Lucélia Santi

Conidial surface proteins of Metarhizium anisopliae: Source of activities related with toxic effects, host penetration and pathogenesis.

Conidial contact with an arthropod surface is the first step of the fungal penetration and infection process. However, conidia of Metarhizium anisopliae have associated components, like enzymes that could be involved in triggering the penetration process and toxic effects that have not yet been well characterized. Fungi produce many enzymes that also are toxic components found in bacteria and animal venoms and thus may be considered as potential virulence factors. In this work, we report several enzymatic activities from spore surface protein extracts. The major proteolytic activities observed in spore surface proteins (SSP) were Pr1 and Pr2 activities, in that order. According to the zymograms obtained, SSP contain different proteases. SSP contain trehalase, exo- and endo-chitinase activities, and seven different chitinase bands which have been observed in zymograms. Activities involved in protection against reactive oxygen species (ROS) were also detected. Two lipolytic enzymes were also detected in lipase zymograms. Phospholipase C activity, closely related to microbial pathogenesis, was detected for the first time in M. anisopliae conidia. These activities described could be an initial step towards understanding the mechanisms involved in the first stage of M. anisopliae infection process and its toxic effects against arthropod hosts.

Metarhizium anisopliae host–pathogen interaction: differential immunoproteomics reveals proteins involved in the infection process of arthropods

Metarhizium anisopliae is an entomopathogenic fungus well characterized for the biocontrol of a wide range of plagues. Its pathogenicity depends on the secretion of hydrolytic enzymes that degrade the host cuticle. To identify proteins involved in the infection process and in host specify, immunoproteomic analysis was performed using antiserum produced against crude extract of M. anisopliae cultured in the presence of Rhipicephalus (Boophilus) microplus and Dysdercus peruvianus cuticles. Spots detected using antisera produced against M. anisopliae cultured in cuticles and spore surface proteins, but not with antiserum against M. anisopliae cultured in glucose, were identified so as to give insights about the infection process. An MS/MS allowed the identification of proteases, like elastase, trypsin, chymotrypsin, carboxypeptidase and subtilisin (Pr1A, Pr1I and PR1J), chitinases, DNase I and proline-rich protein. Chymotrypsin and Pr1I were inferred as host specific, being recognized in D. peruvianus infection only. This research represents an important contribution to the understanding the adaptation mechanisms of M. anisopliae to different hosts.

Metarhizium anisopliae lipolytic activity plays a pivotal role in Rhipicephalus (Boophilus) microplus infection

Lipases secreted by Metarhizium anisopliae, an important biological control agent, could potentially be involved in the host infection process. Here, we present the activity profile during the host infection process and the effect of lipase activity inhibitor ebelactone B on infection. The previous treatment of spores with lipase activity inhibitor, ebelactone B, completely inhibited lipolytic activity and prevented the infection of the Rhipicephalus (Boophilus) microplus host. The results herein presented prove, for the first time, the importance of lipase activity in M. anisopliae host infection process. The filamentous fungus Metarhizium anisopliae is one of the most important and studied biological agents for the control of several arthropod pests, including the cattle tick Rhipicephalus (Boophilus) microplus. Lipases secreted by M. anisopliae could potentially be involved in the host infection process. This work presents the activity profile during the host infection process and the effect of lipase activity inhibitor ebelactone B on infection. During the course of tick exposure to spores (6-120 h) lipase activity increased from 0.03 ± 0.00 U to 0.312 ± 0.068 U using rho NP palmitate as substrate. In zymograms, bands of lipase activity were detected in ticks treated with spores without inhibitor. The previous treatment of spores with lipase activity inhibitor, ebelactone B, completely inhibited lipolytic activity, at all times specified, and prevented the infection of the R. microplus host. Spores treated with the inhibitor did not germinate on the tick, although this effect was not observed in the culture medium. The results herein presented prove, for the first time, the importance of lipase activity in M. anisopliae host infection process.

Characterization of mycoviruses and analyses of chitinase secretion in the biocontrol fungus Metarhizium anisopliae.

