specific developmental structures, e.g., conidia. Some secondary metabolites, such as pigments, polyols and mycosporines, are associated with pathogenicity and/or fungal tolerance to several stress-inducing environmental factors, including temperature and solar radiation extremes. Extracts of M. anisopliae var. anisopliae (strain ESALQ-1037) conidia were purified by chromatographic procedures and the isolated compounds analyzed by (1)H and (13)C nuclear magnetic resonance spectroscopy and high-resolution mass spectrometry. LC-MS analyses were carried out to search for mycosporines (the initial targets), but no compounds of this class were detected. A molecule whose natural occurrence was previously undescribed was identified. It consists of betaine conjugated with tyrosine, and the structure was identified as 2-([1-carboxy-2-(4-hydroxyphenyl)ethyl]amino)-N,N,N-trimethyl-2-oxoethanammonium. mannitol was the predominant compound in the alcoholic conidial extract, but no amino acids other than tyrosine were found to be conjugated with betaine in conidia. The fungal tyrosine betaine was detected also in conidial extracts of three other M. anisopliae var. anisopliae (ARSEF 1095, 5626 and 5749) and three M. anisopliae var. acridum isolates (ARSEF 324, 3391 and 7486), but it was not detected in Aspergillus nidulans conidial extract (ATCC 10074). (C) 2010 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.

Effect of a highly concentrated lipopeptide extract of Bacillus subtilis on fungal and bacterial cells

Lipopeptides produced by Bacillus subtilis are known for their high antifungal activity. The aim of this paper is to show that at high concentration they can damage the surface ultra-structure of bacterial cells. A lipopeptide extract containing iturin and surfactin (5 mg mL-1) was prepared after isolation from B. subtilis (strain OG) by solid phase extraction. Analysis by atomic force microscope (AFM) showed that upon evaporation, lipopeptides form large aggregates (0.1-0.2 mu m2) on the substrates silicon and mica. When the same solution is incubated with fungi and bacteria and the system is allowed to evaporate, dramatic changes are observed on the cells. AFM micrographs show disintegration of the hyphae of Phomopsis phaseoli and the cell walls of Xanthomonas campestris and X. axonopodis. Collapses to fungal and bacterial cells may be a result of formation of pores triggered by micelles and lamellar structures, which are formed above the critical micelar concentration of lipopeptides. As observed for P. phaseoli, the process involves binding, solubilization, and formation of novel structures in which cell wall components are solubilized within lipopeptide vesicles. This is the first report presenting evidences that vesicles of uncharged and negatively charged lipopeptides can alter the morphology of gram-negative bacteria.

Bladder irrigation with amphotericin B and fungal urinary tract infection-systematic review with meta-analysis

Background: Candiduria is a hospital-associated infection and a daily problem in the intensive care unit. The treatment of asymptomatic candiduria is not well established and the use of amphotericin B bladder irrigation (ABBI) is controversial. The aim of this systematic review was to determine the best place for this therapy in practice. Methods: The databases searched in this study included MEDLINE, EMBASE, Web of Science, and LILACS (January 1960-June 2007). We included manuscripts with data on the treatment of candiduria using ABBI. The studies were classified as comparative, dose-finding, or non-comparative. Results: From 213 studies, nine articles (377 patients) met our inclusion criteria. ABBI showed a higher clearance of the candiduria 24 hours after the end of therapy than fluconazole (odds ratio (OR) 0.57, 95% confidence interval (CI) 0.32-1.00). Fungal culture 5 days after the end of both therapies showed a similar response (OR 1.51, 95% CI 0.81-2.80). The evaluation of ABBI using an intermittent or continuous system of delivery showed an early candiduria clearance (24 hours after therapy) of 80% and 82%, respectively (OR 0.87, 95% CI 0.52-1.36). Candiduria clearance at >5 days after the therapy showed a superior response using continuous bladder irrigation with amphotericin B (OR 0.52, 95% CI 0.29-0.94). The use of continuous ABBI for more than 5 days showed a better result (88% vs. 78%) than ABBI for less than 5 days, but without significance (OR 0.55, 95% CI 0.34-1.04). Conclusion: Although the strength of the results in the underlying literature is not sufficient to allow the drawing of definitive conclusions, ABBI appears to be as effective as fluconazole, but it does not offer systemic antifungal therapy and should only be used for asymptomatic candiduria. (C) 2008 International Society for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

