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This work was financially supported by Fundacao para a Ciencia e a Tecnologia (FCT), project MOSTMICRO ITQB with reference no. UIDB/04612/2020 and UIDP/04612/2020, and by project "PinusResina" no. PDR2020-101-031905, funded by PDR2020 through Portugal2020. This research was carried out through a partnership with the RESOLUTION LAB, an infrastructure at NOVA School of Science and Technology. The authors acknowledge and thank the support given by the Portuguese Foundation for Science and Technology (FCT) to CENSE through the strategic project UIDB/04085/2020. T.M. and C.M. are grateful to FCT for the working contract financed by national funds under Norma Transitoria D.L. no. 57/2016 and the fellowship SFRH/BD/118377/2016, respectively. In fungi, salicylate catabolism was believed to proceed only through the catechol branch of the 3-oxoadipate pathway, as shown, e.g., in Aspergillus nidulans. However, the observation of a transient accumulation of gentisate upon the cultivation of Aspergillus terreus in salicylate medium questions this concept. To address this, we have run a comparative analysis of the transcriptome of these two species after growth in salicylate using acetate as a control condition. The results revealed the high complexity of the salicylate metabolism in A. terreus with the concomitant positive regulation of several pathways for the catabolism of aromatic compounds. This included the unexpected joint action of two pathways—3-hydroxyanthranilate and nicotinate—possibly crucial for the catabolism of aromatics in this fungus. Importantly, the 3-hydroxyanthranilate catabolic pathway in fungi is described here for the first time, whereas new genes participating in the nicotinate metabolism are also proposed. The transcriptome analysis showed also for the two species an intimate relationship between salicylate catabolism and secondary metabolism. This study emphasizes that the central pathways for the catabolism of aromatic hydrocarbons in fungi hold many mysteries yet to be discovered. IMPORTANCE Aspergilli are versatile cell factories used in industry for the production of organic acids, enzymes, and pharmaceutical drugs. To date, bio-based production of organic acids relies on food substrates. These processes are currently being challenged to switch to renewable nonfood raw materials—a reality that should inspire the use of lignin-derived aromatic monomers. In this context, aspergilli emerge at the forefront of future bio-based approaches due to their industrial relevance and recognized prolific catabolism of aromatic compounds. Notwithstanding considerable advances in the field, there are still important knowledge gaps in the central catabolism of aromatic hydrocarbons in fungi. Here, we disclose a novel central pathway, 3-hydroxyanthranilate, defying previously established ideas on the central metabolism of the aromatic amino acid tryptophan in Ascomycota. We also observe that the catabolism of the aromatic salicylate greatly activated the secondary metabolism, furthering the significance of using lignin-derived aromatic hydrocarbons as a distinctive biomass source. publishersversion published

In fungi, salicylate catabolism was believed to proceed only through the catechol branch of the 3-oxoadipate pathway, as shown e.g. in Aspergillus nidulans. However, the observation of a transient accumulation of gentisate upon cultivation of Aspergillus terreus in salicylate media questions this concept. To address this we have run a comparative analysis of the transcriptome of these two species after growth in salicylate using acetate as a control condition. The results revealed the high complexity of the salicylate metabolism in A. terreus with the concomitant positive regulation of several pathways for the catabolism of aromatic compounds. This included the unexpected joint action of two pathways: the nicotinate and the 3-hydroxyanthranilate, possibly crucial for the catabolism of aromatics in this fungus. New genes participating in the nicotinate metabolism are here proposed, whereas the 3-hydroxyanthranilate catabolic pathway in fungi is described for the first time. The transcriptome analysis showed also for the two species an intimate relationship between salicylate catabolism and secondary metabolism. This study emphasizes that the central pathways for the catabolism of aromatic hydrocarbons in fungi hold many mysteries yet to be discovered.IMPORTANCEAspergilli are versatile cell factories used in industry for production of organic acids, enzymes and pharmaceutical drugs. To date, organic acids bio-based production relies on food substrates. These processes are currently being challenged to switch to renewable non-food raw materials; a reality that should inspire the use of lignin derived aromatic monomers. In this context, Aspergilli emerge at the forefront of future bio-based approaches due to their industrial relevance and recognized prolific catabolism of aromatic compounds. Notwithstanding considerable advances in the field, there are still important knowledge gaps in the central catabolism of aromatic hydrocarbons in fungi. Here, we disclosed a novel central pathway, defying previous established ideas on the central metabolism of the aromatic amino acid tryptophan in Ascomycota. We also observed that the catabolism of the aromatic salicylate greatly activated the secondary metabolism, furthering the significance of using lignin derived aromatic hydrocarbons as a distinctive biomass source.