Metarhizium anisopliae is the best-characterized entomopathogen and is used to control insect pests in sugar cane plantations in Brazil on a commercial scale. We have previously reported the infection of some M. anisopliae strains by dsRNA mycoviruses. Here we describe the purification and characterization of the viruses (MaV-A1, MaV-M5, MaV-RJ) in terms of dsRNA content, capsid proteins, electron microscopy, Western blot, and hybridization patterns. One spontaneous mutant lost some of the high molecular weight dsRNA components and showed significant alterations in colony morphology and spore production, suggesting that viral genes interfere with fungal phenotype. A comparison between dsRNA mycovirus-free and infected M. anisopliae isolates showed that virus-free isolates have increased endochitinase secretion. By comparing the following parameters: the buoyant density in CsCl of the presumed virions; the number and estimated molecular weight of the dsRNA components and the molecular mass of the capsid proteins to other mycoviruses previously described, we suggest the inclusion of MaV-A1 and MaV-M5 in the family Totiviridae and MaV-RJ in the family Partitiviridae.

Differential immunoproteomics enables identification of Metarhizium anisopliae proteins related to Rhipicephalus microplus infection

Differential immunoproteomics was applied to identify proteins secreted by Metarhizium anisopliae induced by the Rhipicephalus microplus cuticle. In addition, IgG anti-spore surface proteins were used for searching for proteins possibly involved in early stages of fungus versus tick infection. LC-MS/MS of differentially secreted proteins led to the identification of proteases (carboxypeptidase and Pr1A), chitinase, carboxylic acid transport and proline-rich protein. Differential immunoproteomics strategy facilitated the detection and the identification of new proteins related to M. anisopliae host-pathogen interaction and could be used in further works to identify novel proteins related to other microbial infection systems.

Proteomic Profile of Cryptococcus neoformans Biofilm Reveals Changes in Metabolic Processes

Cryptococcus neoformans, a pathogenic yeast, causes meningoencephalitis, especially in immunocompromised patients, leading in some cases to death. Microbes in biofilms can cause persistent infections, which are harder to treat. Cryptococcal biofilms are becoming common due to the growing use of brain valves and other medical devices. Using shotgun proteomics we determine the differences in protein abundance between biofilm and planktonic cells. Applying bioinformatic tools, we also evaluated the metabolic pathways involved in biofilm maintenance and protein interactions. Our proteomic data suggest general changes in metabolism, protein turnover, and global stress responses. Biofilm cells show an increase in proteins related to oxidation–reduction, proteolysis, and response to stress and a reduction in proteins related to metabolic process, transport, and translation. An increase in pyruvate-utilizing enzymes was detected, suggesting a shift from the TCA cycle to fermentation-derived energy acquisition. Additionally, we assign putative roles to 33 proteins previously categorized as hypothetical. Many changes in metabolic enzymes were identified in studies of bacterial biofilm, potentially revealing a conserved strategy in biofilm lifestyle.

A potential source for cellulolytic enzyme discovery and environmental aspects revealed through metagenomics of Brazilian mangroves.

The mangroves are among the most productive and biologically important environments. The possible presence of cellulolytic enzymes and microorganisms useful for biomass degradation as well as taxonomic and functional aspects of two Brazilian mangroves were evaluated using cultivation and metagenomic approaches. From a total of 296 microorganisms with visual differences in colony morphology and growth (including bacteria, yeast and filamentous fungus), 179 (60.5%) and 117 (39.5%) were isolated from the Rio de Janeiro (RJ) and Bahia (BA) samples, respectively. RJ metagenome showed the higher number of microbial isolates, which is consistent with its most conserved state and higher diversity. The metagenomic sequencing data showed similar predominant bacterial phyla in the BA and RJ mangroves with an abundance of Proteobacteria (57.8% and 44.6%), Firmicutes (11% and 12.3%) and Actinobacteria (8.4% and 7.5%). A higher number of enzymes involved in the degradation of polycyclic aromatic compounds were found in the BA mangrove. Specific sequences involved in the cellulolytic degradation, belonging to cellulases, hemicellulases, carbohydrate binding domains, dockerins and cohesins were identified, and it was possible to isolate cultivable fungi and bacteria related to biomass decomposition and with potential applications for the production of biofuels. These results showed that the mangroves possess all fundamental molecular tools required for building the cellulosome, which is required for the efficient degradation of cellulose material and sugar release.

