Ana Maria Mazotto

Genomic and proteomic analyses of the coral pathogen Vibrio coralliilyticus reveal a diverse virulence repertoire.

Vibrio coralliilyticus has been implicated as an important pathogen of coral species worldwide. In this study, the nearly complete genome of Vibrio coralliilyticus strain P1 (LMG23696) was sequenced and proteases implicated in virulence of the strain were specifically investigated. The genome sequence of P1 (5 513 256 bp in size) consisted of 5222 coding sequences and 58 RNA genes (53 tRNAs and at least 5 rRNAs). Seventeen metalloprotease and effector (vgrG, hlyA and hcp) genes were identified in the genome and expressed proteases were also detected in the secretome of P1. As the VcpA zinc-metalloprotease has been considered an important virulence factor of V. coralliilyticus, a vcpA deletion mutant was constructed to evaluate the effect of this gene in animal pathogenesis. Both wild-type and mutant (ΔvcpA) strains exhibited similar virulence characteristics that resulted in high mortality in Artemia and Drosophila pathogenicity bioassays and strong photosystem II inactivation of the coral dinoflagellate endosymbiont (Symbiodinium). In contrast, the ΔvcpA mutant demonstrated higher hemolytic activity and secreted 18 proteins not secreted by the wild type. These proteins included four types of metalloproteases, a chitinase, a hemolysin-related protein RbmC, the Hcp protein and 12 hypothetical proteins. Overall, the results of this study indicate that V. coralliilyticus strain P1 has a diverse virulence repertoire that possibly enables this bacterium to be an efficient animal pathogen.

Keratinolytic activity of Bacillus subtilis AMR using human hair.

Aims:  To determine the ability of a novel Bacillus subtilis AMR isolated from poultry waste to hydrolyse human hair producing peptidases including keratinases and hair keratin peptides.

Keratinase Production by Three Bacillus spp. Using Feather Meal and Whole Feather as Substrate in a Submerged Fermentation

Three Bacillus species (B. subtilis LFB-FIOCRUZ 1270, B. subtilis LFB-FIOCRUZ 1273, and B. licheniformis LFB-FIOCRUZ 1274), isolated from the poultry industry, were evaluated for keratinase production using feathers or feather meal as the sole carbon and nitrogen sources in a submerged fermentation. The three Bacillus spp. produced extracellular keratinases and peptidases after 7 days. Feather meal was the best substrate for keratinase and peptidase production in B. subtilis 1273, with 412 U/mL and 463 U/ml. The three strains were able to degrade feather meal (62–75%) and feather (40–95%) producing 3.9–4.4 mg/ml of soluble protein in feather meal medium and 1.9–3.3 mg/ml when feather medium was used. The three strains produced serine peptidases with keratinase and gelatinase activity. B. subtilis 1273 was the strain which exhibited the highest enzymatic activity.

Natural Products: Insights into Leishmaniasis Inflammatory Response

Leishmaniasis is a vector-borne disease that affects several populations worldwide, against which there are no vaccines available and the chemotherapy is highly toxic. Depending on the species causing the infection, the disease is characterized by commitment of tissues, including the skin, mucous membranes, and internal organs. Despite the relevance of host inflammatory mediators on parasite burden control, Leishmania and host immune cells interaction may generate an exacerbated proinflammatory response that plays an important role in the development of leishmaniasis clinical manifestations. Plant-derived natural products have been recognized as bioactive agents with several properties, including anti-protozoal and anti-inflammatory activities. The present review focuses on the antileishmanial activity of plant-derived natural products that are able to modulate the inflammatory response in vitro and in vivo. The capability of crude extracts and some isolated substances in promoting an anti-inflammatory response during Leishmania infection may be used as part of an effective strategy to fight the disease.

Carbonic anhydrases from Trypanosoma and Leishmania as anti-protozoan drug targets

Trypanosoma cruzi and Leishmania spp. are protozoa of the Trypanosomatidae family, being the etiological agents of two widespread parasitic diseases, Chagas disease and leishmaniasis, respectively. Both parasites are the focus of worldwide research with the aim to find effective and less toxic drugs than the few ones available so far, and for controlling the spread of the diseases. Carbonic anhydrases (CAs, EC 4.2.1.1) belonging to the α- and β-class were recently identified in these protozoans and several studies suggested that they could be new targets for drug development. Sulfonamide, thiol and hydroxamate inhibitors effectively inhibited the α-CA from T. cruzi (TcCA) and the β-CA from L. donovani chagasi (LdccCA) in vitro, and some of them also showed in vivo efficacy in inhibiting the growth of the parasites in animal models of Chagas disease and leishmaniasis. As few therapeutic options are presently available for these orphan diseases, protozoan CA inhibition may represent a novel strategy to address this stringent health problem.

Increased expression of keratinase and other peptidases by Candida parapsilosis mutants

Keratinases are enzymes of great importance involved in pathogenic processes of some fungi. They also have a widespread ecological role since they are responsible for the degradation and recycling of keratin. On the one hand, studying them furthers our knowledge of pathogenicity mechanisms, which has important implications for human health, and on the other hand, understanding their ecological role in keratin recycling has biotechnological potential. Here, a wild-type keratinolytic Candida parapsilosis strain isolated from a poultry farm was treated with ethyl methanesulfonate in order to generate mutants with increased keratinase activity. Mutants were then cultured on media with keratin extracted from chicken feathers as the sole source of nitrogen and carbon. Approximately 500 mutants were screened and compared with the described keratinolytic wild type. Three strains, H36, I7 and J5, showed enhanced keratinase activity. The wild-type strain produced 80 U/mL of keratinolytic activity, strain H36 produced 110 U/mL, strain I7, 130 U/mL, and strain J5, 140 U/mL. A 70% increase in enzyme activity was recorded for strain J5. Enzymatic activity was evaluated by zymograms with proteic substrates. A peptidase migrating at 100 kDa was detected with keratin, bovine serum albumin and casein. In addition, a peptidase with a molecular mass of 50 kDa was observed with casein in the wild-type strain and in mutants H36 and J5. Gelatinase activity was detected at 60 kDa. A single band of 35 kDa was found in wild-type C. parapsilosis and in mutants with hemoglobin substrate.

Antileishmanial activity of sulphonamide nanoemulsions targeting the β-carbonic anhydrase from Leishmania species

Abstract The β-carbonic anhydrase (CA, EC 4.2.1.1) from Leishmania spp. (LdcCA) is effectively inhibited by aromatic/heterocyclic sulphonamides, in the low nanomolar range, but no in vitro antileishmanial activity was detected for such compounds. We formulated some of these sulphonamides as nanoemulsions (NEs) in clove oil, and tested them in vitro against Leishmania infantum MHOM/BR/1974/PP75 and Leishmania amazonensis IFLA/BR/1967/PH8 strains. Interesting inhibitory concentrations IC50 were observed for some of the sulphonamides NEs, with IC50 as low as 3.90 µM (NE-3F) and 2.24 µM (NE-5B) for L. amazonensis and 3.47 µM (NE-5B) for L. infantum. Some of the investigated NEs displayed toxicity for macrophages beyond the parasites. For the same nonoemulsions, a selective index (SI) greater than for Amphotericin B. Haemolytic assay using human red blood cells indicate that the NEs were less cytotoxic than amphotericin B, a widely used antifungal agent. NEs demonstrated to be an excellent strategy for increasing the penetration of these hydrophilic drugs through membranes, with a huge increase of efficacy over the sulphonamide CA inhibitor (CAI) alone.