Molecular phylogeny of animal pathogen Lacazia loboi inferred from rDNA and DNA coding sequences

Lacazia loboi is a geographically restricted, uncultivated fungal pathogen of humans and dolphins. Previous investigations using 18S small unit rDNA, chitin synthase 2 and gp43 DNA sequences positioned L. loboi as a close relative of Paracoccidioides brasiliensis. However, given the few individuals of L. loboi studied and the high degree of genetic variation observed in P. brasiliensis, the existence of L. loboi as an independent species has been questioned. To investigate the phylogenetic position of this species, we conducted a phylogenetic analysis using 20 L. loboi collections (L. loboi was obtained from proven cases of lacaziosis and 14 collections were maintained in mice, the others were analyzed from DNA taken directly from infected human tissue.). L. loboi DNA sequence was compared to that from 17 P. brasiliensis strains that represented the known variation in this species, and outgroup taxa in the Onygenales (Ajellomyces and Coccidioides species). Our analyses used DNA sequence from ITS rRNA, and partial coding sequences of chitin synthase 4, ADP-ribosylation factor, and gp43. Nucleotide variation among strains of L. loboi was minor but numerous nucleotide mismatches and multiple gaps were found for these gene regions among members in the Ajellomycetaceae, including P. brasiliensis. Phylogenies inferred using neighbor-joining, maximum parsimony and Bayesian analyses showed no significant conflict and depicted L. loboi as a well-supported, monophyletic group that was sister to the Paracoccidioides clade. These results argue for maintaining L. loboi as a taxon independent from Paracoccidioides within the Ajellomycetaceae.     

Antigenic relationships between Aracoccidioides loboi and other pathogenic fungi determined by immunofluorescence

Summary The fluorescent antibody technique was used to study antigenic relationships betweenParacoccidioides loboi and other pathogenic fungi. The findings suggest thatP. loboi is more closely related antigenically to certainP. brasiliensis strains than to others and that it has antigens in common with the yeast form ofHistoplasma capsulatum, H. duboisii, Blastomyces dermatitidis, Candida albicans and also the mycelial form ofCoccidioides immitis. Serum globulins from 3 cases of keloidal blastomycosis were labelled with fluorescein isothiocyanate. These conjugates showed slight or no reactivity withP. loboi, the yeast forms ofP. brasiliensis, H. capsulatum, H. duboisii andB. dermatitidis, However, they stained brightlyC. albicans, serotypes A and B, the tissue form ofC. immitis and the yeast form ofSporotrichum schenckii. Adsorption of these reagents withC. albicans eliminated all staining except that forS. schenckii. These patients had no history of clinical sporotrichosis.Deceased. Last address: Fundacão Gonçalo Moniz, Salvador, Bahia, Brazil. Requests for reprints should be sent to Dr.William Kaplan.Dr.Miranda is in private practice in Rio de Janeiro, Brazil.     

Studies on species of Conidiobolus from India-V

Summary Morphological and cultural studies on two new species ofConidiobolus isolated from plant detritus in India are described.C. terrestris is characterized by mycelium differentiating into chains of hyphal bodies, which in older cultures become thick-walled. The morphological similarity of the fungus to the causal organism of Jorge Lobo's disease in Surinam incited byLoboa loboi and the possibility of that being a species ofConidiobolus are discussed.C. lichenicola isolated from decaying lichens is characterized by a vegetative thallus composed of sinuous, lobate and compacted coralloid mycelial mass. The conidial germination is characteristic and organises the coralloid thallus from the very initial stages.     

A secreted serine protease of <Emphasis Type="Italic">Paracoccidioides brasiliensis</Emphasis> and its interactions with fungal proteins

Background  

Paracoccidioides brasiliensis is a thermodimorphic fungus, the causative agent of paracoccidioidomycosis (PCM). Serine proteases are widely distributed and this class of peptidase has been related to pathogenesis and nitrogen starvation in pathogenic fungi.     

Paracoccidioides brasiliensis pancreatic destruction in Calomys callosus experimentally infected

Background  

The wild rodent Calomys callosus is notably resistant to Trypanosoma cruzi infection. In order to better characterize this animal model for experimental infections, we inoculated C. callosus intraperitoneally with Paracoccidioides brasiliensis, a thermally dimorphic fungus that causes a chronic disease with severe granuloma formation in the mouse and humans. The dissemination of P. brasiliensis cells through the lungs, liver, pancreas, and spleen was assessed by histological analysis.     