The diversity and abundance of aromatic compounds in nature is crucial for proper metabolism in all biological systems, and also impacts greatly the development of many industrial processes. Naturally, understanding their catabolism becomes fundamental for many scientific fields of research, from clinical and environmental to technological. The genetic basis of the central pathways for the catabolism of aromatic compounds in fungi, particularly of benzene derivatives, remains however poorly understood largely overlooking their significance. In some Dikarya species the genes of the central pathways are clustered in the genome, often in an array with peripheral pathway genes, even if the existence of a specific pathway does not necessarily mean that the composing genes are clustered. The current availability of many annotated fungal genomes in the postgenomic era creates conditions to reach a more holistic view of these processes through target analysis of the central pathways gene clusters. Inspired by this, we have critically analyzed the established biochemical and genetic data on the catabolism of aromatic compounds in Dikarya after dissecting the presence and distribution of central catabolic gene clusters (at times including also details on gene diversity, order and orientation) and of peripheral genes. Our methodological approach illustrates the multiple degrees of separation in these central pathways gene clusters across Dikarya. Surprisingly, they show a great degree of similarity irrespectively of the Dikarya division, emphasizing that knowledge established on either phyla can guide the identification of clusters of comparable composition (in-cluster plus peripheral genes) in uncharacterized species.

The diversity and abundance of aromatic compounds in nature is crucial for proper metabolism in all biological systems, and also impacts greatly the development of many industrial processes. Naturally, understanding their catabolism becomes fundamental for many scientific fields of research, from clinical and environmental to technological. The genetic basis of the central pathways for the catabolism of aromatic compounds in fungi, particularly of benzene derivatives, remains however poorly understood largely overlooking their significance. In some Dikarya species the genes of the central pathways are clustered in the genome, often in an array with peripheral pathway genes, even if the existence of a specific pathway does not necessarily mean that the composing genes are clustered. The current availability of many annotated fungal genomes in the postgenomic era creates conditions to reach a more holistic view of these processes through target analysis of the central pathways gene clusters. Inspired by this, we have critically analyzed the established biochemical and genetic data on the catabolism of aromatic compounds in Dikarya after dissecting the presence and distribution of central catabolic gene clusters (at times including also details on gene diversity, order and orientation) and of peripheral genes. Our methodological approach illustrates the multiple degrees of separation in these central pathways gene clusters across Dikarya. Surprisingly, they show a great degree of similarity irrespectively of the Dikarya division, emphasizing that knowledge established on either phyla can guide the identification of clusters of comparable composition (in-cluster plus peripheral genes) in uncharacterized species.

