Jean-Luc Gesztesi

Exocellular proteolytic activity of Paracoccidioides brasiliensis: cleavage of components associated with the basement membrane

We have previously characterized an exocellular serine-thiol proteinase activity in Paracoccidioides brasiliensis, using as substrates peptides analogous of the internally quenched fluorogenic peptide Abz-MKRLTL-EDDnp. In this communication, detection of maximal proteinase activity in the culture supernatant fluids followed the abrupt increase in the medium pH, owing to the accumulation of ammonia generated by urease activity. Culture supernatant fluids collected at the peak of proteinase activity against Abz-MRKLTL-EDDnp were able to cleave components of the basal membrane of the extracellular matrix (EM), including laminin, fibronectin, collagen type IV and proteoglycans, and the proteolytic activity was selectively inhibited both by PMSF and p-HMB (sodium 7-hydroxymercuribenzoate), which are also specific inhibitors of the serine-thiol proteinase. Human collagen I, bovine fibrinogen, human immunoglobulin G, BSA or P. brasiliensis gp43 were resistant to proteolysis. The kinetics of appearance of the proteinase activity against EM substrates coincided with that of proteolysis of Abz-MKRLTL-EDDnp. Moreover, chromatographic fractions of culture supernatants containing the serine-thiol proteinase at high specific activity were also active against EM substrates. These data suggest the involvement of this enzyme activity in the degradation of the basement membrane, which is the first step for fungal tissue invasion.

Subcutaneous infection of mice with Paracoccidioides brasiliensis induces a peculiar pattern of inflammatory and immune responses

It is well established that resistance or susceptibility to Paracoccidioidis brasiliensis infection in mice is under strict hosts genetic control. Mice from A/Sn strain inoculated by the ip route are resistant to fungal infection while infection induced in mice from B10.A strain results in a fatal disease. The early cellular events of infection in both strains are characterized by a marked neutrophilic infiltration that is more prominent in B10.A mice. A peculiar characteristic of the Paracoccidioides brasiliensis-mouse model is that the subcutaneous (sc) inoculations of the fungus either in resistant (A/Sn) or susceptible (B10.A) mice is self-curing and turns mice from the B10.A strain able to express typical DTH reaction to fungal antigens, as observed in A/Sn mice. Here we report the investigation on the early events of the inflammatory response induced by the inoculation of live fungus into the hind footpad of A/Sn (resistant) and B10.A (susceptible) mice. The influence of neutrophils on the inflammatory response and antibody titers or DTH response to gp43, the major fungal antigen, was also evaluated. Results showed a different course of the inflammatory response induced by fungal inoculation in A/Sn and B10.A mice. Neutrophil depletion before infection differently influenced the kinetics of the inflammatory process in both mice strains but did not modifythe fungal load in the lesions. In neutrophil depleted mice from both strains, a decrease in DTH response and an increase in total antibody titers to gp43 were observed. The significant increase in the fungal load in lesions seen in nude mice indicates that the self-limited infection evoked by fungal inoculation into the subcutaneous tissue is a T-cell dependent phenomenon. The implications of these observations in the pathogenesis of paracoccidioidomycosis are discussed.

MONOCLONAL ANTIBODIES TO IDENTIFY TOMATO MOSAIC TOBAMOVIRUS (TOMV)

Monoclonal antibodies were obtained against Tomato mosaic tobamovirus (ToMV) isolated in Brazil. One antibody (8G7G2) isotyped as IgG2b (k light chain) showed strong specificity and very low cross reaction with the Tobacco mosaic virus (TMV). It can be used in identification of tomato mosaic virus (ToMV).