Bacterial and fungal pneumonias after lung transplantation

Objective. The aim of this study was to evaluate the epidemiology of bacterial and fungal pneumonia in lung transplant (LT) recipients and to assess donor-to-host transmission of these microorganisms. Materials and Methods. We retrospectively studied all positive cultures from bronchoalveolar lavage (BAL) of 49 lung transplant recipients and their donors from August 2003 to April 2007. Results. There were 108 episodes of pneumonia during a medium follow-up of 412 days (range, 1-1328 days). The most frequent microorganisms were: Pseudomonas aeruginosa (n = 36; 33.3%), Staphylococcus aureus (n = 29; 26.8%), and Aspergillus spp. (n = 18; 16%). Other fungal infections were due to Fusarium spp., Cryptococcus neoformans, and Paracoccidioides brasiliensis. Of the 31 donors with positive BAL, 15 had S. aureus. There were 21 pretransplant colonized recipients (43%) and 16 of them had suppurative underlying lung disease. P. aeruginosa was the most frequent colonizing organism (59% of pretransplant positive cultures). There were 11 episodes of bacteremia and lungs were the source in 5 cases. Sixteen deaths occurred and 6 (37.5%) were due to infection. Statistical analyses showed association between pretransplant colonizing microorganisms from suppurative lung disease patients and pneumonias after lung transplantation (RR = 4.76; P = .04; 95% CI = 1.02-22.10). No other analyzed factor was significant. Conclusions. Bacterial and fungal infections are frequent and contribute to higher mortality in lung transplant recipients. P. aeruginosa is the most frequent agent of respiratory infections. This study did not observe any impact of donor lung organisms on pneumonia after lung transplantation. Nevertheless, we demonstrated an association between pretransplant colonizing microorganisms and early pneumonias in suppurative lung transplant recipients.

Epidemiologic aspects and clinical outcome of fungal keratitis in southeastern Brazil

PURPOSE. Fungal keratitis (FK) is a sight-threatening disease, more prevalent in developing regions. The present retrospective study was conducted in order to evaluate the epidemiologic and clinical aspects and the progression of FK in patients treated at two ophthalmologic reference centers in Southeast Brazil. METHODS. The charts of patients with infectious keratitis treated between 2000 and 2004 were reviewed. For the 66 cases of FK confirmed by microbiological analysis, data related to patient, disease, and therapeutic approaches were obtained. RESULTS. Mean patient age was 40.7 +/- 16 years. Fifty-three were men and 13 were women. Ocular trauma occurred in 40% of cases (27). Previous medications taken by the patients were quinolone in 72.5% and antimycotics in 30%. Visual acuity (VA) at presentation was >0.3 in 16% and <0.1 in 74.5%. Penetrant keratoplasty was performed in 38% and evisceration in 15%. The causing agents were Fusarium sp in 67%, Aspergillus sp in 10.5%, and Candida sp in 10%. Medication alone resolved 39% of cases within a mean period of 24.5 +/- 12 days. Final VA was >0.3 in 28%, and <0.1 in 63%. CONCLUSIONS. Fungal keratitis presented as a disease with severe complications, predominantly among young males, and was mostly caused by filamentous fungi. The present information permits the establishment of preventive strategies. Reducing the time between onset and treatment and using more accessible specific medication would reverse the negative prognosis. (Eur J Ophthalmol 2009; 19: 355-61)

Fungal infection by Paracoccidioides brasiliensis mimicking bone tumor

Paracoccidioides brasiliensis infection causes a systemic mycosis originally described in Latin America but with Current reports of worldwide distribution. The clinical presentation of paracoccidiodomycosis as an isolated long-bone lesion in children is quite unusual. This article describes a 10-year-old male with a lytic femoral bone lesion caused by P. brasiliensis infection that was first suspected of being of neoplasic etiology. The text also emphasizes the importance of including endemic fungal infections in the differential diagnosis of bone lesions.

CCR5-dependent regulatory T cell migration mediates fungal survival and severe immunosuppression

Paracoccidioidomycosis, a debilitating pulmonary mycosis, is caused by the dimorphic fungus Paracoccidioides brasiliensis. The infection results in the formation of granulomas containing viable yeast cells that are the fungal sources for disease reactivation. Because CD4(+)CD25(+) regulatory T cells (Tregs) are in the lesions of patients with paracoccidioidomycosis, the migration of Treg cells is dependent on the axis chemokine-chemokine receptors, and CCR5 ligands are produced in P. brasiliensis-induced lesions, we investigated the role of CCR5 in the control of the infection. The results showed that CCR5(-/-) mice are more efficient in controlling fungal growth and dissemination and exhibited smaller granulomas than wild-type (WT) mice. In the absence of CCR5, the percentage of CD4(+)CD25(+) T cells expressing Foxp3, glucocorticoid-induced TNFR (GITR), CD103, CD45(low), and CTLA-4 in the granulomas was significantly decreased. Interestingly, P. brasiliensis infection resulted in an absence of T cell proliferation in response to Con A in WT but not CCR5(-/-) mice that was abrogated by anti-CTLA-4 mAb and anti-GITR mAb. Moreover, the adoptive transfer of CD4(+)CD25(+) but not CD4(+)CD25(-) T cells from infected WT to infected CCR5(-/-) mice resulted in a significant increase in fungal load. Overall, CCR5 is a key receptor for the migration of Treg cells to the site of P. brasiliensis infections leading to down-modulation of effector immune response and the long-term presence of the fungus in the granulomas. Thus, a tight control of Treg cell migration to the granulomatous lesions could be an important mechanism for avoiding exacerbation and reactivation of the disease.