Pentachlorophenol (PCP) is globally dispersed and contamination of soil with this biocide adversely affects its functional biodiversity, particularly of fungi - key colonizers. Their functional role as a community is poorly understood, although a few pathways have been already elucidated in pure cultures. This constitutes here our main challenge - elucidate how fungi influence the pollutant mitigation processes in forest soils. Circumstantial evidence exists that cork oak forests in N. W. Tunisia - economically critical managed forests are likely to be contaminated with PCP, but the scientific evidence has previously been lacking. Our data illustrate significant forest contamination through the detection of undefined active sources of PCP. By solving the taxonomic diversity and the PCP-derived metabolomes of both the cultivable fungi and the fungal community, we demonstrate here that most strains (predominantly penicillia) participate in the pollutant biotic degradation. They form an array of degradation intermediates and by-products, including several hydroquinone, resorcinol and catechol derivatives, either chlorinated or not. The degradation pathway of the fungal community includes uncharacterized derivatives, e.g. tetrachloroguaiacol isomers. Our study highlights fungi key role in the mineralization and short lifetime of PCP in forest soils and provide novel tools to monitor its degradation in other fungi dominated food webs.

Aspergilli play major roles in the natural turnover of elements, especially through the decomposition of plant litter, but the end catabolism of lignin aromatic hydrocarbons remains largely unresolved. The 3-oxoadipate pathway of their degradation combines the catechol and the protocatechuate branches, each using a set of specific genes. However, annotation for most of these genes is lacking or attributed to poorly- or un-characterised families. Aspergillus nidulans can utilise as sole carbon/energy source either benzoate or salicylate (upstream aromatic metabolites of the protocatechuate and the catechol branches, respectively). Using this cultivation strategy and combined analyses of comparative proteomics, gene mining, gene expression and characterisation of particular gene-replacement mutants, we precisely assigned most of the steps of the 3-oxoadipate pathway to specific genes in this fungus. Our findings disclose the genetically encoded potential of saprophytic Ascomycota fungi to utilise this pathway and provide means to untie associated regulatory networks, which are vital to heightening their ecological significance.

Papain-like cysteine proteases have been shown to have essential roles in parasitic protozoa and are under study as promising drug targets. Five genes were identified by sequence similarity search to be homologous to the cysteine protease family in the ongoing Babesia bigemina genome sequencing project database and were compared with the annotated genes from the complete bovine piroplasm genomes of Babesia bovis, Theileria annulata, and Theileria parva. Multiple genome alignments and sequence analysis were used to evaluate the molecular evolution events that occurred in the C1 family of cysteine proteases in these piroplasms of veterinary importance. BbiCPL1, one of the newly identified cysteine protease genes in the B. bigemina genome was expressed in Escherichia coli and shows activity against peptide substrates. Considerable differences were observed in the cysteine protease family between Babesia and Theileria genera, and this may partially explain the diverse infection mechanisms of these tick-borne diseases.

Malaria remains one of the major human parasitic diseases, particularly in subtropical regions. Most of the fatal cases are caused by Plasmodium falciparum. The rodent parasite Plasmodium chabaudi has been the model of choice in research due to its similarities to human malaria, including developmental cycle, preferential invasion of mature erythrocytes, synchrony of asexual development, antigenic variation, gene sinteny as well as similar resistance mechanisms. Protein disulfide isomerase (PDI) is an essential catalyst of the endoplasmic reticulum in different biological systems with folding and chaperone activities. Most of the proteins exported by parasites have to pass through the endoplasmic reticulum before reaching their final destination and their correct folding is critical for parasite survival. PDI constitutes a potential target for the development of alternative therapy strategies based on the inhibition of folding and chaperoning of exported proteins. We here describe the sequencing of the gene coding for the PDI from P. chabaudi and analyse the relationship to its counterpart enzymes, particularly with the PDI from other Plasmodium species. The model constructed, based on the recent model deduced from the crystallographic structure 2B5E, was compared with the previous theoretical model for the whole PDI molecule constructed by threading. A recombinant PDI from P. chabaudi was also produced and used as an antigen for monoclonal antibody production for application in PDI immunolocalization.