The malate synthase of Paracoccidioides brasiliensis is a linked surface protein that behaves as an anchorless adhesin

Background  

The pathogenic fungus Paracoccidioides brasiliensis is the agent of paracoccidioidomycosis (PCM). This is a pulmonary mycosis acquired by inhalation of fungal airborne propagules that can disseminate to several organs and tissues leading to a severe form of the disease. Adhesion and invasion to host cells are essential steps involved in the internalization and dissemination of pathogens. Inside the host, P. brasiliensis may use the glyoxylate cycle for intracellular survival.     

Polymorphism in the flanking regions of the PbGP43 gene from the human pathogen Paracoccidioides brasiliensis: search for protein binding sequences and poly(A) cleavage sites

Background  

Paracoccidioides brasiliensis is a thermo-dimorphic fungus that causes paracoccidiodomycosis (PCM). Glycoprotein gp43 is the fungal main diagnostic antigen, which can also protect against murine PCM and interact with extracellular matrix proteins. It is structurally related to glucanases, however not active, and whose expression varies considerably. We have presently studied polymorphisms in the PbGP43 flanking regions to help understand such variations.     

Paracoccidioidomycosis in nude mice: Presence of filamentous forms of the fungus

Congenitally athymic nude mice (nu/nu) and their phenotypically normal littermates (nu/+) were intraperitoneally infected with yeast cells of a strain of Paracoccidioides brasiliensis. The nude mice developed a severe and generalized infection with an intense parasitism of several organs, accompanied by a low-grade of tissue reaction. The lesions were characterized by abundant yeast-like cells of the fungus, and in some animals, numerous hyphal forms could be well visualized. In control animals, infection was moderate, almost exclusively restricted to the area of inoculation, and the lesions presented few parasites surrounded by an inflammatory response. Filamentous forms of the fungus were never encountered in these animals.     

Phakopsora schreberae, sp. nov. (Uredinales) occurs onSchrebera swietenioides in India

A rust fungus was described fromSchrebera swietenioides (Oleaceae) collected in India. The fungus possessed densely paraphysate uredinia and subepidermal, crustose telia with irregulary 5–7-layered sessile teliospores. These morphologies are characteristic ofPhakopsora, although noPhakopsora species had been reported from the family Oleaceae. The fungus was concluded to be a new species and a name,Phakopsora schreberae, was proposed for the new species.     

Alkaline phosphatase at the cell wall of the yeast phase ofParacoccidioides brasiliensis

The activity of alkaline phosphatase demonstrated by histochemical techniques was shown at the cell wall of the yeast form ofParacoccidioides brasiliensis at 3, 6, and 9 days of culture. The results showed a very active deposition at the cell wall as early as 9 days of culture of the fungus which made us think an inactive salt precipitate was also present.     

The role of fractions from Paracoccidioides brasiliensis in the genesis of inflammatory response

The influx of inflammatory cells towards the peritoneal cavity in rats inoculated intraperitoneally with subcellular preparations of the fungus Paracoccidioides brasiliensis was studied. In addition to the dead fungus, also fractions F1 of the cell wall, which mainly consisted of polysaccharides and the lipid extract, induced intense cell migration 4 hr after inoculation, with a greatly increased number of polymorphonuclear leucocytes (PMN). Study of the kinetics of cell influx showed that both fraction F1 and the lipid extract initially induced intense PMN migration between the 4th and 24th hr after inoculation of these agents, followed by migration of mononuclear cells (MN) around the 48th hr. We also observed that migration of these cells increased gradually after inoculation of growing doses of fraction F1. The present data suggest that polysaccharides and lipids isolated from P. brasiliensis may participate in the initial phase of the inflammatory response in paracoccidioidomycosis.     