Indoor and outdoor atmospheric fungal spores in the So Paulo metropolitan area (Brazil): species and numeric concentrations

The aim of this study was to estimate the indoor and outdoor concentrations of fungal spores in the Metropolitan Area of Sao Paulo (MASP), collected at different sites in winter/spring and summer seasons. The techniques adopted included cultivation (samples collected with impactors) and microscopic enumeration (samples collected with impingers). The overall results showed total concentrations of fungal spores as high as 36,000 per cubic meter, with a large proportion of non culturable spores (around 91% of the total). Penicillium sp. and Aspergillus sp. were the dominant species both indoors and outdoors, in all seasons tested, occurring in more than 30% of homes at very high concentrations of culturable airborne fungi [colony forming units(CFU) m(-3)]. There was no significant difference between indoor and outdoor concentrations. The total fungal spore concentration found in winter was 19% higher than that in summer. Heat and humidity were the main factors affecting fungal growth; however, a non-linear response to these factors was found. Thus, temperatures below 16A degrees C and above 25A degrees C caused a reduction in the concentration (CFU m(-3)) of airborne fungi, which fits with MASP climatalogy. The same pattern was observed for humidity, although not as clearly as with temperature given the usual high relative humidity (above 70%) in the study area. These results are relevant for public health interventions that aim to reduce respiratory morbidity among susceptible populations.

Fungal community associated with marine macroalgae from Antarctica

Filamentous fungi and yeasts associated with the marine algae Adenocystis utricularis, Desmarestia anceps, and Palmaria decipiens from Antarctica were studied. A total of 75 fungal isolates, represented by 27 filamentous fungi and 48 yeasts, were isolated from the three algal species and identified by morphological, physiological, and sequence analyses of the internal transcribed spacer region and D1/D2 variable domains of the large-subunit rRNA gene. The filamentous fungi and yeasts obtained were identified as belonging to the genera Geomyces, Antarctomyces, Oidiodendron, Penicillium, Phaeosphaeria, Aureobasidium, Cryptococcus, Leucosporidium, Metschnikowia, and Rhodotorula. The prevalent species were the filamentous fungus Geomyces pannorum and the yeast Metschnikowia australis. Two fungal species isolated in our study, Antarctomyces psychrotrophicus and M. australis, are endemic to Antarctica. This work is the first study of fungi associated with Antarctic marine macroalgae, and contributes to the taxonomy and ecology of the marine fungi living in polar environments. These fungal species may have an important role in the ecosystem and in organic matter recycling.

Leukotrienes Target F-actin/Cofilin-1 to Enhance Alveolar Macrophage Anti-fungal Activity

Candida albicans is the most common opportunistic fungal pathogen and causes local and systemic disease in immunocompromised patients. Alveolar macrophages (AMs) are pivotal for the clearance of C. albicans from the lung. Activated AMs secrete 5-lipoxygenase-derived leukotrienes (LTs), which in turn enhance phagocytosis and microbicidal activity against a diverse array of pathogens. Our aim was to investigate the role of LTB(4) and LTD(4) in AM antimicrobial functions against C. albicans and the signaling pathways involved. Pharmacologic and genetic inhibition of LT biosynthesis as well as receptor antagonism reduced phagocytosis of C. albicans when compared with untreated or WT controls. Conversely, exogenous LTs of both classes augmented base-line C. albicans phagocytosis by AMs. Although LTB(4) enhanced mainly mannose receptor-dependent fungal ingestion, LTD(4) enhanced mainly dectin-1 receptor-mediated phagocytosis. LT enhancement of yeast ingestion was dependent on protein kinase C-delta (PKC delta) and PI3K but not PKC alpha and MAPK activation. Both LTs reduced activation of cofilin-1, whereas they enhanced total cellular F-actin; however, LTB(4) accomplished this through the activation of LIM kinases (LIMKs) 1 and 2, whereas LTD(4) did so exclusively via LIMK-2. Finally, both exogenous LTB(4) and LTD(4) enhanced AM fungicidal activity in an NADPH oxidase-dependent manner. Our data identify LTB(4) and LTD(4) as key mediators of innate immunity against C. albicans, which act by both distinct and conserved signaling mechanisms to enhance multiple antimicrobial functions of AMs.