Ana Sofia Duarte

Bacterial collagenases – A review

Abstract Bacterial collagenases are metalloproteinases involved in the degradation of the extracellular matrices of animal cells, due to their ability to digest native collagen. These enzymes are important virulence factors in a variety of pathogenic bacteria. Nonetheless, there is a lack of scientific consensus for a proper and well-defined classification of these enzymes and a vast controversy regarding the correct identification of collagenases. Clostridial collagenases were the first ones to be identified and characterized and are the reference enzymes for comparison of newly discovered collagenolytic enzymes. In this review we present the most recent data regarding bacterial collagenases and overview the functional and structural diversity of bacterial collagenases. An overall picture of the molecular diversity and distribution of these proteins in nature will also be given. Particular aspects of the different proteolytic activities will be contextualized within relevant areas of application, mainly biotechnological processes and therapeutic uses. At last, we will present a new classification guide for bacterial collagenases that will allow the correct and straightforward classification of these enzymes.

Temperature Modulates the Secretome of the Phytopathogenic Fungus Lasiodiplodia theobromae

Environmental alterations modulate host–microorganism interactions. Little is known about how climate changes can trigger pathogenic features on symbiont or mutualistic microorganisms. Current climate models predict increased environmental temperatures. The exposing of phytopathogens to these changing conditions can have particularly relevant consequences for economically important species and for humans. The impact on pathogen/host interaction and the shift on their biogeographical range can induce different levels of virulence in new hosts, allowing massive losses in agricultural and health fields. Lasiodiplodia theobromae is a phytopathogenic fungus responsible for a number of diseases in various plants. It has also been described as an opportunist pathogen in humans, causing infections with different levels of severity. L. theobromae has a high capacity of adaptation to different environments, such as woody plants, moist argillaceous soils, or even humans, being able to grow and infect hosts in a wide range of temperatures (9–39°C). Nonetheless, the effect of an increase of temperature, as predicted in climate change models, on L. theobromae is unknown. Here we explore the effect of temperature on two strains of L. theobromae – an environmental strain, CAA019, and a clinical strain, CBS339.90. We show that both strains are cytotoxic to mammalian cells but while the environmental strain is cytotoxic mainly at 25°C, the clinical strain is cytotoxic mainly at 30 and 37°C. Extracellular gelatinolytic, xylanolytic, amylolytic, and cellulolytic activities at 25 and 37°C were characterized by zymography and the secretome of both strains grown at 25, 30, and 37°C were characterized by electrophoresis and by Orbitrap LC-MS/MS. More than 75% of the proteins were identified, mostly enzymes (glycosyl hydrolases and proteases). The strains showed different protein profiles, which were affected by growth temperature. Also, strain specific proteins were identified, such as a putative f5/8 type c domain protein – known for being involved in pathogenesis – by strain CAA019 and a putative tripeptidyl-peptidase 1 protein, by strain CBS339.90. We showed that temperature modulates the secretome of L. theobromae. This modulation may be associated with host-specificity requirements. We show that the study of abiotic factors, such as temperature, is crucial to understand host/pathogen interactions and its impact on disease.

The Characterisation of the Collagenolytic Activity of Cardosin A Demonstrates its Potential Application for Extracellular Matrix Degradative Processes

Type I collagen is the major fibrous protein of mammals being needed to strengthen and organise the extracellular matrix (ECM). Connective tissue components are modulated by matrix metalloproteinases, which are critical for disintegration and remodelling of ECM under physiological and pathological conditions. Cardosin A is an abundant aspartic proteinase (AP) from Cynara cardunculus L. that has been shown to be able to hydrolyse fibrillar collagen within the alpha-chains. The aim of this work is the characterisation of collagen degradation by cardosin A, since in the native state fibrillar collagen is resistant to most proteolytic enzymes. The pattern of type I collagen hydrolysis by cardosin A is defined and maintained for at least 24 hours of digestion, suggesting that cardosin A can hydrolyse collagen at a small number of specific peptide bonds. N-terminal sequencing of hydrolysis products identified one cleavage site as being Phe464-Gln465 in the alpha2 chain of collagen I. Two peptides were synthesised correspondent to collagen I specific sequences, in order to produce two polyclonal antibodies, that allowed the identification of three collagen fragments following cardosin A cleavage. Defining the mechanism of collagen cleavage by collagenases and other enzymes, like cardosin A, is important for the comprehension of physiological and pathological processes affecting the ECM. To our knowledge, this is the first study of in vitro collagenolytic activity of a plant AP. Therefore, in view of the cardosin A restricted specificity towards collagen this enzyme may be proposed for an eventual medical or technical procedures assisting ECM remodelling.

Immobilisation of Cardosin A in Chitosan Sponges as a Novel Implant for Drug Delivery

Cardosin A is extracted from the pistils of the plant Cynara cardunculus L. and chitosan is a polysaccharide derived from chitin with valuable properties as a biomaterial. In this work we report our experiments on the synthesis of chitosan sponges and immobilisation of cardosin A, by entrapment. We observed that 10-15% of the incorporated cardosin A were released over 6 days of incubation. In addition, we could also note that this immobilisation procedure did not induce any specificity alterations on cardosin A. The specificity study of the enzyme, using beta-chain of oxidised insulin, showed that the immobilised and released enzymes have the same hydrolysis pattern as the free enzyme. The ability of this enzyme to hydrolyse type I collagen was maintained, after the immobilisation procedure. The biocompatibility in vivo of these sponges was evaluated by histological staining after implantation in rats submitted to abdominal surgery. Results of this study demonstrated that these chitosan sponges are very promising vehicles for the application of cardosin A, in abdominal cavity for prevention and reduction of the adhesions formation.