Ornithine decarboxylase in Paracoccidioides brasiliensis

Ornithine decarboxylase in Paracoccidioides brasiliensis, a dimorphic human pathogenic fungus, was more active at 37° C in the yeast phase and at 30° C in the mycelial phase. In contrast to other fungal systems, yeast growth and mycelium-to-yeast transition in P. brasiliensis were accompanied by a high activity of ornithine decarboxylase at the onset of the budding process, the activity of which was inhibited by 1,4-diamino-2-butanone. The activity of ornithine decarboxylase remained at a basal level during vegetative growth of both the mycelial phase and the late stage of yeast phase, and also through the yeast-to-mycelium transition. Received: 18 December 1995 / Accepted: 8 March 1996     

Mitochondrial function in the yeast form of the pathogenic fungus <Emphasis Type="Italic">Paracoccidioides brasiliensis</Emphasis>

Differences between the respiratory chain of the fungus Paracoccidioides brasiliensis and its mammalian host are reported. Respiration, membrane potential, and oxidative phosphorylation in mitochondria from P. brasiliensis spheroplasts were evaluated in situ, and the presence of a complete (Complex I–V) functional respiratory chain was demonstrated. In succinate-energized mitochondria, ADP induced a transition from resting to phosphorylating respiration. The presence of an alternative NADH–ubiquinone oxidoreductase was indicated by: (i) the ability to oxidize exogenous NADH and (ii) the lack of sensitivity to rotenone and presence of sensitivity to flavone. Malate/NAD⁺-supported respiration suggested the presence of either a mitochondrial pyridine transporter or a glyoxylate pathway contributing to NADH and/or succinate production. Partial sensitivity of NADH/succinate-supported respiration to antimycin A and cyanide, as well as sensitivity to benzohydroxamic acids, suggested the presence of an alternative oxidase in the yeast form of the fungus. An increase in activity and gene expression of the alternative NADH dehydrogenase throughout the yeast’s exponential growth phase was observed. This increase was coupled with a decrease in Complex I activity and gene expression of its subunit 6. These results support the existence of alternative respiratory chain pathways in addition to Complex I, as well as the utilization of NADH-linked substrates by P. brasiliensis. These specific components of the respiratory chain could be useful for further research and development of pharmacological agents against the fungus.     

Experimental reproduction of the Jorge Lobo's disease in BALB/c mice inoculated with Lacazia loboi obtained from a previously infected mouse

Long-term maintenance of Lacazia loboi in the laboratory has not been reported. We report here the use BALB/c mice to maintain the Lacazia loboi for extended period of time. Eight to ten week-old mice were inoculated intradermally in both hind footpads with a fungal suspension from a macerated footpad obtained from an original mouse previously infected with the fungi and sacrificed 8 months after inoculation. The inoculated animals were sacrificed at different time intervals, footpads were excised, the right one was submitted to histopathological examination and the left one was macerated in sterile saline for fungal count and viability index determination. The inoculated animals presented the histopathological picture identical to the mice previously inoculated with material from human lesion. Granulomatous infiltrates with predominance of macrophages and giant cells were observed. The granulomas evolved progressively as observed in the different times of sacrifice. After 7 months of inoculation, macroscopic lesions were observed, and the number of fungi obtained from macerated footpads was higher than the number of inoculated fungi. The pattern of lesion development was similar to what was observed in animals infected with a fungal suspension obtained from a human lesion. Considering the histopathological findings, the clinical manifestations, and the finding of a higher number of fungi obtained than the inoculated into footpads of each mice, we believe the BALB/c mice strain is as an excellent way to maintain L. loboi in laboratory. Moreover, even after serial passages of the fungi, the granulomatous lesions are reproduced consistently in laboratory conditions.This revised version was published online in October 2005 with corrections to the Cover Date.     

Polyamines in growth and dimorphism ofParacoccidioides brasiliensis

Putrescine and spermidine were the only polyamines found inParacoccidioides brasiliensis, a dimorphic fungus pathogenic for humans. Free polyamines (putrescine>spermidine) increased during the first 24 h of yeast growth, with a second peak at 42 h, and also during the first 12 h of mycelium-to-yeast transition (spermidine>putrescine). Conjugated and bound polyamines were also quantified. 1,4-Diamino-2-butanone decreased free putrescine and spermidine accumulation by inhibiting the activity of ornithine decarboxylase. The increase in free polyamines corresponds to bud emergence in yeast growth and to the mycelium-to-yeast transition ofP. brasiliensis.Abbreviations DAB 1,4-Diamino-2-butanone - Y Yeasts - M Mycelia - ODC Ornithine decarboxylase     