The entomopathogen Metarhizium anisopliae can modulate the secretion of lipolytic enzymes in response to different substrates including components of arthropod cuticle

The filamentous fungus Metarhizium anisopliae is a well-characterized, arthropod pathogen used in the biological control of arthropod pests. Studies on the regulation of enzymes related to host infection such as proteases and chitinases have been reported but little is known about regulation of lipolytic enzymes in this fungus. Here we present the effects of different carbon sources such as components of the arthropod cuticle on the secretion of lipolytic enzymes by M. anisopliae. Differences in the induction of lipolytic activity were observed between the several carbon sources tested. Higher activities of lipase or lipase/esterase were found in culture media containing the arthropod integument components chitin and cholesteryl stearate. Several bands of lipolytic activity were also detected in zymograms, thus suggesting an important set of lipolytic enzymes secreted by the fungus. These results show that the fungus can modulate the secretion of lipolytic activity in response to host integument components, thus reinforcing the potential role of these enzymes during M. anisopliae infection.

Lonomia obliqua venomous secretion induces human platelet adhesion and aggregation

The caterpillar Lonomia obliqua is a venomous animal that causes numerous accidents, especially in southern Brazil, where it is considered a public health problem. The clinical manifestations include several haemostatic disturbances that lead to a hemorrhagic syndrome. Considering that platelets play a central role in hemostasis, in this work we investigate the effects of L. obliqua venomous secretion upon blood platelets responses in vitro. Results obtained shows that L. obliqua venom directly induces aggregation and ATP secretion in human washed platelets in a dose-dependent manner. Electron microscopy studies clearly showed that the venomous bristle extract was also able to produce direct platelets shape change and adhesion as well as activation and formation of platelet aggregates. Differently from other enzyme inhibitors, the venom-induced platelet aggregation was significatively inhibited by p-bromophenacyl bromide, a specific inhibitor of phospholipases A2. Additional experiments with different pharmacological antagonists indicate that the aggregation response triggered by the venom active components occurs through a calcium-dependent mechanism involving arachidonic acid metabolite(s) of the cyclooxygenase pathway and activation of phosphodiesterase 3A, an enzyme that leads to the consumption of intracellular cAMP content. It was additionally found that L. obliqua-induced platelet aggregation was independent of ADP release. Altogether, these findings are in line with the need for a better understanding of the complex hemorrhagic syndrome resulting from the envenomation caused by L. obliqua caterpillars, and can also give new insights into the management of its clinical profile.

Integrated control of an acaricide-resistant strain of the cattle tick Rhipicephalus microplus by applying Metarhizium anisopliae associated with cypermethrin and chlorpyriphos under field conditions.

The efficacy of the fungus Metarhizium anisopliae to control ticks has been shown in several in vitro experiments. However, few studies have been undertaken in field conditions in order to demonstrate the applicability of its use as a biological control of ticks and its combination with chemical acaricides. The aim of the present study was to evaluate the efficacy of M. anisopliae to control an acaricide-resistant strain of Rhipicephalus microplus under laboratory and field conditions. First, the compatibility of M. anisopliae strain (TIS-BR03) with commercial acaricides and its potential to control the cattle tick were evaluated in vitro. In general, acaricide treatments had mild effects on fungus viability. In the field experiment, the median of treatment efficacy with acaricide only, M. anisopliae only and combination of M. anisopliae with acaricide were 71.1%, 56.3% and 97.9%, respectively. There is no statistical difference between groups treated with M. anisopliae and acaricide alone. Thus, in this work we have demonstrated the applicability of M. anisopliae use associated or not with chemical acaricides on field conditions in order to control an acaricide-resistant strain of the cattle tick R. microplus.