Aeromonas piscicola AH-3 expresses an extracellular collagenase with cytotoxic properties.

The aim of this study was to investigate the presence and the phenotypic expression of a gene coding for a putative collagenase. This gene (AHA_0517) was identified in Aeromonas hydrophila ATCC 7966 genome and named colAh. We constructed and characterized an Aeromonas piscicola AH‐3::colAh knockout mutant. Collagenolytic activity of the wild‐type and mutant strains was determined, demonstrating that colAh encodes for a collagenase. ColAh–collagen interaction was assayed by Far‐Western blot, and cytopathic effects were investigated in Vero cells. We demonstrated that ColAh is a gluzincin metallopeptidase (approx. 100 kDa), able to cleave and physically interact with collagen, that contributes for Aeromonas collagenolytic activity and cytotoxicity. ColAh possess the consensus HEXXH sequence and a glutamic acid as the third zinc binding positioned downstream the HEXXH motif, but has low sequence similarity and distinct domain architecture to the well‐known clostridial collagenases. In addition, these results highlight the importance of exploring new microbial collagenases that may have significant relevance for the health and biotechnological industries.

Lasiodiplodia theobromae as a Producer of Biotechnologically Relevant Enzymes

Phytopathogenic fungi are known to produce several types of enzymes usually involved in plant cell wall degradation and pathogenesis. The increasing of global temperature may induce fungi, such as Lasiodiplodia theobromae (L. theobromae), to alter its behavior. Nonetheless, there is only limited information regarding the effect of temperature on L. theobromae production of enzymes. The need for new, thermostable enzymes, that are biotechnologically relevant, led us to investigate the effect of temperature on the production of several extracellular enzymatic activities by different L. theobromae strains. Fungi were grown at 25 °C, 30 °C and 37 °C and the enzymatic activities were detected by plate assays, quantified by spectrophotometric methods and characterized by zymography. The thermostability (25–80 °C) of the enzymes produced was also tested. Strains CAA019, CBS339.90, LA-SOL3, LA-SV1 and LA-MA-1 produced amylases, gelatinases, caseinases, cellulases, lipases, laccases, xylanases, pectinases and pectin liases. Temperature modulated the expression of the enzymes, and this effect was more visible when fungi were grown at 37 °C than at lower temperatures. Contrary to proteolytic and endoglucanolytic activities, whose highest activities were detected when fungi were grown at 30 °C, lipolytic activity was not detected at this growth temperature. Profiles of proteases and endoglucanases of fungi grown at different temperatures were characterized by zymography. Enzymes were shown to be more thermostable when fungi were grown at 30 °C. Proteases were active up to 50 °C and endoglucanases up to 70 °C. Lipases were the least stable, with activities detected up to 45 °C. The enzymatic profiles detected for L. theobromae strains tested showed to be temperature and strain-dependent, making this species a good target for biotechnological applications.

Production of toxic metabolites by two strains of Lasiodiplodia theobromae, isolated from a coconut tree and a human patient

ABSTRACT Lasiodiplodia theobromae is a phytopathogenic fungus that causes diseases not only in a broad number of plant hosts but also occasionally in humans. The capacity of L. theobromae to produce bioactive metabolites at 25 C (environmental mean temperature) and at 37 C (body mean temperature) was investigated. Two strains, CAA019 and CBS339.90, isolated respectively from a coconut tree and a human patient were characterized. The phytotoxicity and cytotoxicity (on mammalian cells) of the secretomes of both strains of L. theobromae were investigated. Also, phytotoxicity and cytotoxicity of pure compounds were evaluated. The phytotoxicity of the secretome of strain CAA019 was higher than the phytotoxicity of the secretome of strain CBS339.90 at 25 C. However, the phytotoxicity for both strains decreased when they were grown at 37 C. Only the secretome of strain CBS339.90 grown at 37 C induced up to 90% Vero and 3T3 cell mortality. This supports the presence of different metabolites in the secretome of strains CAA019 and CBS339.90. Metabolites typical of L. theobromae were isolated and identified from organic extracts of the secretome of both strains (e.g., 3-indolecarboxylic acid, jasmonic acid, lasiodiplodin, four substituted 2-dihydrofuranones, two melleins, and cyclo-(Trp-Ala)). Also, metabolites such as scytalone, not previously reported for this species, were isolated and identified. Metabolite production is affected by strain and temperature. In fact, some metabolites are strain specific (e.g., lasiodiplodin) and some metabolites are temperature specific (e.g., jasmonic acid). Although more strains should be characterized, it may be anticipated that temperature tuning of secondary-metabolite production emerges as a putative contributing factor in the modulation of L. theobromae pathogenicity towards plants, and also towards mammalian cells.