Morphological, Biochemical and Molecular Approaches for Comparing Typical and Atypical Paracoccidioides brasiliensis Strains

Abstract

We evaluated the morphology of typical and atypical Paracoccidioides brasiliensis strains and the expression of its 43 kDa glycoprotein (GP43). Strains of P. brasiliensis preserved under mineral oil for long periods of time presented different morphological patterns on peptone, yeast-extract and glucose (PYG) agar. The intravenous inoculation in BALB/c mice confirmed that a strain bearing morphological alterations was non-virulent. In contrast, another strain also maintained under mineral oil but which did not exhibit such morphological dysfunction was as virulent as the well characterized Pb 339 and Pb 18 strains. The expression of the main antigen expressed by P. brasiliensis, GP43, was assessed in culture filtrates by western immunoblots. Typical and atypical strains were capable of secreting the glycoprotein, except strain Pb IOC 1059. The identity of the atypical strains was confirmed by PCR using specific primers for gp43, though the single PCR-fragment varied in size for the atypical strains. The PCR fragments from an atypical strain, Pb IOC 1210, and the typical Pb 339 and Pb IOC 3698 strains were sequenced and blasted to the gp43 gene from the Pb 18 strain (GenBank AY005429). These results ensured the identity of the atypical strains as P. brasiliensis, and suggested a relationship between the alteration of morphological differentiation and the virulence factor following storage under mineral oil.

Genetic characterization of morphologically variant strains of Paracoccidioides brasiliensis

“…They were also found to be nonvirulent, as seen from studies using rat and mice models (Mendes da Silva et al 1996, Borba & Schäffer 2002. By detecting the presence of either the GP43 protein or the gp43 gene, we confirmed the identity of the morphologically variant P. brasiliensis, that remained in the transitional phase (Borba et al 2005).…”

“…The fungal cells were stained using Amann's lactophenol cotton blue. DNA was extracted, quantified and assessed as described by Borba et al (2005). RAPDpolymerase chain reaction (PCR) was performed using six different primers named OPF-01 (ACGGATCCTG), OPF-08 (GGGATATCGG), OPF-11 (TTGGTACCCC), OPF-12 (ACGGTACCAG), OPF-13 (GGCTGCAGAA) and OPF-14 (TGCTGCAGGT) (Operon Technologies, Inc., USA).…”

Characteristics of Paecilomyces lilacinus infection comparing immunocompetent with immunosuppressed murine model

“…anaesthesia with 0.04 ml 2.5% sodium thiopental (50 mg kg −1 ), groups of mice, immunocompetent (group 1B) and immunosuppressed (group 2B), for each isolate were inoculated intravenously (i.v.) into the left retro‐orbital plexus, as previously described, 15 with 0.02 ml of sterile PBS containing 4.4 × 10 4 conidia with more than 80% viability. In summary, the mice were manually restrained and the bevelled edge of a 27‐gauge needle attached to a tuberculin syringe was inserted into the orbital eye vein.…”

M2 macrophages and inflammatory cells in oral lesions of chronic paracoccidioidomycosis

“…Paracoccidioidomycosis (PCM) is an infection caused by the dimorphic fungus Paracoccidioides brasiliensis ( Pb ) found in the soil of Latin America countries from Mexico to South of Argentina, and endemic in Brazilian Central‐West, Southeast, and South regions . It represents the main cause of disabilities and death among young adult countryside workers with age varying from 29 to 40 years old, during their most productive lifespan .…”