Plasmodium cysteine proteases have been shown to be immunogenic and are being used as malaria potential serodiagnostic markers and vaccine targets. Genes encoding two Plasmodium chabaudi cysteine proteases chabaupain-1 (CP-1) and chabaupain-2 (CP-2) were identified and further expressed in Escherichia coli. Solubilisation of recombinant CP-1 and CP-2 was achieved by decreasing the temperature of induction. Anopheles gambiae tissues infected with Plasmodium were analyzed by Western blotting using the anti-CP-1 antibody showing that CP-1 is only present in the A. gambiae midguts being absent from other infected mosquito biological material. Anti-CP-1 anti-serum recognized a 30 kDa band in P. chabaudi, Plasmodium berghei and Plasmodium yoelii lysates but does not recognize the recombinant CP-2 extracts suggesting high antibody specificity.

Pentachlorophenol (PCP) is globally dispersed and contamination of soil with this biocide adversely affects its functional biodiversity, particularly of fungi - key colonizers. Their functional role as a community is poorly understood, although a few pathways have been already elucidated in pure cultures. This constitutes here our main challenge - elucidate how fungi influence the pollutant mitigation processes in forest soils. Circumstantial evidence exists that cork oak forests in N. W. Tunisia - economically critical managed forests are likely to be contaminated with PCP, but the scientific evidence has previously been lacking. Our data illustrate significant forest contamination through the detection of undefined active sources of PCP. By solving the taxonomic diversity and the PCP-derived metabolomes of both the cultivable fungi and the fungal community, we demonstrate here that most strains (predominantly penicillia) participate in the pollutant biotic degradation. They form an array of degradation intermediates and by-products, including several hydroquinone, resorcinol and catechol derivatives, either chlorinated or not. The degradation pathway of the fungal community includes uncharacterized derivatives, e.g. tetrachloroguaiacol isomers. Our study highlights fungi key role in the mineralization and short lifetime of PCP in forest soils and provide novel tools to monitor its degradation in other fungi dominated food webs.

Centaurea calcitrapa hairy root cultures were established by infection with Agrobacterium rhizogenes strain LBA 9402. The liquid medium hairy root cultures exhibited a biomass doubling time of approximately 1.5 days in the 20 days exponential growth phase. The effect of the initial sucrose and nitrogen concentrations in biomass and proteinase production of the liquid medium cultures was studied. The highest values for both proteolytic activity and fresh weight were attained between 30 and 50 g/l of sucrose. A low ammonium:nitrate ratio favoured the development of hairy roots and proteolytic activity. The best results for both parameters in terms of nitrogen nutrition were obtained with nitrate (24.7 mM) as the only nitrogen source. The maximum proteolytic activity was found to be at pH 4.0, within the pH range for aspartic proteinases (APs), and the inhibition studies showed that only pepstatin A, specific for that class of enzymes, revealed a significant inhibitory effect. The C. calcitrapa aspartic proteinase (cenprosin) gene was detected in hairy roots using specific polymerase chain reaction (PCR) primers. The specific proteolytic activity present in hairy roots seems to be lower than the reported for flowers but similar to the existent in the untransformed roots and cell suspension cultures.

A collective view of the degradation of monochlorocatechols in fungi is yet to be attained, though these compounds are recognised as key degradation intermediates of numerous chlorinated aromatic hydrocarbons, including monochlorophenols. In the present contribution we have analysed the degradation pathways of monochlorophenols in Aspergillus nidulans using essentially metabolomics. Degradation intermediates herein identified included those commonly reported (e.g. 3-chloro-cis,cis-muconate) but also compounds never reported before in fungi revealing for 4-chlorocatechol and for 3- chlorocatechol unknown degradation paths yielding 3-chlorodienelactone and catechol, respectively. A different 3-chlorocatechol degradation path led to accumulation of 2- chloromuconates (a potential dead-end), notwithstanding preliminary evidence of chloromuconolactones and protoanemonin simultaneous formation. In addition, some transformation intermediates, of which sulfate conjugates of monochlorophenols/ chlorocatechols were the most common, were also identified. This study provides critical information for understanding the role of fungi in the degradation of chlorinated aromatic hydrocarbons; furthering their utility in the development of innovative bioremediation strategies.