The stress responsive and morphologically regulated <Emphasis Type="Italic">hsp90</Emphasis> gene from <Emphasis Type="Italic">Paracoccidioides brasiliensis</Emphasis> is essential to cell viability

Background  

Paracoccidioides brasiliensis is a dimorphic fungus that causes the most prevalent systemic mycosis in Latin America. The response to heat shock is involved in pathogenesis, as this pathogen switches from mycelium to yeast forms in a temperature dependent fashion that is essential to establish infection. HSP90 is a molecular chaperone that helps in the folding and stabilization of selected polypeptides. HSP90 family members have been shown to present important roles in fungi, especially in the pathogenic species, as an immunodominant antigen and also as a potential antifungal therapeutic target.     

Intento de Aislamiento del Paracoccidioides brasiliensis del suelo

Resumen Cuarenta y cinco muestras de tierra, preferentemente de cafetales a más de 500 m de altitud sobre el nivel del mar, de varias regiones de Venezuela fueron inoculadas por vía endovenosa en ratones.No pudo aislarse elParacoccidioides brasiliensis.Se discuten brevemente las posibles causas de los resultados negativos y se proponen otras téchnicas para seguir intentando la determinación del habitat deParacoccidioides brasiliensis.

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Summary Forty five soil samples, most of them from coffee plantations at more than 500 m altitude, from several parts from Venezuela were inoculated intravenously into mice. Paracoccidioides brasilicnsis was not isolated.The authors discuss the possible reasons of the negative results and propose other techniques to apply in the future in order to isolateParacoccidioides brasilicnsis from nature.

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Résumé Quarante cinq échantillons de terre furent inoculés par voie intravéneuse — des souris. Les échantillons provenaient principalement de plantations de café — plus de 500 m d'altitude de plusieurs régions du Venezuela.LeParacoccidioides brasiliensis n'a pu etre isolé.Les auteurs discutent les causes éventuelles de cet échec et proposent des méthodes différentes pour reprendre la recherche duParacoccidioides brasiliensis dans la nature.

     

Paracoccidioides brasiliensis: chemical and molecular tools for research on cell walls, antifungals, diagnosis, taxonomy

Paracoccidioides brasiliensis is a dimorphic fungus, a causative agent of paracoccidioidomycosis, one of the most frequent systemic mycoses that affect the rural population in Latin America, only geographical region in which this fungus is to be found. In this work, we discuss matters related to (a) cell wall studies based on the cloning and analysis of genes involved in the synthesis of cell wall components, and their possible roles in virulence and dimorphism in P. brasiliensis, (b) molecular taxonomy and the molecular classification of P. brasiliensis as an Ascomycete belonging in the Order Onygenales, (c) phylogeny of P. brasiliensis and the possible existence of cryptic species within the genus Paracoccidioides, and (d) new experimental antifungal drugs such as azasterols or sterol hydrazones, compounds that affect the activity of Δ²⁴⁽²⁸⁾ sterol methyl reductase (SMR) and/or Δ⁽²⁴⁾-sterol methyl transferase (SMT), and (e) specific primers for the molecular detection of P. brasiliensis in vitro and in clinical samples.     

Growth of dimorphic human pathogenicfungi on media containing cycloheximide and chloramphenicol

Summary Studies of 24 strains ofBlastomyces dermatitidis confirmed previously published results that the yeast-phase of this fungus is more sensitive than the mycelial-phase to cycloheximide and chloramphenicol.Studies of 5 strains each ofHistoplasma capsulatum, Paracoccidioides brasiliensis andSporotrichum schenckii show that that these species also have a similar yeast-phase mycelial -phase sensitivity differential in regard to these antibiotics.A cycloheximide resistant strain ofB. dermatitidis was developed from a sensitive strain.The experimental results support the general practice of using 0.5 mg/ml cycloheximide and 0.05 mg/ml chloramphenicol in media for the isolation of the four fungi at 25° C. The results indicate, however, that some strains would not be recovered at 37° C with similar concentrations of these antibiotics.It is recommended that a concentration of not more than 0.2 mg/ml chloramphenicol should be used to preserve sputum which is subsequently to be cultured forB. dermatitidis, Histoplasma capsulatum, Paracoccidioides brasiliensis orS. schenckii.