Exp Parasitol. 2011 Oct;129(2):164-74. Epub 2011 Jul 3. Besnoitia besnoiti protein disulfide isomerase (BbPDI): molecular characterization, expression and in silico modelling. Marcelino E, Martins TM, Morais JB, Nolasco S, Cortes H, Hemphill A, Leitão A, Novo C. Source Instituto de Investigação Cientifica Tropical, CIISA, Faculdade de Medicina Veterinária, Universidade Técnica de Lisboa, Av. da Universidade Técnica, 1300-477 Lisboa, Portugal. Abstract Besnoitia besnoiti is an apicomplexan parasite responsible for bovine besnoitiosis, a disease with a high prevalence in tropical and subtropical regions and re-emerging in Europe. Despite the great economical losses associated with besnoitiosis, this disease has been underestimated and poorly studied, and neither an effective therapy nor an efficacious vaccine is available. Protein disulfide isomerase (PDI) is an essential enzyme for the acquisition of the correct three-dimensional structure of proteins. Current evidence suggests that in Neosporacaninum and Toxoplasmagondii, which are closely related to B. besnoiti, PDI play an important role in host cell invasion, is a relevant target for the host immune response, and represents a promising drug target and/or vaccine candidate. In this work, we present the nucleotide sequence of the B. besnoiti PDI gene. BbPDI belongs to the thioredoxin-like superfamily (cluster 00388) and is included in the PDI_a family (cluster defined cd02961) and the PDI_a_PDI_a'_c subfamily (cd02995). A 3D theoretical model was built by comparative homology using Swiss-Model server, using as a template the crystallographic deduced model of Tapasin-ERp57 (PDB code 3F8U chain C). Analysis of the phylogenetic tree for PDI within the phylum apicomplexa reinforces the close relationship among B. besnoiti, N. caninum and T. gondii. When subjected to a PDI-assay based on the polymerisation of reduced insulin, recombinant BbPDI expressed in E. coli exhibited enzymatic activity, which was inhibited by bacitracin. Antiserum directed against recombinant BbPDI reacted with PDI in Western blots and by immunofluorescence with B. besnoiti tachyzoites and bradyzoites.

BbiCPL1 was the first papain-like cysteine protease from a piroplasm to be identified with proteolytic activity. Here we report the improved production of the active recombinant enzyme, and the biochemical characterization of this potential drug target. BbiCPL1 showed characteristic properties of its class, including hydrolysis of papain-family peptide substrates, an acidic pH optimum, requirement of a reducing environment for maximum activity, and inhibition by standard cysteine protease inhibitors such as E-64, leupeptin, ALLN and cystatin. The optimum pH for the protease activity against peptide substrates was 5.5, but enzymatic activity was observed between pH 4.0 and pH 9.0. At slightly basic pH 7.5, BbiCPL1 maintained 83% of maximum activity, suggesting a role in cytosol environment.

SUMMARYMolecular detection ofBabesiaspecies in apparently healthy cattle within an endemic region was carried out in order to determine the prevalence of carriers and the geographical distribution ofBabesia bigeminaandBabesia bovisin Maputo Province, Mozambique. Samples from 477 animals at 5 localities were analysed using 2 techniques, the semi-nested hot-start PCR and the reverse line blot (RLB) assay. With the semi-nested hot-start PCR, detection ofB. bigeminaranged between 30% and 89%, and ofB. bovisbetween 27% and 83%. The RLB assay was comparatively less sensitive in this study and detection ofB. bovisranged from 0% to 17%, andB. bigeminawas not detected at all by this technique. Analysis of new sequences of the 18S rRNA gene revealed that the currentB. bigeminaRLB probe is not specific for the identification of isolates in Mozambique. The RLB assay, however, resulted in the detection of 8 other haemoparasite species belonging to the generaBabesia,Theileria,AnaplasmaandEhrlichia. 18S rRNA gene sequences from theTheileriaspp. were identified, and a phylogenic tree constructed with these sequences yielded a heterogeneousT. mutans-like group. In conclusion, infection withB. bigeminaandB. bovisis endemic in Maputo Province, but rates of transmission vary. Furthermore, mixed infections with the haemoparasites responsible for several tick-borne diseases in cattle are common in Mozambique.

Babesiosis is a tick borne disease (TBD) caused by parasites of the genus Babesia, with considerable worldwide economic, medical, and veterinary impact. Bovine babesiosis and other TBDs were considered responsible for 50% of the deaths of cattle that occurred in Mozambique in the first year after importation from neighbouring countries. Here, we present the detection of Babesia bigemina and Babesia bovis in cattle from Mozambique using two distinct PCR methods. For this study, blood samples were collected in one farm located near Maputo city. The DNA samples were analyzed using a previously described nested PCR and a novel hot-start PCR method. Primers were selected for the hot-start PCR based on the putative gene of an undescribed aspartic protease named babesipsin, present in both B. bovis and B. bigemina. The combination of hot-start polymerase and long primers (29–31 bp) were in this study determinant for the successful amplification and detection in only one PCR. With a seminested approach the sensitivity was further increased. The babesipsin seminested hot-start PCR was in this study more sensitive than the nested PCR. A total of 117 field samples were tested by seminested hot-start PCR, and 104 were positive for B. bigemina (90%), 97 were positive for B. bovis (82%), 86 were mixed infections (52%) and only 2 were negative for both Babesia species (1.7%). The results confirm that this area of Mozambique is endemic for babesiosis, and that this TBD should be regarded as a threat for imported cattle.

The rodent malaria parasite Plasmodium chabaudi encodes one food vacuole plasmepsin—the aspartic proteinases important in haemoglobin degradation. A recombinant form of this enzyme was found to cleave a variety of peptide substrates and was susceptible to a selection of naturally occurring and synthetic inhibitors, displaying an inhibition profile distinct from that of aspartic proteinases from other malaria parasites. In addition, inhibitors of HIV proteinase that kill P. chabaudi in vivo were also inhibitors of this new plasmepsin. P. chabaudi is a widely used model for human malaria species and, therefore, the characterisation of this plasmepsin is an important contribution towards understanding its biology.

Intraerythrocytic malaria parasites degrade haemoglobin to provide nutrients for their own growth and maturation. Plasmodium aspartic proteases known as plasmepsins play an important role on haemoglobin degradation and are being studied as drug targets for chemotherapy of malaria. The rodent model for human malaria, Plasmodium chabaudi, is an experimentally good model for therapy drug design. The gene encoding an aspartic protease precursor (proplasmepsin) from the rodent malaria parasite P. chabaudi was cloned and sequenced. A theoretical 3D structure model was constructed by comparative homology and used for superimposition with other known models. Analysis of the P. chabaudiand Plasmodium yoelligenomes revealed in both the presence of at least seven plasmepsins and each one has sequence similarity to its plasmepsin counterpart of the human malaria Plasmodium falciparum . The predicted proteins were confirmed as plasmepsins by detection on Blocks Database of three characteristic blocks of the eukaryotic and viral aspartic protease family. Analysis of the proline-rich loop amino acid sequence of these plasmepsins suggests that they constitute characteristic motifs of each plasmepsin group suggesting that these sequence variations are related with different substrate specificities. © 2003 Elsevier B.V. All rights reserved.

Papain-like cysteine proteases (CP) have been shown to have essential roles in parasitic protozoa and are under study as promising drug targets. One gene was identified by sequence similarity search to be homologous to the CP family in the ongoing Babesia bigemina genome sequencing project database. The newly identified CP gene, called babesipain-1, was cloned and expressed as a fusion protein, and the effect of different inhibitors on proteolytic activity was tested. A series of new artemisinin-vinyl sulfone hybrid molecules were tested as inhibitors being effective on the range of 0.3-30 microm, depending on the core-containing molecule.